Best Practice Guide for Generating Mass Spectra.
Contents
1. O C Q Q S o D N Best Practice Guide C V 0 for Generating Mass Spectra Authors Vicki Barwick John Langley Tony Mallet Bridget Stein Ken Webb Best Practice Guide oS Setting standards in analytical science VAM valid analytical measurement BEST PRACTICE GUIDE FOR GENERATING MASS SPECTRA December 2006 Vicki Barwick LGC John Langley University of Southampton Tony Mallet University of Greenwich Bridget Stein EPSRC National Mass Spectrometry Service Centre Ken Webb Consultant ISBN 978 0 948926 24 2 Setting standards in analytical science VAM valid analytical measurement LGC Limited 2006 Preface This Guide was prepared as part of the Department of Trade and Industry s VAM Programme which forms part of the UK National Measurement System The Guide arose from discussions held at the VAM Mass Spectrometry Working Group and was prepared by LGC in collaboration with the members In addition to major contributions by the authors other members of the Working Group provided Suggestions and comments The idea for this work came about during preparation of an earlier guidance document concerning accurate mass AccMass applications of mass spectrometry It became clear that users of mass spectrometry instrumentation or services including both specialists and research chemists frequently have little understanding of the instrumentation o2. 2x0 70 Cm 1 212 436 2 438 433 413 5411 371 4715 309 4182 T 373 5467 409 3907 444 6104 i N iy EN A Wl Wp Ni Wella 380 390 410 Bad points of Spectrum 14 ji ALIK d i N i A Ik J i lw W AJ Man lV L P Wn A j Y J V l T at f ees mass 420 430 Wal Inset spectrum shows the theoretical pattern of the expected molecular species In positive ion mode ESI SCAN ES M H would be expected so the major molecular peak in this spectrum should appear at m z 436 2 OS 42 468 5649 437 4371 p 475 4821 if K 501 5791 W 466 8041 441 4617 Wet YH 499 5039 513 5271 e 455 4221 s 11 483 4684 MER i r A igr j 1 A M j A i Mie d i SW LW j Way H W Whe 440 450 e Mass accuracy is poor the mass of the expected molecular ion monoisotopic is m z 436 2 inset spectrum whereas the 1 C peak is observed at m z 434 5 Because of the lack of mass accuracy the number of decimal places used in peak labelling four is inappropriate Inadequate mass resolution The isotope peaks are not completely resolved No information is given on date of analysis or solvents additives used A wider scan range should have been used considering the fact that higher m z ions are present 21 LGC Limited 2006 9 COMMON BACKGROUND IONS e The peaks you observe in your spectr
3. McMaster Wiley 2005 ISBN 0471655317 Liquid Chromatography Mass Spectrometry An Introduction Robert E Ardrey Wiley 2003 ISBN 0 471 49801 7 Mass Spectrometry Desk Reference O David Sparkman Global View Publishing 2000 ISBN 0 9660813 2 3 Mass Spectrometry A Foundation Course K Downard RSC 2004 ISBN 0854046097 Mass Spectrometry Principles and Applications Edmond de Hoffmann amp Vincent Stroobant 2 Ed Wiley 2001 ISBN 0 471 48566 7 Methodology for Accurate Mass Measurement of Small Molecules Ken Webb Tony Bristow Mike Sargent Bridget Stein LGC 2004 available at www vam org uk Quantitative Applications of Mass Spectrometry Pietro Traldi Franco Magno Irma Lavagnini Roberta Seraglia Wiley 2006 ISBN O 470 02516 6 The Expanding Role of Mass Spectrometry in Biotechnology 2nd Edition Gary Siuzdak 2 Ed MCC Press 2006 ISBN 0 9742451 0 0 Journals Journal of Mass Spectrometry Wiley Rapid Communications in Mass Spectrometry Wiley Journal of the American Society for Mass Spectrometry Elsevier Mass Spectrometry Reviews Wiley European Journal of Mass Spectrometry IM Publications International Journal of Mass Spectrometry formerly Journal of Mass Spectrometry and lon Physics Elsevier Websites www vam org uk Information and resources from the Valid Analytical Measurement Programme www spectroscopynow com Free resources for the spectroscopy community www bmss org uk The
4. biopolymers are not covered Examples of applications of mass spectrometry Confirmation of identity of known compounds e g compounds from target synthesis metabolites compounds extracted from a sample matrix Identification of unknowns Assessing the degree of isotope incorporation in labelling studies As a detector linked to a chromatographic system for quantitative and qualitative analysis Accurate mass measurements for the determination of molecular formulae Fundamental studies of the physical chemistry of ions Limitations of mass spectrometry Not all compounds will give a mass spectrum as the compound of interest may not ionise The relative abundances of ions in the mass spectrum obtained for a mixture does not necessarily reflect the proportions of the compounds in the mixture It cannot be used directly to assess purity of a material since impurities may not ionise or may have different ionisation efficiencies In general mass spectrometry cannot easily distinguish between isomers unless it is coupled with a chromatographic technique Spectra may be different on different types of instrument or on different instruments of the same type making library searching difficult Poor quality spectra can mislead Use of a second ionisation technique is often necessary to assist confirmation of identity At all times other supporting analytical and chemical information should be considered 1 LGC Limited 2006 2 1
5. 2 2 2 INSTRUMENT CONFIGURATION A mass spectrometer is an instrument that operates under vacuum and separates charged gas phase species according to their mass and charge A computer is used to control the mass spectrometer and for processing the data Compounds introduced into the spectrometer produce a characteristic mass spectrum which can be used to obtain identity or structural information Key components of a mass spectrometer The key components of a mass spectrometer are shown in Figure 1 The analyst usually has to select the appropriate inlet ionisation source and analyser What is appropriate will depend on the nature of the sample being analysed The main types of inlet ionisation mode and analyser are summarised in Figure 1 Different inlets are appropriate for different ionisation sources as indicated by the colour coding in Figure 1 e g the inlets highlighted in blue can be coupled with the ionisation modes also highlighted in blue Analyser types Magnetic sectors generally use a combination of electrostatic and magnetic fields and can operate as low and high resolution instruments Sector instruments operate with high voltages on the ion source typically 5 10 kV Quadrupoles use a combination of rf radio frequency and dc direct current voltages are generally low resolution instruments and operate at relatively low voltages lt 500V Capable of fast scanning hence are ideally suited to interfacing to
