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LCquan 2.7 Quantitative Analysis User Guide

1. Parameter Description Detector Streams in When a raw file is open displays the list of detector streams that Current Raw File are stored in the current raw file The format of the list items is Generic Detector Stream Name Instrument Name Model Name Channel Name Column Headings Parameter Displays a column of descriptive text for the item Value Displays a column of data for the given parameter Shortcut Menu Open Raw File Create Method view only Opens the Select Raw File dialog box so you can select the raw file to load Lock against All Prevents changes to a raw file or parameters The lock icon is red Change and the lock is closed Unlock Makes the pane editable The lock icon is green and open Copy to Clipboard Copies selected data to the Clipboard You can then paste the data from the Clipboard buffer into a document This view displays the list of unique filters that are stored in the current raw file in table format Figure 158 Figure 158 Filter List view c SRM ms2 309 07 cid 42 00 205 02 205 02 c SRM ms2 314 10 cid 40 00 209 96 209 96 c SRM ms2 330 1 3 cid 20 00 70 15 192 07 c SRM ms2 470 18 cid 26 00 180 07 274 08 The contents of the grid are read only When the view is locked lock icon is red you cannot select any menu item in the shortcut menu that changes the raw file or parameters Menu items that would cause changes are grayed out For more info
2. 142 Generating a Peak List n u u u aw eh a 28 oho ae kee es bee Meee 143 Identifying Gonipoments vs2 cacaatsaueaa eaten rneer 147 Exporting the Active raw File to Qual Browser 1 0 0 0 eee eee eee 149 D ta Views nennou iaon A Sor a ay Sor sesso scr rohie lag panera A eae sens 150 Toolbar for Selectable Views iis c ccsed te gudeen he tated ie ued tee 9 150 Chromatogram View oie sa oats Alok gate ea ache ad ang ae eee 153 Chromatogram List View tis cated st cians ised ayienh doe tesa tk tea bn caret ake Dat 157 Error Report Views vc cc neure eee ee OE oe Se ea SS 158 Filter List View a tele arousal olen Begg id ge IE eat Sse ar 159 General Parameters Plot View lt oehca crapedd ce nthad cescid Vee cies 161 Instrument Method View vs iovs c series tide ware seo nie theraed Awan a ee ate 163 Mass EISE VIEW set Ae wire ig Bs atte adel tae A Redes ca Muti aad A 164 Peak List Views tenir edion ie vei tet e at Mae mais tack 165 SAP le Info View airneisi aca aut a ia a a aa k aE nee ye 169 Spectrum View osse sirita ei ela PAE whe al eee ees UY sal as 170 Status Plot View scd aos Devt s ated a caine Lema eee ang 172 Status Report View sss cite a EE a aoe og ales wae wk 174 Tune Wheto View osc ce alias Yon satin nsive wresauns gu Meets milano Ad seapeca yn el rary 176 Multi Peak Plot View for Review Only nysdicw cadens nner enna 177 Thermo Scientific Thermo Scientific Contents Creating a Processing Method
3. Click Next and Processing Parameters wes i Fi if page Import from existing workbook Select Workbook and Finis i age Do not import Click Next Click Next pag prior data and Finish Click Next and Finish Basic instrument method instrument created for configured No Method lt instruments imported Yes Instrument method updated for configured instruments Instrument Setup view gt Select Legacy Files page Startup Method page Instrument Method page Shutdown Method page Acquisition view y Explore See Acquisition flow diagram view Click Method Create Explore Method page Click Review Review the Explore Results page Click Setup Setup Sequence page Click Acquire Run Sequence dialog box Click OK 412 LCquan User Guide Quantitate view See Quantitate flow diagram Click Method Create Method page i Click Sequence Create Sequence page gt gt Survey page Click Survey gt Review All Results page Click Review Review Reports page Click Reports j Thermo Scientific B LCquan Flow Diagrams LCquan for Web Access Flow Diagram LCquan for Web Access Flow Diagram The LCquan for Web Access flow diagram shows the steps for creating a new study or workbook and the two main windows of the interface Create new study or Study and Workbook Names page Click Next Import Raw Files pag
4. 0ccc cece eee ee eee tenes 181 Creating a Processing Method with the New Method Wizard 182 Statt the Wigan tat stam he haan ab ct Mal nan tale Dai aay a aa My 182 Create of Import a Method oo 3 aft ik bale go Bales Ae bee ae oe 183 Import a Processing Method ieiu d speed each Gs eee eee Roe 184 Specify Calibration Standards acho saa eae ore i hea eae S 184 Select araw Mle gue ao Cpe ee Rae me Rape R elon Van EAT 184 Conmpletethe Wizard pass coe etene he nn oeiebins wane ede 185 Specifying Method Identification i 1 fesevi da dears dude aeeee eames 186 Chromatogram Definition Area ad vue seat Rate eche bad iee eal 187 Retention Wimiea ted sri sa sins ahaa ais ate unnn nurnerr rere 191 Peak Integration Area oy o o s auths sat8 Xe ddd Oe Abie Ashita Lake 192 Peak Identification Area o s a sores Gory nantes Se eine eae Wee es 193 Ion Ratio Confirmation Area wae 2245 oes PAGS a De OS PA AS 194 Summing Ions Dialog Box cio ced heh seid cum ak ce Kh ad oe owing 196 Chromatogram Normalization 0 onic often he Sa ee ee 198 Specifying Component ID Parameters and Integrating the Peaks 201 Defining Peak Integration Parameters onde sce tae te ees wae 204 TRG Detection Method 4 0 leite asnosi enii nea awn nanhe bakit oaks 217 Identification Options Saale saith s ele rnern eere 232 Ion Ratio Confirmation of Analytes Example 00020008 234 Specifying Method Calibration sia su Pes oe depute eek wae wne ke 23
5. Next Row Highlights the sequence row that follows the currently highlighted row Clear All Deletes all samples from the sequence Deletes all empty sequence rows Compress pty seq PERE Acquisition Sequence History The Acquisition Sequence History pane displays the history of all sequences and samples acquired by the current workbook along with other relevant information such as the operator start and end times status and comment You can modify the Acquisition Sequence History pane see Table 13 To populate a sequence from the history to the sequence grid 1 Select a sequence or combination of sequences in the Acquisition Sequence History pane Figure 26 2 Drag the sequence to the Acquisition Sequence grid The LCquan application confirms that you want to replace the sequence Figure 26 Acquisition Sequence History pane E Acquisition Sequence History 4 AcqSequence 1 AcqSequence 2 i Info Operator tester Start Time 9 13 2005 1 38 27 PM End Time 9 13 2005 1 49 18 PM t Startup Method Not Used JI Shutdown Method Not Used I Comment lt None gt Status Complete k Component DA ComponentOl J CalLevels U Qc Levels H steroids02_050913133826 Thermo Scientific LCquan User Guide 35 2 Preparing to Perform Quantitative Analysis Using the Setup Sequence View 36 LCquan User Guide Table 13 Acquisition Sequence History pane parameters Sheet 1 of 2 P
6. 88 LCquan User Guide Thermo Scientific 2 Preparing to Perform Quantitative Analysis Using the Status View Table 29 Chromatogram pane shortcut menu commands Command Description Show Component Displays real time chromatograms corresponding to the Traces components you defined in the processing method Show User Traces Displays real time chromatograms that you defined by using the settings in the Realtime Display Settings dialog box For more information see Realtime Display Settings on page 84 Show Spectrum Trace Displays the mass spectrum in the spectrum pane Filter Spectrum Applies a scan filter to the acquired data The LCquan application creates scan filters from the instrument settings that you specified in the Instrument Setup window Link Trace Time Bases Displays the same time range for all chromatograms Use User Defined Time Displays chromatograms with a time range between the times Range specified in the Min RT and Max RT boxes in the Realtime Display Settings dialog box For more information see Realtime Display Settings on page 84 Realtime Display Displays the Realtime Display Settings dialog box where you can Settings specify the properties of the real time chromatograms that the LCquan application displays in the Chromatogram and Spectrum panes of the Acquisition window Display Raw File Displays the Select Raw File dialog box where you can open a previously acquired
7. Number of The number of component names you want to generate The Components derived names are a concatenate of a base name and an incremental number for each component For example if the base name is Component_ and the number of components is 3 the component names are Component_l Component_2 and Component_3 Component Base Specifies the component base name Name Specify heading labels and displayed values for each user defined column see Table 20 To specify labels and values 1 Type a label for the column heading in the acquisition sequence header and type a value for the column or select a macro from the list Figure 38 When you select a macro name the LCquan application displays the actual text that corresponds to the macro name in the Sequence Header For example when you select Workbook the LCquan application displays the name of the current workbook in the field in the Sequence Header The values you enter here create the user defined column headings in your sequence and specify a static value or macro for each sample in the sequence These names and values are displayed at the top of the Acquisition Setup Sequence view See User Defined Columns Values Examples on page 50 Figure 38 User labels and values page User Label 1 User Label 2 Client User Label 3 Laboratory User Label 4 Company Name User Label 5 Phone 2 Click Next User Value 1 Study v User Value 2 fo
8. 2011 Thermo Fisher Scientific Inc All rights reserved BioWorks Exactive Foundation LCQ LCquan Q Exactive and Web Access are trademarks and Accela LTQ Surveyor TSQ Quantum and Xcalibur are registered trademarks of Thermo Fisher Scientific Inc in the United States The following are registered trademarks in the United States and other countries Adobe and Flash are registered trademarks of Adobe Systems Incorporated Excel Microsoft Vista and Windows are registered trademarks of Microsoft Corporation Oracle is a registered trademark of Oracle Corporation and or its affiliates All other trademarks are the property of Thermo Fisher Scientific Inc and its subsidiaries Thermo Fisher Scientific Inc provides this document to its customers with a product purchase to use in the product operation This document is copyright protected and any reproduction of the whole or any part of this document is strictly prohibited except with the written authorization of Thermo Fisher Scientific Inc The contents of this document are subject to change without notice All technical information in this document is for reference purposes only System configurations and specifications in this document supersede all previous information received by the purchaser Thermo Fisher Scientific Inc makes no representations that this document is complete accurate or error free and assumes no responsibility and will not be liable for any errors omissio
9. A se ae Inst rument Setup LCquan own TE i ie ie a e le Run Application Root Folder File Tracking File Section Configuration Grid Column Settings Acquisition Section F Acquisition Run Dialog Explore Section Quantitate Section Allow changes to selected sample info in Allow changes to column labels in results Prompt user for Comment after manual inl Normalize quan chromatogram plots to d Allow Results Export Allow Manual Integration Allow User Integration Allow Calibration Settings to be changed Create Reports v Allow changes to column labels in proces Permission Level Disallowed Allowed Other requirements oy ent Secure Template Folder il i 3 2 In the Authorization Manager do the following Thermo Scientific Select a user group in the Secure Groups list Click Expand Tree to show the entire list of controlled features for the application From the list click the plus sign before the LCquan folder Click the plus sign before the Quantitate Section folder Select Normalize Quan Chromatogram Plots to Detected Peak The Permission Level options become available Select the Allowed option and click OK LCquan User Guide 199 4 Creating a Processing Method Specifying Method Identification To normalize the chromatogram plot so that the highest peak is 100 1 Choose Start gt All Programs gt Thermo
10. Add or Change Peak Comment 264 LCquan User Guide Opens the Chromatogram Comment dialog box where you can enter a comment to be associated with the selected chromatogram There is a separate comment for each sample component or integration type chromatogram For more information see Adding Comments to the Chromatogram Display on page 374 in Chapter 6 Processing the raw Files and Reviewing the Analytical Results Thermo Scientific Thermo Scientific 4 Creating a Processing Method Data Views Table 102 Chromatogram view parameters Sheet 3 of 4 Parameter User Peak Detection Settings Description Opens the User Identification Settings dialog box where you can change some of the detection and integration parameters without returning to the Identification page of the Create Method view in the Quantitate window For more information see Specifying Additional Peak Detection Criteria on page 346 in Chapter 6 Processing the raw Files and Reviewing the Analytical Results Note Selecting User Identification Settings automatically sets the integration type in the corresponding row in the grid to User Integration Display Options Manually Add Peak Opens the Chromatogram Display Options dialog box where you can select view options For more information see Chromatogram Display Options on page 259 Manually places a baseline on the plot and sets the current integration method t
11. Command Description Keyboard shortcut New Processing Method Opens the New Method Wizard that steps you through a ALT O gt N Wizard procedure for creating a new processing method or importing an existing processing method Display Options Opens the Chromatogram Display Options dialog box where Chromatogram you can label peaks and enter the peak width displayed at both ends of a chromatogram peak For more information see Chromatogram Display Options on page 259 in Chapter 4 Creating a Processing Method Display Options Opens the Spectrum Display Options dialog box where you can Spectrum modify the appearance of spectrum plots For more information see Spectrum Display Options dialog box parameters on page 92 in Chapter 2 Preparing to Perform Quantitative Analysis Display Options Opens the Ion Ratio Chromatogram Display Options dialog box Ion Ratio Chro where you can label peaks and enter the peak width displayed at both ends of a chromatogram peak For more information see Ion Ratio Chromatogram Display Options on page 271 in Chapter 4 Creating a Processing Method Save Chromatograms in Saves chromatogram peak integration information This speeds up ALT O gt S file data processing done after the information is saved because existing chromatograms are not integrated again Commands Specific to Both Explore and Quantitate Windows Table 166 Commands specific to both the Explore and Quan
12. Include C V s 2 238 LCquan User Guide A 50 000 f2 c_Mid 175 000 15 00 75 000 j3 QC_Hi 875 000 15 00 100 000 0 000 0 00 250 000 500 000 750 000 1000 000 0 000 Table 92 Target Compound area parameters Sheet 1 of 3 Parameter General Controls Description ISTD Isotope Specifies the ISTD to associate with the current target compound All defined ISTDs are in the list Opens the Correction for Isotope Contribution dialog box so you can correct for an impurity in the internal standard compound that elutes at the same time as the target compound correct for an impurity in the target compound that elutes at the same time as the internal standard or both For more information see Correction for Isotope Contribution on page 245 Calibration Curve Type Specifies the calibration curve type to be used for the current component Not all Response Origin and Weighting selections are available with every calibration curve type For details see Table 93 Response Area Specifies that this area value will be used in response calculations Height Specifies that this height value will be used in response calculations Thermo Scientific 4 Creating a Processing Method Specifying Method Calibration Table 92 Target Compound area parameters Sheet 2 of 3 Parameter Description Origin Determine how to use the origin when the calibration curve is generated
13. RT 1 91 2 66 3 tm ey t Area Ratio a oO a oa 4 70 2 13 168 18 20 22 26 28 3 0 Time min Time min For Help press F1 Powers Dana NOT SAVED 7 10 2008 9 204M 74 2 85 2 97 o L Relative Intensity Relative Intensity Reviewing the Unknowns To review and rework the results for the unknowns 1 Click the Unknowns tab at the bottom of the Result list to display the peak integration results for the unknown samples 2 In the Component list select the internal standard 3 Inspect each file in the Result list and check for peak detection and integration problems 366 LCquan User Guide Thermo Scientific 6 Processing the raw Files and Reviewing the Analytical Results Reviewing All Results 4 Inspect the chromatogram in the Chromatogram pane for each file a Ensure that the LCquan application found the peak b Ensure that the shaded area accurately represents the contribution of the component to the chromatogram e To modify the peak detection and integration settings go to the topic Modifying the Peak Detection and Integration Settings on page 345 e To integrate a peak manually go to the topic Manually Integrating Peaks on page 363 5 In the Component list select a target compound 6 Repeat step 3 and step 4 for each target compound 7 When you are satisfied that
14. Thermo Scientific LCquan User Guide 83 2 Preparing to Perform Quantitative Analysis Using the Status View Realtime Display Settings 84 LCquan User Guide Use the Realtime Display Settings dialog box Figure 50 to specify the properties of the chromatograms that the LCquan application displays This dialog box consists of two areas the Options area and the User defined traces area Use the settings in the Options area to specify the types and number of chromatograms to display the time range information for the chromatograms and whether or not to display the spectrum trace in the Spectrum pane see Table 28 Many of these options also appear in a shortcut menu that you access by right clicking the Acquisition Status view chromatogram pane Figure 50 Realtime Display Settings dialog box Realtime Display Settings Options 5 Max number of visible chromatograms C Link chromatogram time ranges Show user defined traces C Show spectrum trace C Use user defined time range Auto update spectrum to last scan Min AT 0 000 Max AT 1 000 User defined traces Type Scan filter M Tic M TiC c Full ms2 303 30 40 100 00 310 00 M TIC c Full ms2 315 30 40 100 00 320 00 M TiC c Full ms2 331 30 40 100 00 350 00 M Tic c Full ms2 363 30 40 00 150 00 375 2 Trace properties Title Move Trace Trace type TIC Sean filter c Full
15. __ r ICIS Peak Integration _ p Peak Identification Detector Smoothing Peak Detection Algorithm i MS ficis Advanced HighestPeak Min Peak Height S N g Trace Nearest RT 3 0 Tic Baseline Window fo lon ratio confirmation Mess n c Area Noise Factor E I Enabled lon ratio using Area z Target Window a Pek Noise Factor T0 ECIJA p c SRM ms2 330 13 cid 20 00 70 15 192 0 z I Constrain Peak Width Em m Retention Time Peak Ht J faa xpected min fr g2 Window sec 20 0 ll dow sec Tailing Factor fra I Use as RT Ret View Width min 0 75 Window Relative Qualifier ion coelution IT Adjust Using z f Absolute ozs min Apply Cancel Save As Defaut Help Chromatogram Spectrum APN_004 TIC SM 5 RT 1 52 2 27 NL 9 79E3 APN_004 328 RT 1 91 NL 3 97E3 F c SRM ms2 330 13 ecid 20 00 70 15 192 07 F SRM ms2 330 13 cid 20 00 70 15 192 07 __ 100 400 RT 1 90 90 80 70 60 50 50 40 40 20 30 20 20 10 0 30 80 70 60 Relative Intensity Relative Intensity 19 Time min Powers Dana eal NOT SAVED 6 30 2008 11 02 aM 4 186 LCquan User Guide Thermo Scientific 4 Creating a Processing Method Specifying Method Identification To open the Identification page of the Create Method view When the left navigation pane is displayed a Click Quantitate b Click the Method icon c Click the Identificat
16. LCquan User Guide 91 2 Preparing to Perform Quantitative Analysis Using the Status View 92 LCquan User Guide To customize the spectrum display 1 Right click on the spectrum pane and choose Display Options from the shortcut menu The Spectrum Display Options dialog box opens Figure 54 Figure 54 Spectrum Display Options dialog box Spectrum Display Options Style Label with V Mass Label Styles Offset l Rotated Boxed 2 Decimals Relative to o o r Plotting Automatic Point to point Stick Size foo Axis offset Tx Label threshold 10 0 Cancel Apply Help 2 Specify the labeling plotting and axis display styles you want in the spectrum pane see Table 33 Table 31 Spectrum Display Options dialog box parameters Sheet 1 of 2 Parameter Description Label With Specifies the data attributes to show in the data plots Mass Labels the mass value above the spectral line Relative to Specifies the mass offset value The displayed value is actually the defined chosen mass offset subtracted from the actual mass Decimals Specifies the number of decimal places for the mass label Label Styles Specifies the labeling styles displayed with the data Offset Moves the label a defined distance from the data Rotated Rotates the labels above the peaks to rotate degrees Boxed Displays boxes around the labels above the peaks Size Define t
17. Switches to the Status view of the Acquisition window Process Using Current Acquisition List Switches to the processing view with the current acquired sample list Stop Current Sample and Pause Stops acquiring the current sample and pauses the acquisition Stop Current Sequence and Pause To open the Status view Stops acquiring the current sequence and pauses the acquisition When the left navigation pane is displayed a Click Acquisition b Click the Status icon When the left navigation pane is hidden a Choose View gt Section Selection gt Acquisition Section b Choose View gt Step Selector gt Acquisition Status Using the Status View Control Buttons 82 The control buttons on the Status view of the Acquisition window let you control the acquisition process Figure 49 Figure 49 Status view control buttons To start stop or pause an acquisition e To start an acquisition or restart an acquisition that has been paused click e To pause an acquisition after the current sample has been completely acquired click To restart the acquisition click again To stop an acquisition click and choose one of the following e Stop acquisition for the current sample and pause e Stop acquisition for the current sequence and pause LCquan User Guide Thermo Scientific 2 Preparing to Perform Quantitative Analysis Using the Status View e Stop acquisition for all
18. x 0 0 T T T T 1 0 0 0 5 1 0 1 5 2 0 25 Amount of target compound A Response for target compound response for ISTD Sample Types Each quantitative analysis consists of a number or sequence of samples The sequence represents the order of sample analysis A quantification sequence contains e One or more standards e One or more unknown samples For more demanding applications you can also use optional quality control QC samples and blank samples Standards A calibration standard is a sample containing known amounts of all target components The purpose of a standard is to measure the response of the instrument to the target components so that the LCquan application can generate a calibration curve for each component Unknowns An unknown sample is one containing unknown amounts of the target components The LCquan application performs quantitative analysis on any sample defined as an unknown sample 408 LCquan User Guide Thermo Scientific QCs Blanks Thermo Scientific A Quantitative Analysis Overview Sample Types A QC sample contains a known amount of one or more specific target compounds The LCquan application places QC samples in the sequence so that it can test quantitative analysis results for quality assurance purposes After the QC sample is analyzed it compares the measured quantity with the expected value and an acceptability range The quantitative analysis of a QC sample is classified as
19. Calibrate using Internal Standards C Calibrate using External Standards 2 Click Next Select a raw file Select a raw file to test the peak detection and integration parameters To select a raw file 1 Click Browse 2 Browse to find the raw file in the raw files folder in your workbook 184 LCquan User Guide Thermo Scientific 4 Creating a Processing Method Creating a Processing Method with the New Method Wizard 3 Click Open Note In general open a raw file that corresponds to a low concentration calibration standard Note To help you determine the parameters of the processing method use a raw file from a similar past experiment 4 Click Next Complete the Wizard Click Finish to exit the New Method Wizard The Create Method view in the Quantitate window opens to the Identification page You are now ready to begin building the processing method Thermo Scientific LCquan User Guide 185 4 Creating a Processing Method Specifying Method Identification Specifying Method Identification The LCquan application requires component identification information to associate the internal standard and target compounds with chromatogram peaks Figure 113 Figure 113 Quantitate Create Method Identification window 6 Thermo LCquan 3 Drugs Workbook File Zoom View Apps Options Help BSR SLE FR OOOSCSCSS x i BOlO Instruments Identification Calibration m Chromatogram Definition
20. Ignore Specifies that the origin is not included as a valid point in the calibration curve when the curve is generated When you select this option the calibration curve might or might not pass through the origin Force Specifies that the calibration curve passes through the origin of the data point plot when the calibration curve is generated Include Specifies that the origin is included as a single data point in the calculation of the calibration curve When you select this option the calibration curve might or might not pass through the origin Weighting Specify how the individual data points are weighted in calculating the calibration curve Equal Weights all calibration data points equally during the least squares regression calculation of the calibration curve 1 X Specifies a weighting of 1 X for all calibration data points during the least squares regression calculation of the calibration curve Calibrants are weighted by the inverse of their quantity 1 X 2 Specifies a weighting of 1 X 2 for all calibration data points during the least squares regression calculation of the calibration curve Calibrants are weighted by the inverse of the square of their quantity 1 Y Specifies a weighting of 1 Y for all calibration data points during the least squares regression calculation of the calibration curve Calibrants are weighted by the inverse of their response or response ratio 1 Y 2 Specifies a weig
21. Levels a O a J ao m Component02 Component03 Camponent04 Table 95 Select Component dialog box parameters Sheet 1 of 2 Parameter Description Component Table Component Select the component with the level tables you want to use from the list of components in the sequence Cal Level Table Cal Level Displays the calibration levels for the selected component Amount Displays the amounts of the target compound used for each calibration level LCquan User Guide 243 4 Creating a Processing Method Specifying Method Calibration Table 95 Select Component dialog box parameters Sheet 2 of 2 Parameter Description QC Level Table Use QC samples containing known amounts of a component to help ensure the accuracy of an analysis The LCquan application measures the quantity of the QC component in the same manner as it measures that of unknown components The measured quantity is then compared with a user defined expected quantity and a user defined percent test C Level Displays the quality control levels for the selected component play quality P The LCquan application can accommodate up to 15 QC levels Amount Displays the amounts of the target compound used for each QC quality control level Test Displays a value for the acceptable difference as a percent between the known amount and calculated measured amount of each QC level Button Select Selects the component name yo
22. Parameter Displays a column of descriptive text for the item Value Displays a column of data for the given parameter Shortcut Menu Open Raw File Create Method view only Opens the Select Raw File dialog box where you can select the raw file to load Lock against All Prevents changes to a raw file or parameters The lock icon is red Change and the lock is closed Unlock Makes the pane editable The lock icon is green and the lock is open Copy to Clipboard Copies a chromatogram mass spectrum or calibration curve to the Clipboard You can then paste the data from the Clipboard buffer into a document Tune Method View 288 LCquan User Guide This view displays the tune method stored in the current raw file during acquisition Figure 166 Figure 166 Tune Method view Calibration File Name Tune File Name Auto Peak Width Setting Positive Polarity Q1 MS Spray Voltage Vaporizer Temperature Sheath Gas Pressure lon Sweep Gas Pressure Aux Gas Pressure Capillary Temperature C Xcalibur methods Alpraz lon Source Type C Kcaliburimethods1 0250 E HESI YES 3000 330 70 0 0 30 380 Thermo Scientific 4 Creating a Processing Method Data Views When the view is locked lock icon is red you cannot select any menu item in the shortcut menu that changes the raw file Menu items that would cause changes are grayed out For more information about loc
23. Reset Scaling Resets scaling to the default pane Copy to Clipboard Chromatogram Display Options Copies selected data to the Clipboard You can then paste the data from the Clipboard buffer into a document Use the Chromatogram Display Options dialog box to modify the appearance of the chromatogram Figure 151 Table 101 lists the parameters for the Chromatogram Display Options dialog box LCquan User Guide 259 4 Creating a Processing Method Data Views Figure 151 Chromatogram Display Options dialog box Pa Display Options Style r Label with Platting Retention time 2 Decimals Point To Point IT Name Stick l Base peak l Area gt When no peak found Signaltonoise Height MV Show apex time labels r Label Styles I Offset Size T Rotated r ris offset l Boxed rx Label threshold 4 0 0 OK Cancel Apply Help To display this dialog box When the left navigation pane is displayed a Click Quantitate b Click the Method icon c Right click the data pane of the Chromatogram view and choose Display Options from the shortcut menu When the left navigation pane is hidden a Choose View gt Section Selection gt Quantitate Section b Choose View gt Step Selector gt Quantitate Method c Right click the Chromatogram pane and choose Display Options Table 101 Chromatogram Display Options dialog box parameters Sheet 1 of 2 Parameter Descr
24. Tune Method shortcut menu commands Explore 177 Review 289 U unknowns acquisition setup 31 overview 408 unknowns reviewing 366 upper quantitation limit 405 User Identification Settings Avalon Integration parameters 360 Detection parameters 350 Genesis Advanced parameters 355 ICIS Advanced parameters 359 ICIS Integration parameters 356 Identification parameters 347 user identification settings advanced parameters 347 Avalon integration 360 detection 350 Genesis advanced parameters 354 integration 363 ICIS advanced parameters 363 integration 356 user labels setting 101 V validation Web Access 12 variables quantitation with internal standards 407 separation by LC 402 View menu commands 427 W Web Access flow diagram 413 LCquan for 12 Wizards New Acquisition Sequence 38 New Method 182 New Processing Sequence 296 New Study or Workbook 14 Save As 22 Workbook Summary Information parameters 27 workbooks creating 13 description 8 locking 397 Thermo Scientific Index U locking automatically after creating reports 398 not locking automatically after creating reports 399 saving 400 Z Zoom menu commands 430 zooming in Chromatogram pane 83 in Spectrum pane 90 toolbar buttons 326 LCquan User Guide 445
25. Zooms out the traces along the y axis vertically to display more range and less detail Note This feature is not available in the Instruments window LCquan User Guide 433 c LCquan Menu and Toolbar Reference Toolbar Table 174 Toolbar buttons Sheet 3 of 4 Button Normalize Description Readjusts the display of all peaks in the trace based on the largest peak height Note This feature is not available in the Instruments window Zoom In X Zooms in the traces along the x axis horizontally to display more detail and less range Note This feature is not available in the Instruments window Zoom Out X 6 Zooms out the traces along the x axis horizontally to display more range and less detail Note This feature is not available in the Instruments window Display All Resets the display of the x axis to include the entire displayable range Note This feature is not available in the Instruments window Reset Scaling Resets both x and y axes to display the maximum amount of data Note This feature is not available in the Instruments window Add a Peak k 0 Manually defines a peak in a chromatogram displayed on the Survey or Review All Results views in the Quantitate window The feature is available when no peak is detected To adda peak 1 Select the component which is either listed in the pane to the right of the window or on a tab at the
26. e The Result grid pane contains the peak integration results It has three display tabs at the bottom All Standards and QCs The Result grid also uses a system of color shading to indicate the status of individual samples For information about the Result grid shortcut menu see Result grid shortcut menu on page 327 The Component list pane contains the components that are defined in the current P P P processing method When you select a component in the Component list all the other page elements are updated to display the results for that component Instead of a Component list on the right side of the view you can display the components on tabs above the grid Choose View gt Component Selector Bar from the window menu For information about the Component list shortcut menu see Component List Shortcut Menu on page 332 The Calibration Curve pane displays a plot of area ratio or area versus concentration or amount for the selected component The ISTD Chromatogram pane displays a plot of relative intensity versus retention time of the internal standard in the current sample if available The Chromatogram pane displays a plot of relative intensity versus retention time of the selected component in the current sample Navigation Features In the Chromatogram pane and in the ISTD Chromatogram pane use the toolbar buttons to zoom in and out along the x and y axes to show more detail see Table 124 Table 124 Zoo
27. s response e Analyzing the samples e Reviewing the results This manual does not describe sample preparation and chromatographic method development This manual assumes that you have met these important prerequisites to achieving high quality quantitative analysis For guidance in these areas refer to the getting started and hardware manuals for your autosampler LC pump or MS pump and mass spectrometer Considering the Variables of Quantitative Analysis by LC MS MS The combination of liquid chromatography LC and mass spectroscopy MS sets up a unique set of considerations If you are familiar with quantitative analysis using one or the other of these analytical tools the information in the following topics will prove useful e Using LC for Analyte Separation e Using MS MS for Analyte Detection Using LC for Analyte Separation When working with LC systems you become accustomed to developing methods for analytes of interest These methods take into account a variety of parameters Consider the following LC parameters for optimum separation of compounds e Composition of stationary phase e Column diameter e Column length e Column usability over time e Solvent s or composition of mobile phase e Flow rates e Isocratic or gradient ramped solvent compositions 402 LCquan User Guide Thermo Scientific A Quantitative Analysis Overview Considering the Variables of Quantitative Analysis by LC MS MS Often the only measur
28. 1 0 has no effect Note Valid only when you select the Constrain Peak Width check box Table 80 ICIS Advanced Parameters dialog box Sheet 1 of 2 Parameter Description INCOS Noise A single pass algorithm is used to determine the noise level Repetitive Noise A multiple pass algorithm is used to determine the noise level In general this algorithm is more accurate in analyzing the noise than the INCOS Noise algorithm but it takes longer RMS Noise A root mean square calculation is used to determine signal to noise values instead of the default ICIS noise method 212 LCquan User Guide Thermo Scientific Thermo Scientific 4 Creating a Processing Method Specifying Method Identification Table 80 ICIS Advanced Parameters dialog box Sheet 2 of 2 Parameter Peak Parameters Description Units are in number of scans Min Peak Width The minimum number of scans required in a peak Range 0 to 100 Default 3 Multiplet Resolution Area Tail Extension The minimum separation in scans between the apexes of two potential peaks This is a criterion to determine if two peaks are resolved Use a larger number in a noisy environment when the signal is bouncing around Range 1 to 500 Default 10 scans The number of scans past the peak endpoint to use in averaging the intensity Range 0 to 100 Default 5 scans Area Scan Window The number of allowable scans on each side of the peak apex A zero va
29. Area Tail Extension The number of scans past the peak endpoint to use in averaging the intensity Range 0 to 100 Default 5 scans Area Scan Window The number of allowable scans on each side of the peak apex A zero value defines all scans peak start to peak end to be included in the area integration Range 0 to 100 Default 0 scans Thermo Scientific Avalon Peak Integration 3 Exploring the Data Creating Explore Methods Use the Avalon Peak Integration area to define Avalon peak detection algorithms to be applied to the active raw file To specify Avalon peak integration parameters In the Peak Detection Algorithm box select Avalon Figure 74 Avalon peak identification and integration criteria are applied to the active raw file You can add modify or delete non automated timed events in the Avalon event list but you cannot delete an initial value Figure 74 Avalon Peak Integration area m Avalon Peak Integration Peak Detection Algorithm Avalon Auto Calculate Initial Start Threshold 10000 000 Initial End Threshold 10000 000 Initial Area Threshold 10000 000 Initial P P Threshold 1 000 Initial Bunch Factor 1 000 Initial Negative Peaks Off Initial Tension 1 000 Time min Event Value 0 000 Tension 1 000 Add Delete Change 2 To add a new event to the event list a Specify the new values in the Time min Event and Value boxes b Click Add The v
30. Available Columns Displayed Columns em wn ise Barcode Status 4 dd mj Sample Type Client Sus C FileName Company Name SU Level Dil Factor SA T Vial Pos ISTD Corr Amt T inj Vol Laboratory Comment Phone Move Up Sample ID Sample Name Sample Yol Sample Vt Study r r r r r r E r r r r Cancel Help 2 To hide currently displayed columns select the check box before the column and click Remove This does not delete the column it simply hides it from display on the sequence grid All hidden columns are displayed in the Available Columns list 3 To display currently hidden columns select the check box before the column and click Add The column is added to the Displayed Columns list and is displayed on the sequence grid 4 To change a column position a Select the check box before the column name in the Displayed Columns list b To move the column left in the grid click Move Up c To move the column right in the grid click Move Down LCquan User Guide 59 2 Preparing to Perform Quantitative Analysis Using the Setup Sequence View 5 To change the display precision a In the Places column of the Displayed Columns list select the value you want to change b Type a new value for the number of decimal places to display in the column Note When the Values column is unavailable your LCquan administrator has specified decimal round
31. Data Views Table 57 Error Report view parameters Parameter Arrows Description The left and rights arrows move through the list of detectors and devices incrementing or decrementing by a single item Detector Selector List Options MS Displays the error log of a mass spectrometer Status Displays the error log of a device that does not log data Detector Streams in Current Raw File When a raw file is open displays the list of detector streams that are stored in the current raw file The format of the list items is Generic Detector Stream Name Instrument Name Model Name Channel Name Column Headings Parameter Displays a column of descriptive text for the item Value Displays a column of data for the given parameter Shortcut Menu Open Raw File Opens the Select Raw File dialog box where you can select the raw file to load Lock Against Raw File Prevents changes to a raw file The lock icon is yellow and the Change lock is closed Unlock Makes the pane editable The lock icon is green and the lock is open Copy to Clipboard Copies selected data to the Clipboard You can then paste the data from the Clipboard buffer into a document The Filter List view displays the list of unique filters that are stored in the current raw file in table format Figure 97 Figure 97 Filter List view c SRM ms2 309 07 cid 42 00 205 02 205 02 c SRM ms2 314 10 cid 40 00
32. Emu Units mmu ppm Mass precision Decimals 2 Cancel Help Table 72 Masses dialog box parameters Parameter Description Mass Tolerance Mass Tolerance Type the mass tolerance value Range 0 1 to 50 000 Note Mass mass tolerance is summed for each scan Units Select one unit of measurement for data display e mmu millimass units ppm parts per million Mass Precision Decimals Type the number of decimal places for mass values display 190 LCquan User Guide Thermo Scientific 4 Creating a Processing Method Specifying Method Identification Retention Time Area Use the Retention Time area to specify a search window or corrections for retention time drift Figure 116 Table 73 lists the parameters for the Retention Time area Figure 116 Retention Time area Retention Time Expected min 1 92 Window sec 20 0 M Use as RT Ref View Width min fo 75 r a Table 73 Retention Time area parameters Sheet 1 of 2 Parameter Description Expected min The expected elution time of the component peak apex Range 0 0 to 999 0 Units minutes Window sec The time boundaries for the expected peak apex occurrence Range 1 0 to 999 0 Units seconds Use as RT Ref Specifies that the active component peak is to be used as a retention time reference This component s actual retention time is used to adjust the expected retention times of other components automatically du
33. ISTD Alprazolam d5 Alprazolam Alprazolam d5 Me Isotope a Target Units Calibration Curve Type 75 000 paml Linear Ed E 100 000 Ty 250 000 ISTO Amount e p Weighting 500 000 ea fro Equal E 750 000 pg Height CAR E 1000 000 ISTD Units 0 000 Origin 12 Jro mt m C Ignore D Force r a Include C 1s 2 IRC Chromatogram APN_003 m z 192 07 192 07 RT 1 53 2 28 NL 5 64E3 APN_003 m z 192 07 192 07 El F c SRM ms2 330 13 cid 20 00 70 15 192 07 F c SRM ms2 330 13 c RT 1 90 Relative Intensity Relative Intensity Relative Intensity Relative Intensity u 147 18 19 20 24 22 18 2 0 18 20 Time min Time min Time min For Help press F1 Powers Dana NOT SAVED 7 7 2008 1 40PM 7 To open the Calibration page of the Create Method view When the left navigation pane is displayed a Click Quantitate b Click the Method icon c Click the Calibration tab Thermo Scientific LCquan User Guide 235 4 Creating a Processing Method Specifying Method Calibration When the left navigation pane is hidden a Choose View gt Section Selection gt Quantitate Section b Choose View gt Step Selector gt Quantitate Method c Click the Calibration tab Use this page to enter calibration information for each of your components The Calibration page of the Create Method view includes these areas and functions e Com
34. In the Component list select the internal standard 2 The LCquan application automatically updates the Result list and the Chromatogram pane It does not display a chromatogram in the ISTD Chromatogram pane for this component but displays the response for each internal standard sample in the Calibration Curve pane 3 Check the entries in the Result list for peak detection and integration problems 4 Inspect the chromatogram in the Chromatogram pane for each file a Ensure that the LCquan application found the peak b Ensure that the shaded area accurately represents the contribution of the component to the chromatogram e To modify the peak detection and integration settings that you defined in the Create Method view go to the topic Modifying the Peak Detection and Integration Settings on page 345 e You can manually change the integration starting and ending points and the baseline of the peak To manually integrate a peak go to the topic Manually Integrating Peaks on page 363 When you are satisfied that the LCquan application properly detected and integrated the peaks you are ready to review all the results e For additional information about reworking the data see the following topics Excluding a Calibration Standard from the Calibration Curve on page 342 Modifying the Peak Detection and Integration Settings on page 345 and Manually Integrating Peaks on page 363 e To review all the resul
35. Pre Acquisition Ej Post Acquisition E Run Syncronously l IV Pre Acquisition Post Acquisition After Sequence Set System On Standby C Off Cancel Help 2 To add a comment to the acquisition sequence enter your text in the Comment box The comment will appear for this run in the Acquisition Sequence History 3 Specify the instruments that you want to run your samples a Click Change Instruments to open the Change Instruments In Use dialog box Thermo Scientific 2 Preparing to Perform Quantitative Analysis Using the Run Sequence Dialog Box b To specify the instruments that you want to use and the start instrument for the current acquisition sequence click the In Use and Start Instrument columns Yes appears in the column to indicate that the instrument is selected c Click OK to save changes and close the dialog box 4 Specify whether or not to start the acquisition when the instruments are ready e When you want the LCquan application to start the data acquisition automatically and perform an autosampler injection as soon as all instruments are ready select the Start When Ready check box e When you want the LCquan application to pause the acquisition sequence after all the devices are ready clear the Start When Ready check box You must manually start the acquisition by using the control buttons in the Status view of the Acquisition window 5 Specify the processing a
36. Sample information page parameters Parameter Description Condition Number of Samples Specifies the number of unknown samples to run The total number of unknowns is the number of samples times the number of injections Injections per Sample Specifies the number of injections to be performed for each unknown Base Sample ID Sample ID numbering starts at 001 For example when you enter AB12 the numbers of the first five samples are as follows e AB12001 e AB12002 e AB12003 e AB12004 e AB12005 4 Click Next Specify Tray and Vial Information Specify tray and vial information for the sequence see Table 16 To specify tray and vial information 1 Click the arrow to display the list of vial tray type options and select one of the vial tray types Figure 34 2 Type the first vial position in the new sequence The default first vial position is 1 3 To create a sequence where calibration QC Blank and Unknown samples are drawn from the same vial select the Re Use Vial Positions check box By default each injection is drawn from a new vial 4 To use the injection volume you specified in the instrument method for the autosampler select the Injection Volume Obtained from Autosampler Method check box and enter a new volume 42 LCquan User Guide Thermo Scientific 2 Preparing to Perform Quantitative Analysis Using the Setup Sequence View Figure 34 Tray and vial information page Tray Type Init
37. Semple Nome Integration Type Response ISTO Response Response Ratio Specified Cone 4 methyltestosterone 2 steroidso3 Method Settings 7031020 p progesterone 3 steroids04 Method Settings 3666547 14 steroids05 Method Settings 1782495 5 steroids13 Method Settings 13449804 je steroids14 Method Settings 7160845 steroids15 Method Settings 3574151 e steroids16 Method Settings 34771261 2530482 13 741 D gt AI Standards OCs 7 lei dra Selected steroidz02 TIC SM 7 PT 0 30 1 05 NL 3 7166 steroids02 TIC SM 7 RT 1 63 2 36 ML 3 30E5 hydrocortisone F Full mz2 363 50 40 00 150 00 375 00 F Full mz2 303 30 40 00 100 00 510 00 Y 0 0293758 1 14184 R 2 0 9996 W Equal Chromatogram v 4 gt amp ISTD y Chromatogram v 4 gt amp Calibration Curve FT 0 66 w RT 2 00 30 Retate heast Retatte heast Area Ratio 0 50 os 06 oT i x l l li 20 Time min Time min For Help press F1 L 10 19 2005 12 42 PM Thermo Scientific LCquan User Guide 325 6 Processing the raw Files and Reviewing the Analytical Results Reviewing the Calibration Curve and OCs 326 LCquan User Guide The Survey view includes clockwise from the top the Result grid the Component list the Calibration Curve pane the ISTD Chromatogram pane and the Chromatogram pane
38. Table 40 Chromatogram Definition area parameters Sheet 1 of 2 Parameter Description Detector Specifies the specific data stream Smoothing The number of points used for a moving mean filter to smooth the chromatogram Format whole numbers Range 1 to 15 odd numbers only Note To disable filtering set the parameter to 1 Trace Specifies the type of chromatogram e Mass Range Specify up to 50 mass ranges The masses are added together to form the chromatogram e TIC Full scan acquisition resulting in a Total Ion Current plot e Base Peak In a differential chromatogram the interpolation of the baseline is derived from the distance between the intersections of the tangents drawn to the peak sides and the peak base Note This definition set is the same for the second Trace box The second trace box depends on which operators you select Trace operator used to specify a trace operation This trace operation matrix shows the combinations used to set up a method Mass Range Mass Range Mass Range TIC Mass Range Base Peak Base Peak Mass Range Mass Range Note If the operator box remains empty the second trace choice is disabled 116 LCquan User Guide Thermo Scientific 3 Exploring the Data Creating Explore Methods Table 40 Chromatogram Definition area parameters Sheet 2 of 2 Parameter Description Mass1 m z Specifies the initial mass value Mass2 m z Sp
39. Thermo Scientific 4 Creating a Processing Method Specifying Method Calibration Component List Pane Use the Component List pane to add delete or rename a component Figure 143 Table 94 lists the parameters for the Component List pane Figure 143 Component List pane Component Paroxetine Nefazodone Alprazolam d5 Alprazolam Add Delete Rename Table 94 Component list functions Function Description Buttons add Adds a component Deletes a component Delete P Rename Renames a component Shortcut Menu Add Adds a component name Delete Deletes a component name Rename Renames a component Thermo Scientific LCquan User Guide 241 4 Creating a Processing Method Specifying Method Calibration Calibration Shortcut Menu Right click the anywhere in the Calibration page to display the shortcut menu Command Shortcut Menu Description Delete Selected Rows Deletes the currently selected rows in the Cal or QC grid You must select each row to be deleted by clicking the row indicator to the left of the row Copy Current Component Levels to All Target Components Copies the current Cal and QC level tables to all other defined target components Copy Current Calibration Parameters to All Target Components Copies all calibration parameters defined in this component to other defined target components Copy Current Calibration Parameters amp Le
40. Thermo Scientific LCquan User Guide 75 2 Preparing to Perform Quantitative Analysis Using the Run Sequence Dialog Box Table 26 Run Sequence dialog box parameters Sheet 3 of 4 Parameter Description Post Acquisition Specifies that the Post Acquisition program displayed in the Post Acquisition box runs synchronously in series with data collection The Run Manager waits until the Post Acquisition program can be run after data acquisition For example when you want to convert data from one data type to another data type while you are acquiring data you can select a synchronous Post Acquisition program By default the program runs asynchronously in parallel with data collection For example you can perform operations that do not involve taking data Optional Macro You can use macro arguments when entering the run arguments Arguments Supported macro arguments and their replacements e R Provides the current raw file e Provides the instrument method name e S Provides the sequence name e V Provides the vial or well number in the Position column of the sequence e Provides a single character in the run line After Sequence Set System On Keeps the system in the On state when the current sequence is completed When On is selected you can run another sequence without waiting All power and flows are maintained at operational levels Default On Note This option has the same effect as ch
41. acquire data explore data and process data files Instrument Setup Window Use the LCquan Instrument Setup window to develop instrument methods for the instruments you selected in the Xcalibur Instrument Configuration window An instrument method is a set of experimental parameters and operating settings for a specific instrument such as an autosampler LC pump or MS pump mass spectrometer divert valve or syringe Thermo Scientific LCquan User Guide 1 1 Introduction Quantitative Analysis with LCquan pump Within the Instrument Setup window you can create new methods or modify existing methods You can also import previously collected data files to help you set up time segments and scan events based on the acquired data The Instrument Setup window within LCquan functions similar to the Instrument Setup window of the Xcalibur Home Page The Instrument Setup window contains separate pages for each instrument that you selected in the Xcalibur Instrument Configuration window Figure 1 Note The Instrument Setup window is not available in the LCquan for Web Access Figure 1 Instrument Setup window f LCquan 3 Drugs Workbook File Quantum View Apps Options Help Tae 0 V Starup Method Instrument Method Shutdown Method NIINI AINIIN IE Scan Editor Syringe Pump Divert Valve Method Summary Calibration Correction Method C Run Settings MS Acquire Time min Segments is Current Segment iz To
42. acquisition setup 31 overview 409 Quantitate result grid parameters 386 Quantitate Sequence sample selector 136 Quantitate window 181 description 6 quantitation flow diagram 415 quantitation limits 405 Quantitation Results Sorting Order parameters 331 quantitative analysis described 401 409 internal standards and target compounds 246 LCquan overview 10 sources of error 406 steps of 402 techniques 404 using external standards 406 using internal standards 406 raw files associating with sample types 315 displaying in Chromatogram pane 83 importing into new workbook 17 processing 323 time stamping during remote acquisition always time stamp 109 never time stamp 109 Realtime Display Settings parameters 86 remote acquisition always time stamp raw files 109 never time stamp raw files 109 reports do not lock workbook after creating report 399 lock workbook after creating report 398 response factor 404 restore factory layout configuration 420 result grid shortcut menu commands 327 result grid description 326 Results Export parameters 396 Retention Time area parameters 191 Review All Results page 365 review mode 13 Review page 135 Review Reports page 375 Review Reports parameters 383 Thermo Scientific Index Q reviewing all results 365 reviewing calibration standards 333 Run Sequence dialog box 71 Run Sequence parameters 74 Run Status page 94 Run Status parameters 95 Run Status shortcut menu commands 96 S Sam
43. cid 20 00 70 15 192 07 400 192 07 90 80 70 60 50 pi r a v 2 a w ce 40 30 20 When the view is locked lock icon is red you cannot select any menu item in the shortcut menu that changes the raw file or parameters Menu items that would cause changes are grayed out For more information about locks see Toolbar for Selectable Views on page 252 Table 103 lists the parameters for the Spectrum view Thermo Scientific LCquan User Guide 267 4 Creating a Processing Method Data Views 268 LCquan User Guide Table 103 Spectrum view parameters Parameter Arrows Description The left and right arrows move through scans in the raw file incrementing or decrementing by a single scan Item Selector List Options Survey and Review All Results views only ISTD Displayed spectrum is from the current component s ISTD Component Displayed spectrum is from the current component Shortcut Menu Open Raw File Create Method view only Opens the Select Raw File dialog box where you can select the raw file to load Lock against All Specifies that changes to a raw file or parameters such as Change retention time selection in other panes have no effect on this pane The lock icon is red and the lock is closed Unlock Specifies that changes to raw files and relevant parameters cause an update to occur in this pane using the new settings The lock icon is green and
44. isotopes or multiply charged peptides Note This definition set is the same for the second Trace box The second Trace box depends on which operators you select There is no second Trace box or trace operators for Trace Summed Trace Mass Range TIC or Base Peak a Trace operator used to specify a trace operation This trace operation matrix shows the combinations used to set up a method Mass Range Mass Range Mass Range TIC Mass Range Base Peak Base Peak Mass Range Mass Range Mass1 m z Specifies the initial mass value Mass2 m z Filter 348 LCquan User Guide Specifies the second mass value to define the mass range when you are using trace operator math Specifies an existing filter or a filter from a preloaded filter list obtained from the current raw file Note All filters are validated against the current set of filter entry rules Thermo Scientific 6 Processing the raw Files and Reviewing the Analytical Results Reviewing the Calibration Curve and OCs Table 134 User Identification Settings dialog box Identification page parameters Sheet 3 of 3 Parameter Trace Summed Description Summed Ions table Displays the filters and mass to charge ratios of the ions you specified for ion summing in the Summing Ions Dialog Box on page 196 Retention Time of the component peak and the error window Expected min The expecte
45. of the preview panes and choose User Peak Detection Settings from the shortcut menu The User Identification Settings dialog box includes these tabs e User Identification Settings Identification e User Identification Settings Detection e User Identification Settings Genesis Integration e User Identification Settings Genesis Advanced e User Identification Settings ICIS Integration e User Identification Settings ICIS Advanced e User Identification Settings Avalon Integration Note When you change user peak detection settings and click Apply to All any samples that have been manually integrated will be reintegrated using the new user settings Their Integration Type will be changed from Manual Integration to User Integration 346 LCquan User Guide Thermo Scientific 6 Processing the raw Files and Reviewing the Analytical Results Reviewing the Calibration Curve and OCs User Identification Settings Identification Use this page Figure 202 to set up advanced chromatographic parameters The content of the tabs varies according to the selected peak detection algorithm ICIS Genesis or Avalon Table 134 lists the parameters for the Identification page of the User Identification Settings dialog box Figure 202 User Identification Settings dialog box showing the Identification page User Identification Settings Identification Detection Avalon Integration Smoothing Points 1 c SRM ms2 330 13 c
46. raw file to view its chromatograms and mass spectra Reset Scaling Resets the x axis and y axis ranges in the Chromatogram pane or the Spectrum pane to their default values Copy to Clipboard Copies a chromatogram or mass spectrum to the Clipboard You can then paste it from the Clipboard into a document Thermo Scientific LCquan User Guide 89 2 Preparing to Perform Quantitative Analysis Using the Status View Acquisition Status Spectrum Pane The Spectrum pane displays the data for the currently selected scan in the currently selected chromatogram Use the Show Spectrum Trace command on the shortcut menu to display the Spectrum pane To select a scan Click a chromatogram A red vertical line appears on the selected scan in the Chromatogram pane and the spectrum that corresponds to that scan appears in the Spectrum pane Figure 52 Figure 52 Chromatogram pane top and Spectrum pane bottom 100 60 60 40 20 Relatie heist 0 33 0 393 123 3 27 3 62 187 219 2 45 5 36 5 76 6 25 6 48 0 0 0 5 1 0 1 5 2 0 25 3 0 3 5 4 0 45 5 0 5 5 6 0 6 5 Time min Data file drugx_03 o 424 4 i 80 2 a 60 v E 40 fa 20 423 5 0 423 4 423 6 423 8 424 0 424 2 424 4 424 6 424 8 4 miz To apply a scan filter to the data based on the selected chromatogram 1 Right click the spectrum pane and choose Filter Spectrum from the shortcut menu 2 To remove the filter and show every scan select this
47. raw files and relevant parameters cause an update to occur in this pane using the new settings The lock icon is green and the lock is open 256 LCquan User Guide Thermo Scientific Thermo Scientific 4 Creating a Processing Method Data Views Table 99 Calibration Curve view parameters Sheet 2 of 3 Parameter Exclude Include Description Note This menu item appears only when you click a data point in the curve When the data point you click has already been excluded the menu item is Include instead of Exclude When Exclude is displayed Forces the calibration curve to be recalculated without the selected data point The Exclude entry in the results grid and Cal Exclusion List dialog box are updated to show that this point is excluded and the excluded data point is redrawn as an unfilled square on the calibration curve When Include is displayed Includes the data point and forces the calibration curve to be recalculated The Exclude entry in the results grid and Cal Exclusion List dialog box are updated to show that this point is now included and the included data point is redrawn as a filled square on the calibration curve You can include or exclude samples that are shared between brackets Their status is unique to the bracket that is excluding a shared sample in bracket 1 has no effect on its inclusion status in bracket 2 For more information see Modifying Calibration Settings on page 336 in
48. you cannot select any menu item in the shortcut menu that changes the raw file or parameters Menu items that would cause changes are grayed out For more information about locks see Toolbar for Selectable Views on page 252 Table 106 lists the parameters for the Chromatogram List view Table 106 Chromatogram List view parameters Sheet 1 of 2 Parameter Description Arrows The left and right arrows move through the selection incrementing or decrementing by a single sample peak Item Selector List Options Survey and Review All views only ISTD Displayed chromatogram is from the current component s ISTD Method Displayed chromatogram is the method derived chromatogram regardless of which integration type is currently in use for the current component Selected Displayed chromatogram is from the current component Column Headings RT Displays the retention time of the given data point Intensity Displays the absolute intensity of the given data point Relative Intensity Displays the intensity of the data point relative to the most intense data point in the chromatogram LCquan User Guide 273 4 Creating a Processing Method Data Views Table 106 Chromatogram List view parameters Sheet 2 of 2 Parameter Description Shortcut Menu Open Raw File Create Method view only Opens the Select Raw File dialog box so you can select the raw file to load Lock against All Prevents changes to
49. 0 0 to 999 0 Default multiplier 0 5 Valley Detection Specifies the LCquan valley detection approximation method to Enabled detect unresolved peaks This method drops a vertical line from the apex of the valley between unresolved peaks to the baseline The intersection of the vertical line and the baseline defines the end of the first peak and the beginning of the second peak To turn this method on select the Valley Detection check box To turn this method off clear the check box Expected Width sec This multiplier specifies an expected peak width parameter that controls the minimum width that a peak is expected to have when valley detection is enabled With valley detection enabled any valley points nearer than half the expected width to the top of the peak are ignored If a valley point is found outside the expected peak width the LCquan application ends the peak at that point It always ends a peak when the signal reaches the baseline independent of the value set for the expected peak width Range 0 0 to 999 0 Default multiplier 0 0 Units seconds Note Valid only when you select the Valley Detection Enabled check box Constrain Peak Width Constrains the peak width of a component during the peak integration of a chromatogram You can set values that control when peak integration is turned on and off by specifying a peak height threshold and a tailing factor Selecting this activates the Peak Ht and the Ta
50. 209 96 209 96 c SRM ms2 330 1 3 cid 20 00 70 15 192 07 c SRM ms2 470 18 cid 26 00 180 07 274 08 The contents of the grid are read only LCquan User Guide 159 3 Exploring the Data Data Views When the view is locked lock icon is yellow you cannot select any menu item in the shortcut menu that changes the raw file Menu items that would cause changes are grayed out For more information about locks see Toolbar for Selectable Views on page 150 Table 58 lists the parameters for the Filter List view Table 58 Filter List view parameters Parameter Description Arrows The arrows are not used in Explore window Column Heading Filters Displays the list of unique filters that are stored in the current raw file Shortcut Menu Open Raw File Opens the Select Raw File dialog box where you can select the raw file to load Lock Against Raw File Prevents changes to a raw file The lock icon is yellow and the Change lock is closed Unlock Makes the pane editable The lock icon is green and the lock is open Copy to Clipboard Copies selected data to the Clipboard where you can paste the data from the Clipboard buffer into a document 160 LCquan User Guide Thermo Scientific 3 Exploring the Data Data Views General Parameters Plot View Thermo Scientific The General Parameters Plot view displays the selected parameters for all detected Explore peaks for a sample and Explore method
51. 3 Exploring the Data Data Views T SRM ms2 330 13 cid 20 00 70 15 192 07 100 90 80 70 60 50 wi c a 2 L v Zz E a w ec 40 30 151 01 Table 64 lists the parameters for the Spectrum view Table 64 Spectrum view parameters Parameter Description Arrows The left and right arrows move through the scans incrementing or decrementing by a single scan Shortcut Menu Open Raw File Opens the Select Raw File dialog box where you can select the raw file to load Lock Against Raw File Prevents changes to a raw file The lock icon is yellow and the Change lock is closed Lock against All Prevents changes to a raw file or parameters The lock icon is red Change and the lock is closed Unlock Makes the pane editable The lock icon is green and the lock is open Display Options Opens the Chromatogram Display Options dialog box For more information see Chromatogram Display Options dialog box parameters on page 155 Reset Scaling Resets the x and y axes to display the maximum amount of data Copy to Clipboard Copies selected data to the Clipboard You can then paste the data from the Clipboard buffer into a document LCquan User Guide 171 3 Exploring the Data Data Views Status Plot View The Status Plot view displays status data that the instruments store during the acquisition of the selected raw file Figure 104 The list contains all the parameters for the
52. 4 Edit the columns for the new arrangement and click OK Table 148 Column Arrangement dialog box parameters Parameter Description Available Columns Lists parameters that are not currently selected for display Displayed Columns Lists currently selected parameters which appear in the data grids Add Remove Displays Hides selected columns Move Up Move Up moves columns left in the grid Move Down Move Down moves columns right in the grid Factory Defaults Resets original column values as described in Grid parameters on page 386 380 LCquan User Guide Thermo Scientific 6 Processing the raw Files and Reviewing the Analytical Results Creating and Reviewing Reports To delete an arrangement 1 Select an arrangement from the current list You cannot delete the Quan Result Grid Excel Long Summary or Excel Short Summary column arrangements that ship with the LCquan application 2 Click Delete Selected Arrangement The LCquan application confirms that you want to delete the arrangement XReport Report Selections Thermo Scientific Specify the XReports you want to create Figure 221 1 Select the Use check box of each report that you want to generate using an XReport template 2 To add a row to the table select the Use check box in the last row of the table click in the row and select a report from the list 3 To delete a row select the entire row right click the selection and choose Del
53. 4 Creating a Processing Method Data Views Table 102 Chromatogram view parameters Sheet 4 of 4 Parameter Description Update Expected Updates the expected elution time point of maximum signal of Retention Time all components in the current row from the retention time Update Method specified in the processing method to the detected retention time Settings for All Components Using Current Row Reset Scaling Resets both x and y axes to display the maximum amount of data Copy to Clipboard Copies selected data to the Clipboard You can then paste the data from the Clipboard buffer into a document 266 LCquan User Guide Thermo Scientific 4 Creating a Processing Method Data Views Spectrum View This view displays a spectrum for a sample at the selected retention time Figure 153 In the Survey or Review All Results views in the Quantitate window the Item Selector List options for this view are identical to those for the Mass List view so you can compare the two views side by side To make changes to the spectrum display Right click the spectrum view and choose Display Options from the shortcut menu The Spectrum Display Options dialog box opens For more information see Spectrum Display Options dialog box parameters on page 92 in Chapter 2 Preparing to Perform Quantitative Analysis Figure 153 Spectrum view 4 gt a Component Spectrum APN_O003 327 RT 1 90 NL 5 64E3 T SRM ms2 330 13
54. Adding Comments to the Chromatogram Display on page 374 in Chapter 6 Processing the raw Files and Reviewing the Analytical Results 4 Click OK or Apply as appropriate 5 To draw the new peak drag the peak baseline defining the start and end of the peak 6 To manually adjust the peak baseline and endpoints grab the square blue handles and drag them Figure 152 Chromatogram view for Survey or Review All Results 4i a Selected Chromatogram X APN_OO3 m z 192 07 192 07 RT 1 53 2 28 NL 5 64E3 F SRM ms2 330 13 cid 20 00 70 15 192 07 RT 1 90 100 90 80 70 60 50 40 ia e a E v 2 E 2 w ce 30 1 6 17 1 8 1 9 Time min When the view is locked lock icon is red you cannot select any menu item in the shortcut menu that changes the raw file or parameters Menu items that would cause changes are grayed out For more information about locks see Toolbar for Selectable Views on page 252 Table 102 lists the parameters for the Chromatogram view Thermo Scientific 4 Creating a Processing Method Data Views Table 102 Chromatogram view parameters Sheet 1 of 4 Parameter Arrows Description The left and right arrows move through the chromatogram incrementing or decrementing by a single scan Item Selector List Options ISTD Displays the chromatogram from the current component s ISTD Method Displays the method derived chromatogram regard
55. Amounts The Review Level Amounts dialog box displays the calibration or QC levels depending on the selected sample and the component amounts in each level Figure 183 Figure 183 Review Level Amounts dialog box Review Level Amounts Component amounts for selected level Alprazolam 25 000 pg mL Nefazodone 25 000 pg mL Paroxetine 25 000 pg mL 314 LCquan User Guide Thermo Scientific Using the Create Sequence create View Use the Create Sequence create view Figure 184 to build a processing sequence by 5 Creating a Processing Sequence Editing a Processing Sequence associating each raw file in the data set with a standard calibration level a QC level a blank sample type or an unknown sample type Figure 184 Associate raw files view Create Sequence select files review and edit Available Files Thermo Scientific TH APN_004 RAW APN_OO5 RAW APN_OO6 RAW APN_OO 7 RAW APN_008 RAW APN_009 RAW H E n QuanRoot 3 Drugs Workbook 3 Drugs Workbook 2 Example Study O 3 Drugs Workbook 3 Drugs Workbook 2 3 Drugs Workbook x 3 Drugs Workbook y Exports Imports Rautiles Temp GQ APN_O16 RAW GQ APN_017 RAW GQ APN_O18 RAW i APN_O1S RAW i APN_020 RAW Gj APN_021 RAW Gi APN_022 RAW Gj APN_023 RAW GQ APN_024 RAW Gi APN_025 RAW Gj APN_026 RAW GQAPN_027 RAW i APN_028 RAW Step 1 APN_
56. Chapter 6 Processing the raw Files and Reviewing the Analytical Results Calibration Settings Opens the Calibration Settings dialog box with Type Curve Levels and Isotope tabs For more information see Modifying Calibration Settings on page 336 in Chapter 6 Processing the raw Files and Reviewing the Analytical Results The Type tab is available for both ISTDs and target compounds All other tabs are shown only for target compounds Exclusion List Opens the Cal Exclusion List dialog box which displays all replicates used in creating the current calibration curve and their exclusion status and also is where you can change that status For more information see Excluding a Calibration Standard from the Calibration Curve on page 342 in Chapter 6 Processing the raw Files and Reviewing the Analytical Results Larger Symbols Increase the size of the replicate data points the square markers Five sizes are available The marker size doubles each time you select this command until the maximum size is reached The current size setting is stored in the registry and is used for all workbooks when you open them LCquan User Guide 257 4 Creating a Processing Method Data Views Table 99 Calibration Curve view parameters Sheet 3 of 3 Parameter Description Smaller Symbols Reduces the replicate data points the square markers Five sizes are available The marker size is halved each ti
57. Click Next LCquan User Guide Thermo Scientific 2 Preparing to Perform Quantitative Analysis Using the Setup Sequence View Study and Workbook Names Name the new study or workbook see Table 14 To specify a name for the new study or workbook 1 Type or browse to the new study name Figure 29 2 Type or browse to the new workbook name 3 Optional Create a new root folder or open a new LCquan session in a new window Figure 29 Create new study and workbook page Study Name Sample Stud Browse Workbook Name Sample Workbook Browse C calibur QuanRoot S ample Study S ample Workbook V Change Project Root Folder Open in New Window Table 14 Study and Workbook Names parameters Parameter Description Study Name The LCquan application creates a new folder with this name in the root folder Workbook Name The LCquan application creates a new folder with this name in the Study folder Change Project Root Select to change the Project root folder The default root Folder folder is C Xcalibur QuanRoot Changing the root folder requires authorization from your administrator Open in New Window Opens a new LCquan window using the new workbook or study The original LCquan window remains open 4 Click Next When you are keeping the current root folder go to Import an Acquisition Sequence Thermo Scientific LCquan User Guide 39 2 Preparing to Perform Quantitative Analysis Using the Se
58. Clipboard You can then paste the data from the Clipboard buffer into a document 292 LCquan User Guide Thermo Scientific ee i Creating a Processing Sequence This chapter describes how to create a processing sequence by using the Sequence views of the Quantitate window A processing sequence provides instructions to the LCquan application about what samples to process It consists of a list of sample data files and includes information about sample type and calibration or QC levels Contents e Defining a Processing Sequence e Editing a Processing Sequence e Exporting a Processing Sequence Thermo Scientific LCquan User Guide 293 5 Creating a Processing Sequence Defining a Processing Sequence Defining a Processing Sequence From the Create Sequence views in the Quantitate window you can create a new processing sequence or edit an existing sequence e Ifyou have not defined or imported a processing sequence when you click the Sequence icon the LCquan application opens the New Processing Sequence Wizard When you complete the wizard the Create Sequence create view opens Figure 168 Figure 168 Create Sequence create window bd Thermo LCquan 3 Drugs Workbook y File Zoom View Apps Options Help PSB AAE FSWAOOEOGCOCOS Kii OlO Create Sequence select files review and edit Instruments Available Files Standards OCs Blanks Unknowns Selected Files O GuanRoot 3 Drugs Workboo
59. Editing a Processing Sequence 320 LCquan User Guide 3 Click OK Figure 186 Standard or QC Level Names Association dialog box Standard Level Names Association Associate the level names from the list at left by dragging them to their matching level name at right Sequence Level Names Workbook Level Names standardl standard standardL Add gt gt If any discrepancy remains in the level names the Level Name Association Not Complete dialog box opens Figure 187 Follow the instructions To resolve incomplete level name association Figure 187 Level Name Association Not Complete dialog box Level Name Association Not Complete f Not all levels have been associated between the sequence and the workbook To continue you must do one of the following complete the association process set the samples designated with the remaining levels to Unknown or discard the samples designated with the remaining levels Set to Unknown Discard Help To resolve level names for added samples 1 In the Sample Level Names pane do one of the following e Select the level name and drag it to the appropriate level name in the Workbook Level Names pane or e Select the two level names and click Add 2 Click OK If any discrepancy remains in the level names the Level Name Association Not Complete dialog box opens Figure 187 Follow the instructions to resolve incomplete level
60. Figure 98 Select the parameters you want to see plotted from the Item Selector list You can lock the general parameters plot for one sample and click through the other samples to look for trends or inconsistencies in the data Figure 98 General Parameters Plot view 41 gt a RT General Param gt RT Peak Width 08 0951 1415 1516 1950 2066 2435 2 674 Peak When the view is locked lock icon is red you cannot select any menu item in the shortcut menu that changes the raw file or parameters Menu items that would cause changes are grayed out For more information about locks see Toolbar for Selectable Views on page 150 Table 59 lists the parameters for the General Parameters Plot view Table 59 General Parameters Plot view parameters Sheet 1 of 2 Parameter Description Arrows The left and right arrows move through the item list incrementing or decrementing by a single item Item Selector List Options RT Displays retention time for the detected peaks Area Displays areas for the detected peaks Units count seconds LCquan User Guide 161 3 Exploring the Data Data Views 162 LCquan User Guide Table 59 General Parameters Plot view parameters Sheet 2 of 2 Parameter Description Height Displays heights of the detected peak apexes Units counts Peak Width Displays peak widths for the detected peaks Shortcut Menu Open Raw File Opens the Select Raw File dialog bo
61. Foundation gt Authorization Manager to open the Authorization Manager Figure 123 Figure 123 Authorization Manager showing Normalize Quan Chromatogram Plots to Detected Peak permission set to Disallowed lt UUEUELULU HE Instrument Setup LCquan Coal Run Application File File ni ni Section Configuration ml Grid Column Settings Root Folder Tracking Acquisition Section Acquisition Run Dialog mm Explore Section mi Quantitate Section I Allow changes to selected sample info in Allow changes to column labels in proces Allow changes to column labels in results Prompt user for Comment after manual inl Normalize quan chromatogram plots to d Allow Results Export Allow Manual Integration Allow User Integration Allow Calibration Settings to be changed Create Reports gt 2 In the Authorization Manager do the following 200 LCquan User Guide Select a user group in the Secure Groups list v Permission Level Disallowed Supervisor Password Required Allowed Disallowed State Com ent Secure Template Folder Click Expand Tree to show the entire list of controlled features for the application From the list click the plus sign before the LCquan folder Click the plus sign before the Quantitate Section folder Select Normalize Quan Chromatogram Plots to Detected Peak The Permission Level options become available Selec
62. Information dialog box where you can view peak information that reflects the settings for the currently displayed chromatogram For more information see User Identification Settings ICIS Integration on page 356 in Chapter 6 Processing the raw Files and Reviewing the Analytical Results The compound identification name is displayed in the title bar When the component peak is not found the dialog box displays a single tab labeled No Peak The tabs available and their order depend on the type of peak being examined For typical peaks three tabs provide access to the following Peak Information Info Peak Information Flags and Peak Information Spectrum When IRCs are used an additional tab appears for the Peak Information IRC Tests For more information see User Identification Settings ICIS Integration on page 356 in Chapter 6 Processing the raw Files and Reviewing the Analytical Results Each IRC has a separate menu item only one Peak Info box is allowed for each IRC The selected peak in the plot is updated to reflect the selection in the menu Additional Show Peak Info menu items are available for each qualifier ion Change Peak Baseline Opens the Baseline dialog box where you can enter peak baseline coordinates directly For more information see Manually Integrating Peaks on page 363 in Chapter 6 Processing the raw Files and Reviewing the Analytical Results
63. Multi Peak Plot View for Review Only The Multi Peak Plot view is data view that is available only from the Explore Review view and displays all detected Explore peaks for a sample method combination Figure 107 Figure 107 Multi Peak Plot view Mutti Peak Plot APN_042 TIC RT 1 71 2 22 NL 1 0466 oa pe faa o o o Relative Intensity Relative Intensity W o N oO T 0 86 0 9 J 2 0 Time min Time min APN_042 TIC RT 2 21 2 68 NL 1 0466 APN_042 TIC RT 2 61 2 68 NL 1 0466 100 100 RT 2 42 o o o o oa o oa o Relative Intensity Relative Intensity N o N oO RT 2 66 o Oo 24 J 2 62 2 64 2 66 Time min Time min Thermo Scientific LCquan User Guide 177 3 Exploring the Data Data Views You can set the number of rows and columns define the number of peak graphics displayed on the screen and change chromatographic display options 2 You can define a maximum of 50 cells The x axis range for each peak is 2 0 times the detected peak width 50 on either side The peak baseline endpoints are displayed as blue circles Note When the endpoints appear as blue circles you cannot select and drag them to manually integrate the peak To set the number of rows and columns Right click the plot and choose Options from the shortcut menu The Multi Peak Options dialog box opens For more information see Multi Peak Options dialog
