Structural identification of mouse urinary metabolites of pterostilbene using liquid chromatography/tandem mass spectrometry

Article abstract of DOI:10.1002/rcm.4579

Pterostilbene, the dimethoxy derivative of resveratrol, has drawn much attention recently due to its potential beneficial health effects. The metabolic fate of pterostilbene, however, is not well understood. In the present study, we identified nine novel

Full text of DOI:10.1002/rcm.4579

RAPID COMMUNICATIONS IN MASS SPECTROMETRY
Rapid Commun. Mass Spectrom. 2010; 24: 1770–1778
Published online in Wiley InterScience (www.interscience.wiley.com) DOI: 10.1002/rcm.4579
Structural identification of mouse urinary metabolites of
pterostilbene using liquid chromatography/tandem mass
spectrometry
Xi Shao1,2,3,4, Xiaoxin Chen5, Vladimir Badmaev6, Chi-Tang Ho4 and Shengmin Sang1,2,3
*
1Center for Excellence in Post-Harvest Technologies, North Carolina Agricultural and Technical State University, North Carolina Research
Campus, 500 Laureate Way, Kannapolis, NC 28081, USA
2Human Nutrition Research Program, Julius L. Chambers Biomedical/Biotechnology Research Institute, North Carolina Central University;
North Carolina Research Campus, 500 Laureate Way, Kannapolis, NC 28081, USA
3Food Science Graduate Program, Rutgers University, 65 Dudley Road, New Brunswick, NJ 08901, USA
4Department of Food Science, Rutgers University, 65 Dudley Road, New Brunswick, NJ 08901, USA
5Cancer Research Program, Julius L. Chambers Biomedical/Biotechnology Research Institute, North Carolina Central University,
700 George Street, Durham, NC 27707, USA
6Laboratory of Applied Pharmacology, 1440-6 Forest Hill Road, Staten Island, NY 10314, USA
Received 8 January 2010; Revised 11 April 2010; Accepted 14 April 2010
Pterostilbene, the dimethoxy derivative of resveratrol, has drawn much attention recently due to its
potential beneficial health effects. The metabolic fate of pterostilbene, however, is not well under-
stood. In the present study, we identified nine novel mouse urinary pterostilbene metabolites,
pterostilbene glucuronide, pterostilbene sulfate, mono-demethylated pterostilbene glucuronide,
mono-demethylated pterostilbene sulfate, mono-hydroxylated pterostilbene, mono-hydroxylated
pterostilbene glucuronide, mono-hydroxylated pterostilbene sulfate, and mono-hydroxylated pter-
ostilbene glucuronide sulfate, using liquid chromatography/atmospheric pressure chemical ioniz-
ation and electrospray ionization tandem mass spectrometry. The structures of these metabolites
were confirmed by analyzing the MSn (n ¼ 1–3) spectra. To our knowledge, this is the first report of
the identification of urinary metabolites of pterostilbene in mice. Copyright # 2010 John Wiley &
Sons, Ltd.
Pterostilbene is a naturally occurring stilbenoid compound
originated from several natural plant sources including
deerberries, blueberries, as well as some plants widely used
in traditional medicine, such as Pterocarpus marsupium,
Pterocarpus santalinm, and Vitis vinifera leaves.1–3 As a dime-
thyl ether analogue of resveratrol, pterostilbene has drawn
much attention recently due to its potential health benefits as
an antioxidant, anticancer, antidiabetic and antihypolipi-
demic agent.4–9 It has been reported that the peroxyl-radical
scavenging activity of pterostilbene is the same as that of
resveratrol.8 Suh et al. found that administration of 40 ppm of
pterostilbene for 8 weeks significantly suppressed azoxy-
methane-induced formation of aberrant crypt foci (ACF)
(57% inhibition, P <0.001) and multiple clusters of aberrant
crypts (29% inhibition, P <0.01).6 Dietary pterostilbene also
suppressed azoxymethane-induced colonic cell proliferation
and iNOS expression.6 Cichocki et al. reported that ptero-
stilbene is equally potent as resveratrol in inhibiting 12-O-
tetradecanoylphorbol-13-acetate activated NFkappaB, AP-1,
COX-2, and iNOS in mouse epidermis.5 In addition,
pterostilbene has been reported to significantly lower the
blood glucose level in hyperglycemic rats.7–9
Due to the various potential health benefits of pterostilbene,
a good understanding of its metabolic fate is crucial. However,
only one study has reported that glucuronidated pterostilbene
is the major metabolite in rat liver microsomes; no tandem
mass spectrum is provided in this study to further confirm the
structure of glucuronidated pterostilbene.10 There have been
no reports on the biotransformation of pterostilbene in mice. In
recent years, liquid chromatography/tandem mass spectrom-
etry (LC/MS/MS) has evolved as a valuable tool for structural
analysis of the metabolites of many bioactive polyphenols. The
use of ion traps in metabolite identification takes advantage
of the high sensitivity and multiple stage MS/MS (MSn)
capabilities of this kind of instrument. In the present study, we
analyzed the mouse urinary metabolic profile of pterostilbene
using liquid chromatography/atmospheric pressure chemical
ionization (APCI) and electrospray ionization (ESI) tandem
mass spectrometry. The structures of nine major metabolites
were identified by analyzing the MS2 and MS3 spectra of each
compound.
