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Ion fragmentation of small molecules in mass spectrometry

1J. Prasain 01/19/11 Ion fragmentation of small molecules in mass spectrometryJeevan Prasain 01/19/11 Nomenclature: the main names and acronyms used in mass spectrometry Molecular ion:Ion formed by addition or the removal of oneor several electrons to or from the sample molecules -Electron Impact (EI-MS). M + e- M+ + 2e- Adduct Ion:Ion formed through interaction of two speciesand containing all the atoms of one of them plus one orseveral atoms of them ( alkali, ammonium).2J. Prasain 01/19 Pseudomolecular ion:Ion originating from the analyte molecule by abstraction of a proton [M-H]-or addition of proton [M+H]+ Tandem mass spectrometry (Cooks, 1976): MS/MS (McLafferty, 1978), tandem in space or time Precursor ion/parent ion:Ions undergoing fragmentation .

• Adduct Ion: Ion formed through ... Concentration, uM 0.0 2.0e4 4.0e4 6.0e4 8.0e4 1.0e5 1.2e5 1.4e5 1.6e5 1.8e5 2.0e5 2.2e5 2.4e5 2.6e5 2.8e5 3.0e5 s Sensitivity is an issue in quantification of steroids. 10 J. Prasain 01/19/11 Derivatization of estradiol with dansyl chloride leads to the formation of E 2-dansyl (m/z 506) Source: Nelson et ...

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Transcription of Ion fragmentation of small molecules in mass spectrometry

1 1J. Prasain 01/19/11 Ion fragmentation of small molecules in mass spectrometryJeevan Prasain 01/19/11 Nomenclature: the main names and acronyms used in mass spectrometry Molecular ion:Ion formed by addition or the removal of oneor several electrons to or from the sample molecules -Electron Impact (EI-MS). M + e- M+ + 2e- Adduct Ion:Ion formed through interaction of two speciesand containing all the atoms of one of them plus one orseveral atoms of them ( alkali, ammonium).2J. Prasain 01/19 Pseudomolecular ion:Ion originating from the analyte molecule by abstraction of a proton [M-H]-or addition of proton [M+H]+ Tandem mass spectrometry (Cooks, 1976): MS/MS (McLafferty, 1978), tandem in space or time Precursor ion/parent ion:Ions undergoing fragmentation .

2 Product ion/daughter ion:Ions resulting from parent/precursor ions. Neutral loss:Fragments lost as neutral molecules In positive ionization mode, a trace of formic acid is often added to aid protonation of the sample molecules ; in negative ionization modea trace of ammonia solution or a volatile amine is added to aid deprotonation of the sample molecules . Proteins and peptides are usually analysed under positive ionization conditions and polyphenols and acids under negative ionization conditions. In all cases, the m/zscale must be Prasain 01/19 Ionspraydenotes pneumatically assisted ESI operating at aflow rate of approximately 5 to 50 uL/min. Turboionsprayis ionspray with additional heated gas for flow rate of to 2 ml/min.

3 Heated nebulizeris the trade mark of AB Sciex for APCI. 3J. Prasain 01/19/11 Precursor ionor parent ionActivated ionFragmenting ionNeutral lossProduct ionsSchematic of CID fragmentationWhat is Collision Induced Dissociation (CID)or Collisionally Activated Dissociation (CAD) ?Other activation processes:PSD (post source-decay)ECD (electron capture dissociation)SID (surface-induced dissociationoooooooooooooCollision gasCollision cellJ. Prasain 01/19/11 Various types of MS/MS experimentsHopfgartner etal. J. Mass Spectrom, 20044J. Prasain 01/19/11 Steps involved in fragmentationStep # 1: Creation of ionsStep # 2: Add energy of activationStep # 3:Charge directed fragmentationCN HOCH2CH3CN HOCH2CH3 CIDCN HOCH2CH3 Fragment ion + neutral moleculeESIJ.)

4 Prasain 01/19/11 Applications of MS/MS Identification and characterization drug metabolites Authentification and profiling of chemical components in a crude mixture Substructure analysis of unknown components Quantification of analytes in biological samples5J. Prasain 01/19/11 Ways to approach predicting MS/MS Likely sites of protonation or deprotonation. Likely leaving group. Mobility of protons Literature studyFragmentation always follows the basic rules of chemistryWhere are the sites of Deprotonation/protonation?What is the most likely leaving Group in this molecule?OHOOOOCH2 OHHOHOHOOCOOHOHOHOHJ. Prasain 01/19/11 Ion fragmentation for identification of phase II drug metabolites (glucuronide/sulfate conjugates)6J. Prasain 01/19/11100200300400m/z100500 Relative Intensity (%)5985113133181224269 Neutral loss (176 Da) OOHHOOHCOOH445 Product ion spectrum of genistein glucuronide in ESI-MS/MSGlucosides/glucuronides conjugates are easily cleaved off by higher potential at orificeCharacteristic fragmentsAglyconegenisteinParentionWhat fragment ions are characteristics for glucuronide conjugates?

5 J. Prasain 01/19/11 The loss of 80 Da from the parent ion and the presence of m/z80in the product ion spectra are the indicative of sulfate conjugates of phenolic compounds like daidzein [A] and equol [B] , [A]OHOOOSOOO-m/z, , [B]OOOHSOOO-7J. Prasain 01/19/11 What happens with aliphatic sulfates in MS/MS?Aliphatic and aromatic sulfate conjugates behave differently in MS/MS aliphatic typically show m/z97 (HSO4-) and m/z80 (SO3-.)J. Prasain 01/19/11 Source: Weidolf et al. Biomed. and Environ. Mass Spec. 15, 283-289, 1988 The absence of the m/z97 fragment with the base peakm/z80 makes the distinction between aromatic andaliphatic sulfates 8J. Prasain 01/19/11 Change in mass Methylation14 Demethylation-14 Hydroxylation16 Acetylation42 Epoxidation16 Desulfuration-32 Decarboxylation-44 Hydration18 Dehydration-18 Metabolic rxnChange in mass is associated with possiblemetabolic reactionJ.