64. Peak Width option Range 0 5 to 9 0 Default multiplier 1 0 1 0 has no effect Figure 127 Genesis Advanced Component Options dialog box Genesis Advanced Component Options r Peak Edge Detection m Valley Detection Report Noise As Peak S N Cutoff Rise Percentage Valley S N Thermo Scientific LCquan User Guide 207 4 Creating a Processing Method Specifying Method Identification 208 LCquan User Guide Table 78 Genesis Advanced Component Options parameters Parameter Description Peak Edge Detection Chromatogram peak detection criteria using the peak signal to noise S N cutoff value Peak S N Cutoff The peak edge is set to values below this defined S N This test assumes an edge of a peak is found when the baseline adjusted height of the edge is less than the ratio of the baseline adjusted apex height and the peak S N cutoff ratio When the S N at the apex is 500 and the peak S N cutoff value is 200 the LCquan application defines the right and left edges of the peak when the S N reaches a value less than 200 Range 50 0 to 10 000 0 Valley Detection Valley detection approximation method to detect unresolved peaks Rise Percentage The percentage that the peak trace can rise above the baseline after passing through a minimum before or after the peak This method drops a vertical line from the apex of the valley between unresolved peaks to the baseline The intersection of the v
65. QCs To exclude or include click the grid cell and select or clear the check box presented Selecting checking excludes the data Available Column parameters available for display Acq Date Date when the data file was acquired Area Integrated area under the detected peak count secs The area of the peak in units of counts seconds Barcode Displays the barcode for this sample Barcode Status Displays the status of the barcode reading Cal Eqn Thermo Scientific The calibration curve equation for this component LCquan User Guide 389 6 Processing the raw Files and Reviewing the Analytical Results Creating and Reviewing Reports Table 152 Grid parameters Sheet 5 of 9 Parameter Client Description User defined Displays information that is pertinent to the active sample row in the Acquisition sequence You can use this box to convey information about this sample to others or as a reminder to yourself The User Label 2 default name is Client To change definitions of User Labels and Values Right click the grid on the Setup Sequence view of the Acquisition window and choose one of the following Columns Open the Column Arrangement dialog box For more information see Using the Acquisition Sequence Grid Shortcut Menu on page 55 in Chapter 2 Preparing to Perform Quantitative Analysis User Labels and Values to open the Change Sequence User Labels dialog box For mor
66. RT Identifying Components Thermo Scientific When the LCquan application acquires data it creates unique scan filters according to the type of experiment you specify in the instrument method When you load a raw file it lists the scan filters associated with the raw file in the Filter box 1M To relate the chromatogram peaks with the components From the Filters box select a scan filter and click Apply The LCquan application displays the filtered chromatogram Figure 90 It is important to know the parent ion mass for your components In this example the parent ion mass for methyltestosterone is m z 303 and the scan filter is c Full ms2 303 30 40 100 00 310 00 The methyltestosterone corresponds to the peak named Peak 1 991 in the peak list LCquan User Guide 147 3 Exploring the Data Exploring the Results Figure 90 Filtered chromatogram Steroids Workbook ex View Apps Options Help aj Ha Eis 080e0G0001 0 0 Identification Chromatogram Definition Genesis Peak Integration Limit peaks Acquisition Sequence History Detector Smoothing Peak Detection Algorithm Select top peaks iu Genesis m Advanced Enasi Trace Select by area TIC MM S N Threshold 0 5 O Select by heig
67. SXGA e 250 GB hard drive NTFS file format Software e Microsoft Windows 7 Professional with Service Pack 1 32 bit e Microsoft Office Professional 2010 e Thermo Scientific software Foundation 2 0 SP1 Xcalibur 2 2 0 SP1 LC Devices 2 5 SP1 LCquan 2 7 One of the following mass spectrometer programs Q Exactive 2 0 Exactive 1 1 SP4 TSQ Series 2 3 SP3 Special Notices This guide includes the following types of special notices IMPORTANT Highlights information necessary to prevent damage to software loss of data or invalid test results or might contain information that is critical for optimal performance of the system Note Highlights information of general interest Tip Highlights helpful information that can make a task easier Thermo Scientific LCquan User Guide xi Preface Contacting Us There are several ways to contact Thermo Fisher Scientific for the information you need To contact Technical Support Phone 800 532 4752 Fax 561 688 8736 E mail us techsupport analyze thermofisher com Knowledge base www thermokb com Find software updates and utilities to download at mssupport thermo com To contact Customer Service for ordering information Phone 800 532 4752 Fax 561 688 8731 E mail us customer support analyze thermofisher com Web site www thermo com ms To get local contact information for sales or service Go
68. Second Order lt none gt J Sort in descending order Third Order lt none gt 7 Sort in descending order Save As Default Cancel Help Table 122 Sequence Sorting Order dialog box parameters Sheet 1 of 2 Parameter Description Sorting Options The following sorting options are applicable to all three sort orders File Name Sorts the list so the file names are in alphabetical order Level Name Sorts the list so the calibration standard and QC level names are in alphabetical order Sample ID Sorts the list so the sample IDs are in alphabetical or numerical order Sample Type Sample types are not sorted alphabetically In ascending order the types are Standards QCs Blanks and Unknowns LCquan User Guide 311 5 Creating a Processing Sequence Editing a Processing Sequence Table 122 Sequence Sorting Order dialog box parameters Sheet 2 of 2 Parameter Description First Order Specifies the first order of sorting for the sequence The default first order of sorting is by sample type Second Order Specifies the second order of sorting for the sequence Third Order Specifies the third order of sorting for the sequence Sort in Descending The LCquan application sorts the list in descending n tol order Oorder Clear this option to have it sort the list in ascending order Customizing Column Arrangement 1 From the processing sequence grid shortcut menu choose Columns The Column Arra
69. Sheet 3 of 3 Control Description View Selector list Select from the list of selectable views for the current section Chromatogram Chromatogram List Error Report Filter List Instrument Method List Mass List Chromatogram View Thermo Scientific The Chromatogram View displays a chromatogram and all detected peaks for a sample and Explore method Figure 93 The displayed chromatogram is based on the range specified in the Explore method All detected peaks are displayed but cannot be edited Click the chromatogram to set the Retention Time marker All spectrum spectrum list chromatogram list and multi peak plot views that are not locked receive the selected time and update Figure 93 Chromatogram view APN_OO3 TIC RT 0 01 4 00 NL 1 04E5 100 90 80 70 60 50 gt i D v Zz 5 o w 40 30 20 0 53 RT 088 RT 142 3 02 3 29 3 52 376 2 0 Time min When the view is locked lock icon is yellow or red you cannot select any menu item in the shortcut menu that changes the raw file with the yellow lock or the raw file and parameters with the red lock Menu items that would cause changes are grayed out For more information about locks see Toolbar for Selectable Views on page 150 LCquan User Guide 153 3 Exploring the Data Table 54 lists the parameters for the Chromatogram view Table 54 Chromatogram view parameters Parameters Description Arr
70. Stream Name Instrument Name Model Name Channel Name Column Headings Parameter Displays a column of descriptive text for the item Value Displays a column of data for the given parameter Shortcut Menu Open Raw File Opens the Select Raw File dialog box where you can select the raw file to load Lock Against Raw File Prevents changes to a raw file The lock icon is yellow and the Change lock is closed Lock against All Prevents changes to a raw file or parameters The lock icon is red Change and the lock is closed Unlock Makes the pane editable The lock icon is green and the lock is open Copy to Clipboard Copies selected data to the Clipboard You can then paste the data from the Clipboard buffer into a document LCquan User Guide 175 3 Exploring the Data Data Views Tune Method View 176 LCquan User Guide The Tune Method view displays the tune method stored in the current raw file during acquisition Figure 106 Figure 106 Tune Method view Parameter vane Calibration File Name C Xcaliburimethods 1 0250 Tune File Name C Xcaliburymethods Alpraz lon Source Type HESI Auto Peak Width Setting YES Positive Polarity Q1 MS Spray Voltage Vaporizer Temperature Sheath Gas Pressure 70 lon Sweep Gas Pressure 0 0 Aux Gas Pressure 30 Capillary Temperature Capillary Offset 35 Tube Lens Offset 1 Mass 162 082 Value 6 Tube Lens Offset 2 Mass 50
71. and choose Add or Change Peak Comment The Chromatogram Comment dialog box opens Figure 216 Figure 216 Chromatogram Comment dialog box Chromatogram Comment Paroxetine File Name Integration Type APN_003 Method Settings Comment Cancel Apply Help 2 Type your comment and click OK or Apply 374 LCquan User Guide Thermo Scientific 6 Processing the raw Files and Reviewing the Analytical Results Creating and Reviewing Reports If Apply is used the dialog box remains visible and you can select another sample component or integration type Figure 217 shows an example Figure 217 Example of chromatogram with comment 4 gt a Selected Chromatogram APN_OOS m z 192 07 192 07 RT 1 53 2 28 NL 5 64E3 F SRM ms2 330 13 cid 20 00 70 15 192 07 This is my comment _________________ Comment Area RT 1 90 E Table 146 Chromatogram Comment dialog box parameters Parameter Description File Name View only Displays the name of the current chromatogram Integration Type View only Displays the peak integration types of the current chromatogram e Processing Method Settings The settings specified in the processing method or LCquan method e User Settings Settings specified in the User Identification Settings dialog box e Manual Integration on the selected peak Manually adjust the peak baseline and end points Comment The text you enter as a chromatogram
72. and left edges of the peak when the S N reaches a value less than 200 Range 50 0 to 10 000 0 Note You can test the results of the new criteria by clicking Apply or OK You can apply the new criteria to all files in the Result list by clicking Apply to All Thermo Scientific LCquan User Guide 355 6 Processing the raw Files and Reviewing the Analytical Results Reviewing the Calibration Curve and OCs User Identification Settings ICIS Integration Use this page Figure 206 to set up advanced chromatographic parameters Table 138 lists the parameters for the ICIS Integration page of the User Identification Settings dialog box Figure 206 User Identification Settings dialog box showing the ICIS Integration page User Identification Settings Identification Detection ICIS Integration ICIS Advanced Baseline Window Area Noise Factor Peak Noise Factor I Constrain Peak Width Cancel Apply Apply Toal Help Table 138 User Identification Settings dialog box ICIS Integration page parameters Sheet 1 of 3 Parameter Description Baseline Window The number of scans used when searching for where the minima are occurring A local minimum becomes an anchor point making the entire curve locally reduced until these points are at Zero Range 1 to 500 Default 40 Area Noise Factor The noise level multiplier used to determine the peak edge after the location of the possible peak so that the peak can na
73. and the current selection is at the top of the list so the left backward arrow is grayed out e The yellow or red lock is set so no changes can be made in the red locked pane and only changes other than raw file or component changes can be made in yellow locked panes Both arrows are grayed out Note The actions of the arrows vary in some instances between the Explore and Quantitate windows For example in the Chromatogram pane in Explore the arrows can move through multiple peaks for one sample In the same pane in Quantitate they can move through different samples Lock Allow changes or prevent changes to a raw file or parameters The color of the lock indicates the lock status of a pane Red Locked Against All Changes The lock icon is a closed lock e You cannot make any changes in panes that have red locks e No changes you make are reflected in panes that have red locks e All shortcut menu commands that can change the pane or the data are unavailable Thermo Scientific LCquan User Guide 151 3 Exploring the Data Data Views Table 53 Toolbar for Selectable Views controls Sheet 2 of 3 Control Description Yellow Locked Against raw file or Component Changes The amp lock icon is a closed lock e You cannot make changes to a raw file or to a component in the Calibration Curve pane in panes that have yellow locks e When you make changes other than raw file changes in any pane those change
74. available from the Survey or Review All Results views in the Quantitate window Figure 149 This view displays a calibration curve for the selected component The replicates list is created by adding all the integrated standards from the result rows There is a one to one correspondence between each result row in the Standards tab and the replicates list LCquan User Guide 255 4 Creating a Processing Method Data Views Figure 149 Calibration Curve view Calibration Curve Paroxetine Y 0 017475 0 00224456 K R 2 0 9970 Vi Equal 25 2 T 1a 5 D Ed 400 600 800 1000 When the view is locked lock icon is yellow or red you cannot select any menu item in the shortcut menu that changes the raw file with the yellow lock or the raw file and parameters with the red lock Menu items that would cause changes are grayed out For more information about locks see Toolbar for Selectable Views on page 252 Table 99 lists the parameters for the Calibration Curve view Table 99 Calibration Curve view parameters Sheet 1 of 3 Parameter Description Arrows The left and right arrows move through the sequence list incrementing or decrementing by a single sample Shortcut Menu Lock against All Specifies that changes to a raw file or parameters such as Change retention time selection in other panes have no effect on this pane The lock icon is red and the lock is closed Unlock Specifies that changes to
75. be seeking the maximum possible accuracy from your measurements Time and cost of analysis are less important than achieving the highest possible standards in precision and accuracy This process of measuring the amount of a particular component in a sample is called quantitative analysis In other applications such as in trace analysis you might only want to estimate the quantity of a component It might be sufficient to know that the component is present at a level either significantly higher or significantly lower than a defined threshold For example knowing whether a patient has overdosed 15 or 20 times above a prescribed limit is generally not as important as simply knowing that the limit has been exceeded Such cases would require a rapid measurement rather than a precise one This form of measurement is generally called semi quantitative analysis 1 For further information about the principles of quantitative analysis refer to Mass Spectrometry Principles and Applications de Hoffman E Charette J Stroobant V Wiley New York 1996 and Introduction to Mass Spectrometry 3rd ed Watson J T Lippincott Raven Philadelphia PA 1997 Thermo Scientific LCquan User Guide 401 A Quantitative Analysis Overview Considering the Variables of Quantitative Analysis by LC MS MS Quantitative analysis consists of the following steps e Preparing samples e Developing a suitable chromatographic method e Calibrating the mass spectrometer
76. box parameters on page 180 In the Arrangement box enter the number of columns and rows You can set the normalization level to be the same for all peaks or you can normalize each peak individually To set the normalization levels Right click the plot and choose Options from the shortcut menu The Multi Peak Options dialog box opens For more information see Multi Peak Options dialog box parameters on page 180 In the Intensity Scale box select either the Normalize to Largest Peak or Use Fixed Intensity Range option To set label and plotting styles From the window menu choose Options gt Display Options gt Chromatogram The Chromatogram Display Options dialog box opens These label and plotting styles apply to the multi peak views For more information see Chromatogram Display Options dialog box parameters on page 155 When the view is locked lock icon is yellow you cannot select any menu item in the shortcut menu that changes the raw file Menu items that would cause changes are grayed out For more information about locks see Toolbar for Selectable Views on page 150 Table 68 lists the parameters for the Multi Peak Plot view 178 LCquan User Guide Thermo Scientific 3 Exploring the Data Data Views Table 68 Multi Peak Plot view parameters Parameter Description Arrows The left and right arrows move through the grid incrementing or decrementing by a single sample peak P
77. box to convey information about this sample to others or as a reminder to yourself The User Label 3 default name is Laboratory To change definitions of User Labels and Values for any user defined column refer to the instructions for the Client column LCquan User Guide 391 6 Processing the raw Files and Reviewing the Analytical Results Creating and Reviewing Reports Table 152 Grid parameters Sheet 7 of 9 Parameter Description Left Right Delta drugx_11 TIC SM 5 RT 4 61 5 65 NL 2 32E4 Height F SRM ms2 465 30 419 30 421 30 Left Right Height ss 90 80 70 amp 60 k 50 2 24 30 Left Delta Height 5 0 5 2 Time min Left Delta Height is the vertical distance between the start of the chromatographic peak and the starting point of the chromatographic peak integration Right Delta Height is the vertical distance between the end of the chromatographic peak and the end point of the chromatographic peak integration Left Height is the vertical distance between the starting point of chromatographic peak integration and the x axis of the chromatographic plot Right Height is the vertical distance between the end point of chromatographic peak integration and the x axis of the chromatographic plot Left Time The retention time corresponding to the start of peak integration Phone User defined Displays information that is pertinent to the active sample row in the Acquisition sequence You can use
78. but not yet validated data file has not yet been accepted for acquisition and not yet running e Queued Submitted and validated e In Progress Currently being acquired e Stopped Stopped either at the request of the operator or because of an error in acquisition e Complete Successfully completed e Rejected Failed validation rejected and did not run Components Displays the acquisition component names and their associated level amounts Samples Displays all the sequence row information set at the time of the sample acquisition Shortcut Menu Show Sequence Items Collapses the Acquisition Sequence History tree so that only the first level items sequences are shown Show Displays second level items for each sequence in the Acquisition Info Level Sample Sequence History tree These are Info Levels and Samples Items Show Displays third level items for each sequence in the Acquisition Info Level Sample Sequence History tree These are Info details Level details and Details Sample details Show Info Details Expands the Acquisition Sequence History tree to show details for each Info item and collapse all other branches Show Level Details Show Sample Details Expands the Acquisition Sequence History tree to show details for each Level item and collapse all other branches Expands the Acquisition Sequence History tree to show details for each Sample item and collapse all other branc
79. cave wide eben ttle pln cee lade Aish tae hin elite diate Dac 275 General Parameters Plot View for Survey or Review All Results Only 276 Instrument Method List VieWis lt e 2 sa arsveg dabckars atanbatesanieralbyed tneyuralabe 278 Mass List View sic sis oo ee es Wes oa oO RE Sa NS eee 279 Peak List View for Survey or Review All Results Only 281 Sample Info View arises beh ee ere er kr eee reer tee A RoR 282 Status Plot View sex Mon btdcate toa at staat ah eso ae ca ies ae Ware ep a a heattioe 284 Status Report Views vcewtek dt ae sedi Dia Gh od owe hed ae 287 Mane Method Views irera ain g E wae ale tain E A a A a 288 Multi Peak Plot View for Survey or Review All Results Only 289 Creating a Processing Sequence 0ee eee eee e eee eens 293 Defining a Processing Sequence Jeeps ket de Wi tell ee le ted ews 294 Using the New Processing Sequence Wizard 1 0 0 0 cee eee eee 296 Copying an Acquisition Sequence 0 0 eee eee eee eee 298 Importing a Processing Sequence 1 0 eee eee eee 299 Copying the Last Acquired Sequence votives wire weve eat 300 Editing a Processing Sequence essa eeai rruna 300 Using the Create Sequence edit View n a dena senescent ok 300 Using the Create Sequence create View a cadkiae tated te tee hire oes 315 Resolving Discrepancies in Level Names 0 0 000 cece eee eee 319 Exporting a Processing Sequence 6 ee eee eee 321 Processing the ra
80. command again To navigate the spectrum pane Do one of the following e Use the display buttons in the toolbar or the items in the Zoom menu to zoom in or out along the axes e Drag your cursor in the chromatogram pane to define a region and zoom in When you zoom in the trace does not expand if the data extends out of the pane 90 LCquan User Guide Thermo Scientific Thermo Scientific 2 Preparing to Perform Quantitative Analysis Using the Status View To use the Spectrum pane shortcut menu Right click on the Spectrum pane to display the shortcut menu Figure 53 Figure 53 Spectrum pane shortcut menu Filter Spectrum Display Options Reset Scaling Copy to Clipboard Table 30 Spectrum pane shortcut menu commands Command Filter Spectrum Description Applies a scan filter to the acquired data The LCquan application creates scan filters from the instrument settings that you specified in the Instrument Setup window Display Options Displays the Spectrum Display Options dialog box where you can modify the appearance of the spectrum in the Spectrum pane For more information see Spectrum Display Options dialog box parameters on page 92 Reset Scaling Copy to Clipboard Resets the x axis and y axis ranges in the Chromatogram pane or the Spectrum pane to their default values Copies a chromatogram or mass spectrum to the Clipboard You can then paste it from the Clipboard into a document
81. comment plots below the header info Creating and Reviewing Reports Use the Review Reports view Figure 218 of the Quantitate window to create and review reports describing the quantitative analysis results for your data To create and review reports for your data 1 To open the Review Reports view click the Reports icon in the navigation pane 2 To specify Excel reports do the following a In the Excel Report Selections table select the Use check box in an empty row b Click the Column Arrangement column and select a column arrangement from the list Thermo Scientific LCquan User Guide 375 6 Processing the raw Files and Reviewing the Analytical Results Creating and Reviewing Reports Three column arrangements are available by default Quan Result Grid Excel Long Summary and Excel Short Summary For a description of each report type see Report Options on page 384 When the Quan Result Grid option is selected the report is generated using the column arrangement of the current Quantitate results grid Use the Column Arrangement dialog box to create additional column arrangements c To create additional Excel reports repeat steps a and b d To create sample style Excel reports reports with information on one sample per page rather than the default one component per page select the Sample Style Excel Reports check box 3 To specify reports using the XReport templates do the following a Inthe XRep
82. data Label Threshold Sets a threshold so that peaks above this level can show labels Plotting Select one style to display your data Point to Point Select a graphic style to display the active chromatogram or spectrum using point to point peak profile Stick Select a graphic style to display the active chromatogram using vertical lines On Off Labeling Option When No Peak Found Show Apex Time Selects to always display maxima labels Labels Axis Offset Select one or both to offset the displayed plot from the x axis y axis or both X Select to have the x axis offset move the y axis slightly above the x axis so that you can see baseline details Y Select to have the y axis offset move the x axis slightly to the right of the y axis so that you can see plot details at low x axis values Thermo Scientific 4 Creating a Processing Method Data Views Chromatogram List View Thermo Scientific This view displays the chromatographic data points in table format Figure 156 In the Survey or Review All Results views in the Quantitate window the Item Selector List options for this view are identical to those for the Chromatogram view so that you can compare the two views side by side Figure 156 Chromatogram List view Chromatogram List 003 2 Relative p ew aes 3 4 4 6 8 8 6 5 4 The contents of the grid are read only When the view is locked lock icon is red
83. defined 406 identifying 246 using for quantitation 407 Ion Ratio Chromatogram Display Options parameters 271 ion ratio confirmation IRC Avalon integration 229 description 234 example chromatogram 234 Genesis advanced 223 integration 220 hydrocortisone example 234 ICIS advanced 227 integration 225 Ion Ratio Confirmation area parameters 194 Ion Ratio Confirmation area shortcut menu commands 195 ion summing Summing Ions dialog box 196 trace type 188 348 IRC Chromatogram parameters 269 LCquan User Guide 439 Index K IRC Chromatogram shortcut menu commands 269 IRC Detection Method Avalon Integration parameters 229 Genesis Advanced parameters 223 Genesis Integration parameters 221 ICIS Advanced parameters 227 ICIS Integration parameters 225 Identification parameters 219 isotope calibration settings 339 ISTD Chromatogram pane 326 K keyboard shortcuts Apps menu commands 419 File menu commands 420 Options menu commands 424 toolbar commands 432 View menu commands 427 L levels in processing method 304 resolving names 319 show amounts 314 Limit Peaks parameters 131 liquid chromatography LC analyte separation 402 lock 398 locking plots for comparison 139 locking the workbook 397 locking workbook automatically after creating report 398 locks against raw file change 139 lower quantitation limit 405 Mass List parameters Explore 164 Quantitate 279 Mass List shortcut menu commands Explore 165 Quantitate 280
84. display a chromatogram here use Quantum Open Raw File Se a a a a ee a a es o 4 2 3 4 Retention Time min 6 7 8 Segment 1 Settings Segment Time min 10 00 Tune Method C Xcalibur Methods ex TSOQTune Scan Events 1 Chrom Filter Peak Width s C Collision Gas Pressure mTorr Scan Event 1 Polarity Scan Type SRM Positive Negative Same value for all SAMs 7 Data Type Scan With mya I 1000 __ParentMass Product Mass Scan widtn Centroid Profile Scan Time s i e 1000 000 Le Skimmer Offset Skimmer Offset v 10 Coll Energy V Peak Width 01 FWHM i Micro Scans a 03 FWHM 7 0 M g Use Tuned Tube Lens Value i For Help press F1 dana powers 2 LCquan User Guide Thermo Scientific 1 Introduction Quantitative Analysis with LCquan Acquisition Window In the LCquan Acquisition window you can set up a sequence of samples for acquisition Figure 2 The sequence can contain unknown samples calibration standard samples quality control samples and blank samples After you have defined a sequence you can specify acquisition options and processing action options for the sequence and start running it The Acquisition window also contains a real time display so you can watch updates of the acquisition status and the data being acquired The Acquisition window contains the Setup Sequence
85. e TM pure is the pure target compound Range 0 00 to 100 00 LCquan User Guide 245 4 Creating a Processing Method Specifying Method Calibration Specifying Calibration Options To set global calibration options 1 Choose Options gt Calibration The Calibration Options dialog box opens Table 97 lists the parameters for the Calibration Options dialog box Figure 146 Calibration Options dialog box Calibration Options Calibrate By ntemal Standard C Extemal Standard OK Cancel Save As Default Help 2 Select Internal or External standards 3 Optional Save your calibration standards choice as the default Table 97 Calibration Options dialog box parameters Parameter Description Calibrate By Internal Standard Specifies that the method use internal standard calibration External Standard Specifies that the method use external standard calibration Specifying Internal Standards and Target Compounds An internal standard ISTD component acts as a response reference for the target components in the sample Note When creating an internal standard Processing Method you must define at least one component to be an internal standard before you can define any other components as target compounds To specify the internal standard settings 1 In the Component list Figure 147 select the component that you want to identify as an internal standard 2 In the Component Type area select the ISTD
86. eluate Specifically you must ensure that what goes into the inlet of the mass spectrometer does not suppress ionization of the compound of interest Salt concentrations above 10 mM and strong acids and bases damage the LC column Modifier concentrations greater than 10 mM are not usually necessary for chromatographic stability and can suppress ionization of other compounds For best results whenever possible use volatile modifiers Volatile modifiers include the following e Acetic acid e Ammonium acetate e Ammonium formate e Ammonium hydroxide e Formic acid Trifluoroacetic acid Thermo Scientific LCquan User Guide 403 A Quantitative Analysis Overview Quantitative Analysis Techniques Your analyte solution can contain neutral particles or ions If the solution contains ions the ions can carry a single charge or multiple charges The number of charges depends on the structure of the analyte and the composition and pH of the mobile phase Solvent systems are generally composed of organic solvents water and volatile modifiers The ionization characteristics of your analyte influence your decisions about the optimum pH of your solvent system and the type of probe to use at the interface of the LC and the mass spectrometer With the TSQ Quantum series LTQ series or LCQ series instruments you can use either the H ESI probe the ESI probe the APCI probe or the APCI APPI probe at the interface of the LC The choice of probe depends on
87. evaluate the valley bottom Using this parameter ensures that the surrounding measurements are higher Range 1 0 to 100 0 Default 2 0 Peak S N Cutoff Specifies the value for the signal to noise cutoff below which the LCquan application defines the peak edge the box displays the current cutoff value that it uses for defining the peak edge For example if the signal to noise at the apex is 500 and the Peak S N Cutoff value is 200 the LCquan application defines the right and left edges of the peak when the S N reaches a value of less than 200 Range 50 0 to 10 000 0 224 LCquan User Guide Thermo Scientific IRC Detection Method ICIS Integration 4 Creating a Processing Method Specifying Method Identification Use this page Figure 135 to set up advanced chromatographic parameters Table 86 lists the parameters for the ICIS Integration page of the IRC Detection Method dialog box Figure 135 IRC Detection Method dialog box showing the ICIS Integration page IRC Detection Method Identification ICIS Integration ICIS Advanced Baseline Window IE Area Noise Factor 5 Peak Noise Factor 10 I Constrain Peak Width az Table 86 IRC Detection Method dialog box ICIS Integration page parameters Sheet 1 of 2 Parameter Baseline Window Description The number of scans used when searching for where the minima are occurring A local minimum becomes an anchor point making the entire curve locally reduced un
88. from within the LCquan application Audit Viewer displays only the items associated with the current workbook including uncommitted items You can print the audit trail for an LCquan workbook if the page displays only saved entries For more information refer to the Help within Audit Viewer or refer to the LCguan Administrator Guide Create Locked Version of Workbook Opens the Create Locked Workbook dialog box that you use to ALT F gt L gt L save a copy of the current workbook as a locked workbook Recently opened files Displays a list of recently opened files To open a recently opened Fxit workbook select the workbook file from this list Exit Quits the LCquan application ALT F gt X Commands Specific to Instrument Setup Window Table 158 Commands specific to the Instrument Setup window Command Import Instrument Method Description Keyboard shortcut Opens the Import Instrument Method File dialog box that you use ALT F gt I to import an instrument method file Print Current Instrument Method Thermo Scientific Opens a Print dialog box that you use to print the instrument ALT F gt P method meth LCquan User Guide 421 C LCquan Menu and Toolbar Reference File Menu Commands Specific to Acquisition Window Table 159 Commands specific to the Acquisition window Command Description Keyboard shortcut Import Acquisition Opens the Load Sequence File dialog box that you use to importan A
89. in the current raw file The format of the list items is Generic Detector Stream Name Instrument Name Model Name Channel Name Shortcut Menu Open Raw File Opens the Select Raw File dialog box which you can select the raw file to load Lock Against Raw File Prevents changes to a raw file The lock icon is yellow and the Change lock is closed Unlock Makes the pane editable The lock icon is green and the lock is open Reset Scaling Resets the x and y axes to display the maximum amount of data Copy to Clipboard Copies selected data to the Clipboard You can then paste the data from the Clipboard buffer into a document LCquan User Guide 173 3 Exploring the Data Data Views Status Report View 174 LCquan User Guide The Status Report view displays the status information stored in the current raw file in table format Figure 105 A status report is a list of status information for all parameters at one retention time Use the arrows to move between time points of the chromatogram Notice that status information is acquired less frequently than the chromatogram so the status information does not always change Figure 105 Status Report view 4 gt a MS Quantum F Status Report RT 1 920 Parameter vane 8 kY lonized Voltage 8 kY lonized Current Sheath Gas Flow Rate Aux Gas Flow Rate lon Sweep Gas Flow Rate API Temperature Capillary Temperature Capillary Offset Tube Lens Of
90. in viewbar Acquisition view Run Sequence dialog box Click OK Click Next and Finish Define levels Quantitate view gt 2 LE 2 as g5 e S jg 1 l 2 ij oO 31 feb a ol v o il iq Ti Processing view if Process After Acquisition was selected in the Run Sequence dialog box 414 LCquan User Guide Thermo Scientific LCquan Quantitate Flow Diagram Select Import Existing Method option Import Quantitation Method dialog box Click Next in then click Next Select Import Existing Sequence Import Sequence option dialog box Click Next in New Processing Sequence wizard then click Next New Method wizard Create or import method Select Raw File dialog box Click Next and Finish Create Method Identify Components page Click on Calibrate tab Create Method Define Calibration Settings page Click Sequence in viewbar Create or import sequence Select New Processin then click Next Create Sequence Review and Edit page New Method wizard Sequence wizard Create New Sequence option in B LCquan Flow Diagrams LCquan Quantitate Flow Diagram Select Create New Method option in Calibration Options page Click Next then click Next Select Create New Sequence option Create Sequence in Select Files page New Processing Click Next then click N
91. min 12 01 Right Height 3 06 OK Cancel Apply Help Cancel Any Heb 2 Type the new coordinates for the starting and ending points in the appropriate boxes and click OK Table 142 lists the parameters for the Baseline Paroxetine dialog box Table 142 Baseline dialog box parameters Parameter Description Peak Baseline Endpoints Left min Type the left extreme of the integration baseline for the current component in minutes Right min Type the right extreme of the integration baseline for the current component in minutes Left Height Type the height of the current component peak at the left extreme of the integration baseline in units of counts Right Height Type the height of the current component peak at the right extreme of the integration baseline in units of counts To undo the manual integration 1 In the Integration Type column click the cell containing the words Manual Integration for the sample that you want to change 2 Click the down arrow and select Method Settings from the list to return the integration to its prior settings 364 LCquan User Guide Thermo Scientific 6 Processing the raw Files and Reviewing the Analytical Results Reviewing All Results Reviewing All Results Use the Review All Results view in the Quantitate window to review and rework samples of all types including unknowns and blanks If necessary rework the samples by modifying the peak detection and in
92. name association Thermo Scientific 5 Creating a Processing Sequence Exporting a Processing Sequence To resolve incomplete level name association When you add or import samples or sequences and associate the new level names with the names in the current workbook if any discrepancy remains in the level names the Level Name Association Not Complete dialog box opens Figure 187 You have these options e To return to the Standard or QC Level Names Association dialog box and finish associating the level names click Complete e To assign the unknown sample type to the samples that have unassociated levels click Set to Unknown e To remove the samples that have unassociated levels from the sequence click Discard Resolving Missing Calibration or OC Levels The Workbook Does Not Contain Levels dialog box warns that the samples are associated with level names but the workbook processing method has no calibration or QC levels defined You have these options e To set all samples in the sequence that have a specified QC or calibration level to sample type unknown click Set to Unknown e To remove all samples that have a specified calibration level from the sequence click Discard e To cancel the level names association process click Cancel Make sure you are using the correct sequence and processing method for the workbook To modify the sequence see Editing a Processing Sequence on page 300 To modify the
93. of standard solutions containing known concentrations of the target compound and ISTD and plot the ratio of the target compound and the ISTD detector responses as a function of the corresponding ratio for the two quantities present in the solution 2 Add a fixed amount of the ISTD to each sample prior to any manipulation and after preparing and analyzing the samples obtain the quantity of the target compound present in an unknown sample from the calibration curve Ideally an ISTD is closely related to the target component in terms of its physical and chemical properties It must be pure not present in the sample and inert toward the components of the sample ISTD components are typically analogs homologs or isomers of the target non ISTD component An ideal ISTD is a structural or isotopically labeled analog of one of the target components Stable isotope labeled ISTDs act almost identically to the analyte throughout sample manipulation and with regard to ionization tendencies and fragmentation Internal standards labeled with two or more deuterium D atoms are frequently used for LC MS There can be any number of ISTD components in a sample but each non ISTD component can be calibrated against only one ISTD Figure 232 Thermo Scientific LCquan User Guide 407 A Quantitative Analysis Overview Sample Types Figure 232 Calibration curve generated by using an internal standard Response ratio for 1 24 unknown sample Unknown A
94. on the LCquan window Instruments Acquisition Explore or Quantitate Table 174 Toolbar buttons Sheet 1 of 4 Icon Button Description New Opens the New Study or Workbook Wizard The New Study or Workbook Wizard L prompts you for information that the LCquan application requires to create a new study or workbook For more information see Creating a New Study and Workbook on page 13 in Chapter 2 Preparing to Perform Quantitative Analysis Open Workbook Opens the LCquan Welcome page that you use to open an existing workbook or to create a new study or workbook For more information see Create a New Workbook on page 14 in Chapter 2 Preparing to Perform Quantitative Analysis H Save Saves the current workbook Open Raw Opens the Select Raw File dialog box where you can open an existing data file of extension raw Note This feature is not available in the Instruments window Icon Bar Displays or hides the Icon Bars Use the left navigation panes to navigate between the LCquan windows and the windows within each window For more information see Icon Bars on page 431 Component Bar Displays or hides the Component bar Note This feature is available only for the Quantitate window Status Information Displays or hides the Status pane on the Status view For more information see F e View Using the Status View on page 80 in Chapter 2 Preparing to Perform Quanti
95. option The Internal Standard area becomes active 246 LCquan User Guide Thermo Scientific 4 Creating a Processing Method Specifying Method Calibration 3 Specify the internal standard settings a Inthe ISTD Amount box enter the amount of the internal standard injected into each sample b In the ISTD Units box enter the units of the internal standard injected into each sample 4 Click Apply to accept the internal standard settings 5 To identify other components as internal standards repeat steps 1 through 4 Figure 147 Internal standard interface example Component psoas ae Paroxetine m z Target Compound 1 000 A i g ISTO ISTD Units To specify the calibration curve parameters for the target compounds 1 In the Component list select the component that you want to identify as a target compound 2 In the Component Type area select the Target Compound option The Target Compound area becomes active 3 To create an internal standard method in the ISTD list select the internal standard for the target compound 4 In the Response area specify the LCquan response by selecting the Area option or Height option 5 In the Target Units box enter the units that you want to appear under the x axis of the calibration curve plot Thermo Scientific LCquan User Guide 247 4 Creating a Processing Method Specifying Method Calibration 6 In the Calibration Curve Type list select the cal
96. or hides if it is currently displayed Status Bar Displays or hides the status bar The status bar is the horizontal ALT V gt S box at the bottom of the LCquan window The status bar appears if it was previously hidden or hides if it is currently displayed Icon Bar Displays or hides the Icon Bars For more information see Icon ALT V gt I gt Bars on page 431 ENTER Section Indicator Displays or hides the view title banner For example Explore ALT V gt Review the explore results DOWN ARROW gt ENTER Show Large Toolbar Increases the size of the Toolbar For more information see ALT V gt L Thermo Scientific Toolbar on page 432 LCquan User Guide 427 C LCquan Menu and Toolbar Reference View Menu Table 168 Commands common to all LCquan windows Sheet 2 of 2 Command Description Keyboard shortcut Show Large Icons in Bar Increases the size of the icons in the Icon Bars For more ALT V gt I gt I gt information see Icon Bars on page 431 ENTER Section Selection Choose this command to select a window ALT V gt l DOWN ARROW gt 2 RIGHT ARROW gt cquisition ENTER e Explore e Quantitate or DOWN ARROW gt ENTER Commands Specific to Instruments Window Table 169 Commands specific to the Instruments window Command Description Keyboard shortcut Instrument Selector Selects the instrument to be edited ALT V gt DOWN ARROW gt RIGHT ARROW gt ENTER DOWN ARROW gt ENTER Commands Specific to Ac
97. page in the Create Method view of the Quantitate window Measured Coelution Delta The measured difference in retention time between the apex of the target ion and the apex of the qualifier ion The Measured Coelution Delta must fall within the range of the Allowed Coelution Delta in order for the peak to pass the coelution test lon Ratio Test Passed Indicates the peak passed the ion ratio test Target Ratio Indicates the peak passed the ratio test Allowed Target Range The expected target ratio Measured Ratio The measured ratio LCquan User Guide 373 6 Processing the raw Files and Reviewing the Analytical Results Reviewing All Results Peak Information Spectrum This page Figure 215 displays an unfiltered spectrum plot at the peak apex retention time This spectrum plot is display only Figure 215 Peak Info dialog box showing the Spectrum page Peak Info Alprazolam d5 ISTD for Paroxetine Info Flags Spectrum 492 RT 2 67 NL 1 10E5 T SRM ms2 314 10 cid 40 00 209 96 209 96 400 209 96 80 60 40 w a 2 a 2 P a w c 20 0 209 9600 209 9605 209 9610 209 9615 miz Peak Type Method Adding Comments to the Chromatogram Display Use the Chromatogram Comment dialog box to add a comment to the header in the Chromatogram view Table 146 lists the parameters for the Chromatogram Comment dialog box To add a comment 1 Right click the Chromatogram pane
98. reports The LCquan application does not convert units When you have specified a processing method for your sequence the LCquan application automatically enters the sample volume of the Calibration samples QC samples or both from the processing method settings Thermo Scientific 2 Preparing to Perform Quantitative Analysis Using the Setup Sequence View Table 25 Acquisition sequence grid column parameters Sheet 3 of 3 Parameter Sample Wt Description Displays the amount of a component that has been placed in the sample The unit for this sample weight is specified in the Xcalibur Processing Setup window and is included only in LCquan reports The LCquan application does not convert units When you have specified a processing method for your sequence the LCquan application automatically enters the sample weights of the calibration samples QC samples or both from the processing method settings Sample Name Displays the sample name that you specified when you created the sequence Comment Displays the comment entered for this sample Barcode Displays the barcode for this sample Fill Down cannot be used on Barcode column values Barcode Status Displays the status of the barcode reading User Columns 1 5 User defined Displays information pertinent to the active sample row in the acquisition sequence Use these columns to convey sample information to others or as a reminder to y
99. result grid to display the shortcut menu Figure 189 Table 125 lists the commands for the results grid shortcut menu Figure 189 Result grid shortcut menu Columns Delete Selected Samples Add Sample Duplicate Selected Row Set Sorting Order Color Coding Legend Table 125 Result grid shortcut menu commands Sheet 1 of 2 Command Columns Description Opens the Column Arrangement dialog box where you can customize the column arrangement and display See Customizing Column Arrangement on page 329 Delete Selected Samples Removes the selected samples rows from the Result grid Add Sample Opens the Select Raw File dialog box where you can select samples to add to the Result grid The LCquan application obtains the sample information from the selected files and adds the samples to the sequence Duplicate Selected Row Copies the selected row and adds it to the Result grid The new row is added to the sequence according to the current sort criteria LCquan User Guide 327 6 Processing the raw Files and Reviewing the Analytical Results Reviewing the Calibration Curve and OCs Table 125 Result grid shortcut menu commands Sheet 2 of 2 Command Description Copy Selected Rows to Copies the selected rows to the Thermo LCquan TrackQC TrackQC TrackQC provides calculations for intraday or interday QC run validation The QC samples you copy to TrackQC can include one or more QC le
100. selected instrument Figure 104 Status Plot view 4 gt a 8 k loni Ms Qua Status P APN_011 Device MS 2981 6 2981 4 2981 2 2981 0 2980 8 2980 6 2980 4 2980 2 2980 0 w D gt i gt a N c 2 gt oO 2979 8 2979 6 2979 4 2979 2 2979 0 2978 8 2 Time min The contents of the plot are read only When the view is locked lock icon is yellow or red you cannot select any menu item in the shortcut menu that changes the raw file Menu items that would cause changes are grayed out For more information about locks see Toolbar for Selectable Views on page 150 Table 65 lists the parameters for the Status Plot view Table 65 Status Plot view parameters Sheet 1 of 2 Parameter Description Arrows The left and right arrows move through the item list incrementing or decrementing by a single item Item Selector List Options Parameter Specifies the parameter plotted on the y axis 172 LCquan User Guide Thermo Scientific Thermo Scientific 3 Exploring the Data Data Views Table 65 Status Plot view parameters Sheet 2 of 2 Parameter Description Detector Selector List Options MS Displays status information for a mass spectrometer Status Displays status information for a device that does not log data Detector Streams in Current Raw File When a raw file is open displays the list of detector streams that are stored
101. selected row is highlighted b Click Delete The highlighted row is removed from the list 4 To change the values within a row a Select the row you want to change The selected row is highlighted b Specify the revised settings in some or all of these boxes Time Event Value c Click Change to automatically update the event list and the chromatogram display 214 LCquan User Guide Thermo Scientific Thermo Scientific 4 Creating a Processing Method Specifying Method Identification 5 To estimate the initial values for the detection of peaks and display initial values in the event list click Auto Calculate Table 81 Avalon Peak Integration area parameters Parameter Description Column Headings in the Event List Time min Displays the initial time value in minutes Event Displays descriptions of detection parameters for initial events and timed events For details see Table 89 Note Start Threshold End Threshold Area Threshold P P Resolution Threshold Bunch Factor Negative Peaks and Tension are defined with initial values Value Displays the values associated with initial events or timed events Range Factors are specific to each event Controls to Modify the Event List Auto Calculate Estimates the initial values for the detection of peaks based on the data in the current raw file to display initial values in the event list and searches for the best values of initial
102. sequences Stop acquisition For current sample and pause Aroun ER Stop acquisition For current sequence and pause Stop acquisition For all sequences Riin hd ananar e To pause the Chromatogram and Spectrum displays click 8 To restart the displays click again e To display the Spectrum pane below the Chromatogram pane click To hide the Spectrum pane click again Acquisition Status Chromatogram Pane The chromatogram pane is the upper right pane on the Status view of the Acquisition window While the LCquan application is acquiring data you can display an existing chromatogram in the Chromatogram pane using the current set of filters and settings The LCquan application continues to acquire data while displaying a raw file but the screen is in pause mode To navigate the chromatogram pane Do one of the following e Use the display buttons in the toolbar to zoom in or out along the axes e Drag your cursor in the chromatogram pane to define a region and zoom in When you zoom in the trace does not expand if the data extends out of the pane To display a raw file Do one of the following e Right click the chromatogram pane and choose Display Raw File from the shortcut menu and select an appropriate raw file from the Select Raw File dialog box e Drag a raw file from Windows Explorer to the chromatogram pane e Drag an older sample from the acquisition sequence history to the chromatogram pane
103. side IRC chromatograms with the last rightmost chromatogram being an overlay of all the IRCs and the component chromatogram Figure 154 The chromatograms have color coded borders Green Ion passed both IRC tests Red Ion did not pass at least one of the tests IRC does not support user peak integration settings Figure 154 IRC Chromatogram view IRC Chromatogram APN_OO3 miz 70 15 70 15 F c SRM ms2 330 13 F c SRM ms2 330 13 F c SRM ms2 330 13 Qualifier lon 70 15000 Qualifier lon 151 01000 1 90 h o o 100 100 wv 1A a a a a lt 2 p a a a a ec ec Relative Intensity 0 0 1 8 2 0 1 8 2 0 18 2 0 Time min Time min Time min When the view is locked lock icon is red you cannot select any menu item in the shortcut menu that changes the raw file or parameters Menu items that would cause changes are grayed out For more information about locks see Toolbar for Selectable Views on page 252 Table 104 lists the parameters for the IRC Chromatogram view Table 104 IRC Chromatogram view parameters Sheet 1 of 2 Parameter Description Arrows In the Create Method view the left and right arrows move through the IRC peaks incrementing or decrementing by a single peak In the Survey or Review All Results views the left and right arrows move through the samples incrementing or decrementing by a single sample Note When you open the Peak Info d
104. tae d Uae SO AS OS 397 Saving the Workbook and Exiting the LCquan Application 400 Quantitative Analysis Overview 0cc cece eee eee e eee eee 401 About Quantitative Analysis sy pas sia eg whens wk a ea ee aN 401 Considering the Variables of Quantitative Analysis by LC MS MS 402 Using LC for Analyte Separation s lt iivj 362s 656 heme sn tweed Meals 402 Using MS MS for Analyte Detection 06 6 0 ik de ence wa es 403 Quantitative Analysis Techniques ac cian tothe eta Me tee 404 Using External Standards for Quantitative Analysis 0 406 Using Internal Standards for Quantitative Analysis 0 406 Sample Types sier Teror chew wee wid oat Rede cae eee E the 408 Standards ies a dorra Meee A mae ed ala aad ase a ad Palas ere EO 408 UNKNOWNS aici p a E a wea Sh ae Ewe eee E ee Ree EES 408 OCS aden wa ena Shee apa eea See eee ee Ghee eee ES 409 Blanks apsenin ea Nea arpa ie RGR INCE sph ee Mae RN 409 LCquan Flow Diagrams 00e cece eee eee eee eee eee eaee 411 Le quan Flow Didgram 43 cr ries eae sel ap ieee Deke Ee Ep aE 412 LCquan for Web Access Flow Diagram 0 00 00 ee ences ede ee ee ee 413 Le quan Acquisition Flow Diagram cain owns how encore ae ties 414 LCquan Quantitate Flow Diagrain s asus esate ie ties Oot 415 LCquan Menu and Toolbar Reference 0 eeeeeeeeeeeeees 417 Using the Menusi fax iA Pie hk ies Baa ere ves a ae ine arenas Meas
105. that constrains the peak width of an asymmetric chromatogram peak that has a tailing trace It is the maximum ratio of the trailing edge to the leading side of a constrained peak and calculates the retention time of the maximum extent of the right edge of the tailing peak Range 0 5 to 9 0 Default multiplier 1 0 1 0 has no effect Note Valid only when you select the Constrain Peak Width check box Thermo Scientific 4 Creating a Processing Method Specifying Method Identification IRC Detection Method ICIS Advanced Use this page Figure 136 to set up advanced chromatographic parameters Table 87 lists the parameters for the ICIS Advanced page of the IRC Detection Method dialog box Figure 136 IRC Detection Method dialog box showing the ICIS Advanced page IRC Detection Method Identification ICIS Integration ICIS Advanced Noise Method Incos Noise C Repetitive Noise RMS Noise Min Peak Width 3 Multiplet Resolution fio Area Tail Extension 5 Area Scan Window fo Table 87 IRC Detection Method dialog box ICIS Advanced page parameters Sheet 1 of 2 Parameter Description Noise Method Incos Noise A single pass algorithm is used to determine the noise level Repetitive Noise A multiple pass algorithm is used to determine the noise level In general this algorithm is more accurate in analyzing the noise than the INCOS Noise algorithm but it takes longer RMS Noise A root mean sq
106. the Create Sequence edit view right click and choose Import Processing Sequence gt From File from the shortcut menu to display the Load Sequence File dialog box Note The level names in the processing sequence must match those defined in the processing method Ifthe calibration or quality control level names in the sequence are different from those in the processing method the LCquan application displays the Standard Level Names Association dialog box or the QC Level Names Association dialog box You can use these dialog boxes to associate the level names in the sequence with the level names in the processing method Ifthe imported sequence contains calibration or QC levels but the processing method does not the LCquan application displays a warning box to inform you It then displays the Sample Type Assignment dialog box You can change the sample type of these samples to unknown in the processing sequence or discard the samples that have these levels in the processing sequence LCquan User Guide 299 5 Creating a Processing Sequence Editing a Processing Sequence Copying the Last Acquired Sequence You can replace the defined processing sequence with a shortened sequence that is based on the data already acquired To update the processing sequence with the most recent list of acquired samples do the following 1 Choose Options gt Acquisition Sequence Info to open the Acquisition Sequence dialog box 2 To u
107. the LCquan application has correctly detected and integrated the peaks inspect the Result list again a The calculated amount of components in each unknown sample is displayed in the Calculated Conc column of the list Figure 212 Figure 212 Calculated Concentration column _ Response Ratio Specified Conc Calculated Conc i i 0 337 NA 144 056 0 240 NA 100 450 b Right click the Chromatogram pane and choose Show Peak Info from the shortcut menu The Peak Information dialog box opens where you can review the peak properties for the selected sample For more information see User Identification Settings ICIS Integration on page 356 c Leave the Peak Information dialog box open select the other sample files in the Result list and view the peak properties for them Reviewing Peak Properties To open the Peak Info dialog box right click a Chromatogram view in any of the preview panes and choose Show Peak Info from the shortcut menu The Peak Information dialog box has these tabs e Peak Information Info e Peak Information Flags Thermo Scientific LCquan User Guide 367 6 Processing the raw Files and Reviewing the Analytical Results Reviewing All Results e Peak Information IRC Tests e Peak Information Spectrum The IRC Tests tab is displayed only when showing peak information for an IRC Peak Information Info This page Figure 213 provides information about the selected peak T
108. the Sample Selector pane Thermo Scientific LCquan User Guide 137 3 Exploring the Data Exploring the Results Table 49 Sample Selector pane parameters Parameter Description Rawfile Specifies the current a raw file Explore Sequences Displays both loaded sequences and the acquisition history sequence The acquisition history is updated as samples are acquired Quantitate Sequences Displays a defined set of quantitative experiment parameters that defines operating settings for a single sample or list of samples Each sample can be defined by the following settings sample type file name sample ID QC or Standard level ISTD correction amount dilution factor vial position injection volume sample volume sample weight sample name and additional user defined parameters Acquisition Sequence Displays a history of all sequences and samples that have been History acquired by the current workbook along with other relevant information for the acquisition such as operator start and end times status and comment To use the sample selector shortcut menu Right click on the Sample Selector pane to display the shortcut menu Figure 79 Figure 79 Shortcut menu for the Sample Selector pane Acquisition Sequence k A w AcqSequence 1 APN_001 APN_002 Load New Sequence Show Sequence Items Show Info Level Sample Items Table 50 Sample Selector shortcut menu commands Sheet 1 of 2 Command Desc
109. the class of compound that you want to analyze and to a small extent on the flow rate of your experiment In general the APCI and the APCI APPI probes accommodate higher flow rates and molecules of a less polar nature than does the ESI probe Refer to the introduction in the getting started manual for your mass spectrometer for what factors might influence your decision about flow rates and what probe to use for optimum transfer of ions into the mass spectrometer After the ions are inside the mass spectrometer and the parent ions are isolated collision energy is applied to dissociate the parent ions into product ions MS MS The relative collision energy for a particular analysis depends on the structure of the compound that you are analyzing For this reason you optimize the collision energy on your analyte of interest in a tuning experiment Refer to the getting started manual for your mass spectrometer for the tuning procedure that describes how to optimize the collision energy Quantitative Analysis Techniques 404 LCquan User Guide To carry out quantitative analysis you must evaluate the instrument s response to known amounts of the target component Response is based on either the height of the chromatogram peak or more commonly the area under the peak s profile Figure 230 In both cases you must take into account the baseline of the detected peak Instrument response is generally measured with several samples commonly calle
110. the data file has not yet been accepted for acquisition e Queued submitted and validated e In Progress currently being acquired e Stopped stopped either at the request of the operator or because of an error in acquisition e Complete successfully completed e Rejected failed validation rejected and did not run Components Displays the acquisition component names and their associated level amounts Samples Displays all the sequence row information set when acquisition for that sample was made Shortcut Menu Show Sequence Items Collapses the Acquisition Sequence History tree so only the first level items sequences are shown Show Displays second level items for each sequence in the Acquisition Info Level Sample Sequence History tree These are Info Levels and Samples Items Show Displays third level items for each sequence in the Acquisition Info Level Sample Sequence History tree These are Info details Level details and Details Sample details Show Info Details Expands the Acquisition Sequence History tree to show details for each Info item and collapse all other branches Show Level Details Show Sample Details Expands the Acquisition Sequence History tree to show details for each level item and collapse all other branches Expands the Acquisition Sequence History tree to show details for each sample item and collapse all other branches LCquan User Guide 103 2 Prepa
111. the files sequences and workbooks It can either automatically time stamp raw files or never time stamp raw files when you acquire data to a remote workbook The time stamped raw files have the following properties Remotely stored raw files are time stamped with the submission time All raw files in a sequence share the same time stamp Pausing during acquisition does not change the time stamp The time stamp for the Rawfiles folder and the time stamp for the raw files are not necessarily the same IMPORTANT The LCquan application can overwrite a raw file of the same name if you turn off time stamping LCquan User Guide 109 2 Preparing to Perform Quantitative Analysis Remote Acquisition To time stamp raw files automatically when acquiring data to a remote workbook 1 Choose Start gt All Programs gt Thermo Foundation gt Authorization Manager to open the Authorization Manager 2 In the Authorization Manager do the following Figure 66 a b C f Select a user group in the Secure Groups list Click Expand Tree to show the entire list of controlled features for the application From the list click the plus sign before the LCquan folder Click the plus sign before the Acquisition Section folder Select Prevent Raw File Time Stamping When Doing Remote Workbook Acquisition The Permission Level options become available Select the Disallowed option and click OK Figure 66 Authoriz
112. the lock is open Spectrum at Peak Apex Displays the spectrum at the selected component peak s apex Spectrum at Peak Left Edge Spectrum at Peak Right Edge When more than one chromatogram peak is currently displayed in the pane the first chromatogram in the list is used for setting the RT Displays the spectrum at the selected component peak s left edge When more than one chromatogram peak is currently displayed in the pane the first chromatogram in the list is used for setting the RT Displays the spectrum at the selected component peak s right edge When more than one chromatogram peak is currently displayed in the pane the first chromatogram in the list is used for setting the RT Display Options Reset Scaling Displays the Spectrum Display Options dialog box where you can modify the appearance of the spectrum in the Spectrum pane For more information see Spectrum Display Options dialog box parameters on page 92 in Chapter 2 Preparing to Perform Quantitative Analysis Resets the x axis and y axis ranges in the Chromatogram pane or the Spectrum pane to their default values Copy to Clipboard Copies a chromatogram mass spectrum or calibration curve to the Clipboard You can then paste the data from the Clipboard buffer into a document Thermo Scientific 4 Creating a Processing Method Data Views IRC Chromatogram View Thermo Scientific This view displays up to six side by
113. the sample volume 1 Click the cell in the Sample Vol column 2 Highlight the text and type over it To change the sample weight 1 Click the cell in the Sample Wt column 2 Highlight the text and type over it 70 LCquan User Guide Thermo Scientific 2 Preparing to Perform Quantitative Analysis Using the Run Sequence Dialog Box To change the sample name 1 Click the cell in the Sample Name column 2 Highlight the text and type over it To change user defined column values 1 Right click the grid and choose User Labels and Values The Change Sequence User Labels dialog box opens 2 Edit the values and click OK For details about the parameters see Parameters in the Change Sequence User Labels dialog box on page 34 For an example of how the user labels and values are displayed in the sequence grid see User Defined Columns Values Examples on page 50 Tray Selection Use the Tray Selection dialog box to select the autosampler tray type to be used in the current session If the currently configured Autosampler does not support multiple trays a message confirming this is displayed The selected tray is used to validate sequence vial positions To open this dialog box Choose Change gt Tray Name The Tray Selection dialog box lists autosampler tray types available for use in the current session Using the Run Sequence Dialog Box Use the Run Sequence dialog box to select acquisition options and proces
114. to 10000 0 Valley Detection Valley detection approximation method to detect unresolved peaks Rise Percentage The percentage that the peak trace can rise above the baseline after passing through a minimum before or after the peak This method drops a vertical line from the apex of the valley between unresolved peaks to the baseline The intersection of the vertical line and the baseline defines the end of the first peak and the beginning of the second peak When the trace exceeds rise percentage the LCquan application applies valley detection peak integration criteria This test is applied to both the left and right edges of the peak The rise percentage criteria is useful for integrating peaks with long tails Range 0 1 to 500 0 Valley S N Specifies a value to evaluate the valley bottom Using this parameter ensures that the surrounding measurements are higher Range 1 0 to 100 0 Default 2 0 Report Noise As RMS or Determines if the noise used in calculating S N values is calculated Peak to Peak using an RMS calculation or peak to peak resolution threshold LCquan User Guide 121 3 Exploring the Data Creating Explore Methods ICIS Peak Integration Use the ICIS Peak Integration area to define ICIS peak detection algorithms to be applied to the active raw file Figure 72 To specify ICIS peak integration parameters 1 In the Peak Detection Algorithm box select ICIS Figure 72 ICIS Peak Integrat
115. used in the Explore windows Column Headings Parameter Displays a column of descriptive text for the item Value Displays a column of data for the given parameter Shortcut Menu Open Raw File Opens the Select Raw File dialog box where you can select the raw file to load Lock Against Raw File Change Prevents changes to a raw file The lock icon is yellow and the lock is closed Unlock Makes the pane editable The lock icon is green and the lock is open Copy to Clipboard Spectrum View Copies selected data to the Clipboard You can then paste the data from the Clipboard buffer into a document The Spectrum view displays a spectrum for a sample at the selected retention time Figure 103 To make changes to the spectrum display Right click the spectrum view and choose Display Options from the shortcut menu For more information see Chromatogram Display Options on page 154 When the view is locked lock icon is yellow or red you cannot select any menu item in the shortcut menu that changes the raw file with the yellow lock or the raw file and parameters with the red lock Menu items that would cause changes are grayed out For more information about locks see Toolbar for Selectable Views on page 150 170 LCquan User Guide Thermo Scientific Thermo Scientific Figure 103 Spectrum view Spectrum APN_O27 1 RT 0 01 NL 1 57E1 4 gt a
116. view the Run Sequence dialog box and the Status view Note The Acquisition window is not available in the LCquan for Web Access Figure 2 Acquisition window il Thermo LCquan 3 Drugs Workbook tut5 File Change Actions View Apps Options Help BAR s WL o 0000000 0 0 Instruments Acquisition Explore FileName aPN_001 Acquisition Sequence History SmapeNme Study Tutorial Study 2 Comment Client Client One it Laboratory SanJoseLab ot Company Name Themo Phone 408 555 5555 Prev Row Row f 1 Clear All Help Sample Type Fitettame Sample 1D Levet ISTO Corr Amt Dil Factor Vial Pos inj Vol Sample Sample Sample _Study_ _Ctient z 2 Standard APN_002 APN_002 Cal01 3 Standard APN_003 APN_003 Cal01 Standard APN_004 APN_004 Cal01 j5 Standard APN_005 APN_005 Cal01 je Standard APN_006 APN_006 Cal01 Standard APN_007 APN_OOF Cal02 ja Standard APN_008 APN_008 Cal02 ja Standard APN 009 APN_009 Cal02 10 Standard APN_010 APN_010 Cal02 11 Standard APN_011 APN_O11 Cal02 12 Standard APN_012 APN_012 Cal03 13 Standard APN_013 APN_013 Cal03 Standard APN_O14 APN_014 Cal03 45 Standard APN_015 APN_O15 Cal03 Standard APN_D16 APN_016 Cal03 Standard APN_017 APN_017 Cal04 18 Standard APN_018 APN_018 Cal04 19 Standard APN_019 APN_019 Cal04 ey For Help press F1 Powers Dana NOT SAVED 6 12 2008 12 30PM From AS 0 000 Tutorial Study 2 Client On
117. 029 RAW G9 APN_O30 RAW Gj APN_031 RAW i APN_032 RAW GQ APN_033 RAW Ga APN_034 RAW Gj APN_035 RAW Ga APN_036 RAW Ga APN_037 RAW WJ APN_038 RAW APN_039 RAW WJ APN_O40 RAW i APN_041 RAW i APN_042 RAW Standards acs Blanks Unknowns Selected Files APN_021 Add gt gt lt lt Remove Help F APN_022 APN_023 Cal_05 APN_023 APN_024 APN_025 APN_026 APN_027 Cal_06 APN_030 APN_031 APN_032 Cal_07 APN_033 APN_034 APN_035 APN_036 APN_037 Cal_08 APN_038 APN_039 APN_040 APN_0H APN_042 LCquan User Guide Result Ui K 315 5 Creating a Processing Sequence Editing a Processing Sequence 316 LCquan User Guide To build a new processing sequence In the upper Available Files pane of the Create Sequence create view search for and click the folder containing the raw files to be quantitated The LCquan application displays the names of the raw files in the lower Available Files pane a a Associate the data files with the Standard sample type calibration levels Click the Standards tab in the Selected Files pane and select the appropriate calibration level Figure 185 Figure 185 Date files associated with Standards sample type Standards OCs Blanks Unknowns Selected Files Cal_01 APN_026 Cal_02 APN_008 APN_009 APN_010 APN_O11 APN_012 Cal
118. 03 4 Standard APN_004 5 Standard APN_005 6 Standard APN_006 7 Standard APN_007 a Standard APN_008 9 Standard APN_009 Standard APN_010 Standard APN_011 Vial Pos ISTD Sample ID Level Corr C Xcalibur QuanRooti APNOO1 0 000 1 000 Tray01 0 30 00 C XcaliburvQuanRoot NA 0 000 1 000 TrayO1 0 30 00 C XcaliburiQuanRoot APNOO2 Cal_01 0 000 1 000 Tray01 2 30 00 C XcaliburiQuanRoot APNOOS Cal_01 0 000 1 000 TrayO1 2 30 00 C XcaliburiQuanRoct APNOO4 Cal_01 0 000 1 000 Tray01 2 30 00 C XcaliburiQuanRoot APNOOS Cal_01 0 000 1 000 Tray01 2 30 00 C XcaliburiQuanRoot APNOOG Cal_01 0 000 1 000 TrayO1 2 30 00 C XcaliburiQuanRoot APNOO7 Cal_02 0 000 1 000 TrayO1 3 30 00 C XcaliburiQuanRoot APNOOS Cal_02 0 000 1 000 TrayO1 3 30 00 C XcaliburiQuanRoot APNOOS Cal_02 0 000 1 000 TrayO1 3 30 00 C XcaliburiQuanRoot APNO10 Cal_02 0 000 1 000 Tray01 3 30 00 Thermo Scientific 5 Creating a Processing Sequence Editing a Processing Sequence Table 121 Processing sequence grid column parameters Sheet 1 of 3 Parameter Description Sample Type Displays the type of sample described by the sequence row The sample type defines how the LCquan application processes the sample data Each sample must be classified as one of the following LCquan sample types e Unknown e Blank e QC quality control e Standard FileName Displays the name of the raw file that contains the sample data The file name is a combination of the b
119. 193395 200109 When the view is locked lock icon is red you cannot select any menu item in the shortcut menu that changes the raw file or parameters Menu items that would cause changes are grayed out For more information about locks see Toolbar for Selectable Views on page 252 Table 112 lists the parameters for the Peak List view Table 112 Peak List view parameters Sheet 1 of 2 Parameter Arrows Description The left and right arrows move through the item list incrementing or decrementing by a single peak Column Headings Peak Name The name to apply to the peak column Apex RT The expected RT for the peak column Area The calculated peak area column of Largest Area The area of this peak relative to the area of the peak with the biggest area in the list column LCquan User Guide 281 4 Creating a Processing Method Data Views Sample Info View 282 LCquan User Guide Table 112 Peak List view parameters Sheet 2 of 2 Parameter Description of Total Area The area of this peak relative to the summed total area of all peaks in this list column Shortcut Menu Lock against All Prevents changes to a raw file or parameters The lock icon is red Change and the lock is closed Unlock Makes the pane editable The lock icon is green and the lock is open Copy to Clipboard Copies a chromatogram mass spectrum or calibration curve to the Clipboard You can then paste
120. 2 Identification Calibration m z327 1 50 70 hydrocortisone Component Chromatogram Definition Peak Identificat on Genesis Peak Integration deoxycorticosterone methyltestosterone Detector Smoothing Peak Detection Algorithm progesterone MS wl 7 Genesis a Advanced Highest Peak Min Peak Heigh S N Trace O Nearest AT 3 0 S N Threshold as Mass Hanga Z E lon ratio confirmlstion Mass m z C Valley Detection Enabled Enabled lor ratio using 267 2 309 2 Filter c Full ms2 363 30 150 00 375 00 v C Constrain Peak Width Retention Time Expected min 0 68 Window sec 30 0 A Window Else es AT Rot ee a 1 00 En Qualifier ion coelution Cl Adjust Using O Absolute 0 100 min 0 1 minutes 234 LCquan User Guide Thermo Scientific 4 Creating a Processing Method Specifying Method Calibration Specifying Method Calibration After importing the component names and calibration levels from the acquisition sequence into the processing method use features on the Calibration page to specify the identification settings for the internal standard and target compounds Figure 140 Figure 140 Quantitate Create Method Calibration window E Thermo LCquan 3 Drugs Workbook File Zoom View Apps Options Help PAR SLE PADCOSCOCOS K a0 Instruments m Target Compound Nefazodone
121. 3 Parameter Response Description Response settings Specifies the area or the height of the target compound peak to acquire the data used for the calibration response factor Area Specifies the area of the target compound peak to acquire the data used for the calibration Height Specifies the height of the target compound peak to acquire the Calibration Settings Isotope Thermo Scientific data used for the calibration Use the Isotope page to correct for an impurity in the internal standard compound that elutes at the same time as the target compound correct for an impurity in the target compound that elutes at the same time as the internal standard or correct for both Figure 194 Figure 194 Calibration Settings dialog box showing the Isotope page for Paroxetine Calibration Settings Paroxetine Type Curve Levels Isotope Contribution of ISTD to Target Compound 0 0 Contribution of Target Compound to ISTD o o Table 130 Calibration Settings Paroxetine dialog box Isotope parameters Parameter Contribution of ISTD to Target Compound Description Specify the amount as a percentage of the target compound that the internal standard contributes to the Target Compound s peak Contribution of Target Compound to ISTD Specify the amount as a percentage of the ISTD that the Target compound contributes to the ISTD s peak LCquan User Guide 3
122. 361542 0 046 25 000 13 846 44 62 Alprazolam 3 Standard Method Settings 14481 343213 0 042 25 000 12 225 51 10 Method Settings 18597 317654 0 059 25 000 19 532 21 87 5 Standard Method Settings 17723 299817 0 059 25 000 19 786 20 86 e Standard Method Settings 33231 316871 0 105 50 000 40 236 19 53 Standard Method Settings 35282 323125 0 109 50 000 42 165 15 67 a Standard Method Settings 34312 305110 0112 50 000 43 626 12 75 a Standard Method Settings 41308 258962 0 160 50 000 64 654 29 31 10 Standard Method Settings 34187 263133 0 130 50 000 51 430 2 86 Standard Method Settings 54574 293120 0 186 75 000 76 573 240 12 Standard Method Settings 51754 299387 0 173 75 000 70 621 5 84 13 Standard Method Settings 57117 296990 0 192 75 000 79 314 5 75 Method Settings 56677 326040 0174 75 000 71 055 5 26 15 Standard Method Settings 52306 298244 0175 75 000 71 744 4 34 16 Standard Method Settings 80377 304440 0 264 100 000 111 355 11 36 Standard Method Settings 67274 295463 0 228 100 000 95 121 4 88 18 Standard Method Settings 79642 284954 0 279 100 000 118 271 18 27 19 Standard Method Settings 72937 282186 0 258 100 000 108 877 8 88 20 Standard Method Settings 76823 294590 0 261 100 000 109 908 9 91 Method Settings 194643 320269 0 608 250 000 264 965 5 99 tires Q Q ATAR nena IEJ Ann oR RON EAR F O gt 4 lal Selectec Chromat 4 al isto Chromat 4 lal Calibration Curve
123. 39 6 Processing the raw Files and Reviewing the Analytical Results Reviewing the Calibration Curve and OCs Calibration Settings Levels Use the Levels page Figure 195 to view calibration levels and QC levels that you specified on the Calibration page in the Create Method view of the Quantitate window Figure 195 Calibration Settings dialog box showing the Levels page for Paroxetine Calibration Settings Paroxetine Type Curve Levels Isotope Table 131 Calibration Settings Paroxetine dialog box Levels page parameters Parameter Cal Level Description Displays the calibration levels for the selected component Amount Displays the amounts of the target compound used for each calibration level QC Level Displays the QC quality control levels for the selected component Amount Displays the amounts of the target compound used for each QC quality control level Test Displays a value for the acceptable difference as a percentage between the known amount and calculated measured amount of each QC level Note This is a read only table Compare the measured quantity with a previously defined expected quantity and percent test If the calibration or QC levels must be changed use the Calibration page in the Create Method view of the Quantitate window 340 LCquan User Guide Thermo Scientific 6 Processing the raw Files and Reviewing the Analytical Results Re
124. 4 a E v 2 E 2 w ec Relative Intensity 30 20 10 0 0 32 0 86 3 10 3 56 209 960 209 961 209 962 Time min miz Generating a Peak List Thermo Scientific The LCquan application can generate a peak list a table of peak properties such as retention times and peak areas of the major peaks in the chromatogram You can use the peak list to identify and name quan components and export the component names and properties to the Quantitate window Creating quan components is the first step in creating a processing method The components that the LCquan application creates out of the peak list are based on the Explore method define in the Create view of the Explore window In general you want to filter first to get one peak in the peak list generate its quan component and add quan components to the processing method one at a time LCquan User Guide 143 3 Exploring the Data Exploring the Results 144 LCquan User Guide To create a peak list 1 Select Peak List from the view selector in the right pane Figure 83 Figure 83 Peak List in the right pane Peak List of Total Area i gt 2 406 1 898 84685 31310 2 In the Limit Peaks area specify the peak list parameters a Enable Select Top Peaks Figure 84 Figure 84 Limit Peaks area Limit peaks m Select top peaks MV Enable Select by area C Select by height Num to select E b Select eithe
125. 5 Component Type Area 255 8 ac sectes Were nnsleng de sclarnoy hears essai led Ura Y 236 Tatget Compound Atea nay wate eared ote dour de E 237 Component List Panes serron catered Mee E whee alee 241 Calibration Shortcut Menu acts ct at ce6ieiein dcvere daa ateciaracaneye Alea canaries be 242 Correction for Isotope Contribution 0 cece eee eee 245 Specifying Calibration Options i 0ci e sea bee Se bee A 246 Specifying Internal Standards and Target Compounds 246 Reviewing the Integrated Peaks 0h 00 nen ooee wknd eae as eee eee 250 Saving the Processing Methods is de wane dey mate deen POR a ta 250 Importing a Processing Method ieeew dpeasnise aa brntuee aie bration dip arama 250 Exporting Components and Levels to an Acquisition Sequence 251 LCquan User Guide v Contents vi LCquan User Guide Chapter 5 Chapter 6 Data Views sng ccd sere pase a E A r E wl ded N R E a a ERS 251 Toolbar for Selectable Views onon unune 252 Calibration Curve View for Survey or Review All Results Only 255 Chromatogram View for Create Method Only 0 258 Chromatogram View for Survey or Review All Results Only 262 Spectrum View s n 6 isc a Share he tee Wh aa E NA ean ah alee 267 IRC Chramatecra View nie caus Vac Nate arth s cet Cais 269 Chromatogram List View au 24 pine ba ees Maus alae alkdes 273 Error Report View 6 eee ence eens 274 Filtet List Views sein
