1

Ion fragmentation of small molecules in mass

spectrometry

Jeevan Prasainjprasain@uab.edu

6-2612

Feb 4, 2013

Class overview

• Introduction to tandem mass spectrometry (MS/MS)

• Use of MS/MS for structure elucidation of metabolites

• MS/MS for substructure analysis of taxins

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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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3

Feb 4, 2013 Nielsen et al., J Nat Prod. 2011

Adduct formation in +/–ve ion modes

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Molecules with inherent positive charge- molecular weight and m/z are same

4

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.

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Isotopic distribution and MS

• 1H = 99.9%, 2H = 0.02%

• 12C = 98.9%, 13C = 1.1%

• 35Cl = 68.1%, 37Cl = 31.9%

• Monoisotopic mass - the mass of the most abundant isotope

• Average mass- the abundance weighted mas of all isotopic components.

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Precursor ionor 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) ?

o

o

o

o

o

o

o

o

o

oo

oo

Collision gas

Collision cell

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Applications of MS/MS

• Pharmaceuticals- Identification and quantification of drug metabolites, PK/PD

• Academic/biotechnology- analysis of protein/peptides, authentification and profiling of chemical components in a crude mixture, substructure analysis of unknown components

• Clinical- eg. neonatal screening, steroids in athletes etc.

• Environment- eg. dioxins in fish..• Geological- eg. oil compositions…

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Interpreting MS/MS spectra

• Likely sites of protonation or deprotonation.

• Likely leaving group.

• 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

HOHO

OCOOH

OHOHOH

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O

OH

HO

O

OHO

HO

CH2OHOH

O

OH

O

O

O

OHOH

CH2OH

OH

Puerarinm/z 415

O- and C-glucosides fragment differently in ESI-MS/MS

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

220 280 300 320 360 380 m/z0

%

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

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O

O-

HO

O

OH

O

O-

HO

O

O

O-

HO

O

HO

HO

O

O-

HO

O

OHOCH2OH

OHHO

m/z 415

m/z 325

-90 Da

m/z 295

-120 Da

m/z 267

-28 Da

Possible product ions of puerarin m/z 415 in MS/MS

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Ion fragmentation for identification of phase II drug

metabolites (glucuronide/sulfate conjugates)

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100 200 300 400m/z

100

50

0

Rel

ativ

e In

ten

sity

(%

)

5985

113

133 181 224

269

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

Glucosides/glucuronides conjugates are easily cleaved off by higher potential at orifice

genistein

What fragment ions are characteristics for glucuronide conjugates?

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MSMS of m/z 429 indicate that it may be daidzein glucuronide

m/z 253

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 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

Inten

sity, cps

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

Inten

sity, cps

121.0

119.0

135.0

79.993.0 241.0

147.091.0 320.9

[B]

OO

OH

SO

O

O-

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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-.) Feb 4, 2013

Source: Weidolf et al. Biomed. and Environ. Mass Spec. 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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Ionization mode Scan

Glucuronides pos/neg NL 176 amuHexose sugar pos/neg NL 162 amuPentose sugar pos/neg NL 132 amuPhenolic sulphate pos NL 80 amuPhosphate neg Precursor of m/z 79 GSH- conjugates pos NL 129 amu

taurines Pos Precursor of m/z 126N-acetylcysteins neg NL 129 amu

NL = neutral loss.

Conjugate

Characteristic fragmentation of drug conjugates by MS/MS

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Kostiainen et al., 2003

20 60 100 140 180 220 2605.0e5

1.5e6

2.5e6

3.5e6

4.5e6

5.5e6

6.5e6

Inten

sity, cps

107.1

135.1

159.0

133.0147.1

109.1

173.1

121.2

145.193.181.1

Analysis of steroids by MS/MS

+OH2

HO

HO

+

m/z 159

+HO

m/z 107

20 40 80 120 160 200 2405.0e5

2.5e6

4.5e6

6.5e6

8.4e6

Intensity, cps

133.1

159.1

157.1

197.1107.1 145.1

81.1 213.1 253.2183.0131.1 198.193.1 121.2171.1

141.1105.1 179.1147.179.1

HO

O

OH+

+OH 2

m/z 135

Estradiolm/z 273

Estrone m/z 271

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Estradiol Standard Curve 0.05 – 25 µMr = 0.9959

U ntitled 1 (Estrad io l 273 .2 / 107 .1) : "Quadra tic" Regression ("1 / (x * x)" we igh ting ): y = -215 x^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 25C oncentration , 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

Sensitivity is an issue in quantification of steroids

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Derivatization of estradiol with dansyl chlorideleads to the formation of E2-dansyl (m/z 506)

Source: Nelson et al. Clinical Chemistry, 2004

Easy protonation

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Time, min

1 3 5 7 90

40

100

160

220

280

340

400

460

Intensity, cps

5.62

5.806.26

6.62 7.807.095.50 7.53 8.005.228.494.88

4.37 8.63

MRM chromatogram (m/z 506/171) 50 picomoledansylated E2

Derivatization tremendously helps increase sensitivity of E2

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Calibration curve for dansylated E2 showing linearity from 0.005-100 nM 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

Area, counts

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Substructure analysis in ESI-MS/MS(dereplication and partial identification

of natural products)

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Fragmentation of basic taxoids from T.Wallichiana extract

Prasain et al. Anal Chem, 2001

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

AlkaloidSide chainm/z 210

Diterpenoid Scaffold

Loss of 60 or42

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MS/MS precursor-scan spectra of typical alkaloid side chains to identify the basic taxoids compounds in an ethyl acetate

extract of T. wallichiana.

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600 620 640 660 680 700 720 740 760 780 800 820 840 860 880

605622

714

742

758 800

612 628 668 710

742

806.2 848 862

668684

726

744

758770 786

686710

728

744770

786

800

A

B

C

D

Scaffold (m/z 309)

Side chain (m/z 210)

Side chain (m/z 252)

Side chain (m/z 194)

m/z

Inte

nsi

ty

Comparison of precursor scan spectra obtained from thescaffold m/z 309 and side chain m/z 194, 210 and 252

Taxoids with scaffold m/z 309 and alkaloid side chains are shown by dashed lines

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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 J.K., Wang C.-C., Barnes S. Mass spectrometric analysis of flavonoids in biological samples. Free Radical Biology & Medicine, 37: 1324-1350, 2004.

4. William Griffiths. Tandem mass spectrometry in the study of fatty acids, bile acids and steroids. Mass Spectrometry Reviews, 2003;22:81-152.

5. Yi et al., Anal Bioanal Chem. 2006.

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