EXPERIMENTAL
*Correspondence to: S. Sang, Center for Excellence in Post-Harvest
Technologies, North Carolina Agricultural and Technical State
University, North Carolina Research Campus, 500 Laureate
Way, Kannapolis, NC 28081, USA.
Materials
Pterostilbene was provided by Sarbinsa Corporation (Piscat-
away, NJ, USA). LC/MS-grade solvents and other reagents
were obtained from Fisher Scientific (Pittsburgh, PA, USA).
E-mail: ssang@ncat.edu
Copyright # 2010 John Wiley & Sons, Ltd.

Mouse urinary metabolites of pterostilbene 1771
HPLC-grade water was prepared using a Millipore Milli-Q
purification system (Bedford, MA, USA). Sulfatase from
Aerobacter aerogenes and b-glucuronidase from Helix aspersa
were obtained from Sigma (St. Louis, MN, USA).
centrifugation at 17 ꢂ 1000 rpm for 5 min, the supernatant
was transferred into vials for LC/MS analysis. Enzymatic
deconjugation was performed as described previously with
slight modification.11 In brief, duplicate samples were
prepared in the presence of b-glucuronidase (250 U) and
sulfatase (3 U) for 24 h at 378C and then extracted twice with
ethyl acetate. The ethyl acetate fraction was dried under
vacuum, and the solid was resuspended in 200 mL of 80%
aqueous methanol with 0.1% acetic acid for further LC/MS
analysis.
Treatment of mice and urine collection
Experiments with mice were carried out according to a
protocol approved by the Institutional Review Board for the
Animal Care and Facilities Committee (IACUC No. SS1 08-
08-2008) at North Carolina Central University. Female
C57BL/6J mice (24–30 g) were purchased from the Jackson
Laboratory (Bar Harbor, ME, USA) and allowed to acclimate
for at least 1 week prior to the start of the experiment. The
mice were housed 5 per cage for each group and maintained
in air-conditioned quarters with a room temperature of
20 ꢀ 28C, relative humidity of 50 ꢀ 10%, and an alternating
12-h light/dark cycle. Mice were fed Purina Rodent Chow
#5001 (Research Diets) and water, and were allowed to eat
and drink ad libitum. Pterostilbene in dimethyl sulfoxide
(DMSO) was administered to mice by oral gavage (200 mg/
kg), and urine samples were collected in metabolism cages
(5 mice per case) for 24 h after administration of vehicle
(control group, n ¼ 5) or pterostilbene (treated group, n ¼ 5).
These samples were stored at ꢁ808C before analysis.