6. 3 chlorine atoms er Cl Cl e Elements with distinctive isotopes will manifest themselves by the patterns of the isotope clusters in a spectrum e g 7 Cl and Cl as shown in Figure 11 Naturally occurring chlorine is a mixture of two isotopes Cl 75 5 and Cl 24 5 Thus a compound containing one chorine atom will display two isotope peaks CI and Cl a compound containing two chlorine atoms will display three isotope peaks Cl Cl Cl and Cl and a compound containing three chlorine atoms will display four isotope peaks Cls Cl Cl Cl Cl and Cl3 16 7 3 How to find the molecular species In this document molecular species refers to the molecular ion Or an adduct ion containing the intact molecule The molecular Species observed will depend on the ionisation mode used see Table 1 for example M in El see spectrum of naphthalene in Figure 9 M H in ESI see spectrum of ketoconazole in Figure 10 To find the molecular species e Calculate the nominal molecular mass of the analyte see 7 1 e In MALDI and atmospheric pressure ionisation methods check for cation adducts M Na M Solvent H etc 2M H and M nH or anions if using negative ion mode For example in the ESI spectrum of ketoconazole Figure 10 the peaks at m z 553 1 and m z 555 1 represent sodium adduct ions e Apply the Nitrogen Rule A compound containing C H O N S P o
7. British Mass Spectrometry Society www asms org The American Society for Mass Spectrometry 27 LGC Limited 2006 Setting standards in analytical science Queens Road Teddington Middlesex TW11 OLY UK Tel 44 0 20 8943 7000 Fax 44 0 20 8943 2767 Web www lgc co uk VAM website www vam org uk ISBN 978 0 948926 24 2 Subject to Crown licence Other than as set out below no part of this publication may be reproduced or transmitted in any form or by any means electronic or mechanical including photocopying recording or any retrieval system without the written permission of the copyright holder Download of this document is permitted provided only that i you make no more than one printed copy of such download and no further copies of such printed copy are made ii you make only personal non commercial use of such download and or printed copy and iii you retain on such download and or printed copy all copyright notices and shall remain bound by the terms of such wording and notices Additionally you may not offer for sale or sell or distribute over any other medium including distribution by over the air television or radio broadcast or distribution on a computer network the content or any part thereof LGC Limited 2006 All rights reserved 1472 AS 1006
8. gas is required ve ion reaction with ionised reagent gas e g ammonia or methane lonisation mostly by cation attachment ve ion electron capture or anion attachment ve ion M H M NH ve ion M Much less fragmentation than with El more likely to observe an ion closely related to the original molecule As for El As for El Electrospray l onisation Any compound sufficiently basic in gas phase to accept a proton or other cation positive mode or sufficiently acidic to lose a proton negative mode ve ion addition of cation e g H Na NHq ve ion loss of proton or anion attachment Molecular clusters are common ve ion M H M Na M nH ve ion M H M XT Low energy process few fragments Greater fragmentation by MS MS or increased source voltages Sample must be dissolved in an appropriate solvent Mixture of water organic solvent with optional addition of electrolyte e g formic acid or ammonium acetate frequently typical reversed phase HPLC gradient mixtures Solvent choice may be critical As for ESI Atmospheric Pressure CI APCI Many compounds which will not ionise by ESI will be protonated by APCI as stronger gas phase acids are present in source ve ion addition of proton most common ve ion electron capture ve ion M H ve ion M7 M HI As for ESI As for ESI Also
9. that the peak Shape is satisfactory see section 4 1 Figure 6 Errors arising from poorly tuned instruments Digitonin Res 15 4 Scan ES 1905AM03 1 0 167 Cn Cen 1 80 00 Ar Sm Mn 2x1 00 2 62e7 125 1 6 A 1252 5 1235 6 1236 6 N V 1237 6 Figure 6a I nstrument correctly tuned Digitonin Low Res 12 0 Scan ES 1905AM02 1 0 265 Cn Med 8 Ar 3 84e5 0 1905AM02 1 0 268 Scan ES 1 58e8 y 1267 6 1273 8 ee a ae a a r Figure 6b Instrument incorrectly tuned 12555 42675 1259 7 750S 42835 12055 LGC Limited 2006 5 3 5 4 Mass calibration Calibration of the m z scale of the mass spectrometer is an important step in obtaining reliable mass spectra Calibration typically involves analysing a calibration compound which yields ions of known m z The m z scale is then adjusted to give the correct values for the calibration peaks The exact calibration protocol including the calibration compound to be used will vary with the instrument and the ionisation mode consult the instrument manual Frequency of calibration will depend on the instrument and the reason for acquiring the mass spectrum For example mass calibration is one of the most critical parameters when undertaking accurate mass measurements The mass calibration should cover the complete range of analyte masses Background spectrum Acquire a background spectrum before analysing the sample to check
10. 3 9542 Average molecular mass 608 687 Nominal molecular mass 608 Monoisotopic molecular mass 608 2723 Note that different calculations for molecular mass are used by mass spectrometrists and other chemists Average molecular masses are generally used by chemists to calculate molecular masses nominal molecular masses are used by mass spectrometrists analysing small molecules lt 800 Da on low resolution instruments and exact molecular masses are used by mass spectrometrists using high resolution instruments The term accurate mass is also used This refers to an experimentally determined mass of an ion measured with high precision and accuracy generally to determine an elemental formula For accurate mass measurement the lowest mass isotope is measured whenever possible to avoid measuring isotopically mixed species However when the intensity of the lowest mass peak is too low the monoisotopic or another peak of the cluster must be measured and account taken of the mixed nature of this peak 15 LGC Limited 2006 7 2 Use of stable isotope information Naturally occurring carbon exists principally as a mixture of two isotopes 1 C 98 9 and C 1 1 Hence for every carbon atom present there is a 1 1 chance that it will be C not 1 C e A spectrum of a compound containing 10 carbon atoms will Show a signal with an abundance of 11 of that of the molecular ion at one m z higher Figure 11 Isotope patterns for 1 2 and
