ISOLUTE® SLE+ User Guide
Contents
1. SPE Dry 96 Sample Concentrator System 100 120V SPE Dry 96 Sample Concentrator System 220 240V SPE Dry 96 Sample Concentrator System with with PTFE coated needles Top Head Only 100 120V SPE Dry 96 Sample Concentrator System with with PTFE coated needles Top Head Only 220 240V SPE Dry 96 Dry Dual Sample Concentrator System SPE Dry 96 Dual Concentrator System 110V SPE Dry 96 Dual Concentrator System 220V SPE Dry 96 Dual Concentrator Sample Concentrator System with PTFE coated needles Top Head Only 100 120V SPE Dry 96 Dual Concentrator Sample Concentrator System with PTFE coated needles Top Head Only 220 240V TurboVap TurboVap 96 100 120V TurboVap 96 220 240V TurboVap LV 100 120V TurboVap LV 220 240V Part Number 121 9600 121 9601 121 9602 121 1016 121 2016 PPM 96 PPM 48 SD 9600 DHS NA SD 9600 DHS EU SD 9600 DHS T NA SD 9600 DAS T EU SD2 9600 DHS NA SD2 9600 DHS EU SD2 9600 DHS T NA SD2 9600 DHS T EU C103263 C103264 C103198 C103199 ISOLUTE SLE User Guide 23 Your Complete Partner for Effective Chemistry Biotage is a worldwide supplier of instruments and accessories designed to facilitate the work of laboratory chemists With our deep knowledge of the industry academic contacts and in house R amp D teams we can deliver the best solutions to your challenges We take great pride in our flexibility and ability to meet our customer s i2. allowed to equilibrate prior to any other volume is absorbed onto the extraction bed Itis therefore Sample pre treatment When standards are added in water vital to use a format with sufficient capacity to absorb the miscible organic solvents ensure that the volume added is as whole sample volume low as possible for example 10 uL standard solution in 200 uL sample For optimum performance even when higher sample volumes are used the volume of internal standard solution should be kept as low as possible Note the sample volume refers to the aliquot of raw sample matrix plus any dilution buffer used For example to extract 200 uL of plasma diluted 1 1 with buffer a 400 uL capacity product should be used This will minimize any solvent bridge effect avoiding matrix effects due to increased transfer of water soluble matrix Table 2 below provides guidelines for sample load and elution components into the elution solvent volume for each ISOLUTE SLE format Scalability The recommended workflow for processing ISOLUTE SLE columns and plates is shown below A method developed on one ISOLUTE SLE format can easily be transferred to other formats if larger or smaller sample volumes 1 Pre treat sample as required including internal are to be extracted see below standard addition De Mens CONGCLON VASSELIS IMPlacE B blockers A suite of B blockers Figure 8 was used in Load sample onto ISOLUTE SLE column or plate recove
3. to GC MS Analysis Extraction of Nicotine and Metabolites from Urine Serum Plasma and Whole Blood Using ISOLUTE SLE Prior to LC MS MS Analysis Extraction of Illicit Drugs from Hemolyzed Whole Blood using ISOLUTE SLE Columns Prior to LC MS MS Analysis Extraction of Synthetic Cannabinoids SPICE from Oral Fluid Using ISOLUTE SLE 96 well Plates and Columns Prior to LC MS MS ISOLUTE SLE User Guide Application Number AN793 AN796 AN797 AN798 AN802 AN803 AN805 AN808 AN810 AN811 AN812 AN815 AN816 AN819 AN820 Title Automated Extraction of Synthetic Cannabinoids SPICE from Urine using ISOLUTE SLE Prior to LC MS MS Extraction of Acrylamide from Coffee Using ISOLUTE SLE Prior to LC MS MS Analysis Extraction of Acrylamide from Fried Potato Chips Crisps Using ISOLUTE SLE Prior to LC MS MS Analysis Extraction of Phthalates in Commercial Milk Products Using ISOLUTE SLE Supported Liquid Extraction Columns Prior to LC APCI MS MS Analysis Extraction of Cocaine and Metabolites from Urine Using ISOLUTE SLE Prior to GC MS Analysis Extraction of Cocaine and Metabolites from Oral Fluid Using ISOLUTE SLE Prior to GC MS Analysis Extraction of Antiepileptic Drugs from Serum and Urine Using ISOLUTE SLE Prior to LC MS MS Analysis Extraction of Designer Stimulants from Urine Using ISOLUTE SLE prior to GC MS Analysis Extraction of Mycophenolic Acid MPA and Mycopheno
4. to infringe ISOLUTE SLE Supported Liquid Extraction Products ISOLUTE SLE Supported Liquid Extraction Products FU umi I uj E di is ies ti Las Piria L T cH VacMastor OG This guide describes how to develop supported liquid extraction methods using ISOLUTE SLE products along with hints and tips for optimizing performance and extending the range of analytes that can be extracted ISOLUTE SLE plates and columns contain a modified form of diatomaceous earth and are used for the extraction of a diverse range of analytes from aqueous samples such as biological fluids using a simple load wait elute procedure Various formats both well plates and columns are available for the extraction of aqueous sample volumes ranging from 10 uL to 10 mL See page 16 Hints Tips and Troubleshooting Sample Preparation Using Supported Liquid Extraction The Supported Liquid Extraction SLE process is analogous to traditional liquid liquid extraction LLE and utilizes the same water immiscible solvent systems for analyte extraction However instead of shaking the two immiscible phases together the aqueous phase is immobilized on an inert diatomaceous earth based support material and the water immiscible organic phase flows through the support alleviating many of the liquid handling issues associated with traditional LLE such as emulsion formation As a result recoveries are often higher and demonstrate better reproducibility
5. 0 VacMaster 20 samples in parallel using ISOLUTE SLE columns and are compatible with all 400 uL to 10 mL capacity columns A range of stopcock options and spare parts for VacMaster manifolds are available Contact your local representative or visit www biotage com for further details Biotage 2014 Biotage Pressure Positive Pressure Manifolds Biotage PRESSURE manifolds offer positive pressure parallel processing for all ISOLUTE SLE 96 and 48 well and Array plates and 400 uL 1mL sample capacity ISOLUTE SLE columns The systems utilize a consistent uniform flow of positive pressure to move both samples and solvents on and off ISOLUTE SLE products Each port of the PRESSURE manifold independently maintains constant pressure increasing the overall reproducibility of analyte recoveries This unique design allows for partially populated racks to be used without sacrificing extraction efficiency The intuitive Biotage PRESSURE is easily incorporated into laboratory work flow Biotage PRESSURE 96 The self adjusting upper manifold of the PRESSURE 96 manifold is compatible with all 96 and 48 well plate formats in addition to the popular 1 mL and 2 mL Array modular well formats without the need to purchase supplementary gaskets Biotage collection plates are recommended for the most consistent and reliable results Biotage PRESSURE 48 The same self adjusting technology utilized in the PRESSURE 96 manifold allow
