Identification of Structural Isomers
of Methylated Flavonoids by UHPLC
Coupled with High Resolution Q-TOF
Mass Spectrometry
Application Note
Authors
Abstract
ChengYing Ma, Haipeng Lv,
This application note details the development of a method for identiying flavonoids
Xinzhong Zhang, Zongmao Chen, Jiang
after methylation based on the work published by Ma, et al [1]. The method uses the
Shi, and Zhi Lin
high performance Agilent 1290 Infinity LC System to achieve baseline separation of
Tea Research Institute, CAAS
all the enzymatically-generated methylated flavonol isomers and further applies the
Hangzhou, China
Agilent 6530 Accurate-Mass Q-TOF LC/MS to determine the methylation status and
Meiling Lu, Shan Zhou
methylation patterns. The characteristic fragmentation pattern analysis along with
Agilent Technologies China Ltd.
the retention behavior helped to identify ten compounds. Four of those compounds
Beijing, China
have not been reported in any plants using existing methods.
Introduction
OH
3’
OH
Flavonoids are a class of heterogeneous polyphenols that are
present in foods ubiquitous in the daily diet such as tea, fruits,
vegetables, and wine. They exhibit numerous biological and
pharmaceutical effects, and contribute significantly to human
health [2,3]. Methylated flavonoids can increase metabolic
stability and oral bioavailability, enhance anti-allergic effects,
and destroy cancer cells more effectively than their unmethylated analogs [4-6]. Such methylation is generally catalyzed by
flavonoid o-methyltransferases (OMTs), which are widely present in plants. These OMTs often have distinguished specificities and notable substrate preferences. The methylated isomers produced by the OMT catalysis often differ in physical
and chemical properties, and modulate the biological activities of the compounds. Therefore, identifying and characterizing the structural-related methylated flavonoid isomers are
important. Structural variety and lack of standard compounds
have been obstacles to conduct such studies. This application
note describes the development of a strategy for identifying
one subclass of methylated flavonoids, flavonols, based on
high resolution MS/MS fragmentation patterns, using
kaempferol and quercetin as examples.
HO
7
B
O
C
A
OH
Figure 1.
3
OH
A) Kaempferol
B
O
C
A
5
O
OH
HO
7
3
5
4’
OH
4’
OH
O
B) Quercetin
Chemical structures of flavonols with free hydroxyls for
methylation by flavonol OMTs. A) kaempferol; B) quercetin.
status of the products. Collision induced dissociation (CID)
MS/MS analysis of individual isomers was further conducted
to differentiate these compounds based on the distinct
fragmentation pathways.
Instrumentation
The method was developed using an Agilent high resolution
LC/MS system. It consisted of a 1290 Infinity Binary Pump
with built-in degasser, a 1290 Infinity Autosampler with
thermostat, a 1290 Infinity Thermostatted Column
Compartment, a 1290 Infinity Diode Array Detector, and an
Agilent 6530 Accurate-Mass Q-TOF Mass Spectrometer with
dual JetStream ESI source.
Experimental
Instrument conditions
Sample preparation
The methylated flavonol isomers were identified and characterized with limited standard compounds. Three flavonol OMT
genes (CsFOMT-1, CsFOMT-2, and CsCOMT) were cloned from
C. sinensis and expressed in a prokaryotic expression vector.
The expressed enzymes were extracted and purified for
further use. Kaempferol and quercetin in the amount of
750 µmol each were initially subjected to methylation by
0.3 mg of different flavonol OMTs in a buffer containing
100 mmol/L tris-HCl (pH 7.5), 0.2 mmol/L MgCl2, 0.4 mmol/L
S-adenosyl-L-methionine (SAM), and 2 mmol/L DTT. After
methylation of the substrates for 1 hour at 35 °C, the resultant
reaction mixtures were terminated by addition of 1 N HCl, and
extracted using 25 mL ethyl acetate. Twenty microliters of
organic phase was then transferred to a clean vial and evaporated under nitrogen atmosphere. The resultant residue was
dissolved in 1.5 mL pure methanol, filtrated through a 0.22-µm
membrane, and subjected to reversed phase LC separation
followed with Q-TOF MS detection. A wide gradient elution
using a binary mobile phase was developed for complete
separation of the formed methylated isomers. Accurate mass
extraction was then applied to examine the methylation
Column
Agilent ZORBAX Eclipse Plus C18,
2.1 × 100 mm, 1.8 µm
Column temperature
30 °C
Mobile phase solvent A 0.5% formic acid/2 mM ammonium formate in
water
Solvent B
acetonitrile
Flow rate
0.2 mL/min
Injection volume
5 µL
Post time
3 minutes
Gradient elution profile 0–5 minutes: % B increasing from 12% to 25%
5–20 minutes: % B increasing from 25% to 75%
20–25 minutes: % B decreasing from 75% to 12%
Drying gas temperature 300 °C
Drying gas flow
8 L/min
Sheath gas temperature 300 °C
2
Sheath gas flow
8 L/min
Nebulizer gas
35 psi
Capillary voltage
3,500 V
Fragmentor voltage
130 V
Reference ions
m/z 121.0509, m/z 922.0098
Results and Discussion
Methoxy position-specific methane elimination
for discriminating regioisomers of methylated
kaempferol analogs
Characteristic fragmentation of methylated kaempferol is
mainly achieved through retro Diels-Alder (rDA) fragmentation
pathway and neutral loss pathway (Figure 2) [2,7,8].