11. 8 128 1 Scan ES 9 2 25 0 900 Cn Cen 2 80 00 Ar Sm Mn 2x0 50 Scan El 2 76e6 Ketoconazole ESI ve spectrum C26H28Cl2N40 4 RMM 530 1 Loop injection methanol 0 02 aq formic acid g Se a 2305AMO02 25 0 900 Sm Mn 2x0 50 1 1004 Scan ES 533 1 127 1 ey 126 1 4 0710 syg agaaas7o 0 eao y te mH aira BREA 2 a 90 g i20 125 Figure 9 features of note e The mass calibration is good e the measured mass of the molecular ion M at m z 128 1 is consistent with the molecular mass for Figure 10 features of note naphthalene e The mass calibration is good e significant peaks are mass labelled to an e the measured mass of the M H ion at m z 531 1 is consistent with appropriate number of decimal places the expected molecular mass for ketoconazole e The mass resolution is good the molecular ion is clearly visible together with its C isotope peak at the correct ratio for this compound The molecular ion 13C isotope peak is also present at m z 130 e The mass resolution is good mass peaks appear as resolved isotope clusters The ions at m z 531 1 and m z 533 1 represent the isotope pattern for a molecule containing two chlorine atoms see section 7 2 The ions at m z 553 1 and m z 555 1 are sodium adducts M Na e significant peaks are mass labelled to an appropriate number of decimal places e lonisation mode and polarity are shown El pos
12. GC HPLC CEC and CE inlets Time of Flight TOF measures the time an ion takes to travel a specific distance after acceleration by a high potential Different mass ions travel at different speeds Reflectron systems produce higher resolution than linear systems ES TOF MALDI TOF GC TOF and hybrid instruments are now available lon traps usually the term lon Trap is used to refer to the Quadrupole lon Trap QIT In this analyser ions are produced and stored in the trap and progressively ejected by increasing the rf voltage The trapped ions can be induced to undergo dissociation and hence generate a product ion spectrum within the one analyser The majority of ion traps are bench top instruments used for routine analysis The FT ICR MS is a type of ion trap where the frequencies of the circulating ions are measured and a Fourier transform is performed to give the mass spectrum Unlike a conventional bench top ion trap it is capable of very high mass resolution Another new type of ion trap is the Orbitrap 2 LGC Limited 2006 Figure 1 Components of a mass spectrometer resolution Low medium Quadrupole lon trap Low Time of Flight TOF Medium Magnetic sector High Fourier transform ion cyclotron resonance FT ICR VST high DETECTOR Orbitrap High Examples Combinations of most analysers are possible and allow tandem MS hybrids or MS ion traps FT ICR to be performed Nature of co
13. as follows tune instrument mass calibrate acquire a background spectrum analyse a test compound analyse the sample This sequence requires a number of essential checks on aspects of instrument performance before acquiring a mass spectrum to ensure that spectra obtained for samples will be of acceptable quality Initial instrument performance should be checked by acquiring the mass spectrum of a test compound using a defined protocol Instrument tuning In order to obtain an acceptable quality spectrum the instrument must be tuned according to the instrument protocol to ensure good sensitivity and peak shape and to ensure that the mass resolution is appropriate for the analytical requirements of the sample Figure 6 shows electrospray spectra of a sample containing two triterpene glucosides of relative molecular mass 1228 6 and 1212 6 Each figure shows the centroid spectrum top and continuum spectrum bottom The ions at m z 1251 6 and m z 1235 6 in Figure 6a are the sodiated adducts M Na The spectrum in Figure 6b was obtained for the same sample using an instrument that was badly tuned and gave a non symmetric peak shape and low mass resolution In Figure 6a the centroid spectrum shows the full expected range of the C isotope peaks for the molecular ions but in Figure 6b these are absent Note also that the values of the centroids in Figure 6b differ from those in Figure 6a After the instrument has been tuned check
14. at lower mass 4 Mass range also depends on mass analyser and m z of ion produced 5 The polarity of the sample material can be assessed by investigating which solvents it will dissolve in e g a material that dissolves in hexane but not in water is non polar The solvents listed are for assessment of sample polarity only they are not suggested solvents for dissolution of the sample for analysis by mass spectrometry 5 LGC Limited 2006 Table 1 Summary of ionisation modes l onisation mode Types of compound Nature of ionising mechanisms Typical ions observed Fragmentation Sample introduction Typical solvent Electron l onisation El Non polar and moderately polar species e g hydrocarbons aromatics etc Molecule must be volatile and thermally stable Loss of electron leads to radical cation Excess internal energy may result in significant fragmentation ve ion M M H ve ion El not effective in negative mode Significant fragmentation Very informative about structure of molecule Directly from a temperature controlled probe or via a GC column GCMS requires volatile non polar solvent GC injection temperature can dictate choice Insoluble samples can be introduced as a Solid if compound is sufficiently volatile and thermally Stable Chemical I onisation CI As for El Increased chance of detecting a molecular ion Appropriate choice of reagent
15. cant peaks mass labelled to an appropriate number of decimal places i e appropriate to the mass resolution of the instrument but to at least one decimal place for ions gt m z 500 Mass differences of adjacent related ions consistently equal to 1 Da n for ions of charge n N B Da Dalton is a non SI unit of mass and is the term used to describe a mass unit Mass peaks appear as resolved isotope clusters exceptions ion is multiply charged and instrument is low resolution the species is 100 mono isotopic very unusual J Continuum data show adequate resolution of singly charged ions Spectrum abundance is normalised to the most abundant ion Abundance of largest peak s does not saturate detector peaks are not off scale Spectrum information Should include at least onisation mode and polarity used Good laboratory practice would also require Date instrument name type additives solvents matrices reagents etc All post acquisition data processing e g details of smoothing Labelling of magnified areas with degree of magnification and range to which applied Additional useful information Sample concentration Location name of data file 13 LGC Limited 2006 Figure 9 Mass spectrum centroid for naphthalene Figure 10 Molecular ion region of mass spectrum for ketoconazole CioHs centroid and continuum Naphthalene UE 1151 9 488 Cn Cen 2 80 00 Ht Sm Mn 2x0 75 Cm 1131 1159 1183 12374 998 102
