Collision-Induced Dissociation of Metal Complexes to Identify Isomeric

Collision-Induced Dissociation of Metal Complexes to Identify Isomeric

Collision-Induced Dissociation of Metal Complexes to
Identify Isomeric Flavonoid Glucuronide Metabolites

OVERVIEW
Purpose
To find simple LC-MS/MS methods for differentiating and identifying
isomeric flavonoid monoglucuronide metabolites

Barry D. Davis, Paul W. Needs, Paul A. Kroon, Jennifer S. Brodbelt
The University of Texas at Austin, Department of Chemistry and Biochemistry, Austin TX 78712
The Institute of Food Research, Norwich, United Kingdom NR4 7UA

Methods

Figure 1. Structures and molecular weights of compounds used in this study

Metal complexes of the flavonoid glucuronides were created by mixing the
components, and were analyzed using electrospray ionization quadrupole
ion trap mass spectrometry with collision-induced dissociation (CID)

50

30

Metal complexes for direct infusion experiments were formed by mixing a
flavonoid glucuronide, CoBr2 and an auxiliary ligand (either 4,7-dmphen or
4,7-dpphen) in methanol.

O

O

O

HOOC
HO
HO

O

O

Relative Abundance

OH

HO

O

OH
OH

O

quercetin-7-O-glucuronide
C21H18O13, MW=478
flavonol

O

OH

naringenin-7-O-glucuronide
C21H20O11, MW=448
flavanone

O

HO
HO

O

O

0
100

OH

O

baicalein-7-O-glucuronide
(baicalin), C21H18O11, MW=446
flavone

O
HO

OH

OH

After an intensive search, several types of metal complexes were found that either differentiate
isomeric flavonoid glucuronides or provide consistent indicators of the glucuronidation position.
One type of complex, [Co(II) (M-H) (4,7-dmphen)] +, provides a clear four-way differentiation of
the quercetin glucuronides (Figure 3a). Another type of complex, [Co(II) (M-H) (4,7-dpphen) 2]+,
provides differentiation of isomeric compounds (Figure 3b) while also proving highly amenable
to LC-MS/MS analysis with post-column complex formation. Energy-resolved CID experiments
were performed to show that the distribution of product ions for both types of complexes are
relatively stable over wide ranges of collision energies, which is a significant advantage for
robust structural analysis (Figure 4).
Figure 2. MS/MS spectra of deprotonated flavonoid glucuronides (M-H) kaempferol-3-O-glucuronide

301
-GlcA

175
271
-Agl -GlcA

301
-GlcA

*

quercetin-4-O-glucuronide
175
-Agl

477

Rat plasma was spiked with the four quercetin glucuronide isomers to
achieve 1 M following the consumption M concentrations. Ascorbic acid and acetic acid were added
to stabilize the analytes.7 Proteins were precipitated and the supernatant
was evaporated and reconstituted in water for analysis.

*

343 373
X- 0,3X-

477

*

0,2

150

200

250

300

350
m/z

400

450

445

*

quercetin-7-O-glucuronide

301
-GlcA

baicalein-7-O-glucuronide
(baicalin)

269
-GlcA

500

150

200

250

300

350
m/z

400

3'quglca-dmphe nco-cad744400#1-10 RT: 0.00-0 .37 AV:
10 NL: 1.36E6 T: + p Full
m s 2 744 [email protected] [
200.00-8 00.00]

*

40

8X

10
qu4'g lca-dm phenco-cad744-400
POS, 10
0 msec
100
90

3/5/20 05 7:08:47 PM

568

x8

100
80
90
70

0
100

quercetin-4'-glu curonide / dm phen / Co

3'quglca-dmphe nco-cad744400#1-10 RT: 0.00-0 .37 AV:
10 NL: 1.36E6 T: + p Full
mqu4'glca-d
s 2 744 [email protected]
[
mphenco-cad744200.00-8
400#1-1000.00]
RT: 0.01-0.32 AV:
10 NL: 3.29E5 T: + p Full
m s2 [email protected] [
200.00-800.00]

x8

80
60

692
-(Aux +
GlcA)

