PurduePurdue--UAB Botanicals Center for AgeUAB Botanicals Center for Age--Related DiseaseRelated Disease

MS/MS analysis of MS/MS analysis of PolyphenolsPolyphenolsJeevan Prasain Ph.D.Pharmacology & ToxicologyUAB

OH

O

OHHO

OHO

OHOH

OHOH

OH

OHO

OHOH

OH

OH

O

OHO

OH O OH

OH

OH

HO

OH

CH2O

CH2O

HO

Polyphenols

Phenolic acids�and derivatives

Flavonoids Lignans

Flavanols Flavonols Isoflavones

Caffeic acid

EGC�(Flavanol)

Quercetin�(Flavonol)

Stilbenes

Genistein (Isoflavone)

Resveratrol�(Stilbene)

Enterodiol�(Lignan)

LC-MS Profile of the methanolic extract of KDSColumn: C8 Aquapore; 7µm, 100 x 4.6 mm i.d.Solvent: CH3CN:H2O (10-40%, run time 30 min)

0 4 8 12 16

Time (min)

100

75

50

25

0

Rel

ativ

e In

tens

ity (%

)

m/z 253DZN

m/z 283

m/z 431G’N

m/z 415DZ’N

m/z 415puerarin

m/z 445

m/z 445

m/z 341

m/z 267Formononetin

m/z 547

m/z 431

What is tandem mass What is tandem mass spectrometry?spectrometry?

The ability to induce fragmentation and perform successive mass spectrometry experiments (MS/MS) on those fragments. In MS/MS mode, product ion, precursor ion and constant neutral loss scans are performed. Multiple reaction monitoring (MRM) is useful technique for quantitation.

How does it work?

Tandem in space means having two mass spectrometers in series.It uses two stages of mass analysis, one to pre-select an ion and the Second to analyze fragments induced, for instance, by collision withAn inert gas like argon or helium. This dual analysis can be dual inSpace, or dual in time. The most commonly used tandem mass spectrometry is the triple quadrupole (QqQ).

MS/MS data interpretation

1. Identification of molecular ions or quasi-molecular ions.

2. Origin of product ions.

3. Stability and relative intensity of ions.

m/z

100

50

0

Rel

. Int

. (%

)

150m/z

57

29

0

Rel

. Int

. (%

)

65 107

117

151

225

269

6391

107

133

159 180196

224

240

269

Product ion spectra apigenin vs. genistein

O

OH

HO

OOH

Genistein

OOH

HO

OOH

Apigenin

O

O-

O-

50 100 200 250

220 280 300 320 360 380 m/z0

%

0

100

%

255.050

256.057

297.043

267.037

268.041281.051

307.065321.046

363.046335.061351.044 381.055

100

240 260 340

[B]

Product ion spectra of Product ion spectra of daidzin daidzin [A] and [A] and puerarin puerarin [B] in ESI[B] in ESI--MS/MSMS/MS

-162 Da

Yo+

-120 Da

O

OH

HO

O

OHO

HO

CH2OHOH

O

OH

O

O

O

OHOH

CH2OH

OH

daidzin

[A]

Puerarin

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

100

50

0

Rel

ativ

e In

tens

ity (%

)

5985

113133 181 224

269

Glucuronide loss

O

OHHO

OH

COOH

445Characteristicof Genistein

100 200 300 400m/z

ESIESI--MS/MS Spectra of MS/MS Spectra of Biochanin Biochanin A and A and PrunetinPrunetin

100

50

0

Rel

. Int

. (%

)

138,385

50 100 150 200 250

m/z

14.9

7.4

0.0

Rel

. Int

. (%

)

20,592

132

132

268

239

223211

239

211

O

OOH

H3CO

OH

Prunetin

O

OOCH3

HO

OH

Biochanin A-15 (CH3)

268

283

283

Loss of methyl radical is characteristic for methylated flavonoids

Structural identification of unknown Structural identification of unknown based on product ions based on product ions

150m/z

100

0

Rel

ativ

e In

tens

ity (%

)

91 120

135148

184

211

223

239

268

50

O

O O-OCH3

HO

m/z 283

O

O O-O.