LC/PDA/APCI/ESI-MS method
LC/PDA/MS analysis was carried out with a Thermo-
Finnigan Spectra System which consisted of an Accela high-
speed MS pump, an Accela refrigerated autosampler, an
Accela photodiode array (PDA) detector, and an LCQ Fleet
ion trap mass detector (Thermo Electron, San Jose, CA, USA)
incorporated with atmospheric pressure chemical ionization
(APCI) and electrospray ionization (ESI) interfaces. A Luna
C18 column (50 ꢂ 2.0 mm i.d., 3 mm; Phenomenex, Torrance,
CA, USA) was used for separation at a flow rate of 0.3 mL/
min. The column was eluted with 100% solvent A (5%
aqueous methanol with 0.2% acetic acid) for 5 min, followed
by linear increases in B (95% aqueous methanol with 0.2%
acetic acid) to 50% from 5 to 10 min, to 65% from 10 to 25 min,
to 100% from 25 to 40 min, and then with 100% B from 40 to
45 min. The column was then re-equilibrated with 100% A for
5 min. The LC eluent was introduced into the APCI or ESI
interface. The positive ion polarity mode was set for the APCI
Urine sample preparation
For the metabolic profile, urine samples (50 mL from each
group, control group and pterostilbene-treated group) were
added to 950 mL methanol to precipitate proteins. After
OH
OSO3H
OSO3H
OSO3H
H3CO
H3CO
H3CO
Hydroxylation
Demethylation
OCH3
OH
OCH3
Pt sulfate
Mono-demethylated Pt sulfate (6)
Mono-hydroxylated Pt sulfate
(9)
(3)
COOH
O
OH
OH
O
SULT
PAPS
SULT
PAPS
SULT
PAPS
OH
OSO3H
H3CO
OH
OH
OH
OH
or
OCH3
H3CO
H3CO
H3CO
Hydroxylation
Demethylation
OSO3H
COOH
O
O
OH
OH
OH
OCH3
OCH3
OH
H3CO
Mono-hydroxylated Pt (7)
Pterostilbene (Pt)
Mono-demethylated Pt
(1)
(4)
OCH3
UDPGT
UDPGA
UDPGT
UDPGA
UDPGT
UDPGA
Mono-hydroxylated Pt glucuronide-sulfate
(10)
OH
COOH
COOH
COOH
O
O
O
O
O
O
OH
OH
OH
OH
OH
OH
OH
OH
OH
H3CO
H3CO
H3CO
Hydroxylation
Demethylation
OCH3
OCH3
OH
Pt glucuronide
Mono-demethylated Pt glucuronide
(8)
(2)
(5)
Mono-hydroxylated Pt glucuronide
Figure 1. Structures of pterostilbene and its major metabolites and possible biotransformation pathways of pterostilbene.
SULT: sulfotransferase; PAPS: adenosine 30-phosphate 50-phosphosulfate (sulfuryl group donor); UDPGT: uridine dipho-
sphate glucuronosyltransferase; UDPGA: uridine diphosphate glucuronic acid.
Copyright # 2010 John Wiley & Sons, Ltd.
Rapid Commun. Mass Spectrom. 2010; 24: 1770–1778
DOI: 10.1002/rcm

1772 X. Shao et al.
source and nitrogen gas was used as the sheath gas and
auxiliary gas. Optimized source parameters include APCI
capillary temperature (2808C), APCI vaporizer temperature
(3258C), capillary voltage (43 V), sheath gas flow rate (20
units), auxiliary gas flow rate (5 units), and tube lens (115 V).
These parameters were tuned using authentic pterostilbene.
The negative ion polarity mode was set for the ESI source
with the voltage on the ESI interface maintained at app-
roximately 5 kV. Nitrogen gas was used as the sheath gas and
auxiliary gas. Optimized source parameters include ESI
capillary temperature (3008C), capillary voltage (–50 V), ion
spray voltage (3.6 kV), sheath gas flow rate (30 units),
auxiliary gas flow rate (5 units), and tube lens (–124.65 V).
These parameters were tuned using authentic pterostilbene.
The collision-induced dissociation (CID) for both APCI and
ESI was conducted with an isolation width of 2 Da and
normalized collision energy of 35 for MS2 and MS3. Default
automated gain control target ion values were used for MS,
MS2, and MS3 analyses. The wavelength of the PDA-UV
detector was set at 280 nm. Data acquisition was performed
with Xcalibur version 2.0 (Thermo Electron, San Jose, CA,
USA).
RESULTS AND DISCUSSION
The strategy that we used for structural characterization of
pterostilbene metabolites was knowledge-based metabolic
identification. It has been reported that resveratrol is a good
substrate for phase II biotransformation, such as glucur-
onidation (M þ 176) and sulfation (M þ 80).12 Since pteros-
tilbene has one free hydroxyl group, we predict that ptero-
stilbene is also a substrate for glucuronidation and sulfation.
In our previous study on the biotransformation of poly-
methoxyflavones, we found that demethylation (M–14) was
the major biotransformation pathway for nobiletin, one of the
major polymethoxyflavones in orange peels.13 Therefore, we
predict that demethylation will also be the major biotrans-
formation pathway for pterostilbene. In addition, it has been
reported that resveratrol can be metabolized to form
hydroxylated metabolites, such as piceatannol, and gluta-
thione conjugates under in vitro conditions.14 We used
selected-ion monitoring (SIM) mode to search all the possible
pterostilbene metabolites from mouse urine samples col-
lected after administration of pterostilbene (200 mg/kg, i.g.).
