PurduePurdue--UAB Botanicals Center for AgeUAB Botanicals Center for Age--Related DiseaseRelated Disease
MS/MS analysis of MS/MS analysis of PolyphenolsPolyphenolsJeevan Prasain Ph.D.Pharmacology & ToxicologyUAB
OH
O
OHHO
OHO
OHOH
OHOH
OH
OHO
OHOH
OH
OH
O
OHO
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 KDSColumn: 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
Rel
ativ
e In
tens
ity (%
)
m/z 253DZN
m/z 283
m/z 431G’N
m/z 415DZ’N
m/z 415puerarin
m/z 445
m/z 445
m/z 341
m/z 267Formononetin
m/z 547
m/z 431
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 withAn inert gas like argon or helium. This dual analysis can be dual inSpace, 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.
m/z
100
50
0
Rel
. Int
. (%
)
150m/z
57
29
0
Rel
. Int
. (%
)
65 107
117
151
225
269
6391
107
133
159 180196
224
240
269
Product ion spectra apigenin vs. genistein
O
OH
HO
OOH
Genistein
OOH
HO
OOH
Apigenin
O
O-
O-
50 100 200 250
220 280 300 320 360 380 m/z0
%
0
100
%
255.050
256.057
297.043
267.037
268.041281.051
307.065321.046
363.046335.061351.044 381.055
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/MSMS/MS
-162 Da
Yo+
-120 Da
O
OH
HO
O
OHO
HO
CH2OHOH
O
OH
O
O
O
OHOH
CH2OH
OH
daidzin
[A]
Puerarin
Product ion spectrum of Product ion spectrum of genistein genistein glucuronide glucuronide in ESIin ESI--MS/MSMS/MS
100
50
0
Rel
ativ
e In
tens
ity (%
)
5985
113133 181 224
269
Glucuronide loss
O
OHHO
OH
COOH
445Characteristicof Genistein
100 200 300 400m/z
ESIESI--MS/MS Spectra of MS/MS Spectra of Biochanin Biochanin A and A and PrunetinPrunetin
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
223211
239
211
O
OOH
H3CO
OH
Prunetin
O
OOCH3
HO
OH
Biochanin A-15 (CH3)
268
283
283
Loss of methyl radical is characteristic for methylated flavonoids
Structural identification of unknown Structural identification of unknown based on product ions based on product ions
150m/z
100
0
Rel
ativ
e In
tens
ity (%
)
91 120
135148
184
211
223
239
268
50
O
O O-OCH3
HO
m/z 283
O
O O-O.
HO
OH
m/z 268
O.O
-O
m/z 148
O
O-O
m/z 135
Diagnostic ions
50 100 200 250
Full scan ESIFull 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 componentscomponents. . 100
0
Rel
. Int
. (%
)
100
0
Rel
. Int
. (%
)
200 300 400 500 600 700 800
m/z
100
.0
Rel
. Int
. (%
)
255 360
417
447475
549 579
257
417
447
289 365
417
447 4751 579
433
ESI-MS full scan
Neutral loss scan 120
Neutral loss scan 162
100
75
50
25
0
Rel
. Int
. (%
)
130,325
87
65
43
22
0
Rel
. Int
. (%
)
112,927
63 91 107
167
195 211 239267
303
139
167
195
223251
287
[a]
[b]
-36
-36-28
-28
-28-28
-28-28
-28 -16
Comparison of the product ions obtained in ESIComparison 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
OOH
HO
OH
Cl3'
1
345
7
1'
2
4'
O
OOH
HO
Cl3'
1
345
7
1'
2
4'
50 100 150 200 250 300m/z
Product ion spectra of a reaction Product ion spectra of a reaction product at product at m/zm/z 303 in LC303 in LC--MS/MSMS/MS
m/z
39
29
19
10
0
Rel
. Int
. (%
)
1304
50 100 150 200 250 300
m/z
100
75
50
25
0
Rel
. Int
. (%
)
3358
133
155
167194
211
238
303
6391
107139
167
182
195 210239
267
303
-36
HCl loss
No HCl loss
3’-chlorogenistein
6- or 8-chlorogenistein
HCl loss is diagnostic for positioning chlorine in the aromatic ring.
Urinary metabolites detected in a Urinary metabolites detected in a rat fed with grape seed extractrat fed with grape seed extract
O
OHHO
OH
OH
OHcatechin
O
OMeHO
OH
OH
O
OHHO
OH
OMe
OHOH
4'-O-methylcatechin3'-O-methylcatechin
FullFull--scan ESIscan ESI--MS spectrum of the MS spectrum of the methanolic methanolic extract of grape seed extractextract of grape seed extract
289
287576
305 387
865
580720
652864730
796
866867
577
729 11531017300 600 900 12000
100
%
Deprotonated catechin
m/z
Dimer-H
Trimer-H
Tetramer-HGalloylatedDimer-H
GalloylatedTrimer-H
150 200 250 300m/z
100
50
0
Rel
ativ
e In
tens
ity (%
)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 LCin LC--MS/MS analysis (MS/MS analysis (RtRt. 7.10 min) . 7.10 min)
OOH
OH
HO
OH
OMe
3'-O-methylcatechin
100 150 200 250 300m/z
100
50
0
Rel
ativ
e In
tens
ity (%
)5105
- -
139
137
147
179 221 287111
Product ion spectrum of the ion m/z 305 Product ion spectrum of the ion m/z 305 in LCin LC--MS/MS analysis (MS/MS analysis (RtRt. 7.88 min) . 7.88 min)
OOMe
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
OHO
OH
O
OH
-H2O
H+
H+
m/z 221
m/z 193
m/z 111
H+
m/z 139ring A ion
m/z 137ring Bdignostic of the ring B methylation
H+
H+
H+
ProposedProposed structures of product ions structures of product ions obtained from obtained from m/zm/z 305 in ESI305 in ESI--MS/MSMS/MS
m/z 287
H+
m/z 147
LCLC--MS/MSMS/MS--MRM spectra for MRM spectra for catechin catechin and 3’and 3’--and 4’and 4’--OO--methylcatechin methylcatechin in rat urinein rat urine
ConclusionsConclusionsThe 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 (O- and C-glycosides) generate different product ions in ESI-MS/MS due to the neutral losses of 162 and 120, 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.