6. 50 50 8 0 5 0 95 7 0 0 5 99 5 Therefore for the highest solubility of a weakly basic analyte with pKa of 9 0 in an organic solvent adjust sample to pH 11 0 or higher 2 pH units ABOVE the pKa Note that it is not always necessary to reach the theoretical optimum pH to achieve high recoveries using ISOLUTE SLE products due to the highly efficient extraction process The following pages outline general strategies for method development for extraction of neutral acidic and basic analytes using ISOLUTE SLE A screening approach to method development is also described page 12 When developing methods for LC MS MS analysis we recommend the use of volatile MS friendly buffers wherever possible Recommended Not recommended Hydroxide Phosphate Acetate Carbonate Formate Other non volatile buffers Biotage 2014 Where the use of non volatile buffers is necessary consider reducing the sample load volume to 3 4 of the recommended maximum to minimize any adverse effects This is particularly important when elution solvents in which water is partially soluble e g ethyl acetate ISOLUTE SLE User Guide Method Development Strategies Method Development for Neutral Non lonizable Compounds For true neutral analytes that do not have a pKa value pH control is not a major factor as these analytes should extract across the pH range The choice of the correct extraction solvent is the most important factor A water immiscibl
7. AN776 AN777 AN778 AN779 AN780 AN787 AN790 AN791 Title Extraction of Vitamin D Metabolites From Human Serum Using ISOLUTE SLE in 96 Fixed Well Plate Format Prior to LC MS MS analysis Extraction of a Range of Immunosuppressants From Whole Blood Using ISOLUTE SLE for LC MS MS Analysis Extraction of 1 25 Dihydroxyvitamin D From Human Serum Using ISOLUTE SLE Prior to LC MS MS Analysis Extraction of Low Level Testosterone and Androstenedione From Human Serum Samples Using ISOLUTE SLE Extraction of 22 Pain Management Drugs from Urine using ISOLUTE SLE in 96 Fixed Well Plate Format Prior to LC MS MS Extraction of a range of Opiates and Metabolites from human urine using ISOLUTE SLE columns prior to GC MS analysis Extraction of Cocaine and Metabolites From Hydrolyzed Urine Using ISOLUTE SLE prior to LC MS MS Analysis Extraction of Cocaine and Metabolites From Urine Using ISOLUTE SLE prior to LC MS MS Analysis Extraction of Bath Salts substituted cathinones From Human Urine Using ISOLUTE SLE Columns prior to GC MS Analysis Extraction of Cortisol from Human Urine Using ISOLUTE SLE Plates Prior to LC MS MS Analysis Extraction of Cortisol from Human Saliva Using ISOLUTE SLE Plates Prior to LC MS MS Analysis Extraction of Synthetic Cannabinoids SPICE from Oral Fluid Using ISOLUTE SLE prior to GC MS Analysis Extraction of Synthetic Cannabinoids from Hydrolyzed Urine Using ISOLUTE SLE Prior
8. BE dichloromethane or ethyl acetate are useful For polar molecules that do not elute well in these solvents 5 v v of a polar modifier such as isopropanol can be added to the extraction solvent to enhance extraction efficiency and raise analyte recovery Note however that the use of higher amounts of polar water miscible modifiers in the extraction solvent can lead to co extraction of some matrix components such as phospholipids Typical extraction conditions for acidic analytes using a 200 uL ISOLUTE SLE plate part number 820 0200 Po1 Sample pre treatment Load Wait Elute Post extraction Example Dilute plasma sample 1 1 v v with 1 formic acid approx pH 3 4 Dispense sample 200 uL Apply vacuum 0 2 bar for 2 5 seconds to initiate flow Wait 5 mins for sample to completely absorb Apply extraction solvent 1 mL Allow solvent to flow for 5 mins under gravity Apply vacuum 0 2 bar for 10 30 seconds to complete elution Evaporate to dryness and reconstitute in mobile phase prior to analysis Extraction of NSAIDs from human plasma In this example the plasma sample is adjusted to acidic pH using 1 formic acid CH3 OH Figure 13 Structure of ketoprofen pKa and LogP of selected NSAIDs Analyte pKa Log P Acetaminophen 9 7 0 46 Salicylic acid 2 97 2 21 Naproxen 4 2 2 8 Mefenamic acid 4 2 5 1 Results Recoveries of NSAIDs from human plasma using a 200 uL ISOLUTE SLE p
9. ISOLUTE SLE User Guide Supported Liquid Extraction ISOLUTE SLE User Guide CONTENTS 3 ISOLUTE SLE Supported Liquid Extraction Products 4 Sample Preparation Using Supported Liquid Extraction Extraction Mechanism Extraction Cleanliness Phospholipid and Protein Removal Using ISOLUTE SLE Productivity in Automation Processing ISOLUTE SLE Products Processing Conditions Volume Guidelines Internal Standards Scalability amp Method Development Strategies Sample Pre Treatment Method Development for Neutral Non lonizable Compounds Method Development for Basic Compounds Method Development for Acidic Compounds Streamlined Method Development Recommendations Extending the Range of Extractable Analytes Hints Tips and Troubleshooting 18 Applications Listing 20 Biotage Sample Processing Accessories 2 1 Biotage Evaporation Products 22 Ordering Information Cover image Colored Scanning Electron Micrograph SEM of a Jriceratium sp Diatom BOUTE SLE Anal rare _ rhe ya puncte Fined Walt BE Diatomaceous earth materials are natural products made up of high silica content fossilized biominerals Their properties have been exploited in many industrial processes When packed into columns they have been widely used as a support for chromatographic separations GC and can also be us
10. S a 2 E E ES FFF LS EU ET ET SF R S X S S S S S NS lt Oo C C S we eo Q ep oS A A r Ww m2 HCl 1 formic 0 1 Formic 100mMM NH4OAC H20 0 5M NH4OH ev Figure 6 Levels of phospholipids in sample human plasma 100 uL extracts prepared using ISOLUTE SLE with a variety of sample pre treatment and extraction solvents In figures 4 5 and 6 Phospholipids were monitored by LC MS MS using electrospray ionization in the positive ion mode The most abundant phospholipids selected from full scan SIR and precursor ion scan data were quantified using the 184 product ion in the multiple reaction monitoring mode MRM Productivity in Automation In addition to being easy to use on both vacuum and positive pressure processing manifolds ISOLUTE SLE plates and columns are well suited for use in automated higher throughput applications Procedures are confined to straightforward liquid sample or elution solvent dispensing and vacuum or pressure application No off line shaking or mixing centrifuging flash freeze pour or other manual steps necessary for traditional liquid liquid extraction are required The simple load wait elute procedure allows ISOLUTE SLE plates and columns to be processed in half the time of traditional liquid liquid extraction See table 1 or solid phase extraction procedures ISOLUTE SLE support material gives excellent loading and elution characteristics Due to t
11. TE SLE Extraction of Tamoxifen and Metabolites from Urine Using ISOLUTE SLE Method for the Extraction of Warfarin From Human Plasma Using ISOLUTE SLE Extraction of Methylmalonic Acid MMA and Succinic Acid SA from Human Serum Using ISOLUTE SLE in 96 Well Plates and Columns Extraction of Testosterone and Other Steroid Hormones From Human Plasma Using ISOLUTE SLE 96 Well Plates Extraction of Opiates From Human Urine Using ISOLUTE SLE 96 Well Plates and Columns Extraction of Amphetamines From Urine Using ISOLUTE SLE 96 Well Plates Extraction of Amphetamines From Urine Using ISOLUTE SLE Columns How to Process ISOLUTE SLE Plates and Columns with Pressure 96 amp 48 Positive Pressure Manifolds Extraction of Benzodiazepines From Human Urine Using ISOLUTE SLE 96 Well Plates and Columns Prior to LC MS MS Analysis Extraction of Barbiturates From Human Urine Using ISOLUTE SLE Columns with GC MS Analysis Extraction of Retinol B Carotene Vitamin A and amp Tocopherol Vitamin E from Serum using ISOLUTE SLE 96 Well Plates with APCI LC MS MS Analysis Extraction of Retinol B Carotene Vitamin A and amp Tocopherol Vitamin E from Whole Blood using ISOLUTE SLE 96 Well Plates with APCI LC MS MS Analysis Extraction of Benzodiazepines From Human Urine Using ISOLUTE SLE in Column Format Prior to GC MS analysis Application Number AN757 AN758 AN761 AN762 AN764 AN770 AN771 AN772