16. creases so a compromise often must be sought LGC Limited 2006 Figure 4 Definitions of resolving power e 10 valley definition this is useful only for instruments a 10 valley definition giving Gaussian peaks Two peaks of equal intensity are considered to be resolved when they are separated by a valley which is 10 of the height of each peak made up from a 5 contribution from each component Figure 4a In practice by this definition a resolving power of 1000 means that peaks at m z 1000 and m z 1001 have a 10 valley between them e Full width half maximum FWHM definition the quadrupole FT ICR MS ion trap and TOF definition is based on a peak width Am measured at 50 peak height Figure 4b Figure 5 Mass spectrum of HCI at low broken line and 10 intensity A A higher solid line resolution A 5 intensity Scan El 7 58e12 resolving power m m2 mM1 b FWHM definition 50 intensity Ka 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 mz e The effect of increasing mass resolution can be seen in the example in Figure 5 At low mass resolution the two peaks for the ions at m z 36 and m z 38 merge into one peak mM Increasing the mass resolution enables both ions to be observed resolving power m Am g LGC Limited 2006 5 1 5 2 5 ACQUIRING A MASS SPECTRUM General sequence The general sequence of actions when acquiring a mass spectrum is
17. cular ion Nitrogen rule Orbitrap Quadrupole Quadrupole ion trap Relative molecular mass SALDI SFC S N SP TOF Matrix assisted laser desorption ionisation formation of gas phase ions from molecules that are present in a solid or liquid matrix that is irradiated with a pulsed laser That part of a mass spectrometer that separates a mixture of ions according to their mass to charge ratio through the application of electric and magnetic fields The smallest mass difference Am between two equal magnitude peaks such that the valley between them is a specified fraction of the peak height In a mass spectrum this is the observed mass divided by the difference between two masses that can be separated m Am Plot of the relative abundance of a beam or other collection of ions as a function of their m z values An ion formed by the removal of one or more electrons to form a positive ion or the addition of one or more electrons to form a negative ion The rule stating that an organic molecule containing the elements C H O S P or a halogen has an odd nominal mass if it contains an odd number of nitrogen atoms A type of ion trap lons are orbitally trapped and oscillate harmonically along the trap axis The oscillation frequency is inversely proportional to the square root of m z A type of mass analyser consisting of four parallel rods whose centres form the corners of a square and whose opposing poles are connected t
18. d material packed into a tube column as the stationary phase The sample introduction system of a mass spectrometer The method of ionisation used to produce sample ions for separation in the mass spectrometer analyser Usually this term refers to a Quadrupole lon Trap q v The FT ICR q v and Orbitrap q v are also types of ion trap Atoms of the same element that have the same number of protons but a different number of neutrons in the nucleus and consequently have different masses e g C and C both have 6 protons in the nucleus but t C has 6 neutrons whereas 7 C has 7 Laser desorption ionisation formation of gas phase ions by the interaction of photons from a pulsed laser with a solid or liquid material Liquid secondary ionisation the ionisation of any species by the interaction of a focused beam of ions with a sample that is dissolved in a solvent matrix A type of mass analyser which accelerates ions through a perpendicular magnetic field in a curved flight path to separate ions of different mass momentum For accurate mass measurement a double focussing mass spectrometer is used where the magnetic sector is coupled with an electric sector to minimise energy dispersion and thus achieve higher resolution The term magnetic sector is often used when actually referring to a double focussing mass spectrometer 25 LGC Limited 2006 MALDI Mass analyser Mass resolution Mass resolving power Mass spectrum Mole
19. for contaminants that may be present in the instrument see section 9 for a list of common background ions Figure 7a shows an El spectrum of typical bleed from a non polar GC column Most of the signals arise from the methyl Silicone Compounds bonded in the column Figure 7b is the ESI positive ion spectrum for a 1 1 v v mixture of water and methanol with 0 05 formic acid being introduced at 10 uL min Apart from solvent clusters a number of ions from plasticisers can be identified Figure 7 Typical background spectra gems 200308pm01 970 20 784 1 Figure 7a El spectrum 253 135 0447 1 76 0 7o 1180 1830 5 0 i 84 1 756 0 183 0 1772 405 4 246 0 hal Ni Hell P J jj i 6 80 100 120 140 160 267 0 328 0 Oo igl l mos P 160 200 220 240 260 280 30 320 340 300 380 400 3005AMOZ 21 0 760 Cn Cen 2 80 00 Ar Sm Mn 2x0 50 Cm 14 21 4 6 100 Figure 7b ESI positive ion spectrum x 50 Intensity magnification from m z 250 157 8 214 0 bis 276 1 289 1 B48 gag Raa ea cept prep en ahh cope rre ieie etd i148 140 7 P E R SEER pRREE PRP Rrp eprops Ser pun pen pen gt re nee ee pet deere 60 80 100 120 140 160 180 200 220 240 260 280 300 3200 340 360 300 400 420 440 178 8 2148 qersetretts LGC Limited 2006 10 5 5 Checking instrument performance Figure 8 summarises the aspects of instrument performance that should be checked and lists some of the common cau
20. hat separates ions based on oscillations in an electric field the quadrupole field created with the use of radio frequency and direct current voltages Often called just ion trap A type of mass spectrometer analyser that confines ions using electric and or magnetic fields and then selectively ejects ions of different m z by ramping the rf voltage The ion trap is frequently used for high order fragmentation studies MS because it allows successive series of trapping and fragmentation The mass of one molecule of a compound with specified isotopic composition relative to one twelfth of the mass of one atom of 1 C Surface assisted laser desorption ionisation a type of MALDI using a specially prepared surface to take the role of the matrix Supercritical fluid chromatography chromatographic separation in packed capillary HPLC or GC columns using a supercritical fluid as the mobile phase A pressure reduction device is required at the exit of the column Signal to noise ratio a measure of a signal peak intensity relative to the baseline noise level Solids probe see DIP Time of flight a mass analyser that separates ions of different m z by their time of travel between the ion source and detector through a field free region after acceleration by a constant voltage in the source The ions will have differing velocities depending on their mass 26 LGC Limited 2006 11 Bibliography LC MS A Practical User s Guide Marvin C