536

50
30
40
20
30
10
200
10400

450

500

550

744

600
m/z

650

90
80

700

750

60
50
40
30

8X

20
10
c:\barry\...\qu7glca-dm phenco-cad744-4 00
0 msec
POS, 10
100
90

3/5/20 05 4:01:48 PM

692
-(Aux +
GlcA)

qu4'glca-d mphenco-cad744400#1-10 RT: 0.01-0.32 AV:
10 NL: 3.29E5 T: + p Full
m
s2 [email protected] [
qu7glca-dmphenco-cad744200.00-800.00]
400#1-10 RT: 0.04-0 .41

x8

70
90

AV: 10 NL: 2.7 9E6 T: + p Full
m s2 [email protected] [
200.00-800 .00]

50
70

30
50
20
40
10
30
200
400
10

450

500

550

600
m /z

650

744

568
-GlcA

700

750

800

60
50
40
30

8X

20
10
0
100

0
100

568

qu7glca-dmphenco-cad744400#1-10 RT: 0.04-0 .41
AV: 10 NL: 2.7 9E6 T: + p Full
m s2 [email protected] [
200.00-800 .00]

90

442

80
70
60
50
40
30

442
-Agl

20
10
0
400

450

500

550

600
m /z

650

700

750

800

*

0
400

744

500

600
m/z

700

868
-Aux

1024
-GlcA

qu7glca-dmphenco-cad744400#1-10 RT: 0.04-0 .41
AV: 10 NL: 2.7 9E6 T: + p Full
m s2 [email protected] [
200.00-800 .00]

*

70

1200

400

868
-Aux
898
-Agl

80

m/z 536

60
40
20
0
0.50

450

0.75

1.00

1.25

1.50

1.75

CID Activation Voltage (V)

600

800
m/z

1000

80

m/z 568

60
40
20

m/z 536

0
0.50

0.75

1.00

1.25

1.50

1.75

70

40

40
20
1.00

1.25

1.50

500

The precursor ion is denoted by an asterisk (*).
Fragments are abbreviated as: -GlcA (loss of the
glucuronide moiety); -Agl (loss of the aglycon portion); 0,2X(0,2 cross-ring cleavage of the glucuronide moiety); 0,3X(0,3 cross-ring cleavage of the glucuronide moiety). The
CID activation voltage is 0.48-0.56 V.

100
80

1.75

20
10
0.8 1.0 1.2 1.4 1.6

40

m/z 442
0.75

1.00

1.25

1.50

CID Activation Voltage (V)

1.8 2.0 2.2 2.4

80
70
60
50
40
30
20
10
0

m/z 692
m/z 868

Figure 6. LC-MS/MS results from the
[Co(II) (M-H) (4,7-dpphen)2]+ complexes
of naringenin glucuronides in human urine

0.8 1.0 1.2 1.4 1.6

1.75

60
50

566
-(Aux +
Agl)

1.8 2.0 2.2 2.4

662
838
-(Aux
-Aux
+ GlcA)
898
-Agl

994
-GlcA
1170

*

B, LC-MS/MS (RT=32.1 min)

m/z 868

662
-(Aux
+ GlcA)

40
30

m/z 692

20
10

838
-Aux
1170

*

m/z 1024
naringenin-7-glucuronide standard,
994
MS/MS
838

m/z 898

0
0.8 1.0 1.2 1.4 1.6

1.8 2.0 2.2 2.4

566
-(Aux +
Agl)

CID Activation Voltage (V)

The precursor ion and minor product ions are omitted. Loss of an auxiliary ligand (4,7-dmphen or
4,7-dpphen) is shown in red, loss of the glucuronide moiety is shown in blue, loss of the aglycon
portion is shown in green, and loss of the glucuronide moiety and an auxiliary ligand is shown in
yellow. The brown dashed line indicates the activation voltage used in Figure 3.

