Purdue-UAB Botanicals Center for Age-Related Disease

MS/MSMS/MS analysisanalysis ofof PolyphenolsPolyphenols

Jeevan Prasain Ph.D. Pharmacology & Toxicology UAB Polyphenols

Phenolic acids Flavonoids Stilbenes Lignans and derivatives OH HO CH2O OH

HO CH2O HO Flavanols Flavonols Isoflavones OH Caffeic acid OH OH O OH HO Resveratrol Enterodiol OH O (Stilbene) (Lignan) HO O OH OH O OH OH OH Genistein (Isoflavone) OH EGC (Flavanol) HO O OH

OH OH O Quercetin (Flavonol) 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

m/z 415 m/z 253 DZN DZ’N 75 m/z 547 ) %

( m/z 283 y m/z 445 50 m/z 267 Formononetin

m/z 445 25 m/z 431 m/z 431 G’N Relative Intensit

0 0 4 8 12 16 Time (min) WhatWhat isis tandemtandem massmass 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 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

100 117 HO O OH O-

OH O 50 Apigenin

Rel. Int. (%) 107 65 151 269 225 0 O HO 57 O 269

O- 133 OH OH O 29 Genistein

Rel. Int. (%) 159 224 63 107 180 91 196 240 0 50 100 150 200 250 m/z ProductProduct ionion spectraspectra ofof daidzindaidzin [A][A] andand

puerarinpuerarin [B][B] inin ESIESI--MS/MSMS/MSCH2OH O OH OH O O OH [A] Yo+

100 255.050 O OH daidzin -162 Da

%

256.057

[B] 0 100 297.043 HO -120 Da CH OH OH 2 267.037 HO O % HO O 321.046 268.041 307.065 351.044 281.051 335.061 381.055 363.046

OH 0 O 220 240 260 280 300 320 340 360 380 m/z Puerarin ProductProduct ionion spectrumspectrum ofof genisteingenistein glucuronideglucuronide inin ESIESI--MS/MSMS/MS

269 COOH ) 100 %

( O y HO OH

OH 50 445 Characteristic of Genistein Glucuronide loss Relative Intensit 85 59 113 133 181 224 0 100 200 300 400 m/z ESIESI--MS/MSMS/MS SpectraSpectra ofof BiochaninBiochanin AA andand

PrunetinPrunetin HO O

138,385 100 268 OH O ) OCH3 Biochanin A -15 (CH ) 239 3 50 211 Rel. Int. (% 223 132 283 0 20,592 H CO O 14.9 3 268 )

OH O OH 239 7.4 Prunetin Rel. Int. (% 211

132 283 0.0 50 100 150 200 250 m/z Loss of methyl radical is characteristic for methylated flavonoids StructuralStructural identificationidentification ofof unknownunknown basedbased onon productproduct ionsions

HO O

OH O O- O. 100 211 m/z 268 -O )

% Diagnostic ions 268 ( y

O 239 m/z 148 O. HO O -O O 50 O O O- OCH3 Relative Intensit m/z 135 184 m/z 283 135 223 148 91 120 0 50 100 150 200 250 m/z FullFull scanscan ESIESI--MSMS ofof thethe methanolicmethanolic extractextract ofof kudzukudzu dietarydietary supplement.supplement. ConstantConstant neutralneutral lossloss scansscans toto identifyidentify thethe isoflavoneisoflavone componentscomponents.. 100 ) 417 ESI-MS full scan

Rel. Int. (% 255 360 447 549 433 475 579 0

100 ) Neutral loss scan 120 417 Rel. Int. (% 447 257 0

100

) Neutral loss scan 162 417 Rel. Int. (% 447 289 365 475 1 579 .0 200 300 400 500 600 700 800

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

[a] HO O 1 130,325 2 7 100 3 1' 5 4 ) 4' 75 OH O 303 OH 3' t. (% Cl 50

Rel. In 167 -28 -16 -36 25 195 211 239 -28 267 63 91 107 -28 0 HO O 1 112,927 [b] 2 87 7 3 1' 5 4 ) 65 O 4' OH 3' 195

t. (% -28 Cl 287 43 167 223 -28 -28

Rel. In 251 -36 22 139 -28

0 50 100 150 200 250 300 m/z ProductProduct ionion spectraspectra ofof aa reactionreaction productproduct atat m/zm/z 303303 inin LCLC--MS/MSMS/MS

1304 303 39 211 ) 29 6- or 8-chlorogenistein t. (%

19 133 No HCl loss Rel. In 167 10 194 155 238

0 3358 303 100 167 3’-chlorogenistein ) 75 HCl loss t. (%

50 195 210 239 -36 Rel. In 182 25 63 107 91 139 267

0 50 100 150 200 250 300 m/z HCl loss is diagnostic for positioning chlorine in the aromatic ring. UrinaryUrinary metabolitesmetabolites detecteddetected inin aa ratrat fedfed withwith grapegrape seedseed extractextract

OH OH HO O

OH OH catechin

OH OMe OMe OH HO O HO O OH OH OH OH

3'-O-methylcatechin 4'-O-methylcatechin FullFull--scanscan ESIESI--MSMS spectrumspectrum ofof thethe methanolicmethanolic extractextract ofof grapegrape seedseed extractextract

100

Deprotonated catechin 289

% Dimer-H 577 Trimer-H

Galloylated865 Galloylated Dimer-H Trimer-H Tetramer-H 866 580 729 867 576 720 730 864 1153 287 305 652 796 1017 0 387 300 600 900 1200 m/z ProductProduct ionion spectrumspectrum ofof thethe ionion m/zm/z 305305 inin LCLC--MS/MSMS/MS analysisanalysis ((RtRt.. 7.107.10 min)min)

--

13,847 139 100 OMe

) OH % HO

( O y

OH OH 137 50 3'-O-methylcatechin

Relative Intensit 147

179 221 287 0 150 200 250 300 m/z ProductProduct ionion spectrumspectrum ofof thethe ionion m/zm/z 305305 inin LCLC--MS/MSMS/MS analysisanalysis ((RtRt.. 7.887.88 min)min) --

5105 139 100 OH )

% OMe

( HO

y O

OH OH 137 50 4'-O-methylcatechin

Relative Intensit 147

111 179 221 287 0 100 150 200 250 300 m/z ProposedProposed structuresstructures ofof productproduct ionsions obtainedobtained fromfrom m/zm/z 305305 inin ESIESI--MS/MSMS/MS H+ OH H+ OH HO O OCH O 3 -CO OCH3 m/z 193 OH OH OH m/z 305 m/z 221 H+ H+ -H2O HO O O H+ OH OCH3 HO O OCH3 OH m/z 137 ring B m/z 139 dignostic of the ring OH m/z 287 ring A ion B methylation

H+ H+ HO O

OH

m/z 147 OH m/z 111 LCLC--MS/MSMS/MS--MRMMRM spectraspectra forfor catechincatechin andand 3’3’-- andand 4’4’--OO--methylcatechinmethylcatechin inin ratrat urineurine ConclusionsConclusions 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.