6 Prasain 01/19/11 Ionization modeScanGlucuronidespos/negNL 176 amuHexose sugarpos/negNL 162 amuPentose sugarpos/negNL 132 amuPhenolic sulphateposNL 80 amuPhosphatenegPrecursor of m/z 79 Aryl-GSHposNL 275 amuAliphatic-GSHposNL 129taurinesnegPrecursor of m/z 124N-acetylcysteinsnegNL 129 amuNL = neutral fragmentation of drug conjugates by MS/MS9J. Prasain 01/19 , steroids get fragmented in MS/MS?+OH2 HOHO+m/z 159+HOm/z , ++OH2m/z 135 Estradiolm/z 273 Estrone m/z 271J. Prasain 01/19/11 Estradiol Standard Curve 25 Mr = 1 (Estradiol / ): "Quadratic" Regression ("1 / (x * x)" weighting): y = -215 x^2 + +004 x + -104 (r = )012345678910111213141516171819202122232 425 Concentration, r e a , c o u n tsSensitivity is an issue in quantification of steroids10J.

7 Prasain 01/19/11 Derivatization of estradiol with dansyl chlorideleads to the formation of E2-dansyl (m/z506)Source: Nelson et al. Clinical Chemistry, 2004 Easy protonationJ. Prasain 01/19/11 Time, min13579040100160220280340400460 Intensity, MRM chromatogram (mass transition 506/171) obtained from 50 picomole concentration of dansylated E2 Does derivatization help increase sensitivity?11J. Prasain 01/19/11 Calibration curve for dansylated E2 showing linearity from 50 picomole to 100 nanomole concentration range(r = )0102030405060708090100 Concentration, , countsJ. Prasain 01/19/11 OOHOOOOHOHCH2 OHOHOOHHOOOHOCH2 OHPuerarin; mol. wt. 4 16 Daidzin; mol. wt. 416 OHHOCan MS/MS analysis help distinguish isoflavone glucosides?

8 The Kudzu as a source of isoflavones12J. Prasain 01/19/11 OOHHOOOHOHOCH2 OHOHOOHOOOOHOHCH2 OHOHP uerarinm/z415O-and C-glucosides fragment differently in ESI-MS/MSPrasain et al. J. Agric. Food Chem. 2003220280300320360380m/z0%0100% [A][B]-162 DaYo+-120 DaDaidzinm/z415J. Prasain 01/19/11 Possible product ions of puerarin in ESI-MS/MSin negative ion modeOO-HOOOHOO-HOOOO-HOOHOHOOO-HOOOHOCH2 OHOHHO m/z 415m/z 325-90 Dam/z 295-120 Dam/z 267-28 DaPrasain et al. J Agric Food Chem. 51, 4213, 2003. 13J. Prasain 01/19/11m/z100500100200300400 Int. (%)253109161224253271433433180 Da162 DaNeutral losses (162 and 180) is useful in decidingwhether sugar is attached to an aromatic ring or not OO-HOOOOHOOHHOCH2 OHOO-OOOHOHOHOHOCH2OH162 DaPossiblestructureHexose attachedTo the ring B162 loss is an indicativeof aromatic attachmentJ.

9 Prasain 01/19/11m/z10050050100150200250m/z100500 Rel. Int. (%)6510711715122526963911071331591801962 24240269 OOHHOOOHG enisteinOOHHOOOHA pigeninOO-O-149 Isomers like genistein and apigenin are readily separated by tandem mass spectrometry14J. Prasain 01/19/11M/z 1005005010015020025030010050063107139167 183195211239267303139167195223251287-36- 28-28-28-28-36-28-28 OOO_HOClOOO_HOClOHNeutral loss of HCl (36 Da) is diagnostic for 3 -chloro derivative of genistein and daidzein in ESI-MS/MSbut not in 8-chloro derivativesJ. Prasain 01/19/11 Comparison of product ions help elucidate the unknown structuresOOOHHOOHOOHOHH2 COH+OH150200250300350400100%255268282311 -120431 Monohydroxylatedpuerarin2002503003504001 00500267295\415-120 Puerarin15J.

10 Prasain 01/19/11 fragmentation of taxoids in ESI-MS/MSJ. Prasain 01/19/11 fragmentation of basic taxoids from et al. Anal Chem, 200116J. Prasain 01/19/11 ESI-MS/MS spectra of taxoids (1-3). Peaks m/z194 and 210 represent the intact alkaloid side chain. AlkaloidSide chainm/z 210 Diterpenoid ScaffoldLoss of 60 or42J. Prasain 01/19 Ionization Mass spectrometry by Richard B. P, Prasain JK, Yeboah KF, Konishi Y. Detection and partial structure elucidation of basic taxoids from Taxus wallichianaby electrospray ionization tandem mass spectrometry . Anal Chem. 2001;73 JK, Patel R, Kirk M, Wilson L, Botting N, Darley-Usmar VM, Barnes S. Mass spectrometric methods for the analysis of chlorinated and nitrated isoflavonoids: a novel class of biological metabolites.


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