Figure 7. LC-MS/MS results from the
[Co(II) (M-H) (4,7-dpphen)2]+ complexes
of quercetin glucuronides in rat plasma

A, LC-MS/MS (RT=28.1 min)

m/z 1024

400

600

662
-(Aux -Aux
898
+ GlcA)
-Agl

800
m/z

-GlcA
1170

*
1000

1200

Complexes of flavonoid glucuronides with Co(II) ion and
either 4,7-dmphen or 4,7-dpphen provide sufficiently
distinctive CID product ions to differentiate isomeric
flavonoid metabolites.

The similarity of the CID product ions from complexes of
quercetin, kaempferol, naringenin and baicalein
glucuronides are similar enough to suggest that these
methods may be extended to a wider range of flavonoid
glucuronides.

Strong evidence for the presence of naringenin-4-Oglucuronide was found in human urine, marking the first
time this metabolite has been identified in humans.

The post-column complexation method proved sensitive
enough to identify quercetin glucuronides extracted from
just 500 M following the consumption L of rat plasma that had been spiked with
biologically relevant amounts of these metabolites. This
suggests that use of this method for in vivo plasma
analysis is possible.

Quadrupole ion trap mass
spectrometer

Evidence of naringenin-4-O-glucuronide was found in human urine after drinking grapefruit juice
Human urine samples were obtained from a previous study involving consumption of grapefruit
juice.8 In that study, several glucuronidated and sulfated flavonoid metabolites were partially
identified by LC-MS/MS. However, the positions of glucuronidation could not be determined
conclusively. One of these urine samples was re-analyzed using LC-MS/MS with post-column
complexation (Figure 5) in order to elucidate the structures of the two naringenin glucuronides in the
sample. An isocratic elution consisting of 30:70 water/methanol with 0.05% formic acid was
employed to separate the analytes. Both naringenin glucuronides formed ample complexes of the
type [Co(II) (M-H) (4,7-dpphen)2]+ of m/z 1170. Identification of the glucuronidation position was
achieved by comparing the MS/MS data (Figure 6) to those collected by direct infusion as described
earlier. The fragmentation behavior of the complex of one naringenin glucuronide (A) matched that
of the naringenin-7-O-glucuronide complex. This assignment is consistent with the results of an
alternate identification method based on retention time matching with the authenticated standard.
Unknown B represents the more typical case where an authenticated standard is not available and
the dissociation behavior of the complex is not known beforehand. This complex yields two
significant product ions corresponding to the loss of 4,7-dpphen with and without the glucuronide
moiety. This mirrors the behavior of the quercetin-3-O-glucuronide and quercetin-4-O-glucuronide
complexes. As naringenin does not possess a hydroxyl group at the 3 position, B was assigned as
naringenin-4-O-glucuronide. This assignment is in agreement with the literature, where it is stated
that naringenin-4-O-glucuronide is retained longer by reversed-phase chromatography than the
analogous 7-O-glucuronide.9 This is the first time naringenin-4-O-glucuronide has been identified in
human urine. The similarity of the fragmentation pathways of quercetin and naringenin glucuronides
suggests that consistent product ions may be created by similar metal complexes involving other
flavonoid glucuronides.

Quercetin-7-O-Glucuronide

60

0
0.50

m/z 1024

0

Ion optics

ESI interface
+4.5 kV

CID Activation Voltage (V)

m/z 568

20

m/z 692

Quercetin-4'-O-Glucuronide

60

0.75

m/z 868

CID Activation Voltage (V)

m/z 568

0
0.50

1.8 2.0 2.2 2.4

30

Quercetin-4'-O-Glucuronide
80

m/z 1024

60
50

CID Activation Voltage (V)

100

m/z 692

0.8 1.0 1.2 1.4 1.6

CONCLUSIONS

1200

Quercetin-3'-O-Glucuronide

100

20 L/min

*

CID Activation Voltage (V)

Quercetin-3'-O-Glucuronide

5 M CoBr2 +
5 M 4,7-dpphen

1200

Quercetin-3-O-Glucuronide
90
80
70
60
50
40
30
20
10
0

mixing
tee

Having established that 1 M following the consumption M concentrations can be
analyzed in this way, the quercetin glucuronides were
spiked into 500 M following the consumption L of rat plasma at the 1 M following the consumption M level, and were
then extracted and analyzed. All four compounds could be
observed and identified in this manner (Figure 7). Based
on the similarities between these MS/MS results and those
obtained by direct infusion, the four compounds were
identified as: C quercetin-7-O-glucuronide, D
quercetin-3-O-glucuronide, E quercetin-4-O-glucuronide,
and F quercetin-3-O-glucuronide. The success of this
experiment is promising in terms of using LC-MS/MS
methods to identify flavonoid glucuronides from blood
samples obtained from in vivo studies.