12. TE SLE after Collection with the Oral Eze Collection Device Prior to GC MS Analysis Biotage is constantly developing new applications using ISOLUTE SLE products Visit the literature database on www biotage com 19 20 Sample Processing Accessories ISOLUTE SLE plates and columns can be processed manually using vacuum or positive pressure manifolds For automated processing ISOLUTE SLE plates and columns are compatible with and easy to use on leading automation systems Biotage VacMaster Sample Processing Stations The VacMaster range of manual processing stations offers versatility a small footprint and cost effective sample throughput from 96 and 48 well plates through to large column applications Designed to meet the most demanding criteria for safety extract purity flexibility and ease of use the VacMaster range of vacuum manifolds can be readily incorporated into the laboratory workflow Biotage VacMaster 96 anuit H o The VacMaster 96 manifold is ideal for processing 96 and 48 well plates The compact design and lightweight construction make it suitable for manual processing or for integrating with automated liquid handling system Two control units are available for use with either a vacuum source or for use with lab air to generate the vacuum Biotage VacMaster 10 and 20 nt ys TETO ULU VacMaster 10 and 20 manifolds are ideal for processing up to 10 VacMaster 10 or 2
13. ction describes strategies developed in our R amp D laboratories to enhance recovery of these analytes and extend the range of analytes that can be extracted using supported liquid extraction Example 1 Extraction of small polar acids using advanced pH control Small polar acids may not give high recoveries using standard pre treatment conditions in supported liquid extraction because of their poor solubility in water immiscible solvents when in their ionized state combined with the extreme pH conditions needed to neutralize the analytes and improve their solubility Methylmalonic acid see below is a small extremely polar water soluble acid Using standard sample pre treatment conditions for acids recoveries of lt 40 were achieved see Figure 16 Analyte pKa Log P Methylmalonic acid 3 07 0 17 HO OH Figure 15 Properties and structure of methylmalonic acid Method Development Strategies 100 80 60 m 0 1 Formic Acid 40 m 1 Formic Acid B i _ E DCM 95 5 DCM IPA 90 10 MTBE EtOAc DCM IPA Figure 16 Methylmalonic acid recovery from human plasma using standard sample pre treatment conditions We therefore investigated advanced pH control in order to improve analyte recovery Using the extraction solvents which gave some analyte recovery in the first experiment MTBE and ethyl acetate a range of acidic sample pre treatment conditions were evaluated Sample pre treatment Sa
14. ctional groups OH O Figure 9 Structure of Testosterone Note For neutral analytes that are protein bound pH adjustment may improve recovery See Hints Tips and Troubleshooting page 16 LogP of endogenous steroids Analyte Log P Aldosterone 0 71 21 deoxycortisol 2 07 Androstenedione 2 72 11 deoxycortisol 2 74 17 hydroxyprogesterone 3 04 Testosterone 3 18 DHEA 3 3 Progesterone 3 83 Androsterone 3 93 Results Recoveries of endogenous steroids from human plasma using a 200 uL ISOLUTE SLE plate are shown below For neutral analytes choice of elution solvent is the most important factor affecting analyte recovery 120 0 100 0 0 N wr wr or gt o c ou xe A sf Se x oe 2 ve oo KA x ay D xo X X e amp O X NA mDCM mEtOAC m BuOAC m MTBE EtOEt 50 50 hex DCM Figure 10 Recoveries of endogenous steroids from human plasma using various elution solvents Method Development for Basic Compounds A water immiscible organic solvent in which the analytes are highly soluble should be chosen as extraction solvent For basic analytes recovery may be enhanced by increasing sample pH to deprotonate the analyte increasing the solubility in the water immiscible organic solvent This can be achieved by dilution of the sample typically 1 1 v v with a basic buffer such as 0 5M ammonium hydroxide This will raise pH gt 10 for most biological fluids For strongly basic compounds e
15. e organic solvent in which the analyte is freely soluble is a good choice For very non polar analytes non polar solvents such as heptane hexane or dichloromethane may be most appropriate For more polar compounds more polar extraction solvents such as MTBE dichloromethane or ethyl acetate are useful For polar molecules that do not elute well in these solvents 5 v v of a polar modifier such as isopropanol can be added to the extraction solvent to enhance extraction efficiency and raise analyte recovery Note however that the use of higher amounts of polar water miscible modifiers in the extraction solvent can lead to co extraction of some matrix components such as phospholipids Typical extraction conditions for neutral non ionizable analytes using a 200 uL ISOLUTE SLE plates part number 820 0200 P01 Sample pre treatment Dilute plasma sample 1 1 v v with water Load Dispense sample 200 uL Apply vacuum 0 2 bar for 2 5 seconds to initiate flow Wait Wait 5 mins for sample to completely absorb Elute Apply extraction solvent 1 mL Allow solvent to flow for 5 mins under gravity Apply vacuum 0 2 bar for 10 30 seconds to complete elution Post extraction Evaporate to dryness and reconstitute in mobile phase prior to analysis Example Extraction of endogenous steroids from human plasma In this example pH adjustment of the sample is not required as the analytes do not have any acidic or basic fun
16. ed for sample preparation applications 2014 Biotage Trademark Acknowledgement The following trademarks are owned by Biotage AB Advancer Advancer 350 Advancer Kilobatch AFFINILUTE Biotage Biotage ZIP Endeavor EVOLUTE EVOLUTE EXPRESS ExploraSep Extrahera Firefly design FLASH FlashMaster FlashVac Flash 75 Flash 150 Flash 400 Horizon HPFC HP SIL HP Sphere Initiator Initiator Peptide Workstation Initiator Initiator Alstra Initiator Robot 60 Initiator Robot 8 Initiator SP Wave Isolera Isolera Dalton Isolera Dalton Mass Detector Isolera Dalton Nanolink Isolera Dalton System Isolera Four Isolera LS Isolera One Isolera Prime Isolera Spektra Isolera Spektra Four Isolera Spektra LS Isolera Spektra One ISOLUTE ISOLUTE Myco ISOLUTE QuEChERS IST G IST design KILOPREP KP C18 HS KP C18 WP KP C4 WP KP NH KP Sil KP Sphere MIP Rule of 6 MIP 4 Process MIP 4 Proteins MIP4SPE PathFinder PRESSURE PRESSURE 48 PRESSURE 96 RapidTrace RapidTrace RENSA Resolux Robot 60 Robot 8 Samplet SIM SNAP SNAP Ultra SNAP XL SP Wave SP1 SP4 SPE Dry SPE Dry 96 SPE Dry 96 Dual SPx Syro Wave TurboVap Universal Phase Separator V 10 VacMaster ZIF ZIF SIM ZIP Sphere 1 Point Support Other product and company names mentioned herein may be trademarks or registered trademarks and or service marks of their respective owners and are used only for explanation and to the owners benefit without intent