126. 6 LCquan User Guide Thermo Scientific 5 Creating a Processing Sequence Editing a Processing Sequence Table 119 Processing sequence grid shortcut menu commands Sheet 2 of 2 Command Description Import Processing From File Opens the Load Sequence dialog box where you can Sequence load a sequence sld ipp Iqn or csv Copy from Last Acquired Sequence Overwrites the processing sequence with the last acquisition sequence Copy from Acquisition Sequence Overwrites the processing sequence with the acquisition sequence in the workbook For information about updating the processing sequence with the most recent list of acquired samples see Processing Data While Continuing to Acquire Data on page 104 in Chapter 2 Preparing to Perform Quantitative Analysis Processing Sequence Fill Down Parameters From the processing sequence grid you can duplicate or sequence column values The mechanism is the same as the Fill Down for the acquisition sequence grid but different columns are affected Not all columns can be filled down Figure 179 shows the processing sequence Fill Down dialog box Table 120 lists the parameters for the processing sequence Fill Down dialog box Figure 179 Processing sequence Fill Down dialog box Fill Down Select Columns V Sample Type P Study P FileName P Client p Path I Laboratory Sample ID I Company Name Level I Phone TC ISTD Corr Amt Comment 7 Dil Factor Sampl
127. 8 208 Value 14 Tube Lens Offset 3 Mass 997 398 Value 24 Q0 Q00 RF Amplitude Mass 244 802 Value 21 Source CD 10 Q00 Offset 2 0 Lens 0 Offset 08 Q0 Offset 49 A et a 2 3 5o e C CE 20 22 23 be I When the view is locked lock icon is yellow you cannot select any menu item in the shortcut menu that changes the raw file Menu items that would cause changes are grayed out For more information about locks see Toolbar for Selectable Views on page 150 Table 67 lists the parameters for the Tune Method view Table 67 Tune Method view parameters Sheet 1 of 2 Parameter Description Arrows The arrows are not used in the Explore window Column Headings Parameter Displays a column of descriptive text for the item Value Displays a column of data for the given parameter Thermo Scientific 3 Exploring the Data Data Views Table 67 Tune Method view parameters Sheet 2 of 2 Parameter Description Shortcut Menu Open Raw File Opens the Select Raw File dialog box where you can select the raw file to load Lock Against Raw File Prevents changes to a raw file The lock icon is yellow and the Change lock is closed Unlock Makes the pane editable The lock icon is green and the lock is open Reset Scaling Resets scaling to the default pane Copy to Clipboard Copies selected data to the Clipboard You can then paste the data from the Clipboard buffer into a document
128. 90 o APN_036 A 20 015 020 APN_037 70 Time min APN_038 APN_039 APN_040 APN_041 APN_042 APN_043 20 APN_044 APN_045 APN_046 APN_047 APN_048 APN_049 APN_OSO Spectrum l Time min APN_OS1 APN_052 APN_002 1 RT 0 01 NL 3 65E1 APN 053 T SRM ms2 330 13 cid 20 00 F0 15 192 07 APN_054 100 25100 APN_OSS _ RT 0 54 PENIS 0 50 0 55 APN_057 Time min APN_OS8 APN_059 Acquisition Sequer v mem gt RT 0 87 RT 0 16 60 50 100 ea o 3 gt RT 0 33 30 0 30 0 35 Time min Relative Intensity 10 Time min Relative Intensity 8 8 8 o 0 9 S rime in For Help press F1 Powers Dana a 7 3 2008 511 PM 4 This section includes the following topics e Navigation Features e Opening a raw File and Displaying the Chromatogram e Generating a Peak List e Identifying Components Thermo Scientific LCquan User Guide 135 3 Exploring the Data Exploring the Results To open the Review view of the Explore window When the left navigation pane is displayed a Click Explore b Click the Review icon When the left navigation pane is hidden a Choose View gt Section Selection gt Explore Section b Choose View gt Step Selector gt Explore Review Navigation Features You can use the following features of the Review view of the Explore window to navigate your data e Sample Selector e Locks e Review Displays e Current Review Display list para
129. AI 9 at got 08 a 46 get 338 4 904 4 294 9 Ahh Peak 276 LCquan User Guide Thermo Scientific 4 Creating a Processing Method Data Views When the view is locked lock icon is red you cannot select any menu item in the shortcut menu that changes the raw file or parameters Menu items that would cause changes are grayed out For more information about locks see Toolbar for Selectable Views on page 252 Table 109 lists the parameters for the General Parameters Plot view Table 109 General Parameters Plot view parameters Parameter Arrows Description The left and right arrows move through the item list incrementing or decrementing by a single item Item Selector List Options RT Displays retention times for the current component Area Displays areas for the component peaks Units count seconds Height Displays heights of the current component peak apexes Units counts Response Displays the ratios of sample peak areas or heights to ISTD areas or heights Peak Width Displays peak widths for the current component Calculated Amount Displays the sample amounts as calculated with the response ratio and calibration curve Difference Displays the percent difference as defined by 100 x calculated amount specified amount specified amount RSD Displays the percent relative standard deviation Shortcut Menu Lock against All Prevents changes to a raw
130. Acquisition window reserves a display area for each trace you chose to display Table 28 Realtime Display Settings dialog box parameters Sheet 1 of 3 Parameter Description Options Show Traces Defined Displays real time chromatograms corresponding to the by Components components you defined in the processing method Show User Defined Displays real time chromatograms defined when using the settings Traces in the Trace Properties box in the lower portion of this LCquan view Max Number of The number of chromatograms to display Visible Range 1 to 5 Chromatograms Show Spectrum Trace Displays the mass spectrum in the spectrum pane The LCquan application filters the spectrum in the same manner as the chromatogram Auto Update Spectrum to Last Scan Displays the mass spectrum of the latest scan and the current spectrum updates during each refresh to show the spectrum from the last scan in the selected chromatogram Note Auto Update Spectrum to Last Scan becomes active only when you select Show Spectrum Trace Link Chromatogram Time Ranges Displays the same time range for all chromatograms Any change in the time range displayed on any chromatogram is reflected in all chromatograms When not selected the time ranges for each chromatogram are independent Thermo Scientific 2 Preparing to Perform Quantitative Analysis Using the Status View Table 28 Realtime Display Settings dialog box parameters S
131. Allow Excel Rounding vj Allow Watson File Interface Excel Formal 2i gt Automatically Lock Workbook after crealos Permission Level Disallowed O Allowed Disallowed State Secure Template Folder Saving the Workbook and Exiting the LCquan Application You can save the workbook at any step in the LCquan procedure With this feature you can exit the LCquan application and reenter at the same place at a later time When you reopen a workbook the LCquan application displays the page and view that were open when you exited the workbook To save the workbook and exit the L quan application 1 Choose File gt Save to save the current workbook When you want to save the workbook with a different study or workbook name choose File gt Save As The Save As Wizard guides you through the procedure 2 Choose File gt Exit to exit the LCquan application Note When you acquire data you cannot exit the program unless you save the workbook 400 LCquan User Guide Thermo Scientific ee B Quantitative Analysis Overview This appendix describes some of the basic principles and terminology of quantitative analysis and contains the following topics Contents e About Quantitative Analysis e Considering the Variables of Quantitative Analysis by LC MS MS e Quantitative Analysis Techniques e Sample Types About Quantitative Analysis In some applications such as a clinical trial you might
132. Calculate Determines initial values for only Start Threshold End Threshold Area Threshold P P Resolution Threshold Bunch Factor Negative Peaks and Tension Note Valid only if a raw file is open 360 LCquan User Guide Thermo Scientific Thermo Scientific 6 Processing the raw Files and Reviewing the Analytical Results Reviewing the Calibration Curve and OCs Table 140 User Identification Settings dialog box Avalon Integration page parameters Parameter Description Time Min Displays the initial time value in minutes Event Displays descriptions of detection parameters for initial events and timed events For details see Table 141 Note Start Threshold End Threshold Area Threshold P P Resolution Threshold Bunch Factor Negative Peaks and Tension are defined with initial values Value Displays the values associated with initial events or timed events Range Factors are specific to each event Add Adds the Time Event and Value values to the Avalon Event List Delete Deletes the selected row Change Using the Time Event and Value values updates the event list and the chromatogram display Note You can test the results of the new criteria by clicking Apply or OK You can apply the new criteria to all files in the Result list by clicking Apply to All Table 141 Initial and Timed Events Sheet 1 of 2 Event Start End Threshold Description Half of the result is a good estimate for the St
133. Calibration Curve and OCs Table 135 User Identification Settings dialog box Detection page parameters Sheet 2 of 2 Parameter Mass Target Ratio Description Specifies an ion mass value for the confirmation process Note You can enter up to five qualifier ions New lines appear for each new mass entry Displays the ratio of the Qualifier Ions response to the Quan Ions response Range 0 to 200 Window Specifies the window error value Window The acceptable range from the target window value that is still considered a confirmed ion Relative Absolute Select the Relative option to use the target ratio tolerances in the Window column as relative percentages of the target ratio Select the Absolute option to use the target ratio tolerances in the Window column as absolute percentages of the target ratio Qualifier lon Coelution Min Specifies an acceptable drift from the expected retention time to still be considered a confirmed ion Note You can test the results of the new criteria by clicking Apply or OK You can apply the new criteria to all files in the Result list by clicking Apply to All LCquan User Guide 351 6 Processing the raw Files and Reviewing the Analytical Results Reviewing the Calibration Curve and OCs User Identification Settings Genesis Integration Use this page Figure 204 to set up advanced chromatographic parameters able 136
134. Choose User Labels and Values to open the Change Sequence User Labels dialog box opens 2 Type your label name changes 3 Select variable values or type static values 4 Click OK All rows in the sequence reflect the changes For details about the parameters see Parameters in the Change Sequence User Labels dialog box on page 34 For an example of how the user labels and values are displayed in the sequence grid see User Defined Columns Values Examples on page 50 Standard and OC Levels Use the Standard and QC Levels command to change or import calibration and QC levels see Table 24 on page 63 To change or import calibration and QC levels 1 Choose Standard and QC Levels The Acquisition Levels dialog box opens Figure 43 on page 63 Use this dialog box to modify calibration levels and QC levels specified for the Standard and QC samples in the acquisition sequence You can also import component names and calibration levels from a processing method into the acquisition sequence step 7 2 Edit the component list e To add a component enter the name into the empty row at the end of the component list e To delete a component click the row number and press DELETE e To rename a component select the component name and enter a new name 3 Edit the calibration levels list for the selected component e To add a calibration level enter the name into the empty row at the end of the Cal Level list e
135. Comma Separation m __ tee _ Table 153 Results Export dialog box parameters Sheet 1 of 2 Parameter Description File Selection FileName Specifies a name for the text file of the exported data you want to save When you use a name of an existing file a warning message prompts you to provide another name The file extension csv is automatically appended to the file name Limit 50 characters Invalid characters lt gt One File for All Specifies that data for all components is to be saved in a single file Components One File for Each Specifies that the data for each component is to be saved in a Component separate file The files are named with the component name appended to the end of the file name you provided For example if two components are named C1 and C2 and you provided the file name Export the created file names became Export_Cl csv and Export_C2 csv 396 LCquan User Guide Thermo Scientific 6 Processing the raw Files and Reviewing the Analytical Results Locking the Workbook Table 153 Results Export dialog box parameters Sheet 2 of 2 Parameter Description Format Options Include Item Header Specifies that column headers be exported as a line of data that immediately precedes the results in the exported data file Tab Separation Specifies that individual values in the file are to be separated by tabs Comma Separation Specifies that in the file individual values a
136. Cquan application displays a marker on the chromatogram to show which scan was selected To display the same time range for all chromatograms select the Link Chromatogram Time Ranges check box To display a specific time range for all chromatograms a Select the Use User Defined Time Range check box The Link Chromatogram Time Ranges option is automatically selected b To specify the beginning and ending retention times enter values for Min RT and Max RT To specify and display properties of user defined chromatogram traces To add a chromatogram trace click the box next to a blank line You can enter up to 10 chromatograms In the Trace Properties area define the trace a Type a title for the chromatogram trace b Select mass range chromatograms or total ion current TIC chromatograms as the Trace type c Specify the scan filter to be applied to the acquired data LCquan User Guide 85 2 Preparing to Perform Quantitative Analysis Using the Status View 86 LCquan User Guide d Specify the mass or mass range of a mass range chromatogram e Type the number of smoothing points that the smoothing algorithm applies to the data 3 To select traces for display select the check box before the trace entries 4 Use the Up and Down buttons to change the position of a selected trace 5 When you have finished your display settings click OK The chromatogram pane in the Status view of the
137. Data Data Views 156 LCquan User Guide Table 55 Chromatogram Display Options dialog box parameters Sheet 2 of 2 Parameter Description Signal to Noise Displays the peak signal to noise S N value above the chromatogram peak Note When RMS S N is selected in the Peak Detection Settings options set lt RMS gt appears after the S N value Decimals Sets the number of decimal places for the peak apex retention time label Area Displays the peak area Height Displays the peak height Label Styles Stylize the labeling displayed with the data Offset Moves the label a defined distance from the data Rotated Rotates the labels above the peaks to 90 degrees Boxed Displays boxes around the labels above the peaks Size Defines the distance the label is offset from the data Label Threshold Sets a threshold so that peaks above this level can show labels Plotting Select one style to display your data Point to Point Select a graphic style to display the active chromatogram or spectrum using point to point peak profile Stick Select a graphic style to display the active chromatogram using vertical lines On Off Labeling Option When No Peak Found Show Apex Time Selects to always display maxima labels Labels Axis Offset Select one or both to offset the displayed plot from the x axis y axis or both X Select to have the x axis offset move the y axis slightly abov
138. Editor Import Export Cancel Help 2 To create a new peak a Type a new name on the last line of the grid You can use alphanumeric entries b Specify values for the Apex RT and the Delta RT There is never any range checking on the Apex RT or the Delta RT 3 To import a peak list click Import and browse for your list 146 LCquan User Guide Thermo Scientific 3 Exploring the Data Exploring the Results 4 To export the peak list click Export and enter a name for the file in the Peak Name List File dialog box Figure 89 Figure 89 Peak Name List File dialog box Peak Name List File Export File Name Please enter the name you wish to use for the Exported file No path will be accepted the file will be created in the Export folder of the Workbook and the file extension will be automatically added Name of Exported File MyExportedPeakList Full Name of Exported File C calibur QuanRoot E xample Study 3 Drugs Workbook E xports MyE xportedPeakList csv Cancel Note You can load the Peak Name List from any available location A copy of the file is created in the Import folder and hss tracks entries created for it Table 52 Peak Name List Editor dialog box parameters Parameter Description Peak Name Displays a column of label names for the explore peak Apex RT Displays a column of the expected retention times for the peak Delta RT Displays a column of the allowable ranges from the Apex
139. Explore Methods view of the Explore window opens Figure 3 Explore window 6 Thermo LCquan 3 Drugs Workbook File Zoom View Apps Options Help BAA R CCOSCOSO S 9 0 Instruments Acquisiti Lenton Quantitate Chromatogram Definition PICIS Peak Integration Limit peaks li Acquisition may Histi 4 AcqSequence h Smoothing Peak Detection Algorithm j Select top peaks APN 001 ICIS X Advanced Enable APN_002 Select by area C Select by height Baseline Window jo Area Noise Factor E Peak Noise Factor fi 0 Num to select fa I Constrain Peak Width m Rel peak height threshold Peak Ht 50 I Enable here ne Tailing Factor fi ii Z of highest peak 100 1 8 3 0 Spectrum APN_027 TIC RT 0 01 4 00 NL 1 04E5 APN_027 1 RT 0 01 NL 1 57E1 T SRM ms2 330 1 3 cid 20 00 70 15 192 07 RT 2 44 100 100 24 80 80 70 60 60 50 40 40 Relative Intensity Relative Intensity 30 3 14 3 46 3 84 2 0 25 3 0 3 5 40 120 140 Time min miz For Help press F1 Powers Dana NOT SAVED 6 23 2008 3 47PM 7 4 LCquan User Guide Thermo Scientific 1 Introduction Quantitative Analysis with LCquan Quantitate Window In the Quantitate window the LCquan application steps you through a procedure for batch processing raw files during quantitative analysis Figure 4 Because the LCquan window incorporates an integrated calibration curve pe
140. For detailed descriptions of all these views and the Toolbar for Selectable Views see Data Views on page 150 Opening a raw File and Displaying the Chromatogram 142 Open a raw file that is representative of your data set to determine suitable peak detection and integration parameters To open a raw file Do one of the following in the Explore Review view e Click the Browser button and select a raw file e Choose File gt Open Raw File e Click a sample in the Sample Selector For more information see Sample Selector on page 136 The LCquan application imports the raw data for the raw file sample and displays the raw chromatogram on the Explore Create view To see the mass spectrum 1 In the Current Review Display Layouts select the type of layout to display For more information see Current Review Display list parameters on page 140 In the Chromatogram pane peaks that correspond to the components are displayed with their retention times Figure 82 LCquan User Guide Thermo Scientific 3 Exploring the Data Exploring the Results 2 Click a peak in the chromatogram The Spectrum view displays the corresponding spectrum Figure 82 Chromatogram pane 4 a Chromatogram 4 gt a Spectrum APN_014 TIC RT 0 01 4 00 NL 8 74E4 APN_014 477 RT 2 64 NL 4 50E4 T SRM ms2 314 10 cid 40 00 209 96 209 96 400 RT 2 67 400 209 96 90 80 70 60 50 40 ia
141. Genesis or ICIS only Specifies a value for the minimum S N ratio This criterion specifies that only peaks that meet or exceed this minimum S N are displayed Range all peaks 1 0 to 999 0 LCquan User Guide 193 4 Creating a Processing Method Specifying Method Identification lon Ratio Confirmation Area Use the Ion Ratio Confirmation area to confirm the presence of an analyte by comparing the response of the quantitate ion or ions with the responses of one to five qualifier ions Figure 119 Table 75 lists the parameters for the Ion Ratio Confirmation area Figure 119 lon Ratio Confirmation area lon ratio confirmation MV Enabled 151 01 Window Relative Absolute 25 00 lon ratio using Area Target Ratio Window Qualifier ion coelution 0 100 min Table 75 lon Ratio Confirmation area parameters Sheet 1 of 2 Parameter Enabled Description Places the IRC table in a usable state so that confirmation processing can take place To open the IRC Detection Method dialog box select a row not a cell and right click on the grid For a detailed description of the IRC detection method parameters see IRC Detection Method on page 217 Note When this check box is cleared the data fields are grayed out Mass Specifies the mass of the ion to be used in the confirmation process Type a new mass value into an empty row to generate a new table row Targ
142. LT F gt I gt F Sequence gt From File acquisition sequence Import Acquisition Makes the acquisition sequence identical to the processing ALT F gt I gt P Sequence gt sequence Copy from Processing Sequence Export Acquisition Exports the current acquisition sequence to a sequence file ALT F gt E Sequence Print Sequence Info Opens a Print dialog box that you use to print a copy of the current ALT F gt P sequence in a spreadsheet format Commands Specific to Explore Window Table 160 Commands specific to the Explore window Command Description Keyboard shortcut Import Peak Name List Opens a file selection dialog box where you can import the naming ALT F gt I list to a text file txt or csv A copy of the file is created in the Import folder and both an event record and a file tracking record are created After the file is accepted any peak list or chromatogram is relabeled as required Export Peak Name List Opens a file selection dialog box where you can import the naming ALT F gt E list to a text file txt or csv A copy of the file is created in the Import folder and both an event record and a file tracking record are created After the file is accepted any peak list or chromatogram is relabeled as required Commands Specific to Both Explore and Quantitate Windows Table 161 Commands specific to both the Explore and Quantitative windows Command Description Keyboard shortcut toolbar icon Open Raw Fi
143. Layout Multi Peak View Opens on the left side of the page with the default Chromatogram and Spectrum Opens on the right side of the page with the default Multi Peak Plot Comparison View Displays three stacked panes Chromatogram Spectrum and Peak List as the defaults Peak List View Displays two tall panes side by side Chromatogram and Peak List as the defaults Large Chro View Displays a large single pane Chromatogram as the default Sample Info View Displays four panes one pane in the top row Chromatogram as default and three small side by side panes in the bottom row Sample Info Instrument Method List and Status Plot as the defaults Note You can change the contents of the panes but not their sizes shapes or positions To change the name of the layout 1 Select the name in the Review Display list 2 Type the new name 140 LCquan User Guide Thermo Scientific 3 Exploring the Data Exploring the Results To importa layout 1 Choose Files gt Layout Configuration gt Import Layout Configuration to display the LCquan Config File dialog box 2 Browse to the LCquan configuration file and click Open The LCquan application places the configuration file in the Imports folder To save a layout 1 Choose Files gt Layout Configuration gt Export Layout Configuration to open the LCquan Config File dialog box Figure 81 Figure 81 LCquan Config File dialog box LCquan Config File Expor
144. M 7 2 Inspect the processing sequence and verify that it contains the correct sample data files raw and that each raw file is properly associated with a blank unknown calibration level or QC level 3 Ifyou find an error in the processing sequence correct the errors a Click Create to return to the Create Sequence create view b Click Remove and Add to reassociate the raw files c Click Edit to return to the Create Sequence edit view 302 LCquan User Guide Thermo Scientific Thermo Scientific 5 Creating a Processing Sequence Editing a Processing Sequence Figure 174 Sample raw files and their calibration levels 2 Standard APN_004 Rawfile APNOOS Cal_0 0 000 1 000 TrayO1 2 30 3 ac APN_051 Rawfile Q C_Hi 0 000 1 000 TrayO1 1 30 APN_052 Rawrile C_Hi 0 000 1 000 TrayO1 1 30 APN_001 Rawfile APNOO1 NA 0 000 1 000 TrayO1 0 30 Figure 174 shows an example When you are satisfied that the processing sequence is correct you are ready to process the raw files For more information about processing the raw files see Chapter 6 Processing the raw Files and Reviewing the Analytical Results To save the current sequence in the Exports folder of the workbook choose File gt Export gt Processing Sequence The LCquan application saves the file with the sld file extension To remove a raw file from the processing sequence 1 To select the row in the sequence that you want to delete click the r
145. Menu and Toolbar Reference Zoom Menu Zoom Menu 430 LCquan User Guide Table 173 Zoom menu commands Use this menu to adjust the visual display of your view see able 173 You can increase the size of the view and reset the display to the original state Command Description Keyboard Shortcut Zoom In Y Zooms in on y axis to display more detail ALT Z gt Z and less range Zoom Out Y Zooms out on y axis to display less detail but ALT Z gt O more range Normalize Zooms in on y azis to largest peak height ALT Z gt N Zoom In X Zooms in on x axis to display more detail ALT Z gt X and less range Zoom Out X Zooms out on x axis to display less detail but ALT Z gt m more range Display All Sets x axis to display entire displayable range ALT Z gt D Reset Scaling Resets both x and y axes to display maximum ALT Z gt R amount of data Thermo Scientific C LCquan Menu and Toolbar Reference Icon Bars Icon Bars Use the left navigation panes to navigate between LCquan windows Figure 233 Icon bars Instruments icon bar Acquisition icon bar Explore icon bar Quantitate icon bar Quantitate Quantitate Thermo Scientific LCquan User Guide 431 C LCquan Menu and Toolbar Reference Toolbar Toolbar Use the toolbar buttons to quickly select their corresponding commands see Table 174 To display the LCquan toolbar Choose View gt Toolbar The available toolbar buttons vary depending
146. New Study Wizard gives you the option of importing instrument methods acquisition sequences and processing parameters to initialize the new workbook You can import all this data from an existing workbook or you can import a processing method from an individual legacy file pmd or from an LCquan file lqn e New Method Wizard in the Quantitate window When you use the New Method Wizard in the Quantitate window you have the option of importing a previously saved processing method You can import a processing method from an individual legacy file pmd or from an LCquan file lqn 250 LCquan User Guide Thermo Scientific 4 Creating a Processing Method Exporting Components and Levels to an Acquisition Sequence e Processing Method command from within the Quantitate window From the Create Method view in the Quantitate window you can choose File gt Import gt Processing Method to display the Import Quantitation Method dialog box Use this dialog box to import a processing method from an individual legacy file pmd or from an LCquan file lqn Exporting Components and Levels to an Acquisition Sequence Data Views Thermo Scientific Just as you can import the component names and calibration levels from an acquisition sequence into a processing method you can also do the reverse that is export component names and calibration levels from a processing method into an acquisition sequence To export component names and calibr
147. Peak Integration Limit peaks Acquisition a Hist 4 AcqSequence 1 Detector Smoothing Peak Detection Algorithm p Select top peaks APN_OO1 MS l ICIS X Advanced M Enable APN_002 APN_003 Trace Select by area APN_004 Baseline Window fao APN_OOS Select by height APN _006 Area Noise Factor g APN_OO z s Num to select fa i Oh Peak Noise Factor fi 0 APN_009 b APN_010 T Constrain Peak Width Rel peak height threshold APN_O11 eak Ht 15 0 I Enable APN_O12 eae APN_013 Range min A APN_O14 ailing Facto 0 of highest peal fico TEJO Tailing Factor 17 0 Z of highest peak APN_O1S APN 016 Spectrum APN_027 TIC RT 0 01 4 00 NL 1 04E5 APN_O27 1 RT 0 01 NL 1 57E1 T 0 SRM ms2 330 13 cid 20 00 70 15 192 07 RT 2 44 J p 100 4100 90 80 co 70 60 D 50 i eS o Relative Intensity Relative Intensity 305 3 14 3 46 3 84 2 0 25 3 0 3 5 40 Time min For Help press F1 Powers Dana NOT SAVED 6 23 2008 3 47PM 7 The Create view of the Explore window contains the Chromatogram Definition Peak Integration and Limit Peaks areas that you use to develop an explore method This section includes the following topics e Defining Chromatographic Parameters e Defining Peak Integration Parameters 114 LCquan User Guide Thermo Scientific 3 Exploring the Data Creating Explor
148. Sequence View Sample Sequence Name Specify the base file name and starting sequence number that are used to generate names for the raw files during a sequence run The derived names are a concatenation of a base name and a three digit incremental number for each sample For example if the Base file name is APN_ and 1 is the starting number the LCquan application creates the new sequence with the first file name APN_001 R To specify the sample sequence names 1 Type the Base file name Figure 32 2 Type a starting suffix number Figure 32 Sample sequence name page Base file name APN Starting number f 3 Click Next Specify Sample Information The LCquan application generates sample IDs that uniquely identify the unknown samples in the sequence The derived names are a concatenation of a sample ID and an incremental number for each sample Table 15 lists the parameters for the sample information page 1M To generate sample IDs for the sequence 1 Type the number of unknown samples to include in the new sequence Figure 33 2 Type the number of replicate samples to include in the new sequence 3 Type an alphanumeric prefix to the sample ID for each sample in the new sequence Figure 33 Sample information page Number of Samples E Injections per Sample 1 Base Sample ID APN_ Thermo Scientific LCquan User Guide m 2 Preparing to Perform Quantitative Analysis Using the Setup Sequence View Table 15
149. Specifies the first row to receive new data To Row Specifies the last row to receive new data Using Row Specifies which row the initial data comes from Create Samples in Specifies that a new sample be created in an empty row during a Selected Empty Rows fill down that includes the row in its fill down range When not During Fill Down selected specifies that the empty row is ignored during a fill down that includes the row in its fill down range Buttons Selects all the check boxes in the Select Columns area Clears all the check boxes in the Select Columns area ear All Sort Use the Sort command to reorder the rows in the sequence see Table 22 To reorder a sequence 1 Choose Sort to open the Sort Sequence dialog box Figure 41 Figure 41 Sort Sequence dialog box Sort Sequence Sort Information First Order Fie Name V Sort in descending order Second Order Level Name I Sort in descending order Third Order Sample Type V Sort in descending order lt none gt File Name Level Name Sample ID Sample Type Cancel 2 Select the columns on which to sort and reorder the sequence 3 Select to sort in ascending 1 to n or descending n to 1 order The default is ascending Note To prevent any sorting from occurring select lt none gt in all three sort order fields LCquan User Guide 57 2 Preparing to Perform Quantitative Analysis Using the Setup Sequ
150. To delete a calibration level click the row number and press DELETE Thermo Scientific LCquan User Guide 61 2 Preparing to Perform Quantitative Analysis Using the Setup Sequence View To rename a calibration level select the level name and enter a new name Note When the Cal Level list is empty and the sequence contains some samples with Standard or QC sample type the Sample Type Assignment dialog box opens when you click OK in this dialog box You can change the sample type of these samples to Unknown in the processing sequence or discard the samples that have these levels in the processing sequence e To enter a calibration amount enter the appropriate level in the Amount box 4 Edit the QC levels for the selected component e To adda QC level enter the name into the empty row at the end of the QC Level list e To delete a QC level click the row number and press DELETE e To rename a QC level select the level name and enter a new name e To edit a QC amount enter the appropriate level in the Amount box 5 To display the shortcut menu right click the Acquisition Levels dialog box From the shortcut menu you can e Copy current component levels to all target components e Load default levels for current component e Import processing components 6 To replace the current levels with the defaults stored in the registry click Load Default Levels 7 To import component names and calibration levels from the process
151. User Value 3 fo User Value 4 User Value 5 Thermo Scientific 2 Preparing to Perform Quantitative Analysis Using the Setup Sequence View Table 20 User labels and values parameters Parameter Description User Label 1 to 5 Displays information pertinent to the active sample row in the sequence Use these boxes to convey information about this sample to others or as a reminder to yourself e The User Label 1 default name is Study e The User Label 2 default name is Client e The User Label 3 default name is Laboratory e The User Label 4 default name is Company Name e The User Label 5 default name is Phone User Value 1 to 5 Displays the value in the header of the acquisition sequence Use this user defined box to convey information about a sample to others or as a reminder to yourself The LCquan application automatically substitutes certain selected values in the box e Operator substitutes the name of the operator e Study substitutes the name of the Study e Workbook substitutes the name of the Workbook e Workstation substitutes the name of the Workstation e Do Not Change prevents the LCquan application from substituting anything in the box Complete the Wizard To complete the New Acquisition Sequence Wizard click Finish The LCquan application creates a new acquisition sequence and displays it in the Acquisition Setup Sequence view See Using the Setup Sequence View on page 31 Thermo S
152. V Set the processing sequence path after importing files New processing sequence path C Xcalibur examples data Browse C calibur examples data 2 Click Next Complete the Wizard To complete the New Study or Workbook Wizard click Finish The LCquan application creates the new study and workbook folders and imports the raw files into the raw files folder Saving a Workbook After you create a new workbook or study save the workbook to the current study folder or to a new study or workbook name To save the new workbook to your current location choose File gt Save from the main menu You can also use this wizard to save the new workbook with a new workbook name Start the Wizard Use the Save As Wizard to save the workbook to a new location To save the workbook to a new location 1 Verify that you have read and write privileges for the location where you intend to save the workbook 2 From the main menu choose File gt Save As The Save As Wizard opens Figure 17 22 LCquan User Guide Thermo Scientific 2 Preparing to Perform Quantitative Analysis Creating a New Study and Workbook Figure 17 Create new study and workbook page Study Name Tutorial Study 2 Browse Workbook Name 3 Drugs Workbook tut5 Browse C calibur QuanF oot T utorial Study 243 Drugs Workbook tut5s l Change Study Root Folder Open in New Window Table 7 New study or workbook page parameters Paramete
153. _03 APN_013 APN_O14 To associate a data file with selected calibration level click the raw file in the lower Available Files pane and click Add The LCquan application adds the selected raw file to the selected calibration level To display the amount of standard sample associated with a calibration level right click the calibration level and choose Show Level Amounts from the shortcut menu Associate the data files with the QC sample type levels Click the QCs tab in the Selected Files pane and select the appropriate quality control level To associate a data file with selected QC level click the raw file in the lower Available Files pane and click Add The LCquan application adds the selected raw file to the selected quality control level in the Selected Files pane To display the amount of quality control sample associated with a quality control level right click the quality control level and choose Show Level Amounts from the shortcut menu Thermo Scientific 5 Creating a Processing Sequence Editing a Processing Sequence 4 Associate the data files with the Blanks sample type a Click the Blanks tab in the Selected Files pane b To associate a data file with the Blanks sample type click the raw file in the lower Available Files pane and click Add The LCquan application adds the selected raw file to the Blanks sample type in the Selected Files pane 5 Associate the data files with the Unknowns sampl
154. a raw file or parameters The lock icon is red Change and the lock is closed Unlock Makes the pane editable The lock icon is green and the lock is open Copy to Clipboard Copies selected data to the Clipboard You can then paste the data from the Clipboard buffer into a document Error Report View 274 LCquan User Guide This view displays the error log information stored in the current raw file Figure 157 Figure 157 Error Report view 4 gt a MS Quantum F Error Report APN_OOS detector MS Quantum Parameter vae O The contents of the grid are read only When the view is locked lock icon is red you cannot select any menu item in the shortcut menu that changes the raw file or parameters Menu items that would cause changes are grayed out For more information about locks see Toolbar for Selectable Views on page 252 Table 107 lists the parameters for the Error Report view Table 107 Error Report view parameters Sheet 1 of 2 Parameter Description Arrows The left and right arrows move through the list of detectors incrementing or decrementing by a single detector Detector Selector List Options MS Displays the error log of a mass spectrometer Status Displays the error log of a device that does not log data Thermo Scientific Filter List View Thermo Scientific 4 Creating a Processing Method Data Views Table 107 Error Report view parameters Sheet 2 of 2
155. a combination of the base file name prefix assigned to the sequence and a sequential sequence number When the default sequence starting number 1 was not changed the suffix number is the same as the row number of the sequence 001 002 and so on When the default sequence starting number was changed to another number the first sample has the starting number and subsequent rows in the sequence are incremented by 001 For example if the starting number was 100 the file name for the first sample would have a suffix of 100 the second sample would have a suffix of 101 and so on Sample ID Displays the sample ID for each data file The sample ID is an alphanumeric string of characters that identifies a sample Level Indicates the level defined for a calibration sample or quality control sample Thermo Scientific LCquan User Guide 67 2 Preparing to Perform Quantitative Analysis Using the Setup Sequence View 68 LCquan User Guide Table 25 Acquisition sequence grid column parameters Sheet 2 of 3 Parameter ISTD Corr Amt Description Specifies an updated amount of internal standard for the corresponding sample The default value is 0 0 For each component defined as an internal standard a bulk adjustment factor can be applied to the base response of each internal standard defined in the processing method When no adjustment is required verify that a value of 0 000 is entered in the ISTD Corr Amt box When an adj
156. able 143 lists the parameters for the Info page of the Peak Info dialog box Figure 213 Peak Info dialog box showing the Info page Peak Info Paroxetine Info Flags Spectrum Left min 1 87 Left height 30 81 Apex min fi 93 Peak Height fi 6868 05 Right min 2 04 Right height 235 1 3 Area cts sec 3289 84 ISTD Response 373890 85 Baseline fi 40 16 Response Ratio o1 7 Expected AT min 1 92 Calculated Amount 63 02 Base Peak m z fi 92 07 Signal to Noise fi 184 32 Peak Type Method Close Help Table 143 Peak Info dialog box Info page parameters Sheet 1 of 2 Parameter Description Peak Properties read only Left min Displays the left extreme of the integration baseline for the current component Units minutes Apex min Displays the apex point in minutes for the current component Right min Displays the right extreme of the integration baseline for the current component Units minutes Area cts sec Displays the area of the current component peak Units count seconds 368 LCquan User Guide Thermo Scientific 6 Processing the raw Files and Reviewing the Analytical Results Reviewing All Results Table 143 Peak Info dialog box Info page parameters Sheet 2 of 2 Parameter Baseline Description Displays the baseline height directly below the apex of the current component peak Units counts Expected RT min Displays the expected retention t
157. able 76 Summing lons dialog box parameters Parameter Description Mass m z Specifies one or more mass to charge ratios or ranges of mass to charge ratios of an ion whose chromatogram the LCquan application uses for ion summing Filter Specifies the scan filter of an ion whose chromatogram the LCquan application uses for ion summing add ions Adds an ion for ion summing To add an ion select a scan filter in the Filter box and enter an ion mass to charge ratio in the Mass box You can add the chromatograms of up to five ions subtract ions Removes an ion from the ion summing list When you subtract an ion the LCquan application grays out the entry but does not delete it Apply Populates the Summed Ions table on the Identification page of the Create Method view with the scan filters and mass to charge ratios of the ions you specified in the Summing Ions dialog box The Apply button does not close the dialog box OK Populates the Summed Ions table on the Identification page of the Create Method view with the scan filters and mass to charge ratios of the ions you specified in the Summing Ions dialog box The OK button closes the dialog box To open the Summing lons dialog box In the Chromatogram Definition Area of the Create Method Identification page select Trace Summed and click Edit Summed LCquan User Guide 197 4 Creating a Processing Method Specifying Method Identification Chromatog
158. able result of optical detectors that you can use to identify an analyte is the retention time at a specified wavelength To quantitate compounds accurately the compound peaks must be distinctly separated from each other in time By carefully optimizing the LC variables listed above you can successfully separate and detect compounds with an optical detector such as an ultraviolet UV or photodiode array PDA detector In contrast to optical detectors mass spectrometers can often successfully detect individual analytes even when two or more compounds coelute as a single chromatographic peak Coeluting compounds are ionized in the mass spectrometer and the parent or fragment ions specific to the analyte or analytes are detected and measured for quantitative analysis Because the mass spectrometer detection is so specific you can reduce the chromatographic resolution and shorten the run times Using MS MS for Analyte Detection To optimize detection of ions by MS MS consider the following variables when working with a mass spectrometer interfaced to an LC e Solution chemistry and polarity of the analyte of interest e Probe selection H ESI ESI APPI or APCI e Collision energy to fragment parent ions inside the mass spectrometer optimized by experiment The LC provides a stream of analyte solution eluate into the mass spectrometer where the analyte is detected You must consider the content of modifiers salts and contaminants in the
159. abled lon ratio using Area ic 1 x Delete selected rows Edit selected rows processing info Window Target Ratio The IRC Detection Method dialog box includes these tabs e IRC Detection Method Identification e IRC Detection Method Genesis Integration e IRC Detection Method Genesis Advanced Thermo Scientific LCquan User Guide 217 4 Creating a Processing Method Specifying Method Identification e IRC Detection Method ICIS Integration e IRC Detection Method ICIS Advanced e IRC Detection Method Avalon Integration The tabs are dynamic depending on the selected peak detection algorithm ICIS Genesis or Avalon IRC Detection Method Identification Use this page Figure 132 to set up advanced chromatographic parameters and select the peak detection algorithm Genesis ICIS or Avalon Table 83 lists the parameters for the Identification page of the IRC Detection Method dialog box Figure 132 IRC Detection Method dialog box showing the Identification page IRC Detection Method Identification ICIS Integration ICIS Advanced Smoothing Points Ms fi Trace Mass Range z Mass m z 246 05 Filter c SRAM ms2 470 18 cid 26 180 07 274 08 Peak Detection Algorithm ICIS F 218 LCquan User Guide Thermo Scientific 4 Creating a Processing Method Specifying Method Identification Table 83 IRC Detection Method dialog box Iden
160. ace Figure 62 Disk Space dialog box Disk Space Current directory C calibur QuanRoot E xample Study43 Drugs Workbook 121896 MBytes 79 9 Free 152539 MBytes Total 0K Help Table 37 Disk Space dialog box parameters Parameter Current Directory Description This area at the top of the dialog box displays the path to the current directory For example the path can be C Xcalibur System Programs Free Disk Space This area above the pie chart displays the currently available free disk space on the current drive The current drive is displayed as Current Directory The free disk space is also given as a percentage of the total disk space For example if your drive has a total disk space of 5090 MB and you have 1860 MB free the LCquan application displays the percentage free as 1860 5090 x 100 36 5 Total Storage Area This area below the pie chart displays the total disk space on the current drive The current drive is displayed as Current Directory Graphical Display of Storage Status Directory The pie chart displays the unavailable occupied disk space in red and the available free disk space in green Click Directory to open the Select Directory dialog box Figure 63 where you can change the default path see able 38 Thermo Scientific 2 Preparing to Perform Quantitative Analysis Save Settings Options Figure 63 Select Directory dialog box Select Director
161. ae 417 Actions Menu for Acquisition Window Only 00 00 eee 418 Apps Menun soree een tear anton nea eS Say Golo c tanec st aay Medien 419 Change Menu for Acquisition Window Only 0 0000 e ee eee 419 File Menut aM heads eddy toh E wath mene EEEE nope we atk nea a Ree 420 Help Menun Shirt Pott i hatte Laie Dace At oir 424 Options Mentis nessi opine th ye kee tL wees bap eae bp E a Ep 424 TAY SIE angela eeir uspeo say ce acute cat Rey dhewaceh wees E ESE 427 Zoomi Menut ary erias was Mis Bal cr dah Aleta tear are Aiea deals as ad di ae 430 LCquan User Guide vii Contents TEG HEB AS sek tates t teks tert ele hate gtr es Stata det eal a tendo sae Rta he aed aaa 431 OOM AR veuen aea e E E r a a ote honbstemen Bod dy a Ea AANEEN 432 E ETE OEE E E I E IE nie E E E E T 437 viii LCquan User Guide Thermo Scientific ee i Preface The LCquan application is part of the Thermo Xcalibur mass spectrometry data system This user guide describes how to use LCquan to perform quantitative analysis of compounds The LCquan application is a secure data quantitation package It provides all the tools you need to perform quantitation tasks including importing sequence information from external systems acquiring data developing processing methods reviewing and processing data generating reports and exporting results to external systems Note e With Revision C these instructions change the Microsoft Win
162. ager showing the Prevent Raw File Time Stamping When Doing Remote Workbook Acquisition permission as Allowed Permission Level 4 Instrument Configuration NET a O Disallowed Instrument Setup cient tal LCquan F Run Application zupe Root Folder 3SSWO File Tracking 5 File Allowed Section Configuration Grid Column Settings Acquisition Section DB f Allow changes to column labels in acquis Grayed Prevent raw file timestamping when doing Acquisition Run Dialog Explore Section Other requirements v4 Start Acquisition Dialog 6 Allow changes to selected sample info in Quantitate Section Local Folders The LCquan application creates temporary local folders for the files sequences and workbooks e During remote acquisition the application creates a local temporary folder for the remote workbook C Xcalibur remote acquisition workbook When the workbook is closed the application deletes this temporary workbook folder the instrument method file and the sequence file Thermo Scientific LCquan User Guide 111 2 Preparing to Perform Quantitative Analysis Remote Acquisition e The LCquan application creates a local rawfiles_timestamp folder for each sequence and temporarily stores the raw files C Xcalibur remote acquisition workbook rawfiles_timestamp When the sequence is completed the LCquan application deletes the rawftles_timestamp folder File Transfer The LCquan applica
163. ak integration and results view you can interactively review and rework standards QCs and unknowns in one window You can also enter the Quantitate window to process the acquired data as you acquire it Figure 4 Quantitate window 6 Thermo LCquan 3 Drugs Workbook y DER Fie Zoom View Apps Options Help PER EET EE ENCE EECA Instruments Review All Results review standards QCs blanks and unknowns Acquisition Sample Type Integration Type ISTD Response Specified Cone Calculated Cone Diff pene elazodone Method Settings 17139 338847 0 051 25 000 15 973 36 11 Alprazolam d5 Quantitate Method Settings 16566 361542 0 046 25 000 13 846 44 62 Alprazolam 3 Standard Method Settings 14481 343213 0 042 25 000 12 225 51 10 j4 Standard Method Settings 18597 317654 0 059 25 000 19 532 21 87 j5 Standard Method Settings 17723 299817 0 059 25 000 19 786 20 86 16 Standard Method Settings 33231 316871 0 105 50 000 40 236 19 53 Standard Method Settings 35282 323125 0 109 50 000 42 165 15 67 ja Standard Method Settings 34312 305110 0 112 50 000 43 626 12 75 ja Standard Method Settings 41308 258962 0 160 50 000 64 654 29 31 M0 Standard Method Settings 34187 263133 0 130 50 000 51 430 2 86 11 Standard Method Settings 54574 293120 0 186 75 000 76 573 2 10 12 Standard Method Settings 51754 299387 0 173 75 000 70 621 5 84 13 Standard Method Settings 57117 296990 0 192 75 000 79 314 5 75 14 Standard Me
164. alibration Settings Paroxetine dialog box parameters Sheet 1 of 3 Parameter Calibration Curve Description Specifies a calibration curve type Linear A linear curve that approximates experimental calibration data The LCquan application provides ignore force include origin options for this calibration curve type Quadratic A quadratic curve that approximates experimental calibration data The LCquan application provides ignore force include origin options for this calibration curve type Linear Log Log A linear polynomial curve Quadratic Log Log A quadratic log log curve that approximates experimental calibration data The LCquan application does not provide ignore force include origin options for this calibration curve type Average RF The average response factor of all levels Point to Point Data averages at each calibration level Straight lines are drawn between adjacent averaged calibration points This calibration curve can be used with one or more calibration levels The LCquan application provides ignore force origin options for this calibration curve type Cubic Spline A cubic polynomial curve to fit between each pair of calibration levels so that the slopes of the separate cubic polynomial curves match at common calibration curve points The LCquan application provides ignore force origin options for this calibration curve type Locally Weighted Ther
165. alley detection enabled any valley points nearer than half the expected width to the top of the peak are ignored If a valley point is found outside the expected peak width the LCquan application ends the peak at that point It always ends a peak when the signal reaches the baseline independent of the value set for the expected peak width Range 0 0 to 999 0 Default multiplier 0 0 Units seconds Note Valid only when you select the Valley Detection Enabled check box Constrain Peak Width Constrains the peak width of a component during the peak integration of a chromatogram You can set values that control when peak integration is turned on and off by specifying a peak height threshold and a tailing factor Selecting this activates the Peak Ht and the Tailing Factor parameters LCquan User Guide 119 3 Exploring the Data Creating Explore Methods Table 41 Genesis Peak Integration parameters Sheet 2 of 2 Parameter Description Peak Ht Specifies a percentage of the total peak height This is the minimum that a signal must be above the baseline before integration is turned on or off This option is available only when you select the Constrain Peak Width option Range 0 to 100 Default multiplier 5 0 The peak height percentage is defined as follows 100 Peak Height Tailing Factor Specifies a value for the factor that controls how the LCquan application integrates the tail of a peak This fact
166. alon Integration page of the IRC Detection Method dialog box Note To detect peaks Avalon uses the settings for initial events and user defined timed events in the event list Figure 137 IRC Detection Method dialog box showing the Avalon Integration page IRC Detection Method Identification Avalon Integration Auto Calculate Initial Events Initial Start Threshold 10000 000 Initial End Threshold 10000 000 Initial Area Threshold 10000 000 Initial P P Threshold 1 000 Initial Bunch Factor 1 000 Initial Negative Peaks Off Tension Time Min Event Value 0 000 Tension 7 1 000 Add Delete Change Cancel Table 88 RC Detection Method dialog box Avalon Integration page parameters Sheet 1 of 2 Parameter Description Auto Calculate Initial Estimates the initial values for the detection of peaks based on Events the data in the current raw file to display initial values in the event list and searches for the best values of initial events that detect peaks in the data Any timed event in the event list is unchanged when you click Auto Calculate Determines initial values for only Start Threshold End Threshold Area Threshold P P Resolution Threshold Bunch Factor Negative Peaks and Tension Note Valid only when a raw file is open LCquan User Guide 229 4 Creating a Processing Method Specifying Method Identification 230 LCquan User Guide Table 88 RC Detection Method dialo
167. alues are inserted into the Avalon event list 3 To delete the entire row except for an initial value row Thermo Scientific a Select the row you want to delete The selected row is highlighted b Click Delete The highlighted row is removed from the list LCquan User Guide 127 3 Exploring the Data Creating Explore Methods 128 LCquan User Guide 4 To change the values within a row a Select the row you want to change The selected row is highlighted b Specify the revised settings in some or all of these boxes Time Event Value c Click Change to automatically update the event list and the chromatogram display 5 To estimate the initial values for the detection of peaks and display initial values in the event list click Auto Calculate Table 45 lists the parameters for the Avalon Peak Integration area Table 45 Avalon Peak Integration area parameters Sheet 1 of 2 Parameter Description Column Headings in the Event List Time min Displays the initial time value in minutes Event Value Displays descriptions of detection parameters for initial events and timed events For details see Initial and Timed Events on page 129 Note Start Threshold End Threshold Area Threshold P P Resolution Threshold Bunch Factor Negative Peaks and Tension are defined with initial values Displays the values associated with initial events or timed events Range Factors are specif
168. an application displays Manual Integration in the Integration Type column of the Result list Figure 209 If you have peak area or peak height labeling enabled it labels the peak with AM for area manual or HM for height manual Figure 209 Example of manually integrated peak 4 gt a Selected Chromatogram APN_OO3 m z 192 07 192 07 RT 1 53 2 28 NL 5 64E3 F SRM ms2 330 13 cid 20 00 70 15 192 07 RT 1 90 o oO o Relative Intensity 3 N o Moved Integration Markers Time min Manual Integration Type ee Sample Type Integration Type ISTD Response Standard Manual Integration 12100 338847 Standard Method Settings 48585 361542 Standard Method Settings 14481 343213 Standard Method Settings n 317654 1 To drag a starting or ending point of a peak 1 Position the cursor over one of the integration markers The cursor turns into crosshairs 2 Drag the integration marker to the new location Thermo Scientific LCquan User Guide 363 6 Processing the raw Files and Reviewing the Analytical Results Reviewing the Calibration Curve and OCs To use the Baseline dialog box 1 Right click the Chromatogram pane and choose Change Peak Baseline from the shortcut menu The Baseline dialog box opens Figure 210 Figure 210 Baseline dialog box Baseline Paroxetine Peak Baseline Endpoints Left mink Left Height 3 06 Right
169. arameter Info Description Operator Displays the login ID of the person who is logged onto the system and who is acquiring the sequence Start Time Displays the time when the first sample or startup method began End Time Displays the time when the last sample or shutdown method completed When the LCquan application is closed before acquisition is complete but the acquisition is allowed to complete in the background the end time is displayed as Unknown Comment Status Displays a comment about the sequence as entered in the Run Sequence dialog box For more information see Run Sequence dialog box parameters on page 74 Displays the sequence status one of the following e Waiting Created but not yet submitted e Submitted Submitted but not yet validated data file has not yet been accepted for acquisition and not yet running e Queued Submitted and validated e In Progress Currently being acquired e Stopped Stopped either at the request of the operator or because of an error in acquisition e Complete Successfully completed e Rejected Failed validation rejected and did not run Components Displays the acquisition component names and their associated level amounts Samples Displays all the sequence row information set at the time of the sample acquisition Shortcut Menu Show Sequence Items Collapses the Acquisition Sequence History tree so th
170. ard Apps menu commands 419 File menu commands 420 Options menu commands 424 toolbar commands 432 View menu commands 427 shutdown methods 28 Sort Sequence parameters 58 Spectrum Display Options parameters 92 Spectrum pane shortcut menu commands 91 Spectrum pane Status page 90 Spectrum parameters Explore 171 Review 268 Spectrum shortcut menu commands Explore 171 Review 268 standard samples acquisition setup 31 444 LCquan User Guide standards described 404 external standards 406 internal standards creating 246 overview 406 sample types 408 starting LCquan 13 startup methods defined 28 Status page 80 Status Plot parameters Explore 172 Review 285 Status Plot shortcut menu commands Explore 173 Review 286 status plots Explore window 172 Status Report parameters Explore 174 Review 287 Status Report shortcut menu commands Explore 175 Review 288 status reports Explore window 174 study creating 13 description 8 Summing Ions dialog box 196 survey link xii Survey page 325 Switching to Processing during Acquisition parameters 105 T Target Compound area 237 Target Compound area parameters 238 time stamps always time stamp raw files during remote acquisition 109 never time stamp raw files during remote acquisition 109 toolbar buttons 432 toolbar buttons for zooming 326 trace type base peak 188 348 mass range 188 348 summed 188 348 TIC 188 348 Tune Method parameters Explore 176 Review 289 Thermo Scientific
171. arks the starting and ending points of the peak with integration markers ii Ensure that the shaded area accurately represents the contribution of the component to the chromatogram b Inspect the internal standard peak in the ISTD Chromatogram pane i Ensure that the LCquan application found the peak It shades the peak it finds gray and marks the starting and ending points of the peak with integration markers ii Ensure that the shaded area accurately represents the contribution of the internal standard to the chromatogram e To exclude a calibration standard from the calibration curve go to the next topic Excluding a Calibration Standard from the Calibration Curve e To modify the peak detection and integration settings that you defined in the Create Method view go to the topic Modifying the Peak Detection and Integration Settings on page 345 e You can manually change the integration starting and ending points and the baseline of the peak To manually integrate a peak go to the topic Manually Integrating Peaks on page 363 8 Repeat steps 1 7 for each of the remaining files in the Result list 334 LCquan User Guide Thermo Scientific Thermo Scientific 6 Processing the raw Files and Reviewing the Analytical Results Reviewing the Calibration Curve and OCs To review the calibration standards for the internal standard Review and rework the internal standard results as you did for the target compound 1
172. art Threshold You can modify the Avalon estimates by entering your own values and clicking Add to save your Start or End Threshold The Start Threshold depends on the RMS noise in the chromatogram It is the fundamental control used for peak detection so picking the best Start Threshold is essential for high quality data collection Units Absolute value of peak area counts x seconds Bunch Factor The number of points grouped together during peak detection This method groups several chromatographic points during integration without affecting the final area calculation of the peak Range 1 to 6 Note A high bunch factor groups peaks into clusters Area Threshold Controls the area cutoff Any peak with a final area less than the area threshold is not recorded Format units of area for the data LCquan User Guide 361 6 Processing the raw Files and Reviewing the Analytical Results Reviewing the Calibration Curve and OCs Table 141 Initial and Timed Events Sheet 2 of 2 Event P P Resolution Description Defines how much peak overlap must be present before two or more adjacent peaks create a peak cluster Peak clusters have a baseline drop instead of valley to valley baselines This is specified as a percent of peak height overlap Negative Peaks Automatically resets after a negative peak has been found Tension Controls how closely the baseline follows the overall shape of the chroma
173. ase file name prefix assigned to the sequence and a sequential sequence number When the default sequence starting number 1 was not changed the suffix number is the same as the row number of the sequence 001 002 and so on When the default sequence starting number was changed to another number the first sample has the starting number and subsequent rows in the sequence are incremented by 001 For example if the starting number was 100 the file name for the first sample would have a suffix of 100 the second sample would have a suffix of 101 and so on Path The path in the workbook from which raw files are loaded Sample ID Displays the sample ID for each data file The sample ID is an alphanumeric string of characters that identifies a sample Level Indicates the level defined for a calibration sample or quality control sample For details about calibration or QC levels see Chapter 4 Creating a Processing Method Thermo Scientific LCquan User Guide 309 5 Creating a Processing Sequence Editing a Processing Sequence 310 LCquan User Guide Table 121 Processing sequence grid column parameters Sheet 2 of 3 Parameter ISTD Corr Amt Description Specifies an updated amount of internal standard for the corresponding sample The default value is 0 0 For each component defined as an internal standard a bulk adjustment factor can be applied to the base response of each internal standard defined in
174. at only the first level items sequences are shown Show Info Level Sample Items Displays second level items for each sequence in the Acquisition Sequence History tree These are Info Levels and Samples Thermo Scientific 2 Preparing to Perform Quantitative Analysis Using the Setup Sequence View Table 13 Acquisition Sequence History pane parameters Sheet 2 of 2 Parameter Description Show Displays third level items for each sequence in the Acquisition Info Level Sample Sequence History tree These are Info details Level details and Details Sample details Show Info Details Expands the Acquisition Sequence History tree to show details for each Info item and collapse all other branches Show Level Details Expands the Acquisition Sequence History tree to show details for each Level item and collapse all other branches Show Sample Details Expands the Acquisition Sequence History tree to show details for each Sample item and collapse all other branches Acquisition Sequence Grid Thermo Scientific This section describes the functions you can perform from the Acquisition Sequence grid Creating an Acquisition Sequence Importing or Copying a Sequence Printing or Exporting the Acquisition Sequence Modifying a Sequence Using the Acquisition Sequence Grid Shortcut Menu Editing Column Values from the Sequence Grid This section includes examples of user defined columns and definitions of all the Acqui
175. ation Manager showing the Prevent Raw File Time Stamping When Doing D H Remote Workbook Acquisition permission as Disallowed Permission Level Instrument Configuration NET Disallowed Instrument Setup m LCquan Run Application Supe Root Folder _ File Tracking j File O Allowed Section Configuration Grid Column Settings Disallowed State Acquisition Section v4 Start Acquisition Dialog Allow changes to selected sample info in Allow changes to column labels in acquis m Acquisition Run Dialog Explore Section Quantitate Section gt O E To prevent raw file time stamping when acquiring data to a remote workbook 1 Choose Start gt All Programs gt Thermo Foundation gt Authorization Manager to open the Authorization Manager 2 In the Authorization Manager do the following Figure 67 a Select a user group in the Secure Groups list b Click Expand Tree to show the entire list of controlled features for the application 110 LCquan User Guide Thermo Scientific 2 Preparing to Perform Quantitative Analysis Remote Acquisition c From the list click the plus sign before the LCquan folder d Click the plus sign before the Acquisition Section folder e Select Prevent Raw File Time Stamping When Doing Remote Workbook Acquisition The Permission Level options become available f Select the Allowed option and click OK Figure 67 Authorization Man
176. ation in the Integration Type column of the Result list Figure 201 Figure 201 Integration Type column FileName Sample Type Integration Type Method Settings 16566 Method Settings 14481 Method Settings 18597 Standard Standard Standard LCquan User Guide 345 6 Processing the raw Files and Reviewing the Analytical Results Reviewing the Calibration Curve and OCs To revert back to the original parameters from the processing method 1 In the Integration Type column of the Result list click the cell containing the words User Integration for the sample that you want to change 2 Click the down arrow and select Method Settings from the list The LCquan application reverts to the original peak detection and integration parameters from the processing method Specifying Additional Peak Detection Criteria Use the User Identification Setting dialog box to specify additional peak detection criteria when the standard detection criteria do not provide the expected results Peak detection parameters are also available from the Quantitate window e The Peak Integration area on the Create page of the Explore view e The Peak Integration area on the Identification page of the Create Method view e The IRC Detection dialog box available from the ion ratio confirmation grid on the Identification page of the Create Method view To open the User Identification Settings dialog box right click a Chromatogram view in any
177. ation levels from the processing method to the acquisition sequence follow the instructions To change or import calibration and QC levels on page 61 in Chapter 2 Preparing to Perform Quantitative Analysis This chapter describes the preview panes in the Quantitate window that contain a list of views used to display the data in various forms The preview panes appear on the following views e Create Method e Survey e Review All Results In the preview panes select from the following views to display the data e Calibration Curve View for Survey or Review All Results Only e Chromatogram View for Create Method Only e Chromatogram View for Survey or Review All Results Only e Spectrum View e IRC Chromatogram View e Chromatogram List View e Error Report View e Filter List View e General Parameters Plot View for Survey or Review All Results Only e Instrument Method List View e Mass List View LCquan User Guide 251 4 Creating a Processing Method Data Views Peak List View for Survey or Review All Results Only Sample Info View Status Plot View Status Report View Tune Method View Multi Peak Plot View for Survey or Review All Results Only Each preview pane contains a Toolbar for Selectable Views at the top of the pane Toolbar for Selectable Views 252 LCquan User Guide Each preview pane contains a Toolbar for Selectable Views Figure 148 at the top of the pane Use the Toolbar for Selectable Views to select the vi
178. available columns for the acquisition sequence grid Sampte Type Fittiamel Sample Levet ISTO Corr Amt Di Factor Viat Pos ini vot Sample Vot Sample wi Sample tame j1 Blank APN_O NA 0 000 1 000 1 From AS 0 000 0 000 2 Standard APNO APN_002 Calo 0 000 1 000 2 From AS 0 000 0 000 3 Standard APNO APN_0O3 Cao 10 000 joo 2 From 4S 10 000 0 000 j4 Standard APNO APN_004 Cao 0 000 11 000 2 From AS 0 000 0 000 j5 Standard APNO APN_O0S Cao 0 000 1 000 2 From AS 0 000 0000 e Standard apno APN_006 Cao 0 000 hoo 2 From AS 0 000 0 000 Standard APNO APN_OO7 Cao 0 000 1 000 3 From AS 0 000 0 000 a Standard apno APN_OO08 Cao 10 000 hoo 6 From AS 0 000 0 000 a Standard APNO APN_009 Calo 0 000 11 000 3 From 4S 10 000 10 000 10 Standard APNO APN_O10 Cao 10 000 1 000 3 From AS 10 000 0 000 Standard TAPNO APN_011 Cao 0 000 1 000 3 From AS 0 000 10 000 Standard APNO APND12 Calo 0 000 1 000 4 From AS 0 000 10 000 Table 25 Acquisition sequence grid column parameters Sheet 1 of 3 Parameter Description Sample Type Displays the type of sample described by the sequence row The sample type defines how the LCquan application processes the sample data Each sample must be classified as one of the following LCquan sample types e Unknown Blank e QC quality control e Standard FileName Displays the name of the raw file that contains the sample data The file name is
179. awfie APNOO4 Cal 1 000 Tray01 2 30 00 0 000 0 000 Drugs 4 Standard APN_O06 Rawfile APNOOS Cal 1 000 Tray01 2 30 00 0 000 0 000 3Drugs 5 Standard APN_007 Rawfie APNOO6 Cal_ f 1 000 Tray01 2 30 00 0 000 0 000 Drugs 6 Standard APN 008 Rawfie APNOO7 Cal f 1 000 Tray01 3 30 00 0 000 0 000 Drugs 7 Standard APN_009 Rawfile APNOOB Cal i 1 000 Tray01 3 30 00 0 000 0 000 3Drugs Standard APN_O10Rawtile APNOO9 Cal i 1 000 TrayO1 3 30 00 0 000 0 000 Drugs ja Standard APN_O11 Rawfile APNO10 Cal 1 000 Tray01 3 30 00 0 000 0 000 3Drugs M0 Standard APN_012 Rawfile APNO11 Call f 1 000 Tray01 3 30 00 0 000 0 000 Drugs 1 Standard APN_013 Rawtile APNO12 Cal i 1 000 Tray01 4 30 00 0 000 0 000 3Drugs A2 Standard APN_014 Rawfie APNO13 Call i 1 000 Tray01 4 30 00 0 000 0 000 3Drugs 13 Standard APN_O15 Rawfie APNO14 Cal f 1 000 Tray01 4 30 00 0 000 0 000 Drugs 14 Standard APN_O16 Rawfile APNO1S Cal 1 000 Tray01 4 30 00 0 000 0 000 3Drugs 15 Standard APN_D17 Rawfie APNO16 Cal i 1 000 Tray01 4 30 00 0 000 0 000 Drugs 16 Standard APN_018 Rawfile APNO17 Cal f 1 000 Tray01 5 30 00 0 000 0 000 3Drugs 17 Standard APN_019 Rawfile APNO1S Cal f 1 000 Tray01 5 30 00 0 000 0 000 3Drugs APN_020 Rawfie APNO19 Cal f 1 000 TrayO1 5 30 00 0 000 0 000 Drugs APN_021 Rawtile APNO20 Cal f 1 000 Tray01 5 30 00 0 000 0 000 3Drugs APN_022 Rawfile APNO21 Cal f 1 000 Tray01 5 30 00 0 000 0 000 3Drugs EJ E Powers Dana NOT SAVED 7 8 2008 10 49A
180. bsolute percentages of the target ratio LCquan User Guide 195 4 Creating a Processing Method Specifying Method Identification Summing lons Dialog Box Use the Summing Ions dialog box to specify for ion summing the mass to charge ratios and scan filters of up to five ions Figure 120 lon summing creates a single chromatogram by summing together up to five chromatograms Ion summing can increase sensitivity because it sums the chromatograms of related masses for example water loss adducts isotopes or multiply charged peptides Ion summing provides the best results when chromatographic peaks have similar retention times and peak shapes and are identified by a unique scan filter mass filter or both lon summing is data processing only and does not affect the original raw data The LCquan application can easily reconstruct and report the original chromatograms that are part of the summed chromatogram You cannot use ion summing and ion ratio chromatograms together with the same component Figure 120 Summing lons dialog box Summing lons x mIon 1 Mass m z 205 02 Filter c SRM ms2 309 07 cid 42 00 205 02 x Mass mz 209 96 Filter c SRM ms2 314 10 cid 40 00 209 96 209 OK Cancel Help Table 76 lists the parameters for the Summing Ions dialog box 196 LCquan User Guide Thermo Scientific Thermo Scientific 4 Creating a Processing Method Specifying Method Identification T
181. can Modes MS Mode QIMS OQMS MS MS Mode Parent O Product O Neutral Loss Data Type Centroid Profile Scan Parameters Scan Range Source CID First Mass m z 150 000 Collision Energy W C 10 Accurate Mass Mode Dff Scan Time s 1 Q1 Peak Width FWHM j APPI Lamp Last Mass m z 1500 000 Set Mass m z Q3 Peak Width FWHM On Oodft a Collision Energy V 10 Energy Ramp eV 0 Micro Scans 1 4 Copy ScanEvent Paste ScanEvent 28 LCquan User Guide Thermo Scientific 2 Preparing to Perform Quantitative Analysis Introduction to the Instrument Setup Window Instrument setups are specific to each instrument Refer to your hardware manual for setup instructions for your instrument This section contains the following topics e Creating Instrument Methods e Importing an Existing Instrument Method Creating Instrument Methods You must configure the instrument before you can define an instrument method To configure an instrument close the LCquan application and Xcalibur data system and choose Start gt All Programs gt Thermo Foundation 2 0 gt Instrument Configuration To create instrument methods for your configured instruments 1 Select an instrument by clicking its icon in the navigation pane The LCquan application displays one or more pages of instrument parameters 2 Sel
182. cation automatically adds another row to the table when you click the Test box after filling in the QC Level and Amount boxes 9 To correct for isotope contribution click Isotope Use the Correction for Isotope Contribution dialog box to correct for an impurity in the internal standard compound that elutes at the same time as the target compound correct for an impurity in the target compound that elutes at the same time as the internal standard or both For more information see Correction for Isotope Contribution dialog box parameters on page 245 10 To accept the calibration settings for this component click Apply 248 LCquan User Guide Thermo Scientific Thermo Scientific 11 12 13 4 Creating a Processing Method Specifying Method Calibration To save the current settings as the default settings when settings for the following components are similar to the current settings click Save As Default Peak detection type mass range and scan filters are not saved Repeat steps 1 through 10 for each target component To save the current component identification and calibration settings in a new or existing Processing Method pmd file choose File gt Export gt Processing Method To watch an animation of these procedures Click the play button Note To watch this animation you must have Adobe Flash Player version 10 or later To download the latest Adobe Flash Player go to http get adobe com
183. ccurring 3 In the Area Noise Factor box enter a value to specify the noise level multiplier that determines the peak edge after the location of the possible peak 4 In the Peak Noise Factor box enter a value to specify the noise level multiplier that determines the potential peak signal threshold 5 To constrain the peak width of a component during the peak integration select the Constrain Peak Width check box e To specify the minimum above the baseline before integration is turned on or off enter a percent of the total peak height in the Peak Ht box e To constrain the peak width of an asymmetric chromatogram peak that has a tailing trace enter a peak integration multiplier in the Tailing Factor box Thermo Scientific LCquan User Guide 209 4 Creating a Processing Method Specifying Method Identification 6 To specify advanced component detection criteria click Advanced The LCquan application displays the ICIS Advanced Parameters dialog box Figure 129 Figure 129 ICIS Advanced Parameters dialog box ICIS Advanced Parameters Noise Method INCOS Noise C Repetitive Noise l RMS Noise Peak Parameters Min peak width E Multiplet resolution fi 0 Area tail extension E Area scan window ja OK Cancel Apply Help Use the advanced component detection criteria if the standard detection criteria do not P provide the expected results a Select a noise method e To use a single pass algorithm to
184. ces are queued the power setting of the last submitted sequence is used Note This option has the same effect as choosing Actions gt Devices Off CAUTION The Off state does not guarantee that all voltages are turned off nor does it indicate that all heated components are at room temperature To perform maintenance on an instrument refer to the hardware manual for your instrument LCquan User Guide 77 2 Preparing to Perform Quantitative Analysis Using the Run Sequence Dialog Box Change Instruments Use the Change Instruments In Use dialog box to change the status of instruments in use or select a different start instrument see Table 27 To select the instruments used to run the current acquisition sequence 1 From the Run Sequence dialog box click Change Instruments The Change Instruments In Use dialog box opens Figure 47 Figure 47 Change Instrument In Use dialog box Change Instruments In Use Start Instrument Simulation MS Yes Yes 2 Click the In Use or Start Instrument fields to activate the instrument with Yes for On 3 Click OK to save changes and close the dialog box Table 27 Change Instruments In Use dialog box parameters Parameter Description Instrument list Displays all the instruments that have been configured for operation as LCquan devices In Use list The rows in this list display a Yes or a blank space to indicate whether or not the instrument displayed in the same row is I
185. cessing sequence Keep current processing sequence I Don t tell me about this again Cancel Help 2 Select the Update Sequence with Data Acquired or Keep Current Processing Sequence option Table 36 Switching to Processing During Acquisition dialog box parameters Parameter Description Update Sequence with Specifies that the LCquan application automatically updates the Data Acquired processing sequence with sample information from the acquisition sequence as new sample data is acquired The LCquan application requantitates your data as new samples are completed If you clear this option click FP in the toolbar to force the processing sequence to update using all the samples acquired at that moment Keep Current Specifies that the LCquan application does not modify the Processing Sequence currently defined processing sequence when you switch to the Quantitate window from the Acquisition window during data acquisition If no processing method or processing sequence is available yet the LCquan application displays the appropriate view within the Quantitate window to be completed first Thermo Scientific LCquan User Guide 105 2 Preparing to Perform Quantitative Analysis Verifying Disk Space Verifying Disk Space 106 LCquan User Guide The Disk Space dialog box Figure 62 provides information about the active storage disk see Table 37 To open the Disk Space dialog box choose Actions gt Check Disk Sp
186. ch step In general sample handling errors account for a larger fraction of the total error than do mass spectrometer errors Fortunately the internal standard method can reduce both sources of error For example internal standards can correct for variations in a component s peak area that are caused by the following e Injection irreproducibility e Changes in analyte solution volume 406 LCquan User Guide Thermo Scientific A Quantitative Analysis Overview Quantitative Analysis Techniques e Matrix and coeluter interference both suppression and enhancement e System instability e Variations in the source conditions For maximum precision add the ISTD component as early as possible to the start of the sample work up particularly in those quantitative methods that require sample manipulations such as extraction cleanup and dilution Because the ISTD and non ISTD components are analyzed together the internal standard quantitative analysis approach has the advantage that it corrects for injection and other sample handling errors The ISTD must behave chemically in an identical or similar manner to the target compound through the extraction cleanup and analytical processes You can also add the ISTD component as the last step of sample preparation prior to the sample s use to compensate for fluctuations in the reproducibility of the sample injection Use ISTDs in a quantitative analysis experiment as follows 1 Analyze a series
187. change peak names do one of the following e Select the current name and type a new name e Use the Peak Name List Editor of Total Area 51 567 35 360 Peak List 0 9 Peak Hame Apex RT Area a ih ines a 5 To export all the component names and settings to the Quantitate window right click the peak list and choose Create Quan Components gt For All Peaks in List from the shortcut menu Thermo Scientific LCquan User Guide 145 3 Exploring the Data Exploring the Results 6 To export selected component names and settings to the Quantitate window a Select the peaks for which you want to create quan components b Right click the peak list and choose Create Quan Components gt For Selected Peaks in the List from the shortcut menu Figure 87 Figure 87 Shortcut menu for the peak list Open Raw File Lock against Rawfile Change bf Largest Area of Tot Lock against All Change v Unlock Create Peak Names gt Modify Peak Names Import Peak Name List Export Peak Name List Create Quan Components For Selected Peaks in List For All Peaks in List Copy to Clipboard To use the Peak Name List Editor 1 To open the Peak Name List Editor Figure 88 right click the peak list and choose Modify Peak Names from the shortcut menu Table 52 lists the parameters for the Peak Name List Editor dialog box Figure 88 Peak Name List Editor dialog box Peak Name List