1024
-GlcA

b) [Co(II) (M-H) (4,7-dpphen)2]+

Quercetin-3-O-Glucuronide
100

Waters C18 column UV detector
2.1 x 50 mm
280 nm
3.5 m particles
(urine)
370 nm
100 L/min
(plasma)

1200

Figure 4. Energy-resolved CID data from the quercetin glucuronides complexes
a) [Co(II) (M-H) (4,7-dmphen)]+

HPLC
H2O/MeOH
0.05%
formic acid
10 L inj

*
692
-(Aux +
GlcA)
566
-(Aux + Agl)

0
800

*

*

0
100

60
80

0
100

1024
-GlcA

qu4'glca-d mphenco-cad744400#1-10 RT: 0.01-0.32 AV:
10 NL: 3.29E5 T: + p Full
m s2 [email protected] [
200.00-800.00]

quercetin-7-glucuronide / dm phen / Co

568

x8

100
80

1200

868
-Aux

800

*

70

1024
-GlcA

868
-Aux

800

Quercetin-7-O-Glucuronide

447

*

750

CID Activation Voltage (V)

naringenin-7-O-glucuronide

477

Human urine collected after grapefruit juice consumption was prepared for
analysis by protein precipitation and solid phase extraction on a C18
cartridge.

700

40
60

*

quercetin-3-O-glucuronide

650

744

30

0
100

568
-GlcA

461

*

600
m /z

The precursor ion is denoted by an asterisk (*). Fragments are abbreviated as: -GlcA (loss of the
glucuronide moiety); -Aux (loss of the auxiliary ligand, either 4,7-dmphen or 4,7-dpphen); -Agl (loss
of the aglycon portion). Insets in (a) show magnifications of the region between m/z 400 and 600.
The CID activation voltage used for (a) is 1.31 V and for (b) is 1.67 V.

N

477

5 50

50

60
40

quercetin-7-Oglucuronide

Several metal complexation modes were found that assist in the identification of flavonoid
glucuronides
Before attempting metal complexation, the ability to differentiate isomeric flavonoid glucuronides
based on MS/MS of the deprotonated analytes was evaluated. The MS/MS spectra of the (MH)- ions, however, show few distinguishing features (Figure 2). The four isomeric quercetin
glucuronides yield highly similar product ions, and it is impossible to determine the location of
the glucuronide moiety based on this evidence alone. A secondary goal of obtaining consistent
indicators of the glucuronidation site is not achieved as different product ion profiles are yielded
by the three flavonoid 7-O-glucuronide standards.

285
-GlcA

500

70
50

0
100

COOH

O

X=Ph 4,7-diphenyl-1,10-phenanthroline
(4,7-dpphen) C24H16N2, MW=332

quercetin-3-O-glucuronide

45 0

60

20

536
-Aux

0
100

RESULTS

301
-GlcA

568
-GlcA

quercetin-3-Oglucuronide

X=Me 4,7-dimethyl-1,10-phenanthroline
(4,7-dmphen) C14H12N2, MW=208

HO

20
0
10400

90

X

N

OH

30
10

90

kaempferol-3-O-glucuronide
C21H18O12, MW=462
flavonol

X
HOOC

O

50
30
40
20

0
100

80

OH

OH

692
-(Aux +
GlcA)

qu3 glca-dm phen co-cad744400 #1-10 R T: 0 .03 -0.40
AV: 10 NL: 2 .08E5 T: + p Full
nco-cad744m3'quglca-dmphe
s2 744 .00 @40.00
[
400#1-10
RT: 0.00-0 .37 AV:
200
.0 0-8 00.00]
10 NL: 1.36E6 T: + p Full
m s 2 744 [email protected] [
200.00-8 00.00]