A
0,2 +
A
m/z 165.0182
OH
+
OH
HO
0
HO
A
C
4
O
OH
OH
B
O 1
C 2
3
HO
O +
H
O
0/2
OH
O
+
HO
0,2 +
B
m/z 121.0284
+
C
O
OH
1,3 +
A
m/z 153.0182
-CO
HO
+
OH
OH
0,2
[A-CO]+
m/z 137.0223
B
OH
HO
OH
HO
O
+
OH
+
OH
HO
O
CH3
O
OH
O
O
CH3
HO
OH
OH
OH
HO
O
O
-CH4
OH
+
OH
OH
OH
OH
HO
O
+
H3C
Figure 2.
O
H
O
OH
CH3
OH
-CH4
HO
OH
HO
O
O
H
H
+
O
O
+
O
C
CH3
OH
O
OH
+
O
OH
OH
HO
OH
O
O
+
O
O
O
H
O+
OH
Fragmentation pathway for kaempferol and their methylated analogs. A) rDA fragmentation pathway;
B,C) neutral loss of CH4 pathway with methylation at C3 and C5 positions, respectively.
3
While fragments derived from rDA pathways can provide
hints to the ring on which the methoxy group attaches,
neutral loss of methane can be specific to the proximity of
methylation positions. Methylation at the 4’ and 7 positions
can generate intensive fragments with methyl radical loss
[M+H-CH3•]+ (Figure 3A,B), whereas fragmentation patterns
for the 3,4’-DMK and 3,7-DMK yielded obvious [M+H-CH4]+
fragments (m/z 299.054) in addition to [M+H-CH3•]+
(Figure 3C,D). It is suggested that proper orientation of
C3-OCH3 with a C2’ proton allows the C2’ proton transfer to
the C3-OCH3, leading to the elimination of a methane molecule through the formation of a stable furan ring (Figure 2B).
Besides, the 4-keto group is easily protonated under ESI conditions, and the proton close to 5-methoxy, tends to lose
methane during fragmentation (Figure 2C).
4’-MeK
HO
107.0487
258.0514
O
201.0540
O
[M+H]+
301.0696
Kaempferol
CH3
O
H3C
0,2
O
1,3
MeA+
OH
167.0333
0,2
MeA+
153.0542 179.0332
MeB+
[M+H]+
315.0856
[M+H-CH3•]+
107.0491
257.0438 283.0589
O
C
O
MeB+
108.0566 135.0435
CH3
O
OH
[M+H-CH4]
CH3
229.0488
3,7-DMK
OH
O
H3C
O
150.0306
120
Figure 3.
150
OH
0,2
MeA+
179.0339
121.0282
CsCOMT
[M+H]+
315.0850
243.0643
O
108.0206
MMK3
301.0705
D
272.0670
173.0586 201.0537
173.0591
[M+H-CH3•]+
300.0619
CsFOMT-2
257.0438
0,2
1,3
MeA+
167.0335
+
C
285.0388
O
D
CsFOMT-1
O
E
4’-MeK
[M+H-CH3•]+
300.0622
CH3
[M+H-CH4]+
257.0442
F
229.0492
173.0591 201.0539
180
MMK2
301.0705 DMK1
315.0869
229.0488
3,4’-DMK
HO
244.0723
MMK1
301.0705
B
272.0672
OH
121.0644
A
269.0435
241.0483
7,4’-DMK
O
K
287.0550
230.0564
OH
OH
0,2 +
A
165.0176
B
[M+H-CH3•]+
286.0461
O
1,3 +
A
153.0177
0,2
MeB+
135.0435
CH3
Ultra high performance LC with optimized gradient elution
was initially used to separate as many methylated kaempferol
analogs as possible and identify the methylation status of the
substrate. In the absence of any OMTs, only kaempferol
(m/z 287.0547) was detected (Figure 4A). In comparison,
three monomethyl kaempferols (MMK1, MMK2, MMK3) can
be produced by the OMTs as evidenced by an m/z increase of
14.0158 from kaempferol (Figure 4B-D). In addition, CsFOMT-1
and CsCOMT can produce one dimethyl kaempferol (DMK1)
with an m/z increase of 28.0334 from kaempferol
(Figure 4B,D).