188. changes to a raw file or parameters The color of the lock indicates the lock status of a pane Red Locked Against All Changes The lock icon is a closed lock e You cannot make any changes in panes that have red locks e No changes you make in any panes are reflected in panes that have red locks e All shortcut menu commands that can change the pane or the data are unavailable Thermo Scientific LCquan User Guide 253 4 Creating a Processing Method Data Views Table 98 Toolbar for Selectable Views controls Sheet 2 of 3 Control Description Yellow Locked Against raw file or Component Changes The E lock icon is a closed lock e You cannot make changes to a raw file or to a component in the Calibration Curve pane in panes that have yellow locks e When you make changes other than raw file changes in any panes those changes are reflected in panes that have yellow or green locks For example when you click a Chromatogram pane the focal point moves in a Chromatogram List pane even if the latter pane has a yellow lock e All shortcut menu commands that can change the raw file or components in the pane are unavailable a Green Unlocked The lock icon is an open lock Any changes you make in any panes are reflected in panes that have green locks Note When you apply a yellow or a red lock to a pane you essentially capture take a snapshot of that pane as it was just before you loc
189. cientific LCquan User Guide 49 2 Preparing to Perform Quantitative Analysis Using the Setup Sequence View User Defined Columns Values Examples Example 1 This example shows a set of custom columns and values for Client One at the San Jose Lab as they would appear on the Setup Sequence view These are the user labels and values you specified on the Change Sequence User Labels dialog box or the equivalent page on a wizard Change Sequence User Labels Changes in the labels and values will be applied to all rows New rows will also use these values User Label 1 Study User Value 1 Study X User Label 2 Client User Value 2 Client One User Label 3 Laboratory User Value 3 SanJoseLab User Label 4 Company User Value 4 Therma x User Label 5 Phone User Yalue 5 408 555 5555 v Cancel Help The user labels and values are displayed in the setup sequence header area Study Tutorial Study2 Client Client One Laboratory Sans oseLab Company Name Themo Phone 408 555 5555 The user labels and values are reflected in the user defined columns in the sequence grid Stuy crem Laboratory company Phone Tutorial Study 2 Client One SanJoseLab Thermo 408 555 5555 Tutorial Study 2 Client One SanJoseLab Thermo 408 555 5555 Tutorial Study 2 Client One SanJoseLab Thermo 408 555 5555 Tutorial Study 2 Client One SanJoseLab Thermo 408 555 5555 Tutorial Study 2 Client One SanJoseLab T
190. ck box to indicate when it is selected or not selected The acquisition sequence history that lists all acquisition sequences in the workbook that were submitted to the acquisition queue It provides information about each sequence including the operator the start and end times comments and the status of the sequence and the calibration and QC level data While the LCquan application is acquiring data the history is dynamically updated with samples that have been fully acquired To display the history click the Acquisition History tab The chromatogram pane where you can monitor chromatograms for each of the defined components plus up to ten user defined chromatograms in real time up to five are visible at one time It can also display data from an existing raw file The spectrum pane where you can monitor the mass spectrum in real time or view the mass spectrum of a selected raw file y The acquisition icon in the lower right of your window that lets you control an acquisition while it is running This icon is visible only while an acquisition is running To display the shortcut menu right click the blue vial icon a Loene Dins inaan la a ann l m Acquisition Status View Process Using Current Acquisition List Stop Current Sample and Pause Stop Current Sequence and Pause LCquan User Guide 81 2 Preparing to Perform Quantitative Analysis Using the Status View Command Acquisition Status View Description
191. clicking it The lock can be in one of three different states e Unlocked green Locked against raw file change yellow amp e Absolute lock red amp When a plot is unlocked every change of raw file or parameter is reflected in the plot Parameters such as retention time are sent out from unlocked plots for example when you click a chromatogram Plots that are locked against raw file change respond to changes in parameters but not to a change of raw file Parameters are not sent out from plots that are locked against raw file change Plots that are absolutely locked do not respond to changes in parameters or raw files Parameters are not sent out from plots that are absolutely locked LCquan User Guide 139 3 Exploring the Data Exploring the Results Review Displays You can define the location number and size of the individual graphic or table windows in the results area Select one of the five predefined sets of displays Each choice displays the data in multiple panes in the Explore window To display a window layout Select a layout from the Current Review Display list Figure 80 Figure 80 Current Review Display list Current Review Display G Multi Peak Vie Multi Peak View Rawf Comparison View Table 51 lists the parameters for the Current Review Display list Peak List View il Large Chro View Sample Info View Table 51 Current Review Display list parameters View
192. clude a calibration standard using any of these methods the LCquan application highlights its row in the Result list in dark pink and displays the excluded data point as an empty box in the Calibration Curve pane Figure 197 Figure 197 Excluding a calibration standard from the calibration curve __ Filetlame Sample Type Sample Hame Integration Type Excluded Samples Excluded Data Points Area Ratio 5 in 200 400 600 pg mL To use the Exclude column of the Result list 1 Select the Standards tab or the All tab at the bottom of the Result list 2 For each standard that you want to exclude from the calibration curve select the check box in the Exclude column of its row Figure 198 Figure 198 Exclude column Level Units RT Sampie i Exclude _ M M 3 To include the standard again clear the check box 342 LCquan User Guide Thermo Scientific 6 Processing the raw Files and Reviewing the Analytical Results Reviewing the Calibration Curve and OCs To use the Exclusion list 1 Select the target compound in the Component list 2 Right click the Calibration Curve pane and choose Exclusion List from the shortcut menu to display the Calibration Exclusion List dialog box The Cal Exclusion List dialog box displays a list of all the replicates co located data points used in creating the current calibration curve and their exclusion status Figure 199 Table 133 lists the
193. copy to the new workbook name Figure 19 Figure 19 Copy files page MV Copy Files from Import Folder MV Copy Files from Export Folder V Copy Files from Rawfiles Folder MV Copy Instrument Method Files IV Clear Acquisition History M Start New Audit Trail Table 8 Copy files page parameters Sheet 1 of 2 Option Description Copy Files from Duplicates the files in the Import folder of the original Import Folder workbook Note These files are generally archival copies of imported files Copy Files from Export Duplicates the files in the Export folder of the original Folder workbook Copies of reports and exported files are stored here Note These files are generally archival copies of exported files 24 LCquan User Guide Thermo Scientific Complete the Wizard Thermo Scientific 2 Preparing to Perform Quantitative Analysis Creating a New Study and Workbook Table 8 Copy files page parameters Sheet 2 of 2 Option Copy Files from Rawfiles Folder Description Duplicates the raw files found in the Rawfiles folder of the original workbook Note The raw files are generally files acquired by the original workbook or raw files that were copied when the original workbook was created Copy Instrument Method Files Duplicates up to three instrument method files found in the original workbook folder the Startup Method the instrument method used for acquisition and the Shutdown Method files Clea
194. cquisition Sequence History pane The acquisition sequence history lists all acquisition sequences in the workbook that were submitted to the acquisition queue 1 Select one or more of these sequences and drag them into the sequence grid 2 Hold down the CTRL key to select multiple sequences The LCquan application confirms that you want to replace the current sequence You cannot copy an individual sample you can copy only an entire sequence To use the shortcut menu e Choose Import Acquisition Sequence gt From File and use the dialog box to find a sequence file or e Choose Import Acquisition Sequence gt Copy from Processing Sequence 52 LCquan User Guide Thermo Scientific 1M kod KD kod 2 Preparing to Perform Quantitative Analysis Using the Setup Sequence View To copy the processing sequence in the current workbook From the window File menu or the shortcut menu choose Import Acquisition Sequence gt Copy From Processing Sequence The LCquan application asks if you want to modify the user labels and values of the imported sequence Keep the current user defined labels and values or create new ones for the sequence e Ifyou click No the LCquan application overwrites your acquisition sequence with the imported processing sequence without keeping your user defined columns and values e Ifyou click Yes the Change Sequence User Labels dialog box opens See Parameters in the Change Sequence User Labe
195. ction dialog box where you can select the ALT C gt T autosampler tray type to be used in the current session For more information see Tray Selection on page 71 in Chapter 2 Preparing to Perform Quantitative Analysis LCquan User Guide 419 c LCquan Menu and Toolbar Reference File Menu File Menu Commands Common to All LCquan Windows Table 157 File menu commands common to all LCquan windows Sheet 1 of 2 You can use the mouse or keyboard to activate File menu commands see Table 157 A few of the most commonly used commands can also be activated by clicking a Toolbar button Command Description Keyboard shorten toolbar icon New Opens the New Study or Workbook Wizard that you use to create ALT F gt Nor a new study or workbook For more information see Creating a New Study and Workbook on page 13 in Chapter 2 Preparing to Perform Quantitative Analysis Open Opens the LCquan Welcome page that you use to open an existing ALT F gt Oor Go workbook or to create a new study or workbook For more information see Creating a New Study and Workbook on page 13 in Chapter 2 Preparing to Perform Quantitative Analysis Save Saves the currently open workbook CTRL S or fA Save As Opens the Save As Wizard that you use to name a workbook and to ALT F gt A select the storage location folder For more information see Saving a Workbook on page 22 in Chapter 2 Preparing to Per
196. ction volume Select Get Injection Volume from AS This sets the currently selected row to get an injection volume from the autosampler method at run time using the default injection volume that you set in the Instrument Setup window for the autosampler The Inj Vol column value displays From AS in place of a numeric value Insert Rows Use the Insert Rows command to insert rows in the grid To insert rows in the grid 1 Select the rows in the sequence below where you want the new rows samples to be located 2 Right click the sequence and choose Insert Rows Empty rows are added above the selected or highlighted rows The LCquan application adds as many empty rows as the number of selected rows Delete Rows Use the Delete Rows command to delete rows from the grid To delete rows from the grid 1 Select the row in the sequence that you want to delete 2 Right click the sequence and choose Delete Rows Duplicate Selected Samples Use the Duplicate Selected Samples command to duplicate rows in the grid To duplicate rows in the grid 1 Select the rows in the sequence that you want to duplicate 2 Right click the sequence and choose Duplicate Selected Samples The selected rows are copied and added to the end of the sequence Thermo Scientific LCquan User Guide 65 2 Preparing to Perform Quantitative Analysis Using the Setup Sequence View Import Acquisition Sequence Use the Import Acquisition Sequence co
197. ctions e When you want the LCquan application to automatically process the data after it is acquired select the Process after Acquisition check box Note For the LCquan application to automatically process the data after it is acquired a processing method must already be defined and the acquisition component names must match the processing component names When you want to have the LCquan application automatically print Sample Reports and Summary Reports after the data is processed select the Generate Selected Reports check box e When you do not want the LCquan application to automatically process the data after it is acquired clear the Process after Acquisition check box 6 Specify any optional startup or shutdown instrument methods e When you want the startup method to run before the sequence starts select the Use Startup Method check box e When you want the shutdown method to run after the sequence is completed select the Use Shutdown Method check box 7 Specify any optional pre acquisition or post acquisition programs a In the Pre Acquisition box specify the program to run prior to running the sequence Use the Browse button to find the program b In the Post Acquisition box specify the program to run after the sequence is completed Use the Browse button to find the program 8 To synchronously run the pre or post acquisition programs a Select the Pre Acquisition check box b Select the Post Acquis
198. current sequence in the Create Sequence edit view Figure 169 From this view you can access the Create Sequence create view in the Quantitate window Figure 169 Create Sequence edit window El Thermo LCquan 3 Drugs Workbook y File Zoom View Apps Options Help PSBSILE FZFRIOOOSCOOS Kis OlO Create Sequence select files review and edit FileName Ravwtiles APN_0O03 raw Acquisition Sequence History Sample Name Study 3Drugs Standard 1 Conc 25 pg ml Client Laboratory Quans quad Company Thermo Electron Phone O bro non 1 we cm Pea ee Be swe N E 004 Rawle APNOO3 1 000 TrayO1 2 30 00 0 000 0 000 3Drugs a APN_O0S Rawle APNOO4 Cal 1 000 TrayO1 2 30 00 0 000 0 000 Drugs j4 Standard APN_O06 Rawfie APNOOS Cal i 1 000 TrayO1 2 30 00 0 000 0 000 3Drugs 5 Standard APN_OO7 Rawfie APNOO6 Cal if 1 000 Tray01 2 30 00 0 000 0 000 Drugs 6 Standard APN 008 Rawfie APNOO7 Cal i 1 000 Tray01 3 30 00 0 000 0 000 Drugs 7 Standard APN 009 Rawfie APNOOB Cal_ i 1 000 Tray01 3 30 00 0 000 0 000 3Drugs B Standard APN_DIO Rawfie APNOOS Cal f 1 000 Tray01 3 30 00 0 000 0 000 Drugs 9 Standard APN_O11 Rawfile APNO10 Cal i 1 000 Tray01 3 30 00 0 000 0 000 3Drugs Mo Standard APN_012 Rawfie APNO11 Cal 1 000 Tray01 3 30 00 0 000 0 000 3Drugs 11 Standard APN_013 Rawfie APNO12 Cal I 1 000 Tray01 4 30 00 0 000 0 000 Drugs A2 Standard APN_014 Rawfie APNO13 Cal i 1 000 Tray01 4 30 00 0 000 0 000 3Drugs A3 Stan
199. d elution time of the component peak apex Range 0 0 to 999 0 Units minutes Window sec The time boundaries for the expected peak apex occurrence Range 1 0 to 999 0 Units seconds Use as RT Reference Specifies that the active component peak is to be used as a retention time reference This component s actual retention time is used to adjust the expected retention times of other components automatically during processing All RT References appear in the Adjust Using list View Width The amount of time to display the chromatogram pane Range Expected RT View Width 2 through Expected RT View Width 2 Range 0 10 to 999 0 Units minutes Adjust Using Specifies adjusting the retention time for this component based on the actual RT of another component that has been designated an RT Reference Retention time references are automatically created when you select the Use as RT Reference option when the component is active The processing method must have at least one retention time reference for this box to be active All RT References appear in the Adjust Using list Note You can test the results of the new criteria by clicking Apply or OK You can apply the new criteria to all files in the Result list by clicking Apply to All Thermo Scientific LCquan User Guide 349 6 Processing the raw Files and Reviewing the Analytical Results Reviewing the Calibration Curve and OC
200. d standards or calibration standards These standards must cover a suitably wide range of concentrations or amounts and must bracket the range of expected concentrations in the unknown Responses to these standards are plotted in a graph called a calibration curve Ideally this curve corresponds to the equation of a straight line to ensure the highest degree of precision Fitting an equation to the calibration curve with a user specified method for example a least squares regression provides a response factor a comparative measure of the response of the mass spectrometer to a component It is based on the amount of sample injected and the resulting peak area or peak height The response factor gives an intuitive and quantitative measure of how responsive or sensitive the mass spectrometer is to a certain component 2 Mass Spectrometry Principles and Applications de Hoffman E Charette J Stroobant V Wiley New York 1996 p 162 Thermo Scientific A Quantitative Analysis Overview Quantitative Analysis Techniques Figure 230 Integrated chromatographic peak 10 80 60 Relative Intensity 20 0 2 0 3 0 4 0 5 0 6 0 7 0 8 0 9 1 0 Time min To perform quantitative analysis of samples containing unknown amounts of the target component calculate the peak area or height and compute and apply the appropriate response to the equation derived from the calibration curve These steps provide an estimate of the amount o
201. dard APN_D15 Rawfie APNO14 Cal O00 1 000 TrayO1 4 30 00 0 000 0 000 Drugs 14 Standard APN_016 Rawfie APNOI5 Call 000 1 000 Tray01 4 30 00 0 000 0 000 3Drugs A5 Standard APN_017 Rawfie APNO16 Cal I 1 000 Tray01 4 30 00 0 000 0 000 Drugs A6 Standard APN_018 Rawfie APNO17 Call 1 000 Tray01 5 30 00 0 000 0 000 3Drugs 17 Standard APN_019 Rawfie APNO18 Cal i 1 000 Tray01 5 30 00 0 000 0 000 3Drugs 8 Standard APN_020 Rawfie APNO1I9 Cal i 1 000 Tray01 5 30 00 0 000 0 000 Drugs 19 Standard APN_021 Rawfile APNO20 Cal i 1 000 Tray01 5 30 00 0 000 0 000 3Drugs 20 Standard APN_022 Rawfie APNO21 Cal f 1 000 Tray01 5 30 00 0 000 0 000 SDrugs B For Help press F1 Powers Dana MOT SAVED 7 8 2008 10 49AM 7 Opens the create view Use any of these methods to create a processing sequence e Using the New Processing Sequence Wizard e Copying an Acquisition Sequence e Importing a Processing Sequence e Copying the Last Acquired Sequence Thermo Scientific LCquan User Guide 295 5 Creating a Processing Sequence Defining a Processing Sequence Using the New Processing Sequence Wizard If you have not defined or imported a processing sequence use the New Processing Sequence Wizard to create a new sequence Start the Wizard Use the New Processing Sequence Wizard to create a new sequence To open the New Processing Sequence Wizard 1 Do one of the following e Click Quantitate and then click Sequence e Choose View gt S
202. determine the noise level select the INCOS Noise option e To use a multiple pass algorithm to determine the noise level select the Repetitive Noise option b To use an RMS calculation instead of the default ICIS noise method select the RMS Noise check box c To specify the minimum number of scans required in a peak enter a value in the Min Peak Width box d To specify the minimum separation in scans between the apexes of two potential peaks enter a value in the Multiplet Resolution box e To specify the number of scans past the peak endpoint to use in averaging the intensity enter a value in the Area Tail Extension box f To specify the number of allowable scans on each side of the peak apex enter a value in the Area Scan Window box 210 LCquan User Guide Thermo Scientific Thermo Scientific 4 Creating a Processing Method Specifying Method Identification Table 79 ICIS Peak Integration area parameters Sheet 1 of 2 Parameter Advanced Description Opens the ICIS Advanced Parameters dialog box see Table 80 Baseline Window The number of scans used when searching for where the minima are occurring A local minimum becomes an anchor point making the entire curve locally reduced until these points are at zero Range 1 to 500 Default 40 Area Noise Factor The noise level multiplier used to determine the peak edge after the location of the possible peak so that the peak can narrow or broad
203. displayed columns select the check box before the column and click Remove This does not delete the column it simply hides it from display on the sequence grid All hidden columns are displayed in the Available Columns list 3 To display currently hidden columns select the check box before the column and click Add The column is added to the Displayed Columns list and is displayed on the sequence grid 4 To change a column position a Select the check box before the column name in the Displayed Columns list b To move the column left in the grid click Move Up c To move the column right in the grid click Move Down Thermo Scientific LCquan User Guide 329 6 Processing the raw Files and Reviewing the Analytical Results Reviewing the Calibration Curve and OCs 5 To change the display precision a In the Places column of the Displayed Columns list select the value you want to change b Type a new value for the number of decimal places to display in the column Note When the Values column is unavailable your LCquan administrator has specified decimal rounding for exported Excel reports In this case the number of decimal places is fixed and cannot be edited 6 To change a column width a In the Width column of the Displayed Columns list select the value you want to change b Type the new value Tip You can also resize a column width by dragging the column boundary in the heading r
204. dn amen de nets 82 Acquisition Status Chromatogram Pane 2 0 0 0 eee eee 83 Acquisition Status Spectrum Pane 0 cee eee 90 Acquisition Status Run Status 1 eee cee ee eee 94 Acquisition Status Acquisition Queue 6 6 eee cece 97 Acquisition Status Acquisition History 0 0 0 6 cee eee ee 101 Processing Data While Continuing to Acquire Data 4 104 Verifying Disk Space sty seo Pee hoe de NG Key ots Mom won gases eats 106 Save Settings Options es nese ie ane dae eile Mie eee oie ot Sle tee erp 107 Emergency Shutdown lt 2 s1033 aiest gees bets ata eee ar Batee wees 108 Shutting Down LCquan While Acquiring 0 0 0 00 0 0 00 eee eee 109 Remote Acquisition desee eis nna e ee ee nes Mes tees Shee ees 109 Exploring the Data 000 e cece e eee eee eee eens 113 Introduction to the Explore Window 0 ccc e eee e ee 113 Creating Explore Methodsiis c2 Seaaes wae pavers koh weeded kee 114 Defining Chromatographic Parameters ss view ts Soe ete 115 Defining Peak Integration Parameters scot ves ioe ras awn DA eoe 62 117 Acquisition Sequence History 0 cee eee 132 Preyiew Paies nai cytes carne Kata aeaa aa ABW Slr aa klar nein ok eh 134 Exploring the Results t 4 lade Pal a a e ce alas ste lala had aia is 135 Navigation Features oie eva 3 Wah to o s One wea es Lee tte 136 Opening a raw File and Displaying the Chromatogram
205. dows operating system from Windows XP or Vista to Windows 7 e The LCquan for Web Access does not include the Acquisition feature Contents e Related Documentation e System Requirements e Special Notices e Contacting Us To suggest changes to documentation or to Help Complete a brief survey about this document by clicking the button below Thank you in advance for your help SURVEY Thermo Scientific LCquan User Guide ix Preface Related Documentation The following LCquan manuals are available on the LCquan software CD as PDF files e LCquan Administrator Guide describes how to configure the software for compliance and security e LCquan User Guide describes how to use LCquan to perform quantitative analysis of compounds e LCgquan Tutorial describes step by step procedures to perform quantitative analysis with sample data To view the product manuals Go to Start gt All Programs gt Thermo Xcalibur gt Manuals gt LCquan To open Help From the LCquan window choose Help gt LCquan Help To find a particular topic use the Help Contents Index or Search panes For more information visit www thermo com X LCquan User Guide Thermo Scientific Preface System Requirements In addition your system must meet these minimum requirements System Requirements Computer e 2 4 GHz processor with 4 GB RAM use 4 GB for enhanced speed e DVD CD ROM drive e Display resolution 1280 x 1024
206. dvanced page parameters Parameter Description Noise Method INCOS Noise A single pass algorithm is used to determine the noise level Repetitive Noise A multiple pass algorithm is used to determine the noise level In general this algorithm is more accurate in analyzing the noise than the INCOS Noise algorithm but it takes longer RMS Noise A root mean square calculation is used to determine signal to noise values instead of the default ICIS noise method Min Peak Width The minimum number of scans required in a peak Range 0 to 100 Default 3 Multiplet Resolution The minimum separation in scans between the apexes of two potential peaks This is a criterion to determine if two peaks are resolved Use a larger number in a noisy environment when the signal is bouncing around Range 1 to 500 Default 10 scans Area Tail Extension Area Scan Window The number of scans past the peak endpoint to use in averaging the intensity Range 0 to 100 Default 5 scans The number of allowable scans on each side of the peak apex A zero value defines all scans peak start to peak end to be included in the area integration Range 0 to 100 Default 0 scans Note You can test the results of the new criteria by clicking Apply or OK You can apply the new criteria to all files in the Result list by clicking Apply to All LCquan User Guide 359 6 Processing the raw Files and Reviewing the Analytical Results R
207. e e Specifies the retention time corresponding to the selected scan in the Expected box in the Retention Time Area Display the starting and ending points and shaded area of the component peak The LCquan application shades the peak gray and displays the current baseline blue with square handles at the starting and ending points of the peak To adjust the x axis and y axis ranges to improve the display of the peak activate the Chromatogram pane by clicking the square button in the upper right corner of the pane Adjust the x axis range by doing one of the following e Drag the cursor horizontally over the x axis range that you want displayed Use the and buttons Adjust the y axis range by doing one of the following e Drag the cursor vertically over the y axis range that you want displayed e Usethe Q O and buttons Inspect the component peak Verify that the peak has the proper symmetry and that the grayed area between the blue handles and above the blue baseline accurately represents the contribution of the component to the chromatogram e When necessary repeat steps a through c e When you have problems with noise in the peak unresolved peaks or peak tailing see Defining Peak Integration Parameters on page 204 e When baseline noise is interfering with peak identification or integration see the advanced features for Genesis or ICIS in Defining Peak Integration Parameters on page 204 Ther
208. e Click Open and Next Import from legacy files Import Instrument Method Click Next Acquisition Sequence and Processing Parameters page Import from existing workbook gt Select Legacy Files page Select Instrument Method optional Acquisition Processing Sequence Click Next Click Next and Finish Do not import prior data Thermo Scientific rf and Processing Method Select Workbook page Click Next Click Next and Finish and Finish r lt x Quantitate view Explore See Quantitate flow diagram view Click Method Click Method Create Method page Click Sequence Create Explore Method page Click Review Create Sequence page Explore Results page gt see Click Survey gt Review AT Rese Click Review gt Review Reports page Click Reports LCquan User Guide 413 B LCquan Flow Diagrams LCquan Acquisition Flow Diagram LCquan Acquisition Flow Diagram Base acquisition on a Create Import Sequence File previously saved file Create new new method Generate Sample Names dialog box in acquisition sequence in page Click Next New Acquisition New Acquisition Click Next Sample Information page ck Next Vial Positions page ck Next Add Standards page Next Add OCs page Next c c QD l omponent Name page Next Acquisition view User Label page Setup Sequence page Click Acquire
209. e S From AS 0 000 Tutorial Study 2 Client One S From 4S 0 000 Tutorial Study 2 Client One S From AS 0 000 Tutorial Study 2 Client One S From AS 0 000 Tutorial Study 2 Client One S From AS 0 000 Tutorial Study 2 Client One S From AS 0 000 Tutorial Study 2 Client One S From AS 0 000 Tutorial Study 2 Client One S From AS 0 000 Tutorial Study 2 Client One S From AS 0 000 Tutorial Study 2 Client One S S 5 S 5 S S S S gt From AS 0 000 Tutorial Study 2 Client One From AS 0 000 Tutorial Study 2 Client One From AS 0 000 Tutorial Study 2 Client One From AS 0 000 Tutorial Study 2 Client One From AS 0 000 Tutorial Study 2 Client One From AS 0 000 Tutorial Study 2 Client One From AS 0 000 Tutorial Study 2 Client One From AS 0 000 Tutorial Study 2 Client One nn pb Bb BB oul ul ul WW NN wil Thermo Scientific LCquan User Guide 3 1 Introduction Quantitative Analysis with LCquan Explore Window In the Explore window you can display a multi peak chromatogram for a single sample and experiment with peak detection and integration parameters to see how they affect the chromatogram Figure 3 Use the Explore window to determine the optimum parameters used in the processing method The LCquan application can also generate a peak list that you use to generate the quan components in the processing method The Create
210. e information see Parameters in the Change Sequence User Labels dialog box on page 34 in Chapter 2 Preparing to Perform Quantitative Analysis Right click the sequence grid on any view of the Quantitate window and choose Columns Open the Column Arrangement dialog box For more information see Customizing Column Arrangement on page 312 in Chapter 5 Creating a Processing Sequence Comment Displays the comment entered for this sample Company User defined Displays information that is pertinent to the active sample row in the Acquisition sequence You can use this box to convey information about this sample to others or as a reminder to yourself The User Label 4 default name is Company To change definitions of User Labels and Values for any user defined column refer to the instructions for the Client column Delta RT The difference between the Expected RT and the Actual RT Dil Factor Displays the dilution factor that was used to prepare the sample The valid range is 0 000 to 10000 000 The LCquan application interprets a value of 0 000 as no dilution To change this factor double click the Dil Factor box The cursor changes to the vertical bar cursor Type the correct factor If you have specified a processing method for the current sequence the LCquan application automatically enters the Dil Factor value from the processing method settings Duration Length of the acquisit
211. e shortcut menu that changes the raw file with the yellow lock or the raw file and parameters with the red lock Menu items that would cause changes are grayed out For more information about locks see Toolbar for Selectable Views on page 150 Table 60 lists the parameters for the Instrument Method view Table 60 Instrument Method view parameters Sheet 1 of 2 Parameter Description Arrows Switches between instrument methods in the current raw file Instrument Selector List Options Instrument Name Displays the method for the selected instrument Shortcut Menu Open Raw File Opens the Select Raw File dialog box where you can select the raw file to load LCquan User Guide 163 3 Exploring the Data Data Views Mass List View 164 LCquan User Guide Table 60 Instrument Method view parameters Sheet 2 of 2 Parameter Description Lock Against Raw File Prevents changes to a raw file The lock icon is yellow and the Change lock is closed Unlock Makes the pane editable The lock icon is green and the lock is open Copy to Clipboard Copies selected data to the Clipboard You can then paste the data from the Clipboard buffer into a document The Mass List view displays the spectral mass intensity list for a sample at the selected retention time Figure 100 Figure 100 Mass List view 4 gt a Component Mass List APN_003 RT 1 90 Standard 1 Conc 25 pg ml The contents o
212. e the left peak edge retention time in minutes They start at the peak apex and test measured S N values at successively higher retention times to determine the right peak edge retention time in minutes Sample Vol Displays the volume of a component that has been placed in the sample The unit for this volume is specified in the Processing Setup window and is only included in LCquan reports The LCquan application does not convert units To change the sample volume Double click the Sample Volume box The cursor changes to the vertical bar cursor Type the correct sample volume If you have specified a processing method for your sequence the LCquan application automatically enters the sample volume of the Calibration or Quality Control samples or both from the processing method settings Sample Wt Displays the amount of a component that has been placed in the sample The unit for this sample weight is specified in the Processing Setup window and is only included in LCquan reports The LCquan application does not convert units To change the sample weight Double click the Sample Weight box The cursor changes to the vertical bar cursor Type the correct sample weight If you have specified a processing method for your sequence the LCquan application automatically enters the sample weights of the Calibration or Quality Control samples or both from the processing method settings Search Window The retention time windo
213. e Autosampler dialog box in the Instrument Setup window The minimum and maximum injection volumes you can use depend on the autosampler you select The usable range is dependent on the injection mode and might be smaller than the range displayed in the status bar Refer to your autosampler manual 5 Click Next Specify Calibration Standards Add calibration standards to the acquisition sequence see Table 17 To add calibration standards 1 Select the Add Standards check box Figure 35 Figure 35 Standards information page V Add Standards Based on existing cal levels from method Based on automatically generated cal levels Number of cal levels E Cal level base name Cal Number of Injections per Level fi 0 V Add Blanks V Fillin Sample ID for Standards 2 Select either the Based on Existing Cal Levels from Method or Based on Automatically Generated Cal Levels option 3 If you selected the Based on Automatically Generated Cal Levels option specify the number of calibration levels and the calibration level base name 44 LCquan User Guide Thermo Scientific Thermo Scientific 2 Preparing to Perform Quantitative Analysis Using the Setup Sequence View 4 Specify the Number of Injections per Level 5 Optional Select the Add Blanks check box 6 Optional Select the Fill in Sample ID for Standards check box When your sequence contains Standard samples the order used is as follows e Optional B
214. e Methods e Acquisition Sequence History e Preview Panes To open the Create view of the Explore window When the left navigation is displayed a Click Explore b Click Method When the left navigation pane is hidden a Choose View gt Section Selection gt Explore Section b Choose View gt Step Selector gt Explore Method Defining Chromatographic Parameters Use the Chromatogram Definition area Figure 69 to define your chromatogram see Table 40 To define chromatographic parameters 1 In the Detector box select the specific data stream Figure 69 Chromatogram Definition area Identification m Chromatogram Definition Detector Smoothing MS v fi Trace Mass Range x z Mass Range Massi mz fa Filter Range min fo 0 993 9 2 In the Smoothing box enter the number of points to use for a moving mean filter to smooth the chromatogram 3 In the Trace box select a type of chromatogram Thermo Scientific LCquan User Guide 115 3 Exploring the Data Creating Explore Methods 4 To use trace math select an addition or a subtraction trace operator and select another type of chromatogram in the second Trace box 5 In the Mass1 m z box enter an initial mass value 6 When you are using trace operator math enter a second mass value in the Range min box to define the mass range 7 In the Filter box enter an existing filter or select a filter from a preloaded filter list
215. e Vol I Sample vt Sample Name Fill From Row fi To Row fig Using Row 1 Select Al Clear All Cancel Help Thermo Scientific LCquan User Guide 307 5 Creating a Processing Sequence Editing a Processing Sequence Table 120 Processing sequence Fill Down dialog box parameters Parameter Select Columns Description See Processing Sequence Grid Column Parameter Definitions on page 308 for descriptions of all column names and values Row Controls Fill From Row Specifies the first row to receive new data To Row Specifies the last row to receive new data Using Row Specifies which row the initial data comes from Create Samples in Specifies that a new sample be created in an empty row during a Selected Empty Rows fill down that includes the row in its fill down range When not During Fill Down selected specifies that the empty row is ignored during a fill down that includes the row in its fill down range Buttons Select All Selects all the check boxes in the Select Columns area Clears all the check boxes in the Select Columns area Processing Sequence Grid Column Parameter Definitions 308 LCquan User Guide Figure 180 shows the processing sequence grid columns able 121 lists the available columns for the processing sequence grid Figure 180 Processing sequence grid columns a panes Feta Blank APN_001 2 Blank APN_002 3 Standard APN_0
216. e advanced criteria if the standard detection criteria do not provide the expected results a To specify a peak signal to noise cutoff enter a Peak S N Cutoff value b To use a valley detection approximation method to detect unresolved peaks enter Rise Percentage value and a Valley S N value Thermo Scientific Thermo Scientific 3 Exploring the Data Creating Explore Methods c Select whether the noise used in calculating S N values is calculated using an RMS calculation or Peak to Peak resolution threshold Table 41 Genesis Peak Integration parameters Sheet 1 of 2 Parameter Advanced Description Opens the Genesis Advanced Component Options dialog box Figure 71 S N Threshold This multiplier specifies a signal to noise threshold for peak integration Only peaks with a signal to noise ratio greater than this value are integrated Range 0 0 to 999 0 Default multiplier 0 5 Valley Detection Enabled Detects unresolved peaks with the valley detection approximation method The LCquan application drops a vertical line from the apex of the valley between unresolved peaks to the baseline The intersection of the vertical line and the baseline defines the end of the first peak and the beginning of the second peak Expected Width sec This multiplier specifies an expected peak width parameter that controls the minimum width that a peak is expected to have when valley detection is enabled With v
217. e file options in the FileName box 386 LCquan User Guide Thermo Scientific 6 Processing the raw Files and Reviewing the Analytical Results Creating and Reviewing Reports Table 152 Grid parameters Sheet 2 of 9 Parameter Sample Type Description Displays the sample type defined in the sequence row The sample type defines how the LCquan application processes the sample data Each sample must be classified as one of the following LCquan sample types e Unknown A sample type that is not a Calibration Sample Calibration Blank Quality Control Sample or Quality Control Blank The LCquan application performs quantitative analysis of unknown sample types by using internal or external standards e Blank A sample type that contains no target components You can add internal standard components to calibration blanks You can include blanks in the sequence to assist in purging all residual components from the LCquan application prior to the analysis of the next sample or sample sequence Use LCquan analysis of a blank sample to confirm that there are no residual components in the solvent system that can cause erroneous results This is especially important during quantitation You can expect quantitative analysis of a blank sample to result in the detection of no target compounds e QC quality control A type of sample that contains known amounts of one or more known components QC samples are placed in the sequence so that quantita
218. e maximum ratio of the trailing edge to the leading side of a constrained peak and calculates the retention time of the maximum extent of the right edge of the tailing peak Range 0 5 to 9 0 Default multiplier 1 0 1 0 has no effect Note Valid only when you select the Constrain Peak Width check box Table 44 ICIS Advanced Parameters dialog box Sheet 1 of 2 Parameter INCOS Noise Description A single pass algorithm is used to determine the noise level Repetitive Noise A multiple pass algorithm is used to determine the noise level In general this algorithm is more accurate in analyzing the noise than the INCOS Noise algorithm but it takes longer RMS Noise Thermo Scientific A root mean square calculation is used to determine signal to noise values instead of the default ICIS noise method LCquan User Guide 125 3 Exploring the Data Creating Explore Methods 126 LCquan User Guide Table 44 ICIS Advanced Parameters dialog box Sheet 2 of 2 Parameter Peak Parameters Description Units are in number of scans Min Peak Width The minimum number of scans required in a peak Range 0 to 100 Default 3 Multiplet Resolution The minimum separation in scans between the apexes of two potential peaks This is a criterion to determine if two peaks are resolved Use a larger number in a noisy environment when the signal is bouncing around Range 1 to 500 Default 10 scans
219. e name e Additional user defined parameters such as Study Client Laboratory Each row of the sequence corresponds to one sample injection You must have an acquisition sequence to acquire data and a processing sequence to process data The acquisition sequence and the processing sequence can be the same Each sequence has a limit of 5000 samples From the Sequence Setup view you can e Create a new acquisition sequence e Create a new processing sequence e Import view and modify an existing acquisition sequence e Import view and modify an existing processing sequence To open the Setup Sequence view When the left navigation pane is displayed a Click Acquisition b Click the Setup icon When the left navigation pane is hidden a Choose View gt Section Selection gt Acquisition Section b Choose View gt Step Selector gt Acquisition Setup 32 LCquan User Guide Thermo Scientific 2 Preparing to Perform Quantitative Analysis Using the Setup Sequence View Acquisition Sequence Header The Sequence Header area Figure 24 displays information about the current sequence and function buttons for navigating the sequence grid Figure 24 Sequence header page FileName JAPN_001 Tutorial Study 2 Sample Name Sample123 Study utonal Study Client CientOne Comment Laboratory SanJoseLab Company Name Thermo Phone 408 555 5555 Fone T cout ue To modify the sequence user labels 1 To enter comment
220. e new study name Figure 8 2 Type the new workbook name 3 Optional Select the Change Study Root Folder check box Figure 8 Create new study and workbook page Study Name Example Study Browse Workbook Name M y Workbook Browse C Xcalibur QuanRoot E xample Study My Workbook Change Study Root Folder Table 3 Options for study and workbook name page Parameter Description Study Name The LCquan application creates a new folder with this name in the root folder Workbook Name The LCquan application creates a new workbook with this name in the Study folder Change Study Root Select to change the project root folder The default root Folder folder is C Xcalibur QuanRoot Changing the root folder requires authorization from your administrator 4 Click Next Note You cannot overwrite existing data in the LCquan application When you specify a new study and workbook set with the same name as an existing set it displays an error message In this case enter another name for your study or workbook e To change the root folder go to Specify the Root Folder e To keep the current root folder go to Import raw Files 16 LCquan User Guide Thermo Scientific 2 Preparing to Perform Quantitative Analysis Creating a New Study and Workbook Specify the Root Folder Specify a root folder for the study The default root folder is C Xcalibur QuanRoot Figure 9 To specify the root folder f
221. e of peak area counts x seconds Bunch Factor The number of points grouped together during peak detection This method groups several chromatographic points during integration without affecting the final area calculation of the peak Range 1 to 6 Note A high bunch factor groups peaks into clusters Area Threshold Controls the area cutoff Any peak with a final area less than the area threshold is not recorded Format units of area for the data P P Resolution Defines how much peak overlap must be present before two or more adjacent peaks create a peak cluster Peak clusters have a baseline drop instead of valley to valley baselines This is specified as a percent of peak height overlap Negative Peaks Automatically resets after a negative peak has been found Tension Controls how closely the baseline follows the overall shape of the chromatogram A lower tension traces the baseline to follow changes in the chromatogram more closely A high baseline tension follows the baseline less closely over longer time intervals Units minutes Tangent Skim 216 LCquan User Guide Enables tangent skim on any peak clusters By default the LCquan application selects the tallest peak in a cluster as the parent solvent and detects peaks on either side or both sides of the tallest peak You can also identify which peak in the cluster is the parent Tangent skim automatically resets at the end o
222. e or import method page Parameter Description Condition Create New Method Creates a new processing method Initialize with Creates default processing method components with the Acquisition names from the acquisition sequence component list Capone Maias Note This check box is enabled only when the components are defined in the acquisition sequence Import Fxisting Select to specify an existing processing method Method 2 Click Next e Ifyou chose to create a new method go to Specify Calibration Standards e Ifyou chose to import an existing method go to Import a Processing Method Thermo Scientific LCquan User Guide 183 4 Creating a Processing Method Creating a Processing Method with the New Method Wizard Import a Processing Method Import a saved processing method file To import a processing method file 1 Type the path to a processing method file or click Browse and find a file Figure 111 Figure 111 Import processing method page Processing Method le Study3 Drugs Workbook 3 Drugs Workbook Iq Browse 2 Click Next Specify Calibration Standards Specify the type of standards you are using to calibrate Figure 112 To specify the standards 1 Select a standard e To use an internal standard select the Calibrate Using Internal Standards option e To use an external standard select the Calibrate Using External Standards option Figure 112 Internal or external standards page
223. e parameters Parameter Description Last used workbook The name of the most recently opened workbook Open Last Used Opens the most recently opened viewed workbook Workbook Open Existing Opens a dialog box where you can select any workbook Workbook Create a New Opens the New Study or Workbook Wizard that walks you Workbook through the steps to create a new LCquan workbook Most recently used Displays up to eight of the most recently opened workbooks Workbooks Summary Info Displays created modified saved by information about the highlighted workbook Hold the cursor over the workbook name without clicking to see the summary information Open in Review Mode Opens a workbook in read only mode 3 Click Create a New Workbook The Welcome page of the New Study or Workbook Wizard opens Figure 7 Figure 7 Welcome page of the New Study or Workbook Wizard New Study or Workbook Wizard x Welcome to the New Study or Workbook Setup Wizard LCauan 2 7 This wizard helps you set up a new Study or Workbook A New Era in Mass Spectrometry You can initialize a workbook by importing data from another workbook or from legacy files 4 Click Next Thermo Scientific LCquan User Guide 15 2 Preparing to Perform Quantitative Analysis Creating a New Study and Workbook Name the New Study or Workbook Name the new study or workbook see Table 3 To specify a name for the new study or workbook 1 Type th
224. e the x axis so that you can see baseline details Y Select to have the y axis offset move the x axis slightly to the right of the y axis so that you can see plot details at low x axis values Thermo Scientific 3 Exploring the Data Data Views Chromatogram List View Thermo Scientific The Chromatogram List view displays the chromatographic data points in tabular format Figure 95 Figure 95 Chromatogram List view T m Q ao Chromatogram List RT 0 01 4 00 ee ee ao 3 4 5o 6 B Bo g3 The contents of the grid are read only When the view is locked lock icon is yellow or red you cannot select any menu item in the shortcut menu that changes the raw file with the yellow lock or the raw file and parameters with the red lock Menu items that would cause changes are grayed out For more information about locks see Toolbar for Selectable Views on page 150 Table 56 lists the parameters for the Chromatogram List view Table 56 Chromatogram List view parameters Sheet 1 of 2 Parameter Description Arrows The left and right arrows move through the chromatogram incrementing or decrementing by a single scan Column Headings RT Displays the retention time of the given data point Intensity Displays the absolute intensity of the given data point Relative Intensity Displays the intensity of the data point relative to t
225. e type a Click the Unknowns tab in the Selected Files pane b To associate a data file with the Unknowns sample type click the raw file in the lower Available Files pane and click Add The LCquan application adds the selected raw file to the Unknowns sample type in the Selected Files pane Thermo Scientific LCquan User Guide 317 5 Creating a Processing Sequence Editing a Processing Sequence To watch an animation of these procedures Click the play button Note To watch this animation you must have Adobe Flash Player version 10 or later You can download the latest Adobe Flash Player from http get adobe com flash player To select raw files by using other methods e With the cursor drag raw files to the Selected Files pane e Hold down SHIFT or CTRL to select multiple data files before dragging or clicking Add Tip Use the sample type information in the file information pane at the bottom of the window to identify the data files 318 LCquan User Guide Thermo Scientific 5 Creating a Processing Sequence Editing a Processing Sequence Resolving Discrepancies in Level Names When you add samples to a sequence if the LCquan application detects a discrepancy in the level names one of these dialog boxes opens e The Standard or QC Level Names Association dialog box opens when the samples are associated with calibration or QC level names that do not match the level names of the workbook sequence Fo
226. e window to display a multi component chromatogram and experiment with peak detection and integration parameters to see how they affect the chromatogram In the Explore window you can also create quan components and export them to the processing method The Explore window of LCquan is similar to the Qual window of Processing Setup in Xcalibur From the Explore window you can do the following e Automatically generate quan components by using the peak list function Display the acquisition sequence history and select a sample from it e Import several sequences to review and select samples from any of the imported sequences Although you cannot edit sequences in the Explore window you can import them e Create methods that can later be applied to raw files in the Quantitate window e Use common tools to investigate acquired data and to produce a processing method e Perform actions that do not become part of the audit trail except for exporting quan components to the Quantitate window Thermo Scientific LCquan User Guide 113 3 Exploring the Data Creating Explore Methods Creating Explore Methods Use the Create view of the Explore window to develop explore methods by specifying peak integration and detection criteria Figure 68 Figure 68 Explore Create window El Thermo LCquan 3 Drugs Workbook File Zoom View Apps Options Help PAA SH A OCOSCSCOS L B O Identification m Chromatagram Definition ICIS
227. eak Integration area r Genesis Peak Integration Peak Detection Algorithm Genesis 7 Advanced S N Threshold 05 Valley Detection Enabled Constrain Peak Width Type a multiplier value in the S N Threshold box To detect unresolved peaks with the valley detection approximation method select the Valley Detection Enabled check box e To set the expected peak width parameter and control the minimum width that a peak is expected to have enter a multiplier value in the Expected Width sec box To constrain the peak width of a component during the peak integration of a chromatogram select the Constrain Peak Width check box e To specify the minimum above the baseline before integration is turned on or off enter a percent of the total peak height in the Peak Ht box e To constrain the peak width of an asymmetric chromatogram peak that has a tailing trace enter a peak integration multiplier in the Tailing Factor box To specify advanced component detection criteria click Advanced Use these advanced criteria if the standard detection criteria do not provide the expected results a To specify a peak signal to noise cutoff enter a Peak S N Cutoff value b To use a valley detection approximation method to detect unresolved peaks enter Rise Percentage value and a Valley S N value LCquan User Guide 205 4 Creating a Processing Method Specifying Method Identification 206 LCquan Use
228. ecific data stream Smoothing The number of points used for a moving mean filter to smooth the chromatogram Format whole numbers Range 1 to 15 odd numbers only Note To disable filtering set the parameter to 1 Trace Specifies the type of chromatogram e Mass Range Specify up to 50 mass ranges The masses are added together to form the chromatogram e TIC Full scan acquisition resulting in a Total Jon Current plot e Base Peak In a differential chromatogram the interpolation of the baseline is derived from the distance between the intersections of the tangents drawn to the peak sides and the peak base e Summed Uses ion summing to create a chromatogram that is the sum of the chromatograms of related compounds Ion summing can increase sensitivity because it sums up to five chromatograms of related masses for example water loss adducts isotopes or multiply charged peptides Note This definition set is the same for the second Trace box The second Trace box depends on which operators you select There is no second Trace box or trace operators for Trace Summed 188 LCquan User Guide Thermo Scientific 4 Creating a Processing Method Specifying Method Identification Table 71 Chromatogram Definition area parameters Sheet 2 of 2 Parameter Description Trace Mass Range TIC or Base Peak da Trace operator used to specify a trace operation This trace operation matrix shows the combina
229. ecifies the second mass value to define the mass range used for trace operator math Filter Specifies a scan filter to be applied to the acquired data Enter a filter or select a filter from a preloaded filter list obtained from the current raw file Note All filters are validated against the current set of filter entry rules Defining Peak Integration Parameters Use the Peak Integration area to select and define peak integration parameters Use the Peak Integration area to define Genesis ICIS and Avalon peak detection algorithms to be applied to the active raw file Peak detection parameters are also available from the Quantitate window e The Peak Integration area on the Identification page e The IRC Detection dialog box available from the ion ratio confirmation grid on the Identification page e The User Identification Settings dialog box available from the Chromatogram view in the preview panes This section contains instructions for specifying parameters for the following e Genesis Peak Integration e ICIS Peak Integration e Avalon Peak Integration Note e The Genesis peak detection algorithm has been provided for backward compatibility with Xcalibur 1 0 studies e The ICIS peak detection algorithm has been designed for MS data and has superior peak detection efficiency at low MS signal levels e The Avalon peak detection algorithm has been designed for UV data Avalon also supports negative peaks The
230. ect Instrument Method 3 Set the instrument settings that are appropriate for your experiment The Instrument Setup window might contain multiple views or dialog boxes 4 For each instrument that you want to set up repeat steps 1 through 3 5 When you want to create an optional startup method that the LCquan application will run before it runs the first sample in an acquisition do the following a Select the appropriate instrument by clicking its icon in the navigation pane b Click Startup Method c Specify the startup settings for the instrument 6 When you want to create an optional shutdown method that the LCquan application will run after it runs the last sample in an acquisition do the following a Select the appropriate instrument by clicking its icon in the navigation pane b Click Shutdown Method c Specify the shutdown settings for the instrument When you create or modify an instrument method the LCquan application automatically saves it when you leave the Instrument Setup window The file name is based on the workbook name for example 3 Drugs Workbook meth After you save a method in the workbook it remains part of the workbook even if the configured instrument is removed Thermo Scientific LCquan User Guide 29 2 Preparing to Perform Quantitative Analysis Introduction to the Acquisition Window Importing an Existing Instrument Method Instead of creating a new method you can import an existing ins
231. ection Selection gt Quantitate Section and then choose View gt Step Selector gt Quantitate Sequence The New Processing Sequence Wizard opens Figure 170 Figure 170 Welcome page for the New Processing Sequence Wizard New Processing Sequence Wizard x Welcome to the New Processing Sequence Wizard LCauan 27 This wizard helps you set up the Processing A New Era in Mass Spectrometry Sequence 2 Click Next Create or Import a Sequence Create a new processing sequence or import an existing sequence file see Figure 171 and Table 118 To create a processing sequence 1 Do one of the following e Select the Create New Sequence option e Select the Import Existing Sequence from File option Figure 171 Create or import sequence page Create new sequence C Import existing sequence from file Import from acquisition sequence 296 LCquan User Guide Thermo Scientific 5 Creating a Processing Sequence Defining a Processing Sequence Table 118 Create or import sequence page parameters Option Description Condition Create New Sequence Select this to specify that you want to create a new processing sequence Import Existing Select this to specify that you want to import an existing Sequence from File processing sequence Import from Select this to create default processing sequence components Acquisition Sequence with the names from the acquisition sequence component list and import the sequence defin
232. ed in the acquisition setup Note This option is enabled only if the acquisition sequence exists 2 Click Next e Ifyou selected the Create New Sequence option go to Complete the Wizard e Ifyou selected the Import Existing Sequence from File option go to Import a Sequence File Import a Sequence File Import a saved sequence file To import a sequence file 1 Type the path for the sequence file or click Browse and find a file Figure 172 Figure 172 Sequence file path page Sequence File C Xcalibu G uanRoot Example Study 3 Drugs Workbook 3 2 Click Next Complete the Wizard On the final Wizard page click Finish to exit the New Processing Sequence Wizard and apply all your settings Note If you receive a warning about missing or mismatched level names see Resolving Discrepancies in Level Names on page 319 When you complete the New Processing Sequence Wizard the Create Sequence create view in the Quantitate window opens You are now ready to begin refining your processing sequence Thermo Scientific LCquan User Guide 297 5 Creating a Processing Sequence Defining a Processing Sequence Copying an Acquisition Sequence Usually instead of creating a processing sequence it is more convenient to use your acquisition sequence as your processing sequence You can do this in either of two ways e Copying from the Acquisition Sequence History e Copy from the Acquisition Sequence Copyin
233. el LCquan User Guide 45 2 Preparing to Perform Quantitative Analysis Using the Setup Sequence View Add Quality Controls Add quality control samples to the sequence see able 18 To add quality controls 1 Select the Add QCs check box Figure 36 Figure 36 Add Quality Controls page MV Add OCs C Based on existing OC levels from method Based on automatically generated OC levels Number of QC levels 1 OC level base name fa C_ MV Add Blanks IV Fillin Sample ID for OCs 2 Select either the Based on Existing QC Levels from Method or Based on Automatically Generated QC Levels option 3 Optional Select the Add Blanks option 4 Optional Select the Fill in Sample ID for QCs option When your sequence contains QC samples the order used is e Optional Blank samples e First half of the calibration Standard samples e Optional Blank samples QC samples e Optional Blank samples e Unknown samples e Optional Blank samples e Second half of the calibration Standard samples e Optional Blank samples 5 Click Next 46 LCquan User Guide Thermo Scientific 2 Preparing to Perform Quantitative Analysis Using the Setup Sequence View Table 18 Add Quality Controls page parameters Parameter Description Add QCs Adds quality control samples QCs to your acquisition sequence Based on Existing QC Select this option to use QC levels from an existing processing Levels from Method method Based on Automatically Gen
234. elect and define peak integration parameters Use the Peak Integration area to define Genesis ICIS and Avalon peak detection algorithms to be applied to the active raw file The Peak Integration area is dynamic displaying criteria for the selected peak detection method Figure 117 Genesis ICIS or Avalon Figure 117 Peak Detection Algorithm list Peak Detection Algorithm Genesis Ez Genesis ICIS Avalon The parameters for each of these peak integration methods is described in detail in Defining Peak Integration Parameters on page 204 192 LCquan User Guide Thermo Scientific 4 Creating a Processing Method Specifying Method Identification Peak Identification Area Thermo Scientific Use the Peak Identification area to specify criteria for identifying peaks in other raw files of the data set that correspond to the current component Table 74 lists the parameters for the Peak Identification area Figure 118 Peak Identification area Peak Identification C Highest Peak Min Peak Height S N Gi 3 0 Table 74 Peak Identification area parameters Parameter Description Highest Peak Specifies that the highest most intense peak in the spectrum is used as the component identification criterion Nearest RT Specifies that the peak with the retention time in the chromatogram that is closest to the expected retention time is used as the component identification criterion Min Peak Height S N For
235. eline level Valley V edge type The edge of the peak is in a peak valley Manual M edge type The edge of the peak has been adjusted manually Stripe S edge type The edge of the peak reached the Constrain Peak Height Percent specified in the method Tail T The edge of the peak reached the Constrain Peak Height Trailing Factor limit before the Height Percent Tilt An error occurred before the edge of the peak could be determined Unknown An unknown error occurred Valid Indicates whether the peak was successfully detected The LCquan application indicates the peak was detected by displaying the check mark in the Valid check box Right Edge Type The criteria that the LCquan application uses to detect the right edge of a peak The following edge types are defined Baseline B edge type The edge of the peak is at baseline level Valley V edge type The edge of the peak is in a peak valley Manual M edge type The edge of the peak has been adjusted manually Stripe S edge type The edge of the peak reached the Constrain Peak Height Percent specified in the method Tail T The edge of the peak reached the Constrain Peak Height Trailing Factor limit before the Height Percent Tilt An error occurred before the edge of the peak could be determined Unknown An unknown error occurred LCquan User Guide 371 6 Processing the raw Files and Reviewing the Analytical Resul
236. en without affecting the baseline Range 1 to 500 Default multiplier 5 Peak Noise Factor The noise level multiplier a minimum S N ratio used to determine the potential peak signal threshold Range 1 to 1000 Default multiplier 10 Constrain Peak Width Constrains the peak width of a component during the peak integration of a chromatogram You can set values that control when peak integration is turned on and off by specifying a peak height threshold and a tailing factor Selecting this activates the Peak Ht and the Tailing Factor parameters LCquan User Guide 211 4 Creating a Processing Method Specifying Method Identification Table 79 ICIS Peak Integration area parameters Sheet 2 of 2 Parameter Description Peak Ht A percent of the total peak height This is the minimum that a signal must be above the baseline before integration is turned on or off Range 0 to 100 Default multiplier 5 0 The peak height percentage is defined as follows 100 Peak Height Note Valid only when you select the Constrain Peak Width check box Tailing Factor A peak integration multiplier that constrains the peak width of an asymmetric chromatogram peak that has a tailing trace It is the maximum ratio of the trailing edge to the leading side of a constrained peak and calculates the retention time of the maximum extent of the right edge of the tailing peak Range 0 5 to 9 0 Default multiplier 1 0
237. ence View 58 LCquan User Guide Table 22 Sort Sequence dialog box parameters Parameter Sorting Options Description The following sorting options are applicable to all three sort orders File Name Sorts the list so the file names are in alphabetical order Level Name Sorts the list so the calibration standard and QC level names are in alphabetical order Sample ID Sorts the list so the sample IDs are in alphabetical or numerical order Sample Type Sample types are not sorted alphabetically In ascending order the types are Standards QCs Blanks and Unknowns First Order Specifies the first order of sorting for the sequence For the LCquan application the default first order of sorting is by sample type Second Order Specifies the second order of sorting for the sequence Third Order Specifies the third order of sorting for the sequence Sort in Descending Order The LCquan application sorts the list in descending n tol order Clear this option to have the LCquan application sort the list in ascending order Thermo Scientific Thermo Scientific 2 Preparing to Perform Quantitative Analysis Using the Setup Sequence View Columns Use the Columns command to customize the column arrangement see Table 23 1M To customize column arrangement 1 Choose Columns to open the Column Arrangement dialog box opens Figure 42 Figure 42 Column Arrangement dialog box Column Arrangement
238. ent e The User Label 3 default name is Laboratory e The User Label 4 default name is Company Name e The User Label 5 default name is Phone Comment Displays comments about the sample selected from the acquisition sequence Note By default the dialog box is pinned in place you see the pin icon in the upper left corner of the dialog box meaning the dialog box stays open when you select a new sample The content of the dialog box fields is updated for the new sample Acquisition Status Acquisition History The Acquisition History page in the Status view of the Acquisition window displays a history of all sequences and samples that have been acquired by the current workbook along with other relevant information such as the operator start and end times status and comment Table 34 lists the parameters for the Acquisition History tab Thermo Scientific LCquan User Guide 101 2 Preparing to Perform Quantitative Analysis Using the Status View 102 LCquan User Guide 1M Kod To open the Status Acquisition History page When the left navigation pane is displayed a Click Acquisition b Click the Status icon c Click the Acquisition History tab When the left navigation pane is hidden a Choose View gt Section Selection gt Acquisition Section b Choose View gt Step Selector gt Acquisition Status c Click the Acquisition History tab Figure 59 Figure 59 Acquisitio
239. erates names for the QC levels The derived names are a Generated QC Levels concatenate of a base name and an incremental number for each level For example if the base name is QC and the number of levels is 3 QC levels are named QC1 QC2 and QC3 e Number of QC Levels QC Level Base Name Add Blanks Adds quality control QC blanks to your sequence The LCquan application places one blank after each series of quality control samples in the new sequence Fill in Sample ID for The LCquan application automatically fills in the quality control QCs QC sample ID in the new sequence This information is defined in the processing method for each quality control level Specify Components Names Name the components used in the sequence see Table 19 To name the components 1 Select the Use Processing Method Component Names check box Figure 37 Figure 37 Processing method component names page l Use processing method component names Number of components 4 Component base name Component 2 Type the number of component names you want to generate 3 Type component base name 4 Click Next Thermo Scientific LCquan User Guide 47 2 Preparing to Perform Quantitative Analysis Using the Setup Sequence View Specify User Labels 48 LCquan User Guide Table 19 Component names parameters Parameter Description Use Processing Method Use the component names from an existing processing method Component Names
240. ers Parameter Description Import Instrument Enables the import options Method Acquisition Sequence and Processing Parameters Import from existing Imports the instrument method acquisition sequence or workbook processing methods for a particular workbook Import legacy files Imports existing instrument methods acquisition sequences or processing methods that reside in different workbooks or Imports methods and sequences not created by the LCquan application 3 Click Next Depending on the options you chose do one of the following e When you are not importing files the final page of the wizard opens Go to Complete the Wizard e To import files from an existing workbook go to Import Files From Workbook e To import legacy files go to Import Legacy Files Import Files From Workbook Import existing instrument methods acquisition sequences or processing methods from existing workbooks To import files from an existing workbook 1 Click Browse to find the workbook whose files you want to copy Figure 14 Figure 14 Existing workbook path page Path to existing workbook C calibur QuanRoot Example Study3 Browse 2 Click Next and go to Specify the Processing Sequence 20 LCquan User Guide Thermo Scientific 2 Preparing to Perform Quantitative Analysis Creating a New Study and Workbook Import Legacy Files Import existing instrument methods acquisition sequences or processing
241. ertical line and the baseline defines the end of the first peak and the beginning of the second peak When the trace exceeds rise percentage the LCquan application applies valley detection peak integration criteria This test is applied to both the left and right edges of the peak The rise percentage criteria is useful for integrating peaks with long tails Range 0 1 to 500 0 Valley S N Specifies a value to evaluate the valley bottom Using this parameter ensures that the surrounding measurements are higher Range 1 0 to 100 0 Default 2 0 Report Noise As RMS or Determines if the noise used in calculating S N values is calculated Peak to Peak using an RMS calculation or peak to peak resolution threshold Thermo Scientific 4 Creating a Processing Method Specifying Method Identification ICIS Peak Integration Use the ICIS Peak Integration area to define ICIS peak detection algorithms to be applied to the active raw file Table 79 lists the parameters for the ICIS Peak Integration area To specify ICIS peak integration parameters 1 In the Peak Detection Algorithm box select ICIS Figure 128 Figure 128 ICIS Peak Integration area ICIS Peak Integration Peak Detection Algorithm ICIS Advanced Baseline Window 20 Area Noise Factor 5 Peak Noise Factor fi 0 I Constrain Peak Width 2 In the Baseline Window box enter the number of scans to use when searching for where the minima are o
242. es e mae Recent Places Desktop Libraries iy Computer Da Es Network File name APN_059 RAW APN_001 RAW APN_0027 Riles of type Raw Files raw Ba Cancel 18 LCquan User Guide Thermo Scientific 2 Preparing to Perform Quantitative Analysis Creating a New Study and Workbook 5 To return to the wizard click Open The wizard displays the selected raw files Figure 12 Figure 12 Import raw files page J Import rawfiles File location C lt calibur QuanRoot Example Study Rawfiles File name A APN_059 raw 2 2 2007 8 07 04 PM z APN_OO1 raw 2 2 2007 8 07 00 PM APN_002 raw 2 2 2007 8 07 00 PM APN_003 raw 2 2 2007 9 07 00 PM v gt Number of rawfiles selected for import 59 Browse 6 Click Next Import Data Import existing instrument methods acquisition sequences or processing methods see Table 5 1M To import methods or sequences 1 Select the Import Instrument Method Acquisition Sequence and Processing Parameters check box Figure 13 2 Select either the Import from Existing Workbook or the Import Legacy Files option Figure 13 Import data page V Import Instrument Method Acquisition Sequence and Processing Parameters Import from existing workbook C Import legacy files Thermo Scientific LCquan User Guide 19 2 Preparing to Perform Quantitative Analysis Creating a New Study and Workbook Table 5 Import data page paramet
243. estarts at the beginning of the list until stopped manually Switching panes also stops the timed display Item Selector List Options All Samples for Selected Component Select this option so chromatographic peaks for each sample in the given tab All Standards QCs Blanks and Unknowns are produced and displayed in the grid like arrangement All Components for Select this option so that chromatographic peaks for each Selected Sample component for the selected sample are produced and displayed in the grid like arrangement Shortcut Menu Lock against All Prevents changes to a raw file or parameters The lock icon is red Change and the lock is closed Unlock Makes the pane editable The lock icon is green and the lock is open Use User Defined Resets the time scale to the user defined range as entered in the Time Range Realtime Display Settings dialog box For more information see Realtime Display Settings on page 84 in Chapter 2 Preparing to Perform Quantitative Analysis Options Opens the Multi Peak Options dialog box For more information see Multi Peak Plot parameters on page 291 LCquan User Guide 291 4 Creating a Processing Method Data Views Table 117 Multi Peak Plot parameters Sheet 2 of 2 Parameter Description Reset Scaling Resets x and y axes to show the maximum amount of data Copy to Clipboard Copies a chromatogram mass spectrum or calibration curve to the
244. et Ratio Specifies the ratio of the qualifier ion response to the quan ion response Range 0 to 200 Window 194 LCquan User Guide Specifies the amount as a percentage that the measured ratio can vary from the target window value for the ion to still be considered confirmed Thermo Scientific Thermo Scientific 4 Creating a Processing Method Specifying Method Identification Table 75 lon Ratio Confirmation area parameters Sheet 2 of 2 Parameter Qualifier Ion Coelution Shortcut Menu Delete Selected Rows Description Specifies the time the retention time can vary from the expected retention time for the ion to still be considered confirmed Range 0 000 to 0 100 Units minutes Deletes all currently selected rows You must select entire rows by clicking the leftmost column before you can delete them Edit Selected Rows Processing Info Opens the IRC Detection Method dialog box where you can change IRC peak detection settings The settings for each IRC are independent When multiple rows are selected when you choose this command the first IRC in the list is selected for editing For detailed information about IRC peak detection settings see IRC Detection Method on page 217 Window Relative Uses the target ratio tolerances in the Window column as relative percentages of the target ratio Absolute Uses the target ratio tolerances in the Window column as a
245. ete Selected Rows from the shortcut menu Figure 221 XReport report selections page Report Report Selections iv CAX caliburitemplates LC quan LC quanQu M oi CAXcaliburitemplates LC quan LC quanPea H C Table 149 lists the parameters for the XReport report selections page Table 149 XReport report selections page parameters Sheet 1 of 2 Parameter Description Use Specifies which reports are created when you click Create Reports Select the check box of each report you want to generate Note If your administrator has set up secure reporting you can only access the templates in the secure templates folder LCquan User Guide 381 6 Processing the raw Files and Reviewing the Analytical Results Creating and Reviewing Reports Table 149 XReport report selections page parameters Sheet 2 of 2 Parameter Description Save Type Specifies the report file type which includes these options e txt plain text e doc Microsoft Word Document e html HTML pdf PDF e rtf Rich Text Format e xls Microsoft Excel spreadsheet Note If your administrator has set up secure reporting you can only select the PDF option XReport Template Specifies the XReport template to use in generating the report Click the empty cell and click to browse for the template file Note If your administrator has set up secure reporting you can only access the templates in the secure templates folder Launch XRe
246. etermine how the peaks are arranged and to change chromatographic display options Figure 108 Table 69 lists the parameters for the Multi Peak Options dialog box Figure 108 Multi Peak Options dialog box Multi Peak Options Arrangement Intensity scale Number of display columns Normalize to largest peak Number of display rows 5 C Use fixed intensity range fi 0 Playback Time scale Playback interval secs 15 0 J Use user defined time range V Repeat playback f Cancel Help Table 69 Multi Peak Options dialog box parameters Parameter Description Arrangement Range 1 to 50 Note The product of columns and rows must be equal to or less than 50 Number of Display Number of peaks displayed left to right Columns Number of Display Number of peaks displayed top to bottom Rows Playback Playback options are unavailable in the Explore window Intensity Scale Normalize to Largest Overrides the settings defined in the Display Options dialog box Peak This option disables the fixed intensity range edit box Use Fxed Intensity Overrides the settings defined in the Display Options dialog box Range This option disables the fixed intensity range edit box Time Scale Use User Defined Time Select to enter a user specified time range to use for all plots Range Time Range mins Sets the time base in minutes for all plots 180 LCquan User Guide Thermo Scientific ee i Creating a Pr
247. events that detect peaks in the data Any timed event in the event list is unchanged when you click Auto Calculate Determines initial values for only Start Threshold End Threshold Area Threshold P P Resolution Threshold Bunch Factor Negative Peaks and Tension Note Valid only if a raw file is open Time Min Initial value or a time value in minutes Event Descriptions of detection parameters for initial events and timed events Value Values associated with the initial timed events described in the Event box The range of factors allowed for each value is specific to each event Buttons Add Adds the Time Event and Value values to the Avalon Event List Delete Deletes the selected row Change Using the Time Event and Value values updates the event list and the chromatogram display LCquan User Guide 215 4 Creating a Processing Method Specifying Method Identification Table 82 Initial and Timed Events Sheet 1 of 2 Event Start End Threshold Description Half of the result is a good estimate for the Start Threshold You can modify the Avalon estimates by entering your own values and clicking Add to save your Start or End Threshold The Start Threshold depends on the RMS noise in the chromatogram It is the fundamental control used for peak detection so picking the best Start Threshold is essential for high quality data collection Units Absolute valu
248. eviewing the Calibration Curve and OCs User Identification Settings Avalon Integration Use this page Figure 208 to specify advanced component detection criteria Use these additional criteria when the standard detection criteria do not provide the expected results Table 140 lists the parameters for the Identification page of the User Identification Settings dialog box Note To detect peaks Avalon uses the settings for initial events and user defined timed events in the event list Figure 208 User Identification Settings dialog box showing the Avalon Integration page User Identification Settings Identification Detection Avalon Integration Auto Calculate Initial Events Initial Start Threshold 10000 000 Initial End Threshold 10000 000 Initial Area Threshold 10000 000 Initial P P Threshold 1 000 Initial Bunch Factor 1 000 Initial Negative Peaks Off Tension Time Min Event Value 0 000 Tension 7 1 000 Add Delete Change Cancel Apply Apply Toan Help Table 140 User Identification Settings dialog box Avalon Integration page parameters Parameter Description Auto Calculate Initial Estimates the initial values for the detection of peaks based on Events the data in the current raw file to display initial values in the event list and searches for the best values of initial events that detect peaks in the data Any timed event in the event list is unchanged when you click Auto
249. ew views let you display your data in any of the following views e Chromatogram View e Chromatogram List View e Error Report View e Filter List View e General Parameters Plot View e Instrument Method View e Mass List View e Peak List View e Sample Info View e Spectrum View e Status Plot View e Status Report View e Tune Method View e Multi Peak Plot View for Review Only Each preview pane contains a Toolbar for Selectable Views at the top of the pane Toolbar for Selectable Views 150 Use the Toolbar for Selectable Views Figure 92 to select the view you want to display in the preview pane Not all views display all the toolbar options At a minimum the toolbar contains the View Selector list and a lock icon Figure 92 Toolbar for Selectable Views gt 83 Arrows Lock LCquan User Guide Peak Width General Parz Item Selector View Selector Thermo Scientific 3 Exploring the Data Data Views Table 53 lists the controls for the Toolbar for Selectable Views Table 53 Toolbar for Selectable Views controls Sheet 1 of 3 Control Description Arrows Moves forward or backward through a range of possible selections 4 gt for example to display the next or previous chromatogram peak The arrows are not selectable grayed out when either of these cases exists e A particular directional move is not possible for example the arrows move through a list