Those metabolites included pterostilbene glucuronide (m/z
Before enzyme hydrolysis
After enzyme hydrolysis
12.95
100
14.24
100
APCI
4
1
(A)
(B)
5
2
21.27
50
17.80
50
3, 9
7
29.66
32.08
34.56
26.88
0
0
0
0
0
2
4
6
8
10 12 14 16 18 20 22 24 26 28 30 32 34 36
0
2
4
6
8
10 12 14 16 18 20 22 24 26 28 30 32 34
26.79
13.03
100
50
0
100
5
ESI
(C)
3, 9
(D)
2
14.14
17.41
18.32
7
12.50
4
50
1
6
10
30.64
8
21.19
20.96
0
2
4
6
8
10 12 14 16 18 20 22 24 26 28 30 32 34
0
2
4
6
8
10 12 14 16 18 20 22 24 26 28 30 32 34
14.13
(F)
4
2000000
1800000
1600000
1400000
1200000
1000000
800000
600000
400000
200000
0
UV at 280 nm
700000
600000
500000
400000
300000
200000
100000
0
(E)
5
1
12.85
2
21.19
7
3, 9
18.12
32.13
30.40
2
4
6
8
10 12 14 16 18 20 22 24 26 28 30 32 34
Time (min)
0
2
4
6
8
10 12 14 16 18 20 22 24 26 28 30 32 34
Time (min)
Figure 2. LC chromatograms of urine samples collected from pterostilbene-treated mice obtained using the positive APCI-MS
interface: (A) before and (B) after enzymatic hydrolysis; by negative ESI-MS interface: (C) before and (D) after enzymatic
hydrolysis; and by PDA-UV detector (280 nm): (E) before and (F) after enzymatic hydrolysis.
Copyright # 2010 John Wiley & Sons, Ltd.
Rapid Commun. Mass Spectrom. 2010; 24: 1770–1778
DOI: 10.1002/rcm

Mouse urinary metabolites of pterostilbene 1773
Table 1. Major mouse urinary metabolites of pterostilbene
glucuronide (m/z 448), mono-sulfate (m/z 352), and glucur-
onide sulfate (m/z 528) metabolites.
Retention
We analyzed the urine samples collected both from control
mice and mice treated with 200 mg/kg pterostilbene through
oral gavage using positive APCI-MS, negative ESI-MS, and
UV (280 nm) detection (Figs 1 and 2 and Supplementary Fig.
S1, see Supporting Information). Among all the possible
metabolites that we searched, we identified pterostilbene
glucuronide, pterostilbene sulfate, mono-demethylated pter-
ostilbene, mono-demethylated pterostilbene glucuronide,
mono-demethylated pterostilbene sulfate, mono-hydroxyl-
ated pterostilbene, mono-hydroxylated pterostilbene glucur-
onide, mono-hydroxylated pterostilbene sulfate, and mono-
hydroxylated pterostilbene glucuronide sulfate as the
urinary metabolites of pterostilbene in mice (Figs. 1 and 2
and Table 1). This indicates that glucuronidation, sulfation,
demethylation, and hydroxylation are the major biotrans-
formation pathways of pterostilbene in mice. We found that
APCI-MS detection was more sensitive to free pterostilbene
and its mono-demethylated metabolite than ESI-MS detec-
tion (Fig. 2). This may be due to the fact that APCI-MS is more
sensitive to less polar compounds, such as pterostilbene and
mono-demethylated pterostilbene, than ESI-MS. We could
only detect the molecular ions of the sulfate metabolites with
ESI-MS detection because ESI is a softer ionization process
than APCI. The LC chromatograms obtained under UV
Compound/metabolite name
Peak #
time (min)
Pterostilbene (Pt)
Pt glucuronide
Pt sulfate
1
2
3
4
5
6
7
8
9
21.27
17.80
30.64
14.24
12.95
20.96
17.41
12.32
30.64
27.49
Mono-demethylated Pt
Mono-demethylated Pt glucuronide