17. edure compared with the equivalent liquid liquid extraction procedure Because the same water immiscible solvents are used in SLE proteins and phospholipids are efficiently removed from the final extract and no additional steps such as protein crash precipitation are required Using a fast simple load wait elute procedure supported liquid extraction using ISOLUTE SLE products provides inherently cleaner extracts than other simple sample preparation techniques such as dilute and shoot or protein precipitation The efficient extraction process combining high analyte recoveries elimination of emulsion formation and complete removal of matrix interferences such as proteins phospholipids and salts results in lower limits of quantitation compared to traditional LLE Step 3 Elute Analytes partition into elution solvent and are collected A y ISOLUTE SLE User Guide Supported Liquid Extraction Mechanism of Action ISOLUTE SLE products contain a modified form of diatomaceous earth which provides a support for the liquid liquid extraction process to occur but does not interact chemically with the aqueous sample Application of the sample to the column results in the aqueous sample spreading over the surface of the material forming an immobilized layer of small droplets held in place by a network of pores Figure 2 When the water immiscible extraction solvent is applied for the elution step it flows
18. effect it was necessary to under load the extraction plate We loaded a maximum of 300 uL of pre treated sample per well onto a 400 uL plate This afforded extra capacity to the plate and avoided aqueous breakthrough as well as maintaining excellent extract cleanliness In addition the samples demonstrated excellent flow characteristics through the plate Method Development Strategies Optimized procedure for extraction of highly protein bound 25 OH Vitamin D from serum using a 400 UL supported liquid extraction plate part number 820 0400 P01 Sample pre treatment Dilute serum 1 1 v v using water isopropanol 1 1 v v Load Dispense sample 300 uL Apply vacuum 0 2 bar for 2 5 seconds to initiate flow Wait Wait 5 mins for sample to completely absorb Elute Apply heptane 750 uL Allow solvent to flow for 5 mins under gravity Apply a further aliquot of heptane 750 uL Apply vacuum 0 2 bar for 10 30 seconds to complete elution Post extraction Evaporate to dryness at room temperature and reconstitute in a Suitable solvent prior to analysis Results Using the optimized procedure described high recoveries were achieved and excellent extract cleanliness was maintained 80 60 40 20 25 hydroxyvitamin D2 25 hydroxyvitamin D3 Optimized 50 50 IPA H20 Figure 21 Recovery of 25 OH Vitamin D2 and D3 using sample pre treatment conditions optimized to disrupt protein binding of strongly
19. f total well column capacity Elute with first aliquot of elution solvent Add 10 uL conc ammonium hydroxide NH OH and wait 5 mins then elute with second aliquot of elution solvent Alternatively increase sample pH using ammonium hydroxide 1 2 load sample then elute using solvent containing up to 1 v v trifluoroacetic acid TFA Evaporate to dryness gt 40 C to remove any traces of TFA and avoid any ion pair effect in subsequent chromatography Alternatively evaluate the use of ion pair reagents see page 14 Evaluate the use of a series of aliquots of different extraction elution solvents or blended solvents Ensure maximum sample volume is not exceeded See page 7 load volumes for further details and reduce load volume if necessary If a polar modifier is used evaluate the effect of reducing the of polar modifier or reduce load volume if recoveries are adversely affected 17 18 Applications Application Number AN601 AN602 AN603 AN721 AN734 AN738 AN740 AN741 AN742 AN746 AN747 AN751 AN752 AN753 AN754 AN756 Biotage 2014 Title Extraction of Tricyclic Anti depressants from Plasma Using ISOLUTE SLE Supported Liquid Extraction Plates Extraction of Corticosteroids from Plasma using ISOLUTE SLE Supported Liquid Extraction Plates Extraction of Non steroidal Anti inflammatory Drugs NSAIDs from Plasma using ISOLU
20. from sample to sample Figure 1 In sample preparation the principles of traditional LLE partitioning of analytes between aqueous and water immiscible organic solvents are well known and understood Traditionally analytes are extracted from aqueous samples through the addition of an appropriate water immiscible organic solvent The two immiscible phases are shaken or mixed thoroughly in a Separating funnel and based on relative solubility of the analytes in the two phases analytes will partition into the organic solvent The efficiency of the extraction is enhanced by the shaking which creates a high surface area for the extraction interface allowing partitioning to occur Liquid liquid extraction can give particularly clean extracts of biological fluids since matrix components such as proteins and phospholipids are not soluble in typical LLE solvents and are therefore excluded from the final extract The same benefits are true for supported liquid extraction SLE procedures Step 1 Load A Analyte ea Matrix components e g phospholipids salts and proteins SLE Support material diatoms Aqueous sample flows onto extraction bed and is dispersed in small droplets lt Figure 2 Supported Liquid Extraction Mechanism 4 OBiotage 2014 Step 2 Wait Analyte Recovery Nortryptiline Imipramine Trimipramine Figure 1 Recovery of tricyclic antidepressants from plasma typical ISOLUTE SLE proc
21. g quaternary amines consider the use of ion pair reagents see page 14 pH control is most important when dealing with polar analytes For very non polar analytes non polar solvents such as heptane hexane or dichloromethane may be most appropriate For more polar compounds more polar extraction solvents such as MTBE dichloromethane or ethyl acetate are useful For polar molecules that do not elute well in these solvents 5 v v of a polar modifier such as isopropanol can be added to the extraction solvent to enhance extraction efficiency and raise analyte recovery Note however that the use of higher amounts of polar water miscible modifiers in the extraction solvent can lead to co extraction of some matrix components such as phospholipids Typical extraction conditions for basic analytes pKa and LogP of selected B blockers using a 200 ULISOLUTE SLE plate Analyte pK Log P part number 820 0200 Po1 Atenolol 9 5 0 57 Sotalol 8 3 9 2 0 85 Sample pre treatment Dilute plasma sample 1 1 v v with 0 5M NH4OH approximate pH 10 4 Nadolol 9 67 1 23 Pindolol 8 8 2 17 Load Dispense sample 200 uL Apply Propranolol 9 45 3 17 vacuum 0 2 bar for 2 5 seconds to initiate flow Wait Wait 5 mins for sample Results to completely absorb Recoveries of B blockers from human plasma using a 200 uL Elute Apply extraction solvent 1 mL ISOLUTE SLE plate are shown below Allow solvent to flow for 5 mins under gravity Apply vacuu
22. he unique extraction mechanism of supported liquid extraction with the whole Biotage 2014 Protein Removal Using ISOLUTE SLE To demonstrate the effectiveness of ISOLUTE SLE in removing proteins from biological fluid samples serum was prepared using ISOLUTE SLE protein precipitation and solid phase extraction Residual protein in the extracts was analyzed using gel electrophoresis Complete protein removal was seen with ISOLUTE SLE zZ 2 ep 4 E 5 lt 5 u g a gt Ww 2 D E D o D a D s Zo T o g 5 Fam E rN 8 Sg X n X M Sp X i a 220 7 220 220 120 3 120 id E 120 B 7 70 ru 70 50 50 50 40 f 40 x E x 30 4 30 20 20 20 _ 10 10 10 A ISOLUTE SLE Rat serum pre treated 1 1 v v with water loaded onto ISOLUTE SLE plate and extracted with MTBE All extracts were evaporated to dryness for gel electrophoresis work up B Protein precipitation rat serum precipitated with acetonitrile 1 3 and 1 6 v v C Solid phase extraction 100 uL rat serum extracted using manufacturers recommended generic methods Gel electrophoresis NUPAGE Novex 12 Bis Tris mini gel with MOPS SDS running buffer at 200V 120mA and 12 5V Gels were run for approximately 65 minutes to ensure complete protein migration Figure 7 Protein removal ISOLUTE SLE vs protein precipitation and solid phase extraction sample being absorbed onto the support there is no
