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
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)
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
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.
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
MS/MS data interpretation
1. Identification of molecular ions or quasi-molecular ions. 2. Origin of product ions.
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
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
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
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 283Structural 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
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 433ESI-MS full scan
Neutral loss scan 120
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
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-chlorogenisteinUrinary 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-methylcatechinFull
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
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
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
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+
LC
LC
-
-
MS/MS
MS/MS
-
-
MRM spectra for
MRM spectra for
catechin
catechin
and 3’
and 3’
-
-and 4’
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.