Mono-demethylated Pt sulfate
Mono-hydroxylated Pt
Mono-hydroxylated Pt glucuronide
Mono-hydroxylated Pt sulfate
Mono-hydroxylated Pt
glucuronide sulfate
10
432), pterostilbene sulfate (m/z 336), pterostilbene gluta-
thione conjugate (m/z 561), pterostiblene cysteine conjugate
(m/z 375), mono-demethylated pterostilbene (m/z 242), mono-
demethylated pterostilbene mono-glucuronide (m/z 418),
mono-demethylated pterostilbene mono-sulfate (m/z 322),
mono-demethylated pterostilbene mono-glucuronide and
mono-sulfate (m/z 498), mono-demethylated pterostilbene
di-glucuronide (m/z 594), mono-demethylated pterostilbene
di-sulfate (m/z 402), resveratrol (m/z 228) and its reported
mono-glucuronide (m/z 404) and mono-sulfate (m/z 308), and
hydroxylated pterostilbene (m/z 272) and its related mono-
13.03
18.00
100
100
SIM: m/z 419 [M+H]+
SIM: m/z 433 [M+H]+
(A)
(B)
Mono-demethylated Pt
Pt glucuronide
80
80
glucuronide
Peak-5
Peak-2
60
40
20
0
60
40
20
0
0
2
4
6
8
10 12 14 16 18 20 22 24 26 28 30 32 34 36
0
2
4
6
8
10 12 14 16 18 20 22 24 26 28 30 32 34 36
Time (min)
Time (min)
257.14
382.92
243.13
100
MS2: 419 [M+H]+
RT: 13.03
100
MS2: 433 [M+H]+
RT: 18.00
400.97
255.13
50
50
267.12
397.02
269.17
308.15
365.04
414.89
338.98
285.09
281.12
323.04
0
0
120 140 160 180 200 220 240 260 280 300 320 340 360 380 400 420 440 460
m/z
120 140 160 180 200 220 240 260 280 300 320 340 360 380 400 420 440
m/z
149.16
MS3: 243/419
RT: 13.03
133.15
100
100
MS3: 257/433
RT: 18.00
225.11
133.10
121.20
145.12
50
50
111.14
197.12
105.16
225.15
211.05
197.16
159.17
229.14
121.13
239.20
179.21
91.17
105.11
91.21
183.15
207.14
200 220
0
0
60
80
100
120
140
160
180
m/z
240
260
280
300
80
100
120
140
160
m/z
180
200
220
240
260
Figure 3. LC/APCI-MS2 and -MS3 (positive ion) spectra of (A) pterostilbene glucuronide (peak 2 in Fig. 2(A)) and (B) mono-
demethylated pterostilbene glucuronide (peak 5 in Fig. 2(A)). Pt stands for pterostilbene.
Copyright # 2010 John Wiley & Sons, Ltd.
Rapid Commun. Mass Spectrom. 2010; 24: 1770–1778
DOI: 10.1002/rcm

1774 X. Shao et al.
detection (280 nm) were similar to those obtained under
APCI-MS detection (Fig. 2).
identical retention time (RT) and MS/MS spectrum, sugg-
esting that peak 1 was pterostilbene (Figs. 4(A) and 4(B)).
Under negative ESI-MS detection, peak 2 was shown as
one of the major peaks in the LC chromatogram of urine
before treatment with b-glucuronidase and sulfatase
(Fig. 2(C)). Peak 2 had a molecular ion m/z 431 [M–H]–
(255 þ 176), further suggesting that this peak was the
glucuronide metabolite of pterostilbene (m/z 256). This was
also confirmed by observing m/z 255 [431–176–H]– as one of
the major product ions in the MS2 spectrum of the molecular
ion m/z 431 [M–H]– (Fig. 5(A)). In addition, the MS3 spectrum
of product ion m/z 255 [M–H]– (MS3: m/z 255/431) was
identical to the MS2 spectrum of authentic pterostilbene
(Figs. 5(A) and 5(C)). All of these features suggest that peak 2
was pterostilbene glucuronide. Since pterostilbene has only
one hydroxyl group at position 40, peak 2 was identified as
pterostilbene 40-glucuronide (Fig. 1).