23. holipid contamination of the final extract when using polar extraction solvent such as ethyl acetate Extraction of amphoteric analytes To increase recovery of strongly acidic analytes e g SO PO or strongly basic analytes To increase recovery of very water soluble analytes To extract both acidic and basic analytes in the same procedure I To extract both acidic and basic analytes in the same procedure II To extract analytes with a wide range of polarity or solubility characteristics in a single procedure To avoid breakthrough of aqueous phase into final extract Method Development Strategies Solution Centrifuge sample following sample pre treatment to lyse red blood cells Decrease load volume For example load a maximum of 300 uL pre treated sample onto a 400 uL capacity plate or column and optimize extraction solvent volume to obtain desired extract cleanliness and or analyte recovery Evaluate loading at pH corresponding to the isoelectric point of the analyte Note that this approach is highly dependent on analyte polarity and pKa values Evaluate the use of an ion pair reagent during sample pre treatment See page 14 for further details on ion pair reagents Evaluate the use of a saturated salt solution in sample pre treatment Note that this may reduce extract cleanliness Load aqueous sample pH5 50 o
24. hydrophobic analytes 15 16 Hints Tips and Troubleshooting Situation To maximize the volume of raw sample that can be loaded onto an ISOLUTE SLE product To maximize the volume of raw sample that can be loaded onto an ISOLUTE SLE product for NEUTRAL analytes such as steroids and amides To load low sample volumes lt 150 pL To disrupt protein binding of hydrophobic analytes in serum or plasma To improve extract cleanliness for very low concentration analytes in biological fluids To improve extract cleanliness where a higher polarity extraction solvent is required To remove lipids and improve extract cleanliness for low level analysis To improve extract cleanliness when extracting whole blood and avoid breakthrough of red blood cells Sample pre treatment tips for whole blood samples Biotage 2014 Solution The standard sample pre treatment recommendation is a 1 1 v v sample dilution prior to sample loading However to load a larger volume of raw sample without exceeding Capacity we recommend the addition of a smaller volume e g 1 3 or 1 9 v v of more concentrated buffer or pH control reagent Sample pre treatment may not be required as neutral analytes often extract well at physiological pH so raw sample can be loaded directly with internal standard as required Use a high dilution factor E
25. late are shown below Recoveries of more polar analytes are enhanced by the use of more polar elution solvents 100 mDCM m95 5 DCM IPA mBMTBE mEtOAc Figure 14 Recoveries of NSAIDs from human plasma using various elution solvents 11 Streamlined Method Development Recommendations Biotage have developed a simple screening approach based on the principles described in this section according to analyte s functionality By simply screening 2 pHs combined with 4 extraction solvents you can develop a method in minutes ISOLUTE SLE Supported Liquid Extraction Method Selection ACIDIC DRUG NEUTRAL DRUG BASIC DRUG Buffer 1 Buffer 2 Buffer 3 Buffer 4 1 v v formic acid 0 1 v v formic acid Water 0 5M ammonium hydroxide Mix plasma or urine sample 1 1 v v Mix plasma or urine sample 1 1 v v Mix plasma or urine sample Mix plasma or urine sample 1 1 v v with 1 formic acid aq v v with 0 1 formic acid aq v v 1 1 v v with water with 0 5M ammonium hydroxide Urine pH 2 7 Plasma pH 3 5 Urine pH 4 0 Plasma pH 6 2 Urine pH 7 0 Plasma pH 8 2 Urine pH 10 8 Plasma pH 10 7 Extraction Solvent 1 Extraction Solvent 2 Extraction Solvent 3 Extraction Solvent 4 MTBE DCM DCM IPA 95 5 v v Ethyl Acetate Using the Chart Recommendations can be applied to any format of ISOLUTE SLE product Appropriate load and elution volumes are detailed on page 7 y M Analyte functionality according to whether
26. lic Acid Glucuronide MPAG from Serum Using ISOLUTE SLE prior to LC MS MS Extraction of Antiepileptic Drugs from Oral Fluid Using ISOLUTE SLE Prior to LC MS MS Analysis Extraction of 1 25 di OH Vitamin D2 1 a 25 di OH Vitamin D3 25 OH Vitamin D2 and 25 OH Vitamin D3 from Serum Using ISOLUTE SLE Prior to LC MS MS Analysis Analysis of THC and an Extended Metabolite Suite from Oral Fluid Using ISOLUTE SLE Supported Liquid Extraction Columns Prior to LC MS MS Extraction of Propofol from Whole Blood Using ISOLUTE SLE Prior to GC MS Analysis Extraction of THC THCA and Carboxy THC from Oral Fluid by ISOLUTE SLE after Collection with the Intercept Oral Fluid Drug Test Kit Prior to GC MS Analysis Extraction of Barbiturates from Oral Fluid Using ISOLUTE SLE after Collection with the Intercept Oral Fluid Drug Test Kit Prior to GC MS Analysis Application Number AN821 AN822 AN824 AN825 Applications Title Extraction of Barbiturates from Oral Fluid using ISOLUTE SLE after Collection with the Quantisal Collection Device prior to GC MS Analysis Extraction of THC THCA and Carboxy THC from Oral Fluid by ISOLUTE SLE after Collection with the Quantisal Collection Device prior to GC MS Analysis Extraction of Barbiturates from Oral Fluid Using ISOLUTE SLE after Collection with the Oral Eze Collection Device Prior to GC MS Analysis Extraction of THC THCA and Carboxy THC from Oral Fluid Using ISOLU
27. lute with analytes of interest causing significant ion suppression Elimination of phospholipids from sample extracts is therefore essential for reliable analyte quantitation O O Na Na O O O r O a TA O 7 a 7 7 de ae ei o Phosphatidylcholine Lysophosphatidylcholine Figure 3 Examples of phospholipid structures Sample Preparation Using Supported Liquid Extraction Simple sample preparation techniques such as dilute and shoot and protein precipitation do not significantly reduce phospholipid concentration in sample extracts meaning analytical sensitivity and extract cleanliness can be compromised Figure 4 compares the levels of phospholipids PL and lysophospholipids lysoPL in sample extracts prepared using protein precipitation PPT and ISOLUTE SLE 9 00E 07 4 8 00E 07 7 00E 07 6 00E 07 m PPT 5 00E 07 E ISOLUTE SLE 4 00E 07 3 00E 07 2 00E 07 4 1 00E 07 0 00E 00 F PL Lyso PL Figure 4 Comparison of phospholipids and lysophospholipids present in sample extracts prepared using protein precipitation PPT 100pL human plasma precipitated with 300uL acetonitrile and ISOLUTE SLE 100 uL human plasma extracted using MTBE Phospholipid depletion products are designed to remove phospholipids from sample extracts Figure 5 compares phospholipid PL and lysoPL levels in sample extracts prepared using two popular phospholipid depletion products with tho
