MS/MS analysis of Polyphenols

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

2 Polyphenols H Caffeic acid Phenolic acids and derivatives H EGC (Flavanol) Flavanols H Flavonoids H H Flavonols Isoflavones Stilbenes Genistein (Isoflavone) Lignans Resveratrol (Stilbene) H CH 2 CH 2 Enterodiol (Lignan) Quercetin (Flavonol)

3 LC-MS Profile of the methanolic extract of KDS Column: C 8 Aquapore; 7µm, 1 x 4.6 mm i.d. Solvent: CH 3 CN:H 2 (1-4%, run time 3 min) Relative Intensity (%) m/z 341 m/z 547 m/z 415 puerarin m/z 445 m/z 445 m/z 431 m/z 415 DZ N m/z 431 G N m/z 253 DZN m/z 283 m/z 267 Formononetin Time (min)

4 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.

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 mass spectrometry is the triple quadrupole (QqQ).

6 MS/MS data interpretation 1. Identification of molecular ions or quasi-molecular ions. 2. rigin of product ions. 3. Stability and relative intensity of ions.

7 Product ion spectra apigenin vs. genistein H 151 Apigenin H m/z Genistein

8 Product ion spectra of daidzin [A] and puerarin [B] in ESI-MS/MS CH 2 [A] Yo Da daidzin % H H H CH 2 [B] 1 % Da Puerarin m/z

9 Product ion spectrum of genistein glucuronide in ESI-MS/MS Relative Intensity (%) 1 5 Characteristic of Genistein 269 C H 445 Glucuronide loss m/z

10 ESI-MS/MS Spectra of Biochanin A and Prunetin H H 3 C Biochanin A 132 CH , (CH 3 ) 283 2, Prunetin m/z Loss of methyl radical is characteristic for methylated flavonoids

11 Structural identification of unknown based on product ions H Relative Intensity (%) 1 5 Diagnostic ions - m/z m/z m/z m/z H - m/z 283 CH 3

12 m/z Full scan ESI-MS of the methanolic extract of kudzu dietary supplement. Constant neutral loss scans to identify the isoflavone components ESI-MS full scan Neutral loss scan 12 1 Neutral loss scan

13 Comparison of the product ions obtained in ESI-MS/MS of 3 -chlorogenistein [a] and 3 -chlorodaidzein [b] [a] [b] H H ' 5 1' 3' Cl 4' ' 3' Cl , , m/z

14 Product ion spectra of a reaction product at m/z 33 in LC-MS/MS or 8-chlorogenistein No HCl loss 1 3 -chlorogenistein HCl loss HCl loss is diagnostic for positioning chlorine in the aromatic ring. m/z

15 Urinary metabolites detected in a rat fed with grape seed extract H catechin H Me H Me 3'--methylcatechin 4'--methylcatechin

16 Full-scan ESI-MS spectrum of the methanolic extract of grape seed extract 1 Deprotonated catechin 289 % Dimer-H 577 Trimer-H Galloylated865 Galloylated Dimer-H Trimer-H 729 Tetramer-H m/z

17 Product ion spectrum of the ion m/z 35 in LC-MS/MS analysis (Rt( Rt min) Me 13,847 Relative Intensity (%) H 3'--methylcatechin m/z

18 Product ion spectrum of the ion m/z 35 in LC-MS/MS analysis (Rt( Rt min) Relative Intensity (%) H Me 4'--methylcatechin m/z

19 Proposed structures of product ions obtained from m/z 35 in ESI-MS/MS H+ H+ H H m/z 35 -H2 m/z 287 CH 3 CH 3 H+ H+ H m/z 221 m/z 139 ring A ion CH 3 -C m/z 193 CH 3 H+ m/z 137 ring B dignostic of the ring B methylation H+ H+ H m/z 147 m/z 111

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

21 Conclusions 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 (- and C-glycosides) generate different product ions in ESI-MS/MS due to the neutral losses of 162 and 12, 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.