Identification of the conjugated metabolites of
pterostilbene
Mono-glucuronidated pterostilbene (2) and mono-sulfatated
pterostilbene (3) were identified as the two conjugated
metabolites of pterostilbene (1) using LC/APCI and LC/ESI
tandem mass spectrometry (Figs. 1 and 2 and Table 1). We
observed one major peak (peak 2 at 17.80 min) in the LC
chromatogram obtained from APCI-MS detection (Fig. 2(A))
with the molecular ion m/z 433 [M þ H]þ (257 þ 176)
indicating that this peak was the glucuronide metabolite
of pterostilbene. This was further confirmed by observing
m/z 257 [433–176 þ H]þ (loss of one glucuronide moiety from
m/z 433) as the major product ion in the MS2 spectrum of
molecular ion m/z 433 [M þ H]þ (Fig. 3(A)). The MS3
spectrum of product ion m/z 257 [M þ H]þ (MS3: m/z 257/
433) was identical to the MS2 spectrum of the authentic
pterostilbene (Figs. 3(A) and 4(A)), indicating that peak 2 is
the glucuronide metabolite of pterostilbene. The presence
of the glucuronide metabolite of pterostilbene was also
confirmed by the observation that peak 2 disappeared and
one new peak (peak 1 at 21.27 min) was shown in the LC
chromatogram of urine after treatment with b-glucuronidase
and sulfatase (Fig. 2(B)). Peak 1 and authentic pterostilbene
had the same molecular ions (m/z 257 [M þ H]þ) and almost
The sulfate metabolite of pterostilbene could not be
detected under APCI-MS detection. Peak 3 (pterostilbene
sulfate) has the molecular ion m/z 257 [M þ H]þ (instead of
the expected molecular ion m/z 337 [M þ H]þ), which was the
ion that lost the sulfate group [337–80]þ, under positive
APCI-MS detection. Fortunately, negative ESI-MS detection
could provide the molecular ion for pterostilbene sulfate
(peak 3 at 31.19 min, m/z 335 [M–H]– (255 þ 80)) (Fig. 2(C)). In
SIM mode for pterostilbene sulfate (m/z 335 [M–H]– (255 þ 80)),
there was one major peak (peak 3, RT: 31.19 min) that had m/z
20.86
133.12
100
m/z 257 M+H]+
Pt Standard
100
Pt Standard
(A)
MS2: 257 [M+H]+
80
225.13
60
40
20
0
50
145.14
159.14
111.14
105.17
197.15
239.16
240
121.14
91.17
229.09
179.22
180
207.10
200
0
80
100
120
140
160
220
260
280
300
0
2
4
6
8
10 12 14 16 18 20 22 24 26 28 30 32 34 36
Time (min)
m/z
20.82
133.16
100
80
60
40
20
0
100
SIM: m/z 257 [M+H]+
Pterostilbene (Pt)
Peak-1
MS2: 257 [M+H]+
RT: 20.82
(B)
225.09
50
145.10
111.15
121.12
197.15
159.13
239.14
242.15
179.19
180
91.17
210.11
14.44
15
3.17
0
80
100
120
140
160
200
220
240
260
280
300
0
5
10
20
25
30
35
Time (min)
m/z
13.99
149.10
100
80
60
40
20
0
SIM: m/z 243 [M+H]+
Mono-demethylated Pt
Peak-4
MS2: 243 [M+H]+
RT: 13.99
100
(C)
133.12
121.11
50
0
225.12
211.14
197.16
105.20
100
91.19
183.13
180
80
120
140
160
200
220
240
260
0
2
4
6
8
10 12 14 16 18 20 22 24 26 28 30 32 34 36
Time (min)
m/z
Figure 4. APCI-MS2 (positive ion) spectrum of (A) pterostilbene standard and LC/APCI-MS2 (positive ion) spectra of (B)
pterostilbene (peak 1 in Fig. 2(B)) and (C) mono-demethylated pterostilbene (peak 4 in Fig. 2(B)). Pt stands for pterostilbene.
Copyright # 2010 John Wiley & Sons, Ltd.
Rapid Commun. Mass Spectrom. 2010; 24: 1770–1778
DOI: 10.1002/rcm

Mouse urinary metabolites of pterostilbene 1775
31.19
18.48
SIM: m/z 335 [M-H]-
Pt sulfate
Peak-3
SIM: m/z 431 [M-H]-
Pt glucuronide
Peak-2
100
50
100
50
(A)
(B)
0
0
0
5
10
15
20
25
30
35
0
5
10
15
20
255.10
25
30
35
Time (min)
Time (min)
175.01
100
100
COOH
O
MS2: 335 [M-H]-
RT: 31.19
MS2: 431 [M-H]-
RT: 18.48
O
O
SO3H
OH
OH
H3CO
H3CO
OH
255
255
175
50
50
OCH3
OCH3
255.06
250
412.98
256.23
250
0
0
100
150
200
300
350
150
200
300
m/z
350
400
450
m/z
240.22
240.18
100
100
MS3: 255/335
RT: 31.19
MS3: 255/431
RT: 18.48
50
50
0
0
100
150
200
250
m/z
300
350
400
450
100
150
200
250
300
350
400
m/z
240.09
OH
100
Pt Standard
MS2: 255 [M-H]-
(C)
H3CO
240
50
0
O
CH3
80
100
120
140
160
180
m/z
200
220
240
260
280
300
Figure 5. LC/ESI-MS2 and -MS3 (negative ion) spectra of (A) pterostilbene glucuronide (peak 2 in Fig. 2(C)) and (B)
pterostilbene sulfate (peak 3 in Fig. 2(C)), and MS2 spectrum of (C) pterostilbene standard. Pt stands for pterostilbene.
