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Ion Fragmentation of Small Molecules in Mass Spectrometry

Ion Fragmentation of Small Molecules in Mass Spectrometry

fragmentation of small in spectrometry

Jeevan Prasain [email protected] 6-2612

J. Prasain 01/19/11

Nomenclature: the main names and acronyms used in

• Molecular ion: Ion formed by addition or the removal of one or several to or from the sample molecules- Impact (EI-MS). M + e-  M+• + 2e-

• Adduct Ion: Ion formed through interaction of two species and containing all the of one of them plus one or several atoms of them (e.g. alkali, ammonium).

J. Prasain 01/19/11

1 Contd.. • Pseudomolecular ion: Ion originating from the analyte by abstraction of a [M-H]- or addition of proton [M+H]+ • (Cooks, 1976): MS/MS (McLafferty, 1978), tandem in space or time

• Precursor ion/parent ion: undergoing fragmentation. • Product ion/daughter ion: Ions resulting from parent/precursor ions. • Neutral loss: Fragments lost as neutral molecules

• In positive mode, a trace of formic acid is often added to aid protonation of the sample molecules; in negative ionization mode a trace of ammonia solution or a volatile amine is added to aid deprotonation of the sample molecules. and are usually analysed under positive ionization conditions and polyphenols and acids under negative ionization conditions. In all cases, the m/z scale must be calibrated. J. Prasain 01/19/11

Terminology..

• Ionspray denotes pneumatically assisted ESI operating at aflow rate of approximately 5 to 50 uL/min.

• Turboionspray is ionspray with additional heated gas for flow rate of 0.1 to 2 ml/min.

• Heated nebulizer is the trade mark of AB Sciex for APCI.

J. Prasain 01/19/11

2 What is Collision Induced Dissociation (CID) or Collisionally Activated Dissociation (CAD) ?

Fragmenting ion Collision gas

Collision cell

o o o o o o o o o o o o Product ions Precursor ion o or parent ion

Activated ion Neutral loss Schematic of CID fragmentation

Other activation processes: PSD (post source-decay) ECD (electron capture dissociation) SID (surface-induced dissociation J. Prasain 01/19/11

Various types of MS/MS experiments

Hopfgartner etal. J. Mass Spectrom, 2004 J. Prasain 01/19/11

3 Steps involved in fragmentation

Step # 1: Creation of ions

C N OCH2CH3 ESI H

Step # 2: Add energy of activation

CID

Step # 3: Charge directed fragmentation

Fragment ion + neutral molecule

J. 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 samples

J. Prasain 01/19/11

4 Ways to approach predicting MS/MS

• Likely sites of protonation or deprotonation. • Likely leaving group. • Mobility of

CH OH • Literature study HO 2 O HO HO OH O OH Where are the sites of HO OH Deprotonation/protonation? O O COOH What is the most likely leaving O Group in this molecule?

Fragmentation always follows the basic rules of chemistry

J. Prasain 01/19/11

Ion fragmentation for identification of phase II drug metabolites (glucuronide/sulfate conjugates)

J. Prasain 01/19/11

5 What fragment ions are characteristics for glucuronide conjugates? Product ion of genistein glucuronide in ESI-MS/MS

100 269 COOH Aglycone O genistein HO OH

OH 445 50 Characteristic fragments Parent Neutral loss (176 Da) Relative Relative Intensity (%) ion 85 59 113 133 181 224 0 100 200 300 400 m/z Glucosides/glucuronides conjugates are easily cleaved off by higher potential at orifice

J. Prasain 01/19/11

The loss of 80 Da from the parent ion and the presence of m/z 80 in the product ion spectra are the indicative of sulfate conjugates of phenolic compounds like daidzein [A] and equol [B]

116.9 O O 5.5e6 S O O- HO O 4.5e6 [A]

Intensity, cps 3.5e6 253.0 O 2.5e6

1.5e6 134.9 224.0 80.091.0 132.9 252.0 5.0e5 207.9 96.9 160.0 197.0 225.0 20 60 100 140 180 220 260 300