x8

60
40

quercetin-4-Oglucuronide

OH

100

quercetin-3'-glucuronid e / dm phen / Co

536

80
60
70
50

80

OH

quercetin-3-O-glucuronide
C21H18O13, MW=478
flavonol

O

8X

3/5/2005 3:04:00 PM

x8

90
70

70

OH
O

8X

90
100
80

% of Total Ion Current

O

HO
HO

A Finnigan LCQ Duo quadrupole ion trap mass spectrometer with
electrospray ionization (ESI) was used, and CID tandem mass
spectrometry was employed to find characteristic fragmentation pathways
for each compound.

Urine and plasma samples were analyzed by LC-MS/MS with post-column
complexation. A Waters Alliance 2695 HPLC system was used with a
Waters Symmetry C18 column, 2.1 x 50 mm. Details of the separation are
given in the Results section.

OH

quercetin-3-O-glucuronide
C21H18O13, MW=478
flavonol
OH

HOOC

Sufficient sensitivity for in vivo plasma analysis at realistic concentrations
was achieved

All flavonoid glucuronides in Figure 1 were synthesized4-6 at the Institute of
Food Research except kaempferol-3-O-glucuronide and baicalin, which
were purchased from commercial sources.

O

O
HO

COOH

20
10
3'quglca-dm
phenco-cad744-400
POS, 10
0 msec
100

536
-Aux

quercetin-3-Oglucuronide

OH

Evidence of naringenin-4-O-glucuronide was found in human urine after
drinking grapefruit juice

METHODS

O

HO

40
30

% of Total Ion Current

O

100

60
50

% of Total Ion Current

Several metal complexation modes were found that assist in the
identification of flavonoid glucuronides

OH

Relative Abundance

OH

70

OH

OH

quercetin-4-O-glucuronide
C21H18O13, MW=478
flavonol

INTRODUCTION

O

80

OH

O

Figure 5. Instrumental setup for LC-MS/MS experiments with post-column complexation

qu3 glca-dm phen co-cad744400 #1-10 R T: 0 .03 -0.40
AV: 10 NL: 2 .08E5 T: + p Full
m s2 744 .00 @40.00 [
200 .0 0-8 00.00]

90

OH
OH

20
10

0
100

O

HO

OH

OH
COOH

O

OH

Relative Abundance

HO
O

O

HO

HO
HO
HOOC

HO

OH

Methods for identifying unknown flavonoid glucuronides were found and
applied to the on-line LC-MS/MS analysis of human urine and rat plasma

Recent work has shown that although flavonols, flavanones and flavones are
consumed in the diet as glycoside conjugates, it is mainly the glucuronidated,
sulfated and methylated derivatives that circulate in blood plasma following
flavonoid-rich meals.1 However, most research on flavonoid bioactivity has
involved either flavonoid aglycons or glycosides that are not present for any
appreciable amount of time in the body. It has therefore been suggested that
in vitro studies of flavonoids should focus on the metabolic forms rather than
the glycosides found in foodstuffs or commercially available aglycons. 2
However, the problem remains that far less is known about the in vivo
metabolites than about their precursors, and relatively few analytical methods
have been developed for studying these metabolites. NMR methods have the
potential to definitively identify unknown compounds, but are not amenable to
low-level dietary metabolites due to low sensitivity. Another method based on
UV-Vis spectroscopy has been applied to flavonoid identification, but it is an
inconvenient and time-consuming method involving the use of several UV shift
reagents.3 The current project explores a convenient alternative strategy for
qualitative analysis of flavonoid metabolites by taking advantage of the
specificity, sensitivity and ease of mass spectrometry. Metal complexes of
flavonoid glucuronides provide useful structural information under CID
conditions, allowing isomeric metabolites to be differentiated. The methods
were adapted to LC-MS/MS analysis of human urine and rat plasma.

qu3 glca-dm phen co-cad744400 #1-10 R T: 0 .03 -0.40
AV: 10 NL: 2 .08E5 T: + p Full
m s2 744 .00 @40.00 [
200 .0 0-8 00.00]