28. m 0 2 bar for 10 30 seconds to complete elution 120 5 100 Post extraction Evaporate to dryness and reconstitute in mobile phase prior to analysis 80 60 40 Example 20 Extraction of B blockers from human plasma 2 soe a soo oo es ro o gt D In this example the plasma sample is adjusted to basic pH dr m using 0 5 M ammonium hydroxide pe we y ogo wee mMTBE mDCM m95 5 DCM IPA mEtOAc Figure 12 Recoveries of endogenous steroids from human plasma using various elution solvents O N H OH Note that Sotalol is an amphoteric compound so further method optimization is required to achieve high analyte recoveries See Hints Tips and Troubleshooting Page 16 Figure 11 Structure of propranolol 10 Biotage 2014 ISOLUTE SLE User Guide Method Development Strategies Method Development for Acidic Compounds A water immiscible organic solvent in which the analytes are highly soluble should be chosen For acidic analytes recovery may be enhanced by lowering sample pH to suppress ionization of the analyte allowing it to exist in the neutral form This can be achieved through the addition of an acidic buffer such as 1 v v formic acid pH control is more critical when dealing with more polar analytes For very non polar analytes non polar solvents such as heptane hexane or dichloromethane may be most appropriate For more polar compounds more polar extraction solvents such as MT
29. mple pH Dilution 1 1 v v with HCI 2 1 71 Dilution 1 1 v v with HCI 5 0 89 Dilution 1 1 v v with H3PO4 4 1 71 Dilution 1 1 v v with H3PO4 5 155 120 0 100 0 80 0 60 0 40 0 i 20 0 0 0 T t 1 T t 1 Ca QC amp amp amp cS S L gF gF x o oe a oe Roy Figure 17 Methylmalonic acid recovery from human plasma using advanced sample pre treatment conditions Using advanced pH control for plasma pre treatment acceptable recoveries of methymalonic acid were achieved However use of strong non volatile acids for sample pre treatment did lead to increased ion suppression when using solvents with higher water solubility e g ethyl acetate To minimize this effect the use of solvents with low water solubility e g MTBE are recommended Alternatively consider the use of a decreased load volume ora larger capacity product Hints Tips and Troubleshooting page 16 13 14 Example 2 Use of ion pair reagents The use of ion pair reagents can be a very useful approach especially when dealing with matrices and or analytes that are susceptible to hydrolysis or other degradation when exposed to extreme pH conditions This approach can also be used to help with the simultaneous extraction of acid neutral and basic drugs in a single extraction protocol In this example the optimized simultaneous extraction of 11 nor 9 carboxyA THC and 11 nor 9 carboxyA THC glucuronide fr
30. ndividual needs With strong foundations in both analytical and organic chemistry we can offer the widest range of solutions available on the market EUROPE Main Office 46 18 565900 Toll Free 800 18 565710 Fax 46 18 591922 Order Tel 46 18 565710 Order Fax 46 18 565705 order biotage com 2014 Biotage All rights reserved All brand and product names are trademarks or registered trademarks of their respective companies NORTH amp LATIN AMERICA Main Office 1 704 654 4900 Toll Free 1 800 446 4752 Fax 1 704 654 4917 Order Tel 1 704 654 4900 Order Fax 1 434 296 8217 ordermailbox biotage com The information contained in this document is subject to change without notice E amp OE Part Number U1304 V 2 JAPAN Tel 81 3 5627 3123 Fax 81 3 5627 3121 jp_order biotage com CHINA Tel 86 21 2898 6655 Fax 86 21 2898 6153 cn_order biotage com To locate a distributor please visit our website at www biotage com Biotage
31. need to collect waste The extract collection plate can be in place throughout the procedure The organic elution solvent is the only solvent that flows through the well column ensuring even flows and minimizing the possibility of well or column blockage In addition to 96 well plate formats for low volume biological fluid samples ISOLUTE SLE columns are available in both 400 uL and 1 mL sample capacity with the tabless format option compatible with most automation systems Table 1 Comparison of Automated Extraction 12 5 min 22 5 min Automated ISOLUTE SLE Automated liquid liquid extraction Using the Quadra 96 liquid handling system typical ISOLUTE SLE procedure compared with the equivalent liquid liquid extraction procedure ISOLUTE SLE User Guide Processing ISOLUTE SLE Products Processing ISOLUTE SLE Products Processing Conditions Processing ISOLUTE SLE columns and well plates is largely performed under gravity with a pulse of vacuum or positive pressure used to initiate loading of the sample and to maximize solvent recovery leading to more reproducible analyte recovery after elution Both manual and automated vacuum or positive pressure systems can be used Volume Guidelines Internal Standards Unlike SPE where the sample is loaded onto the column and When using internal standards they should be added to the flows through the sorbent to waste in SLE the ENTIRE sample raw sample mixed and
32. not affect subsequent chromatography Biotage 2014 Using the ion pair reagent as described gave high recoveries of both analytes and no losses of the glucuronide metabolite were observed 25mM dibutylammonium acetate demonstrated optimum recovery see Figure 19 THC COOH THC COOH glc 10mM 8m25mM m200mM Figure 19 Recovery of THC and THC Glucuronide from urine using dibutylammonium acetate as an ion pair reagent 10mM 25mM 200mM Ion pair pre treatment recommendations Functional Recommended Final group to ion pair reagent sample be paired pH Basic ve charge Heptafluorobutyric acid Acidic 10 100mM 1 1 v v Basic ve charge Trifluoroacetic acid Acidic TFA up to 1 aq v v 1 1 v v Acidic ve charge Dibutyl ammonium acetate Neutral 25mM 1 1 v v Acidic ve charge Tetrabutylammonium acetate Basic 25mM 1 1 v v For best results volatile ion pair reagents should be used These are removed at the extract evaporation stage and will not therefore impact on the subsequent chromatographic separation ISOLUTE SLE User Guide Example 3 Analytes with extreme protein binding Because proteins are insoluble in typical SLE elution solvents strongly protein bound analytes cannot partition freely into the organic elution solvent leading to reduced analyte recovery Where analytes are strongly protein bound it may therefore be necessary to disrupt this binding prior to sample load in o
33. ns ISOLUTE SLE 2 mL Sample Volume columns Bulk Pack ISOLUTE SLE 5 mL Sample Volume Columns ISOLUTE SLE 10 mL Sample Volume Columns Array Accessories Description ISOLUTE Array base plate ISOLUTE base plate sealing strips strips of 8 Luer Adaptors to fit any vacuum manifold Well removing tool Pack Qty 1 50 25 Deep Well Collection Plates Description Collection plate 1 mL Collection plate 2 mL Collection plate 5 mL 48 well 22 Biotage 2014 Pack Qty 50 50 20 Pack Qty 100 100 50 500 50 30 500 1000 30 1000 20 1000 20 16 Part Number 820 0200 P01 820 0400 P01 820 1000 Q01 820 0200 T 820 0400 T 820 0055 B 820 0055 B 500 820 0055 BG 820 0140 C 820 0055 BG 500 820 0140 C 1000 820 0140 CG 820 0140 CG 1000 820 0290 D 820 0290 D 1000 820 0690 E 820 1420 F Part Number 120 1000 P01 120 1200 120 1201 120 1202 Part Number 121 5202 121 5203 121 5210 Sample Processing Manifolds and Evaporation Products Description VacMaster 96 VacMaster 96 Sample Processing Manifold Vacuum Control Unit VCU 1 Vacuum Control and Generation Unit VCU 2 VacMaster 10 and 20 VacMaster 10 Sample Processing Manifold VacMaster 20 Sample Processing Manifold Pressure 96 and 48 PRESSURE 96 Positive Pressure Manifold PRESSURE 48 Positive Pressure Manifold SPE Dry 96 Sample Concentrator System