21. he expected masses Scan range used was not sufficiently high higher m z ions are evident and although the mass range is sufficiently above the expected molecular ion because the limit is close to the highest observed ions at m z 585 587 it is not possible to know whether these ions represent molecular or fragment species if this occurs and there is ambiguity analysis should be repeated to a higher m z LGC Limited 2006 Figure 13b Improved mass spectrum of C 5 H23 0SiBr 100 346 raai g 24 Oct 2000 Inset spectrum shows the CI NH3 45 0 942 Cm 44 48 theoretical isotope pattern of 2 Scan Cl 100 134 2 the expected molecular 1 45e5 212 1 species M NH4 i oy 151 2 346 3 117 2 ees 0 41 M2 M 344 a MGT IM M 30 266 3 69 2 91 2 106 2 r 251 3 174 2 288 2 329 2 Olean blab AN ER wlll vee 304 3 MM E eee wee so ds 60 80 100 120 140 160 180 200 220 240 260 280 300 320 340 360 380 400 420 440 460 480 500 This is an example of a better quality mass spectrum of the same sample used for Figure 13a obtained using a different ionisation mode i e ammonia Cl rather than APCI Note how the higher m z species are no longer present indicating that they were artefacts and thus show another symptom of a poor quality spectrum The molecular species observed is M NH LGC Limited 2006 20 Figure 14 Poor quality mass spectrum of C25H29N30 nominal molecular mass 435 47 0 871 Sm Mn
22. hydrocarbon alcohol mixtures as for normal phase HPLC As for ESI Atmospheric Pressure Photoionisation APPI Optimised for non polar compounds New technique range of applications being evaluated ve ion addition of proton ve ion electron capture ve ion M M H ve ion M As for ESI As for APCI lons formed in a vacuum lons formed at atmospheric pressure Lons rormeniin vacuum P P or atmospheric pressure Matrix Assisted Laser Desorption l onisation MALDI Wide range from non polar to ionic can be analysed Good for large molecules ve ion radical cation or addition of proton Molecular clusters also formed ve ion electron capture or loss of proton ve ion M M H M gt H M 2H 2 ve ion M M HI Little or no fragmentation Greater fragmentation requires MS MS Sample needs to be applied in an appropriate matrix Solvent from which sample will form crystalline mixture with matrix Choice of matrix and sample preparation can be critical LGC Limited 2006 4 INSTRUMENT SET UP KEY DEFINITIONS 4 1 Peak shape Each instrument will have its own optimum peak shape In order to achieve an accurate peak centroid and reproducible intensity measurements a peak without any spikes or Shoulders on the sides is essential For all systems excluding TOFs the peak should be symmetrical about the centroid Two comm
23. in Figure 2 and the information in Table 1 to help you select the most appropriate method of ionisation for your Sample this will enable you to obtain the most useful spectrum to assist in compound identification Figure 2 shows the polarity of the sample increasing down the y axis sample types can range from e g aromatic compounds non polar to salts and peptides polar Typical solvents for the polarity range are also shown The polarity of the sample can be assessed by investigating which solvents it will dissolve in Where a choice of ionisation methods is indicated those coloured green are the ideal first choice those coloured red could be used if there is no other choice As an example consider toluene C7Hg this is a non polar molecule with a relative molecular mass of 92 Using Figure 2 the ionisation method of first choice would be El As a further example consider the complex vitamin folic acid Ci9Hi9N70 this is a polar molecule with a relative molecular mass of 441 which is insoluble in most organic solvents but slightly soluble in water So from the chart the most appropriate ionisation method would be ESI or APCI Other methods not shown in Figure 2 or Table 1 include fast atom bombardment FAB liquid secondary ion mass spectrometry LSIMS both similar to MALDI field desorption field ionisation FD FI used for non volatile molecules instead of El CI 4 LGC Limited 2006 Figure 2 Chart to assist with ton
24. isation mode selection reproduced from EPSRC National Mass Spectrometry Service Centre Summer School B K Stein 2006 with permission from EPSRC National Mass Spectrometry Service Centre SELECTION of IONI SATION MODE for MASS SPECTROMETRY ANALYSIS of COMMON ORGANIC MOLECULES TYPICAL RELATIVE MOLECULAR MASS 4 SOLVENT 0 100 200 300 400 600 800 1000 gt 1200 hydrocarbons polycyclic aromatic hydrocarbons PAHs polychlorinated biphenyls PCBs z oluene C 182 common organics alcohols amines organometallics functionalised species wn dichloromethane El 3 chloroform CI I xo o D a methanol ESI acetonitrile MALDI MALDI Sugars peptides nucleotides salts multiply charged species water APCI acid base ESI DN ISVJYON I ALTYV10d FIdNVS MALDI3 MALDI KEY Examples of compounds of differing polarity are shown in italics Primary technique most likely to work well Secondary technique should work and will give complementary information to the primary technique Tertiary technique try it if you have no choice 1 For all El and Cl a suitable inlet will also be required to match sample volatility e g GC or solids probe or desorption probe 2 Cl suitability will depend on gas selection e g ammonia is unsuitable for hydrocarbons which will need e g methane 3 MALDI matrix ions cause interference at lower m z So MALDI without matrix LDI or surface assisted SALDI can be used more readily
25. itive e lonisation mode and polarity are shown ESI positive 14 LGC Limited 2006 7 MOLECULAR SPECIES RECOGNITION 7 1 Definitions of molecular mass There are different approaches to calculating the molecular mass Average calculated using the average mass of each element weighted for its natural isotopic abundance This is the value chemists usually use when calculating molecular masses Nominal calculated using the mass of the most abundant isotope of each element rounded to the nearest integer value Monoisotopic calculated using the mass of the most abundant isotope of each element Exact calculated using the exact mass of a single isotope most frequently the lightest isotope of each element present in the molecule Note that in each case it is actually the relative molecular mass that is being calculated using the appropriate relative masses of the elements that make up the molecule The relative mass of an element is defined as the mass of one atom relative to one twelfth of the mass of one atom of 1 C Relative masses therefore have no units As an example consider a compound of formula C33H49N2O0o Using Average Atomic Masses Using Nominal Masses Using Monoisotopic Masses C 33x 12 011 396 363 C 33x12 396 C 33 x 12 0000 396 0000 H 40x 1 0079 40 316 H 40x1 40 H 40 x 1 0078 40 3120 N 2x 14 0067 28 013 N 2x14 28 N 2x 14 0031 28 0062 O 9x 15 9994 143 995 O 9x16 144 O 9x 15 9949 14