250. ew you want to display in the preview pane Not all views display all the toolbar options At a minimum the toolbar contains the View Selector list and a lock icon Figure 148 Toolbar for Selectable Views 4 gt Peak Width v General Par Arrows Item Selector View Selector Lock Thermo Scientific 4 Creating a Processing Method Data Views Table 98 lists the controls for the Toolbar for Selectable Views Table 98 Toolbar for Selectable Views controls Sheet 1 of 3 Control Description Arrows Moves forward or backward through a range of possible selections 4 gt for example to display the next or previous chromatogram peak The arrows are not selectable grayed out when either of these cases exists e A particular directional move is not possible for example the arrows move through a list and the current selection is at the top of the list so the left backward arrow is grayed out e The yellow or red lock is set so no changes can be made in the red locked pane and only changes other than raw file or component changes can be made in yellow locked panes Both arrows are grayed out Note The actions of the arrows vary in some instances between the Explore and Quantitate windows For example in the Chromatogram pane in Explore the arrows can move through multiple peaks for one sample In the same pane in Quantitate they can move through different samples Lock Allow changes or prevent
251. ext g Sequence wizard Survey standards and QCs Click Survey in viewbar Survey page Thermo Scientific Review all results Click Review All in viewbar Review All Results page Click Reports in viewbar Review Reports page LCquan User Guide 415 es i LCquan Menu and Toolbar Reference This appendix provides a reference to the LCquan menus icon bars and toolbars Contents e Using the Menus e Actions Menu for Acquisition Window Only e Apps Menu e Change Menu for Acquisition Window Only e File Menu e Help Menu e Options Menu e View Menu e Zoom Menu e Icon Bars Toolbar Using the Menus To use the keyboard to activate a command 1 Press the ALT or F10 key to activate the menu bar 2 Type the character underlined in the menu title such as F in File 3 Type the character underlined in the command name such as N in New If you do not select a command you can close the menu by pressing the ESC key Thermo Scientific LCquan User Guide 417 C LCquan Menu and Toolbar Reference Actions Menu for Acquisition Window Only Actions Menu for Acquisition Window Only Table 154 Actions menu commands of the Acquisition window Use this menu to check disk space and start stop and pause data acquisition see Table 154 Command Description Keyboard shortcut Check Disk Space Opens the Disk Space dialog box where you can determinehow ALT A gt D
252. f the grid are read only When the view is locked lock icon is yellow or red you cannot select any menu item in the shortcut menu that changes the raw file with the yellow lock or the raw file and parameters with the red lock Menu items that would cause changes are grayed out For more information about locks see Toolbar for Selectable Views on page 150 Table 61 lists the parameters for the Chromatogram List view Table 61 Mass List view parameters Sheet 1 of 2 Parameter Description Arrows The left and right arrows move through the chromatogram incrementing or decrementing by a single scan Column Headings Mass Displays the mass value above the spectral line column Intensity Displays the intensity of the spectral data point column Base Displays the relative percentage this data point has to the largest data point in the Intensity column Thermo Scientific 3 Exploring the Data Data Views Table 61 Mass List view parameters Sheet 2 of 2 Parameter Description Shortcut Menu Open Raw File Opens the Select Raw File dialog box where you can select the raw file to load Lock Against Raw File Prevents changes to a raw file The lock icon is yellow and the Change lock is closed Lock against All Prevents changes to a raw file or parameters The lock icon is red Change and the lock is closed Unlock Makes the pane editable The lock icon is green and the lock is open Copy
253. f the peak cluster Thermo Scientific 4 Creating a Processing Method Specifying Method Identification Table 82 Initial and Timed Events Sheet 2 of 2 Event Description Integrate On Off Turns integration on or off at the set time Shoulders On Turns on the detection of shoulders Shoulders Off Turns off the detection of shoulders Force Cluster On Turns on the grouping of peaks into a single peak Force Cluster Off Turns off the grouping of peaks into a single peak Disable Cluster On Enables the grouping effect in the specified time range Disable Cluster Off Disables the grouping effect in the specified time range IRC Detection Method Use the IRC Detection Method dialog box to specify additional peak detection criteria when the standard detection criteria on the Identification page do not provide the expected results Peak detection parameters are also available from these locations e The Peak Integration area in the Create Method view of the Explore window e The Peak Integration area on the Identification page in the Create Method view of the Quantitate window e The User Identification Settings dialog box available from the Chromatogram view in the preview panes To open the IRC Detection Method dialog box Figure 131 right click the Ion ratio confirmation grid and choose Edit Selected Rows Processing Info from the shortcut menu Figure 131 lon Ratio Confirmation area lon ratio confirmation V En
254. f the target component in the samples The precision of the measurement depends on the quality and to a lesser extent the quantity of the calibration data The detection limit of the quantitative analysis method is the lowest concentration of analyte in a sample that can be detected but not necessarily quantitated as an exact value The lower and upper quantification limits are the lowest and highest concentrations of analytes in a sample that can be measured with an acceptable level of accuracy and precision In an analytical method the highest concentration calibration standard defines the upper quantification limit The quantitative analysis technique you use determines the manner in which the response is calculated both for generating the calibration curves and for subsequent quantitative analysis The two basic methods are described in these topics e Using External Standards for Quantitative Analysis e Using Internal Standards for Quantitative Analysis Thermo Scientific LCquan User Guide 405 A Quantitative Analysis Overview Quantitative Analysis Techniques Using External Standards for Quantitative Analysis An external standard is a separate sample that contains the compound of interest at a known concentration in solution In the quantitative analysis that uses external standards a series of standards are analyzed and a calibration curve is constructed by plotting the magnitude of the detector response as a function of the exter
255. f the target compound used for each QC quality control level Test Displays a value for the acceptable difference as a percentage between the known amount and calculated measured amount of each QC level Shortcut Menu Copy Current Component Levels to All Target Components Copies the current Calibration Levels and QC Levels tables to all other target components Load Default Levels for Current Component Import Processing Components Loads the default level tables for the current component Imports the component names and levels tables from the processing method Functions exactly the same as the Import Processing Components button Buttons Import Processing Components Imports the component names and levels tables from the processing method Functions exactly the same as the Import Processing Components menu command Load Default Levels 64 LCquan User Guide Loads the default level tables for the current component replacing the current levels with the defaults stored in the registry The defaults are set on the calibration page as described in Specifying Method Calibration on page 235 and in Chapter 4 Creating a Processing Method Thermo Scientific 2 Preparing to Perform Quantitative Analysis Using the Setup Sequence View Get Injection Volume from AS Use the Get Injection Volume from AS command to use an autosampler injection volume To use an autosampler inje
256. figured for operation as LCquan devices Shows In Use status in the Change Instruments In Use dialog box in a read only list Start Instrument This read only list can have either one Yes in one of the instrument rows or all blanks in all instrument rows no Yes entries Start When Ready 74 LCquan User Guide Automatically begins the LCquan application performs an autosampler injection acquisition when all devices are ready When you clear this check box the sequence waits until all devices are ready and pauses so you can check device statuses To continue the acquisition click Start in the Status view of the Acquisition window See Using the Status View on page 80 This ability to pause is useful if any external equipment is in use that cannot return status to the LCquan application This feature applies only to the first sequence row Thermo Scientific 2 Preparing to Perform Quantitative Analysis Using the Run Sequence Dialog Box Table 26 Run Sequence dialog box parameters Sheet 2 of 4 Parameter Description Change Instruments Opens the Change Instruments In Use dialog box where you can change the status of instruments in use or select a different start instrument Processing Actions Process After Specifies that the LCquan application runs the processing Acquisition sequence at the end of data acquisition Generate Selected Specifies that the LCquan application creates report
257. file or parameters The lock icon is red Change and the lock is closed Unlock Makes the pane editable The lock icon is green and the lock is open Reset Scaling Resets scaling to the default pane Copy to Clipboard Copies selected data to the Clipboard You can then paste the data from the Clipboard buffer into a document LCquan User Guide 277 4 Creating a Processing Method Data Views Instrument Method List View This view displays the instrument method parameters for the selected sample Figure 160 When you select Instrument Method List an instrument selector list appears in the toolbar of the preview pane To view an instrument s method select the instrument from the instrument selector list When you select a TSQ Quantum the LCquan application appends the source parameters from the tune method to the end of the instrument method parameters Figure 160 Instrument Method List view gt a TSQ Quantum v Instrument Method List v APN_003 Creator Quantum Last modified 11 16 2005 by Quantum MS Run Time min 4 00 a TSQ MS Method Settings Segment 1 2 Scan Events 1 1 2 Segment 1 Tune Method C Xcalibur methods AlprazolamMfaurizioMethod TSQTune Chrom filter 5 lt Note Use the arrows 4 gt to change the preview to display the parameters for the other types of instrument methods associated with the current sample raw file To display the shortcut menu for the Inst
258. flash player LCquan User Guide 249 4 Creating a Processing Method Saving the Processing Method Reviewing the Integrated Peaks When you have finished entering the component identification and integration settings for the target compounds and the internal standard review the integrated peaks To review the integrated peaks e When the peaks have been correctly integrated go to Ion Ratio Confirmation of Analytes Example on page 234 e When there is excessive noise in a peak unresolved peaks or peak tailing return to Summing Ions Dialog Box on page 196 and change the peak identification and integration parameters e When baseline noise is interfering with peak identification or integration change the settings in the Advanced Parameters dialog box for the appropriate peak detection method For details about displaying and viewing data in the preview panes see Data Views on page 251 Saving the Processing Method When you have finished creating the processing method choose File gt Save to save the settings in the workbook Importing a Processing Method When you are doing routine quantitative analysis experiments you might save time by importing an existing processing method into your current workbook instead of creating a new method You can use the method as is or you can edit it Use any of these options to import a processing method e New Study Wizard When you create a new workbook the
259. form Quantitative Analysis Layout Configuration gt Applies the factory defined view layouts and column arrangement ALT F gt L gt Restore Factory Layout information to all windows RIGHT ARROW Configuration Layout Configuration gt Loads a view layout and column arrangement configuration file ALT F gt L gt Import Layout and applies it to the current workbook RIGHT ARROW gt Configuration I Layout Configuration gt Saves the view layout and column arrangement configuration ALT F gt L gt Export Layout information to a file in the Export folder with a user supplied RIGHT ARROW gt Configuration name This data is file tracked and an event is made stating that E the file was exported Summary Info Opens the Workbook Summary Information dialog box that you ALT F gt U 420 LCquan User Guide can use to obtain information and provide reference information about the current workbook For more information see Workbook Summary Info dialog box parameters on page 27 in Chapter 2 Preparing to Perform Quantitative Analysis Thermo Scientific Command C LCquan Menu and Toolbar Reference File Menu Table 157 File menu commands common to all LCquan windows Sheet 2 of 2 Description Keyboard shortcut toolbar icon Opens the Audit Viewer utility which is shared by other ALT F gt T Audit Trail Xcalibur based applications Audit Viewer provides a chronological listing of user initiated events When you start Audit Viewer
260. fset Forepressure QO RF Power Q0 RF Voltage QOO Offset Lens 0 Offset Q0 Offset Lens 11 Offset Lens 12 Offset Q1 Rod Driver Voltage Q1 Rod Driver voltage i Q1 RF Detected Q1 RF Power Q1 Amplifier Temperature Q1 Thermal Hat Temperatu Lens 21 Offset Ai E B6 C CE N nN pzs When the view is locked lock icon is yellow or red you cannot select any menu item in the shortcut menu that changes the raw file with the yellow lock or the raw file and parameters with the red lock Menu items that would cause changes are grayed out For more information about locks see Toolbar for Selectable Views on page 150 Table 66 lists the parameters for the Status Report view Table 66 Status Report view parameters Sheet 1 of 2 Parameter Description The left and right arrows move through the status item list incrementing or decrementing by a single status item Arrows Detector Selector List Options MS Displays status information for a mass spectrometer Status Displays status information for a device that does not log data Thermo Scientific Thermo Scientific 3 Exploring the Data Data Views Table 66 Status Report view parameters Sheet 2 of 2 Parameter Detector Streams in Current Raw File Description When a raw file is open displays the list of detector streams that are stored in the current raw file The format of the list items is Generic Detector
261. fset Q0 Offset Lens 11 Offset Lens 12 Offset Q1 Rod Driver Voltage Q1 Rod Driver Voltage Q1 RF Detected Q1 RF Power Q1 Amplifier Temperature a Thermal Hat Temperatu Lens 21 Offset ia 3 S e C CE be I The contents of the grid are read only When the view is locked lock icon is red you cannot select any menu item in the shortcut menu that changes the raw file Menu items that would cause changes are grayed out For more information about locks see Toolbar for Selectable Views on page 252 Table 115 lists the parameters for the Status Report view Table 115 Status Report view parameters Sheet 1 of 2 Parameter Description Arrows The left and right arrows move through the status items in the raw file incrementing or decrementing by a single status item Detector Selector List Options MS Displays status information for a mass spectrometer Status Displays status information for a device that does not log data Thermo Scientific LCquan User Guide 287 4 Creating a Processing Method Data Views Table 115 Status Report view parameters Sheet 2 of 2 Parameter Description Detector Streams in When a raw file is open displays the list of detector streams that Current Raw File are stored in the current raw file The format of the list items is Generic Detector Stream Name Instrument Name Model Name Channel Name Column Headings
262. g box Avalon Integration page parameters Sheet 2 of 2 Parameter Description Time Min Displays the initial time value in minutes Event Displays descriptions of detection parameters for initial events and timed events For details see Table 89 Note Start Threshold End Threshold Area Threshold P P Resolution Threshold Bunch Factor Negative Peaks and Tension are defined with initial values Value Displays the values associated with initial events or timed events Range Factors are specific to each event Add Adds the Time Event and Value values to the Avalon Event List Delete Deletes the selected row Change Using the Time Event and Value values updates the event list and the chromatogram display Table 89 Initial and Timed Events Sheet 1 of 2 Event Start End Threshold Description Half of the result is a good estimate for the Start Threshold You can modify the Avalon estimates by entering your own values and clicking Add to save your Start or End Threshold The Start Threshold depends on the RMS noise in the chromatogram It is the fundamental control used for peak detection so picking the best Start Threshold is essential for high quality data collection Units Absolute value of peak area counts x seconds Bunch Factor The number of points grouped together during peak detection This method groups several chromatographic points during integration without affecting the final area calcu
263. g from the Acquisition Sequence History Use the acquisition sequence history list on the Create Sequence edit view to copy sequences to your sequence grid When you have already acquired data in the workbook the acquisition sequence or sequences that you used are listed in the acquisition sequence history list To copy an acquisition sequence select it from the list and drag it into the processing sequence grid To select multiple sequences hold down the SHIFT or CTRL keys The LCquan application will ask if you want to replace the current sequence and if you want to overwrite the levels for matching component names Copy from the Acquisition Sequence When an acquisition sequence already exists in the current workbook you can use it to define the processing sequence in the following ways e From the File gt Import menu Choose Processing Sequence gt Copy From Acquisition Sequence to copy the acquisition sequence from the current workbook Choose Processing Sequence gt Copy From Last Acquired Sequence to copy the last acquired sequence e Right click anywhere in the Create Sequence edit view and use the commands on the shortcut menu Choose Import Processing Sequence gt Copy From Acquisition Sequence to copy the acquisition sequence from the current workbook Choose Import Processing Sequence gt Copy From Last Acquired Sequence to copy the last acquired sequence 298 LCquan User Guide Thermo Scien
264. ge IRC Detection Method Identification Genesis Integration Genesis Advanced m Report Noise 4s C RMS Peak to Peak Rise Percentage 10 0 Valley S N 20 Peak S N Cutoff 200 0 Table 85 IRC Detection Method Genesis Advanced page parameters Sheet 1 of 2 Parameter Description Report Noise As RMS Specifies that noise is calculated using an RMS calculation based on the noise data points Peak to Peak Specifies that noise is calculated using the peak to peak variation of the noise data points Thermo Scientific LCquan User Guide 223 4 Creating a Processing Method Specifying Method Identification Table 85 IRC Detection Method Genesis Advanced page parameters Sheet 2 of 2 Parameter Description Rise Percentage The percentage that the peak trace can rise above the baseline after passing through a minimum before or after the peak This method drops a vertical line from the apex of the valley between unresolved peaks to the baseline The intersection of the vertical line and the baseline defines the end of the first peak and the beginning of the second peak When the trace exceeds rise percentage the LCquan application applies valley detection peak integration criteria This test is applied to both the left and right edges of the peak The rise percentage criteria is useful for integrating peaks with long tails Range 0 1 to 500 0 Valley S N Specifies a value to
265. ght a In the Select Top Peaks area select the Enable check box b Select either option Select By Area or Select By Height c Type the maximum number of peaks to select 2 To specify a threshold minimum a In the Rel Peak Height Threshold area select the Enable check box b Specify the percentage of the highest peak to be used for classifying detected peaks Table 47 Limit Peaks area parameters Sheet 1 of 2 Parameter Description Select Top Peaks Enable Specifies to use only specific top peaks specific criteria defined by either area or height Select by Area Specifies peak areas as the only criteria for determining which peaks to use Select by Height Specifies peak heights as the only criteria for determining which peaks to use Thermo Scientific LCquan User Guide 131 3 Exploring the Data Creating Explore Methods Table 47 Limit Peaks area parameters Sheet 2 of 2 Parameter Description Num to Select The maximum number of peaks to include for consideration Rel Peak Height Threshold Enable Excludes all peaks that fall below the threshold percentage set for example the maximum peak height of a detected group of peaks of Highest Peak The percentage of the highest peak to be used for classifying detected peaks Range 0 to 100 Acquisition Sequence History 132 LCquan User Guide The Acquisition Sequence History pane Figure 76 displays the history of all sequences and sample
266. gin is set to Include all Weighting values are grayed out and Weighting is set to Equal Quadratic All settings are allowed with this exception When Origin is set to Include all Weighting values are grayed out and Weighting is set to Equal Linear Log Log No Weighting or Origin selections are allowed All Weighting and Origin values are grayed out Weighting is set to Equal and Origin is set to Ignore Quadratic Log Log No Weighting or Origin selections are allowed All Weighting and Origin values are grayed out Weighting is set to Equal and Origin is set to Ignore Average RF No Weighting or Origin selections are allowed All Weighting and Origin values are grayed out Weighting is set to Equal and Origin is set to Ignore Point to Point Cubic Spline Locally Weighted No Weighting selections are allowed All Weighting values are grayed out Weighting is set to Equal Only the Ignore and Force Origin options are allowed The Include Origin option is grayed out When Origin is set to Include it changes to Ignore No Weighting selections are allowed All Weighting values are grayed out Weighting is set to Equal Only the Ignore and Force Origin options are allowed The Include Origin option is grayed out When Origin is set to Include it changes to Ignore No Weighting or Origin selections are allowed All Weighting and Origin values are grayed out Weighting is set to Equal and Origin is set to Ignore
267. gration Report LCquanPeakIntegration xrt Provides the chromatogram traces of the confirmation ion and qualifier ions for each component The report also lists sample information peak status and calculated concentrations for each sample Provides the Results list information for example peak areas and calculated amounts for all components the sample information and the instrument method name and processing method name if applicable The report includes plots of the chromatogram peaks for all components Processing Method Report Avalon Genesis or ICIS algorithm LCquanProcessingMethod xrt Lists all the peak detection and integration parameters in the processing method for each component The report also lists level amounts IRC data and the mass to charge ratios and intensities of the peaks in the mass spectrum for each component Quan Components Peak Results Report LCquanQuanPeakResults_ESTD xrt LCquanQuanPeakResults_ISTD xrt Lists the sample information acquisition conditions and quantitative analysis results for a single sample The report also displays the integrated chromatograms of each target compound and the standard and the calibration curves Quantify Component Report LCquanQuantifyComponentReport xrt Provides a summary by component of the samples for each component The report includes sample information and quantitative analysis results for example peak areas response and calculated am
268. grid to the Displayed and Available categories as shown in the following table Figure 224 Column Arrangement dialog box Column Arrangement Available Columns Displayed Columns C nem winn Places Acq Date 250 dd FileName Area 100 z Sample Type Barcode 150 5 Response Barcode Status 75 soit Response Ratio Cal Eqn 250 T ISTD Area T Client 250 Specified Conc Comment 250 n Calculated Conc T Company Name 250 mj Deta RT T Dil Factor I Duration I Height Inj Vol ctory Defaul Current Arrangement Name Ouan Result Grid i Cancel Help Table 152 Grid parameters Sheet 1 of 9 Parameter Description Displayed Column parameters currently displayed in the grid FileName Displays the name of the raw file that contains the sample data The file name is a combination of the base file name prefix assigned to the sequence and a sequential sequence number If the default sequence starting number 1 has not changed the suffix number is the same as the row number of the sequence 001 002 and so on If the default sequence starting number has changed to another number the first sample will have the starting number and subsequent rows in the sequence are incremented by 001 For example if the starting number is 100 the file name for the first sample will have the suffix 100 the second sample will have the suffix 101 and so on To select a new file name select from th
269. he distance the label is offset from the data Label Threshold Set a threshold so that peaks above this level can show labels Thermo Scientific Thermo Scientific 2 Preparing to Perform Quantitative Analysis Using the Status View Table 31 Spectrum Display Options dialog box parameters Sheet 2 of 2 Parameter Description Plotting Automatic Lets the LCquan application determine the best spectrum display The graphic style chosen is based upon the data acquisition method used for the active spectrum Point to Point Displays the active chromatogram or spectrum using point to point peak profile Stick Displays the active chromatogram using vertical lines Axis Offset Offsets the displayed plot from the x axis y axis or both X Displays the y axis slightly above the x axis so you can see baseline details Y Displays the x axis slightly to the right of the y axis so you can see plot details at low x axis values LCquan User Guide 93 2 Preparing to Perform Quantitative Analysis Using the Status View Acquisition Status Run Status The Run Status page in the Status view of the Acquisition window provides an overall summary of LCquan status Figure 55 The LCquan application updates the information as conditions change Table 32 lists the parameters for the Run Status tab Figure 55 Acquisition Run Status Tab view 6 Thermo LCquan 3 Drugs Workbook File Change Actions View Ap
270. he most intense data point in the chromatogram LCquan User Guide 157 3 Exploring the Data Data Views Table 56 Chromatogram List view parameters Sheet 2 of 2 Parameter Description Shortcut Menu Open Raw File Opens the Select Raw File dialog box where you can select the raw file to load Lock Against Raw File Prevents changes to a raw file The lock icon is yellow and the Change lock is closed Lock against All Prevents changes to a raw file or parameters The lock icon is red Change and the lock is closed Unlock Makes the pane editable The lock icon is green and the lock is open Copy to Clipboard Copies selected data to the Clipboard You can then paste the data from the Clipboard buffer into a document Error Report View 158 LCquan User Guide The Error Report view displays the error log information stored in the current raw file Figure 96 Figure 96 Error Report view 4 gt a MS Quantum F Error Report APN_OO3 detector MS Quantum The contents of the grid are read only When the view is locked lock icon is yellow you cannot select any menu item in the shortcut menu that changes the raw file Menu items that would cause changes are grayed out For more information about locks see Toolbar for Selectable Views on page 150 Table 57 lists the parameters for the Error Report view Thermo Scientific Filter List View Thermo Scientific 3 Exploring the Data
271. heet 2 of 2 Parameter Description Area Height Ratio Increasing ratio of the peak area or height of the selected component to that of the internal standard Sorts by peak area ratio when you selected Response Area in the Create Method view in the Quantitate window or the dialog box Sorts by peak height ratio when you selected Response Height in the Create Method view in the Quantitate window or the dialog box Exclude Lists excluded samples first File Name IDs are in alphabetical or numerical order Integration Type Lists Method Settings integration type first User Settings integration type second and Manual Integration integration type last Level Name Calibration standard and QC level names are in alphabetical order Peak Status Groups low responses high response excluded and related status markers by this tagging style Sample ID IDs are in alphabetical or numerical order Sample Type Type of sample Standard QC Blank and Unknown Sample RT Displays retention time in chronological order Sample Original Order Displayed in original order Acquisition Time Date Component List Shortcut Menu 332 LCquan User Guide Displayed in chronological order The Component list shortcut menu is available from the Survey view and the Review All Results view Right click on the Component list and choose one of the sort orders from the shortcut menu see Table 128 Table 128 Component list s
272. heet 2 of 3 Parameter Use User Defined Time Range Description Displays chromatograms with a time range between the times specified by Min RT and Max RT and enforces the link time range feature When not selected the time base limits come from the raw file being acquired Note Use User Defined Time Range becomes active only when you select Link Chromatogram Time Range Min RT The beginning time of the chromatogram traces Note The Minimum Retention Time box becomes active only when you select Use User Defined Time Range Max RT The ending time of the chromatogram traces Note The Maximum component Retention Time box becomes active only when you select Use User Defined Time Range User Defined Traces Trace Table Trace Properties Title Trace Type Displays the properties of the user defined chromatogram traces The title for the chromatogram trace Maximum length 60 characters Default User Trace trace number Mass range chromatograms or total ion current TIC chromatograms Scan Filter Thermo Scientific The scan filter to be applied to the acquired data The LCquan application creates scan filters from the instrument settings that you specified in the Instrument Setup view For more information about filter formats refer to Xcalibur Help LCquan User Guide 87 2 Preparing to Perform Quantitative Analysis Using the Status View Table 28 Realtime Display Setti
273. hermo 408 555 5555 Tutorial Study 2 Client One SanJoseLab Thermo 408 555 5555 50 LCquan User Guide Thermo Scientific 2 Preparing to Perform Quantitative Analysis Using the Setup Sequence View Example 2 This example shows a set of custom columns and values for a New Client at the West Palm Beach Lab as they would appear on the Setup Sequence view These are the user labels and values you specified on the Change Sequence User Labels dialog box or the equivalent page on a wizard Change Sequence User Labels Changes in the labels and values will be applied to all rows New rows will also use these values User Value 1 Tes User Value 2 NewClient x User Label 1 Study User Label 2 Client User Value 3 WPBLab x User Value 4 ThermaFisher User Label 3 Laboratory User Label 4 Company Name User Label 5 Phone User Value 5 Cancel Heb 408 555 5555 v The user labels and values are displayed on the setup sequence header area Study Te Client NewClient Laboratory WPBLabo S Company Name ThermoFisher Phone 408 555 5555 00 The user labels and values are reflected in the user defined columns in the sequence grid Study Client Laboratory Company Hame Phone Test NewClient WPBLab ThermoFisher 408 555 5555 Test NewClient VWPBLab ThermoFisher 408 555 5555 Testt NewClie
274. hermo Scientific The LCquan application provides a direct link to Qual Browser from the Explore view Qual Browser provides for more sophisticated qualitative review than what is available in the LCquan application which is mainly concerned with quantitative rather than qualitative analysis With Qual Browser you can perform library searches formula searches for spectra background subtraction and other data manipulation To open the Qual Browser Click the Qual Browser icon in the navigation pane When you open Qual Browser Figure 91 the LCquan application retrieves the currently selected raw file Figure 91 Qual Browser window f Thermo Xcalibur Qual Browser APN_004 File Edit View Displa Grid Actions Tools Window Help sme S ee gt x 141 4 te oe m eleg F APN_004 raw i IED 4 LEA O F BID CAXealibun RawfilesAPN_004 Standard 1 Conc 25 pg ml Mg TIC APN_004 i 4 9K Mass Range no file See noe 4 9K Mass Range no file 100 9K Mass Range no file 1 9K Mass Range no file 2 APN_004 JK Mass Range no file 80 9K Mass Range no file JK Mass Range no file 70 60 50 40 r 3 g 3 5 3 Ia 3 3 3 ec 30 0 86 1 17 1 44 20 Time min LCquan User Guide 149 3 Exploring the Data Data Views Data Views The Explore Create and Explore Revi
275. hes LCquan User Guide 133 3 Exploring the Data Creating Explore Methods Preview Panes 134 LCquan User Guide For each sample you can display various types of data The preview panes in the Explore window contain a list of views to display the data in various forms To explore the data in the preview panes Use the Toolbar for Selectable Views to select any of these views Chromatogram View Chromatogram List View Error Report View Filter List View General Parameters Plot View Instrument Method View Mass List View Peak List View Sample Info View Spectrum View Status Plot View Status Report View Tune Method View Multi Peak Plot View For detailed descriptions of all these views and the Toolbar for Selectable Views see Data Views on page 150 Thermo Scientific 3 Exploring the Data Exploring the Results Exploring the Results In the Review view of the Explore window Figure 77 you can create an explore method apply it to the raw data to create a peak list and use the peak list to create components for the processing method Exploring the data helps you to construct a quantitative processing method Figure 77 Explore Review window E Thermo LCquan 3 Drugs Workbook File Zoom View Apps Options Help 3B 8 4 4 2 0000000 4 0 0 Instruments Acquisiti Chromatogram 4 gt lal Multi Peak Plot z APN 002 TIC RT 0 01 4 00 NL 1 10E5 Quantitate 1005 100
276. hlight a cell Highlighted rows are not selected You must click the number of the row to select the row To insert rows in the grid 1 Select the row in the sequence below where you want the new row sample to be located 2 Right click the sequence and choose Insert Rows An empty row is added above the selected or highlighted row To duplicate rows in the grid 1 Select the rows in the sequence that you want to duplicate 2 Right click the sequence and choose Duplicate Selected Samples The selected rows are copied and added to the end of the sequence To move rows 1 Select the row in the sequence below where you want the row sample to be moved 2 Right click the sequence and choose Insert Rows An empty row is added above the selected or highlighted row 3 Select the row you want to move 4 While holding the mouse button down on the selected rows in the row number column drag your cursor to the location in the grid above the empty row you added 5 Release the mouse button 6 The selected row overwrites the empty row When you move a row it leaves an empty row behind 7 Click Compress to remove all empty rows 54 LCquan User Guide Thermo Scientific 2 Preparing to Perform Quantitative Analysis Using the Setup Sequence View To delete rows 1 Select the entire row or rows that you want to delete e To select a series of rows position the cursor over the starting row number and drag the cursor o
277. hortcut menu commands Sort Order Description Unsorted Ascending order of times when components were added to the list By RT Ascending order of Expected Retention Times for the components Alphabetically Alphabetical order of the components When you make a selection the information is saved in the registry All workbooks use the current setting until the user changes it The setting is independently saved for each user Thermo Scientific 6 Processing the raw Files and Reviewing the Analytical Results Reviewing the Calibration Curve and OCs Note To change the order of the Component tabs set the sort order in the Component list and choose View gt Component Selector Bar to display the Component tabs Reviewing the Calibration Standards Use the Survey view to confirm that the LCquan application properly detected and integrated the peaks To review the calibration standards for a target compound 1 In the Component list on the right side of the view select the target compound The LCquan application automatically updates the Result list the Chromatogram and ISTD Chromatogram panes and the Calibration Curve pane 2 Ensure that the calibration standards are displayed in the Result list If necessary click the Standards tab at the bottom of the Result list to display the calibration standards 3 In the Calibration Curve pane inspect the calibration curve at all concentrations Evaluate the calibration cur
278. ht Mass m 2 C Valey Detection Enabled Expected Width sec 0 0 Num to select 4 dike CI Constrain Peak Width Rel peak height threshold c Full ms2 303 30 40 100 00 310 00 na Peak Ht 5 0 CI Enable R in Emad aRar 10 of highest peak 100 0 0 999 9 Apply Cancel Save As Default gt a Chromatogram Hidr a Peak List 5 steroids04 TIC SM 7 RT 0 01 3 79 NL 3 73E5 prorated F c Full ms2 303 30 40 00 100 00 310 00 A Peak Hame Apex RT Area of Largest Area of TotalArea 100 RT 1 99 1 Peak 1 991 i 1 991 2605128 100 000 100 000 B 30 B E 80 II BI 70 wd e PE Em 10 m E 50 m o 2 12 a 13 40 Mee 14 15 he 17 i me 19 10 no 49 1 i a a fl SONA SS BA S SO a O J SG LL RL 2 Os 1 0 15 20 25 3 0 35 23 Time min 4 z SFL NOT SAVED 11 17 2005 5 07 Ph To review the filtered chromatograms and mass spectra 1 Click the Review icon The LCquan application displays the Review view of the Explore window 2 Select Multi Peak Plot in the right pane 3 Click a chromatogram peak in the multipeak plot The LCquan application displays the product ion mass spectrum at the maximum intensity and the mass to charge ratios of the first several ions of highest intensity in the mass spectrum 148 LCquan User Guide Thermo Scientific 3 Exploring the Data Exporting the Active raw File to Qual Browser Exporting the Active raw File to Qual Browser T
279. ht endpoint minus the Apex RT The Peak Apex and Delta values are used to determine which label to apply to the peak When a peak is located within the range e Peak Apex minus Delta to Peak Apex plus Delta the corresponding label is used Note Delta RT is the range that is allowed from the Apex RT Thermo Scientific LCquan User Guide 167 3 Exploring the Data Data Views Table 62 Peak List view parameters Sheet 3 of 4 Parameter Modify Peak Names Description Opens the Peak Name List Editor dialog box where you can manage the current peak name list Entries are not validated in this dialog box You can create duplicate names or location parameters For more information see Generating a Peak List on page 143 The LCquan application use the first item in the list that matches the criteria of a peak Import Peak Name List Opens a file selection dialog box where you can import the naming list to a text file txt or csv A copy of the file is created in the Import folder and both an event record and a file tracking record are created After the file is accepted the LCquan application relabels any peak list or chromatogram as required Export Peak Name List Create Quan Components For Selected Peaks in List 168 LCquan User Guide Opens an export name dialog box where you can export a naming list to a text file You specify the file name in the export name dialog box The file is created in
280. hting of 1 Y 2 for all calibration data points during the least squares regression calculation of the calibration curve Calibrants are weighted by the inverse of the square of their response or response ratio 1 s 2 Specifies a weighting of 1 s 2 for all calibration data points during the least squares regression calculation of the calibration curve Calibrants at a given level are weighted by the inverse of the standard deviation of their responses or response ratios For this weighting factor to be used there must be two or more replicates at each level When only one calibrant is available for any level 1 s 2 weighting cannot be used Thermo Scientific LCquan User Guide 239 4 Creating a Processing Method Specifying Method Calibration 240 LCquan User Guide Table 92 Target Compound area parameters Sheet 3 of 3 Parameter Table Headings Description Cal Level Displays calibration levels for this compound Amount Displays the amount of the calibration level compound QC Level Displays QC levels for this compound Amount Displays the amount of the QC level compound Test Displays the acceptable difference as a percentage between the known amount and calculated measured amount of each QC level Table 93 Calibration Curve Type area parameters Calibration curve type Allowable settings for response origin and weighting Linear All settings are allowed with this exception When Ori
281. i Preparing to Perform Quantitative Analysis This chapter describes procedures for starting the LCquan application and creating a new study and workbook in preparation for analyzing pre existing data This chapter also describes the Instrument Setup window and the Acquisition window and outlines procedures to create instrument methods and acquire data however these steps are not required to perform quantitative analysis Contents e Creating a New Study and Workbook e Introduction to the Instrument Setup Window e Introduction to the Acquisition Window e Using the Setup Sequence View e Using the Run Sequence Dialog Box e Using the Status View e Emergency Shutdown Creating a New Study and Workbook When you start the LCquan application you can open an existing study and workbook or you can create a new study and workbook set You can also open a workbook in review mode where you can analyze existing data and create methods but you cannot acquire data or save any changes you make to the workbook This section includes instructions for using an LCquan workbook e Create a New Workbook e Saving a Workbook e File Tracking Results e Workbook Summary Information To start your quantitative analysis create a new study or workbook and import raw data Thermo Scientific LCquan User Guide 13 2 Preparing to Perform Quantitative Analysis Creating a New Study and Workbook Create a New Workbook To get started create a new workbo
282. ial Vial Position il V Re Use Vial Positions V Injection Volume obtained from Autosampler Method a Table 16 Tray and vial information page parameters Sheet 1 of 2 Parameter Description Tray Type Displays the tray type that must be installed in your autosampler to use the current sequence Initial Vial Position Specifies the first vial position in the new sequence The default first vial position is 1 For example when you enter 50 the first three vial positions are e 50 e 51 e 52 Re Use Vial Positions Controls whether or not you reuse vial positions for replicate samples When you select the Re Use Vial Positions check box the LCquan application creates a sequence where replicate calibration QC Blank and Unknown samples are drawn from the same vial When you clear the Re Use Vial Positions check box the LCquan application creates a sequence where each sample is drawn from a different vial Thermo Scientific LCquan User Guide 43 2 Preparing to Perform Quantitative Analysis Using the Setup Sequence View Table 16 Tray and vial information page parameters Sheet 2 of 2 Parameter Description Injection Volume Uses the injection volume specified in the instrument method Obtained from for the autosampler Autosampler Method Injection Volume Displays the injection volume in microliters of sample to be injected When you are using an autosampler you can set the default injection volume in th
283. ialog box Genesis Integration page parameters Sheet 3 of 3 Parameter Description Tailing Factor Specifies a value for the factor that controls how the LCquan application integrates the tail of a peak This factor is the maximum ratio of the trailing edge to the leading side of a constrained peak and calculates the retention time of the maximum extent of the right edge of the tailing peak This option is available only when you select the Constrain Peak Width option Range 0 5 to 9 0 Default multiplier 1 0 1 0 has no effect Note You can test the results of the new criteria by clicking Apply or OK You can apply the new criteria to all files in the Result list by clicking Apply to All User Identification Settings Genesis Advanced Use this page Figure 205 to set up advanced chromatographic parameters able 137 lists the parameters for the Genesis Advanced page of the User Identification Settings dialog box Figure 205 User Identification Settings dialog box showing the Genesis Advanced page User Identification Settings Identification Detection Genesis Integration Genesis Advanced Report Noise s C RMS Peak to Peak Rise Percentage 10 0 Valley S N 2 0 Peak S N Cutoff 1200 0 Cancel Apply Apply Toal Help 354 LCquan User Guide Thermo Scientific 6 Processing the raw Files and Reviewing the Analytical Results Reviewing the Calibration Curve and OCs Table 137 User Identification Sett
284. ialog box its content reflects the current selection Shortcut Menu Lock against All Prevents changes to a raw file or parameters The lock icon is red Change and the lock is closed Unlock Makes the pane editable The lock icon is green and the lock is open LCquan User Guide 269 4 Creating a Processing Method Data Views Table 104 IRC Chromatogram view parameters Sheet 2 of 2 Parameter Show Peak Info Description Opens the Peak Information dialog box The selected peak in the plot is updated to reflect the selection in the menu Additional Show Peak Info menu items are available for each qualifier ion For more information see User Identification Settings ICIS Integration on page 356 in Chapter 6 Processing the raw Files and Reviewing the Analytical Results Load Raw File Create Method view only Opens the Select Raw File dialog box All panes are updated using the new raw file Method Settings Manual Integration Survey or Review All Results views only Selects the Method peak for the currently selected IRC chromatogram When Method Settings is selected a check mark appears to the left of the menu item Survey or Review All Results views only Selects the Manual peak for the currently selected IRC chromatogram When Manual Integration is selected a check mark appears to the left of the menu item This item is only available when there is a manually selected peak otherwi
285. ibration curve type When you select Linear or Quadratic the Weighting area becomes active Do the following e Inthe Weighting area select the weighting option that the LCquan application applies to the correct regression weighting method when it calculates the least squares regression calibration curve e In the Origin area select how to treat the origin in the calibration curve calculation e To exclude the origin in the calibration curve calculation select the Ignore option e To require that the calibration curve pass through the origin select the Force option e To include the origin as one data point select the Include option When you select any of the other curve types the Weighting area is not active 7 Specify calibration level data e Use the Cal Level boxes to enter calibration levels e Use the Amount boxes to enter the amount of internal standard added at each level The LCquan application automatically adds another row to the table when you click the Amount box after filling in the Cal Level box 8 Specify quality control level data e Use the QC Level boxes to enter quality control levels Use the down arrow key to add more levels e Use the Amount boxes to enter the amount of internal standard added at each level e Use the Test boxes to enter a value for the acceptable difference as a percent between the known amount and calculated measured amount of each quality control level The LCquan appli
286. ic to each event Controls to Modify the Event List Auto Calculate Estimates the initial values for the detection of peaks based on the data in the current raw file to display initial values in the event list and searches for the best values of initial events that detect peaks in the data Any timed event in the event list is unchanged when you click Auto Calculate Determines initial values for only Start Threshold End Threshold Area Threshold P P Resolution Threshold Bunch Factor Negative Peaks and Tension Note Valid only if a raw file is open Time Min Initial value or a time value in minutes Event Descriptions of detection parameters for initial events and timed events Value Values associated with the initial timed events described in the Event box The range of factors allowed for each value is specific to each event Thermo Scientific 3 Exploring the Data Creating Explore Methods Table 45 Avalon Peak Integration area parameters Sheet 2 of 2 Parameter Buttons Description Add Adds the Time Event and Value values to the Avalon Event List Delete Deletes the selected row Change Using the Time Event and Value values updates the event list and the chromatogram display Table 46 Initial and Timed Events Sheet 1 of 2 Event Start End Threshold Description Half of the result is a good estimate for the Start Threshold You ca
287. ick the sequence grid and choose Fill Down from the shortcut menu c In the Fill Down dialog box Figure 177 specify the columns to be included and the range of rows 304 LCquan User Guide Thermo Scientific Thermo Scientific Figure 177 Fill Down dialog box Select Columns Sample Type P FileName TV Path Sample ID 7 Level TC ISTD Corr Amt Dil Factor Sample Vol T Sample Vt 7 Sample Name 5 Creating a Processing Sequence Editing a Processing Sequence P Study Client I Laboratory Company Name I Phone Comment Fill From Row fil To Row fio Using Row fi during fill dowr IV Create sar iples in selected empty row Select Al Clear All Cancel Help For detailed descriptions of the fill down parameters see Fill Down dialog box parameters on page 57 in Chapter 2 Preparing to Perform Quantitative Analysis To modify the arrangement of the processing sequence Use the shortcut menu commands For example you can change the sorting order of the sequence For detailed descriptions of the processing sequence grid shortcut menu commands see the Processing Sequence Shortcut Menu LCquan User Guide 305 5 Creating a Processing Sequence Editing a Processing Sequence Processing Sequence Shortcut Menu Right click on the sequence grid to display the shortcut menu Figure 178 Table 119 lists the commands for the processing sequence shortcut menu Figure 178 Process
288. icrosoft Excel For more information see Excel Short Summary on page 394 Long Excel Summary Displays the Result list of the Quantitate window processing sequence and component identification and calibration information in a Microsoft Excel spreadsheet Each component is placed on a separate page worksheet in the main workbook This arrangement can be used as a template to create a new arrangement from the Review Reports view You can review analyze and graph the data by using all the functions and features of Microsoft Excel For more information see Excel Long Summary on page 395 If your administrator has set up secure reporting the Create XReport Templates feature is unavailable You have access only to the templates in the secure template folder The only output option is Save Only which saves the file as a PDF Thermo Scientific LCquan User Guide 385 6 Processing the raw Files and Reviewing the Analytical Results Creating and Reviewing Reports Quan Result Grid The Quantitate Results grid can contain any of the following column parameters To select which parameters to display in the grid see Table 152 right click on the grid choose Columns and use the Column Arrangement dialog box Figure 224 The parameters displayed in the Quantitate Results grid are used to create a Quan Result Grid Excel report Note Click Factory Default in the Column Arrangement dialog box to reset the columns in the
289. id 20 70 15 192 07 r Retention Time Expected min 92 Window sec 20 0 V Use as RT Reference View Width min 0 75 Adjust Using E E Cancel Apply Apply Toan Help Table 134 User Identification Settings dialog box Identification page parameters Sheet 1 of 3 Parameter Description Smoothing Specifies the number of points to use for a moving mean filter to smooth the chromatogram Format integers Range 1 to 15 odd numbers only Note Set the parameter to 1 to disable filtering Thermo Scientific LCquan User Guide 347 6 Processing the raw Files and Reviewing the Analytical Results Reviewing the Calibration Curve and OCs Table 134 User Identification Settings dialog box Identification page parameters Sheet 2 of 3 Parameter Trace Description Specifies the type of chromatogram e Mass Range Specify up to 50 mass ranges The masses are added together to form the chromatogram e TIC Full scan acquisition resulting in a Total Ion Current plot e Base Peak In a differential chromatogram the interpolation of the baseline is derived from the distance between the intersections of the tangents drawn to the peak sides and the peak base e Summed Uses ion summing to create a chromatogram that is the sum of the chromatograms of related compounds Ion summing can increase sensitivity because it sums up to five chromatograms of related masses for example water loss adducts
290. ified Conc Calculated Conc Units 1 Diff Level RSD 19 CY Peak Status Response Ratio These parameters are displayed in the Excel Long Summary column arrangement FileName e Sample Type e Sample Name e Sample ID e Specified Conc e Calculated Conc e Units e Diff e Level e RSD e CV e Peak Status e Response Ratio e Response Type e Cal Eqn e Area e Height ISTD Area ISTD Height RT Integration Type Response ISTD Response Rel RT Delta RT S N Left Time Right Time Left Height Right Height Intg Code Search Window S N Threshold Smoothing e Acq Date Duration e Vial Pos Inj Vol Sample Wt Sample Vol ISTD Base Amt ISTD Calc Amt Dil Factor Study Client e Laboratory Company Phone Comment LCquan User Guide 395 6 Processing the raw Files and Reviewing the Analytical Results Exporting Results Exporting Results From the Survey Review All Results or Review Reports views you can export your results to a file Figure 227 shows the Results Export dialog box Table 153 lists the parameters for the Results Export dialog box Figure 227 Results Export dialog box Results Export File Selection Select Manage Export Column Arrangement Select an Arrangement uan Result Grid One File for all Components q C One File for each Component Create Edit View an Arrangement Format Options IV Include Item Header r t Tab Separation C
291. ile Tracking Results parameters 26 Fill Down parameters Acquisition 57 Quantitate 308 Filter List parameters Explore 160 Quantitate 276 Filter List shortcut menu commands 276 flow diagrams acquiring data 414 creating study or workbook 412 creating study or workbook Web Access 413 processing data 415 G General Parameters Plot parameters Explore 161 Quantitate 277 General Parameters Plot shortcut menu commands Explore 162 Quantitate 277 Genesis Advanced Component Options parameters Explore 121 Quantitate 208 Genesis peak detection advanced 223 baseline 232 integration 220 user identification settings advanced 354 integration 352 Genesis Peak Integration parameters Explore 119 Quantitate 206 H Help menu commands 424 H ESI probe 404 history viewing acquisition 101 Thermo Scientific Index F ICIS Advanced parameters Explore 125 Quantitate 212 ICIS peak detection advanced 227 integration 225 user identification settings advanced 363 integration 356 ICIS Peak Integration parameters Explore 124 Quantitate 211 importing layout configuration 420 processing method 250 processing sequence 298 raw files 17 Initial and Timed Events Explore 129 IRC detection 230 Quantitate 216 Instrument Method List parameters 278 Instrument Method parameters 163 instrument methods creating 29 defined 28 Instrument Setup window 28 Instrument Setup window overview 28 integrating peaks manually 363 internal standards ISTDs
292. iling Factor parameters Thermo Scientific LCquan User Guide 221 4 Creating a Processing Method Specifying Method Identification Table 84 IRC Detection Method Genesis Integration page parameters Sheet 2 of 2 Parameter Description Peak Ht Specifies a percentage of the total peak height This is the minimum that a signal must be above the baseline before integration is turned on or off This option is available only when you select the Constrain Peak Width option Range 0 to 100 Default multiplier 5 0 The peak height percentage is defined as follows 100 Peak Height Tailing Factor Specifies a value for the factor that controls how the LCquan application integrates the tail of a peak This factor is the maximum ratio of the trailing edge to the leading side of a constrained peak and calculates the retention time of the maximum extent of the right edge of the tailing peak This option is available only when you select the Constrain Peak Width option Range 0 5 to 9 0 Default multiplier 1 0 1 0 has no effect 222 LCquan User Guide Thermo Scientific 4 Creating a Processing Method Specifying Method Identification IRC Detection Method Genesis Advanced Use this page Figure 134 to set up advanced chromatographic parameters Table 85 lists the parameters for the Genesis Advanced page of the IRC Detection Method dialog box Figure 134 IRC Detection Method dialog box showing the Genesis Advanced pa
293. ime labels m Axis offset Tx Cancel Apply Help Table 105 lon Ratio Chromatogram Display Options dialog box parameters Sheet 1 of 2 Parameter Label With Description Select the data attributes you want to show in the data plots Retention Time Displays the peak apex retention time RT above the chromatogram peaks RT is displayed on all peaks that meet the selection criteria set in the Label Threshold box Name Displays the peak name above the peak Base Peak Displays the base peak mass for the scan above the chromatogram peak Signal to Noise Displays the peak signal to noise S N value above the chromatogram peak Note When RMS S N is selected in the Peak Detection Settings options set rms appears after the S N value Decimals Sets the number of decimal places for the peak apex retention time label Area Displays the peak area Height Displays the peak height LCquan User Guide 271 4 Creating a Processing Method Data Views 272 LCquan User Guide Table 105 lon Ratio Chromatogram Display Options dialog box parameters Sheet 2 of 2 Parameter Description Label Styles Stylize the labeling displayed with the data Offset Moves the label a defined distance from the data Rotated Rotates the labels above the peaks to 90 degrees Boxed Displays boxes around the labels above the peaks Size Defines the distance the label is offset from the
294. ime of the peak Units minutes Base Peak m z Displays the mass to charge ratio of the ion with the largest response in the current component peak Left height Displays the height intensity of the current component peak at the left extreme of the integration baseline Units counts Peak Height Displays the height amplitude of the current component peak apex Units counts Right height Displays the height intensity of the current component peak at the right extreme of the integration baseline Units counts ISTD Response Displays the integrated area in units of count minutes or the height of the apex Units counts Response Ratio Calculated Amount Displays the ratio of the peak sample peak area or height to the internal standard area or height Displays the amount in the sample as calculated with response ratio and cal curve Signal To Noise Displays the measured signal to noise ratio at the apex of the current component peak LCquan User Guide 369 6 Processing the raw Files and Reviewing the Analytical Results Reviewing All Results Peak Information Flags This page Figure 214 provides information about how the peak was detected and integrated Table 144 lists the parameters for the Flags page of the Peak Info dialog box Figure 214 Peak Info dialog box showing the Flags page Peak Info Alprazolam d5 ISTD for Paroxetine Info Flags Spect
295. ing for exported Microsoft Excel reports In this case the number of decimal places is fixed and cannot be edited 6 To change a column width a In the Width column of the Displayed Columns list select the value you want to change b Type the new value Tip To resize a column width drag the column boundary in the heading row of the sequence grid 7 To change an item name a In the Item column of the Displayed Columns list select the item name you want to change b Type the new name 8 To reset column values click Factory Defaults There is no undo for this function Table 23 Column Arrangement dialog box parameters Parameter Description Available Columns Lists parameters that are not currently selected for display Displayed Columns Lists currently selected parameters that appear in the data grids Add Remove Displays Hides selected columns Move Up Move Up moves columns left in the grid Move Down Move Down moves columns right in the grid Factory Defaults Resets original column values as described in Grid parameters on page 386 in Chapter 6 Processing the raw Files and Reviewing the Analytical Results 60 LCquan User Guide Thermo Scientific 2 Preparing to Perform Quantitative Analysis Using the Setup Sequence View User Labels and Values Use the User Labels and Values command to change user labels and values To change user labels and values 1
296. ing method click Import Processing Components or right click and choose Import Processing Components from the shortcut menu 62 LCquan User Guide Thermo Scientific 2 Preparing to Perform Quantitative Analysis Using the Setup Sequence View Figure 43 Acquisition Levels dialog box Acquisition Levels Component i Component02 Table 24 Acquisition Levels dialog box parameters Sheet 1 of 2 Parameter Description Components Component l n Lists the components in the sequence Levels Cal Level Cal Level Displays calibration levels for the selected component The LCquan application can accommodate up to 50 calibration levels Amount Displays the amounts of the target compound used for each calibration level Thermo Scientific LCquan User Guide 63 2 Preparing to Perform Quantitative Analysis Using the Setup Sequence View Table 24 Acquisition Levels dialog box parameters Sheet 2 of 2 Parameter Levels OC Level Description QC Level Displays the QC quality control levels for the selected component Use QC samples containing known amounts of a component to help ensure the accuracy of an analysis The LCquan application measures the quantity of the QC component in the same manner as for unknown components The measured quantity is then compared with a user defined expected quantity and a user defined percent test Value up to 15 QC levels Amount Displays the amounts o
297. ing sequence shortcut menu Fill Down Set Sorting Order Columns Add Sample Delete Selected Samples Duplicate Selected Row Import Processing Sequence gt From File Copy From Last Acquired Sequence Copy From Acquisition Sequence Table 119 Processing sequence grid shortcut menu commands Sheet 1 of 2 Command Description Fill Down Opens the Fill Down dialog box where you can duplicate column data from a selected row to other rows in the sequence For more information see Processing Sequence Fill Down Parameters on page 307 Set Sorting Order Opens the Sequence Sorting Order dialog box where you can change the sorting order of the rows in the sequence For detailed parameter definitions see Modifying the Column Sort Order on page 311 Columns Opens the Column Arrangement dialog box where you can customize the column arrangement and display See Customizing Column Arrangement on page 312 Add Sample Opens the Select Raw File dialog box where you can select samples to add to the Result list Delete Selected Removes the selected samples rows from the Result list Samples Duplicate Selected Copies the selected row and adds it to the Result list The new row Row is added to the sequence according to the current sort criteria Show Level Amounts Opens the Review Level Amounts dialog box that displays the amount of sample in a calibration level or quality control level 30
298. ings dialog box Genesis Advanced page parameters Parameter Report Noise As Description RMS or Peak to Peak Determines if the noise used in calculating S N values is calculated using an RMS calculation or peak to peak resolution threshold Rise Percentage The percentage that the peak trace can rise above the baseline after passing through a minimum before or after the peak This method drops a vertical line from the apex of the valley between unresolved peaks to the baseline The intersection of the vertical line and the baseline defines the end of the first peak and the beginning of the second peak When the trace exceeds rise percentage the LCquan application applies valley detection peak integration criteria This test is applied to both the left and right edges of the peak The rise percentage criteria is useful for integrating peaks with long tails Range 0 1 to 500 0 Valley S N Peak S N Cutoff Specifies a value to evaluate the valley bottom Using this parameter ensures that the surrounding measurements are higher Range 1 0 to 100 0 Default 2 0 The peak edge is set to values below this defined S N This test assumes an edge of a peak is found when the baseline adjusted height of the edge is less than the ratio of the baseline adjusted apex height and the peak S N cutoff ratio When the S N at the apex is 500 and the peak S N cutoff value is 200 the LCquan application defines the right
299. ion about all components on each worksheet Specifies that the selected column arrangement is used to generate the reports but the File Name box is replaced by the Component Name box A header on each sheet of the Sample Style report displays the Sample File Name for each sample The row number of the sample in the processing sequence is displayed in parentheses next to the file name When not selected the LCquan application generates the Excel reports with one worksheet per component and with information about all samples on each worksheet Thermo Scientific LCquan User Guide 379 6 Processing the raw Files and Reviewing the Analytical Results Creating and Reviewing Reports Manage Excel Column Arrangements To create a new arrangement 1 Select an arrangement from the Select an Arrangement list Figure 220 You cannot edit the predefined arrangements that ship with the LCquan application but you can use one of these standard arrangements as a template to create your own custom arrangement Figure 220 Manage Excel Column Arrangements page m Manage Excel Column Arrangements Select an Arrangement Quan Result Grid x Create Edit View an Arrangement 2 Click Create Edit View an Arrangement The Column Arrangement dialog box for the selected arrangement opens Table 148 lists the parameters for the Column Arrangement dialog box 3 In the Column Arrangement dialog box change the Current Arrangement Name
300. ion area ICIS Peak Integration Peak Detection Algorithm ICIS v Advanced Baseline Window 20 Area Noise Factor 5 Peak Noise Factor fi 0 I Constrain Peak Width 2 In the Baseline Window box enter the number of scans to use when searching for where the minima are occurring 3 In the Area Noise Factor box enter a value to specify the noise level multiplier that determines the peak edge after the location of the possible peak 4 In the Peak Noise Factor box enter a value to specify the noise level multiplier that determines the potential peak signal threshold 5 To constrain the peak width of a component during the peak integration select the Constrain Peak Width check box e To specify the minimum above the baseline before integration is turned on or off enter a percent of the total peak height in the Peak Ht box e To constrain the peak width of an asymmetric chromatogram peak that has a tailing trace enter a peak integration multiplier in the Tailing Factor box 122 LCquan User Guide Thermo Scientific 3 Exploring the Data Creating Explore Methods 6 To specify advanced component detection criteria click Advanced to open the ICIS Advanced Parameters dialog box Figure 73 Use the advanced component detection criteria if the standard detection criteria do not provide the expected results Figure 73 ICIS Advanced Parameters dialog box ICIS Advanced Parameters Noise Method INCOS Noise C Re
301. ion places a copy of the report in the Exports folder of the workbook 376 LCquan User Guide Thermo Scientific 6 Processing the raw Files and Reviewing the Analytical Results Creating and Reviewing Reports Figure 218 Review Reports view Excel Report Selections Manage Excel Column Arrangements Use Column Arrangement Select an Arrangement x M Excel Short Summary FS Excel Long Summary Excel Long Summary v Create Edit View an Arrangement V Sample Style Excel Reports Launch XReport Template Generator XReport Report Selections Select Report Options Print Onl Save Only Cc sn cm __ Generating Reports Use the following options to generate reports in Excel or XReport e Excel Report Selections e Manage Excel Column Arrangements e XReport Report Selections e Launch XReport Template Generator e Select Report Options e Function Buttons Excel Report Selections Specify the Excel reports you want to create 1 Select the Use check box for each report that you want created in the Excel format Figure 219 2 To create a new column arrangement use the features in the Manage Excel Column Arrangements area 3 To add a row to the table select the Use check box in the last row of the table click in the row and select a report from the list Thermo Scientific LCquan User Guide 377 6 Processing the raw Files and Reviewing the Analytical Results Creating and Revie
302. ion run defined in the run header of the raw file Height 390 LCquan User Guide Integrated height under the detected peak counts Thermo Scientific 6 Processing the raw Files and Reviewing the Analytical Results Creating and Reviewing Reports Table 152 Grid parameters Sheet 6 of 9 Parameter Description Inj Vol Displays the injection volume in microliters of sample to be injected To change the volume type the new volume in the Injection Volume box When you are using an autosampler you can set the default injection volume in the Autosampler dialog box in the Instrument Setup window The minimum and maximum injection volumes that you can use depend on the autosampler you select The usable range depends on the injection mode and might be smaller than the range displayed in the status bar For more details consult your autosampler manual Intg Code Two character code representing baseline status of the peak s left edge and right edge respectively B Base V Valley M Manual S Stripe T Tail _ Tilt ISTD Area Integrated area under the Internal Standard peak count secs ISTD Base Amt Internal Standard expected amount ISTD Calc Amt Internal Standard calculated amount ISTD Height Integrated height under the Internal Standard peak counts Laboratory User defined Displays information that is pertinent to the active sample row in the Thermo Scientific Acquisition sequence You can use this
303. ion tab When the left navigation pane is hidden a Choose View gt Section Selection gt Quantitate Section b Choose View gt Step Selector gt Quantitate Method c Click the Identification tab The Identification page of the Create Method view includes these functional areas e Chromatogram Definition Area e Retention Time Area e Peak Integration Area e Peak Identification Area e Jon Ratio Confirmation Area This section contains instructions for performing the following e Summing Ions Dialog Box e Defining Peak Integration Parameters Chromatogram Definition Area Use the Chromatogram Definition area to specify the chromatographic parameters used to create a chromatogram Figure 114 Table 71 lists the parameters for the Chromatogram Definition area Figure 114 Chromatogram Definition area Identification Calibration Identification Calibration Chromatogram Definition Chromatogram Definition Detector Smoothing Detector Smoothing Ms MS ft Trace Summed zE c SAM ms2 309 07 cid 42 00 205 02 c SAM ms2 314 10 cid 40 00 203 96 Trace Mass Range Mass Rane v a 2 192 07 Filter c SRM ms2 330 13 cid 20 00 70 15 192 0 Thermo Scientific LCquan User Guide 187 4 Creating a Processing Method Specifying Method Identification Table 71 Chromatogram Definition area parameters Sheet 1 of 2 Parameter Description Detector Specifies the sp
304. ions dialog box Style r Label with T Name l Base peak r Label Styles Offset T Rotated l Boxed Chromatogram Display Options Signaltonoise l Height Plotting Retention time 2 Decimals Point To Point C Stick Area i gt When no peak found MV Show apex time labels Size oo m Axis offset J E Label threshold 0 0 OK Cancel Apply Help To display this dialog box When the left navigation pane is displayed a Click Explore b Click the Method or Review icon c Right click the data pane of the Chromatogram View and choose Display Options from the shortcut menu When the left navigation pane is hidden a Choose View gt Section Selection gt Explore Section b Choose View gt Step Selector gt Explore Method or Explore Review c Right click the Chromatogram pane and choose Display Options Table 55 Chromatogram Display Options dialog box parameters Sheet 1 of 2 Parameter Label With Description Select the data attributes you want to show in the data plots Retention Time Displays the peak apex retention time RT above the chromatogram peaks RT is displayed on all peaks that meet the selection criteria set in the Label Threshold box Name Displays the peak name above the peak Base Peak Displays the base peak mass for the scan above the chromatogram peak LCquan User Guide 155 3 Exploring the
305. iption Label With Select the data attributes you want to show in the data plots Retention Time Displays the peak apex retention time RT above the chromatogram peaks RT is displayed on all peaks that meet the selection criteria set in the Label Threshold box Name Displays the peak name above the peak 260 LCquan User Guide Thermo Scientific Thermo Scientific 4 Creating a Processing Method Data Views Table 101 Chromatogram Display Options dialog box parameters Sheet 2 of 2 Parameter Description Base Peak Displays the base peak mass for the scan above the chromatogram peak Signal to Noise Displays the peak signal to noise S N value above the chromatogram peak Note When RMS S N is selected in the Peak Detection Settings options set rms appears after the S N value Decimals Sets the number of decimal places for the peak apex retention time label Area Displays the peak area Height Displays the peak height Label Styles Stylize the labeling displayed with the data Offset Moves the label a defined distance from the data Rotated Rotates the labels above the peaks to 90 degrees Boxed Displays boxes around the labels above the peaks Size Defines the distance the label is offset from the data Label Threshold Sets a threshold so that peaks above this level can show labels Plotting Select one style to display your data Point to Point Select a graphic
306. ition check box Thermo Scientific LCquan User Guide 73 2 Preparing to Perform Quantitative Analysis Using the Run Sequence Dialog Box By default these programs run asynchronously in parallel with data collection 9 Specify the status of the system after data acquisition by selecting one of the After Sequence Set System options On Standby or Off Note On is the default setting 10 Click OK to save the settings and close the dialog box The LCquan application places the selected samples at the end of the acquisition queue and opens the Status view of the Acquisition window Note When you acquire data the LCquan application requires that you save the workbook Table 26 Run Sequence dialog box parameters Sheet 1 of 4 Parameter General Controls Description User Displays the name of the operator who ran the acquisition sequence This box is non editable Run Rows The list of rows must be consecutive For example you can run samples 1 through 10 by using the Run Sequence dialog box only one time However to run samples 1 through 3 and samples 5 through 10 you must use the Run Sequence dialog box twice The first time select samples 1 through 3 the second time select samples 5 through 10 Comment Optional A comment that identifies the sequence The comment appears in the Acquisition Sequence History Instrument Displays all the instruments that have been con
307. k 3 Drugs Workbook 2 APN_021 Example Study APN_022 3 Drugs Workbook APN_023 FE 3 Drugs Workbook 2 Cal_05 H 3 Drugs Workbook x APN_023 3 Drugs Workbook y 3 APN_024 Exports Ld APN_025 Imports APN_026 Rawtiles APN_027 Temp z Cal_06 APN_028 APN_029 APN_030 For Help press F1 APN_OO7 RAW HAPN 021 RAw I APN_035 RAW APN_035 APN_OOS RAW GQJAPN_022 RAW APN_036 RAW APN_036 APN_OOSRAW GJAPN_O23 RAW GQ APN_037 RAW APN_037 APN_OIORAW MQAPN_024 RAW QAPN_038 RAW Cal_08 File Name C calibur QuanRoot Example Study 3 Drugs Workbook y Rawfiles 4PN_039 RAW GG APN_O1E RAW i APN_017 RAW Ga APN_018 RAW Ga APN_019 RAW Ga APN_020 RAW Gi APN_025 RAW i APN_026 RAW i APN_027 RAW i APN_028 RAW Sample Type Std Bracket Comment Standard 8 Conc 1000 pg ml LCquan User Guide ij 4PN_030 RAW Wj 4PN_031 RAW i APN_032 RAW WAPN_033 RAW Gj APN_034 RAW i APN_O40 RAW GAPN_041 RAW WaPN_042 Raw Ta eta APN_031 APN_032 E Cal_07 APN_033 APN_034 APN_038 APN_039 APN_040 APN_041 APN_042 e4 Powers Dana NOT SAYED 7 8 2008 9 38AM Opens the edit view Thermo Scientific 5 Creating a Processing Sequence Defining a Processing Sequence e Ifyou already created a processing sequence when you click the Sequence icon the LCquan application opens the
308. ked it Play Pause Runs an automated action for the current pane Item Selector list Displays a list of selectable items for the current pane Source Current uA Yaporizer Temp C Sheath Gas Flow Rate Aux Gas Flow Ratef Capillary Voltage Capillary Temp C lon Gauge x10e 5 Torr Convectron Gauge Torr 254 LCquan User Guide Thermo Scientific 4 Creating a Processing Method Data Views Table 98 Toolbar for Selectable Views controls Sheet 3 of 3 Control Description Detector Selector list The detector stream for the data type of interest MS Quantum When a raw file is currently selected for the pane the detector box contains a list of the detector streams in the raw file in the form Generic Detector Stream Name Instrument Name Model Name Channel Name When no raw file is available when specifying the detector stream MS is the only option available When a new selection is made from the list the selection list continues to have the initial selection in the list until the current pane is changed When redisplaying only the current selection and any other selection available from the raw file is displayed View Selector list Select from the list of selectable views for the current section Chromatogram Chromatogram List Error Report Filter List Instrument Method List Mass List Calibration Curve View for Survey or Review All Results Only Thermo Scientific The Calibration Curve view is
309. ks see Toolbar for Selectable Views on page 252 Table 116 lists the parameters for the Tune Method view Table 116 Tune Method view parameters Parameter Description Arrows Not used in the Create Method view The left and right arrows move through the sequence list incrementing or decrementing by a single status item Column Headings Parameter Displays a column of descriptive text for the item Value Displays a column of data for the given parameter Shortcut Menu Open Raw File Create Method view only Opens the Select Raw File dialog box where you can select the raw file to load Lock against All Prevents changes to a raw file or parameters The lock icon is red Change and the lock is closed Unlock Makes the pane editable The lock icon is green and open Copy to Clipboard Copies a chromatogram mass spectrum or calibration curve to the Clipboard You can then paste the data from the Clipboard buffer into a document Multi Peak Plot View for Survey or Review All Results Only The Multi Peak Plot view is available from the Survey or Review All Results views on the Quantitate window Figure 167 This view displays all detected peaks for a sample method combination You can set the number of rows and columns and define the number of peak graphics displayed on the screen e You can define a maximum of 50 cells e The x xis range for each peak is 2 0 times the detected peak width 50 on ei
310. l necessary files are contained within the workbook In addition it uses a file tracking feature to maintain a secure file system Check with your administrator if you have problems with the security configuration IMPORTANT Always use the LCquan application to open and modify the workbook and its associated files To maintain secure file operations it tracks the files in the workbook to ensure that none has been modified by an external process If you modify a file in the workbook by using another software application including Xcalibur it prevents you from reopening the workbook LCquan User Guide 7 1 Introduction Quantitative Analysis with LCquan LCquan Applications Menu LCquan User Guide To provide quick access to external applications you can add them to the Apps menu Figure 5 Table 1 lists the App Selection dialog box parameters To add an application to the Apps menu 1 Choose Apps gt Add Apps from any view The App Selection dialog box opens 2 Click Add to open the Add App dialog box Click Browse to open the Locate Programs to be Added dialog box and find the program A o Select the program to add to the menu and click Open 5 Click OK in the Add App dialog box 6 Click Close in the App Selection dialog box Figure 5 Apps menu Step 2 Step 3 m Apps a Program Add f C Xcalibur system pragrams xReport exe Browse Remove Move Up cnenenanene Move Dow
311. l of the LCquan application This value is not displayed if your LC is under contact closure control of the LCquan application Raw File Displays a data file with the raw extension created by the mass spectrometer when a sample is run that contains raw analysis data Instrument Method Displays a defined set of experiment parameters that define operating settings for the autosampler liquid chromatograph LC pump mass spectrometer divert valve and syringe pump Instrument methods are defined by using the Instrument Setup window Instrument methods are saved as the file type meth Instruments The readback status of each LCquan configured instrument appears on the Run Status page Shortcut Menu Each instrument s shortcut menu contains the following commands Turn Device On Puts an instrument in the On state Turn Device into Puts an instrument in the Standby state Standby Turn Device Off Puts an instrument in the Off state To select Help for a particular instrument 1 Click Instruments in the left navigation pane 2 Click the icon for the instrument 3 Choose Help gt Instrument Name Help Thermo Scientific 2 Preparing to Perform Quantitative Analysis Using the Status View Acquisition Status Acquisition Queue The Acquisition Queue page in the Status view of the Acquisition window displays all the sequences that are queued including those from other workbooks Figure 57 The ac