Ion intensity
A
O
Application of the fragmentation pattern for
identification of enzymatically synthesized
methylated kaempferol analogs
210
m/z
3,7-DMK
284.0311 315.0855
240
270
300
330
0
Figure 4.
Characteristic MS/MS patterns for available standard compounds
including rDA and neutral loss fragmentation pathways.
(A) 4’-MeK, (B) 7,4’-DMK, (C) 3,4’-DMK, and (D) 3,7-DMK.
4
5
10
15
Retention time (min)
20
25
Extracted ion chromatograms demonstrating the formation of
mono- and dimethylated kaempferol compounds catalyzed by
cloned OMTs from tea. (A) without OMT; (B) CsFOMT-1,
(C) CsFOMT-2, and (D) CsCOMT; (E) 4’-MeK standard, and
(F) 3,7-DMK standard.
Identification of methylated products
Extension of the approach to characterize
methylated quercetin analogs derived from
OMT catalysis
Presence of methylated rDA ions related to the A-ring
(1,3MeA+) and absence of fragments due to methane loss for
MMK3 indicate that MMK3 correspond to 7-MeK (Figure 5C).
MMK1 and MMK2 with fragments of [M+H-CH4]+ suggest
that both are either 5-MeK or 3-MeK. The presence of visible
methylated rDA ions related to the A-ring suggests that
MMK1 should be 5-MeK, and MMK2 then correspond to
3-MeK (Figure 5-A,B). Presence of 1,3MeA+ and [M+H-CH4]+
and absence of 0,2MeB+ suggest that DMK1 should be
3,7-DMK, which matched well with the standard compound
(Figure 3D). The retention behavior under reversed phase elution of these four compounds is consistent with the tested
result for the available standard compounds (Figure 4E,F), the
hydrophobicity of the compounds by calculation (Table 1) [9],
and those reported in the literatures [10].
Structural analysis of methylated quercetin analogs indicated
that C4’-OCH3 with neighboring C3-OH can generate
[M+H-CH4]+ during collision induced dissociation (CID) in
addition to methylation at C3 and C5 positions. LC separation
demonstrates that four monomethylated and two dimethylated quercetin anologs can be produced with the applied
OMTs (Figure 6). Analysis of the Q-TOF MS/MS fragmentation
patterns (Figure 7) suggests that MMQ3 and MMQ4 correspond to 3’-MeQ and 7-MeQ with the absence of [M+H-CH4]+.
The presence of 1,3MeA+ indicates that MMQ4 corresponds to
7-MeQ, therefore MMQ3 should be 3’-MeQ. The presence of
[M+H-CH4]+ and absence of 1,3MeA+ fragments indicate that
MMQ1 and MMQ2 are 3-MeQ and 4’-MeQ, which can be further differentiated based on corresponding hydrophobicity
(Table 1).
Q
303.0499
A
Quercetin
A
MMK1
[M+H-CH4]+
285.0380
1,3
MeA+
167.0335
MeA+
179.0374
[M+H-CH3•]+
0,2
B+
121.0279
153.0225
[M+H]+
301.0667
258.0497
229.0488
184.0492
CsFOMT-1
Relative intensity
[M+H-CH3•]+
0,2
B+
121.0279
258.0512
153.0180
C
MMK3
+
[M+H]
301.0656
229.0484
184.0513
C
DMQ1
331.0812
MMQ4
317.0656
CsFOMT-2
MMQ3
317.0656
D
258.0511
1,3
MeA+
167.0331
DMQ2
331.0812
CsCOMT
[M+H]+
301.0695
107.0489
B+
MMQ1
317.0656
B
[M+H-CH4]+
285.0385
B
MMK2
0,2
MMQ2
317.0656
0,2
• +
0,2
MeA+
[MeA-CO]+
151.0381 179.0326
[M+H-CH3 ]
286.0459
0,2
212.0457
241.0481 272.0666
E
3’-MeQ
D
DMK1
1,3
MeA+
167.0310
0,2
MeA+
179.0374
121.0281
150.0305
108.0189
90
120
Figure 5.
[M+H-CH3•]+
300.0612
+
150
[M+H-CH4]
257.0434
229.0486
173.0586 201.0536
180
210
240
m/z
F
271.0587
300.0622
270
300
[M+H]+
315.0835
7-MeQ
0
330
Figure 6.
