1

J. Prasain 01/19/11

Ion fragmentation of small

molecules in mass

spectrometry

Jeevan Prasain

jprasain@uab.edu

6-2612

J. Prasain 01/19/11

Nomenclature: the main names and

acronyms used in mass spectrometry

• Molecular ion: Ion formed by addition or the removal of one

or several electrons to or from the sample molecules-

Electron Impact (EI-MS). M + e- M+• + 2e-

• Adduct Ion: Ion formed through interaction of two species

and containing all the atoms of one of them plus one or

several atoms of them (e.g. alkali, ammonium).

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J. Prasain 01/19/11

Contd..• 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.

• 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 mode a 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/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.

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J. Prasain 01/19/11

Precursor ion

or parent ion

Activated ion

Fragmenting ion

Neutral loss

Product ions

Schematic of CID fragmentation

What is Collision Induced Dissociation (CID)

or Collisionally Activated Dissociation (CAD) ?

Other activation processes:

PSD (post source-decay)

ECD (electron capture dissociation)

SID (surface-induced dissociation

o

o

o

o

o

o

o

o

o

oo

oo

Collision gas

Collision cell

J. Prasain 01/19/11

Various types of MS/MS experiments

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J. Prasain 01/19/11

Steps involved in fragmentation

Step # 1: Creation of ions

Step # 2: Add energy of activation

Step # 3: Charge directed fragmentation

C N H

OCH2CH3

C N H

OCH2CH3

CID

C N H

OCH2CH3

Fragment ion + neutral molecule

ESI

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

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J. Prasain 01/19/11

Ways to approach predicting MS/MS

• Likely sites of protonation or deprotonation.

• Likely leaving group.

• Mobility of protons

• Literature study

Fragmentation always follows the basic

rules of chemistry

Where are the sites of

Deprotonation/protonation?

What is the most likely leaving

Group in this molecule?

OHO

O

O

O

CH2OHHO

HO

HO

OCOOH

OH

OHOH

J. Prasain 01/19/11

Ion fragmentation for identification of phase

II drug metabolites (glucuronide/sulfate

conjugates)

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J. Prasain 01/19/11

100 200 300 400

m/z

100

50

0

Rela

tiv

e I

nte

nsit

y (

%)

59

85

113

133 181 224

269

Neutral loss (176 Da)

O

OHHO

OH

COOH

445

Product ion spectrum of genistein glucuronide in ESI-MS/MS

Glucosides/glucuronides conjugates are easily

cleaved off by higher potential at orifice

Characteristic fragments

Aglycone

genistein

Parent

ion

What fragment ions are characteristics

for glucuronide conjugates?

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]

20 60 100 140 180 220 260 300

5.0e5

1.5e6

2.5e6

3.5e6

4.5e6

5.5e6

Inte

ns

ity, c

ps

116.9

253.0

134.9

252.0132.991.0224.0

207.980.0

96.9 225.0197.0160.0

[A] OHO

O

O

SO

O O-

m/z, amu

20 60 100 140 180 220 260 300 340

2.0e6

4.0e6

6.0e6

8.0e6

1.0e7

1.2e7

1.4e7

Inte

ns

ity, c

ps

121.0

119.0

135.0

79.9

93.0 241.0147.0

91.0 320.9

[B]

OO

OH

SO

O

O-

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J. 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/z 97 (HSO4-) and m/z 80 (SO3-.)

J. Prasain 01/19/11

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

The absence of the m/z 97 fragment with the base peak

m/z 80 makes the distinction between aromatic and

aliphatic sulfates

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J. Prasain 01/19/11

Change in mass

Methylation 14

Demethylation -14

Hydroxylation 16

Acetylation 42

Epoxidation 16

Desulfuration -32

Decarboxylation -44

Hydration 18

Dehydration -18

Metabolic rxn

Change in mass is associated with possible

metabolic reaction

J. Prasain 01/19/11

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

NL = neutral loss.

Conjugate

Characteristic fragmentation of drug

conjugates by MS/MS

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J. Prasain 01/19/11

20 60 100 140 180 220 2605.0e5

1.5e6

2.5e6

3.5e6

4.5e6

5.5e6

6.5e6

Inte

ns

ity, c

ps

107.1

135.1

159.0

133.0147.1

109.1

173.1

121.2

145.1

93.181.1

How steroids get fragmented in MS/MS?