255 [M–H]– as the base product ion (Fig. 5(B)). The MS3
spectrum of the product ion m/z 255 [M–H]– (MS3: m/z 255/
335) was identical to the MS2 spectrum of authentic
pterostilbene (Figs. 5(B) and 5(C)), further suggesting that
peak 3 was pterostilbene sulfate. The presence of sulfate
metabolites was also confirmed by the observation that
peak 3 disappeared in the LC chromatogram of urine after
treatment with b-glucuronidase and sulfatase (Fig. 2(D)).
Similar to the glucuronide metabolite, we identified peak 3
as pterostilbene 40-sulfate since pterostilbene has only one
hydroxyl group at the 40 position (Fig. 1).
of molecular ion m/z 419 [M þ H]þ (Fig. 3(B)). The presence of
glucuronide metabolites was also confirmed by the obser-
vation that peak 5 disappeared and one new peak (peak 4 at
14.02 min) was shown in the LC chromatogram of urine after
treatment with b-glucuronidase and sulfatase (Fig. 2(B)).
Peak 4 had a molecular ion m/z 243 [M þ H]þ, which was 14
mass units less than the molecular ion of pterostilbene (m/z
257 [M þ H]þ), suggesting that peak 4 was mono-demethy-
lated pterostilbene. The MS2 spectrum of peak 4 was identical
to the MS3 spectrum of the product ion m/z 243 [M þ H]þ of
peak 5 (MS3: m/z 243/419) (Figs. 3(B) and 4(C)), which further
confirmed that peak 5 was the mono-glucuronide metabolite
of mono-demethylated pterostilbene.
Identification of the conjugated metabolites of
mono-demethylated pterostilbene
Under negative ESI-MS detection, peak 5 had molecular
ion m/z 417 [M-H]- (241 þ 176) (Fig. 2(C)), further suggesting
that this peak was the glucuronide metabolite of mono-
demethylated pterostilbene (m/z 242). This was also con-
firmed by observing m/z 241 [417–176–H]– as one of the major
product ions in the MS2 spectrum of molecular ion m/z 417
[M–H]– (Fig. 6(A)). In addition, the MS3 spectrum of product
ion m/z 241 [M–H]– (MS3: m/z 241/417) was almost identical
to the MS2 spectrum of peak 4 (Figs. 6(A) and 6(C)).
Therefore, peak 5 was identified as the mono-demethylated
pterostilbene mono-glucuronide. We observed only one
glucuronide metabolite of the mono-demethylated pteros-
Mono-demethylated pterostilbene glucuronide and mono-
demethylated pterostilbene sulfate were identified as the
major conjugated metabolites of mono-demethylated pter-
ostilbene using LC/APCI- and LC/ESI-MS/MS. In the LC
chromatogram obtained from APCI-MS detection (Fig. 2(A)),
there was one major peak (peak 5 at 12.95 min) with
molecular ion m/z 419 [M þ H]þ (243 þ 176), indicating that
this peak was the glucuronide metabolite of mono-demethy-
lated pterostilbene. This was further confirmed by observing
m/z 243 [419–176 þ H]þ (loss of one glucuronide moiety from
m/z 419) as one of the major product ions in the MS2 spectrum
Copyright # 2010 John Wiley & Sons, Ltd.
Rapid Commun. Mass Spectrom. 2010; 24: 1770–1778
DOI: 10.1002/rcm

