MS analysis of Polyphenols - UAB

Purdue-UAB Botanicals Center for Age-Related Disease MS/MS analysis of Polyphenols. Jeevan Prasain Ph.D. Pharmacology & Toxicology. UAB...

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Purdue-UAB Botanicals Center for Age-Related Disease

MS/MS analysis of Polyphenols Jeevan Prasain Ph.D. Pharmacology & Toxicology UAB

Polyphenols

Phenolic acids and derivatives

Flavonoids

Stilbenes

Lignans

OH

HO

CH2 O

OH CH2 O

HO HO

Flavanols Flavonols Isoflavones

OH

Caffeic acid

OH

OH

O

HO

OH HO

O

OH OH

OH

O

OH

Genistein (Isoflavone)

OH

EGC (Flavanol)

Resveratrol (Stilbene)

O

OH HO

O

OH OH

OH

O

Quercetin (Flavonol)

OH

Enterodiol (Lignan)

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

m/z 415 puerarin

100 m/z 341

Relative Intensity (%)

75

m/z 253 DZN

m/z 415 DZ’N

m/z 547

m/z 283 m/z 445

50

m/z 267 Formononetin m/z 445

25

m/z 431 G’N

m/z 431

0 0

4

8

Time (min)

12

16

What is tandem mass 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 with An inert gas like argon or helium. This dual analysis can be dual in Space, 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.

Product ion spectra apigenin vs. genistein 117

Rel. Int. (%)

100

HO

O OH

O-

O

OH

50

Apigenin

107

65

151

269 225

0

O HO

Rel. Int. (%)

57 O-

133 OH

OH

O

Genistein

29 63 91 0

269

O

50

224 159 180 196 240

107

100

150 m/z m/z

200

250

Product ion spectra of daidzin [A] and puerarin [B] in ESI-MS/MS CH2 OH OH

O

OH

100

O

OH

Yo+

[A]

O

255.050

O

OH

daidzin

-162 Da % 256.057

[B]

0

100

297.043

HO CH2 OH

OH HO

267.037

O

% HO

-120 Da

321.046 307.065 268.041 351.044 281.051 335.061 381.055 363.046

O

O

Puerarin

OH

0

220

240

260

280

300

320

340

360

380

m/z

Relative Intensity (%)

Product ion spectrum of genistein glucuronide in ESI-MS/MS 269

100

COOH O HO

OH

OH

50

Characteristic of Genistein 59

0

445

Glucuronide loss

85 113 133

100

181

224

200

m/z

300

400

ESI-MS/MS Spectra of Biochanin A and Prunetin HO

O

268

100 O

Rel. Int. (%)

OH

138,385

OCH3

Biochanin A

-15 (CH3)

239

50

211 132

223 283

0

Rel. Int. (%)

14.9

H3 CO

268

O

OH

7.4

20,592

O

OH

Prunetin

239

211

132 0.0

50

100

150

m/z

283 200

250

Loss of methyl radical is characteristic for methylated flavonoids

Structural identification of unknown based on product ions HO

O

OH O

Relative Intensity (%)

100

Diagnostic ions

211

-O

m/z 268

O.

268

m/z 148

239

O O.

HO

O

-O

O-

O

50 O

O m/z 135

135

91

0

50

100

184 148

m/z 283

223

120

150 m/z

200

250

OOCH3

Rel. Int. (%)

Full scan ESI-MS of the methanolic extract of kudzu dietary supplement. Constant neutral loss scans to identify the isoflavone components. 100

ESI-MS full scan

417

255

360

447 433 475

0

549

100

579

Rel. Int. (%)

Neutral loss scan 120 417

0

447

257

Neutral loss scan 162

Rel. Int. (%)

100 417

289 .0 200

300

447 475

365 400

1

500 m/z

579 600

700

800

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

OH

75

1'

3

4

5

130,325

2

7

100

Rel. Int. (%)

O 1

HO

4'

O

303

OH

3' Cl

50 167 -28

-16

-36

195 211

25 63

-28

91 107

239

-28

0

[b] Rel. Int. (%)

87

O 1

HO

65

3

4

112,927

2

7 5

1' 4'

O

195

OH

3' Cl

43

167

-28 22 0

-28

100

150

287

223

-28

251

-36

-28

139

50

267

m/z

200

250

300

Product ion spectra of a reaction product at m/z 303 in LC-MS/MS 1304 303

Rel. Int. (%)

39 29

211

6- or 8-chlorogenistein

19 133

No HCl loss

167 194

10

155

238

0 3358

Rel. Int. (%)

100

303

167

3’-chlorogenistein

75

HCl loss 195

50 63

25 0

91

50

107

100

210

239

-36

182 267

139

150 m/z m/z

200

250

300

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

Urinary metabolites detected in a rat fed with grape seed extract OH OH HO

O OH OH

catechin

OH OMe OMe OH HO

HO

O

O OH OH

OH

OH

3'-O-methylcatechin

4'-O-methylcatechin

Full-scan ESI-MS spectrum of the methanolic extract of grape seed extract 100 Deprotonated catechin 289

%

Dimer-H 577 Trimer-H

576

0

287 305

387

300

Galloylated865 Galloylated Dimer-H Trimer-H Tetramer-H 729 1017 1153 866

580

652

720 730

864 867

796

600

900

m/z

1200

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

13,847

Relative Intensity (%)

100

139

OMe OH HO

O OH OH

50

137

3'-O-methylcatechin 147 221

179 0

150

200

m/z

287 250

300

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

5105

139

100

Relative Intensity (%)

-

OH OMe HO

O OH OH

137

50

4'-O-methylcatechin 147 111 0

100

221

179 150

200

m/z

287 250

300

Proposed structures of product ions obtained from m/z 305 in ESI-MS/MS H+

OH HO

O

H+ OH

OCH3

O

OCH3

OH OH

m/z 221 H+

-H2O

H+ HO

O

OH

H+ O

OH

m/z 193

OH

m/z 305

HO

-CO

OCH3

OCH3

OH

m/z 139 ring A ion

m/z 287

H+

H+ O

HO

OH

m/z 147

O

OH

m/z 111

m/z 137 ring B dignostic of the ring B methylation

LC-MS/MS-MRM spectra for catechin and 3’and 4’-O-methylcatechin in rat urine

Conclusions z

z

z

z

The 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.