+OH2

HO

HO

+

m/z 159

+HO

m/z 107

20 40 80 120 160 200 240

5.0e5

2.5e6

4.5e6

6.5e6

8.4e6

Inte

nsity

, cps

133.1

159.1

157.1

197.1

107.1 145.181.1 213.1 253.2183.0131.1 198.193.1 121.2

171.1

141.1105.1 179.1147.179.1

HO

O

OH+

+OH2

m/z 135

Estradiol

m/z 273

Estrone m/z 271

J. Prasain 01/19/11

Estradiol Standard Curve 0.05 – 25 µM

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

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 25Concentration, 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

Are

a,

cou

nts

Sensitivity is an issue in quantification of steroids

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J. Prasain 01/19/11

Derivatization of estradiol with dansyl chloride

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

Source: Nelson et al. Clinical Chemistry, 2004

Easy

protonation

J. Prasain 01/19/11

Time, min

1 3 5 7 90

40

100

160

220

280

340

400

460

Inte

nsity

, cps

5.62

5.806.26

6.62 7.807.095.50 7.53 8.005.22 8.494.884.37 8.63

Representative MRM chromatogram (mass transition 506/171)

obtained from 50 picomole concentration of dansylated E2

Does derivatization help increase sensitivity?

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J. Prasain 01/19/11

Calibration curve for dansylated E2 showing linearity

from 50 picomole to 100 nanomole concentration range

(r = 0.999)

0 10 20 30 40 50 60 70 80 90 100

Concentration, nM

5.0e5

2.0e6

3.5e6

5.0e6

6.5e6

8.0e6

Are

a, c

ounts

J. Prasain 01/19/11

O

OH

O

O

O

OHOH

CH2OH

OH

O

OH

HO

O

OHO

CH2OH

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

OH

HO

Can MS/MS analysis help distinguish isoflavone

glucosides?

The Kudzu as a

source of isoflavones

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J. Prasain 01/19/11

O

OH

HO

O

OHO

HO

CH2OHOH

O

OH

O

O

O

OHOH

CH2OH

OH

Puerarin

m/z 415

O- and C-glucosides fragment

differently in ESI-MS/MS

Prasain et al. J. Agric. Food Chem. 2003

220 280 300 320 360 380m/z

0

%

0

100

%

255.050

256.057

297.043

267.037

268.041281.051

307.065321.046

363.046335.061

351.044381.055

100

240 260 340

[A]

[B]

-162 Da

Yo+

-120 Da

Daidzin m/z 415

J. Prasain 01/19/11

Possible product ions of puerarin in ESI-MS/MS

in negative ion mode

O

O-

HO

O

OH

O

O-

HO

O

O

O-

HO

O

HO

HO

O

O-

HO

O

OHO

CH2OH

OH

HO

m/z 415

m/z 325

-90 Da

m/z 295

-120 Da

m/z 267

-28 Da

Prasain et al. J Agric Food Chem. 51, 4213, 2003.

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J. Prasain 01/19/11

m/z

100

50

0

100 200 300 400

m/z

100

50

0.0

Rel. I

nt.

(%

)253

109

161

224

253

271

433

433

180 Da

162 Da

Neutral losses (162 and 180) is useful in deciding

whether sugar is attached to an aromatic ring or not

O

O-

HO

O

O

OHO

OH

HO

CH2OH

O

O-

O

O

OH

OHO

HO

HO CH2OH

162 Da

Possiblestructure

Hexose attached

To the ring B

162 loss is an indicative

of aromatic attachment

J. Prasain 01/19/11

m/z

100

50

0

50 100 150 200 250m/z

100

50

0

Rel. I

nt.

(%

) 65107

117

151

225

269

63

91107

133

159 180196

224

240

269O

OH

HO

OOH

Genistein

O

OH

HO

OOH

Apigenin

O

O-

O-

149

Isomers like genistein and apigenin are readily

separated by tandem mass spectrometry

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J. Prasain 01/19/11

M/z

100

50

0

50 100 150 200 250 300

100

50

0

63 107 139

167

183

195211 239

267

303

139

167

195

223

251

287-36

-28

-28

-28

-28

-36

-28

-28

O

OO_

HO

Cl

O

OO_

HO

Cl

OH

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

J. Prasain 01/19/11

Comparison of product ions help

elucidate the unknown structures

O

OOH

HO

O

HO

OH

OH

H2C

OH

+OH

150 200 250 300 350 400

100

%

255268

282

311

-120431

Monohydroxylated

puerarin

200 250 300 350 400

100

50

0

267 295

\

415

-120

Puerarin

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J. Prasain 01/19/11

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

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J. Prasain 01/19/11

ESI-MS/MS spectra of taxoids (1-3). Peaks m/z 194

and 210 represent the intact alkaloid side chain.

Alkaloid

Side chain

m/z 210

Diterpenoid

Scaffold

Loss of 60 or

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J. Prasain 01/19/11

References

1. Electrospray Ionization 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.