312. lank samples e First half of the calibration Standard samples e Optional Blank samples e Unknown samples e Optional Blank samples e Second half of the calibration Standard samples e Optional Blank samples 7 Click Next Table 17 Add Standards page parameters Parameter Description Add Standards Adds calibration standards to your acquisition sequence Based on Existing Cal Uses the calibration levels from an existing processing Levels from Method method Based on Automatically Generate names for the calibration levels The derived names Generated Cal Levels consist of a base name concatenated with an incremental number for each level For example if the base name is Cal and the number of levels is 5 the calibration levels are named Cal01 Cal02 Cal03 Cal04 and Cal05 e Number of Cal Levels e Cal Level Base Name Number of Injections Specifies the number of replicate calibration Standard samples per Level that are to be run at each defined calibration level The LCquan application groups replicate calibration samples in the new sequence Add Blanks Adds Blanks to your sequence The LCquan application places one Blanks before and one Blanks after each series of calibration standard samples in the new sequence Fill in Sample ID for Automatically fills in the calibration sample ID in the new Standards sequence This information is defined in the processing method for each calibration standard lev
313. lation of the peak Range 1 to 6 Note A high bunch factor groups peaks into clusters Area Threshold Controls the area cutoff Any peak with a final area less than the area threshold is not recorded Format units of area for the data Thermo Scientific 4 Creating a Processing Method Specifying Method Identification Table 89 Initial and Timed Events Sheet 2 of 2 Event P P Resolution Description Defines how much peak overlap must be present before two or more adjacent peaks create a peak cluster Peak clusters have a baseline drop instead of valley to valley baselines This is specified as a percent of peak height overlap Negative Peaks Automatically resets after a negative peak has been found Tension Controls how closely the baseline follows the overall shape of the chromatogram A lower tension traces the baseline to follow changes in the chromatogram more closely A high baseline tension follows the baseline less closely over longer time intervals Units minutes Tangent Skim Integrate On Off Shoulders On Enables tangent skim on any peak clusters By default the LCquan application selects the tallest peak in a cluster as the parent solvent and detects peaks on either side or both sides of the tallest peak You can also identify which peak in the cluster is the parent Tangent skim automatically resets at the end of the peak cluster Turns integration on or off at the se
314. lay Pause Starts or pauses the timed review of the samples Play causes the peaks for the given sample to be displayed for the specified time interval see Multi Peak Options on page 180 then the next set of peaks is displayed for the specified time interval until there are no more peaks in the list When the Repeat Playback box is selected the playback restarts at the beginning of the list until manually stopped Switching panes also stops the timed display Shortcut Menu Lock Against Raw File Prevents changes to a raw file The lock icon is yellow and the Change lock is closed Lock against All Prevents changes to a raw file or parameters The lock icon is red Change and the lock is closed Unlock Makes the pane editable The lock icon is green and the lock is open Use User Defined Time Resets the time scale to the user defined range as entered in the Range Realtime Display Settings dialog box For more information see Realtime Display Settings on page 84 Options Opens the Multi Peak Options dialog box For more information see Multi Peak Options on page 180 Reset Scaling Resets x and y axes to show the maximum amount of data Copy to Clipboard Copies selected data to the Clipboard You can then paste the data from the Clipboard buffer into a document Thermo Scientific LCquan User Guide 179 3 Exploring the Data Data Views Multi Peak Options Use the Multi Peak Options dialog box to d
315. le Opens the Select Raw File dialog box where you can open an ALT F gt F or jj existing data file of extension raw 422 LCquan User Guide Thermo Scientific c LCquan Menu and Toolbar Reference File Menu Commands Specific to Quantitate Window Table 162 File menu commands specific to the Quantitate window Command Description Keyboard shortcut Import gt Opens the Import Quantitation Method dialog box where you ALT F gt I Processing Method import a sequence from an existing file on disk If an acquisition sequence exists the Initialize with Acquisition Component Names box is selected and default method components are created with the names in the acquisition component list These components use the acquisition component level tables Import gt Opens the Load Sequence File dialog box where you can importa ALT F gt I gt Processing Sequence gt sequence from a file on disk A copy of the imported file is DOWN ARROW gt From File maintained in the import folder and an audit log entry is created RIGHT ARROW gt for this event The entire sequence is replaced with the imported ENTER sequence Import gt Replaces the defined processing sequence with a shortened Processing Sequence gt sequence that is based on the data already acquired Copy from Last Acquired Sequence Import gt Replaces the processing sequence with a copy of the acquisition ALT F gt I1 gt Processing Sequence gt sequence DOWN ARROW gt Copy fro
316. lector List options for this view are identical to those for the Chromatogram view so you can compare the two views side by side Figure 161 Mass List view 4 gt a Component Mass List APN_003 RT 1 90 Standard 1 Conc 25 pg ml The contents of the grid are read only When the view is locked lock icon is red you cannot select any menu item in the shortcut menu that changes the raw file or parameters Menu items that would cause changes are grayed out For more information about locks see Toolbar for Selectable Views on page 252 Table 111 lists the parameters for the Mass List view Table 111 Mass List view parameters Sheet 1 of 2 Parameter Description Arrows The left and right arrows move through the chromatogram incrementing or decrementing by a single scan Item Selector List Options Survey and Review All Results views only ISTD Displayed values are from the current component s ISTD Component Displayed values are from the current component Column Headings Mass Displays the mass value above the spectral line column Intensity Displays the intensity of the spectral data point column LCquan User Guide 279 4 Creating a Processing Method Data Views 280 LCquan User Guide Table 111 Mass List view parameters Sheet 2 of 2 Parameter Description Base Displays the relative percentage this data point has to the largest data point in the Intensity col
317. less of which integration type is currently in use for the current component Selected Displays the chromatogram from the current component Shortcut Menu Lock against All Prevents changes to a raw file or parameters The lock icon is red Change and the lock is closed Unlock Makes the pane editable The lock icon is green and the lock is open Method Settings Resets the integration method to those methods specified in the User Settings original processing method Applies a set of user defined peak detection parameters to the integration of this peak For more information see Specifying Additional Peak Detection Criteria on page 346 in Chapter 6 Processing the raw Files and Reviewing the Analytical Results Note Selecting this menu item automatically places the current sample into User Integration mode Manual Integration Thermo Scientific Denotes that you have adjusted the integration baseline by dragging the control boxes on the plot or by typing new numbers in the Baseline dialog box This option is grayed out until you have made baseline changes at least one time For more information see Manually Integrating Peaks on page 363 in Chapter 6 Processing the raw Files and Reviewing the Analytical Results LCquan User Guide 263 4 Creating a Processing Method Data Views Table 102 Chromatogram view parameters Sheet 2 of 4 Parameter Show Peak Info Description Opens the Peak
318. leted or modified The button changes to Un Filter after you click it click again to restore the full list of files Allow Opens the workbook if all currently required files are available This item can be configured by the administrator in the Authorization Manager application Thermo Scientific 2 Preparing to Perform Quantitative Analysis Creating a New Study and Workbook Workbook Summary Information Use this dialog box to find information about the current workbook and to enter information about the current workbook see Table 10 To open the Workbook Summary Info dialog box Figure 21 choose File gt Summary Info from any window Figure 21 Workbook Summary Info dialog box Workbook Summary Info Header Created Thursday July 17 2008 1 15 15 PM Modified Tuesday July 22 2008 1 16 14 PM Last saved by dana powers Number of saves TE Comment This is the summary information for workbook 3 Drugs Table 10 Workbook Summary Info dialog box parameters Parameter Description Header Displays the workbook creation date and time the last modified date and time the user name of the last person to save the workbook and the number of times the workbook has been saved Comment Type or edit information about the active workbook Thermo Scientific LCquan User Guide 27 2 Preparing to Perform Quantitative Analysis Introduction to the Instrument Setup Window Introduction to the Instrument Setup Wind
319. lgorithms to be applied to the active raw file Peak detection parameters are also available from these locations e The Peak Integration area in the Create view of the Explore window e The IRC Detection dialog box available from the ion ratio confirmation grid on the Identification page in the Create Method view of the Quantitate window e The User Identification Settings dialog box available from the Chromatogram view in the preview panes This section contains instructions for specifying parameters for the following e Genesis Peak Integration e ICIS Peak Integration e Avalon Peak Integration Note e The Genesis peak detection algorithm has been provided for backward compatibility with Xcalibur 1 0 studies e The ICIS peak detection algorithm has been designed for MS data and has superior peak detection efficiency at low MS signal levels e The Avalon peak detection algorithm has been designed for UV data Avalon also supports negative peaks Genesis Peak Integration Use the Genesis Peak Integration area to define Genesis peak detection algorithms to be applied to the active raw file Figure 126 Table 77 lists the parameters for the Genesis Peak Integration area 204 LCquan User Guide Thermo Scientific Thermo Scientific 4 Creating a Processing Method Specifying Method Identification To specify Genesis peak integration parameters In the Peak Detection Algorithm box select Genesis Figure 126 Genesis P
320. lists the parameters for the Genesis Integration page of the User Identification Settings dialog box Figure 204 User Identification Settings dialog box showing the Genesis Integration page User Identification Settings Identification Detection Genesis Integration Genesis Advanced S N Threshold 0 5 Valley Detection Enabled I Constrain Peak Width Cancel Apply Apply To All Help Table 136 User Identification Settings dialog box Genesis Integration page parameters Sheet 1 of 3 Parameter S N Threshold Description This multiplier specifies a signal to noise threshold for peak integration Only peaks with a signal to noise ratio greater than this value are integrated Range 0 0 to 999 0 Default multiplier 0 5 Valley Detection Enabled 352 LCquan User Guide Detects unresolved peaks with the valley detection approximation method The LCquan application drops a vertical line from the apex of the valley between unresolved peaks to the baseline The intersection of the vertical line and the baseline defines the end of the first peak and the beginning of the second peak Thermo Scientific 6 Processing the raw Files and Reviewing the Analytical Results Reviewing the Calibration Curve and OCs Table 136 User Identification Settings dialog box Genesis Integration page parameters Sheet 2 of 3 Parameter Description Expected Width sec This multiplier specifies an expected peak width pa
321. lossary Opens the glossary How to use Online Help Opens an HTML Help system that describes how to use the Help viewer About LCquan Opens the About LCquan dialog box where you can check the Options Menu version of each installed Xcalibur application including LCquan and view a list of the configured instruments The LCquan application groups commands that deal with identification calibration and calibration levels and graphic display control commands in the Options menu see Table 164 Options Commands Common to All LCquan Windows Table 164 Options menu commands common to all LCquan windows Sheet 1 of 2 Command Enable Warnings Description Enables the display of warning messages You can disable warnings for example by selecting the Don t Tell Me About This Again box in the Save Settings Options dialog box For more information see Save Settings Options on page 107 in Chapter 2 Preparing to Perform Quantitative Analysis Keyboard shortcut ALT O gt E Wizards gt Show the New Acquisition Sequence Wizard when no sequence is defined 424 LCquan User Guide Opens the New Acquisition Wizard if there is no acquisition sequence defined when you enter the Acquisition window The New Acquisition Wizard steps you through a procedure for creating a new acquisition sequence or importing an existing acquisition sequence For more information see Creating an Acquisition Sequence on
322. ls dialog box on page 34 When you complete the Change Sequence User Labels dialog box the LCquan application overwrites your acquisition sequence with the imported processing sequence To import an acquisition sequence from a file From the window File menu choose Import Acquisition Sequence gt From File Printing or Exporting the Acquisition Sequence Thermo Scientific Print the acquisition sequence grid or export the sequence information to an sld file 1M kod To print the acquisition sequence Choose File gt Print Sequence Info The Print dialog box opens where you select the format of the printing output To export the acquisition sequence Choose File gt Export Acquisition Sequence The LCquan acquisition sequence dialog box opens Type a name for the sequence file Do not enter a path or an extension The extension defaults to sld and the LCquan application creates a copy of the file in the Exports folder for the workbook LCquan User Guide 53 2 Preparing to Perform Quantitative Analysis Using the Setup Sequence View Modifying a Sequence Use keyboard keys and the acquisition sequence grid shortcut menu to modify the sequence See Using the Acquisition Sequence Grid Shortcut Menu on page 55 To select rows and cells by using the arrow keys 1 Click the grid to make it active 2 Use your keyboard up and down arrow keys to highlight a row Use the right and left arrow keys to hig
323. lue defines all scans peak start to peak end to be included in the area integration Range 0 to 100 Default 0 scans LCquan User Guide 213 4 Creating a Processing Method Specifying Method Identification Avalon Peak Integration Use the Avalon Peak Integration area to define Avalon peak detection algorithms to be applied to the active raw file Table 81 lists the parameters for the Avalon Peak Integration area To specify Avalon peak integration parameters 1 In the Peak Detection Algorithm box select Avalon Figure 130 Avalon peak identification and integration criteria are applied to the active raw file You can add modify or delete non automated timed events in the Avalon event list but you cannot delete an initial value Figure 130 Avalon Peak Integration area Avalon Peak Integration Peak Detection Algorithm Avalon X Auto Calculate Initial Start Threshold 10000 000 Initial End Threshold 10000 000 Initial Area Threshold 10000 000 Initial P P Threshold 1 000 Initial Bunch Factor 1 000 Initial Negative Peaks Off Initial Tension 1 000 Time min Event Value 0 000 Tension 1 000 Add Delete Change 2 To add a new event to the event list a Specify the new values in the Time min Event and Value boxes b Click Add The values are inserted into the Avalon event list 3 To delete the entire row except for an initial value row a Select the row you want to delete The
324. m Acquisition RIGHT ARROW gt Sequence DOWN ARROW gt ENTER Export gt Saves the current processing sequence in the Export folder witha ALT F gt E gt Processing Sequence name you supply An export event is logged and the exported file is ENTER tracked in the audit trail for the workbook Export gt Results Opens the Results Export dialog box where you can save the ALT F gt E gt current results information to a csv file in the Export folder This DOWN ARROW gt data is file tracked and an event is made stating that the filewas ENTER exported For more information see Results Export dialog box parameters on page 396 in Chapter 6 Processing the raw Files and Reviewing the Analytical Results Export gt QuickCalc Data Thermo Scientific Saves QuickCalc versions of the current LCquan processing sequence sld and processing method pmd in the Export folder using the file names that you supply An export event is logged and the exported QuickCalc files are tracked in the audit trail for the workbook LCquan User Guide 423 c LCquan Menu and Toolbar Reference Help Menu Help Menu Use this menu to access Help topics for LCquan see Table 163 Table 163 Help menu commands Command Description Keyboard shortcut LCquan Help Opens the LCquan Help system at the first Help topic Current View Help Opens the LCquan Help system and displays the topic for the F1 currently displayed LCquan view G
325. m buttons Sheet 1 of 2 Icon Button Description Zoom In Y Zooms in the traces along the y axis vertically to display more detail and less range Zoom OutY Zooms out the traces along the y axis vertically to display more range and less detail a Normalize Readjusts the display of all peaks in the trace based on the largest peak height Zoom In X Zooms in the traces along the x axis horizontally to display more detail and less range Zoom OutX Zooms out the traces along the x axis horizontally to display more range and less detail Thermo Scientific 6 Processing the raw Files and Reviewing the Analytical Results Reviewing the Calibration Curve and OCs Table 124 Zoom buttons Sheet 2 of 2 Icon Button Display All Description Resets the display of the x axis to include the entire displayable range Reset Scaling Resets both x and y axes to display the maximum amount of data You can also use the arrow keys on the keyboard to zoom The left arrow key zooms in along the x axis by a factor of two 2 The right arrow key zooms out along the x axis by a factor of two after you have zoomed in Similarly the up arrow zooms in along the y axis by a factor of two Result Grid Shortcut Menu Thermo Scientific and the down arrow zooms out The result grid shortcut menu is available from the Survey view and the Review All Results view Right click on the
326. mass range 249 mass spectrometry analyte detection 403 Masses parameters 190 method level names 321 modifiers volatile 403 Multi Peak Plot parameters Explore 179 Quantitate 291 440 LCquan User Guide Multi Peak Plot shortcut menu commands Explore 179 Quantitate 291 Multi Peak Options parameters 180 New Acquisition Sequence Wizard 38 New Method Wizard 182 New Processing Sequence Wizard 296 New Study or Workbook Wizard 14 normalization chromatogram with respect to detected peak 198 with respect to highest peak 198 0 Options menu commands 424 overview of the LCquan application 1 11 P parameter tables Acquisition Levels 63 Acquisition Sequence 104 Acquisition Sequence History Explore 132 Status 102 Acquisition Sequence History shortcut menu Acquisition 36 Explore 133 Status 103 App Selection 10 Avalon Peak Integration Explore 128 Quantitate 215 Baseline 364 Cal Exclusion List 344 Calibration Curve 256 Calibration Curve shortcut menu 256 Calibration Curve Type area 240 Calibration Options 246 Calibration page shortcut menu 242 Calibration Settings Curve 337 Isotope 339 Levels 340 Type 341 Change Instruments In Use 78 Change Sequence User Labels 34 Chromatogram Explore 154 Quantitate 259 Review 263 Thermo Scientific Chromatogram Comment 375 Chromatogram Definition area Explore 116 Quantitate 188 Chromatogram Display Options Explore 155 Quantitate 260 Chromatogram List Explore 157 Q
327. me you select this command until the minimum size is reached The current size setting is stored in the registry and is used for all workbooks when you open them Reset Scaling Resets the graph axes to the maximum limits so the entire plot is displayed The limits for the calibration plot are set to allow the display of all measured samples not just the calibration replicates When you select an Unknown sample the intersection lines drawn on the plot are visible Copy to Clipboard Copies the current calibration curve graph to the Clipboard buffer You can then paste it from the buffer into a document Chromatogram View for Create Method Only 258 LCquan User Guide This view is available from the Create Method view in the Quantitate window This view displays a chromatogram and all detected peaks for a sample component integration type and method Figure 150 The displayed chromatogram is based on the range specified in the method All detected peaks are displayed but cannot be edited Click the chromatogram to set the Retention Time marker All spectrum and spectrum list views that are not locked receive the selected time and update Figure 150 Chromatogram view for Create Method Chromatogram z APN_OO3 m z 192 07 192 07 RT 1 53 2 28 NL 5 64E3 F c SRM ms2 330 13 cid 20 00 70 15 192 07 400 RT 1 90 0 o Q o 40 ia a 2 E v 2 E 2a w ec N o 2 08 2 14 o Time min Whe
328. meters e Preview Panes Sample Selector Three categories of sequences appear in the sample selector e Explore Sequences sequences that you imported into Explore by using Load You can use Remove or Rename to delete or rename the selected sequence e Quantitate Sequences processing sequences that you previously created in the Quantitate window e Acquisition Sequence History historical information about the sequences that were used to acquire the data in the current workbook Note Raw files use absolute paths When you move the workbook the LCquan application will not find the samples and will create an empty graph 136 LCquan User Guide Thermo Scientific 3 Exploring the Data Exploring the Results To process a raw file against the Explore Method Click a raw file in the Sample Selector area Figure 78 The LCquan application automatically processes the data using the Explore method and displays the results in the panes in the Explore window Figure 78 Sample Selector area Current Review Display Multi Peak View v Rawfile soen Explore Sequences E W reserpinecali sid reserpine03 reserpine04 reserpine0S reserpine06 reserpine0 Quantitate Sequence W Processing Sequence steroids02 steroids03 steroids04 steroids05 steroids13 steroids14 steroids15 steroids16 sampleA sampleB samplec sampleD sampleE Acquisition Sequence History Table 49 lists the parameters for
329. meters Sheet 1 of 2 Parameter Description Baseline Baseline amp Noise Set the width of the chromatographic piece that is sent to the Window analysis routines during peak detection and integration The piece created is centered around the Expected Retention Time window width Genesis Baseline These global parameters apply to all samples in a sequence They apply only when you use the Genesis peak detection algorithm Baseline Noise This value controls how the baseline is drawn in the noise data Tolerance The higher the baseline noise tolerance value the higher the baseline is drawn through the noise data Range 0 0 to 100 0 Thermo Scientific Thermo Scientific 4 Creating a Processing Method Specifying Method Identification Table 90 Identification Options parameters Sheet 2 of 2 Parameter Minimum Number of Scans in Baseline Description Minimum number of scans to calculate a baseline A larger number includes more data in an averaged baseline Range 2 to 100 0 Baseline Noise Rejection Factor Current baseline noise rejection factor This factor controls the width of the RMS noise band above and below the peak detection baseline and is applied to the raw RMS noise values to raise the effective RMS noise during peak detection The left and right peak boundaries are assigned above the noise and therefore are closer to the peak apex value in minutes This action effectively raises
330. methods from legacy files see Table 6 To import legacy files 1 Click Browse to display the Import Instrument Method File dialog box and find the instrument method file Figure 15 Figure 15 Import legacy files page Instrument Method Browse Acquisition Processing Sequence Browse Processing Method Browse Table 6 Import legacy files page parameters Parameter Description Instrument Method Specifies the instrument method to import Acquisition Processing Specifies the sequence to be both the acquisition sequence and Sequence the processing sequence Note When you do not specify a processing method or the processing method is unavailable the LCquan application does not make the processing sequence the same as the sequence specified with this wizard Processing Method Specifies the processing method to import 2 Click Browse to display the Load Sequence File dialog box and find the sequence file 3 Click Browse to open the Import Quantitation Method dialog box and find the processing method file 4 Click Next Thermo Scientific LCquan User Guide 21 2 Preparing to Perform Quantitative Analysis Creating a New Study and Workbook Specify the Processing Sequence Specify the processing sequence path To specify a processing sequence path 1 Click Browse and find a folder in which to store processing sequences Figure 16 Figure 16 Set the processing sequence path page
331. mmand to import a sequence from a file or the processing sequence To import an acquisition sequence e From the shortcut menu choose Import Acquisition Sequence gt From File and use the dialog box to find a sequence file or e From the shortcut menu choose Import Acquisition Sequence gt Copy from Processing Sequence The LCquan application asks if you want to modify the user labels and values of the imported sequence Keep the current user defined labels and values or create new ones for the imported sequence Ifyou click No the LCquan application overwrites your acquisition sequence with the imported processing sequence without keeping your user defined columns and values Ifyou click Yes the Change Sequence User Labels dialog box opens See Creating an Acquisition Sequence on page 38 When you complete the Change Sequence User Labels dialog box the LCquan application overwrites your acquisition sequence with the imported processing sequence New Acquisition Sequence Wizard Use the New Acquisition Sequence Wizard command to open the wizard and define a new sequence To create a new acquisition sequence Choose New Acquisition Sequence Wizard See Creating an Acquisition Sequence on page 38 66 LCquan User Guide Thermo Scientific 2 Preparing to Perform Quantitative Analysis Using the Setup Sequence View Acquisition Sequence Grid Column Parameter Definitions Table 25 defines the
332. mo Scientific 4 Creating a Processing Method Specifying Method Identification To specify peak detection criteria The Peak Integration Area is dynamic displaying criteria for the selected peak detection method Genesis ICIS or Avalon Figure 125 Figure 125 Peak Detection Algorithm list Peak Detection Algorithm Genesis v Genesis ICIS Avalon For detailed descriptions of the peak detection parameters see Defining Peak Integration Parameters on page 204 To automatically identify the current component in other raw files 1 In the Peak Identification area select criteria for identifying peaks in the other raw files of the data set that correspond to the current component e Select the Highest Peak option default to identify the highest peak as the peak that corresponds to the current component after the filters and settings of the current component have been applied to the raw file e Select the Nearest RT option to identify the peak with a retention time nearest the value shown in the Expected box in the Retention Time area as the peak that corresponds to the current component after the filters and settings of the current component have been applied to the raw file The peak must have a height greater than the value you specify in the Min Peak Height box 2 To add qualifier ions select the Enabled check box in the Jon Ratio Confirmation Area and enter settings for up to five qualifier io
333. mo Scientific A calibration curve constructed from individual line segments At multiple points across the calibration region a weighted linear regression is performed Lines are drawn between point slopes forming a continuous curve At any point on the curve the calculated amount is based on the nearest weighted linear regression The LCquan application does not provide ignore force include origin options for this calibration curve type LCquan User Guide 337 LCquan User Guide 6 Processing the raw Files and Reviewing the Analytical Results Reviewing the Calibration Curve and OCs Table 129 Calibration Settings Paroxetine dialog box parameters Sheet 2 of 3 Parameter Description Origin Origin Settings Specifies how to use the origin in your calibration curve Ignore Excludes the origin as a valid data point for calculation or plotting Force Forces the calibration curve plot to pass through the origin Include Includes the origin as a single data point in the calculation of the calibration curve but the calibration curve is not required to pass through the origin Weighting Weighting settings Defines how the calibration data is weighted during the least squares regression calculation of the calibration curve Equal Use to weight all calibration data points equally during the least squares regression calculation of the calibration curve 1 X Specifies a weighting of 1 X for all calibra
334. ms2 363 30 40 150 00 375 00 Rangefs Smoothing points To open the Realtime Display Settings dialog box Right click in the chromatogram pane and choose Realtime Display Settings Use this dialog box to specify the properties of the real time chromatograms that the LCquan application displays in the Chromatogram and Spectrum panes of the Acquisition window Thermo Scientific Thermo Scientific 2 Preparing to Perform Quantitative Analysis Using the Status View To specify the chromatogram pane display options To filter the chromatograms according to the mass range and scan filters associated with each component select the Show Traces Defined by Components check box To display user defined real time chromatograms a Specify the traces in the Trace Properties box b Select the Show User Defined Traces check box To limit the number of displayed chromatograms enter the number of chromatograms to display in the Max Number of Visible Chromatograms box To display the mass spectrum in the spectrum pane a Select the Show Spectrum Trace check box The Auto Update Spectrum to Last Scan check box becomes active b To automatically update during each refresh to show the spectrum from the last scan select the Auto Update Spectrum to Last Scan check box To display the mass spectrum for a particular chromatogram and scan click the displayed chromatogram The L
335. much disk space is available on your disk drives Restart Acquisition Restarts an acquisition that has been paused ALT A gt R Stop Acquisition Stops the acquisition of the current sample and pauses ALT A gt T Pause Acquisition Pauses the acquisition after the current sample has been completely ALT A gt P acquired Devices On Puts all devices in the On state ALT A gt O Devices Standby Puts all devices in the Standby state ALT A gt A Devices Off Puts all devices in the Off state The Off state indicates that all ALT A gt F power to the instrument that can be controlled by the LCquan application is turned off CAUTION The Off state does not guarantee that all voltages are turned off nor does it indicate that all heated components are at room temperature To perform maintenance on an instrument refer to the hardware maintenance manual Automatic Devices On Use this option to automatically turn on all devices controlled by ALT A gt U 418 LCquan User Guide the LCquan application prior to starting a data acquisition If this command has a check mark to its left the LCquan application automatically turns on all devices that are in the Off or Standby states If this command does not have a check mark to its left the LCquan application posts the following message when you have one or more devices in the Standby or Off states One or More devices to be used by this sequence are not On The sequence will not start until all the reques
336. n Look in e programs X ane excelexp exe F lxconvert exe lFinss_Server exe US yreport exe F HomePage exe SS Result 5 Menu Text My Recent xReport Documents EE InstSetup exe Elintradaygc exe Program fi icquan exe fe Xcalibur system pragrams Report exe Desktop LibraryManager exe T ProcSetup exe Arguments i Qualbrowser exe iy Decunienis QuanBrowser exe Initial Directory A ce Plqueserv exe PF SubBackground exe versioninfo exe P alxcaliburConfig exe fe Xcalibur system programs Browse My Computer Programs exe 7 rr r 7 IDE IIIS ne cee ee ee ee ae IS oe Places Files of type Thermo Scientific 1 Introduction Quantitative Analysis with LCquan Table 1 App Selection dialog box parameters Parameter Description Add Adds an application to the Apps menu Select the executable file exe of the application that you want to add to the menu Remove Removes the selected application from the menu list Move Up Moves the selected application up the menu list When the top is reached the button is disabled Move Down Moves the selected application down the menu list When the bottom is reached the button is disabled Menu Text Lists the text that appears in the Apps menu list Program Lists the full path to the program to be run When you click Add the Program field automatically fills No validation is made on u
337. n Tailing Factor A peak integration multiplier that constrains the peak width of an asymmetric chromatogram peak that has a tailing trace It is the maximum ratio of the trailing edge to the leading side of a constrained peak and calculates the retention time of the maximum extent of the right edge of the tailing peak Range 0 5 to 9 0 Default multiplier 1 0 1 0 has no effect Note Valid only when you select the Constrain Peak Width check box Note You can test the results of the new criteria by clicking Apply or OK You can apply the new criteria to all files in the Result list by clicking Apply to All User Identification Settings ICIS Advanced Use this page Figure 207 to set up advanced chromatographic parameters able 139 lists the parameters for the ICIS Advanced page of the User Identification Settings dialog box Figure 207 User Identification Settings dialog box showing the ICIS Advanced page User Identification Settings Identification Detection ICIS Integration ICIS Advanced Noise Method Incos Noise C Repetitive Noise T RMS Noise Min Peak Width 3 Multiplet Resolution fio Area Tail Extension Bo Area Scan Window fo Cancel Apply Apply To All Help 358 LCquan User Guide Thermo Scientific Thermo Scientific 6 Processing the raw Files and Reviewing the Analytical Results Reviewing the Calibration Curve and OCs Table 139 User Identification Settings dialog box ICIS A
338. n History tab 0000 Run Status Acquisition Queue Acquisition History Acquisition Sequence History w AcqSequence 1 Fl z Info Operator Quantum Start Time 11 16 2005 4 37 02 PM 4 End Time 11 16 2005 7 02 51 PM tU Startup Method Not Used J Shutdown Method Not Used I Comment lt None gt El Status Complete H Component 4 Nefazodone J fees Table 34 Acquisition History page parameters Sheet 1 of 2 Parameter Description Info Operator Displays the login ID of the person who is logged onto the system and who is acquiring the sequence Start Time Displays the time when the first sample or startup method began End Time Displays the time when the last sample or shutdown method completed When the LCquan application is closed before acquisition is complete but the acquisition is allowed to complete in the background the end time is displayed as Unknown Thermo Scientific Thermo Scientific 2 Preparing to Perform Quantitative Analysis Using the Status View Table 34 Acquisition History page parameters Sheet 2 of 2 Parameter Comment Description Displays a comment about the sequence as entered in the Run Sequence dialog box For more information see Run Sequence dialog box parameters on page 74 Status Displays the sequence status e Waiting created but not yet submitted e Submitted submitted but not yet validated and not yet running that is
339. n Use Yes or Not In Use a blank space When you configure an instrument using the Instrument Configuration view the default status of the instrument is In Use Yes When you do not want to use an instrument for the current sequence click the In Use Yes entry to change it to a blank space Instruments with a blank space in the In Use row are not available for the current sequence For example if a sample is to be manually injected by syringe into a mass spectrometer or MS detector the In Use entries for all instruments except the mass spectrometer or MS detector must show blank spaces All instruments to be used for the sequence that you are about to submit for processing must display In Use Yes Note The Automatic Devices On command in the Actions menu applies only to In Use devices Start Instrument list Note This list has either one Yes in one of the instrument rows or all blanks for all instrument rows no Yes entries 78 LCquan User Guide Thermo Scientific 2 Preparing to Perform Quantitative Analysis Using the Run Sequence Dialog Box To add an instrument to the instruments list 1 Close all running LCquan programs 2 From the Windows taskbar choose Start gt All Programs gt Thermo Foundation 2 0 gt Instrument Configuration to open the Instrument Configuration view gt Select and configure the instrument you want to add 4 From the Run Sequence dialog box click Change Instruments The newly configu
340. n an autosampler tray if appropriate For more information see Chapter 2 Preparing to Perform Quantitative Analysis LCquan User Guide 9 1 Introduction Quantitative Analysis with LCquan 10 LCquan User Guide Acquiring data not available for the Web Access environment With LCquan you can run one sample or a series of samples from the current acquisition sequence For more information see Chapter 2 Preparing to Perform Quantitative Analysis Exploring your data The Explore window is a sandbox where you can open a raw file and play with peak detection and integration parameters that you use in the Quantitate window For more information see Chapter 3 Exploring the Data Creating a processing method The LCquan application uses a processing method to identify detect and integrate components in a chromatogram generate calibration curves quantify unknowns and produce reports For more information see Chapter 4 Creating a Processing Method Building a processing sequence A processing sequence consists of a list of sample data files and includes information about sample type and calibration or QC level For more information see Chapter 5 Creating a Processing Sequence Processing the raw files and reviewing the calibration and quantitative analysis results After the LCquan application processes the raw file you can evaluate and rework the calibration standard
341. n before the LCquan folder Click the plus sign before the Quantitate Section folder Select Automatically Lock Workbook After Creating Reports The Permission Level options become available Select the Allowed option and click OK Figure 228 Thermo Scientific 6 Processing the raw Files and Reviewing the Analytical Results Locking the Workbook Figure 228 Authorization Manager showing the Automatically Lock Workbook After Creating kes SM LCquan E _ 0 2 Be ml Root Folder Report permission set to Allowed E Run Application File Tracking Acquisition Run Dialog Explore Section Quantitate Section v4 Allow changes to selected sample info in Allow changes to column labels in proces Allow changes to column labels in results v Prompt user for Comment after manual inl Normalize quan chromatogram plots to de 4 Allow Results Export Allow Manual Integration Allow User Integration Allow Calibration Settings to be changed Create Reports Remove signature line from excel report Allow Watson File Interface Excel Formal Secure Report Template Allow Excel Rounding Automatically Lock Workbook after cr vA vA v4 y lv E lt Permission Level O Disallowed iqnature List ot if jer sord Required Allowed Disallowed State Comment To not automatically lock the workbook after you create a report 1 Choose Start gt All Programs gt Thermo Foundation gt Au
342. n is red Change and the lock is closed Unlock Makes the pane editable The lock icon is green and the lock is open Thermo Scientific 3 Exploring the Data Data Views Table 62 Peak List view parameters Sheet 2 of 4 Parameter Description Create Peak Names Create peak names for the selected peaks in the list based on the For Selected Peaks in current detected set of peaks Each peak name is auto generated in List the form Peak x xxxx where x xxxx is the RT at the peak apex A peak apex value is created corresponding to the detected peak s apex and a delta value defined by the lesser value of either of these two calculations e Apex RT minus the Peak Left endpoint e Peak Right endpoint minus the Apex RT The Peak Apex and Delta values are used to determine which label to apply to the peak When a peak is located within the range e Peak Apex minus Delta to Peak Apex plus Delta the corresponding label is used Note Delta RT is the range that is allowed from the Apex RT Create Peak Names Create peak names for all peaks in the list based on the current For All Peaks in List detected set of peaks Each peak name is auto generated in the form Peak x xxxx where x xxxx is the RT at the peak apex A peak apex value is created corresponding to the detected peak s apex and a delta value defined by the lesser value of either of these two calculations e Apex RT minus the Peak Left endpoint e Peak Rig
343. n modify the Avalon estimates by entering your own values and clicking Add to save your Start or End Threshold The Start Threshold depends on the RMS noise in the chromatogram It is the fundamental control used for peak detection so picking the best Start Threshold is essential for high quality data collection Units Absolute value of peak area counts x seconds Bunch Factor Area Threshold The number of points grouped together during peak detection This method groups several chromatographic points during integration without affecting the final area calculation of the peak Range 1 to 6 Note A high bunch factor groups peaks into clusters Controls the area cutoff Any peak with a final area less than the area threshold is not recorded Format units of area for the data P P Resolution Defines how much peak overlap must be present before two or more adjacent peaks create a peak cluster Peak clusters have a baseline drop instead of valley to valley baselines This is specified as a percent of peak height overlap Negative Peaks Thermo Scientific Automatically resets after a negative peak has been found LCquan User Guide 129 3 Exploring the Data Creating Explore Methods Table 46 Initial and Timed Events Sheet 2 of 2 Event Description Tension Controls how closely the baseline follows the overall shape of the chromatogram A lower tension traces the baseline to follow changes in the chr
344. n the view is locked lock icon is red you cannot select any menu item in the shortcut menu that changes the raw file or parameters Menu items that would cause changes are grayed out For more information about locks see Toolbar for Selectable Views on page 252 Table 100 lists the parameters for the Calibration Curve view Thermo Scientific 4 Creating a Processing Method Data Views Table 100 Chromatogram parameters Parameter Description Arrows The left and right arrows move through the chromatogram incrementing or decrementing by a single peak Shortcut Menu Open Raw File Opens the Select Raw File dialog box where you can select the raw file to load Lock against All Prevents changes to a raw file or parameters The lock icon is red Change and the lock is closed Unlock Makes the pane editable The lock icon is green and the lock is open Display Options Opens the Chromatogram Display Options dialog box where you can select pane options For more information see Chromatogram Display Options dialog box parameters on page 260 Set Spectrum to Peak Apex Select this menu item to set Selected RT at the peak apex Other panes might be updated depending on status Auto Update Expected RT From Spectrum Marker When selected makes the cursor active within a chromatogram by copying the RT position to the Expected RT box The menu item is selected indicating it is enabled
345. nal standard concentration Figure 231 You analyze the unknown sample and determine the concentration by matching the magnitude of the detector response with the magnitude on the calibration curve Using external standards is advantageous when you are analyzing various components in a sample and you can assay all compounds of interest by using a single set of external standards Although this approach offers time and cost effective quantitative analysis it cannot achieve the very highest precision and accuracy Variations in analyte and solution stability injection reproducibility and matrix interference lead to lower precision levels in the external standard method than in the internal standard method Figure 231 Calibration curve generated by using an external standard 500000 Response for unknown sample 400000 yg 300000 4 200000 4 Amount A in unknown sample 0 1 0 20 40 60 80 100 Response for target compound Amount of target compound A Using Internal Standards for Quantitative Analysis An internal standard ISTD is a component that is added to a sample to act as a response reference for one or more non ISTD components in the sample The concentration or amount of an ISTD in any standard or unknown sample remains constant Because quantitative mass spectrometric analysis usually involves multiple steps the total error in the analysis results from the accumulation of the errors at ea
346. nd to blue and places a large X in a circle in the selection box to the left of each numbered sequence row Thermo Scientific LCquan User Guide 97 2 Preparing to Perform Quantitative Analysis Using the Status View 98 LCquan User Guide To expand or collapse a folder Click the icon to expand a folder Click the icon to collapse a folder To remove a list or sequence row from the queue Click the sequence or sequence row to select it The LCquan application places a small check mark in the box to the left of the sequence or sequence row To delete the selected sequence or sequence row press the DELETE key To view specific information about an individual sample Right click the row in the queue Choose Properties from the shortcut menu To remove a sequence or sequence row that has not been acquired Select the sequence and press DELETE You cannot delete a sequence that another workbook or application submitted To view sample specific information for a selected sequence row Right click a sample in the acquisition queue Choose Sample Info from the shortcut menu to open the Sample Information dialog box Figure 58 Thermo Scientific 2 Preparing to Perform Quantitative Analysis Using the Status View Figure 58 Sample Information dialog box Sample Information C Xcalibur QuanRoot Example Study 3 Drugs W x SampleT ype Bik t lt CstsOS lt lt Sam
347. ne gt J Sort in descending order Third Order lt none gt J Sort in descending order Save As Default Cancel Help Table 127 Quantitation Results Sorting Order dialog box parameters Sheet 1 of 2 Parameter Description First Order Select a sorting topic as the first order of sorting Second Order Select a sorting topic as the second order of sorting Third Order Select a sorting topic as the third order of sorting Sort in Descending Select this check box to sort the list in descending reverse order Order Difference Increasing negative to positive percent difference The percent difference is defined as 100 x calculated amount specified amount specified amount RSD Increasing percent relative standard deviation RSD The percent RSD is the standard deviation of the difference of the calculated amount and the specified amount expressed as a percentage of the average value Area Height Increasing peak area or height Sorts by peak area when you selected Response Area in the Create Method view in the Quantitate window or the dialog box Sorts by peak height when you selected Response Height in the Create Method view in the Quantitate window or the dialog box Thermo Scientific LCquan User Guide 331 6 Processing the raw Files and Reviewing the Analytical Results Reviewing the Calibration Curve and OCs Table 127 Quantitation Results Sorting Order dialog box parameters S
348. new acquisition sequence LCquan User Guide 55 2 Preparing to Perform Quantitative Analysis Using the Setup Sequence View Fill Down Use the Fill Down command to fill sequential rows with duplicate or sequential values see Table 21 To fill sequential rows 1 Choose Fill Down to open the dialog box Figure 40 2 Select the columns whose data you want to duplicate 3 Specify the rows that you want to fill For example Fill From Row 1 To Row 10 4 Specify the number of the row you want to duplicate For example Using Row 1 5 Click OK The LCquan application duplicates or appropriately sequences column entries Figure 40 Fill Down dialog box Fill Down Select Columns Sample Type Sample Name P FileName P Study Sample ID p Client Level P Laboratory TV ISTO Corr Amt I Company Name P Dil Factor P Phone V vial Pos I Comment Ini vol p Barcode Sample Vol T Sample vt Fill From Row fi To Row E Using Row fi IV Create samples in selected empty rows during fill down OK Select Al Clear Al Cancel Help 56 LCquan User Guide Thermo Scientific Thermo Scientific 2 Preparing to Perform Quantitative Analysis Using the Setup Sequence View Table 21 Fill Down dialog box parameters Parameter Select Columns Description See Acquisition Sequence Grid Column Parameter Definitions on page 67 for descriptions of all column names and values Row Controls Fill From Row
349. ng Explore Methods on page 114 in Chapter 3 Exploring the Data Note This feature is available only for the Quantitate and Explore windows iW Sequence View When you are in the Acquisition window displays the Acquisition Setup Sequence view For more information see Using the Setup Sequence View on page 31 in Chapter 2 Preparing to Perform Quantitative Analysis When you are in the Quantitate window displays the Create Sequence view in the Quantitate window For more information see Defining a Processing Sequence on page 294 in Chapter 5 Creating a Processing Sequence Note This feature is available only for the Acquisition and Quantitate windows Results View Ea When you are in the Quantitate window displays the Review All Results view in the Quantitate window For more information see Reviewing All Results on page 365 in Chapter 6 Processing the raw Files and Reviewing the Analytical Results When you are in the Explore window displays the Explore Review view For more information see Exploring the Results on page 135 in Chapter 3 Exploring the Data Note This feature is available only for the Quantitate and Explore windows Zoom In Y Zooms in the traces along the y axis vertically to display more detail and less range Note This feature is not available in the Instruments window O Zoom Out Y Thermo Scientific
350. ngement dialog box opens Figure 182 Table 123 lists the parameters for the Column Arrangement dialog box Figure 182 Column Arrangement dialog box Column Arrangement Available Columns Displayed Columns Barcode Status P Sample Type FileName Path Sample ID Level STD Corr Amt Dil Factor Vial Pos Inj ol Sample Yol Sample Vt Sample Name Study Factory Defaults Current Arrangement Name Ouan Sequence Gri Cancel Help 2 To hide currently displayed columns select the check box before the column and click Remove mi F F F z F F F F This does not delete the column it simply hides it from display on the sequence grid All hidden columns are displayed in the Available Columns list 3 To display currently hidden columns select the check box before the column and click Add The column is added to the Displayed Columns list and is displayed on the sequence grid 312 LCquan User Guide Thermo Scientific Thermo Scientific 5 Creating a Processing Sequence Editing a Processing Sequence To change a column position a Select the check box before the column name in the Displayed Columns list b To move the column left in the grid click Move Up c To move the column right in the grid click Move Down To change the display precision a In the Places column of the Displayed Columns list select the value you want to change b Type a new value for the number
351. ngs dialog box parameters Sheet 3 of 3 Parameter Description Range s The mass or mass range of a mass range chromatogram You can enter multiple mass ranges separated by commas Up to 50 mass ranges are summed to form a chromatogram For a single mass the chromatogram is generated from that mass the mass tolerance For a mass range the chromatogram includes all masses between the exact masses entered Format Low Mass High Mass Units mass charge z Example for the ranges m through z and x through y enter m n x y Note The Range s box is inactive when you select trace type mE Smoothing Points The number of smoothing points that the smoothing algorithm applies to the data By reducing the level of noise through smoothing you improve the graphical appearance of data Format integers Range 1 to 15 odd numbers only Note To disable filtering set the parameter to 1 Move Trace Up Moves the selected trace up by one position in the Trace table Move Trace Down Moves the selected trace down by one position in the Trace table Chromatogram Pane Shortcut Menu Right click on the chromatogram pane to display the shortcut menu Figure 51 Figure 51 Chromatogram pane shortcut menu Show Component Traces Show User Traces Show Spectrum Trace Filter Spectrum Link Trace Time Bases Use User Defined Time Range Realtime Display Settings Display Raw File Reset Scaling Copy to Clipboard
352. ns damage or loss that might result from any use of this document even if the information in the document is followed properly This document is not part of any sales contract between Thermo Fisher Scientific Inc and a purchaser This document shall in no way govern or modify any Terms and Conditions of Sale which Terms and Conditions of Sale shall govern all conflicting information between the two documents Release history Revision A January 2009 Revision B April 2011 Revision C September 2011 Software version Thermo Scientific Foundation 2 0 SP1 and later Xcalibur 2 2 0 SP1 and later LCquan 2 7 and later LC Devices 2 5 SP1 and later Q Exactive 2 0 and later Exactive 1 1 SP4 and later TSQ Quantum 2 3 SP3 and later Microsoft Windows 7 Professional SP1 32 bit and Office 2010 For Research Use Only Not for use in diagnostic procedures ee Contents Prelate sicscuddauawtnrnealenennte raed a EEEE ix Related Documentanoni s0o 200 sds LO Gee es be SW ede RS as OO Oe x System Requirements sssr 0 cee cece eee eee ees xi Special Notices ederin a hea beew died eka eee chawns ee xi Contacting Us sds aaa Mie ede Sha ads Mg Hae Maes be Gals xii Chapter Introduction nnsa cecacadedveacineesdetedesoeseesebdaduscees 1 Quantitative Analysis with LCquan o ciise ide b ied ee ees ag eee epee 1 Overview of LCquan civ a dictoeatiesie etd eheneeeehivesennries 2 LCqguan Folder Structure ts out evet e E AAE O 8 LCquan A
353. ns into the table For an example of ion ratio confirmation see Ion Ratio Confirmation of Analytes Example on page 234 3 In the Retention Time Area specify a search window or retention time criteria e To specify a search window enter the value for the peak detection time search in the Window sec box When the apex of the detected peak is outside this window the peak will not be detected using retention time peak detection criteria A search window is defined as Expected min Window sec 2 e To use the retention time of the current component to adjust for retention time drift of other components select the Use as RT Ref check box e To correct for retention time drift select the Adjust Using check box and select a previously defined retention time reference from the list 4 Click Apply to save the settings for the current component Thermo Scientific LCquan User Guide 203 4 Creating a Processing Method Specifying Method Identification To finish identifying components 1 Optional Save the current settings as the default settings when settings for the following components are similar to the current settings by clicking Save As Default 2 Repeat the previous steps for all components in the raw file Defining Peak Integration Parameters Use the Peak Integration Area to select and define peak integration parameters Use the Peak Integration area to define Genesis ICIS and Avalon peak detection a
354. ns the Acquisition Sequence dialog box where you can specify ALT O gt A Info that the LCquan application update the processing sequence with acquisition sequence information whenever a sample is run In the Acquisition Sequence dialog box you can also specify the maximum number of rows in the acquisition sequence For more information see Acquisition Sequence dialog box parameters on page 104 in Chapter 2 Preparing to Perform Quantitative Analysis Options Commands Specific to Quantitate Window Table 165 Option menu commands specific to the Quantitate window Sheet 1 of 2 Command Description Keyboard shortcut Identification Opens the Identification Options dialog box where you can selecta ALT O gt I baseline noise window and void time for peak identification purposes For more information see Identification Options parameters on page 232 in Chapter 4 Creating a Processing Method Calibration Opens the Calibration Options dialog box where you can select ALT O gt C internal or external calibration for the current processing method For more information see Calibration Options dialog box parameters on page 246 in Chapter 4 Creating a Processing Method Delete Component Removes the selected component from the processing method For ALT O gt D LCquan User Guide 425 C LCquan Menu and Toolbar Reference Options Menu Table 165 Option menu commands specific to the Quantitate window Sheet 2 of 2
355. nt VWPBLab ThermoFisher 408 555 5555 Testi NewClient VYPBLab ThermoFisher 408 555 5555 Test NewClient WWPBLab ThermoFisher 408 555 5555 Testi NewClient VWPBLab ThermoFisher 408 555 5555 Testi NewClient WPBLab ThermoFisher 408 555 5555 Test NewClient WPBLab ThermoFisher 408 555 5555 Thermo Scientific LCquan User Guide 51 2 Preparing to Perform Quantitative Analysis Using the Setup Sequence View Importing or Copying a Sequence When an imported acquisition sequence is also used as the processing sequence the level names must match those defined in the processing method e When the calibration or quality control level names in the sequence are different from those in the processing method the LCquan application displays the Standard Level Names Association dialog box of the QC Level Names Association dialog box Use these dialog boxes to associate the level names in the sequence with the level names in the processing method e When the imported sequence contains calibration or QC levels but the processing method does not the LCquan application displays a warning box to inform you and then displays the Sample Type Assignment dialog box Change the sample type of these samples to Unknown in the processing sequence or discard the samples that have these levels in the processing sequence There are several ways to import or copy an acquisition sequence To drag and drop sequences from the A
356. o Manual Integration Use this option if no peak has been detected for the currently selected compound and there is no integration baseline on the chromatogram plot for you to adjust manually Set Peak to Not Found Status Discards the currently detected peak All panes are updated to reflect the fact that no peak exists When no peak is currently available this command is grayed out ISTD peaks cannot be set to Not Found status the command is grayed out After you use this command the Manually Add Peak command is available Update Expected Retention Time Update User Settings for Component in Current Row Updates the expected elution time point of maximum signal of the current component in the current row from the retention time specified in the user defined peak detection settings to the detected retention time Update Expected Retention Time Update User Settings for All Components in Current Row Updates the expected elution time point of maximum signal of all components in the current row from the retention time specified in the user defined peak detection settings to the detected retention time Update Expected Retention Time Update Method Settings for Component Using Current Row Updates the expected elution time point of maximum signal of the current component in the current row from the retention time specified in the processing method to the detected retention time LCquan User Guide 265
357. o Scientific LCquan User Guide 219 4 Creating a Processing Method Specifying Method Identification Table 83 IRC Detection Method dialog box Identification page parameters Sheet 2 of 2 Parameter Description Filter Specifies an existing filter or a filter from a preloaded filter list obtained from the current raw file This parameter is read only Note All filters are validated against the current set of filter entry rules Peak Detection Specifies the peak detection algorithm used Avalon Genesis or Algorithm ICIS The tabs in this dialog box reflect the value chosen here IRC Detection Method Genesis Integration Use this page Figure 133 to set up advanced chromatographic parameters Table 84 lists the parameters for the Genesis Integration page of the IRC Detection Method dialog box Figure 133 IRC Detection Method dialog box showing the Genesis Integration page IRC Detection Method Identification Genesis Integration Genesis Advanced S N Threshold 0 5 Valley Detection Enabled fi 0 J Constrain Peak Width 220 LCquan User Guide Thermo Scientific 4 Creating a Processing Method Specifying Method Identification Table 84 IRC Detection Method Genesis Integration page parameters Sheet 1 of 2 Parameter Description S N Threshold This multiplier specifies a signal to noise threshold for peak integration Only peaks with a signal to noise ratio greater than this value are integrated Range
358. ocessing Method A processing method provides instructions to the LCquan application about how to perform 8 q quantitative analysis on raw data A processing method contains component identification detection integration and calibration information This chapter describes how to create a 8 processing method by using the Create Method views of the Quantitate window Contents e Creating a Processing Method with the New Method Wizard e Specifying Method Identification e Specifying Method Calibration e Saving the Processing Method e Importing a Processing Method e Exporting Components and Levels to an Acquisition Sequence e Data Views Thermo Scientific LCquan User Guide 181 4 Creating a Processing Method Creating a Processing Method with the New Method Wizard Creating a Processing Method with the New Method Wizard From the Create Method view in the Quantitate window you can construct a processing method The LCquan application uses a processing method to perform automatic quantitation and report generation Start the Wizard Use the New Method Wizard to create a processing method To start the Wizard 1 From any window click Quantitate in the navigation pane The Create Method view opens 2 Open the New Method Wizard e When you have not previously defined a processing method the New Method Wizard automatically opens and leads you through the initial steps of creating a method e When you have already defined a p
359. ocessing Setup window and is included only in LCquan reports The LCquan application does not convert units Sample Wt Displays the amount of a component that has been placed in the sample The unit for this sample weight is specified in the Xcalibur Processing Setup window and is included only in LCquan reports The LCquan application does not convert units Sample Name Displays the sample name that you specified when you created the sequence Thermo Scientific 5 Creating a Processing Sequence Editing a Processing Sequence Table 121 Processing sequence grid column parameters Sheet 3 of 3 Parameter Description Comment Displays the comment entered for this sample Barcode Displays the barcode for this sample Fill Down cannot be used on Barcode column values Barcode Status Displays the status of the barcode reading User Columns User defined Displays information pertinent to the active 1 5 sample row in the acquisition sequence Use these columns to convey sample information to others or as a reminder to yourself Modifying the Column Sort Order Thermo Scientific Use the Sequence Sorting Order dialog box to can change the sorting order of the rows in the sequence Figure 181 Table 122 lists the parameters for the Sequence Sorting Order dialog box Figure 181 Sequence Sorting Order dialog box Sequence Sorting Order Sort Information First Order I lt none gt O 7 I Sort in descending order
360. of decimal places to display in the column Note When the Values column is unavailable your LCquan administrator has specified decimal rounding for exported Excel reports In this case the number of decimal places is fixed and cannot be edited To change a column width a In the Width column of the Displayed Columns list select the value you want to change b Type the new value Tip You can also resize a column width by dragging the column boundary in the heading row of the sequence grid To change an item name a In the Item column of the Displayed Columns list select the item name you want to change b Type the new name To reset column values click Factory Defaults There is no undo for this function Table 123 Column Arrangement dialog box parameters Parameter Description Available Columns Lists parameters that are not currently selected for display Displayed Columns Lists currently selected parameters which appear in the data grids Add Remove Displays Hides selected columns Move Up Move Move Up moves columns left in the grid Down Move Down moves columns right in the grid Factory Defaults Resets original column values as described in Grid parameters on page 386 in Chapter 6 Processing the raw Files and Reviewing the Analytical Results LCquan User Guide 313 5 Creating a Processing Sequence Editing a Processing Sequence Review Level
361. oints displayed in the Calibration Curve pane Diff Displays percent difference values for the data points displayed in the Calibration Curve pane These values are the percentage difference between the Calculated amount and the Expected amount Exclude e Yes Replicate is excluded from the data points displayed in the Calibration Curve pane e Blank Replicate is included in the calibration curve plot To use the Calibration Curve pane 1 In the Calibration Curve pane click the data point that you want to exclude Figure 200 2 Right click and choose Exclude from the shortcut menu 3 To include the standard again click the data point right click and choose Include from the shortcut menu Figure 200 Calibration curve showing excluded data points Excluded Data Points Area Ratio 0 5 0 0 pginL 344 LCquan User Guide Thermo Scientific 6 Processing the raw Files and Reviewing the Analytical Results Reviewing the Calibration Curve and OCs Modifying the Peak Detection and Integration Settings Thermo Scientific You can modify the peak detection and integration settings from the Survey or the Review All Results views in the Quantitate window Note If you modify the user peak detection settings and if you have peak area or peak height labeling enabled the LCquan application labels the peak with AU for area user as opposed to AA for area automatic or HU for height user as opposed to HA for heigh
362. ok after you create a report When you open a locked workbook the LCquan application displays Locked in the title bar next to the workbook name and in the status bar LCquan User Guide 397 6 Processing the raw Files and Reviewing the Analytical Results Locking the Workbook 398 LCquan User Guide 1M kod To create a locked version of the current workbook From any window choose File gt Create Locked Version of Workbook The LCquan application creates a copy of the workbook and its associated files under the current study folder The locked workbook is renamed by appending _locked_nnn to the original workbook name where nnn is the number of the locked workbook For example the first time that you create a locked workbook from the workbook Trial1 the LCquan application names the locked workbook Trial1_locked_001 The second time that you lock the workbook Triall it names the locked workbook Trial1_locked_002 You can create up to 999 locked versions of a workbook You cannot unlock a locked workbook To automatically lock the workbook after you create a report Choose Start gt All Programs gt Thermo Foundation gt Authorization Manager to open the Authorization Manager a b C In the Authorization Manager do the following Select a user group in the Secure Groups list Click Expand Tree to show the entire list of controlled features for the application From the list click the plus sig
363. ok in an existing study or create both a new study and a new workbook Start the Wizard 14 Start the New Study or Workbook Wizard To open the New Study or Workbook Wizard 1 Choose Start gt All Programs gt Thermo Xcalibur gt LCquan to open the LCquan application Figure From the LCquan startup dialog box you can open the last used workbook open an existing workbook or create a new workbook see Table 2 Figure 6 LCquan Open Workbook LCauan 2 7 A New Era in Mass Spectrometry SC pVENT Le ec Last used workbook C Xcalibur T utorial Study 343 Drug Workbook tut5 C lt calibur 4 3 Drug Workbook tut5 Most recently used Workbooks r SSID E AA a C Xcalibur 3 Drug Workbook tut5 C Xcalibur new project new Workbook1 7 C Xcalibur new project new Workbook Open an Existing Workbook E Create a New Workbook Summary Info for C calibur Tutorial Study 343 Drug Workbook tutS Created Monday August 01 2011 10 41 51 AM Modified Monday August 01 2011 10 41 51 AM Last saved by robert heather Number of saves 1 Ty Open in Review Mode Exit Help Open in Review Mode check box 2 Ensure that the Open in Review Mode check box is cleared You cannot create a new workbook in read only mode LCquan User Guide Thermo Scientific 2 Preparing to Perform Quantitative Analysis Creating a New Study and Workbook Table 2 New Study or Workbook Wizard pag
364. olders Each workbook folder holds all the information used by the LCquan application for an individual quantitative analysis project The workbook folder contains the LCquan file lqn the instrument method files meth and the audit log files In addition the workbook folder contains these folder names Exports Imports Rawfiles and Temp The Exports folder stores copies of all files that are exported from the LCquan application such as report files The Imports folder stores a copy of legacy files that you import into the workbook such as instrument method files or sequence files The Rawfiles folder contains acquired data files raw and any imported raw files The Temp folder contains backup and temporary files that it uses When you create a new project the LCquan application requires you to name the study folder and the workbook folder You can have as many different workbooks in the same study folder as you want but each workbook must have a unique name This is convenient for organizing your quantitative analysis projects For example you can use one study folder to organize all the separate quantitative analysis projects that you are doing for a particular client You cannot rename the folders that the LCquan application creates in the workbook folder The LCquan application manages all the files associated with the project You do not have to remember what individual files are named or where they reside on your hard drive Al
365. omatogram more closely A high baseline tension follows the baseline less closely over longer time intervals Units minutes Tangent Skim Enables tangent skim on any peak clusters By default the LCquan application selects the tallest peak in a cluster as the parent solvent and detects peaks on either side or both sides of the tallest peak You can also identify which peak in the cluster is the parent Tangent skim automatically resets at the end of the peak cluster Integrate On Off Turns integration on or off at the set time Shoulders On Turns on the detection of shoulders Shoulders Off Turns off the detection of shoulders Force Cluster On Turns on the grouping of peaks into a single peak Force Cluster Off Turns off the grouping of peaks into a single peak Disable Cluster On Enables the grouping effect in the specified time range Disable Cluster Off Disables the grouping effect in the specified time range 130 LCquan User Guide Thermo Scientific 3 Exploring the Data Creating Explore Methods Peak Limitations In the Limit Peaks area Figure 75 you can limit the number of peaks based on user defined thresholds see Table 47 Figure 75 Limit Peaks area Limit peaks Select top peaks V Enable Select by area Select by height Num to select 4 Rel peak height threshold l Enable To limit the number of peaks 1 To use only specific peaks defined by either area or hei
366. on made in the calibration section LCquan User Guide 387 6 Processing the raw Files and Reviewing the Analytical Results Creating and Reviewing Reports Table 152 Grid parameters Sheet 3 of 9 Parameter Response Ratio Description The ratio of the analytes area to its associated internal standards area or the ratio of the analytes height to its associated internal standards height The numerical value of the ratio Response ISTD Response The Xcalibur data system and the LCquan application do not report the response ratio for an internal standard because the ratio is exactly 1 0 by definition If the Response is Area in the Component Calibration window of the Processing Setup window the units of both Response and ISTD Response are counts sec If the Response is Height in the Component Calibration window of the Processing Setup window units of both Response and ISTD Response are counts Specified Conc The user entered amount of the component at the Cal or QC level Calculated Conc Amount of component as determined by the response ratio and calibration curve Diff Percentage difference between the calculated amount and the specified amount Undefined for internal standards RSD Relative Standard Deviation based on calculated amounts or response values response ratios when Internal standards are used CV Coefficient of Variation Peak Status 388 LCquan User Guide Displa
367. on peak detection baseline 232 integration 227 user identification settings integration 360 Avalon Peak Integration parameters Explore 128 Quantitate 215 LCquan User Guide 437 Index B Baseline parameters 364 Blank samples acquisition setup 31 defined 409 C Cal Exclusion List parameters 344 calibration curve described 404 excluding standards 342 inspecting at low concentrations 333 using external standard 406 using internal standard illustrated 408 weighting 7 Calibration Curve pane Survey page 326 Calibration Curve parameters 256 Calibration Curve shortcut menu commands 256 Calibration Curve Type area parameters 240 calibration levels matching names 52 Calibration Options parameters 246 Calibration page shortcut menu commands 242 Calibration Settings Curve parameters 337 Isotope parameters 339 Levels parameters 340 Type parameters 341 calibration standards defined 408 Change Instruments In Use parameters 78 Change menu commands Acquisition window 419 Change Sequence User Labels parameters 34 Chromatogram Comment parameters 375 Chromatogram Definition area parameters Explore 116 Quantitate 188 Chromatogram Display Options parameters Explore 155 Quantitate 260 Chromatogram List parameters Explore 157 Quantitate 273 Chromatogram List shortcut menu commands 158 chromatogram normalization with respect to detected peak 198 with respect to highest peak 198 Chromatogram pane Review All Results
368. onent Paroxetine Component Paroxetine RT 1 90 i o Relative Intensity Relative Intensity 2 0 2 0 Time min Time min When the view is locked lock icon is red you cannot select any menu item in the shortcut menu that changes the raw file Menu items that would cause changes are grayed out For more information about locks see Toolbar for Selectable Views on page 252 290 LCquan User Guide Thermo Scientific Thermo Scientific 4 Creating a Processing Method Data Views Table 117 Multi Peak Plot parameters Sheet 1 of 2 Parameter Arrows Description When you select All Samples for Selected Component in the Item Selector list you can click the left right arrow to move the selection to the previous next component until the first last component is selected When you select All Components for Selected Sample in the Item Selector list you can click the left right arrow to move the selection to the previous next sample in the sequence grid until the first last sample is selected Play Pause o Click to start or pause the timed review of the samples Click the play button to cause the peaks for the given component or sample to be displayed for the specified time interval see Multi Peak Plot parameters on page 291 The next set of peaks is displayed for the specified time interval until there are no more peaks in the list When you select the Repeat Playback check box the playback r
369. oosing Actions gt Devices On 76 LCquan User Guide Thermo Scientific Thermo Scientific 2 Preparing to Perform Quantitative Analysis Using the Run Sequence Dialog Box Table 26 Run Sequence dialog box parameters Sheet 4 of 4 Parameter Standby Description Keeps the system in the Standby state when the current sequence is completed When you select Standby you can run another sequence with only a short delay between runs Some devices do not have a Standby feature For devices with this feature a power saving or consumable saving mode is entered and the devices can be switched back on in approximately 15 minutes or less Depending the instrument this state turns gas and liquid flows to Off but maintains heaters and other subsystems in an On state so that there is no warm up time required when you change from Standby to On Note This option has the same effect as choosing Actions gt Devices Standby Off Keeps the system in the Off state when the current sequence is completed The Off state indicates that all power to the instrument which can be controlled by the LCquan software is turned Off This includes power to all heaters and most subassemblies but in some cases not all subassemblies Some devices do not have an Off feature For devices that do have this feature a power saving or consumable saving mode is entered and you can switch the devices back on in an undetermined time When several sequen
370. or is the maximum ratio of the trailing edge to the leading side of a constrained peak and calculates the retention time of the maximum extent of the right edge of the tailing peak This option is available only when you select the Constrain Peak Width option Range 0 5 to 9 0 Default multiplier 1 0 1 0 has no effect Table 42 lists the parameters for the Genesis Advanced Component Options dialog box Figure 71 Genesis Advanced Component Options dialog box Genesis Advanced Component Options m Peak Edge Detection Peak S N Cutoff r Valley Detection 20o Rise Percentage j 0 0 Pe Valley S N Report Noise As C RMS Peak to Peak oo Cancel I Help 120 LCquan User Guide Thermo Scientific Thermo Scientific 3 Exploring the Data Creating Explore Methods Table 42 Genesis Advanced Component Options dialog box parameters Parameter Description Peak Edge Detection Chromatogram peak detection criteria using the peak signal to noise S N cutoff value Peak S N Cutoff The peak edge is set to values below this defined S N This test assumes an edge of a peak is found when the baseline adjusted height of the edge is less than the ratio of the baseline adjusted apex height and the peak S N cutoff ratio When the S N at the apex is 500 and the peak S N cutoff value is 200 the LCquan application defines the right and left edges of the peak when the S N reaches a value less than 200 Range 50 0
371. or the study 1 Type the new study root folder name or click Browse to find the folder Figure 9 Study root folder page Folder where studies will be located C Xcalibur QuanFoot Browse 2 Click Next Import raw Files Import raw files into the workbook see Table 4 To import raw files into the new workbook 1 Select the Import Raw Files check box Figure 10 2 Click Browse to open the Select Raw File dialog box Figure 10 Import raw files option page J Import rawfiles File location C lt calibur QuanFoot Example Study Rawfiles Modified Number of rawfiles selected for import 0 Browse Thermo Scientific LCquan User Guide 17 2 Preparing to Perform Quantitative Analysis Creating a New Study and Workbook Table 4 Import Rawtiles page parameters Parameter Description Condition Import Rawfiles Activates the File Location list File Location Displays the location of the raw files listed in the File name list File name Lists the raw files in the selected file location Modified Lists the dates the raw files were modified Browse Click Browse to find or change the files 3 Browse to the location of your raw files For example C Xcalibur QuanRoot examples data 4 Select your raw files To highlight a group of raw files click the first file name press SHIFT and click the last file name Figure 11 Figure 11 Select Rawfile dialog box x Lookin jj Raw l
372. orrectly calculated 372 LCquan User Guide Thermo Scientific 6 Processing the raw Files and Reviewing the Analytical Results Reviewing All Results Table 144 Peak Info dialog box Flags page parameters Sheet 4 of 4 Parameter Response Low Description Indicates the calculated peak amount is lt the lowest specified standard amount of the component in the calibration curve When selected this indicates the amount was calculated by extrapolation Note Forcing or including the origin defines the lowest level to be 0 0 Response High Peak Information IRC Tests Thermo Scientific Indicates the calculated peak amount is gt the highest specified standard amount of the component in the calibration curve When selected this indicates the amount was calculated by extrapolation This information is displayed only when showing peak information for an IRC and lets you review the acceptable test levels to still be considered a confirmed ion Table 145 Peak Information IRC Tests parameters Parameter lon Coelution Test Description Passed Indicates that the peak passed the coelution test If it did not pass no further tests are performed and the Ion Ratio Test section is empty Allowed Coelution Delta The allowed difference in retention time between the apex of the target ion and the apex of the qualifier ion as specified by the Qualifier Ion Coelution parameter on the Identification
373. ort Report Selections table select the Use check box in an empty row b In the Save Type column select the file format that you want to save the report in c Inthe XReport Template column type the name of an XReport template or click the empty cell and click the down arrow to browse for a template The standard report templates contain preselected methods and results summaries For more information see Report Options on page 384 Or click Create Xreport Templates to create a custom report template Tip To preview reports with your data open a template and choose Report gt Simulate Report For information about XReport refer to User Guide Designing and Generating Custom Reports with XReport d To create additional reports with XReport repeat steps a through c Note If your administrator has set up secure reporting the Create XReport Templates feature is unavailable and you have access only to the templates in the secure template folder The only output option is Save Only which saves the file in PDE The secure reports contain a serial number in the footer of each page and a watermark appears on the background of each page 4 In the Select Report Options area select the output option You can select Print Only Save Only or Print and Save 5 To generate the specified reports click Create Reports Note When you save a report using the Save Only or the Print and Save option the LCquan applicat