26. lydimethylsiloxane phthalate C6H4 CO gt OH 446 EE polyphenol ether diffusion pump oil 9 2 1 Oligomeric series 74 m z units apart dimethylsiloxane e g from septa 23 LGC Limited 2006 Accurate mass Analyser APCI API APPI CE CEC Centroid Cl Da DCI DEI Detector DIP EI ES 10 GLOSSARY OF TERMS Experimentally determined mass of an ion used for the determination of an elemental formula See mass analyser Atmospheric pressure chemical ionisation chemical ionisation that takes place using a nebulised liquid and atmospheric pressure corona discharge c f chemical ionisation which takes place at reduced pressure Atmospheric pressure ionisation any ionisation process in which ions are formed in the gas phase at atmospheric pressure Atmospheric pressure photoionisation atmospheric pressure chemical ionisation in which the reactant ions are generated by photoionisation Capillary electrophoresis separation of dissolved ionic species by migration under the influence of a voltage gradient in a capillary containing a buffer Capillary electrochromatography separation as for CE but using a capillary packed with an HPLC stationary phase with the added impetus of a flowing buffer The centre of mass of a peak It is the point at which the m z for the peak is measured A centroid histogram or stick spectrum shows the m z value x axis and the ion abundance y axis Chemical ionisation For
27. m 13 1 2 Examples of applications of mass spectrometry 1 6 2 Spectrum information 13 1 3 Limitations of mass spectrometry 1 7 MOLECULAR SPECIES RECOGNITION 15 2 INSTRUMENT CONFIGURATION 2 7 1 Definitions of molecular mass 15 2 1 Key components of a mass spectrometer 2 7 2 Use of stable isotope information 16 2 2 Analyser types 2 Laa How to find the molecular species 16 7 4 Questions to ask about the validity of the proposed molecular species 17 3 CHOICE OF IONISATION MODE 4 7 5 What if the expected molecular species is not visible in the spectrum 17 4 INSTRUMENT SET UP KEY DEFINITION 7 aie me 8 FURTHER EXAMPLES OF MASS SPECTRA 18 4 1 Peak shape 7 4 2 Mass resolution 7 9 COMMON BACKGROUND IONS 22 9 1 Electrospray ionisation 22 5 ACQUIRING A MASS SPECTRUM 9 9 2 Electron ionisation 23 5 1 General sequence 9 5 2 Instrument tuning 9 10 GLOSSARY OF TERMS 24 5 3 Mass calibration 10 11 BIBLIOGRAPHY 27 5 4 Background spectrum 10 5 5 Checking instrument performance 11 LGC Limited 2006 1 1 1 INTRODUCTION Aims of the guide To enable those unfamiliar with mass spectrometry to generate mass spectra that are fit for purpose To help users recognise poor quality spectra and to understand some of the common causes for such spectra To enable users to understand some of the limitations of mass spectrometry Note that the guidance in the document is restricted to the qualitative analysis of small molecules i e larger molecules such as
28. mation of a new ion in the gas phase by the reaction of a neutral species with an ion The process may involve the transfer of an electron a proton or other charged species between the reactants Dalton non SI unit of mass equal to the unified atomic mass unit u u one twelfth of the mass of one atom of 1 C Desorption chemical ionisation chemical ionisation of a solid sample by vaporisation from a conductive filament in the presence of a reagent gas Desorption electron ionisation electron ionisation of a solid sample by vaporisation from a conductive filament in the presence of an electron beam That part of a mass spectrometer that detects the ions after mass separation Direct insertion probe a system for introducing a single sample of a solid or a liquid usually contained in a quartz or other non reactive holder into a mass spectrometer ion source Electron ionisation ionisation of an atom or molecule by electrons that are typically accelerated to energies between 10 and 150 electron volts eV in order to remove one or more electrons from the molecule 70eV is the accepted norm for acquisition of El spectra Electrospray see ESI 24 LGC Limited 2006 ESI FAB FD FI FIA FT ICR GC HPLC Inlet lonisation mode lon trap Isotopes LDI LSI Magnetic sector Electrospray ionisation a process in which ionised species in the gas phase are produced from a solution via highly charged fine droplets b
29. mpound being Nature of compound being Mass range analysed analysed Mass resolution solubility polarity Scan speed volatility solubility Limit of detection thermal stability thermal stability Dynamic range molecular mass Key factors in Is fragmentation required As above Voltages applied Mass calibration Memory effects Solvents used Mass resolution Additives used Energy settings Quantity and concentration of the sample Choice of ionisation mode is discussed in detail in section 3 key factors affecting performance 3 LGC Limited 2006 3 CHOICE OF IONISATION MODE The principal factors that affect the choice of ionisation mode are the polarity molecular mass and thermal stability of the compound being analysed If an inappropriate ionisation mode is selected then no spectrum or a poor quality spectrum will be obtained Most samples will be initially analysed to obtain positive ion spectra however negative ion spectra can be useful where the negatively charged molecule may be more stable than the corresponding positively charged one and may also provide complementary structural information through different fragmentation processes In general a hard ionisation process such as electron ionisation can produce many fragment ions and possibly poor abundance of the molecular species a soft ionisation process such as electrospray produces few fragment ions with abundant molecular species Use the chart shown
30. nsoluble for ESI Is ionisation of the expected molecular species being Suppressed by other species present e g because the sample is a mixture Has an appropriate amount of sample been used for the analysis Remember too much sample can be an issue Are memory effects being observed due to carry over of previous sample s analysed Was the spectrometer performing to specification at the time of measurement Was the mass calibration correct LGC Limited 2006 3 FURTHER EXAMPLES OF MASS SPECTRA Figure 12 Good quality mass spectrum of toluene C H File U1lL02AM03 70SEQ EI Magnet BpM 91 BpI 6147862 TIC 31149138 Flags HALL Sample Text 290861 39314 II File Text EI SCAN TOLUENE 100 gI 903 80 T T repre J Co bi J tl oh iG WI ON om Oo 3 503 p J i a repre a a ee E E O h This spectrum of toluene is good because The mass calibration is good Significant peaks are mass labelled to an appropriate number of decimal places The measured mass of the molecular ion M at m z 92 is consistent with the expected molecular mass for toluene Mass difference of adjacent related ions consistently equal to 1 m z unit The mass resolution is good Mass peaks appear as resolved isotope clusters the ions at m z 93 and m z 94 are the C containing isotope ions lonisation mode and polarity are shown El positive In the case of electron ionisation the molecular ion cluster is the highes