121.0 1.4e7 O- O 1.2e7 [B] O S O

1.0e7 O Intensity, cps 8.0e6 119.0 6.0e6 135.0

4.0e6 79.9 OH 2.0e6 93.0 241.0 147.0 91.0 320.9 20 60 100 140 180 220 260 300 340 m/z, amu J. Prasain 01/19/11

6 What happens with aliphatic sulfates in MS/MS?

Aliphatic and aromatic sulfate conjugates behave differently in MS/MS aliphatic typically show m/z 97 (HSO4-) and m/z 80 (SO3-.) J. Prasain 01/19/11

The absence of the m/z 97 fragment with the base peak m/z 80 makes the distinction between aromatic and aliphatic sulfates

Source: Weidolf et al. Biomed. and Environ. Mass Spec. 15, 283-289, 1988

J. Prasain 01/19/11

7 Change in mass is associated with possible metabolic reaction

Metabolic rxn Change in mass

Methylation 14 Demethylation -14 Hydroxylation 16 Acetylation 42 Epoxidation 16 Desulfuration -32 Decarboxylation -44 Hydration 18 Dehydration -18

J. Prasain 01/19/11

Characteristic fragmentation of drug conjugates by MS/MS

Conjugate Ionization mode Scan

Glucuronides pos/neg NL 176 amu Hexose sugar pos/neg NL 162 amu Pentose sugar pos/neg NL 132 amu Phenolic sulphate pos NL 80 amu Phosphate neg Precursor of m/z 79 Aryl-GSH pos NL 275 amu Aliphatic-GSH pos NL 129 taurines neg Precursor of m/z 124 N-acetylcysteins neg NL 129 amu

J. Prasain 01/19/11 NL = neutral loss.

8 How steroids get fragmented in MS/MS?

+OH2 +OH2 6.5e6 107.1

5.5e6 HO + + m/z 135

4.5e6 m/z 107 135.1 HO Intensity, cps

3.5e6 m/z 159 HO

2.5e6 159.0 Estradiol

1.5e6 109.1121.2133.0 m/z 273 81.1 93.1 147.1 173.1 5.0e5 145.1 20 60 100 140 180 220 260

133.1 8.4e6 O

OH+ 159.1 6.5e6

157.1 Intensity, Intensity, cps 4.5e6 HO

2.5e6 197.1 Estrone m/z 271 145.1 107.1 171.1 81.1 213.1 93.1 121.2131.1 183.0 198.1 253.2 5.0e5 79.1 105.1 141.1147.1 179.1 20 40 80 120 160 200 240 J. Prasain 01/19/11

Estradiol Standard Curve 0.05 – 25 µM r = 0.9959

Untitled 1 (Estradiol 273.2 / 107.1): "Quadratic" Regression ("1 / (x * x)" weighting): y = -215 x^2 + 1.7e+004 x + -104 (r = 0.9959)

3.0e5

2.8e5

2.6e5

2.4e5

2.2e5

2.0e5

1.8e5

1.6e5

1.4e5

Area, counts 1.2e5

1.0e5

8.0e4

6.0e4 4.0e4 Sensitivity is an issue in quantification of steroids 2.0e4

0.0

0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 Concentration, uM

J. Prasain 01/19/11

9 Derivatization of estradiol with dansyl chloride

leads to the formation of E2-dansyl (m/z 506)

Easy protonation

Source: Nelson et al. Clinical Chemistry, 2004

J. Prasain 01/19/11

Does derivatization help increase sensitivity?

Representative MRM chromatogram (mass transition 506/171) obtained from 50 picomole concentration of dansylated E2

5.62 460

400

340

Intensity, Intensity, cps 280

220

160

5.80 100 6.26 6.627.09 7.80 5.225.50 7.53 8.00 4.88 8.49 40 4.37 8.63

0 1 3 5 7 9

Time, min

J. Prasain 01/19/11

10 Calibration curve for dansylated E2 showing linearity from 50 picomole to 100 nanomole concentration range (r = 0.999)

8.0e6

6.5e6

5.0e6 Area, counts

3.5e6

2.0e6

5.0e5

0 10 20 30 40 50 60 70 80 90 100

Concentration, nM

J. Prasain 01/19/11

Can MS/MS analysis help distinguish isoflavone glucosides?