374. ount and displays the integrated chromatogram peaks Quantify Sample Report LCquanQuantifySampleReport xrt Provides a summary by sample of the components for each sample The report includes sample information and quantitative analysis results for example peak areas response and calculated amount and displays the integrated chromatogram peak Quan Results Report LCquanQuanResults xrt 384 LCquan User Guide Provides quantitative results for a single sample Thermo Scientific Table 151 Report Options Sheet 2 of 2 Report option Excel Report Options 6 Processing the raw Files and Reviewing the Analytical Results Creating and Reviewing Reports Description Quan Result Grid Generates an Excel report using the current Quantitate Results column arrangement as specified on the Survey and Review All Results views of the Quantitate window This arrangement cannot be used as a template to create a new arrangement from the Review Reports view For detailed descriptions of the displayed columns see Quan Result Grid on page 386 Short Excel Summary Displays the Result list of the Quantitate window in a Microsoft Excel spreadsheet Each component is placed on a separate page worksheet in the main workbook This arrangement can be used as a template to create a new arrangement from the Review Reports view You can review analyze and graph the data by using all the functions and features of M
375. ourself Editing Column Values from the Sequence Grid Thermo Scientific There are several ways to change column values from the sequence grid To change a sample type 1 Click the cell in the Sample Type column Figure 44 Figure 44 Sample Type column Sample Type 2 Select from the list of sample type options To change a file name 1 Click the cell in the File Name column 2 To open a dialog box where you can select a new file click the arrow 3 Navigate to the new file select it and click Open LCquan User Guide 69 2 Preparing to Perform Quantitative Analysis Using the Setup Sequence View To change a calibration level 1 Click the cell in the Level column Figure 45 Figure 45 Level column Level ac_01 Cal01 v 2 Select from the list of calibration levels To change an ISTD correlation amount 1 Click the cell in the ISTD Corr Amt column 2 Highlight the text and type over it To change the dilution factor 1 Click the cell in the Dil Factor column 2 Highlight the text and type over it To change the vial position 1 Click the cell in the Vial Pos column 2 Highlight the text and type over it To change the injection volume 1 Click the cell in the Inj Vol column 2 Highlight the text and type over it 3 When you are using an autosampler set the default injection volume in the Autosampler dialog box in the Instrument Setup window To change
376. ow Use the Instrument Setup window Figure 22 to define a set of experimental parameters for each configured instrument These parameters include the operating settings for the instrument collectively referred to as the instrument method and any optional instrument settings to be used before or after the LCquan application runs a sequence of samples startup method and shutdown method respectively The parameters that are displayed in the Instrument Setup window depend on the instrument that you select in the pane on the left side of the window Note The Instrument Setup window is not available in the LCquan application for Web Access Figure 22 Instrument Setup window Sd LCquan Steroids Workbook File Quantum View Apps Options Help BA 4 e e Startup Method V instrument Method Shutdown Method Quantitate a 2 Sean Editor Syringe Pump Divert Valve Accurate Mass Method Summary Run Settings MS Acquire Time min 10 00 Segments Current Segment To display a chromatogram here use Quantum Open Raw File ft 4 4 Retention Time min Segment 1 Settings Segment Time min 10 00 Tune Method C XcalibursMethodssex TSOTune Scan Events 1 Chrom Filter Peak Width s C 10 Collision Gas Pressure mT orr C Current Scan Event 1 Scan Event 1 Polarity Pa Z Positive O Negative
377. ow number You can select multiple rows to delete 2 Right click the sequence grid to display the shortcut menu 3 Choose Delete Selected Samples To add a raw file to the processing sequence 1 Right click the sequence grid to display the shortcut menu 2 Choose Add Sample The Select Raw File dialog box opens 3 In the Select Raw File dialog box select the files you want to add and click Open The LCquan application obtains the sample information from the selected files and adds the samples to the last rows of the sequence LCquan User Guide 303 5 Creating a Processing Sequence Editing a Processing Sequence To change the sample type 1 Click the Sample Type box to display the down arrow Figure 175 Figure 175 Sample Type list sample pe __Filetame Sample C oc 7 4 2 Select the new sample type from the list to open the Select Level dialog box Figure 176 Figure 176 Select Level dialog box Select Level Component amounts for selected level Alprazolam 0 000 pg mL Nefazodone 0 000 pg mL Paroxetine 0 000 pg mL 3 Select the level you want to associate with the sample 4 Click OK Note If no levels are defined in the selected processing method the list of levels appears blank To change a range of cells Fill selected columns of selected rows with duplicate text entries or appropriately sequenced number entries a Select the row that you want to copy b Right cl
378. ow of the sequence grid 7 To change an item name a In the Item column of the Displayed Columns list select the item name you want to change b Type the new name 8 To reset column values click Factory Defaults There is no undo for this function Table 126 Column Arrangement dialog box parameters Parameter Description Available Columns Lists parameters that are not currently selected for display Displayed Columns Lists currently selected parameters which appear in the data grids Add Remove Displays Hides selected columns Move Up Move Up moves columns left in the grid Move Down Move Down moves columns right in the grid Factory Defaults Resets original column values as described in Grid parameters on page 386 in Chapter 6 Processing the raw Files and Reviewing the Analytical Results 330 LCquan User Guide Thermo Scientific 6 Processing the raw Files and Reviewing the Analytical Results Reviewing the Calibration Curve and OCs Customizing the Results Sorting Order From the Quantitation Results Sorting Order dialog box Figure 191 change the sorting order of the rows in the sequence lable 127 lists the parameters for the Quantitation Results Sorting Order dialog box Figure 191 Quantitation Results Sorting Order dialog box Quantitation Results Sorting Order Sort Information First Order LevelName O O J Sort in descending order Second Order lt no
379. ows The left and right arrows move through the chromatogram incrementing or decrementing by a single sample peak Shortcut Menu Open Raw File Opens the Select Raw File dialog box where you can select the raw file to load Lock Against Raw File Prevents changes to a raw file The lock icon is yellow and the Change lock is closed Lock Against All Prevents changes to a raw file or parameters The lock icon is red Change and the lock is closed Unlock Makes the pane editable The lock icon is green and the lock is open Display Options Opens the Chromatogram Display Options dialog box For more information see Chromatogram Display Options on page 154 Reset Scaling Resets both x and y axes to display the maximum amount of data Copy to Clipboard Copies selected data to the Clipboard You can then paste the data Chromatogram Display Options from the Clipboard buffer into a document Use Chromatogram Display Options dialog box to modify the appearance of the chromatogram Figure 94 These display options are also used when creating a processing method For more information about using these options to create a processing method see Chromatogram Display Options on page 259 in Chapter 4 Creating a Processing Method Table 55 lists the parameters for the Chromatogram Display Options dialog box Thermo Scientific Thermo Scientific 3 Exploring the Data Data Views Figure 94 Chromatogram Display Opt
380. page 367 Survey page 326 Chromatogram pane shortcut menu commands 89 438 LCquan User Guide Chromatogram parameters Explore 154 Quantitate 259 Review 263 Chromatogram shortcut menu commands Quantitate 259 Review 263 Column Arrangement parameters Acquisition 60 Quantitate 313 Review All Results 330 comparing plots 139 Component list shortcut menu commands Quantitate 241 Review 332 Component Type area parameters 237 components Component List pane 241 component types specifying 236 Survey page component list 326 contact us xii copying acquisition sequence to processing sequence 298 Correction for Isotope Contribution parameters 245 Create page 114 Create Sequence create page 315 Create Sequence edit page 301 Current Review Display view layouts 140 detection limit 405 detection peak See algorithms peak detection Disk Space parameters 106 documentation survey xii E Emergency Shutdown parameters 108 Error Report parameters Explore 159 Quantitate 274 Error Report shortcut menu commands 275 ESI probe 404 Excel long summary report 395 Excel short summary report 394 Explore Sequence sample selector 136 Explore window about 5 Create page 114 introduction 113 locks 139 Thermo Scientific Review page 135 status plots 172 status reports 174 export layout configuration 420 exporting an acquisition sequence 53 external standards defined 406 F file and folder structure 8 File menu commands 420 F
381. page 38 in Chapter 2 Preparing to Perform Quantitative Analysis ALT O sW gt A Thermo Scientific C LCquan Menu and Toolbar Reference Delete All Components Thermo Scientific more information see Specifying Method Identification on page 186 in Chapter 4 Creating a Processing Method Removes all components from the processing method For more information see Specifying Method Identification on page 186 in Chapter 4 Creating a Processing Method Options Menu Table 164 Options menu commands common to all LCquan windows Sheet 2 of 2 Command Description Keyboard shortcut Wizards gt Show the New Opens the New Method Wizard if there is no processing method ALT O gt W gt M Processing Method defined when you enter the Quantitate window The New Method Wizard when no method Wizard steps you through a procedure for creating a new is defined processing method or importing an existing processing method Wizards gt Show the New Opens the New Processing Sequence Wizard if there is no ALT O gt W gt P Processing Sequence processing sequence defined when you display the Sequence view Wizard when no of the Quantitate window The New Processing Sequence Wizard sequence is defined steps you through a procedure for creating a new processing sequence For more information see Defining a Processing Sequence on page 294 in Chapter 5 Creating a Processing Sequence Acquisition Sequence Ope
382. parameters Sheet 2 of 2 Parameter Description Detector Streams in When a raw file is open displays the list of detector streams that Current Raw File are stored in the current raw file The format of the list items is Generic Detector Stream Name Instrument Name Model Name Channel Name Shortcut Menu Open Raw File Create Method view only Opens the Select Raw File dialog box where you can select the raw file to load Lock against All Prevents changes to a raw file or parameters The lock icon is red Change and the lock is closed Unlock Makes the pane editable The lock icon is green and the lock is open Reset Scaling Resets scaling to the default pane Copy to Clipboard Copies a chromatogram mass spectrum or calibration curve to the Clipboard You can then paste the data from the Clipboard buffer into a document 286 LCquan User Guide Thermo Scientific 4 Creating a Processing Method Data Views Status Report View This view displays the status information stored in the current raw file in table format Figure 165 Figure 165 Status Report view 4 gt al Ms Quantum F Status Report RT 1 920 Parameter vate 8 kY lonized Voltage 8 k lonized Current Sheath Gas Flow Rate Aux Gas Flow Rate lon Sweep Gas Flow Rate API Temperature Capillary Temperature Capillary Offset Tube Lens Offset Forepressure QO RF Power Q0 RF Voltage Q00 Offset Lens 0 Of
383. parameters for the Cal Exclusion List dialog box Figure 199 Cal Exclusion List dialog box Cal Exclusion List Paroxetine 25 000 pg mL 25 000 pg mL 25 000 pg mL 25 000 pg mL 25 000 pg mL 50 000 pg mL 50 000 pg mL 50 000 pg mL 50 000 pg mL 50 000 pg mL 75 000 pg mL 75 000 pg mL 75 000 pg mL 75 000 pg mL 75 000 pg mL 100 000 pg mL 100 000 pg mL 100 000 pa mL Mi OK Cancel Apply Help e When points are co located they cannot be graphically individuated as plot points and the granularity of the cursor is not fine enough to manually change the individual exclusion status e These points can be included or excluded in the Results grid if they are listed here Note When data is co located the plotted data appears to be mismatched to the text entry lists 3 For each standard that you want to exclude from the calibration curve click the Exclude column of its row The word Yes appears in the Exclude column 4 Click Apply to incorporate the change To include the standard again click the word Yes and click Apply Thermo Scientific LCquan User Guide 343 6 Processing the raw Files and Reviewing the Analytical Results Reviewing the Calibration Curve and OCs Table 133 Cal Exclusion List dialog box parameters Parameter Description Level Displays calibration level names for the sample data points displayed in the Calibration Curve pane Expected Displays expected quantity values for the data p
384. passed if the difference between the observed and expected quantities is within the user defined tolerance A QC sample is classified as failed if the difference between the observed and expected quantities is outside the defined tolerance A blank sample contains no target components but might contain an ISTD when you use the internal standard quantitative analysis technique By analyzing a blank sample you can confirm that there are no residual components in the solvent system that can cause erroneous results LCquan User Guide 409 ee i LCquan Flow Diagrams Use this set of LCquan flow diagrams as a reference The flow diagrams show step by step progressions through the different LCquan windows Contents e LCquan Flow Diagram e LCquan for Web Access Flow Diagram e LCquan Acquisition Flow Diagram e LCquan Quantitate Flow Diagram Thermo Scientific LCquan User Guide 411 B LCquan Flow Diagrams LCquan Flow Diagram LCquan Flow Diagram The LCquan flow diagram shows the steps for creating a new study or workbook and the four main windows of the interface Create new study or Study and Workbook Names page Click Next Import Raw Files page Click Open and Next Import from legacy files Import Instrument Method Open existing or workbook Create new study or workbook last used workbook J a LCquan or open existing workbook opens last used view i Acquisition Sequence Click Next
385. pdate the sequence select Auto Update the Processing Sequence When Acquiring If you are currently acquiring data when you select this option a message box prompts you to update the current processing sequence with the most recent list of samples acquired If you do not respond to the message box after five seconds the LCquan application automatically updates the sequence or Right click the sequence grid and choose Import Processing Sequence gt Copy from Last Acquired Sequence from the shortcut menu For details about the Acquisition Sequence parameters see Processing Data While Continuing to Acquire Data on page 104 in Chapter 2 Preparing to Perform Quantitative Analysis Editing a Processing Sequence If you already created a processing sequence use the Create Sequence views to edit the current sequence or create a new one e Using the Create Sequence edit View you can review the sequence information and edit the information in many of the sequence columns e Using the Create Sequence create View you can associate new raw files with sample types or levels Using the Create Sequence edit View 300 LCquan User Guide When you use the LCquan application for quantitative analysis you do not always have to create a new processing sequence Instead you can copy the acquisition sequence from the current workbook to use as the processing sequence or import an existing sequence from another workbook o
386. petitive Noise l RMS Noise Peak Parameters Min peak width 3 Multiplet resolution fi 0 Area tail extension 5 Area scan window ja OK Cancel Apply Help a Select a noise method e To use a single pass algorithm to determine the noise level select the INCOS Noise option e To use a multiple pass algorithm to determine the noise level select the Repetitive Noise option b To use an RMS calculation instead of the default ICIS noise method select the RMS Noise check box c To specify the minimum number of scans required in a peak enter a value in the Min Peak Width box d To specify the minimum separation in scans between the apexes of two potential peaks enter a value in the Multiplet Resolution box e To specify the number of scans past the peak endpoint to use in averaging the intensity enter a value in the Area Tail Extension box f To specify the number of allowable scans on each side of the peak apex enter a value in the Area Scan Window box Thermo Scientific LCquan User Guide 123 3 Exploring the Data Creating Explore Methods Table 43 lists the parameters for the ICIS Peak Integration area Table 43 ICIS Peak Integration area parameters Sheet 1 of 2 Parameter Description Baseline Window The number of scans used when searching for where the minima are occurring A local minimum becomes an anchor point making the entire curve locally reduced until these points are at ze
387. ple Info parameters Explore 170 Review 283 Sample Info shortcut menu commands Explore 170 Review 283 Sample Information parameters 99 Sample Selector parameters 138 Sample Selector shortcut menu commands 138 sample types blanks 409 quality controls QCs 409 standards 408 unknowns 408 Save As Wizard 22 saving workbook 400 scan filters matching with components 201 Select Component parameters 243 Select Directory parameters 107 semi quantitative analysis definition 401 sequence See acquisition sequence processing sequence sequence grid function buttons 35 Sequence Sorting Order parameters 311 shortcut menus Acquisition Sequence History Acquisition 36 Explore 133 Status 103 Calibration Curve 256 Calibration page 242 Chromatogram Quantitate 259 Review 263 Chromatogram List 158 Chromatogram pane 89 Component list Quantitate 241 Review 332 Error Report 275 Filter List 276 LCquan User Guide 443 Index T General Parameters Plot Explore 162 Quantitate 277 Ion Ratio Confirmation area 195 IRC Chromatogram 269 Mass List Explore 165 Quantitate 280 Multi Peak Plot Explore 179 Quantitate 291 Peak List Explore 166 Review 282 processing sequence grid 306 result grid 327 Run Status 96 Sample Info Explore 170 Review 283 Sample Selector 138 Spectrum Explore 171 Review 268 Spectrum pane 91 Status Plot Explore 173 Review 286 Status Report Explore 175 Review 288 Tune Method Explore 177 Review 289 shortcuts keybo
388. ple Name B File Name APN _001 Sample ID JAPNOO Path C Xcalibur QuanRoot Example Study 3 Drugs Workbook R awtiles Instrument File C Xcalibur QuanFioot E sample Study 3 Drugs Workbook 3 Drugs Position TrayOlOT ss Inj Volume t Cst s lt lt tr lt it i SS Level er Samp Weight f0O E Sample Volume fO ISTD Corr Amt D_i Dill Factor E o Study Dus i itsti S SO O OOO Client _ Laboratory Quans SS Company Thermo Electon i S C Phone PE Comment Bik Table 33 Sample Information dialog box parameters Sheet 1 of 3 Parameter Description Sample Type Displays the type of sample described by the acquisition sequence row The sample type defines how the LCquan application processes the sample data Each sample must be classified as one of these LCquan sample types e Unknown Blank e QC quality control e Standard Sample Name When this box is in a sequence or Result grid row Displays the sample name that you specified when you created the sequence Thermo Scientific LCquan User Guide 99 2 Preparing to Perform Quantitative Analysis Using the Status View Table 33 Sample Information dialog box parameters Sheet 2 of 3 Parameter File Name Description When this box is in an acquisition sequence Displays the name of the raw file that contains the sample data The File Name is a combination of the Base File Name prefi
389. ponent Type Area e Target Compound Area e Component List Pane e Calibration Shortcut Menu e Correction for Isotope Contribution This section contains instructions for performing the following e Specifying Calibration Options e Specifying Internal Standards and Target Compounds e Reviewing the Integrated Peaks Component Type Area Use the Component Type area Figure 141 to specify the type of component internal standard or target Table 91 lists the parameters for the Component Type area Figure 141 Component Type area Component Type Component Type Target Compound ISTD Target Units ret Units pa mL pg mL STD Amour ISTD Amount fi 70 1 000 ISTD Units tae 236 LCquan User Guide Thermo Scientific 4 Creating a Processing Method Specifying Method Calibration Table 91 Component Type area parameters Parameter Description Target Compound Specifies that the component you are calibrating is a target compound This option disables ISTD Amount and ISTD Units and enables all elements in the Target Compound box ISTD Specifies that the component you are calibrating is an internal standard This option enables ISTD Amount and ISTD Units and disables all elements in the Target Compound box except Isotope and the response group Note ISTD ISTD Amount and ISTD Units are not available when you perform an external calibration Target Units The unit type for the target compound This option is a
390. port Template Generator Figure 222 Launch XReport Template Generator area Launch Report Template Generator To start the XReport template generation feature click Create XReport Templates Figure 222 For more information about XReport refer to the Help for XReport Note If your administrator has set up secure reporting the Create XReport Templates feature is unavailable Select Report Options Figure 223 Select Report Options area Select Report Options C Print Only Save Only Print and Save 382 LCquan User Guide Thermo Scientific Function Buttons 6 Processing the raw Files and Reviewing the Analytical Results Creating and Reviewing Reports Use the following commands to save print and create reports Parameter Description Print Only Prints the report without saving Save Only Saves the reports without printing Print and Save Prints and saves the reports Note If your administrator has set up secure reporting you can use only the Save Only option Create Reports Creates the reports that are selected in the Use column check boxes When no Use check boxes are selected the feature is unavailable Apply Saves any changes to the Excel report or XReport grids on the Review Reports view When you do not click Apply and close the view the grid reverts back to the last saved status Review Reports Warning Thermo Scientific The Review Reports dialog box warns that you selected report op
391. pplications Menu ci2ctescssbied ee reee 9 Acquiring and Processing Data with LCquan 0 00000 10 LGguan for Weh ACESS eros ener misen or rE EEEE EEE here Kaede Kees 12 Chapter2 Preparing to Perform Quantitative Analysis n 13 Creating a New Study and Workbook lt cise c2csc aed see asdewnee dees 13 Create a New Workbook ouccclnntudedeadencbe como niu agen duet 14 Saving a WOrk0OU i sce ctaeeeatabbbesdeedace ddeardicerdchensts 22 File Tracking Resultsi 45 i0s tata ctv aciex ieee e nip e be eee Kes 26 Workbook Summary Information o n ananuna 27 Introduction to the Instrument Setup Window 0 00000 e ee eee 28 Creating Instrument Methads Jaca toured eo annae 29 Importing an Existing Instrument Method 000 0 00 aee 30 Introduction to the Acquisition Window si cus 4s ses cee gees eens 30 Using the Setup Sequence View oink cd dine edd bee reed eRe eee eae neds 31 Acquisition Sequence Header cts cep es co 2 hee eed ROS eRe CESS 33 Acquisition Sequence History 0 6 eee eee 35 Acquisition Sequence Grid crac eee eee eee Rew Ee hae ees 37 Using the Run Sequence Dialog Baiy 205 uscVjaSaheseusatiidksubead es 71 Change INStEUmErntS rosae he aia eheada E sad eee eae kee 78 Thermo Scientific LCquan User Guide iii Contents iv LCquan User Guide Chapter 3 Using th Status WHEW oiee chon Seated ath arate hk E EE EA bt Ra e we 80 Using the Status View Control Buttons csistn eaein
392. processing method see Chapter 4 Creating a Processing Method Exporting a Processing Sequence Thermo Scientific To save the current sequence to a file in the workbook Exports folder choose File gt Export gt Processing Sequence The LCquan application saves the file with the sld file extension You can later import this file using a command such as File gt Import gt Processing Sequence gt From File LCquan User Guide 321 es i Processing the raw Files and Reviewing the Analytical Results This chapter describes how to process raw data by using the processing method and the processing sequence that you created in the previous chapters It also explains how to review the results for each component in each raw file and how to produce reports Contents e Processing the raw Files e Reviewing the Calibration Curve and QCs e Reviewing All Results e Creating and Reviewing Reports e Exporting Results e Locking the Workbook e Saving the Workbook and Exiting the LCquan Application Processing the raw Files The LCquan application processes the raw files in the processing sequence when you open the Survey view the Review All Results view or the Review Reports view of the Quantitate window It uses the peak detection and integration parameters in the processing method to detect and integrate peaks for all components Using the calibration parameters set in the processing method it builds a calibration cu
393. ps Options Help Tanja waa O000000 90 e Instruments a w O 0 89O E Quantitate is Acquisition Queue Acquisition History E Run Manager lt Run Manager Status gt Sequence Sample Name Working On Position Raw File Inst Method Waiting for Acquisition to Start Waiting for Acquisition to Start Waiting for Acquisition to Start Waiting for Acquisition to Start Data file Number of traces 4 For Help press F1 Powers Dana 7 7 2008 9 46AM To open the Run Status tab When the left navigation pane is displayed a Click Acquisition b Click the Status icon c Click the Run Status tab When the left navigation pane is hidden a Choose View gt Section Selection gt Acquisition Section b Choose View gt Step Selector gt Acquisition Status c Click the Run Status tab Figure 56 94 LCquan User Guide Thermo Scientific Thermo Scientific Figure 56 Run Status tab 2 Preparing to Perform Quantitative Analysis Using the Status View Run Status Acquisition Queue Acquisition History Run Manager Ready To Download Sequence Sample Name Working On Pasition Raw File Inst Method Simulation MS Ready to Download a Status iv Scan speed x Last scan Start time min Real time elapsed min Status First scan Scan number Repeat count Stopped 5 1 1145 1145 4 001333 0 805733 0 To di
394. quisition Window Table 170 Commands specific to acquisition window Command Description Keyboard shortcut Step Selector Switches to the selected Acquisition view e Acquisition Setup e Acquisition Acquire e Acquisition Status 428 LCquan User Guide Thermo Scientific c LCquan Menu and Toolbar Reference View Menu Commands Specific to Explore Window Table 171 Commands specific to the Explore window Command Description Keyboard Shortcut Select Review Display Selects a review layout for the current display ALT V gt Multi Peak Vi DOWN ARROW gt aa RIGHT ARROW gt e Comparison View ENTER e Peak List View e Large Chro View or e Sample Info View DOWN ARROW gt ENTER Commands Specific to Quantitate Window Table 172 Commands specific to Quantitate window Command Description Keyboard shortcut Component Selector Bar Only in Survey and Review All Results views Displays or hides the Component list at the far right of the screen When this option is cleared the components are listed as tabs above the Results grid Step Selector Switches to the selected Quantitate view e Quantitate Method e Quantitate Sequence e Quantitate Survey e Quantitate Review All e Quantitate Reports Select Review Display Selects a review layout for the current display ALT V gt e Trivle P DOWN ARROW gt np i a RIGHT ARROW gt Dis Pane ENTER e Single Pane DOWN ARROW gt ENTER Thermo Scientific LCquan User Guide 429 C LCquan
395. quisition queue proceeds from the top sequence to the bottom sequence and from the top sequence row to the bottom sequence row for each sequence The LCquan application places a large X to the left of each completed sequence row as samples are acquired Each sequence is labeled with the client doing the acquisition Sequences are stored in the All Sequences folder and sequence rows are stored in the Sequence folder specified by their paths for example C methods Test sld The sequences and sequence rows are organized in a tree view that displays the folders as an indented outline Table 33 lists the parameters for the Sample Information dialog box To open the Status Acquisition Queue page When the left navigation pane is displayed a Click Acquisition b Click the Status icon c Click the Acquisition Queue tab When the left navigation pane is hidden a Choose View gt Section Selection gt Acquisition Section b Choose View gt Step Selector gt Acquisition Status c Click the Acquisition Queue tab Figure 57 Acquisition Queue tab Status Acquisition Queue All Sequences _ E 8YT methodsT est4S T eat Sequence Row 1 Sequence Row 2 Sequence Row 3 Sequence Row 4 Sequence Row 5 Sequence Row 6 3 0 When the LCquan application is acquiring a sample the background of its test tube is green After the sample is acquired the LCquan application changes the backgrou
396. r 28 un2002 WB_28Jun2002 W B_28 e sn Fie side 500 SRM med 110 00 40 00 127 0428 595 AS Statut Oven Tray Enor log Target inection Vial postion A14 Ingection volume uL 10 00 AS state Waking for pump Doce state Close No For Help press F1 __ 10 3 2005 9 57 al The following topics provide information about the Status view of the Acquisition window e Using the Status View Control Buttons e Acquisition Status Chromatogram Pane e Acquisition Status Spectrum Pane e Acquisition Status Run Status e Acquisition Status Acquisition Queue 80 LCquan User Guide Thermo Scientific Thermo Scientific 2 Preparing to Perform Quantitative Analysis Using the Status View e Acquisition Status Acquisition History e Processing Data While Continuing to Acquire Data The Status view of the Acquisition window contains the following e A set of control buttons The run manager status that provides a summary of the acquisition progress It lists the sequence and sample information and the readback status of each configured instrument The instrument status that provides the complete readback status of each configured instrument The acquisition queue that displays all the sequences that have been submitted for acquisition including those for other workbooks The sequences and their individual sequence rows are organized in a tree view Each row of the tree view contains a che
397. r Acquisition Clears the Acquisition Sequence History information from History the workbook The Acquisition Sequence History is intended to show how the workbook acquired data The original workbook contains this information Start New Audit Trail Creates a new Audit Trail An Audit Trail entry states that this 2 Click Next workbook was created from another workbook and includes the original workbook file path Note If this check box is cleared the LCquan application copies the Audit Trail first and makes the creation entry To complete the Save As Wizard click Finish After you create a new workbook the LCquan application opens the Instrument Setup window When you have not created instrument methods for the configured devices the LCquan application creates default methods LCquan User Guide 25 2 Preparing to Perform Quantitative Analysis Creating a New Study and Workbook File Tracking Results 26 LCquan User Guide For validation purposes the LCquan application keeps track of whether files in a workbook have been deleted or modified since the last LCquan session with that workbook If you attempt to open an existing workbook and a validation error message about a file tracking failure appears click OK in the message dialog box The File Tracking Results dialog box opens Figure 20 It lists the files in your workbook and flags the files that have been deleted or modified Figure 20 File Tracking Resul
398. r Description Study Name Creates a new study folder with this name in the root folder for example C Xcalibur QuanRoot Workbook Name Creates a new workbook with this name in the study folder Change Study Root Changes the root folder for the study The default root folder Folder is C Xcalibur QuanRoot Changing the root folder requires authorization from your administrator Open in New Window Opens a new LCquan window using the new workbook or study The original LCquan window remains open 3 Type the study and workbook names for this workbook 4 Click Next Depending on the options you chose do one of the following e When you are changing the root folder go to Change the Root Folder e When you are keeping the current root folder go to Complete the Wizard Thermo Scientific LCquan User Guide 23 2 Preparing to Perform Quantitative Analysis Creating a New Study and Workbook Change the Root Folder Save the study to a new root folder The default root folder is C Xcalibur QuanRoot To specify a new root folder for the study 1 Type the new study root folder name or click Browse to find the folder Figure 18 Figure 18 Root folder path page Folder where studies will be located C calibur QuanF oot Browse 2 Click Next Copy the Files Select the files you want to copy from the current workbook to the new workbook see Table 8 To copy the files 1 Select the files you want to
399. r Guide c Select whether the noise used in calculating S N values is calculated using an RMS calculation or Peak to Peak resolution threshold For a detailed description of advanced parameters see Genesis Advanced Component Options parameters on page 208 Table 77 Genesis Peak Integration area parameters Sheet 1 of 2 Parameter Advanced Description Opens the Genesis Advanced Component Options dialog box Figure 127 S N Threshold This multiplier specifies a signal to noise threshold for peak integration Only peaks with a signal to noise ratio greater than this value are integrated Range 0 0 to 999 0 Default multiplier 0 5 Valley Detection Enabled Detects unresolved peaks with the valley detection approximation method The LCquan application drops a vertical line from the apex of the valley between unresolved peaks to the baseline The intersection of the vertical line and the baseline defines the end of the first peak and the beginning of the second peak Expected Width sec This multiplier specifies an expected peak width parameter that controls the minimum width that a peak is expected to have when valley detection is enabled With valley detection enabled any valley points nearer than half the expected width to the top of the peak are ignored If a valley point is found outside the expected peak width the LCquan application ends the peak at that point It always ends a peak when
400. r legacy file With these techniques you can quickly define your processing sequence Thermo Scientific 5 Creating a Processing Sequence Editing a Processing Sequence After you copy or import a sequence use the Create Sequence edit view to review the processing sequence to ensure that it is correct and if necessary alter the processing sequence in several different ways e To review the processing sequence e To remove a raw file from the processing sequence e To add a raw file to the processing sequence e To change the sample type e To change a range of cells e To modify the arrangement of the processing sequence Thermo Scientific LCquan User Guide 301 5 Creating a Processing Sequence Editing a Processing Sequence To review the processing sequence 1 To open the Create Sequence edit view Figure 173 and display the new processing sequence click Edit Figure 173 Create Sequence edit window El Thermo LCquan 3 Drugs Workbook y File Zoom View Apps Options Help BPSBSAE FFAOOKOSCSCSOS Kis OlO Create Sequence select files review and edit Acquisition FileName Rawrfiles 4PN_003 raw Acquisition Sequence History Sample Name OO Study Dus t S Standard 1 Conc 25 pg ml A Client f i y y Laboratory fOuanSqud Company Themo Electon Phones s lt isi s S Prev Row Row 1 NextRow Help ee nman 004 Rawfile APNOO3 1 000 Tray01 2 30 00 0 000 0 000 3Drugs Slew APN_005 R
401. r more information see Resolving Mismatched Level Names on page 319 e Workbook Does Not Contain Levels dialog box opens when the samples are associated with level names but the workbook processing method has no calibration or QC levels defined For more information see Resolving Missing Calibration or QC Levels on page 321 Resolving Mismatched Level Names The Standard or QC Level Names Association dialog box warns you of a discrepancy in the calibration level names or QC level names under the following conditions e Importing an acquisition sequence The level names in the acquisition sequence do not match the level names in the workbook e Importing a processing sequence The level names in the imported processing sequence do not match the level names in the workbook processing method e Adding sample files to a processing sequence The level names associated with the added samples do not match the level names in the workbook To resolve the level names for an imported sequence 1 Verify that you imported the correct sequence 2 To associate the level names do one of the following Figure 186 e Select the level name in the Sequence Level Names pane and drag it to the appropriate level name in the Method Level Names pane processing method or the Workbook Level Names pane or e Select the two level names and click Add Thermo Scientific LCquan User Guide 319 5 Creating a Processing Sequence
402. r the Select By Area or Select By Height option c Specify a number to select This limits the listed peaks to the n most significant d Click Apply The LCquan application generates a peak list for the peaks in the chromatogram Figure 85 Note In Explore only the peak name is editable in the peak list Thermo Scientific Figure 85 Generated peaks list 3 Exploring the Data Exploring the Results dra Chromatogram gt a Peak List v steroids04 TIC RT 0 01 3 79 NL 1 08E6 RT 0 68 Relative Intensity so 4 a i RT 1 98 RT 3 15 stercnds04 Explore Peak Name Apex RT Area of Largest Area of Total Area 0 675 3154 1 983 Hi 382 30 35 0875 3451 1 983 ESN 3621463 2715440 2633421 4569926 700 000 34358 74 982 25 763 72 717 24 984 43 351 14 885 NOT SAVED 11 17 2005 2 28 PF 3 To generate names for the peaks right click the peak list and choose Create Peak Names gt For All Peaks in List from the shortcut menu The LCquan application generates peak names in the form Peak x xxxx where x xxxx is the retention time at the peak apex Figure 86 The component name is the same as the peak name Figure 86 Peak list A Peak 2 649 2 649 123502 2 408 84685 100 000 68 570 4 To
403. ram Normalization In the Quantitate window the LCquan application can normalize the chromatogram plot so either the height of the detected peak is 100 or the height of the highest peak is 100 Figure 121 You select which normalization to use in the Authorization Manager Note In the Explore window the LCquan application normalizes the chromatogram so that the height of the highest peak is 100 Figure 121 Multi Peak Plot view steroids02 TIC SM 7 RT 0 40 2 40 NL 1 23E6 ce a sesrooo Normalized to detected peak 905 804 705 Relative Intensity go Q 6 Duis pN Lusi w I N S J 050 1 22 pig Baa oa anata nd aa aS ne nasi SS GAL UN LIL lta at a 04 06 08 1 0 12 14 16 18 20 22 Time min steroids02 TIC SM 7 RT 0 40 2 40 NL 3 71E6 mn 068 Normalized to highest peak 90 80 70 2 60 E 50 gt 5 ga RT 1 40 AA 6657090 0 4 0 6 0 8 1 0 1 2 14 16 18 2 0 22 Time min 198 LCquan User Guide Thermo Scientific 4 Creating a Processing Method Specifying Method Identification To normalize the chromatogram plot so that the detected peak is 100 1 Choose Start gt All Programs gt Thermo Foundation gt Authorization Manager to open the Authorization Manager dialog box Figure 122 Figure 122 Authorization Manager showing Normalize Quan Chromatogram Plots to Detected Peak permission set to Allowed l
404. rameter that controls the minimum width that a peak is expected to have when valley detection is enabled With valley detection enabled any valley points nearer than half the expected width to the top of the peak are ignored If a valley point is found outside the expected peak width the LCquan application ends the peak at that point It always ends a peak when the signal reaches the baseline independent of the value set for the expected peak width Range 0 0 to 999 0 Default multiplier 0 0 Units seconds Note Valid only when you select Valley Detection Enabled Constrain Peak Width Constrains the peak width of a component during the peak integration of a chromatogram You can set values that control when peak integration is turned on and off by specifying a peak height threshold and a tailing factor Selecting this activates the Peak Ht and the Tailing Factor parameters Peak Height Specifies a percentage of the total peak height This is the minimum that a signal must be above the baseline before integration is turned on or off This option is available only when you select the Constrain Peak Width option Range 0 to 100 Default multiplier 5 0 The peak height percentage is defined as follows 100 Peak Height Thermo Scientific LCquan User Guide 353 6 Processing the raw Files and Reviewing the Analytical Results Reviewing the Calibration Curve and OCs Table 136 User Identification Settings d
405. re separated by commas Select Manage Export Column Arrangement Select an Arrangement Select a previously defined column arrangement from this list to edit or delete Note You cannot delete the Quantitate Results Grid Excel Long Summary and Excel Short Summary column arrangements that ship with the LCquan application Create Edit View an Opens the Column Arrangement dialog box to edit the currently Arrangement selected column arrangement You can create a new column arrangement by editing an existing arrangement and changing its name Delete Selected Deletes the selected arrangement Anangament Note You cannot delete the Quan Results grid Excel Long Summary and Excel Short Summary column arrangements that ship with the LCquan application Button Export Exports the data to the text file or files Locking the Workbook Thermo Scientific You can create a locked version of a workbook at any time except when you are acquiring data by using the File gt Create Locked Version of Workbook command This locked workbook is essentially a copy of the workbook and its associated files that cannot be overwritten You cannot save any changes made to a locked workbook and you cannot acquire data in a locked workbook You can create new reports but the LCquan application does not save the report selections Alternatively you can have the LCquan application automatically lock the workbook not a copy of the workbo
406. red instrument appears on the Instrument list To specify the Start Instrument 1 From the Run Sequence dialog box click Change Instruments 2 Click the Start Instrument list in the row of the instrument you want to be the start instrument The blank space changes to display a Yes The autosampler is usually selected to be the start instrument because this is the instrument that controls when a run starts In this case all instruments to be used for the sequence submission including the autosampler display In Use Yes This means they are waiting for a contact closure event to start operation When this status has been achieved by all devices used in the run the start instrument initiates the run Thermo Scientific LCquan User Guide 79 2 Preparing to Perform Quantitative Analysis Using the Status View Using the Status View The Status view of the Acquisition window Figure 48 provides a real time display of the data acquisition Use the Status view to monitor data acquisition and display chromatograms and mass spectra in real time Figure 48 Status view of the Acquisition window AN LCquan Steroids Workbook TES Change Actions Yiew Apps Options Help Heb G4 Wi gt OOOSSOO 0 Aca amp Run Manager Acquiing Sequence C Xcalbur 28Jun2002 WB_28 un2002i T emp Acg Sample Name Working On Seq Row 42 Position A 14 Raw File C Xcalibur 28 un2002 W B_28 un2002 R awfiles SCI Inst Method C Xcalibu
407. ring processing All RT References appear in the Adjust Using list View Width min The amount of time to display the chromatogram pane Range Expected RT View Width 2 through Expected RT View Width 2 Range 0 10 to 999 0 Units minutes Thermo Scientific LCquan User Guide 191 4 Creating a Processing Method Specifying Method Identification Table 73 Retention Time area parameters Sheet 2 of 2 Parameter Description Adjust Using Specifies that the retention time for this component be adjusted based on the actual RT of another component that has been designated an RT Reference Retention time references are automatically created if you select the Use as RT Reference option when the component is active The processing method must have at least one retention time reference for this box to be active All RT References appear in the Adjust Using list Adjust Using list Specifies the RT Reference from the list to be used to adjust the expected retention time of the current component This list is active only when you select the Adjust Using check box The actual retention time of the RT Reference component is used to adjust the retention time of the active component automatically during processing The adjustment to the expected retention time Corrected RT Component Expected RT Component Expected x RT Reference Actual RT Reference Expected Peak Integration Area Use the Peak Integration area to s
408. ring to Perform Quantitative Analysis Using the Status View Processing Data While Continuing to Acquire Data 104 LCquan User Guide While acquiring data you can enter the Quantitate window to process the data you have already acquired You must first complete the procedures for defining a processing method and a processing sequence For more information see Chapter 4 Creating a Processing Method and Chapter 5 Creating a Processing Sequence You can use the defined processing sequence or you can replace the defined processing sequence with a shortened sequence that is based on the data already acquired To update the processing sequence with the most recent list of acquired samples 1 To enter the Quantitate window click Quantitate in the navigation pane 2 Choose Options gt Acquisition Sequence Info to open the Acquisition Sequence dialog box Figure 60 Table 35 lists the parameters for the Acquisition Sequence dialog box Figure 60 Acquisition Sequence dialog box Acquisition Sequence Auto update the processing sequence when acquiring Maximum number of rows allowed in acquisition sequence 500 Cancel Help 3 To update the sequence select the Auto Update the Processing Sequence When Acquiring check box If you are currently acquiring data when you select this option a message box prompts you to update the current processing sequence with the most recent list of samples acquired If yo
409. ription Load New Sequence Selects a sequence to load to the Explore Sequence area even if the Acquisition History is the active sequence Remove Active Deletes the selected sequence The command is effective only if Sequence the Explore Sequence is active 138 LCquan User Guide Thermo Scientific Locks Thermo Scientific 3 Exploring the Data Exploring the Results Table 50 Sample Selector shortcut menu commands Sheet 2 of 2 Command Description Rename Active Renames the selected sequence The command is effective only if Sequence the Explore Sequence is active Replace Active Replaces the currently selected sequence A file selection dialog Sequence box opens where you can select the replacement file This command is not available when any of the sequences under Quantitate Sequence or Acquisition Sequence History are selected The contents of the sequence are replaced but the name does not change Show Sequence Items Collapses the currently selected sequence to display the sequence name only Show Expands the currently selected sequence to display the sample Info Level Sample names Items Use locks to simultaneously compare data from different raw files for example to see how the acquisition progressed from sample to sample You can compare a plot from one sample to the plots of other samples by locking one plot and clicking down the sequence in the sample selector Change the state of the lock by
410. rmation about locks see Toolbar for Selectable Views on page 252 Table 108 lists the parameters for the Filter List view LCquan User Guide 275 4 Creating a Processing Method Data Views Table 108 Filter List view parameters Parameter Description Arrows Not used in the Create Method view The left and right arrows move through the sequence list incrementing or decrementing by a single sample Column Heading Filters Displays the list of unique filters that are stored in the current raw file Shortcut Menu Open Raw File Create Method view only Opens the Select Raw File dialog box so you can select the raw file to load Lock against All Prevents changes to a raw file or parameters The lock icon is red Change and the lock is closed Unlock Makes the pane editable The lock icon is green and the lock is open Copy to Clipboard Copies selected data to the Clipboard You can then paste the data from the Clipboard buffer into a document General Parameters Plot View for Survey or Review All Results Only This data view is available from the Survey or Review All Results views in the Quantitate window Figure 159 This data view displays the selected parameters over the sequence for all detected peaks in the current view All Standards QCs Blanks or Unknowns Select the parameters you want to see plotted from the Item Selector list Figure 159 General Parameters Plot view Ot ga gat
411. rmo Scientific LCquan User Guide 117 3 Exploring the Data Creating Explore Methods Genesis Peak Integration 118 LCquan User Guide Use the Genesis Peak Integration area to define Genesis peak detection algorithms to be applied to the active raw file see Table 41 To specify Genesis peak integration parameters In the Peak Detection Algorithm box select Genesis Type a multiplier value in the S N Threshold box To detect unresolved peaks with the valley detection approximation method select the Valley Detection Enabled check box To set the expected peak width parameter and control the minimum width that a peak is expected to have enter a multiplier value in the Expected Width sec box To constrain the peak width of a component during the peak integration of a chromatogram select the Constrain Peak Width check box e To specify the minimum above the baseline before integration is turned on or off enter a percent of the total peak height in the Peak Ht box e To constrain the peak width of an asymmetric chromatogram peak that has a tailing trace enter a peak integration multiplier in the Tailing Factor box To specify advanced component detection criteria click Advanced Figure 70 Figure 70 Genesis Peak Integration area Genesis Peak Integration Peak Detection Algorithm Gensis B Advanced S N Threshold 05 Valley Detection Enabled m l Constrain Peak Width m m Use thes
412. ro Range 1 to 500 Default 40 Area Noise Factor The noise level multiplier This determines the peak edge after the location of the possible peak so that the peak can narrow or broaden without affecting the baseline Range 1 to 500 Default multiplier 5 Peak Noise Factor The noise level multiplier a minimum S N ratio This determines the potential peak signal threshold Range 1 to 1000 Default multiplier 10 Constrain Peak Width Constrains the peak width of a component during the peak integration of a chromatogram You can set values that control when peak integration is turned on and off by specifying a peak height threshold and a tailing factor Selecting this activates the Peak Ht and the Tailing Factor parameters 124 LCquan User Guide Thermo Scientific 3 Exploring the Data Creating Explore Methods Table 43 ICIS Peak Integration area parameters Sheet 2 of 2 Parameter Peak Ht Description A percent of the total peak height This is the minimum that a signal must be above the baseline before integration is turned on or off Range 0 to 100 Default multiplier 5 0 The peak height percentage is defined as follows 100 Z TN Peak Height Note Valid only when you select the Constrain Peak Width check box Tailing Factor A peak integration multiplier that constrains the peak width of an asymmetric chromatogram peak that has a tailing trace It is th
413. rocessing method use the Options gt New Processing Method menu command to open the wizard Figure 109 Welcome page for the New Method Wizard New Processing Method Wizard xi Welcome to the New Processing Method Wizard LCQuANn 2 7 This wizard helps you set up a new Processing A New Era in Mass Spectrometry Method 3 Click Next 182 LCquan User Guide Thermo Scientific 4 Creating a Processing Method Creating a Processing Method with the New Method Wizard Create or Import a Method Create a new processing method or importing an existing one from a file see Table 70 To define a processing method 1 Do one of the following e Select the Create New Method option and the Initialize with Acquisition Component Names check box e Select the Import Existing Method option Note The Initialize with Acquisition Component Names check box is available only when you have defined component names Figure 110 When you create a new acquisition sequence you can assign temporary component names using a sequenced base component name such as Comp1 Comp4 To assign custom component names use the Acquisition Levels dialog box available from the Acquisition Sequence grid shortcut menu Refer to the Standard and QC Levels command on the shortcut menu Figure 110 Create or import method page Create new method I Initialize with acquisition component names C Import existing method Table 70 Options for the creat
414. rrow or broaden without affecting the baseline Range 1 to 500 Default multiplier 5 356 LCquan User Guide Thermo Scientific 6 Processing the raw Files and Reviewing the Analytical Results Reviewing the Calibration Curve and OCs Table 138 User Identification Settings dialog box ICIS Integration page parameters Sheet 2 of 3 Parameter Description Peak Noise Factor The noise level multiplier a minimum S N ratio used to determine the potential peak signal threshold Range 1 to 1000 Default multiplier 10 Constrain Peak Width Constrains the peak width of a component during the peak integration of a chromatogram You can set values that control when peak integration is turned on and off by specifying a peak height threshold and a tailing factor Selecting this activates the Peak Ht and the Tailing Factor parameters Peak Height A percent of the total peak height This is the minimum that a signal must be above the baseline before integration is turned on or off Range 0 to 100 Default multiplier 5 0 The peak height percentage is defined as follows 100 Peak Height Note Valid only when you select Constrain Peak Width Thermo Scientific LCquan User Guide 357 6 Processing the raw Files and Reviewing the Analytical Results Reviewing the Calibration Curve and OCs Table 138 User Identification Settings dialog box ICIS Integration page parameters Sheet 3 of 3 Parameter Descriptio
415. rum Integration info Detected By Highest Peak w Valid Left Edge Type Baseline Right Edge Type Baseline r Flags E Saturated E GC Failed E Response OK O Calculated Amount w RT Ref OK Response Low E valley Det Response High Peak Type Method Table 144 Peak Info dialog box Flags page parameters Sheet 1 of 4 Parameter Description Integration Info read only Detected By Displays the method the LCquan application uses to detect the peak It displays one of the following detection methods e Spectrum the LCquan application uses the user defined mass intensity pairs and applies a spectral matching algorithm to find the peak that contains the closest match to the comparison spectrum Up to 50 entries are allowed to define the comparison spectrum e Highest Peak the LCquan application searches for the highest peak within the search window e Nearest RT the LCquan application searches for the peak nearest to the expected retention time 370 LCquan User Guide Thermo Scientific Thermo Scientific 6 Processing the raw Files and Reviewing the Analytical Results Reviewing All Results Table 144 Peak Info dialog box Flags page parameters Sheet 2 of 4 Parameter Left Edge Type Description Displays how the LCquan application detected the left baseline edge of the current peak It displays one of the following peak baseline detection methods Baseline B edge type The edge of the peak is at bas
416. rum pane that correspond to the selected component Only these ions are included in the chromatogram b In the Filter list select a scan filter for the selected component and click Apply The LCquan application applies the scan filter to the data in the raw file and displays the resulting filtered chromatogram data in the Chromatogram pane Note The LCquan application created these scan filters from the instrument method parameters that were defined and used to obtain the selected raw file It displays all scan filters in the raw file For a detailed description of the Chromatogram Definition area see Chromatogram Definition Area on page 187 Thermo Scientific LCquan User Guide 201 4 Creating a Processing Method Specifying Method Identification 3 In the Chromatogram pane a 202 LCquan User Guide To specify the expected retention time and retention time window for the selected component right click and choose Auto Update Expected RT from Spectrum Marker from the shortcut menu Right click again and choose Set Spectrum to Peak Apex from the shortcut menu The LCquan application automatically does the following e Determines the peak apex scan maximum or selected scan and draws a vertical red bar in the component peak in the Chromatogram pane e Displays the spectrum for the apex peak scan or selected scan in the Spectrum pane e Displays the chromatogram for all ion masses displayed in the Spectrum pan
417. rument Method List right click the Instrument Method preview pane When the view is locked lock icon is red you cannot select any menu item in the shortcut menu that changes the raw file or parameters Menu items that would cause changes are grayed out For more information about locks see Toolbar for Selectable Views on page 252 Table 110 lists the parameters for the Instrument Method List view Table 110 Instrument Method List view parameters Sheet 1 of 2 Command Description Open Raw File Create Method view only Opens the Select Raw File dialog box where you can select the sample file to load Lock against All Disables the Open Raw File command for the Instrument Method Change preview pane The lock icon is red and the lock is closed 278 LCquan User Guide Thermo Scientific Mass List View Thermo Scientific 4 Creating a Processing Method Data Views Table 110 Instrument Method List view parameters Sheet 2 of 2 Command Description Unlock Enables the Open Raw File command for the Instrument Method preview pane The lock icon is green and the lock is open Copy to Clipboard Copies the contents of the Instrument Method preview pane to the Clipboard To create a report you can paste the copy into other software This view displays the spectral mass and intensity list for a sample at the selected retention time Figure 161 In the Survey or Review All Results views in the Quantitate window the Item Se
418. rve to quantitate the QCs and unknowns Note You need to define a processing method and a processing sequence before you can open the Survey view the Review All Results view or the Review Reports view You can review the results in any order Thermo Scientific LCquan User Guide 323 6 Processing the raw Files and Reviewing the Analytical Results Reviewing the Calibration Curve and OCs Reviewing the Calibration Curve and OCs Use the Survey view in the Quantitate window to review and rework the calibration standards The process of reviewing and reworking the calibration standards involves these steps 1 Opening the Survey View 2 Reviewing the Calibration Standards Oo Excluding a Calibration Standard from the Calibration Curve optional A Modifying the Peak Detection and Integration Settings optional 5 Manually Integrating Peaks optional 324 LCquan User Guide Thermo Scientific 6 Processing the raw Files and Reviewing the Analytical Results Reviewing the Calibration Curve and OCs Opening the Survey View After creating the processing sequence you are ready to review the calibration curves To process the raw files and open the Survey view Figure 188 click the Survey icon in the navigation pane Figure 188 Quantitate Survey window A LCquan Steroids Workbook ile Zoom View Apps Options Help HEHE RES PR COOGHOOO KHL VO Survey review standards and QCs ___Faename Semple Type
419. s User Identification Settings Detection 350 LCquan User Guide Use this page Figure 203 to specify advanced component detection criteria Use these additional criteria when the standard detection criteria do not provide the expected results Table 135 lists the parameters for the Detection page of the User Identification Settings dialog box Figure 203 User Identification Settings dialog box showing the Detection page User Identification Settings Identification Detection ICIS Integration ICIS Advanced Peak Detection Algorithm x i Highest Peak Min Peak Height S N C Nearest RT 2o 2s Target Ratio Window Cancel Apply Apply Toal Help Table 135 User Identification Settings dialog box Detection page parameters Sheet 1 of 2 Parameter Description Peak Detection Genesis ICIS or Avalon the peak integration parameters Algorithm Highest Peak The LCquan application searches for the highest peak within the search window Nearest RT The LCquan application searches for the peak nearest to the expected retention time lon Ratio Confirmation Enable When Ion ratio confirmation is enabled the IRC grid is available Note When disabled all entry areas within the Ion ratio confirmation group are disabled Ion ratio using Displays area or height Thermo Scientific Thermo Scientific 6 Processing the raw Files and Reviewing the Analytical Results Reviewing the
420. s Reports automatically after data acquisition and processing completion Instrument Method Use Startup Method Specifies that the LCquan application runs the optional startup method before the sequence starts No raw file is acquired by this method and no autosampler injection takes place This feature is not available for all devices Use Shutdown Method Specifies that the LCquan application runs the optional shutdown method after the sequence has completed No autosampler injection takes place This feature is not available for all devices Programs Pre Acquisition Displays the current Pre Acquisition program exe or bat Browse program that runs before data acquisition for every sequence row Post Acquisition Displays the current Post Acquisition program exe or bat that Browse runs after data acquisition for every sequence row Run Synchronously Pre Acquisition Specifies that the Pre Acquisition program displayed in the Pre Acquisition box runs synchronously in series The Run Manager waits until the Pre Acquisition program can be run prior to data acquisition For example when you want to switch the divert valve before a run you can select a synchronous Pre Acquisition program By default the program runs asynchronously in parallel with data collection For example you can use the XConvert exe program to perform file conversions from one data type to another data type during processing
421. s aki Integration Type Cal Eqn 2 Response Client ISTD Response Comment Mi Response Ratio Company Name N Response Type Delta RT j Movel TO Specified Conc Dil Factor Calculated Conc Duration Diff Exclude 1 RSD Height CV F F mi E T m r F The Excel Short Summary column arrangement displays the following parameters e FileName e Response Ratio CV e Sample Type e Response Type e Peak Status e Sample Name e Specified Conc e Level e Integration Type e Calculated Conc e Units e Response e Diff e RT e ISTD Response e RSD e Sample ID Thermo Scientific Excel Long Summary Thermo Scientific 6 Processing the raw Files and Reviewing the Analytical Results Creating and Reviewing Reports The Excel long summary is a column arrangement for creating Microsoft Excel formatted reports from the Reports view in the Quantitate window You can use the Excel Long Summary column arrangement as a template to create a new arrangement Figure 226 shows Excel Long Summary selected in the Column Arrangement dialog box Figure 226 Column Arrangement dialog box Column Arrangement Available Columns iem J wian Paces Barcode Barcode Status Exclude Integration Type ISTD Response Lett Delta Height Response Right Delta Height NA NA NA NA Displayed Columns FileName Sample Type Sample Name Sample ID Spec
422. s acquired by the current workbook along with other relevant information such as the operator start and end times status and comment You can modify the Acquisition Sequence History pane see Table 48 Figure 76 Acquisition Sequence History pane Acquisition Sequence History Sequence_1 Sequence_2 4PN_005 4PN_006 4PN_OO7 4PN_008 Table 48 Acquisition Sequence History pane parameters Sheet 1 of 2 Parameter Description Info Operator Displays the login ID of the person who is logged onto the system and who is acquiring the sequence Start Time Displays the time when the first sample or startup method began End Time Displays the time when the last sample or shutdown method completed When the LCquan application is closed before acquisition is complete but the acquisition is allowed to complete in the background the end time is displayed as Unknown Thermo Scientific Thermo Scientific 3 Exploring the Data Creating Explore Methods Table 48 Acquisition Sequence History pane parameters Sheet 2 of 2 Parameter Comment Description Displays a comment about the sequence as entered in the Run Sequence dialog box For more information see Run Sequence dialog box parameters on page 74 in Chapter 2 Preparing to Perform Quantitative Analysis Status Displays the sequence status one of the following e Waiting Created but not yet submitted e Submitted Submitted
423. s type them in the Comment box 2 Change a User Label name or value a Right click anywhere on the sequence to display the shortcut menu b Choose User Labels and Values to open the Change Sequence User Labels dialog box Figure 25 c Type your label name changes and select values for those labels d To return to the Sequence view click OK Your changes are reflected in the sequence header and in the user defined columns on the grid For an example of user defined columns and values see User Defined Columns Values Examples on page 50 Figure 25 Change Sequence User Labels dialog box Change Sequence User Labels Changes in the labels and values will be applied to all rows New rows will also use these values User Label 1 Study User Value 1 Study User Label 2 Client User Value 2 Client One User Label 3 Laboratory User Value 3 SanJoseLab User Label 4 Company Name User Value 4 Thermo User Label 5 Phone User Value 5 408 555 5555 Cancel Help Thermo Scientific LCquan User Guide 33 2 Preparing to Perform Quantitative Analysis Using the Setup Sequence View Table 11 Parameters in the Change Sequence User Labels dialog box Parameter Description User Label 1 to 5 Displays information pertinent to the selected sample row in the sequence Use these boxes to convey information about this sample to others or as a reminder to yourself e The User Label 1 default name is S
424. s are reflected in panes that have yellow or green locks For example if you click a Chromatogram pane the focal point moves in a Chromatogram List pane even if the latter pane has a yellow lock e All shortcut menu commands that can change the raw file or components in the pane are unavailable al Green Unlocked The lock icon is an open lock Any changes you make in any panes are reflected in panes that have green locks Note When you apply a yellow or a red lock to a pane you essentially capture take a snapshot of that pane as it was just before you locked it Play Pause Runs an automated action for the current pane Item Selector list Displays a list of selectable items for the current pane Source Current uA Yaporizer Temp C Sheath Gas Flow Rate Aux Gas Flow Rate Capillary Voltage Capillary Temp C lon Gauge x10e 5 Torr Convectron Gauge Torr Detector Selector list The detector stream for the data type of interest MS Quantum When a raw file is currently selected for the pane the detector box contains a list of the detector streams in the raw file in the form Generic Detector Stream Name Instrument Name Model Name Channel Name When redisplaying only the current selection and any other selection available from the raw file is displayed 152 LCquan User Guide Thermo Scientific 3 Exploring the Data Data Views Table 53 Toolbar for Selectable Views controls
425. s by modifying the peak detection and integration settings You can also manually integrate peaks For more information see Chapter 6 Processing the raw Files and Reviewing the Analytical Results Creating reports The LCquan application provides both standard and custom printed reports Standard reports contain preselected methods and results summaries Custom reports contain methods and results summaries that you select from a list of options For more information see Chapter 6 Processing the raw Files and Reviewing the Analytical Results Thermo Scientific 1 Introduction LCquan for Web Access LCquan for Web Access The LCquan application supports the Thermo Scientific Web Access Server which is hosted ona Web page Web Access is used for application virtualization to manage LCquan configuration and maintenance The LCquan virtual application runs on centralized servers only keystrokes and mouse clicks travel over the network from the user s computer and only screen images travel over the network to the user s computer Validate LCquan only on the server LCquan 2 7 and later versions support the Web Access environment for data processing LCquan for Web Access includes only the Explore and Quantitate windows of LCquan You can perform certain actions within the LCquan application such as data acquisition only on a local validated computer and not the server Thermo Scientific LCquan User Guide 11 es
426. s selected data to the Clipboard You can then paste the data from the Clipboard buffer into a document The Sample Info view displays sample information stored in the current raw file during acquisition Figure 102 Figure 102 Sample Info view Row Number when Acquir Sample Type Std Bracket Filename APN_003 RAVY Sample ID APNOO2 Path C Xcaliburi uanRoot 3Dru Instrument Method C KcaliburiQuanRoot 3Dru Processing Method LCquan Internal Processing Method Level ISTD Correction Amount Dilution Factor Vial Position Injection Volume Sample Yolume Sample Weight Sample Name Study Client Laboratory Company Name Phone Comment Barcode Barcode Status Cal_01 0 00 4 00 TrayO1 2 30 00 0 00 0 00 3Drugs QuanSquad Thermo Electron Standard 1 Conc 25 pgitn J The contents of the grid are read only Thermo Scientific LCquan User Guide 169 3 Exploring the Data Data Views When the view is locked lock icon is yellow you cannot select any menu item in the shortcut menu that changes the raw file Menu items that would cause changes are grayed out For more information about locks see Toolbar for Selectable Views on page 150 Table 63 lists the parameters for the Sample Info view Table 63 Sample Info view parameters Parameter Arrows Description The arrows are not
427. se it is grayed out Change Peak Baseline Survey or Review All Results views only Opens the Baseline dialog box so you can review and modify peak baseline properties For more information see Manually Integrating Peaks on page 363 in Chapter 6 Processing the raw Files and Reviewing the Analytical Results Display Options Reset Scaling Opens the Ion Ratio Chromatogram Display Options dialog box where you can select view options For more information see lon Ratio Chromatogram Display Options on page 271 Resets scaling to the default pane Copy to Clipboard 270 LCquan User Guide Copies a chromatogram mass spectrum or calibration curve to the Clipboard You can then paste the data from the Clipboard buffer into a document Thermo Scientific lon Ratio Chromatogram Display Options Thermo Scientific 4 Creating a Processing Method Data Views Use this dialog box Figure 155 to modify the appearance of the IRC chromatogram view Table 105 lists the parameters for the Ion Ratio Chromatogram Display Options dialog box Figure 155 lon Ratio Chromatogram Display Options dialog box lon Ratio Chromatogram Display Options Style r Label with Plotting V Retention time 2 Decimals Point To Point Name Base peak l Area C Stick When no peak found I Signaltonoise Height Label Styles J Offset Size joo I Rotated Boxed Label threshold 0 0 I Show apex t
428. se changes are grayed out For more information about locks see Toolbar for Selectable Views on page 252 Table 114 lists the parameters for the Status Plot view 284 LCquan User Guide Thermo Scientific 4 Creating a Processing Method Data Views Table 114 Status Plot view parameters Sheet 1 of 2 Parameter Arrows Description The left and right arrows move through the item list incrementing or decrementing by a single item Item List Forepressul MS Quantu 7 Forepressure Q0 RF Power Q0 RF Yoltage Q00 Offset Lens 0 Offset Lens 12 Offset Q1 Rod Driver Voltage Q1 Rod Driver Voltage Q1 RF Detected Q1 RF Power Q1 Amplifier Temperature Q1 Thermal Hat Temperature Lens 21 Offset Item Selector List Options Parameter Create Method view only Select the parameter you want to plot on the y axis e 8 KV Ionized Voltage e 8 kV Ionized Current e Sheath Gas Flow Rate e Aux Gas Flow Rate Ion Sweep Gas Flow Rate e API Temperature e Capillary Temperature e Capillary Offset e Tube Lens Offset e Forepressure e QO RF Power e QO RF Voltage QO00 Offset Lens 0 Offset QO Offset Detector Selector List Options MS Displays status information for a mass spectrometer Status Displays status information for a device that does not log data Thermo Scientific LCquan User Guide 285 4 Creating a Processing Method Data Views Table 114 Status Plot view
429. ser specified data Arguments Lists arguments that are to be passed to the application when it is started No validation is made to the argument list Initial Directory Defines the starting directory You can manually enter a path or use Browse to select a location No validation is made to the initial directory Browse Browses for an initial directory When you select an application with Browse the Initial Directory field automatically fills Close Closes the dialog box and accepts the changes Acquiring and Processing Data with LCquan Thermo Scientific With the LCquan application quantitative analysis usually involves the following processes The order of some of these processes is not rigid For example before you acquire data you can create the processing method and processing sequence and use these to define the acquisition sequence 1 Developing an instrument method not available for the Web Access environment The LCquan application uses an instrument method to store the specific set of parameters that operate the autosampler LC pump or MS pump mass spectrometer divert valve or syringe pump during analysis For more information see Chapter 2 Preparing to Perform Quantitative Analysis 2 Building an acquisition sequence of samples not available for the Web Access environment An acquisition sequence defines each sample as a standard unknown QC or blank and identifies its position o
430. several known standards the LCquan application constructs a calibration curve that it uses to accomplish the quantitative analysis of unknown samples It provides a number of options for fitting the calibration data to generate the calibration curve You can have it fit the calibration data to the following curve types e Linear e Quadratic e Linear log log e Quadratic log log e Average response factor RF e Point to point e Cubic spline e Locally weighted You can also have the LCquan application weight the calibration data with the following weighting functions when performing the least squares fit to the calibration data Equal e 1 X e 1 xX e 1 Y e 1 Y e 1 S where S X Y In addition you can have it ignore the origin use the origin as a data point or force the calibration curve to include the origin The Quantitate window contains these views Create Method Create Sequence Survey Review All Results and Review Reports Thermo Scientific 1 Introduction Quantitative Analysis with LCquan LCquan Folder Structure Thermo Scientific When you start a new quantitative analysis project the LCquan application creates a hierarchical folder structure for the project on the hard drive C Example Study E Example Workbook C Exports C Imports C Rawfiles C Temp The top most level in the file structure is the study folder This folder is used to organize one or more workbook f
431. sing actions for the acquisition sequence see able 26 You can run one sample or a sequence of samples After you have created your acquisition sequence click the Acquire icon in the navigation pane to open the Run Sequence dialog box Use this dialog box to run one sample or a sequence of samples and select acquisition options and processing action options for the acquisition sequence To open the Run Sequence dialog box When the left navigation pane is displayed a Click Acquisition b Click the Acquire icon Thermo Scientific LCquan User Guide 1 2 Preparing to Perform Quantitative Analysis Using the Run Sequence Dialog Box 72 LCquan User Guide When the left navigation pane is hidden a Choose View gt Section Selection gt Acquisition Section b Choose View gt Step Selector gt Acquisition Acquire To run a series of samples in the current acquisition sequence 1 In the Run Rows box enter the range of row numbers of the samples that you want to run Figure 46 Figure 46 Run Sequence dialog box Run Sequence m Acquisition Options User dana powers Run Rows he Sequence Name Sequence_8 Comment Instrument E Start Instrument Simulation MS Yes M Start When Ready Change Instruments m Processing Actions I Process after Acquisition T Generate Selected Reports m Instrument Method I Use Startup Method P Use Shutdown Method Programs
432. sition Sequence grid columns User Defined Columns Values Examples e Acquisition Sequence Grid Column Parameter Definitions LCquan User Guide 37 2 Preparing to Perform Quantitative Analysis Using the Setup Sequence View Creating an Acquisition Sequence Use the New Acquisition Sequence Wizard to create a new acquisition sequence and optionally a new workbook or new study folder for the sequence Start the Wizard Use the New Acquisition Sequence Wizard to create a new acquisition sequence To open the New Acquisition Sequence Wizard 1 Do one of the following e When you have a sequence open Right click the acquisition sequence and choose New Acquisition Sequence Wizard from the shortcut menu Figure 27 e When you do not have a sequence open In the left navigation pane click Acquisition Figure 27 Welcome page of the New Acquisition Sequence Wizard New Acquisition Sequence Wizard x Welcome to the New Acquisition Sequence Wizard LCauan 2 7 This wizard helps you set up an Acquisition Sequence A New Era in Mass Spectrometry and if requested a new Study Workbook 2 Click Next Create a Workbook 38 When you do not want to create a new workbook click Next on this wizard page and go to the instructions to Import an Acquisition Sequence To create a new workbook 1 Select the Create a New Workbook check box Figure 28 Figure 28 Create a new workbook page IV Create a New Workbook 2
433. splay an instrument s status Click the instrument name in the list of instruments The status displays below the Run Manager status in the lower left area of the page Table 32 Run Status page parameters Sheet 1 of 2 Parameter Run Manager lt Run Manager Status gt Description Acquiring or Acquiring in another Workbook displays the latter if the currently acquiring sample is being acquired by another client Sequence Displays which sequence is currently being acquired Sample Name Displays a unique alphanumeric name assigned to each sample In the LCquan application the assigned name can be up to 50 characters long LCquan User Guide 95 2 Preparing to Perform Quantitative Analysis Using the Status View 96 LCquan User Guide Table 32 Run Status page parameters Sheet 2 of 2 Parameter Description Working On Displays a sequence spreadsheet row that describes the characteristics of a single sample The LCquan application numbers all sequence rows with a row number You can select multiple row numbers to define a sample sequence Rows samples are created modified and deleted using the Setup Sequence view For more information see Acquisition Sequence Header on page 33 Position Also called the vial number displays the vial position in the autosampler tray of the current sample This readback value is displayed only if your LC provides this information under direct contro
434. stem Thermo Scientific LCquan User Guide 435 ee ie Index Symbols raw files locked and unlocked 139 A acquiring data acquisition sequence 31 flow diagrams 414 overview 10 procedure 72 remote acquisition 109 time stamping raw files during remote acquisition always time stamp 109 never time stamp 109 Acquisition Levels parameters 63 acquisition queue navigation 97 acquisition sequence copying to processing sequence 298 definition 31 exporting 53 grid parameter definitions 67 printing 53 Acquisition Sequence History sample selector 136 viewing 101 Acquisition Sequence History parameters Explore 132 Status 102 Acquisition Sequence History shortcut menu commands Acquisition 36 Explore 133 Status 103 Acquisition Sequence parameters 104 Acquisition window overview 30 Run Sequence dialog box 71 Run Status page 94 Status page 80 Thermo Scientific algorithms peak detection Avalon baseline 232 integration 227 Genesis advanced 223 baseline 232 integration 220 ICIS advanced 227 baseline 232 integration 225 user identification settings Avalon integration 360 Genesis advanced 354 integration 363 ICIS advanced 363 integration 356 analyte detection using mass spectrometry 403 animations create method 249 create sequence 318 APCI probe 404 APCI APPI probe 404 App Selection parameters 10 Apps menu commands 419 associating raw sample files with sample types 315 audit trail opening the utility 421 Aval
435. style to display the active chromatogram or spectrum using point to point peak profile Stick Select a graphic style to display the active chromatogram using vertical lines On Off Labeling Option When No Peak Found Show Apex Time Selects to always display maxima labels Labels Axis Offset Select one or both to offset the displayed plot from the x axis y axis or both X Select to have the x axis offset move the y axis slightly above the x axis so that you can see baseline details Y Select to have the y axis offset move the x axis slightly to the right of the y axis so that you can see plot details at low x axis values LCquan User Guide 261 4 Creating a Processing Method Data Views Chromatogram View for Survey or Review All Results Only 262 LCquan User Guide This view is available from the Survey or Review All Results views in the Quantitate window This view displays a chromatogram and all detected peaks for a sample Figure 152 The Item Selector List options for this view are identical to those for the Chromatogram List view so you can compare the two views side by side To adda peak 1 Right click the pane and choose Set Peak to Not Found Status 1 Right click the pane and choose Manually Add Peak The pointer changes to Ria 2 Right click the pane and choose Add or Change Peak Comment The Chromatogram Comment dialog box opens 3 To enter comments about the manually added peak see