31. on ideal peak shapes are Gaussian and Lorentzian Shown in Figure 3 Note that peaks produced by TOF analysers tend to be asymmetric but the asymmetry should be minimised Poor peak shapes are often due to poor tuning and will result in incorrect identification of the peak centroid This can lead to errors in mass assignment which could result in misidentification of samples Poor peak shapes will also cause problems with mass resolution and sensitivity problems can arise Figure 3 Examples of ideal peak shapes 4 2 Mass resolution The resolving power is the ability of a mass spectrometer to separate ions of two different m z values It is defined as m Am where m is the m z value of a single charged ion and Am is the difference between m and the next highest m z value ion that can be separated from m Two different approaches to calculating the resolving power are used routinely depending on the type of instrument employed These are illustrated in Figure 4 The term mass resolution is also used This is defined as the smallest mass difference Am between two equal magnitude peaks such that the valley between them is a specified fraction of the peak height The mass resolution should be correct for the analytical requirements of the sample For instruments where mechanical slits are used e g sector instruments there is a trade off between sensitivity and resolution as resolution is increased the sensitivity de
32. oo high Is the instrument calibrated correctly according to the instrument protocol Have the wrong peaks been chosen for processing Is the instrument correctly tuned and are peak shapes satisfactory Poor tuning and distorted peaks will lead to poor mass calibration due to incorrect identification of peak centroids N B Symmetry and resolution are both important in particular Smooth symmetrical peaks with no noise lead to good mass calibration Is the source clean Is there carry over from previous samples causing a memory effect Are the solvents gases pure 12 LGC Limited 2006 6 1 6 EVALUATING SPECTRUM APPEARANCE In a mass spectrum the x axis represents the mass to charge ratio m z and the y axis ion abundance usually shown as O to 100 There are two popular forms of mass spectrum In one the continuum or analogue spectrum the full profile of each ion peak is shown Such data recording takes up a lot of storage space and is commonly simplified as the centroid histogram or stick spectrum Here only two pieces of data are recorded the peak centroid i e the m z value and the maximum intensity i e the ion abundance These are plotted with the ion abundance of the most intense peak base peak normalised to 100 The two types of spectrum are shown in Figure 10 What to look for in a good quality mass spectrum a valid and good quality mass spectrum the following should all be apparent Signifi
33. r a halogen will have an odd numbered molecular mass if it contains an odd number of nitrogen atoms The molecular mass will be an even number if there is zero or an even number of nitrogen atoms e Look for the peak with highest m z value allowing for isotope patterns e The m z value of the molecular ion should be accurate to better than 0 2 m z units e In El check that fragment ions have possible m z values i e not differing by 3 13 and 21 25 units Be prepared for a vanishingly small signal with increasing mass e g hydrocarbons e Note that for El mass spectra some compounds do not show a molecular ion at all e g many phthalate esters LGC Limited 2006 7 4 Questions to ask about the validity of the proposed molecular species Does the observed isotope pattern match the theoretical one E g in the ketoconazole spectrum Figure 10 the ton at m z 531 1 is the M H Cl The presence of the ion at m z 533 1 and its abundance is consistent with the Cl Cl isotope peak for a molecule containing two chlorine atoms Is the measured mass of the molecular ion consistent with the expected mass within the expected precision and mass calibration of the instrument Is the signal noise S N ratio of major isotopes at least 3 1 Are the proposed molecular species the highest m z species in the spectrum Possible exceptions predictable adducts and or dimer and fragments of dimer may occur with soft ioni
34. r the meaning of the spectra they produce Often they will obtain or request an accurate mass determination for confirmation of identity on the basis of spectra which are meaningless or which could not possibly have originated from the target molecule Discussion of this problem highlighted the changes which have taken place in teaching chemistry and analytical science and the rapid expansion in the application of mass spectrometry The latter has been fuelled by a number of factors including advances in the automation and performance of instrumentation and recent rapid growth in the use of mass spectrometry for the biosciences The outcome has been widespread use of complex instrumentation often as a walk up service by staff with little education or training relevant to the task The main aim of the Guide is to enable those unfamiliar with mass spectrometry to generate mass spectra that are fit for purpose primarily for qualitative analysis of small molecules We have done this by providing a clear and concise summary of the essential steps in obtaining reliable spectra In addition the reader should obtain a better understanding of the limitations of different types of Spectrometer and the particular precautions which are necessary in setting up the instrument and acquiring a spectrum Advice is also given on how to assess the quality of the spectrum from its appearance and locating the target molecular species within the spectrum The emphasis i
35. s on giving practical advice which is specific easy to follow and in a format which will encourage its use on the job With this in mind we have set out the Guide in a number of short targeted sections and made extensive use of bullet points tables illustrations and flow charts We have also included a wide range of examples to illustrate key points and make it easier to identify LGC Limited 2006 common problems We hope that these features of the Guide will also facilitate its use for private study and training courses away from the laboratory With this in mind a glossary of the terms used has also been included Mike Sargent Chairman VAM Mass Spectrometry Working Group LGC September 2006 Methodology for Accurate Mass Measurement of Small Molecules Best Practice Guide Ken Webb Tony Bristow Mike Sargent and Bridget Stein Coordinating Editors LGC November 2004 ISBN 0 948926 22 8 Available from www vam org uk Acknowledgements The authors would like to thank the members of the VAM Mass Spectrometry Working Group for their valuable comments on the content of the guide Production of this guide was supported under contract with the Department of Trade and Industry as part of the National Measurement System Valid Analytical Measurement VAM programme LGC Limited 2006 Contents 1 INTRODUCTION 1 6 EVALUATING SPECTRUM APPEARANCE 13 1 1 Aims of the guide 1 6 1 What to look for in a good quality mass spectru