The Kudzu as a

OH source of isoflavones HO CH2OH CH2OH HO O O OH OH O O HO O OH

O OH O OH

Puerarin; mol. wt. 416 Daidzin; mol. wt. 416

J. Prasain 01/19/11

11 O- and C-glucosides fragment differently in ESI-MS/MS CH2OH O OH O [A] + OH O Yo OH 100 255.050

O OH -162 Da Daidzin m/z 415

%

256.057

[B] 0 100 297.043 HO -120 Da CH OH OH 2 267.037 HO O % HO O 321.046 307.065 268.041 351.044 281.051 335.061 381.055 363.046 OH O 0 220 240 260 280 300 320 340 360 380 m/z Puerarin J. Prasain 01/19/11 m/z 415 Prasain et al. J. Agric. Food Chem. 2003

Possible product ions of puerarin in ESI-MS/MS in negative ion mode

OH Prasain et al. J Agric Food Chem. 51, 4213, 2003. HO CH2OH HO O OH

HO O -120 Da HO O

O O-

O- m/z 415 O m/z 295 -90 Da

HO -28 Da

HO HO O HO O

O O- O O- m/z 325 m/z 267

J. Prasain 01/19/11

12 Neutral losses (162 and 180) is useful in deciding whether sugar is attached to an aromatic ring or not

OH 100 HO 253 Hexose attached CH2OH HO O To the ring B

HO O Possible 50 O structure 180 Da

O- O 433 0 HO

HO CH2OH 253 100 162 loss is an indicative

Rel. Rel. Int. (%) O HO O O OH of aromatic attachment 161 271

50 O O- 109 162 Da162 Da 224 433 0.0 100 200 300 400 m/z J. Prasain 01/19/11

Isomers like genistein and apigenin are readily separated by tandem mass spectrometry

100 117 HO O OH O-

OH O 50 Apigenin 107 65 151 269 149 225 0 O HO Rel. Rel. Int. (%) 100 O 269

O- 133 OH OH O 50 Genistein 159 224 63 107 180 91 196 240 0 50 100 150 200 250 m/z m/z

J. Prasain 01/19/11

13 Neutral loss of HCl (36 Da) is diagnostic for 3’-chloro derivative of genistein and daidzein in ESI-MS/MS but not in 8-chloro derivatives

100 HO O 303

OH O O_ 167 50 -28 -36 Cl 195 211 239 267 63 107 183 139 -28 0

100 195 HO O -28 167 -28 -36 287 223 -28 O 251 50 O_ -28 Cl 139

0 50 100 150 200 250 300 M/z

J. Prasain 01/19/11

Comparison of product ions help

elucidate the unknown structuresOH

H2C

267 295 O 100 Puerarin OH

OH -120 HO 50 HO O 415

\ 0 200 250 300 350 400 O OH +OH 100 311 282 Monohydroxylated puerarin

% 431 -120 268 255

150 200 250 300 350 400

J. Prasain 01/19/11

14 Fragmentation of taxoids in ESI-MS/MS

J. Prasain 01/19/11

Fragmentation of basic taxoids from T. Wallichiana extract

Prasain et al. Anal Chem, 2001

J. Prasain 01/19/11

15 ESI-MS/MS spectra of taxoids (1-3). Peaks m/z 194 and 210 represent the intact alkaloid side chain.

Loss of 60 or 42

Diterpenoid Alkaloid Scaffold Side chain m/z 210

J. Prasain 01/19/11

References

1. Mass Spectrometry by Richard B. Cole. 2. Stefanowicz P, Prasain JK, Yeboah KF, Konishi Y. Detection and partial structure elucidation of basic taxoids from Taxus wallichiana by electrospray ionization tandem mass spectrometry. Anal Chem. 2001;73:3583-9. 3. Prasain 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. J Mass Spectrom. 2003;38:764-71. 4. William Griffiths. Tandem mass spectrometry in the study of fatty acids, bile acids and steroids. Mass Spectrometry Reviews, 2003;22:81-152.

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