436. t automatic To modify the peak detection and integration settings Select a sample by clicking its row in the Result list Right click the Chromatogram pane and choose User Peak Detection Settings from the shortcut menu The User Identification Settings dialog box opens For details of user identification settings see Specifying Additional Peak Detection Criteria on page 346 To modify the peak detection settings click the Detection tab and change the settings as necessary To modify the peak integration settings click the Integration tab Change the settings as necessary to correct problems with noise in the peak unresolved peaks or peak tailing To modify the advanced peak detection parameters click the Advanced tab Adjust the settings as necessary to improve peak detection and integration if baseline noise causes interference Apply the changes e To apply the changes to the selected sample in the Result list click Apply e To apply the changes to all the samples on the current page of the Result list for example to all samples on the Standards page click Apply To All When you are finished click OK to close the dialog box If you changed any parameters since you last clicked Apply or Apply To All clicking OK applies the new changes to the sample selected in the Result list When you modify the peak detection and integration parameters the LCquan application displays User Integr
437. t File Name Please enter the name you wish to use for the Exported file No path will be accepted the file will be created in the Export folder of the Workbook and the file extension will be automatically added Name of Exported File MyExportConfig Full Name of Exported File C Xcalibur QuanRoot E xample Study3 Drugs Workbook Exports MyE xportConfig Ict Cancel 2 Type a name for the exported configuration file Type only the file name You cannot change or enter the path The LCquan application saves the exported file in the Exports folder for the current workbook 3 Click OK The LCquan application places the config file in the Exports folder Note When you change the properties of a display and save the workbook the new properties will be active when you reopen the workbook Preview Panes For each sample you can display various types of data The preview panes in the Explore window contain a list of views to display the data in various forms Use the Toolbar for Selectable Views to select any of these views e Chromatogram View e Chromatogram List View e Error Report View Thermo Scientific LCquan User Guide 141 3 Exploring the Data Exploring the Results e Filter List View e General Parameters Plot View e Instrument Method View e Mass List View e Peak List View e Sample Info View e Spectrum View e Status Plot View e Status Report View e Tune Method View e Multi Peak Plot View
438. t menu Explore 165 Quantitate 280 Masses 190 Multi Peak Plot Explore 179 Quantitate 291 Multi Peak Plot shortcut menu Explore 179 Quantitate 291 Multi Peak Options 180 Peak Identification area 193 Peak Information Flags 370 Info parameters 368 IRC Tests 373 Peak List Explore 166 Quantitate 281 Peak List shortcut menu Explore 166 Review 282 Peak Name List Editor 147 processing sequence grid columns 308 processing sequence grid shortcut menu 306 Quantitation Results Sorting Order 331 Realtime Display Settings 86 result grid shortcut menu 327 Results Export 396 LCquan User Guide 441 Index P Retention Time area 191 Review Reports 383 Run Sequence 74 Run Status 95 Run Status shortcut menu 96 Sample Info Explore 170 Review 283 Sample Info shortcut menu Explore 170 Review 283 Sample Information 99 Sample Selector 138 Sample Selector shortcut menu 138 Select Component 243 Select Directory 107 Sequence Sorting Order 311 Sort Sequence 58 Spectrum Explore 171 Review 268 Spectrum Display Options 92 Spectrum pane shortcut menu 91 Spectrum shortcut menu Explore 171 Review 268 Status Plot Explore 172 Review 285 Status Plot shortcut menu Explore 173 Review 286 Status Report Explore 174 Review 287 Status Report shortcut menu Explore 175 Review 288 Switching to Processing during Acquisition 105 Target Compound area 238 Tune Method Explore 176 Review 289 Tune Method shortcut menu Explore 177 Review 289 User Iden
439. t the Disallowed option and click OK Figure 123 Thermo Scientific 4 Creating a Processing Method Specifying Method Identification Specifying Component ID Parameters and Integrating the Peaks When the LCquan application acquires data it creates unique scan filters according to the type of experiment you specify in the instrument method When you load a raw file it lists the scan filters associated with the raw file in the Filter list To quantitate components you must filter the chromatogram Use the Peak Detection Algorithm list in the Peak Integration Area to specify the type of peak detection algorithm ICIS Genesis or Avalon you want to use to analyze the raw data These algorithms apply smoothing construct a chromatogram using the scan or mass filters assign peak numbers generate a peak list and determine the peak start and peak end points All algorithms provide component peak detection and chromatographic peak detection For detailed instructions on selecting peak detection algorithms see Defining Peak Integration Parameters on page 204 To detect and integrate a component peak in the current raw file 1 In the Component list select a component The Component list is ordered by increasing retention time Figure 124 Figure 124 Component list Component Paroxetine Nefazodone 2 Specify these parameters in the Chromatogram Definition Area a In the Mass box enter ion masses from the Spect
440. t time Turns on the detection of shoulders Shoulders Off Turns off the detection of shoulders Force Cluster On Turns on the grouping of peaks into a single peak Force Cluster Off Turns off the grouping of peaks into a single peak Disable Cluster On Enables the grouping effect in the specified time range Disable Cluster Off Thermo Scientific Disables the grouping effect in the specified time range LCquan User Guide 231 4 Creating a Processing Method Specifying Method Identification Identification Options 232 LCquan User Guide Use the Identification Options dialog box to select a baseline and noise window for peak identification purposes The Genesis Baseline parameters in the dialog box apply to all samples in a sequence not just component by component They apply only when you use the Genesis peak detection algorithm Table 90 lists the parameters for the Identification Options dialog box To open the Identification Options dialog box From the window menu choose Options gt Identification The Identification Options dialog box opens Figure 138 Figure 138 Identification Options dialog box Identification Options Baseline Baseline and Noise Window min Genesis Baseline Baseline Noise Tolerance fi 0 0 Minimum Number of Scans in Baseline fi 6 Baseline Noise Rejection Factor 20 Cancel Save s Default Help Table 90 Identification Options para
441. tative Analysis Note This feature is available only for the Acquisition window Realtime Plot View Displays the Status view For more information see Using the Status View on a page 80 in Chapter 2 Preparing to Perform Quantitative Analysis Note This feature is available only for the Acquisition window 432 LCquan User Guide Thermo Scientific C LCquan Menu and Toolbar Reference Toolbar Table 174 Toolbar buttons Sheet 2 of 4 Icon Button j gt Run Sample Description Displays the Run Sequence dialog box where you can run a single sample For more information see Run Sequence dialog box parameters on page 74 in Chapter 2 Preparing to Perform Quantitative Analysis Note This feature is available only for the Acquisition window Run Sequence oy Method View Displays the Run Sequence dialog box where you can run a sequence of samples For more information see Run Sequence dialog box parameters on page 74 in Chapter 2 Preparing to Perform Quantitative Analysis Note This feature is available only for the Acquisition window When you are in the Quantitate window displays the Create Method view in the Quantitate window For more information see Specifying Method Calibration on page 235 in Chapter 4 Creating a Processing Method When you are in the Explore window displays the Explore Create view For more information see Creati
442. ted devices are ready Do you want all the devices to be switched On Press Yes to switch devices On or No to continue with devices in the Off or in Standby state When you select No you must select the Devices On command from the Actions menu before the sequence will proceed Thermo Scientific Apps Menu C LCquan Menu and Toolbar Reference Apps Menu Use this menu to add external applications to the Apps menu for quick access and to select those applications see Table 155 Table 155 Apps menu commands Command Add Apps Description Keyboard shortcut Displays the App Selection dialog box where you can add external ALT A gt A applications to the Apps menu for quick access For additional information see App Selection dialog box parameters on page 9 in Chapter 1 Introduction Change Menu for Acquisition Window Only Use this menu to modify user labels and values and select the autosampler tray type see Table 156 Table 156 Change menu commands of the Acquisition window Command User Labels Description Keyboard shortcut Opens the Change Sequence User Labels dialog box where you can ALT C gt U modify the user labels associated with a sequence For more information see Parameters in the Change Sequence User Labels dialog box on page 34 in Chapter 2 Preparing to Perform Quantitative Analysis Tray Name Thermo Scientific Opens the Tray Sele
443. tegration settings and by manually integrating peaks For calibration standards and QCs the Review All Results view functions exactly as the Survey view does For more information see Reviewing the Calibration Curve and QCs on page 324 Opening the Review All Results View Thermo Scientific To process the raw files and open the Review All Results view Figure 211 click the Review All icon in the navigation pane The Review All Results view includes clockwise from the top the Result list the Component list the Calibration Curve pane the ISTD Chromatogram pane and the Chromatogram pane These elements function exactly as they do on the Survey view and have an additional display tab in the Result list the Unknowns tab along with the All Standards QCs and Blanks tabs Clicking the All tab displays the results for every sample in the sequence LCquan User Guide 365 6 Processing the raw Files and Reviewing the Analytical Results Reviewing All Results Figure 211 Quantitate Review All Results window E Thermo LCquan 3 Drugs Workbook y File Zoom View Apps Options Help PSB SISEFZBR OGOOSCOCOO Ki OlO Instruments Review All Results review standards QCs blanks and unknowns Acquistion Sample Type Integration Type ISTD Response Response Ratio Specified Cone Calculated Cone Diff Explore ft Method Settings 17139 338847 0 051 25 000 15973 36 11 Apone Guantitate Method Settings 16566
444. the Export folder of the current workbook The LCquan application creates both an event and a file tracking record Creates a quantitation component for each selected peak in the list If no peaks are selected the LCquan application uses all peaks The component name is the same as the peak name The component parameters are defaulted to a common set of values with the exception of the following Expected RT and RT Window are peak dependent the Mass Filter and Scan Filter are obtained from the Explore method All components are assumed to be target components and the level tables are copied from existing components After the components have been created a confirmation dialog box is displayed Thermo Scientific 3 Exploring the Data Data Views Table 62 Peak List view parameters Sheet 4 of 4 Parameter Create Quan Components For All Peaks in List Description Creates a quantitation component for each peak in the list The component name is the same as the peak name The component parameters are defaulted to a common set of values with the exception of the following Expected RT and RT Window are peak dependent the Mass Filter and Scan Filter are obtained from the Explore method All components are assumed to be target components and the level tables are copied from existing components After the components have been created a confirmation dialog box is displayed Copy to Clipboard Sample Info View Copie
445. the data from the Clipboard buffer into a document This view displays sample information stored in the current raw file during acquisition Figure 163 Figure 163 Sample Info view 4 gt al 3 a 5 e e C Filename APN_OOS RAVY Sample ID APNOO2 Path C XcaliburiQuanRoot 3Dru Instrument Method C XcaliburiQuanRoot 3Dru Processing Method LCquan Internal Processing Method Level Cal_01 ISTD Correction Amount 0 00 Dilution Factor 1 00 Vial Position TrayO1 2 Injection Volume 30 00 Sample Volume 0 00 Sample Weight 0 00 Sample Name Study 3Drugs Client Laboratory QuanSquad Company Name Thermo Electron Phone Comment Standard 1 Conc 25 pgi Barcode Barcode Status 0 The contents of the grid are read only Sample Info Parameter va OE Row Number when Acquir Sample Type _ Std Bracket Thermo Scientific Thermo Scientific 4 Creating a Processing Method Data Views When the view is locked lock icon is red you cannot select any menu item in the shortcut menu that changes the raw file or parameters Menu items that would cause changes are grayed out For more information about locks see Toolbar for Selectable Views on page 252 Table 113 lists the parameters for the Sample Info view Table 113 Sample Info view parameters Parameter Arrows Description Not used in the Create Method vie
446. the peak integration baseline above the RMS noise level Range 0 1 to 10 0 Default 2 0 LCquan User Guide 233 4 Creating a Processing Method Specifying Method Identification lon Ratio Confirmation of Analytes Example With Ion Ratio Confirmation IRC you can confirm the presence of an analyte by comparing the response of the quantitate ion or ions with the responses of one to five qualifier ions When any of the qualifier ion quantitate ion response ratios are outside of the upper or lower bounds that you specify or if the quantitate and qualifier ions do not coelute the LCquan application marks the analyte as Not Found in the Review All Results view in the Quantitate window Specify the masses target area ratios and area ratio windows of the qualifier ions in the Ion Ratio Confirmation table of the processing method The LCquan application displays the mass chromatograms of the qualifier and quantitate ions in the IRC Chromatogram pane In the following example the LCquan application divides the area of the m z 327 1 mass chromatogram peak by the area of the m z 309 2 267 2 mass chromatogram peak and multiplies this value by 100 If the value is less than 50 or greater than 70 the LCquan application marks hydrocortisone as Not Found Also the peak maximum of the qualifier ion chromatogram must be within 0 1 minutes of the peak maximum of the quantitate ion chromatogram Figure 139 Identification page m z 309 2 267
447. the processing method When no adjustment is required verify that a value of 0 000 is entered in the ISTD Corr Amt box When an adjustment is required enter the actual value of all internal standard amounts or concentrations in the sample into the ISTD Corr Amt box for the sample row or rows requiring adjustment The new value entered must use the same units as specified in the processing method Do not enter the units of measurement into the box For example for 20 ng type 20 Dil Factor Displays the dilution factor used to prepare the sample The valid range is 0 000 to 10 000 000 The LCquan application interprets a value of 0 000 as no dilution Vial Pos Inj Vol Displays the sample s position number in the autosampler This column value is read only Displays the injection volume in microliters of sample to be injected When you are using an autosampler you can set the default injection volume in the Autosampler dialog box in the Instrument Setup window The minimum and maximum injection volumes that you can use depends upon the Autosampler you select The usable range is dependent upon the injection mode and might be smaller than the range displayed in the status bar For more details consult your Autosampler manual Fill Down cannot be used on Inj Vol column values Sample Vol Displays the volume of a component that has been placed in the sample The unit for this volume is specified in the Xcalibur Pr
448. the signal reaches the baseline independent of the value set for the expected peak width Range 0 0 to 999 0 Default multiplier 0 0 Units seconds Note Valid only when you select Valley Detection Enabled Constrain Peak Width Constrains the peak width of a component during the peak integration of a chromatogram You can set values that control when peak integration is turned on and off by specifying a peak height threshold and a tailing factor Selecting this activates the Peak Ht and the Tailing Factor parameters Thermo Scientific 4 Creating a Processing Method Specifying Method Identification Table 77 Genesis Peak Integration area parameters Sheet 2 of 2 Parameter Peak Ht Description Specifies a percentage of the total peak height This is the minimum that a signal must be above the baseline before integration is turned on or off This option is available only when you select the Constrain Peak Width option Range 0 to 100 Default multiplier 5 0 The peak height percentage is defined as follows 100 Peak Height Tailing Factor Specifies a value for the factor that controls how the LCquan application integrates the tail of a peak This factor is the maximum ratio of the trailing edge to the leading side of a constrained peak and calculates the retention time of the maximum extent of the right edge of the tailing peak This option is available only when you select the Constrain
449. ther side e The peak baseline endpoints are displayed as blue circles Note When the endpoints appear as blue circles you cannot select and drag them to manually integrate the peak Thermo Scientific LCquan User Guide 289 4 Creating a Processing Method Data Views KD To set the number of rows and columns 1 Right click the plot and choose Options from the shortcut menu The Multi Peak Plot View dialog box opens where you can determine how peaks are arranged and change chromatographic display options For more information see Multi Peak Plot parameters on page 291 2 In the Arrangement box type the number of columns and rows You can set the normalization level to be the same for all peaks or you can normalize each peak individually To set the normalization levels 1 Right click the plot and choose Options from the shortcut menu The Multi Peak Plot View dialog box opens Figure 167 Table 117 lists the parameters for the Sample Info view 2 In the Intensity Scale list select Normalize to Largest Peak or Use Fixed Intensity Range Figure 167 Multi Peak Plot view for Survey or Review All Results 4 gt jo a fan Samples fc Multi Peak Ple F SRM ms2 330 13 F SRM ms2 330 13 Component Paroxetine Component Paroxetine o oO Relative Intensity Relative Intensity 2 0 2 0 Time min Time min F c SRM ms2 330 13 F SRM ms2 330 13 Comp
450. this box to convey information about this sample to others or as a reminder to yourself The User Label 5 default name is Phone To change definitions of User Labels and Values for any user defined column refer to the instructions for the Client column Rel RT Ratio of Actual RT to Expected RT Response Type Area or height Right Delta Height Refer to Left Right Delta Height described earlier Right Height Right Time The retention time corresponding to the end of peak integration 392 LCquan User Guide Thermo Scientific 6 Processing the raw Files and Reviewing the Analytical Results Creating and Reviewing Reports Table 152 Grid parameters Sheet 8 of 9 Parameter S N Description The calculated signal to noise measured The ratio of the signal height S to the noise height N The signal height is the baseline corrected peak height The noise height is the peak to peak height of the baseline noise S N Threshold Threshold signal to noise S N value for peak to be considered A user specified chromatogram peak edge detection criterion used for peak integration The Xcalibur data system and the LCquan application determine the retention time when the measured signal to noise S N value first becomes less than the S N threshold value The default value is 0 5 The Xcalibur data system and the LCquan application start at the peak apex and test measured S N values at successively lower retention times to determin
451. thod Settings 56677 326040 0 174 75 000 71 055 5 26 15 Standard Method Settings 52306 298244 0175 75 000 71 744 4 34 Method Settings 80377 304440 0 264 100 000 111 355 11 36 Standard Method Settings 67274 295463 0 228 100 000 95 121 4 88 18 Standard Method Settings 79642 284954 0 279 100 000 118 271 18 27 19 Standard Method Settings 72937 282186 0 258 100 000 108 877 8 88 20 Standard Method Settings 76823 294590 0 261 100 000 109 908 9 91 21 Standard Method Settings 194643 320269 0 608 250 000 264 965 5 99 Standard Mati ATAR NRNS IEN nnn oR RAN EAR od All Standards hace i Blanks i Unknowns lai q 4 8 Selectec gt Chromat 4 gt alisto Chromat 4 gt lal Calibration Curve a t Area Ratio 1 70 2 13 2 85 2 97 1 6 18 2 0 2 2 26 28 3 0 Time min Time min For Help press F1 Powers Dana NOT SAVED 7 10 2008 9 20AM _7 Relative Intensity Relative Intensity The LCquan application uses a processing method to automatically determine the concentration or amount of the sample being analyzed The processing method measures the response of the detection system to the compounds in your sample by integrating the area under each peak Thermo Scientific LCquan User Guide 5 1 Introduction Quantitative Analysis with LCquan 6 LCquan User Guide After measuring
452. thorization Manager to open the Authorization Manager Figure 229 2 In the Authorization Manager do the following a b Thermo Scientific Select a user group in the Secure Groups list Click Expand Tree to show the entire list of controlled features for the application From the list click the plus sign before the LCquan folder Click the plus sign before the Quantitate Section folder Select Automatically Lock Workbook After Creating Reports The Permission Level options become available Select the Disallowed option and click OK Figure 229 LCquan User Guide 399 6 Processing the raw Files and Reviewing the Analytical Results Saving the Workbook and Exiting the LCquan Applicat ion Figure 229 Authorization Manager showing the Automatically Lock Workbook After Creating Report permission set to Disallowed LCquan amp amp 7 E3 E Exp vj a m Run Application me Root Folder Acquisition Section Acquisition Run Dialog lore Section Quantitate Section vil Allow changes to selected sample info in Allow changes to column labels in proces Allow changes to column labels in results vj Prompt user for Comment after manual inl Normalize quan chromatogram plots to de Allow Results Export Allow Manual Integration Allow User Integration Create Reports vij Allow Calibration Settings to be changed Remove signature line from excel report Secure Report Template
453. tific 5 Creating a Processing Sequence Defining a Processing Sequence Importing a Processing Sequence Thermo Scientific Use any of these methods to import an existing processing sequence into your current workbook e New Study Wizard When you create a new workbook the New Study Wizard gives you the option of importing instrument methods acquisition sequences and processing parameters to initialize the new workbook You can import all this data from an existing workbook or you can import an acquisition sequence from an individual legacy file sld ipp or csv or from an LCquan file lqn When you import an acquisition sequence from an individual legacy file into a workbook that does not contain a processing sequence the LCquan application uses the same sequence for the acquisition sequence and the processing sequence in the current workbook New Processing Sequence Wizard in the Quantitate window When you use the New Processing Sequence Wizard you have the option to import a previously saved sequence from an individual legacy file sld ipp or csv or from an LCquan file lqn Menu commands from within the Quantitate window From the Create Sequence views of the Quantitate window choose File gt Import gt Processing Sequence gt From File to display the Load Sequence File dialog box and import a sequence from an individual legacy file sld ipp or csv or from an LCquan file lqn From anywhere in
454. tification Settings Avalon Integration 360 Detection 350 Genesis Advanced 355 ICIS Advanced 359 ICIS Integration 356 Identification 347 Workbook Summary Information 27 442 LCquan User Guide peak detection See algorithms peak detection peak detection and integration settings editing 345 peak detection type Avalon 117 Genesis 117 ICIS 117 Peak Identification area parameters 193 Peak Information Flags parameters 370 Info parameters 368 IRC Tests parameters 373 peak integration 354 Peak List parameters Explore 166 Quantitate 281 Peak List shortcut menu commands Explore 166 Review 282 peak lists generating 143 Peak Name List Editor parameters 147 peak parameters 232 peaks integrated illustrated 405 integrating manually 363 modifying integration parameters 345 undoing manual integration of 364 peaks limiting 131 printing acquisition sequence 53 probes choosing 404 processing overview 10 while acquiring data 104 processing method creating 181 importing 250 saving 250 processing raw files 323 processing sequence copying acquisition sequence 298 creating 293 defined 293 editing 301 grid parameter definitions 308 importing 298 reviewing 301 switching to processing during acquisition 105 processing sequence grid column parameters 308 processing sequence grid shortcut menu commands 306 Thermo Scientific Q QC levels matching names 52 Qual Browser starting from Explore window 149 quality control QC samples
455. tification page parameters Sheet 1 of 2 Parameter Description Component Specifies the mass value of the selected compound This parameter is read only Smoothing Points Specifies the number of points to use for a moving mean filter to smooth the chromatogram Format integers Range 1 to 15 odd numbers only Note Set the parameter to 1 to disable filtering Trace Specifies the type of chromatogram TIC Mass Range or Base Peak This parameter is read only e Mass Range Specify up to 50 mass ranges The masses are added together to form the chromatogram e TIC Full scan acquisition resulting in a Total lon Current plot e Base Peak In a differential chromatogram the interpolation of the baseline is derived from the distance between the intersections of the tangents drawn to the peak sides and the peak base Note This definition set is the same for the second Trace field The second trace field depends on which operators are chosen Specifies an addition or a subtraction trace operator when you are using trace math This parameter is read only This Trace Operation matrix shows the combinations used to set up an Explore Method Mass Range Mass Range Mass Range TIC Mass Range Base Peak Base Peak Mass Range Mass Range Note If the Operator box is empty the second trace choice is disabled Mass m z Specifies the initial mass value This parameter is read only Therm
456. til these points are at zero Range 1 to 500 Default 40 Area Noise Factor Thermo Scientific The noise level multiplier used to determine the peak edge after the location of the possible peak so that the peak can narrow or broaden without affecting the baseline Range 1 to 500 Default multiplier 5 LCquan User Guide 225 4 Creating a Processing Method Specifying Method Identification 226 LCquan User Guide Table 86 IRC Detection Method dialog box ICIS Integration page parameters Sheet 2 of 2 Parameter Peak Noise Factor Constrain Peak Width Peak Ht Description The noise level multiplier a minimum S N ratio used to determine the potential peak signal threshold Range 1 to 1000 Default multiplier 10 Constrains the peak width of a component during the peak integration of a chromatogram You can set values that control when peak integration is turned on and off by specifying a peak height threshold and a tailing factor Selecting this activates the Peak Ht and the Tailing Factor parameters A percent of the total peak height This is the minimum that a signal must be above the baseline before integration is turned on or off Range 0 to 100 Default multiplier 5 0 The peak height percentage is defined as follows 100 Peak Height Note Valid only when you select the Constrain Peak Width check box Tailing Factor A peak integration multiplier
457. tion data points during the least squares regression calculation of the calibration curve Calibrants are weighted by the inverse of the square of their quantity 1 X 2 Specifies a weighting of 1 X 2 for all calibration data points during the least squares regression calculation of the calibration curve Calibrants are weighted by the inverse of their quantity 1 Y Specifies a weighting of 1 Y for all calibration data points during the least squares regression calculation of the calibration curve Calibrants are weighted by the inverse of their response or response ratio 1 Y 2 Specifies a weighting of 1 Y 2 for all calibration data points during the least squares regression calculation of the calibration curve Calibrants are weighted by the inverse of the square of their response or response ratio 1 s 2 Specifies a weighting of 1 s 2 for all calibration data points during the least squares regression calculation of the calibration curve Calibrants at a given level are weighted by the inverse of the standard deviation of their responses or response ratios For this weighting factor to be used there must be two or more replicates at each level If only one calibrant is available for any level then 1 s 2 weighting cannot be used Thermo Scientific 6 Processing the raw Files and Reviewing the Analytical Results Reviewing the Calibration Curve and OCs Table 129 Calibration Settings Paroxetine dialog box parameters Sheet 3 of
458. tion results can be compared with known component amounts or concentrations for quality assurance purposes The LCquan application performs quantitation on these samples using the same calibration settings as it uses on Unknown sample types It places Quality Control Samples after Calibration Samples and before Unknown Samples in the sequence e Standard A sample with a known amount of calibration reference material To change the sample type select from the list of sample type options The new sample type appears in the Sample Type box Sample Name Displays the sample name that you specified when you created the sequence Integration Type The type of integration used is displayed in the results grid but can be overridden The three types are Method User and Manual Integration When you select Method the LCquan application uses the integration parameters embedded within the processing method You can enter new integration parameters by right clicking the chromatogram plot and selecting User Peak Detection Settings This sets the integration type to User Dragging the integration control handles on the integration baseline sets the integration type to Manual Response Area or height of the detected peak The value stored depends on the selection made in the calibration section ISTD Response Thermo Scientific Area or height of the detected peak associated Internal Standard The value stored depends on the selecti
459. tion transfers raw files to the remote location e Raw files are tracked in the global database the same as raw files used in local acquisition e When a sample is completed the raw file is transferred to the remote location e When the user closes the LCquan application and no acquisition is running it deletes the local workbook folder The LCquan application cannot transfer files when it is not running Importing a Processing Sequence During remote acquisition use any of these methods to import a processing sequence e Import a saved sequence file or copy an acquisition sequence For detailed descriptions of the various commands and methods see Chapter 5 Creating a Processing Sequence e Update the processing sequence with the most recently acquired samples For detailed instructions for copying files as they are acquired see Processing Data While Continuing to Acquire Data on page 104 112 LCquan User Guide Thermo Scientific ee ie Exploring the Data This chapter describes the features of the Explore window You can use the Explore window to display a multi component chromatogram and experiment with peak detection and integration parameters to see how they affect the chromatogram Contents e Introduction to the Explore Window e Creating Explore Methods e Exploring the Results e Exporting the Active raw File to Qual Browser e Data Views Introduction to the Explore Window Use the Explor
460. tions in the Review Reports view in the Quantitate window and exited the view before creating reports Table 150 lists the parameters for the Review Reports dialog box Table 150 Review Reports dialog box parameters Parameter Description Save Data Applies the changes you have made to the reports settings Your Automatically default changes are not saved until you save the workbook Discard Any Dhanges _ Discards the changes you have made to the reports settings Don t Tell Me About Prevents this dialog box from appearing again This Again LCquan User Guide 383 6 Processing the raw Files and Reviewing the Analytical Results Creating and Reviewing Reports Report Options Table 151 lists the report options Table 151 Report Options Sheet 1 of 2 Report option Report Selections Description Acquisition Sequence Report LCquanAcgSequence xrt Displays the sample information that is in the acquisition sequence for example sample ID raw file names sample types and cal levels Component Calibration Report Avalon Genesis or ICIS algorithm LCquanCompCalReport xrt Provides the component identification and calibration parameters calibration curves calibration and QC levels and amounts and Results list information for example peak areas and calculated amounts for all components in all samples Ion Ratio Confirmation Results Report LCquanlonRatioConfirmationGraphical xrt Peak Inte
461. tions used to set up a method Mass Range Mass Range Mass Range TIC Mass Range Base Peak Base Peak Mass Range Mass Range Note If the operator box remains empty the second trace choice is disabled Mass1 m z Specifies the initial mass value Mass2 m z Specifies the second mass value to define the mass range used for trace operator math Filter Trace Summed Edit Summed Specifies a scan filter to be applied to the acquired data Enter a filter or select a filter from a preloaded filter list obtained from the current raw file Note All filters are validated against the current set of filter entry rules Opens the Summing Ions Dialog Box Use the Summing Ions dialog box to specify the mass to charge ratios and scan filters of up to five ions for ion summing Summed Ions table Thermo Scientific Displays the filters and mass to charge ratios of the ions you specified for ion summing in the Summing Ions Dialog Box LCquan User Guide 189 4 Creating a Processing Method Specifying Method Identification Mass Tolerance and Precision Use the Masses dialog box to specify tolerance and precision settings for the mass data displayed in the Chromatogram and Spectrum panes Figure 115 Table 72 lists the parameters for the Masses dialog box To open the Masses dialog box choose Options gt Masses Figure 115 Masses dialog box Mass tolerance Mass tolerance
462. titate windows Command Description Keyboard shortcut Masses Opens the Masses dialog box where you can specify tolerance and ALT O gt M precision settings for the mass data displayed in the Chromatogram view For more information see Masses dialog box parameters on page 190 in Chapter 4 Creating a Processing Method 426 LCquan User Guide Thermo Scientific c LCquan Menu and Toolbar Reference Commands Specific to Acquisition Window Table 167 Commands specific to the Acquisition window Command Realtime Display Settings View Menu Description Opens the Realtime Display Settings dialog box where you can specify the properties of the real time chromatograms that the LCquan application displays in the Chromatogram and Spectrum panes of the Acquisition window For more information see Realtime Display Settings dialog box parameters on page 86 in Chapter 2 Preparing to Perform Quantitative Analysis menu commands Commands Common to All LCquan Windows Table 168 Commands common to all LCquan windows Sheet 1 of 2 View Menu Keyboard shortcut ALT O gt R The View menu has group commands that you can use to display or hide the toolbar or status bar in the View menu You can also change the visual display of the toolbar using the View Command Description Keyboard shortcut Toolbar Displays or hides the LCquan window toolbar The toolbar appears ALT V gt T if it was previously hidden
463. to Clipboard Copies selected data to the Clipboard You can then paste the data from the Clipboard buffer into a document Peak List View The Peak List view displays a list of detected Explore peaks for a sample and an Explore method Figure 101 You can edit the peak name in the view You can also modify the peak names using the Peak Name List Editor dialog box Figure 101 Peak List view Table 62 lists the parameters for the Peak List view Thermo Scientific LCquan User Guide 165 3 Exploring the Data Data Views 166 LCquan User Guide Table 62 Peak List view parameters Sheet 1 of 4 Parameter Arrows Description The left and right arrows move through the peak list incrementing or decrementing by a single peak Column Headings Peak Name The name to apply to the explore peak column Apex RT The expected RT for the peak column Area The calculated peak area column of Largest Area The area of this peak relative to the area of the peak with the biggest area in the list column of Total Area The area of this peak relative to the summed total area of all peaks in this list column Shortcut Menu Open Raw File Opens the Select Raw File dialog box where you can select the raw file to load Lock Against Raw File Prevents changes to a raw file The lock icon is yellow and the Change lock is closed Lock against All Prevents changes to a raw file or parameters The lock ico
464. to www thermoscientific com wps portal ts contactus To copy manuals from the Internet Go to mssupport thermo com agree to the Terms and Conditions and then click Customer Manuals in the left margin of the window To suggest changes to documentation or to Help e Fill out a reader survey online at www surveymonkey com s PQMG6P62 e Send an e mail message to the Technical Publications Editor at techpubs lcms thermofisher com xii LCquan User Guide Thermo Scientific M Introduction This chapter provides a brief overview of quantitative analysis with the LCquan application and an overview of using the LCquan application with Thermo Scientific Web Access Suite Contents e Quantitative Analysis with LCquan e LCquan for Web Access Quantitative Analysis with LCquan The LCquan application is a complete quantitative analysis software package that lets you acquire data specifically for an analyte of interest to quantitate the results and to produce reports For more information about quantitative analysis concepts see Appendix A Quantitative Analysis Overview This section contains the following topics e Overview of LCquan e LCquan Folder Structure e LCquan Applications Menu e Acquiring and Processing Data with LCquan Overview of LCquan The LCquan application is divided into four main windows Instrument Setup Acquisition Explore and Quantitate By using these windows you can develop instrument methods
465. togram A lower tension traces the baseline to follow changes in the chromatogram more closely A high baseline tension follows the baseline less closely over longer time intervals Units minutes Tangent Skim Integrate On Off Shoulders On Enables tangent skim on any peak clusters By default the LCquan application selects the tallest peak in a cluster as the parent solvent and detects peaks on either side or both sides of the tallest peak You can also identify which peak in the cluster is the parent Tangent skim automatically resets at the end of the peak cluster Turns integration on or off at the set time Turns on the detection of shoulders Shoulders Off Turns off the detection of shoulders Force Cluster On Turns on the grouping of peaks into a single peak Force Cluster Off Turns off the grouping of peaks into a single peak Disable Cluster On Enables the grouping effect in the specified time range Disable Cluster Off 362 LCquan User Guide Disables the grouping effect in the specified time range Thermo Scientific 6 Processing the raw Files and Reviewing the Analytical Results Reviewing the Calibration Curve and OCs Manually Integrating Peaks You can manually change the baseline and the starting and ending points of a peak in either of two ways e Drag the starting or ending points e Use the Baseline dialog box When you manually integrate a peak the LCqu
466. top of the Results grid 2 In the Item Selector list select Selected 3 Click the Add a Peak icon L on the LCquan toolbar The pointer changes to Ruk 4 Drag the peak baseline defining the start and end of the peak You can manually adjust the peak baseline and endpoints by grabbing the square blue handles and dragging them Note This feature is available only for the Survey or Review All Results views in the Quantitate window with the Item Selector set to Selected Update Raw File tes Explore Update Sequence Quantitate 434 LCquan User Guide Only when you are in Explore while acquiring data Updates the raw file with a partial sequence created from the acquired samples Only when you are in Quantitate while acquiring data Updates the current processing sequence with a partial sequence created from the acquired samples Thermo Scientific c LCquan Menu and Toolbar Reference Toolbar Table 174 Toolbar buttons Sheet 4 of 4 Icon Button Description Shut Down Instrument Displays the Emergency Shutdown dialog box where you can shut down all devices Q and stop the acquisition of the current sample and pause stop the acquisition of the current sequence and pause or stop the acquisition of all sequences For more information see Emergency Shutdown on page 108 in Chapter 2 Preparing to Perform Quantitative Analysis Help Displays the Help topic for the current view or displays the Help sy
467. trument method into your current workbook You can use the method as is or you can edit it Import an instrument method in one of the following ways To use the New Study or Workbook Wizard When you create a new workbook the New Study or Workbook Wizard gives you the option of importing instrument methods acquisition sequences and processing parameters to initialize the new workbook Import this data from an existing workbook or import an instrument method from an individual legacy file meth See Creating a New Study and Workbook on page 13 To use the Import Instrument Method File dialog box Choose File gt Import Instrument Method to open the Import Instrument Method File dialog box Use this dialog box to import an instrument method from an individual legacy file meth Introduction to the Acquisition Window 30 LCquan User Guide Use the Acquisition window to define an acquisition sequence run individual samples and sequences and monitor real time data acquisition This section describes the features of the following views e Using the Setup Sequence View e Using the Run Sequence Dialog Box e Using the Status View Use the navigation pane on the left side of the window to select the view or function Thermo Scientific 2 Preparing to Perform Quantitative Analysis Using the Setup Sequence View Using the Setup Sequence View Use the Sequence Setup view Figure 23 to modify an acquisition seq
468. ts Reviewing All Results Table 144 Peak Info dialog box Flags page parameters Sheet 3 of 4 Parameter Description Flags read only Saturated When selected this indicates one or more saturated scans were detected Calculated Amount When selected this indicates a quantitation calculation was performed The calculated value is displayed in the Result grid view When cleared this indicates that quantitation calculation was not successful Valley Detect When selected this indicates valley detection is enabled in the processing method QC Failed When selected this indicates that the sample type failed the QC test If the calculated amount is greater than the specified percentage difference from the expected amount the sample fails the QC test For example if the tolerance level is 10 and the expected amount is 100 calculated amounts less than 90 or greater than 110 fail RT Ref OK Indicates whether the retention time reference component was found and whether it was used correctly by the processing method When selected this indicates the retention time reference peak was found or there is no correction at all because there is no retention time reference When cleared this indicates the peak was searched for and not found Response OK Indicates whether a response factor was calculated When selected this indicates the peak was found its internal standard was found and the response ratio was c
469. ts go to Reviewing All Results on page 365 LCquan User Guide 335 6 Processing the raw Files and Reviewing the Analytical Results Reviewing the Calibration Curve and OCs Modifying Calibration Settings You can use the Calibration Settings dialog box to change the calibration curve parameters that you specified on the Calibration page in the Create Method view of the Quantitate window including curve type and level information To open the Calibration Settings dialog box right click a Calibration Curve view in any of the preview panes and choose Calibration Settings from the shortcut menu The Calibration Settings dialog box includes these tabs e Calibration Settings Curve Calibration Settings Type Calibration Settings Levels Calibration Settings Isotope Calibration Settings Curve Use the Curve page to modify calibration curve settings Figure 193 Table 129 lists the parameters for the Calibration Settings Paroxetine dialog box Figure 193 Calibration Settings dialog box for Paroxetine Calibration Settings Paroxetine Type Curve Levels Isotope Calibration Curve Linear v Origin m Weighting C Ignore Equal C Force CK Include C 1 2 C14 Response So Area C Height C 1 s 2 Cancel pl Help 336 LCquan User Guide Thermo Scientific 6 Processing the raw Files and Reviewing the Analytical Results Reviewing the Calibration Curve and OCs Table 129 C
470. ts dialog box W File Tracking Results a _ C Xcalibur QuanRoot Example Study 3 Drugs VWorkbook_3 Rawfiles 4PN 9 _ C Xcalibur QuanRoot Example Study 3 Drugs Workbook_3RawfilestAPN 10 C Xcalibur QuanRoot Example Study 3 Drugs VWorkbook_3 Rawrfiles 4PN 11 C Xcalibur QuanRoot Example Study 3 Drugs Workbook_3RawfilestAPN C XcaliburiQuanRoot Example Study 3 Drugs Vorkbook_3 Rawiles APN 43 C Xcalibur QuanRoot Example Study 3 Drugs Workbook_3RawfilestAPN 44 C Xcalibur QuanRoot Example Study 3 Drugs Workbook _3 Rawfiles 4PN 45 C Xcalibur QuanRoot Example Study 3 Drugs Vorkbook_3 Rawrfiles 4PN 146 C Xcalibur QuanRoot Example Study 3 Drugs VWorkbook_3 Rawrfiles 4PN C Xcaliburi uanRoot Example Study 3 Drugs Vorkbook_3 RawrilesV4PN 48 C Xcalibur QuanRoot Example Study 3 Drugs Vorkbook_3 Rawfiles 4PN 49 C Xcalibur QuanRoot Example Study 3 Drugs VWorkbook_3 Rawfiles 4PN 20 C Xcalibur QuanRoot Example Study 3 Drugs VWorkbook_3 Rawfiles 4PN 21 C XcaliburtQuanRoot Example Study 3 Drugs Workbook_3RawfilestAPN ede CV eslih ri Ninan Rart Evamnla Chidi Prine Wierkhonk 2PReswmfilaci ADA Filter Allow VAUD TTT TT TTT TT TTT TT FOOD OOOO OO OOOO Table 9 File Tracking Results dialog box parameters Parameter Description Highlighting in the grid Red highlighting Signifies that the file has been modified Yellow highlighting Signifies that the file is missing Filter Sets the grid to display only the files that have been de
471. tudy e The User Label 2 default name is Client e The User Label 3 default name is Laboratory e The User Label 4 default name is Company Name e The User Label 5 default name is Phone User Value 1 to 5 Displays the value in the header of the acquisition sequence Use this user defined box to convey information about a sample to others or as a reminder to yourself The LCquan application automatically substitutes certain selected values in the box e Operator substitutes the name of the operator e Study substitutes the name of the Study e Workbook substitutes the name of the Workbook e Workstation substitutes the name of the Workstation e Do Not Change prevents the LCquan application from substituting anything in the box To view or edit the sequence with the function buttons Select a row in the sequence grid e To move up or move down a row in the sequence click Prev Row or Next Row As you move through the rows the sequence header information for each sample is displayed e To remove all empty rows in the sequence grid click Compress e To remove all samples from the sequence click Clear All Table 12 describes each of the function buttons 34 LCquan User Guide Thermo Scientific 2 Preparing to Perform Quantitative Analysis Using the Setup Sequence View Table 12 Sequence grid function buttons Buttons Description Prev Row Highlights the sequence row that precedes the currently highlighted row
472. tup Sequence View Specify a Root Folder Specify a root folder for the study The default root folder is C Xcalibur QuanRoot To specify the root folder for the study 1 Type the new study root folder name or click Browse to find the folder Figure 30 The default root folder is C Xcalibur QuanRoot Figure 30 Root folder path page Folder where studies will be located C Xcalibur QuanR oot Browse 2 Click Next e To import an acquisition sequence go to Import an Acquisition Sequence e When you are not importing an acquisition sequence go to Sample Sequence Name Import an Acquisition Sequence When you are not importing an acquisition sequence ensure that the Base Acquisition on a Previously Saved File check box is cleared click Next and go to Sample Sequence Name To import an acquisition sequence 1 Select the Base Acquisition on a Previously Saved File check box Note Ensure the Create a New Workbook check box is cleared on the first page of the wizard 2 In the Acquisition Sequence box Figure 31 enter the sequence name or click Browse to find the sequence Figure 31 Acquisition sequence page V Base acquisition on a previously saved file Acquisition Sequence c calibur QuanF oot Tutorial Study 243 Drugs Workbook tut 3 3 Click Next and go to Complete the Wizard 40 LCquan User Guide Thermo Scientific 2 Preparing to Perform Quantitative Analysis Using the Setup
473. u do not respond to the message box after five seconds the LCquan application automatically updates the sequence For more information about defining a processing sequence see Chapter 5 Creating a Processing Sequence Table 35 Acquisition Sequence dialog box parameters Parameter Description Auto Update the Select to automatically add sample information to the processing Processing Sequence sequence as samples are run When Acquiring Maximum Number of Type a value between 1 and 5000 for the maximum number of Rows Allowed in the rows in the acquisition sequence Acquisition Sequence Thermo Scientific 2 Preparing to Perform Quantitative Analysis Using the Status View To switch to processing during acquisition Use the Switching to Processing during Acquisition features to determine what processing sequence you want the LCquan application to use when you switch to the Quantitate window from the Acquisition window during an acquisition see able 35 1 During data acquisition click Quantitate The Switching to Processing during Acquisition dialog box opens Figure 61 Figure 61 Switching to Processing During Acquisition dialog box Switching to Processing during Acquisition 4 partial sequence based on the acquired data to this point can be created It will overwrite any existing processing sequence that is currently defined Update sequence with data acquired You may also continue without modifying the pro
474. u highlighted in the Component table 244 LCquan User Guide Thermo Scientific 4 Creating a Processing Method Specifying Method Calibration Correction for Isotope Contribution Thermo Scientific Use the Correction for Isotope Contribution dialog box to correct for an impurity in the internal standard compound that elutes at the same time as the target compound correct for an impurity in the target compound that elutes at the same time as the internal standard or correct for both Figure 145 Table 96 lists the parameters for the Correction for Isotope Contribution dialog box Figure 145 Correction for Isotope Contribution dialog box Correction for Isotope Contribution Contribution of ISTD to Target Compound 0 0 Contribution of Target Compound to ISTD 0 0 OK Cancel Help Table 96 Correction for Isotope Contribution dialog box parameters Parameter Description Contribution of ISTD Displays the ratio ISTD impurity ISTD pure x 100 to Target C d i ala e ISTD impurity is an impurity compound in the internal standard reagent that elutes at the same time as the target compound e ISTD pure is the pure internal standard compound Range 0 00 to 100 00 Contribution of Target Displays the ratio TM impurity TM pure x 100 C d to ISTD ompound to e TM impurity is an impurity compound in the target molecule reagent that elutes at the same time as the internal standard
475. uantitate 273 Chromatogram pane shortcut menu 89 Chromatogram shortcut menu Quantitate 259 Review 263 Column Arrangement Acquisition 60 Quantitate 313 Review All Results 330 Component list shortcut menu Quantitate 241 Review 332 Component Type area 237 Correction for Isotope Contribution 245 Current Review Display 140 Disk Space 106 Emergency Shutdown 108 Error Report Explore 159 Quantitate 274 Error Report shortcut menu 275 File Tracking Results 26 Fill Down Acquisition 57 Quantitate 308 Filter List Explore 160 Quantitate 276 Filter List shortcut menu 276 General Parameters Plot Explore 161 Quantitate 277 General Parameters Plot shortcut menu Explore 162 Quantitate 277 Genesis Advanced Component Options Explore 121 Quantitate 208 Genesis Peak Integration Explore 119 Quantitate 206 ICIS Advanced Explore 125 Quantitate 212 Thermo Scientific Index P ICIS Peak Integration Explore 124 Quantitate 211 Initial and Timed Events Explore 129 IRC detection 230 Quantitate 216 Instrument Method 163 Instrument Method List 278 Ion Ratio Chromatogram Display Options 271 Ion Ratio Confirmation area 194 Ion Ratio Confirmation area shortcut menu 195 IRC Chromatogram 269 IRC Chromatogram shortcut menu 269 IRC Detection Method Avalon Integration 229 Genesis Advanced 223 Genesis Integration 221 ICIS Advanced 227 ICIS Integration 225 Identification 219 Limit Peaks 131 Mass List Explore 164 Quantitate 279 Mass List shortcu
476. uare calculation is used to determine signal to noise values instead of the default ICIS noise method Min Peak Width The minimum number of scans required in a peak Range 0 to 100 Default 3 scans Thermo Scientific LCquan User Guide 227 4 Creating a Processing Method Specifying Method Identification 228 LCquan User Guide Table 87 IRC Detection Method dialog box ICIS Advanced page parameters Sheet 2 of 2 Parameter Multiplet Resolution Description The minimum separation in scans between the apexes of two potential peaks This is a criterion to determine if two peaks are resolved Use a larger number in a noisy environment when the signal is bouncing around Range 1 to 500 Default 10 scans Area Tail Extension Area Scan Window The number of scans past the peak endpoint to use in averaging the intensity Range 0 to 100 Default 5 scans The number of allowable scans on each side of the peak apex A zero value defines all scans peak start to peak end to be included in the area integration Range 0 to 100 Default 0 scans Thermo Scientific 4 Creating a Processing Method Specifying Method Identification IRC Detection Method Avalon Integration Thermo Scientific Use this page Figure 137 to specify advanced component detection criteria Use these additional criteria when the standard detection criteria do not provide the expected results Table 88 lists the parameters for the Av
477. ued Pauses the current acquisition and deletes all sequences in the Sequences queue Use this when you want to restart all queued acquisitions Before you can run a deleted acquisition sequence you must resubmit the sequence Thermo Scientific 2 Preparing to Perform Quantitative Analysis Shutting Down LCquan While Acquiring Shutting Down LCquan While Acquiring When you need to shut down the LCquan application while an acquisition is running the Shutdown While Acquiring dialog box Figure 65 prompts you to select how the application handles the acquisition Figure 65 Shutdown While Acquiring dialog box Shutdown while acquiring Exit LCquan but continue acquisition C Exit LCquan and stop the acquisition I Don t tell me about this again OK Cancel e To quit the LCquan application and continue acquiring select the Exit LCquan but Continue Acquisition option To quit the LCquan application and stop acquiring select the Exit LCquan and Stop the Acquisition option The acquisition for all submitted sequences stops When you restart the LCquan application and open the latest used workbook the Acquisition Queue does not show any sequences and the Acquisition History shows the Stop status for the sequence that was acquiring when you exited the application Remote Acquisition Time Stamps Thermo Scientific When acquiring raw files in a remote workbook the LCquan application creates temporary local folders for
478. uence containing Unknown samples calibration Standard samples quality control QC samples and Blank samples Figure 23 Acquisition Setup Sequence dialog box E Thermo LCquan 3 Drugs Workbook tut5 File Change Actions View Apps Options Help DaA G4a FL al 0000000 0 0 Acquisition FileName APN_0O01 Acquisition Sequence History SamleName Study Tutorial Study 2 Client Client One Laboratory SanJoseLab Company Name hem SsS S Phone 408 555 5555 Eev Row Row 1 Nex Row Clear All Help ii Comment APN_002 APN_002 From AS 0 000 0 000 Tutorial Study 2 Client One APN_003 APN_003 f From as 0 000 0 000 Tutorial Study 2 Client One TAPN_004 TAPN_004 A From AS 0 000 0 000 Tutorial Study 2 Glient One APN_OOS APN_005 From AS 0000 0 000 Tutorial Study 2 Client One APN_006 APN 006 f i From AS 0 000 0 000 Tutorial Study 2 Client One APN_007 APN_0O7 C f From AS 0 000 0 000 Tutorial Study 2 Client One JAPN_008 APN_008 f From AS 0 000 0 000 Tutorial Study 2 Client One APN_009 APN_009 f j From AS 0000 0 000 Tutorial Study 2 Client One APN_01 0 APN_010 K f From AS 0 000 0 000 Tutorial Study 2 Client One APN_O11 APN_011 f From AS 0 000 0 000 Tutorial Study 2 Client One APN_012 APN_012 f From AS 0 000 0 000 Tutorial Study 2 Client One JAPN_013 APN_013 f r Fromas 0 000 0 000 Tutorial Study 2 Client One APN_O14 APN_014 f From AS 0 000 0 000 Tutorial Stud
479. ume in the Autosampler dialog box in the Instrument Setup window The minimum and maximum injection volumes that you can use depends upon the Autosampler you select The usable range depends on the injection mode and can be smaller than the range displayed in the status bar For more details consult your Autosampler manual Thermo Scientific 2 Preparing to Perform Quantitative Analysis Using the Status View Table 33 Sample Information dialog box parameters Sheet 3 of 3 Parameter Level Description Displays the calibration level whenever the acquisition sequence row corresponds to a calibration sample or a quality control QC sample for which a level is defined Sample Weight Displays the amount of a component that has been placed in the sample Sample Volume Displays the volume of a component that has been placed in the sample ISTD Corr Amt Specifies an updated amount of internal standard for the corresponding sample The default value is 0 0 Dil Factor Displays the dilution factor that was used to prepare the sample The valid range is 0 000 to 10 000 000 The LCquan application interprets a value of 0 000 as no dilution User Label 1 to 5 Displays information pertinent to the active sample row in the sequence Use these boxes to convey information about this sample to others or as a reminder to yourself e The User Label 1 default name is Study e The User Label 2 default name is Cli
480. umn Shortcut Menu Open Raw File Create Method view only Opens the Select Raw File dialog box where you can select the raw file to load Lock against All Prevents changes to a raw file or parameters The lock icon is red Change and the lock is closed Unlock Makes the pane editable The lock icon is green and the lock is open Spectrum at Peak Apex Selects the spectrum at the selected component peak s apex The first chromatogram in the list sets the RT Spectrum at Peak Left Edge Selects the spectrum at the selected component peak s left edge The first chromatogram in the list sets the RT Spectrum at Peak Right Selects the spectrum at the selected component peak s right edge Edge The first chromatogram in the list sets the RT Copy to Clipboard Copies a chromatogram mass spectrum or calibration curve to the Clipboard You can then paste the data from the Clipboard buffer into a document Thermo Scientific 4 Creating a Processing Method Data Views Peak List View for Survey or Review All Results Only Thermo Scientific This view is available from the Survey or Review All Results views in the Quantitate window The Peak List view displays a list of detected peaks for a sample Figure 162 Figure 162 Peak List view Peak List 4 al Paroxetine 16566 14481 18597 17723 33231 35282 34312 41308 34187 54574 51754 57117 56677 52306 80377 67274 79642 72937 76823 194643
481. ustment is required enter the actual value of all internal standard amounts or concentrations in the sample into the ISTD Corr Amt box for the sample row or rows requiring adjustment The new value entered must use the same units as specified in the processing method Do not enter the units of measurement into the box For example for 20 ng type 20 Dil Factor Vial Pos Inj Vol Displays the dilution factor used to prepare the sample The valid range is 0 000 to 10 000 000 The LCquan application interprets a value of 0 000 as no dilution When you have specified a processing method for the current sequence the LCquan application automatically enters the Dil Factor value from the processing method settings Displays the sample s position number in the autosampler Displays the injection volume in microliters of sample to be injected When you are using an autosampler you can set the default injection volume in the Autosampler dialog box in the Instrument Setup window The minimum and maximum injection volumes that you can use depend upon the Autosampler you select The usable range is dependent upon the injection mode and might be smaller than the range displayed in the status bar For more details consult your Autosampler manual Sample Vol Displays the volume of a component that has been placed in the sample The unit for this volume is specified in the Xcalibur Processing Setup window and is included only in LCquan
482. uts all instruments into their Stop condition see Table 39 To define the emergency shutdown procedures 1 In the toolbar click the shutdown icon O open the Emergency Shutdown dialog box Figure 64 Figure 64 Emergency Shutdown dialog box Emergency Shutdown Emergency Shutdown Confirmation This will shut down all devices and Pause the acquisition C Delete the current acquisition sequence and pause C Delete all queued sequences Cancel Help 2 Select one of the following To pause the current acquisition select the Pause the Acquisition option You can restart the acquisition sequence later e To quit the current acquisition and delete the sequence from the acquisition queue select the Delete the Current Acquisition Sequence and Pause option 3 To quit the acquisition and delete all queued acquisition sequences select the Delete All Queued Sequences option Table 39 Emergency Shutdown dialog box parameters Parameter Description Pause the Acquisition Pauses the current acquisition and date time stamps the current raw file When you restart the acquisition you must rerun this raw file from the beginning Delete the Current Pauses the current acquisition and deletes the current sequence Acquisition Sequence Use this when you want to restart the sequence from the and Pause beginning Before you can run a deleted acquisition sequence you must resubmit the sequence Delete All Que
483. vailable only when Target Compound is the compound type ISTD Amount The amount of the selected internal standard This option is available only when ISTD is the compound type Range 0 001 to 100 000 000 ISTD Units The unit type for the internal standard This option is available only when ISTD is the compound type Target Compound Area Use the Target Compound area to specify parameters for the target compound Figure 142 Table 92 lists the parameters for the Target Component area To edit the level tables 1 To remove a level select the entire row and click Delete You can select multiple rows and delete them all at once 2 To change a level name select the name and type the new one over it When you enter a name that already exists a warning message is displayed and you must enter a different name 3 To move from cell to cell press TAB To move backward through the cells press SHIFT TAB 4 To enter a new level click the empty row at the end of the grid enter a new name and enter an amount Note For QCs you can also enter the Test amount Thermo Scientific LCquan User Guide 237 4 Creating a Processing Method Specifying Method Calibration Figure 142 Target Compound area m Target Compound ISTD Calibration Curve Type Alprazolam d5 v Isotope Linear s Response r Weighting gt Area Equal C Height c 1K Origin C 1 2 C Ignore Hi C Force Boe
484. ve according to the criteria used in your laboratory 4 To inspect the calibration curve at low concentrations Figure 192 a With the mouse pointer draw a rectangle around low concentration data points in the calibration curve When you release the mouse button the LCquan application displays only the selected low concentration data points Figure 192 Calibration curve at low concentrations hydrocortisone hydrocortisone Y 0 0293758 1 14184 K R 2 0 9996 Wi Equal Y 0 0293758 1 14184 X R 2 0 9996 Wi Equal 2 T oe pA o T Area Ratio 0 04 a 1 2 3 ng Thermo Scientific LCquan User Guide 333 6 Processing the raw Files and Reviewing the Analytical Results Reviewing the Calibration Curve and OCs b To reset the scaling of the x and y axes of the calibration curve to full scale right click and choose Reset Scaling from the shortcut menu or click the Reset Scaling icon Q 5 Inspect the Result list a Check the entries in the Result list for peak detection and integration problems Use the scroll bar at the bottom of the list to see all the columns b Ensure that the data files correspond to the correct levels and sample types 6 Select a data file by clicking its row in the Result list 7 Inspect the target compounds and internal standard peaks a Inspect the component peak in the Chromatogram pane i Ensure that the LCquan application found the peak It shades the peak it finds gray and m
485. vel Tables to all Target Components Copies all calibration parameters defined in this component and in the current Cal and QC level tables to other defined target components Copy Selected Acquisition Levels to All Target Components Opens the Select Component dialog box where you can copy the Cal and QC level tables defined for the acquisition components to all defined target components The level tables are a composite of all levels for all acquisition components For more information see Select Component Dialog Box on page 243 Load Default Levels for This Component Loads the default level tables for the current component Load Selected Acquisition Levels for this Component Opens the Select Component dialog box where you can select acquisition components The levels from the selected component are copied for both the Cal and QC levels For more information see Select Component Dialog Box on page 243 Thermo Scientific 4 Creating a Processing Method Specifying Method Calibration Select Component Dialog Box Thermo Scientific Use the Select Component dialog box to select the component with the level tables you want to use to copy acquisition levels to all target components or load acquisition levels for the selected component Figure 144 Table 95 lists the parameters for the Select Component dialog box Figure 144 Select Component dialog box Select Component r Components A a
486. vels for a single component and can be from one or more LCquan workbooks For details refer to the TrackQC online Help Set Sorting Order Opens the Quantitation Results Sorting Order dialog box where you can change the sorting order of the rows in the sequence See Customizing the Results Sorting Order on page 331 Color Coding Legend Displays the color coding legend Color Legend 328 LCquan User Guide Thermo Scientific Customizing Column Arrangement 6 Processing the raw Files and Reviewing the Analytical Results Reviewing the Calibration Curve and OCs 1 From the results grid shortcut menu select Columns The Column Arrangement dialog box opens Figure 190 Table 126 lists the parameters for the Column Arrangement dialog box Figure 190 Column Arrangement dialog box Column Arrangement Available Columns Acq Date Area Barcode with Piac 250 100 150 Barcode Status 75 Cal Eqn Client Comment 250 250 250 250 Delta RT Dil Factor Duration Height Inj Yol E F m E F Company Name m m 4 F mi F Factory Defaults Current Arrangement Name Displayed Columns tem J wiam Places r FileName Sample Type Response Response Ratio zlz ISTD Area Specified Conc Calculated Conc 1 cv RSD Diff Level NIM NY wlw owo Quan Result Grid qa FAFA Units RT 2 To hide currently
487. ver the rows e Use the CTRL key to select non contiguous rows 2 Press DELETE or right click the sequence and choose Delete Rows from the shortcut menu Using the Acquisition Sequence Grid Shortcut Menu Thermo Scientific You can perform many functions from the shortcut menu Figure 39 Some of these commands are also found on the window menus and others are unique to the shortcut menu Figure 39 Acquisition Sequence Grid shortcut menu 000 1 000 z From AS 0 000 000 ER a amda 000 ooo Fill Down 00 000 P 00 300 Columns 00 000 User Labels and Values 00 300 Standard and QC Levels 00 000 Get Injection Volume from 4S 00 000 00 000 Insert Rows o0 J00 Delete Rows 50 000 Duplicate Selected Samples 00 000 00 000 Import Acquisition Sequence 00 00 New Acquisition Sequence Wizard 00 000 1 000 5 From 4S 0 000 e Fill Down fills sequential rows with duplicate or sequential values e Sort reorders the rows in the sequence e Columns customizes column arrangement e User Labels and Values changes user labels and values e Standard and QC Levels changes or imports calibration and QC levels e Get Injection Volume from AS uses an autosampler injection volume e Insert Rows inserts rows in the grid e Delete Rows deletes rows from the grid e Duplicate Selected Samples duplicates rows in the grid e Import Acquisition Sequence imports an acquisition sequence e New Acquisition Sequence Wizard creates a
488. viewing the Calibration Curve and OCs Calibration Settings Type Use the Type page Figure 196 to modify the component type settings that you specified on the Calibration page in the Create Method view of the Quantitate window Figure 196 Calibration Settings dialog box showing the Type page for Paroxetine Calibration Settings Paroxetine Type Curve Levels Isotope ISTD FAEraeeniee a Table 132 Calibration Settings Paroxetine dialog box Type page parameters Parameter Description Component Type Target Compound The sample you are measuring ISTD This button is active only when at least one component is defined as an internal standard and another component is selected as Target Compound ISTD Select any available internal standard calibration to be associated with the target compound Note This selection is not available when the current target component is selected Thermo Scientific LCquan User Guide 341 6 Processing the raw Files and Reviewing the Analytical Results Reviewing the Calibration Curve and OCs Excluding a Calibration Standard from the Calibration Curve With LCquan you can exclude specific calibration standards from the calibration curve Follow the procedures of your laboratory for proper treatment of calibration data Use any of these options to exclude calibration data e The Exclude column of the Result list e The Exclusion list e The Calibration Curve pane When you ex
489. w The left and right arrows move through the sequence list incrementing or decrementing by a single sample Column Headings Parameter Displays a column of descriptive text for the item Value Displays a column of data for the given parameter Shortcut Menu Open Raw File Create Method view only Opens the Select Raw File dialog box where you can select the raw file to load Lock against All Prevents changes to a raw file or parameters The lock icon is red Change and the lock is closed Unlock Makes the pane editable The lock icon is green and the lock is open Copy to Clipboard Copies a chromatogram mass spectrum or calibration curve to the Clipboard You can then paste the data from the Clipboard buffer into a document LCquan User Guide 283 4 Creating a Processing Method Data Views Status Plot View This view displays the status information stored in the current raw file in graphical form Figure 164 Figure 164 Status Plot view 4 gt a 8 k loni Ms Qua Status P APN_O11 Device MS 2981 6 2931 4 2981 2 2981 0 2980 8 2980 6 2980 4 2980 2 2980 0 a a S gt a a N c 2 gt oO 2979 8 2979 6 2979 4 2979 2 2979 0 2978 8 2 Time min The contents of the plot are read only When the view is locked lock icon is red you cannot select any menu item in the shortcut menu that changes the raw file Menu items that would cau
490. w Files and Reviewing the Analytical Results 323 Processing the Taw Piles es tithe toerien e ee ae Semen n eh 323 Reviewing the Calibration Curve and QCs 0 eee eee 324 Opening the Survey View sicher cok ceive biweeak tea hine he ees 325 Reviewing the Calibration Standards 2 un deka wivetaeeionies new kes 333 Modifying Calibration Settings yy lac ek ee hee ets A 336 Excluding a Calibration Standard from the Calibration Curve 342 Modifying the Peak Detection and Integration Settings 345 Specifying Additional Peak Detection Criteria 0 000 000 0 346 Manually Integrating Peaks so endo actnsoy dacdas bale aleealeu 363 Thermo Scientific Thermo Scientific Appendix A Appendix B Appendix C Contents Reviewing All Results ires tats coc Rap a hint a dont fa e a hae 365 Opening the Review All Results View 0 0c cee cece eee eee 365 Reviewing the Unknowns fae Sse sears Me han Ve ee le 366 Reviewing Peak Properties n siete ced aiden Ab acd aA ate aS ee 367 Adding Comments to the Chromatogram Display 44 374 Creating and Reviewing Reportsss 00 50 hai eka tas tan whe oiSAl Sawes 375 Generating Reports secsoii ont oak E ek E E ne EE AN 377 Review Reports Warning 0 0 cece cece eens 383 Report Options 0 0 i e eea cc cee ee eee eee 384 Fxp rting Res lts wns aera eGR tether sae tue hee we hie tds 396 Locking the Workbook si tics osetia Sol
491. w over which the LCquan application searches the peak Smoothing Thermo Scientific Number of smoothing points used in the chromatogram LCquan User Guide 393 6 Processing the raw Files and Reviewing the Analytical Results Creating and Reviewing Reports Table 152 Grid parameters Sheet 9 of 9 Parameter Study Description User defined Displays information that is pertinent to the active sample row in the Acquisition sequence You can use this box to convey information about this sample to others or as a reminder to yourself The User Label 1 default name is Study To change definitions of User Labels and Values for any user defined column refer to the instructions for the Client column Vial Pos Excel Short Summary 394 LCquan User Guide Displays the sample s position number in the autosampler To change the position number Type the new position number in the Vial Position box The Excel Short Summary is a column arrangement for creating Microsoft Excel formatted reports from the Reports view in the Quantitate window You can use the Excel Short Summary column arrangement as a template to create a new arrangement Figure 225 shows Excel Short Summary selected in the Column Arrangement dialog box Figure 225 Column Arrangement dialog box Column Arrangement Available Columns Displayed Columns a Acq Date FileName Area s Sample Type Barcode Sample Name Barcode Statu
492. wing Reports 4 To delete a row select the entire row right click the selection and choose Delete Selected Rows from the shortcut menu Figure 219 Excel report selection page Excel Report Selections a cama 1 M Quan Result Grid M Excel Short Summary s O l Sample Style Excel Reports 378 LCquan User Guide Thermo Scientific 6 Processing the raw Files and Reviewing the Analytical Results Creating and Reviewing Reports Table 147 Excel report selections page parameters Parameter Description Use Specifies which reports the LCquan application creates when you click Create Reports Select the check box of each report you want to generate Column Arrangement Specifies the column arrangement to use for the Excel report The LCquan application comes with the following predefined column arrangements e Quan Result Grid Generates an Excel report using the current Quan Results column arrangement as specified on the Survey and Review All Results views of the Quantitate window You cannot modify this column arrangement from the Review Reports view e Excel Long Summary Generates a detailed Excel report You can use this column arrangement to create your own column arrangement e Excel Short Summary Generates a brief Excel report You can use this column arrangement to create your own column arrangement Sample Style Excel Generates the Excel reports with one worksheet per sample and Reports with informat
493. x assigned to the sequence and a sequential sequence number When the default sequence starting number 1 is not changed the suffix number is the same as the row number of the sequence 001 002 and so on When the default sequence starting number is changed to another number the first sample has the starting number and subsequent rows in the sequence are incremented by 001 For example if the starting number is 100 the File Name for the first sample has a suffix of 100 the second sample has a suffix of 101 and so on Sample ID Displays the sample ID for the data file The sample ID is an alphanumeric string of characters that uniquely identifies a sample Path Displays the path to the raw files that the LCquan application creates for the sample data The LCquan application creates these files with extension raw A path contains the drive and one or more folders A typical path is C Xcalibur data Instrument File Specify the path and file name of the instrument method to be used to analyze the samples in the active sequence A path contains the drive and one or more folders A typical path for instrument method file ABC is C Xcalibur methods ABC Position Displays the sample s position number in the autosampler Inj Volume 100 LCquan User Guide Displays the injection volume in microliters of sample to be injected When you are using an autosampler you can set the default injection vol
494. x where you can select the raw file to load Lock against All Prevents changes to a raw file or parameters The lock icon is red Change and the lock is closed Unlock Makes the pane editable The lock icon is green and the lock is open Reset Scaling Resets x and y axes to display the maximum amount of data Copy to Clipboard Copies selected data to the Clipboard You can then paste the data from the Clipboard buffer into a document Thermo Scientific 3 Exploring the Data Data Views Instrument Method View Thermo Scientific The Instrument Method view displays the instrument method parameters Figure 99 To view an instrument s method select the instrument from the Item Selector list Figure 99 Instrument Method view 4 gt 3 Durandal Instrument Meti gt Durandal ThermoPal Creator O _LegacyDualPump bt modified 11 16 2005 by Quantum MS Run Time min 4 00 TSQ MS Method Settings Segment 1 Duration min 2 15 Scan Events 1 Segment 1 Tune Method C Xcalibur methods AlprazolamMaurizioMethod TSQTune Chrom filter 5 Q2 Gas Pressure 1 5 Syringe Pump Off Scan Events 1 c SRM Accurate Mass Off Parent Center Width Time CE Q1PW Q3 PW Tube Lens 330 130 70 150 0 002 0 10 35 0 70 0 70 96 330 130 151 010 0 002 0 10 25 0 70 0 70 96 2270 4290 1409 NATA anand aan on ATH The contents of the grid are read only When the view is locked lock icon is yellow you cannot select any menu item in th
495. y Directory selected oo c xcalibur system programs OK Sct Cancel E Xcalibur E gt system Disk Space gt gt Help i Network Table 38 Select Directory dialog box parameters Parameter Directory Selected Description Displays the current directory Drives Disk Space No Chart Opens the Map Network Drive dialog box where you can change the drive location You can gain access to additional drives by connecting to a network Displays or hides a pie chart that displays the unavailable occupied disk space in red and the available free disk space in green Network Save Settings Options Opens the Map Network Drive dialog box where you can connect to a shared network drive or folder and assign a drive letter to the connection This dialog box warns you that you changed components or views without saving the latest setting changes Select Discard Any Changes if you do not want the latest changes saved in the settings when you change components or views Default The latest changes are saved in the settings when you change components or views Note After the default is set up select the Don t Tell Me About This Again check box LCquan User Guide 107 2 Preparing to Perform Quantitative Analysis Emergency Shutdown Emergency Shutdown 108 LCquan User Guide From the Emergency Shutdown dialog box specify what you want the LCquan application to do before it p
496. y 2 Client One APN_O1S APN_O15 f From AS 0000 0 000 Tutorial Study 2 Client One APN_016 APN_016 1 From AS 0 000 0 000 Tutorial Study 2 Client One APN_O17 APN_O17 f From AS 0 000 0 000 Tutorial Study 2 Client One APN_018 APN 018 f From AS 0 000 0 000 Tutorial Study 2 Client One TAPN_O19 APN_019 J From AS 0 000 0 000 Tutorial Study 2 Client One AOA AAD A aan aa aaawua bd Powers Dana NOT SAVED 6 12 2008 12 30PM A The Setup Sequence view includes the following e Acquisition Sequence Header e Acquisition Sequence History e Acquisition Sequence Grid A sequence is a defined set of experiment parameters that define operating settings for a single sample or list of samples The acquisition sequence is a list of samples and defined settings that the LCquan application uses to acquire data The acquisition sequence can contain Unknown samples calibration Standard samples quality control QC samples and Blank samples Each sample can be defined by some or all of the following settings e Sample type e File name e Sample identification e QC or Standard level Thermo Scientific LCquan User Guide 31 2 Preparing to Perform Quantitative Analysis Using the Setup Sequence View e Internal standard correction amount e Dilution factor e Vial position e Injection volume e Sample volume e Sample weight e Sampl
497. ys peak status e If peak is excluded display Excluded e If this peak is an unknown display Peak Status e If this peak is from a QC and it fails use QC Failed e Ifthe peak is not found use Not Found e For all others use peak status Peak Status is one of the following ePeakNotFound eNotCalibrated eCalibExcluded ePeakFound eQCFailed eQCPassed eResponseHigh eResponseLow Thermo Scientific 6 Processing the raw Files and Reviewing the Analytical Results Creating and Reviewing Reports Table 152 Grid parameters Sheet 4 of 9 Parameter Description Level Indicates the level defined for a Calibration Sample or Quality Control Sample To change this level click the grid cell and select another level Units Displays the calibration or QC level of the sample To change this value click the grid cell and select another level RT Retention time in minutes at the peak maximum time when an analyte elutes after injection This is the total time that the analyte is retained on the chromatographic column If the maximum signal from an analyte is detected 5 minutes and 14 seconds after injection the analyte has a retention time of 5 14 Sample ID Displays the sample ID for each data file The sample ID is an alphanumeric string of characters that identifies a sample Exclude Indicates if the sample point can be included or excluded from the calibration curve Standards or RSD

LCquan 2.7 Quantitative Analysis User Guide

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