Purdue-UAB Botanicals Center for Age-Related Disease MS/MS analysis of Polyphenols Jeevan Prasain Ph.D. Pharmacology & Toxicology UAB
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)
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 (%) 1 75 5 25 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 4 8 12 16 Time (min)
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. rigin of product ions. 3. Stability and relative intensity of ions.
Product ion spectra apigenin vs. genistein 1 5 65-17 117 H 151 Apigenin 269 57 H 225 269 29-133 63 91 17 159 18 224 196 24 5 1 15 2 25 m/z Genistein
Product ion spectra of daidzin [A] and puerarin [B] in ESI-MS/MS CH 2 [A] Yo + 1 255.5-162 Da daidzin % 256.57 H H H CH 2 [B] 1 % 267.37 297.43-12 Da 268.41 281.51 37.65321.46 335.61 351.44 381.55 363.46 Puerarin 22 24 26 28 3 32 34 36 38 m/z
Product ion spectrum of genistein glucuronide in ESI-MS/MS Relative Intensity (%) 1 5 Characteristic of Genistein 269 C H 445 Glucuronide loss 59 85 113 133 181 224 1 2 3 4 m/z
ESI-MS/MS Spectra of Biochanin A and Prunetin H 1 5 14.9 H 3 C Biochanin A 132 CH 3 239 211 223 268 268 138,385-15 (CH 3 ) 283 2,592 7.4 Prunetin 211 239 132 283. 5 1 15 2 25 m/z Loss of methyl radical is characteristic for methylated flavonoids
Structural identification of unknown based on product ions H Relative Intensity (%) 1 5 Diagnostic ions - m/z 148. - m/z 135-211 m/z 268. 268 239 184 135 223 148 91 12 5 1 15 2 25 m/z H - m/z 283 CH 3
m/z Full scan ESI-MS of the methanolic extract of kudzu dietary supplement. Constant neutral loss scans to identify the isoflavone components. 1 255 36 417 447 549 433 475 579 ESI-MS full scan 1 257 417 447 Neutral loss scan 12 1 Neutral loss scan 162 417 289 365 447 475 1 579. 2 3 4 5 6 7 8
Comparison of the product ions obtained in ESI-MS/MS of 3 -chlorogenistein [a] and 3 -chlorodaidzein [b] [a] 1 75 5 25 [b] 87 65 43 22 H H 7 7 1 2 5 4 3 63 91 17 1 2 4 3 1' 5 1' 3' Cl 4' 167-28 -16 195 211 239-28 -28 4' 3' 195-28 Cl 167-28 223-28 139-28 13,325 33-36 267 112,927 287 251-36 5 1 15 2 25 3 m/z
Product ion spectra of a reaction product at m/z 33 in LC-MS/MS 39 211 134 33 29 19 1 6- or 8-chlorogenistein 133 167 155 194 238 No HCl loss 1 3 -chlorogenistein 167 3358 33 75 5 25 63 91 17 139 195 21 182 239 HCl loss -36 267 5 1 15 2 25 3 HCl loss is diagnostic for positioning chlorine in the aromatic ring. m/z
Urinary metabolites detected in a rat fed with grape seed extract H catechin H Me H Me 3'--methylcatechin 4'--methylcatechin
Full-scan ESI-MS spectrum of the methanolic extract of grape seed extract 1 Deprotonated catechin 289 % Dimer-H 577 Trimer-H 58 576 287 35 652 387 Galloylated865 Galloylated Dimer-H Trimer-H 729 Tetramer-H 866 72 73 864867 117 1153 796 3 6 9 12 m/z
Product ion spectrum of the ion m/z 35 in LC-MS/MS analysis (Rt( Rt.. 7.1 min) - - 1 139 Me 13,847 Relative Intensity (%) 5 137 147 H 3'--methylcatechin 179 221 287 15 2 25 3 m/z
Product ion spectrum of the ion m/z 35 in LC-MS/MS analysis (Rt( Rt.. 7.88 min) - - 1 139 515 Relative Intensity (%) 5 137 147 H Me 4'--methylcatechin 111 179 221 287 1 15 2 25 3 m/z
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
LC-MS/MS MS/MS-MRM MRM spectra for catechin and 3 - and 4 --methylcatechin in rat urine
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.