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MS/MS analysis of Polyphenols

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Purdue

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

MS/MS analysis of

MS/MS analysis of

Polyphenols

Polyphenols

Jeevan Prasain Ph.D.

Pharmacology & Toxicology

UAB

(2)

OH O OH HO O HO OH OH OH OH OH O HO OH OH OH OH O O HO 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)

(3)

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)

0 4 8 12 16 Time (min) 100 75 50 25 0 Relative Intensit y ( % ) m/z253 DZN m/z283 m/z431 G’N m/z415 DZ’N m/z415 puerarin m/z445 m/z445 m/z341 m/z267 Formononetin m/z547 m/z431

(4)

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.

(5)

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

(6)

MS/MS data interpretation

1. Identification of molecular ions or quasi-molecular ions. 2. Origin of product ions.

(7)

m/z 100 50 0 Rel. Int. (%) 150 m/z 57 29 0 Rel. Int. (%) 65 107 117 151 225 269 63 91107 133 159 180 196 224 240 269

Product ion spectra apigenin vs. genistein

O OH HO O OH Genistein O OH HO O OH Apigenin O O-50 100 200 250

(8)

220 280 300 320 360 380 m/z 0 % 0 100 % 255.050 256.057 297.043 267.037 268.041 281.051307.065 321.046 363.046 335.061351.044 381.055 100 240 260 340 [B]

Product ion spectra of

Product ion spectra of

daidzin

daidzin

[A] and

[A] and

puerarin

puerarin

[B] in ESI

[B] in ESI

-

-

MS/MS

MS/MS

-162 Da Yo+ -120 Da O OH HO O O HO HO CH2OH OH O OH O O O OH OH CH2OH OH daidzin [A] Puerarin

(9)

Product ion spectrum of

Product ion spectrum of

genistein

genistein

glucuronide

glucuronide

in ESI

in ESI

-

-

MS/MS

MS/MS

100 50 0 Relative Intensit y ( % ) 59 85 113 133 181 224 269 Glucuronide loss O OH HO OH COOH 445 Characteristic of Genistein 100 200 300 400 m/z

(10)

ESI

ESI

-

-

MS/MS Spectra of

MS/MS Spectra of

Biochanin

Biochanin

A and

A and

Prunetin

Prunetin

100 50 0 Rel. Int. (% ) 138,385 50 100 150 200 250 m/z 14.9 7.4 0.0 Rel. Int. (% ) 20,592 132 132 268 239 223 211 239 211 O O OH H3CO OH Prunetin O O OCH3 HO OH Biochanin A -15 (CH3) 268 283 283
(11)

Structural identification of unknown

Structural identification of unknown

based on product ions

based on product ions

150 m/z 100 0 Relative Intensit y ( % ) 91 120 135 148 184 211 223 239 268 50 O O O-OCH3 HO m/z283 O O O-O. HO OH m/z268 O. O -O m/z 148 O O -O m/z135 Diagnostic ions 50 100 200 250

(12)

Full scan ESI

Full 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

components

components

.

.

100 0 Rel. Int. (% ) 100 0 Rel. Int. (% ) 200 300 400 500 600 700 800 m/z 100 .0 Rel. Int. (% ) 255 360 417 447 475 549579 257 417 447 289 365 417 447 475 1 579 433

ESI-MS full scan

Neutral loss scan 120

(13)

100 75 50 25 0 Rel. In t. (% ) 130,325 87 65 43 22 0 Rel. In t. (% ) 112,927 63 91 107 167 195 211 239 267 303 139 167 195 223 251 287 [a] [b] -36 -36 -28 -28 -28 -28 -28 -28 -28 -16

Comparison of the product ions obtained in ESI

Comparison 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 O OH HO OH Cl 3' 1 3 4 5 7 1' 2 4' O O OH HO Cl 3' 1 3 4 5 7 1' 2 4' 50 100 150 m/z 200 250 300

(14)

Product ion spectra of a reaction

Product ion spectra of a reaction

product at

product at

m/z

m/z

303 in LC

303 in LC

-

-

MS/MS

MS/MS

m/z 39 29 19 10 0 Rel. In t. (% ) 1304 50 100 150 200 250 300 m/z 100 75 50 25 0 Rel. In t. (% ) 3358 133 155 167 194 211 238 303 63 91 107 139 167 182 195 210 239 267 303 -36 HCl loss No HCl loss 3’-chlorogenistein 6- or 8-chlorogenistein
(15)

Urinary metabolites detected in a

Urinary metabolites detected in a

rat fed with grape seed extract

rat fed with grape seed extract

O OH HO OH OH OH catechin O OMe HO OH OH O OH HO OH OMe OH OH 4'-O-methylcatechin 3'-O-methylcatechin
(16)

Full

Full

-

-

scan ESI

scan ESI

-

-

MS spectrum of the

MS spectrum of the

methanolic

methanolic

extract of grape seed extract

extract of grape seed extract

289 287305 387 576 865 580 720 652 730 796 864 866 867 577 729 1153 1017 300 600 900 1200 0 100 % Deprotonated catechin m/z Dimer-H Trimer-H Tetramer-H Galloylated Dimer-H Galloylated Trimer-H

(17)

150 200 250 300 m/z 100 50 0 Relative Intensit y ( % ) 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 LC

in LC

-

-

MS/MS analysis (

MS/MS analysis (

Rt

Rt

. 7.10 min)

. 7.10 min)

O OH OH HO OH OMe 3'-O-methylcatechin

(18)

100 150 200 250 300 m/z 100 50 0 Relative Intensit y ( % ) 5105 - -139 137 147 179 221 287 111

Product ion spectrum of the ion m/z 305

Product ion spectrum of the ion m/z 305

in LC

in LC

-

-

MS/MS analysis (

MS/MS analysis (

Rt

Rt

. 7.88 min)

. 7.88 min)

O OMe OH HO OH OH 4'-O-methylcatechin

(19)

O OH OCH3 HO OH O OH OCH3 HO m/z 305 O OH OCH3 OH -CO O OCH3 OH HO OH OH O HO OH O OH -H2O H+ H+ m/z 221 m/z 193 m/z 111 H+ m/z 139 ring A ion m/z 137 ring B

dignostic of the ring B methylation

H+

H+ H+

Proposed

Proposed

structures of product ions

structures of product ions

obtained from

obtained from

m/z

m/z

305 in ESI

305 in ESI

-

-

MS/MS

MS/MS

m/z 287

H+

(20)

LC

LC

-

-

MS/MS

MS/MS

-

-

MRM spectra for

MRM spectra for

catechin

catechin

and 3’

and 3’

-

-and 4’

(21)

Conclusions

Conclusions

z The CID of isoflavones in ESI-MS generates a series of cluster ions due to the subsequent losses of carbonyl/aldehyde or oxygen.

z 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.

z Isoflavones glycosides (O- and C-glycosides) generate different product ions in ESI-MS/MS due to the neutral losses of 162 and 120,

respectively.

z 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.

References

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