Figure 3. MS/MS spectra from metal complexes of the quercetin glucuronides
b) [Co(II) (M-H) (4,7-dpphen)2]+
a) [Co(II) (M-H) (4,7-dmphen)]+
70
60

% of Total Ion Current

x8

80

Relative Abundance

qu ercetin-3-glucuron ide / dm phen / Co

90

40

Results

3/5/2005 1:26:04 PM

x8
100

% of Total Ion Current

c:\barry\...\qu3glca-dm phenco-cad 744-400
POS, 50 ms ec

% of Total Ion Current

Flavonoid glucuronides were synthesized at the Institute of Food
Research

% of Total Ion Current

% of Total Ion Current

Sufficient sensitivity for in vivo plasma analysis at realistic
concentrations was achieved
The concentration of flavonoid glucuronides in human blood
is typically between 0.1 to 2 M following the consumption M following the consumption
of flavonoid-rich foods.1,7 In order to determine whether
metal complexation methods are effective at these low
concentrations, a solution of the four quercetin glucuronide
standards (1 M following the consumption M each) was prepared and analyzed by LCMS/MS with post-column complexation. 10 M following the consumption L of this
solution was injected, and a 20 minute gradient (35% to
75% methanol with 0.05% formic acid) was used to elute
the analytes. Each quercetin glucuronide was identified
based on the fragmentation of [Co(II) (M-H) (4,7-dpphen) 2]+
(m/z 1200).

692
-(Aux +
GlcA)

The precursor ion
is denoted by an
asterisk (*).
Fragments are
abbreviated as:
-GlcA (loss of the
glucuronide
moiety); -Aux (loss
of the auxiliary
ligand, either 4,7dmphen or 4,7dpphen); -Agl (loss
of the aglycon
portion). The CID
activation voltage
used for Figure 6
is 1.59 V and for
Figure 7 is 1.62 V.

692
-(Aux +
GlcA)

692
-(Aux +
GlcA)

692
-(Aux +
GlcA)

400

600

868
-Aux

C, LC-MS/MS
(RT= 10.5 min)
1024
- GlcA
898
-Agl

1200

*

D, LC-MS/MS
(RT= 14.1 min)
868
-Aux

868
-Aux

868
-Aux

1024
- GlcA

m/z

*

E, LC-MS/MS
(RT= 17.4 min)
1024
- GlcA

1200

*

F, LC-MS/MS
(RT= 20.5 min)
1024
- GlcA

800

1200

1000

1200

*
1200

ACKNOWLEDGEMENTS
This work was supported by a National Science
Foundation Graduate Research Fellowship to BDD, and a
Food Standards Agency contract (N05051) to PWN and
PAK. The authors also acknowledge support from the
National Institutes of Health (NIH RO1 GM63512) and the
Welch Foundation (F-1155).

REFERENCES
1. Williamson G, Barron D, Shimoi K, Terao J. Free Radical
Research. 2005, 39, 457.
2. Kroon PA, Clifford MN, Crozier A, Day AJ, Donovan JL,
Manach C, Williamson G. American Journal of Clinical
Nutrition. 2004, 80, 15.
3. Day AJ, Bao YP, Morgan MRA, Williamson G. Free
Radical Biology and Medicine. 2000, 29, 1234.
4. O'Leary KA, Day AJ, Needs PW, Sly WS, O'Brien NM,
Williamson G. FEBS Letters. 2001, 503, 103.
5. Davis BD, Needs PW, Kroon PA, Brodbelt JS. Journal of
Mass Spectrometry. 2006, (in press).
6. Needs PW, Kroon PA. Tetrahedron. 2006, (in press).
7. Day AJ, Mellon F, Barron D, Sarrazin G, Morgan MRA,
Williamson G. Free Radical Research. 2001, 35, 941.
8. Zhang J, Brodbelt JS. Analyst. 2004, 129, 1227.
9. Abe K, Katayama H, Suzuki A, Yumioka E. Shoyakugaku
Zasshi. 1993, 47, 402.

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