The characteristic MS/MS pattern for the monomethyl and
dimethyl kaempferol compounds derived from OMTs. The identified compounds were 5-MeK for MMK1 (A); 3-MeK for MMK2 (B);
7-MeK for MMK3 (C); and 3,7-DMK for DMK1 (D), respectively.
5
5
10
15
Retention time (min)
20
25
Extracted ion chromatograms demonstrating the formation of
mono- and dimethylated quercetin compounds catalyzed by OMTs
(A) without OMTs; (B) CsFOMT-1, (C) CsFOMT-2, (D) CsCOMT,
(E) 3’-MeQ standard, and (F) 7-MeQ standard.
A
[M+H-CH3•]+
302.0406
MMQ1
[M+H-CH4]+
B+
1,3 +
A
110.0367 137.0234 153.0174
0,2
228.0409
[M+H]+
317.0648
274.0480
200.0447
[M+H-CH3•]+
302.0403
B
MMQ2
[M+H-CH4]+
0,2
110.0359
B+
137.0224
166.0241
C
121.0282
0,2
[M+H-CH3•]+
302.0412
274.0461
[M+H]+
1,3
MeA+
167.0331
+
B
[M+H]+
317.0661
274.0456
137.0221
90
229.0485
203.0333
D
109.0269
274.0457
1,3 +
A
153.0174
MMQ3
MMQ4
[M+H]+
317.0624
228.0412
200.0458
243.0618
0,2
MeA+
179.0311
228.0404
151.0434
120
150
180
210
[M+H-CH3•]+
302.0311
257.0442
240
270
300
330
m/z
Figure 7.
Characteristic MS/MS patterns for the four monomethylated
quercetin isomers catalyzed by three OMTs and their identified
structures. The identified compounds are 3-MeQ for MMQ1 (A),
4'-MeQ for MMQ2 (B); 3'-MeQ for MMQ3 (C); 7-MeQ for MMQ4 (D),
respectively.
Table 1.
Identified Methylated Flavonoids Compounds and their Retention Times
Observed compound
Theoretical candidates
Observed characteristic fragment ions
logP*
0.2B+
1.3A+
0.2A+
[MH-CH4]+
[MH-CH3•]+
5-MeK
2.989
121.0276
153.0223
–
285.0379
286.0454
11.59
3-MeK
3.008
121.0279
153.0182
–
285.0385
286.0453
14.70
7-MeK
3.498
121.0281
167.0331
179.0327
–
286.0464
Std 1
14.70
4’-MeK
3.499
135.0436
153.0175
165.0178
–
286.0462
DMK1
15.29
3,7-DMK
4.011
121.0284
167.0333
–
299.0541
300.0612
Std 2
15.30
3,7-DMK
4.011
121.0282
167.0336
–
299.0547
300.0662
MMQ1
7.25
3-MeQ
1.765
–
153.0170
–
301.0327
302.0407
MMQ2
9.95
4’-MeQ
2.67
–
153.0174
–
301.0332
302.0403
MMQ3
11.40
3’-MeQ
2.787
–
153.0174
–
–
302.0413
MMQ4
12.72
7-MeQ
2.802
137.0222
167.0337
179.0335
–
302.0418
Std 3
11.38
3’-MeQ
2.787
–
153.0177
–
–
302.0413
Std 4
12.82
7-MeQ
2.802
137.0235
167.0333
179.0338
–
302.0406
DMQ1
11.83
3,3’-DMQ
2.681
–
153.0187
165.0184
315.0492
316.0562
DMQ2
15.00
7,3’-DMQ
3.600
–
167.0332
179.0324
–
316.0566
ID
tR/min
Name
MMK1
8.67
MMK2
MMK3
*logP values were obtained from www.chemspider.com (ACD/logP ).
6
Similarly, DMQ1 and DMQ2 were identified as 3,3’-MeQ and
7,3’-MeQ respectively. Table 1 summarized the identified
methylated flavonoid compounds with their retention times,
corresponding hydrophobicity (logP), and the observed
characteristic fragment ions. In total, ten compounds were
identified, with four as new compounds that have not been
previously reported in any plants.
Conclusions
Agilent 1290 Infinity LC System coupled with the high
resolution and accuracy of the Agilent 6530 Accurate-Mass
Q-TOF LC/MS provides sufficient separation and accurate
determination of the methylation status of flavonol derivatives. This study details the development of a practical
approach for identifying and characterizing isomers of
individual methylated flavonol analogs based on accurate and
specific MS/MS patterns. The developed method identifies
methylated flavonols with very limited standards. It can
potentially be applied to the identification of other methylated
flavonoids and can be used for the rapid characterization of
OMTs in plants.
7
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August 21, 2014
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