34. nsure load volume is sufficient to completely cover the top frit of the column or plate This will facilitate reliable flows during sample processing For plate formats the minimum volume required to cover the frit is 150 uL For example for a 10 uL sample dilute to a minimum of 150 uL with a suitable pre treatment buffer Dilution of the sample with 1 1 v v using water isopropanol 1 1 v v can help to disrupt the protein without causing full precipitation of proteins Decrease load volume For example load 300 or 350 uL pre treated sample onto a 400 uL capacity plate or column Decrease load volume For example load 300 or 350 uL pre treated sample onto a 400 uL capacity plate or column Once sample is loaded and absorbed use a 2 step elution process Selectively elute non polar lipids using hexane discard then elute analytes of interest with a Suitable solvent Decrease load volume use approximately 34 of maximum recommended volume For example load 300 uL pre treated sample onto a 400 uL capacity plate or column Avoid precipitation of proteins with precipitating agents Avoid the use of high concentrations of ZnSO4 Vortex samples thoroughly after addition of pre treatment reagents ISOLUTE SLE User Guide Situation To increase recovery of analytes present inside red blood cells To improve extract cleanliness and avoid phosp
35. om urine is described The parent compound extracts well under acidic conditions but the highly water soluble glucuronide metabolite is not recovered see Figure 18 100 THC COOH THC COOH glu mDCM MTBE amp DCM DCM MTBE mDCM MTBE DCM amp DCM MTBE MTBE Figure 18 Recovery of THC and THC Glucuronide from urine using standard 1 formic acid pre treatment and a variety of extraction solvents The use of a neutral ion pair reagent allows ion pairing of the carboxylic acid groups increasing their solubility in water immiscible solvents Procedure for simultaneous extraction of 11 nor 9 carboxy A THC and 11 nor 9 carboxy A THC glucuronide from urine using a 200 uL supported liquid extraction plate part number 820 0200 Po1 Sample pre treatment Dilute urine 1 1 v v using 25mM dibutylammonium acetate Load Dispense sample 200 uL Apply vacuum 0 2 bar for 2 5 seconds to initiate flow Wait Wait 5 mins for sample to completely absorb Elute Apply ethyl acetate 1 mL Allow solvent to flow for 5 mins under gravity Apply vacuum 0 2 bar for 10 30 seconds to complete elution Post extraction Evaporate to dryness at 40 C and reconstitute in mobile phase prior to analysis 1 These pre treatment conditions allow ion pairing of the carboxylic acid groups while avoiding any pH related hydrolysis of the glucuronide or the matrix 2 Blowdown at 40 C ensures that the ion pair reagent does
36. oretical considerations For acidic analytes it is often preferential to acidify the sample with an appropriate buffer prior to loading to ensure that the analyte charge is suppressed non ionized and therefore more soluble the organic phase Conversely for basic analytes higher recoveries may be obtained by adding a small amount of basic modifier to raise the sample pH prior to loading pH control is most important for polar analytes that are only sparingly soluble in water immiscible solvents when charged Buffer Recommendations for LC MS MS Analysis The 2 pH Unit Rule The pK of an ionizable functional group is defined as the pH at which 50 of this group in solution is charged and 50 is uncharged neutral Each pH unit change affects the percentage of charged or uncharged groups by a factor of 10 so it is optimal to perform extractions at a pH at least 2 pH units from the pKa value to ensure that 99 5 of the functional groups are in the desired state of ionization e g Effect of pH on the dissociation of weak acid with a pKa value of 4 0 2 0 99 5 0 5 3 0 95 5 0 ACID pK 4 0 50 50 5 0 5 0 95 6 0 0 5 99 5 Therefore for the highest solubility of a weakly acidic analyte with pKa of 4 0 in an organic solvent adjust sample to pH 2 0 or less 2 pH units BELOW the pKa e g Effect of pH on the dissociation of the conjugate acid of a weak base with a pKa value of 9 0 11 0 99 5 0 5 10 0 95 5 0 ACID pK 9 0
37. over the aqueous droplets allowing efficient analyte partitioning The large surface area of the extraction interface and flow through nature of the technique leads to avery efficient extraction procedure because analytes come into contact with fresh solvent as the organic phase travels through the bed mimicking a repeat LLE mechanism Extract Cleanliness Excellent removal of phospholipids and proteins Although supported liquid extraction is a very simple sample preparation technique it provides much cleaner extracts than techniques such as dilute and shoot and protein precipitation Extract cleanliness can often be comparable with more selective SPE approaches In supported liquid extraction analytes are extracted by partitioning into a water immiscible organic solvent Endogenous components such as salts proteins peptides and phospholipids present in blood related matrices which cause matrix effects in LC MS MS are not soluble in these solvents As a result they remain in the aqueous phase on the sorbent and are therefore eliminated from the final extract High analyte recoveries along with low matrix components and reduced ion suppression result in better quantitation and method performance Phospholipid Removal Using ISOLUTE SLE Phospholipids are matrix components found in blood based biological fluids such as plasma and serum Their presence in sample extracts to be analyzed by LC MS MS is problematic as they often co e
38. r freely soluble analytes 8 Method Development Strategies Supported Liquid Extraction is based on the partitioning of the desired analyte s from an aqueous sample phase into a water immiscible organic extraction solvent Key factors affecting analyte partitioning are Analyte functionality pKa and LogD Solubility of the desired analyte s in the water immiscible extraction solvents Typical extraction solvents used in supported liquid extraction are MTBE ethyl acetate dichloromethane hexane or mixed solvents Many other water immiscible extraction solvents are also applicable Solvent selection depends largely on the range of analytes to be extracted in terms of polarity LogP or LogD and their solubility characteristics The range of analytes that can be extracted is extended using some of the strategies described in this section If a liquid liquid extraction method exists similar extraction conditions solvent and pH conditions are a good starting point for method development using ISOLUTE SLE products Sample Pre Treatment In order to promote partitioning of analytes into the organic phase in supported liquid extraction the charge on any acidic or basic groups should be suppressed wherever possible This is particularly important for more polar analytes Extraction efficiency can be optimized based on drug functionality by modification of the aqueous sample pH see 2 pH unit rule opposite for the
39. ration system for simultaneous automated concentration of multiple samples with unattended operation convenience and speed It can accommodate one or two 96 and 48 well collection plates TurboVap LV The TurboVap LV Concentration Evaporator Workstation provides simultaneous evaporation of up to 50 samples This evaporation system offers many interchangeable tube racks giving you the flexibility for automated low volume sample preparation ranging in volumes size from 1 5 mL to 30 mL The microprocessor control provides monitoring of the timed operation and water bath temperature It also provides automatic gas shutoff and operational diagnostics 21 Ordering Information ISOLUTE SLE Well Plates and Columns Description ISOLUTE SLE 200 uL Supported Liquid Extraction Plate ISOLUTE SLE 400 uL Supported Liquid Extraction Plate ISOLUTE SLE 1 mL Supported Liquid Extraction Plate 48 well ISOLUTE SLE 200 uL Array Wells ISOLUTE SLE 400 uL Array Wells ISOLUTE SLE 400 uL Sample Volume Columns ISOLUTE SLE 400 uL Sample Volume Columns Bulk Pack ISOLUTE SLE 400 uL Sample Volume Columns tabless ISOLUTE SLE 1 mL Sample Volume Columns ISOLUTE SLE 400 uL Sample Volume Columns tabless Bulk Pack ISOLUTE SLE 1 mL Sample Volume columns Bulk Pack ISOLUTE SLE 1 mL Sample Volume Columns tabless ISOLUTE SLE 1 mL Sample Volume Columns tabless Bulk Pack ISOLUTE SLE 2 mL Sample Volume Colum