36. sation such as Cl ESI APCI APPI and MALDI where little or no fragmentation occurs In the ketoconazole spectrum the ions at m z 553 1 and m z 555 1 are sodiated adducts Was the scan range to a sufficiently high mass to allow the point above to be properly answered 100 m z units above the highest observed mass should generally be adequate Are the proposed molecular species separated from other lower m z tons by masses corresponding to feasible chemical entities In the spectrum of toluene Figure 12 the loss of one m z unit from the molecular ion is acceptable H loss and the next fragment ion 15 m z units below 92 m z is also reasonable CH3 loss 17 7 5 What if the expected molecular species is not visible in the spectrum Has the correct sample been analysed Are expectations wrong Check the molecular formula and calculated mass Is the Nitrogen Rule obeyed Has an inappropriate ionisation mode been used Has the wrong polarity positive or negative ionisation been applied Are the MS ionisation conditions too harsh thus causing Significant fragmentation e g cone voltage in ESI use of hard ionisation like El Are the MS physical conditions causing sample decomposition e g source temperature in El Cl Are analyte additives e g solvent acid decomposition or other changes causing sample Is the sample not reaching the ionisation source e g too volatile for probe analysis i
37. ses of problems Figure 8 Instrument performance troubleshooting Is sensitivity response If no peaks are observed check the following satisfactory Is the vacuum system OK check gauges Is the isolation valve open if there is one Are all the electrical signals present check the readbacks Is the ion source contaminated visually check and clean if needed Is the ionisation mode appropriate for the sample type See also section 3 Is there a blockage in the spray capillary check HPLC back pressure If the sensitivity is low few background peaks few isotope peaks seen see sections 5 4 and 7 2 check the following Is the source tuned Is the detector failing Is the signal peak detection threshold set too high Is ion source contaminated Is a wrong MALDI matrix being used Is the mass resolution too high see section 4 2 Is the instrument tuning correct see section 5 2 Is peak shape l satisfactory see section Is the ion source contaminated 4 1 Is vacuum system OK Are the applied energies too high Laser in MALDI ion energy of quadrupole 11 LGC Limited 2006 Figure 8 continued YES Is mass resolution NO satisfactory see section 4 2 YES Is mass calibration NO satisfactory see section 5 3 YES Is background spectrum NO satisfactory see section 5 4 Is the source contaminated Is there a good vacuum when using EIl CI Are the applied voltages t
38. t m z species in the spectrum The molecular ion cluster is separated from other lower m z ions by masses corresponding to feasible chemical entities 18 LGC Limited 2006 Figure 13a Poor quality mass spectrum of C 5 H230SiBr nominal molecular mass 326 using Br 327 5 485 Rf 7 3 000 Inset spectrum shows the theoretical isotope pattern of the expected molecular species In positive ion mode APCI M H would be expected so the major peaks should appear at m z 327 and m z 329 for the Br and Br isotopes respectively 249 22246 200 225 250 Good points of Spectrum 13a e Significant peaks are mass labelled to an appropriate number of decimal places e Header information includes ionisation mode and polarity used AP APCI ve ion and post acquisition processing information Rf 7 3 000 e Signal noise ratio of major isotopes is at least 3 1 Bad points of Spectrum 13a e Mass difference of adjacent related ions is not consistently equal to 1 m z unit e g peaks at m z 389 4 391 5 392 7 peaks at 195 1 196 9 197 5 19 SCAN AP 1 3 5 Isotope clusters are not resolved e g m z 524 6 has no isotope peaks There is no information given on date of analysis or solvents additives Isotope pattern incorrect isotope ratio should be ca 1 1 at m z 327 and 329 M H of Br and Br isotopes respectively Also the measured masses of these two ions are not consistent with t
39. um may not be from your compound of interest they can arise from impurities in your sample e g residual solvent phthalate plasticisers or may be present in the spectrometer e g contamination of the instrument from the analysis of previous samples acquire a background spectrum before running your sample see section 5 4 9 1 Electrospray ionisation 9 1 1 Positive ion m z lon Compound Cz ttn Compound M HCl 9 Cl 64 M Na acetonitrile 4M tetrabbutylammonium C4Ho 4N 102 M H triethylamine TEA 273 AE Na t Meon monomethoxytrityl cation MMT 122 7 F tris buffer tris hydroxymethyl 338 M H erucamide aminomehtane 130 c a sacs be a 391 a i diisooctyl phthalate plasticiser Ht A _iniprgpvtamine TPA __1 408_1 M Na __ sip agent from plastic tips acetonitrile _ P T aa DMSO PMO ao H O OM E pp ienei urea DCU emm 22 LGC Limited 2006 9 1 2 Negative ton 9 1 3 Oligomeric series cluster ions positive or negative ionisation mz ion compouna series in 9 1 3 249 2M Na sodium trifluoroacetate see cluster series in 9 1 3 9 2 Electron ionisation m z _ lon_ Compound m z__ lon_ Compound phthalate butylated hydroxytoluene BHT butylated hydroxytoluene BHT 40 Mt argon 207 F polydimethylsiloxane 44 Mi carbon dioxide polydimethylsiloxane F fragment trimethylsilyl TMS polydimethylsiloxane F methyisi polydimethylsiloxane i po
40. y means of spraying the solution from a narrow bore needle tip at atmospheric pressure in the presence of a high electric field 1000 to 10000 eV Fast atom bombardment the ionisation of any species by the interaction of a focused beam of neutral atoms having a translational energy of several thousand eV with a sample that is typically dissolved in a solvent matrix Field desorption the formation of gas phase ions from a material deposited on a solid surface Field ionisation ionisation by the removal of electrons from any species usually in the gas phase by interaction with a high electric field Flow injection analysis a sample introduction system by which a sample is injected into a continuous liquid flow that enters into the mass spectrometer Fourier transform ion cyclotron resonance a type of ion trap mass analyser based on the principle of ion cyclotron resonance in which an ion in a very strong magnetic field moves in a circular orbit at a frequency characteristic of its m z value Gas chromatograph a system for separating the volatile components of a mixture after conversion to the gas phase using a suitable column as the stationary phase and a carrier gas as the mobile phase When used with a mass spectrometer the carrier gas is usually helium High performance liquid chromatography a system for separating the components of a solution or liquid mixture using a suitable solvent as the mobile phase and chemically coate
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