HO

OH

m/z 268

O.O

-O

m/z 148

O

O-O

m/z 135

Diagnostic ions

50 100 200 250

Full scan ESIFull scan ESI--MS of the MS of the methanolic methanolic extract of extract of kudzu dietary supplement. Constant neutral loss kudzu dietary supplement. Constant neutral loss

scans to identify the scans to identify the isoflavone isoflavone componentscomponents. . 100

0

Rel

. Int

. (%

)

100

0

Rel

. Int

. (%

)

200 300 400 500 600 700 800

m/z

100

.0

Rel

. Int

. (%

)

255 360

417

447475

549 579

257

417

447

289 365

417

447 4751 579

433

ESI-MS full scan

Neutral loss scan 120

Neutral loss scan 162

100

75

50

25

0

Rel

. Int

. (%

)

130,325

87

65

43

22

0

Rel

. Int

. (%

)

112,927

63 91 107

167

195 211 239267

303

139

167

195

223251

287

[a]

[b]

-36

-36-28

-28

-28-28

-28-28

-28 -16

Comparison of the product ions obtained in ESIComparison of the product ions obtained in ESI--MS/MS MS/MS of 3’of 3’--chlorogenistein chlorogenistein [a] and 3’[a] and 3’--chlorodaidzein chlorodaidzein [b][b]

O

OOH

HO

OH

Cl3'

1

345

7

1'

2

4'

O

OOH

HO

Cl3'

1

345

7

1'

2

4'

50 100 150 200 250 300m/z

Product ion spectra of a reaction Product ion spectra of a reaction product at product at m/zm/z 303 in LC303 in LC--MS/MSMS/MS

m/z

39

29

19

10

0

Rel

. Int

. (%

)

1304

50 100 150 200 250 300

m/z

100

75

50

25

0

Rel

. Int

. (%

)

3358

133

155

167194

211

238

303

6391

107139

167

182

195 210239

267

303

-36

HCl loss

No HCl loss

3’-chlorogenistein

6- or 8-chlorogenistein

HCl loss is diagnostic for positioning chlorine in the aromatic ring.

Urinary metabolites detected in a Urinary metabolites detected in a rat fed with grape seed extractrat fed with grape seed extract

O

OHHO

OH

OH

OHcatechin

O

OMeHO

OH

OH

O

OHHO

OH

OMe

OHOH

4'-O-methylcatechin3'-O-methylcatechin

FullFull--scan ESIscan ESI--MS spectrum of the MS spectrum of the methanolic methanolic extract of grape seed extractextract of grape seed extract

289

287576

305 387

865

580720

652864730

796

866867

577

729 11531017300 600 900 12000

100

%

Deprotonated catechin

m/z

Dimer-H

Trimer-H

Tetramer-HGalloylatedDimer-H

GalloylatedTrimer-H

150 200 250 300m/z

100

50

0

Rel

ativ

e In

tens

ity (%

)13,847

- -

137

139

147

179 221 287

Product ion spectrum of the ion m/z 305 Product ion spectrum of the ion m/z 305 in LCin LC--MS/MS analysis (MS/MS analysis (RtRt. 7.10 min) . 7.10 min)

OOH

OH

HO

OH

OMe

3'-O-methylcatechin

100 150 200 250 300m/z

100

50

0

Rel

ativ

e In

tens

ity (%

)5105

- -

139

137

147

179 221 287111

Product ion spectrum of the ion m/z 305 Product ion spectrum of the ion m/z 305 in LCin LC--MS/MS analysis (MS/MS analysis (RtRt. 7.88 min) . 7.88 min)

OOMe

OH

HO

OH

OH

4'-O-methylcatechin

O

OH

OCH3

HO

OH

O

OH

OCH3

HO

m/z 305

O

OH

OCH3

OH

-CO

O

OCH3

OH

HO

OH

OH

OHO

OH

O

OH

-H2O

H+

H+

m/z 221

m/z 193

m/z 111

H+

m/z 139ring A ion

m/z 137ring Bdignostic of the ring B methylation

H+

H+

H+

ProposedProposed structures of product ions structures of product ions obtained from obtained from m/zm/z 305 in ESI305 in ESI--MS/MSMS/MS

m/z 287

H+

m/z 147

LCLC--MS/MSMS/MS--MRM spectra for MRM spectra for catechin catechin and 3’and 3’--and 4’and 4’--OO--methylcatechin methylcatechin in rat urinein rat urine

ConclusionsConclusionsThe CID of isoflavones in ESI-MS generates a series of cluster ions due to the subsequent losses of carbonyl/aldehyde or oxygen.The presence of prominent ions at m/z 133 and 135 in MS/MS of genistein and daidzein indicates that they are not originated from the ring A.Isoflavones glycosides (O- and C-glycosides) generate different product ions in ESI-MS/MS due to the neutral losses of 162 and 120, respectively.The major fragmentation of isoflavones with chlorinated ring B in MS/MS starts with elimination of HCl, whereas the loss from product ions was observed with those chlorine in the ring A.