40. rder to achieve high analyte recoveries Protein binding disruption strategies include pH adjustment for example addition of trichloroacetic acid addition of ZnSO solutions aqueous or aqueous solvent combination addition of various concentrations of water miscible organic solvent In this example the extraction of 25 OH Vitamin D2 and D3 from human serum is described Due to the strongly protein bound nature of the analytes standard aqueous pre treatment conditions only resulted in analyte recoveries of approximately 65 77 n t w n HO 25 Hydroxyvitamin D3 we HO 25 Hydroxyvitamin D Figure 20 Structure of 25 OH Vitamins D2 and D3 LogP of 25 OH Vitamin D2 D3 Analyte Log P 25 OH Vitamin D2 7 0 25 OH Vitamin D3 7 0 In the final optimized procedure serum was pre treated by dilution 1 1 v v with a mixture of water isopropanol 1 1 v v resulting in a total solvent concentration of 25 v v in the load This relatively high organic content was sufficient to induce disruption of the 25 OH Vitamin D from its binding without Causing precipitation of the proteins in the sample Due to the high organic content in the treated sample a solvent bridge effect is possible potentially leading to increased solubility of the aqueous portion of the sample in the extraction solvent and ultimately breakthrough of some matrix components into the final extract In order to counteract this
41. ry experiments from plasma using the relevant sample Apply vacuum 0 2 bar or pressure 3 psi for volumes diluted 1 1 v v 0 5M NH OH and eluted with 2 5 seconds to initiate loading recommended elution volumes As can be seen consistent 5 Wait 5 minutes for sample to completely absorb and form recoveries in excess of 80 were evident across all formats extraction layer 6 Apply water immiscible extraction solvent and allow to flow 100 0 for 5 minutes under gravity 80 0 7 Apply vacuum 0 2 bar or pressure 10 psi for 10 30 60 0 _ seconds to complete elution Ree 8 Evaporate eluate to dryness and reconstitute as required 200 0 0 Table 2 Maximum Sample and Elution Volumes Er RE 1 es ue 2 ei Le us for ISOLUTE SLE Products Elution Elution Elution Elution Elution Product Capacity Maximum Elution Protocol Load Volume M Oxprenolol M Propranolol M Alprenolol Volume Figure 8 Scalability of columns using B Blocker analyte suites 200 uL 96 well plate 200 uL 1ximL 400 uL 96 well plate 400 uL 2 x 900 uL 1 mL 48 well plate 1 mL 5x1 mL 200 uL Array column plate 200 uL 2 x 600 uL 400 uL Array column plate 400 uL 3 x 750 uL 400 uL column 400 uL 2 x 900 uL 1 mL column 1 mL 2x2 5 mL 2 mL column 2 mL 2x5mL 5 mL column 5 mL 3x6 ML 10 mL column 10 mL 2x 20 mL Table 2 The elution volumes described represent a starting point for method development and may be reduced during method optimization particularly fo
42. s the PRESSURE 48 to utilize all columns up to 1 mL volume ISOLUTE SLE columns without the need to purchase supplementary gaskets In addition the unique design allows for between 1 and 48 columns to be processed in parallel without empty ports affecting flow rates Tabless or flangeless columns should be used for full population and optimum sealing The modular rack system accommodates most popular collection vessels ISOLUTE SLE User Guide Evaporation Products Biotage SPE Dry 96 and 96 Dual Microplate Sample Evaporation System Designed for high throughput laboratories the SPE Dry 96 and SPE Dry 96 Dual Sample Concentrator Systems provide efficient solvent evaporation in microplate format and are compatible with 96 and 48 well collection plates Heated gas flow from above and below the collection plate ensures efficient solvent evaporation An easy to use front panel display allows accurate temperature programming and gas flow control The systems are supplied as standard with stainless steel needles and are also available with PTFE coated needles for applications using aggressive solvents acids and bases Biotage delivers the most efficient solvent evaporation laboratory equipment for method development sample extractions and much more Evaporation Products TurboVap Automated Solvent Evaporation Systems TurboVap 96 The TurboVap 96 Concentration Evaporator Workstation is a microprocessor controlled evapo
43. se in ISOLUTE SLE extracts Using the fast simple load wait elute procedure ISOLUTE SLE reduces phospholipid concentration to significantly lower levels than phospholipid depletion products 6 00E 05 5 00E 05 4 4 00E 05 J EPL L 00E 05 Aa E Lyso PL 2 00E 05 1 00E 05 0 00E 00 r Depletion P Depletion 0 ISOLUTE SLE Figure 5 Comparison of phospholipids and lysophospholipids present in sample extracts prepared using phospholipid depletion 100 uL human plasma prepared using manufacturer s instructions and ISOLUTE SLE 100 pL human plasma extracted using MTBE 6 In supported liquid extraction using ISOLUTE SLE plates we have found that water immiscible solvents such as MTBE work effectively to extract a broad range of analytes However for analytes which do not elute well in these solvents 5 v v of a polar modifier can be added without sacrificing extract cleanliness The data below Figure 6 illustrates the removal of phospholipids from plasma samples under various combinations of pre treatment and extraction solvent conditions using ISOLUTE SLE plates Similar results were seen for lysophospholipids All extracts were compared to protein precipitated plasma samples as the 100 standard 3 0E 06 ry 2 5E 06 2 0E 06 1 5E 06 1 0E 06 5 0E 05 1 0 0E 00 ee ae a ae Foa i P x g gt amp amp g
44. your analyte is acidic neutral or basic various pH conditions are recommended For best results analytes should be in their non ionized form if possible M For acidic drugs low and intermediate sample pH conditions should be evaluated For basic drugs intermediate and high sample pH conditions should be evaluated y Due to the rapid partition and equilibration of analytes into fresh solvent as the elution solvent passes through the ISOLUTE SLE column extraction efficiency compared to LLE is increased Precise pH control adjustment may not always be required for low polarity analytes Optimized pH control is more important for more polar analytes M Neutral analytes with no pKa value can be extracted across the pH range If protein binding is apparent then the use of acid or base in the sample pre treatment can help to disrupt protein binding or minimize extraction of unwanted sample components The 4 extraction solvents selected give a wide range of solvent characteristics and polarity 12 Biotage 2014 ISOLUTE SLE User Guide Extending the Range of Extractable Analytes For some analytes standard extraction conditions may not be suitable This is often due to the low solubility of certain analytes in non water miscible solvents For example small polar acids and analytes with strongly acidic or basic groups which cannot be neutralized by pH control may need further method optimization This se
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