1776 X. Shao et al.
13.15
21.42
100
100
50
SIM: m/z 321 [M-H]-
Mono-demethylated Pt
sulfate
SIM: m/z 417 [M-H]-
Mono-demethylated
Pt glucuronide
Peak-5
Peak-3
(B)
(A)
Peak-6
50
17.38
0
0
0
5
10
15
Time (min)
MS2: 417 [M-H]-
RT: 13.15
20
25
30
35
0
5
10
15
20
25
30
35
Time (min)
241.07
175.02
100
MS2: 321 [M-H]-
RT: 17.38
100
COOH
O
O
OH
OH
OH
50
H3CO
241.12
121.17
50
241
175
119.22 135.17
201.04
200
0
OH
399.05
100
150
250
241.09
300
350
m/z
156.38
150
100
MS2: 321 [M-H]-
RT: 21.42
0
O
200
250
300
350
400
450
SO3H
m/z
H3CO
225.09
MS3: 241/417
RT: 13.15
100
50
241
226.02
OH
0
50
100
150
200
250
300
350
m/z
121.11
100
MS3: 241/321
RT: 17.38
0
50
100
150
200
250
m/z
300
350
225.14
400
450
135.16
119.16
100
100
MS2: 241 [M-H]-
RT: 13.96
Mono-demethylated
Pt
Peak-8
0
(CC))
150
200
225.13
250
300
350
400
m/z
226.17
OH
100
255
MS3: 241/321
RT: 21.42
226.05
O
50
0
H3C
50
0
OH
100
150
200
250
300
350
400
0
80
100
120
140
160
m/z
180
200
220
240
260
m/z
Figure 6. LC/ESI-MS2 and -MS3 (negative ion) spectra of (A) mono-demethylated pterostilbene glucuronide (peak 5 in
Fig. 2(C)) and (B) mono-demethylated pterostilbene sulfate (peak 6 in Fig. 2(C)), and MS2 spectrum of (C) mono-
demethylated pterostilbene (peak 4 in Fig. 2(D)). Pt stands for pterostilbene.
tilbene even though it has two hydroxyl groups. The mono-
glucuronide metabolite can be generated from the glucur-
onidation of mono-demethylated pterostilbene and/or the
demethylation of pterostilbene 40-glucuronide. Therefore,
we tentatively identify it as shown in Fig. 1.
(Figs. 6(B) and 6(C)), indicating that this peak was not the
sulfate metabolite of mono-demethylated pterostilbene, even
though it had almost identical MS2 spectra as those of the
sulfate metabolite of mono-demethylated pterostilbene. In
addition, this metabolite was also observed in the urine
sample obtained from mice in the control group. The peak
observed in the control sample has identical MS2 (m/z 321)
and MS3 (m/z 241/321) spectra as those of the peak at RT
17.38 min in the pterostilbene-treated sample (data not
shown). Therefore, this metabolite is an endogenous meta-
bolite instead of a metabolite of pterostilbene. This obser-
vation supports the conclusion that structural elucidation of
conjugated metabolites cannot simply reply on the MS2
spectrum, which has been widely used as the major tool for
predicting the structures of the conjugated metabolites of
dietary polyphenols in the literature. The presence of sulfate
metabolites was also confirmed by the observation that
peak 6 disappeared in the LC chromatogram of urine after
treatment with sulfatase (Fig. 2(D)). Similar to the glucur-
onide metabolites, we also observed only one sulfate
metabolite of the mono-demethylated pterostilbene even
though it has two hydroxyl groups. The mono-sulfate
metabolite can be generated from the sulfation of mono-
demethylated pterostilbene and/or the demethylation of
The sulfate metabolites of mono-demethylated pterostil-
bene could not be detected under APCI-MS detection.
However, negative ESI-MS detection provided the molecular
ions for mono-demethylated pterostilbene sulfate (peak 6 at
21.51 min, m/z 321 [M–H]– (241 þ 80)). In SIM mode for mono-
demethylated pterostilbene sulfate (m/z 321 [M–H]–
(241 þ 80)) there were two peaks that had m/z 241 [M–H]–
as the base product ion. In order to further confirm that those
compounds were the metabolites of mono-demethylated
pterostilbene, we compared the MS3 spectra of product ion
m/z 241 [M–H]– of those two peaks with the MS2 spectrum
of mono-demethylated pterostilbene (peak 4 in Fig. 2(D))
obtained from urine samples after enzymatic hydrolysis
(Figs. 6(B) and 6(C)). We found that only the peak at
21.42 min (peak 6) had a similar fragment ion mass spectrum
as that of mono-demethylated pterostilbene (Figs. 6(B)
and 6(C)). The MS/MS spectrum of the product ion m/z
241 [M–H]– of the peak at 17.38 min was completely different
from the MS2 spectrum of mono-demethylated pterostilbene
Copyright # 2010 John Wiley & Sons, Ltd.
Rapid Commun. Mass Spectrom. 2010; 24: 1770–1778
DOI: 10.1002/rcm

Mouse urinary metabolites of pterostilbene 1777
pterostilbene 40-sulfate. Therefore, we tentatively identified
the mono-sulfate metabolite of mono-demethylated pteros-
tilbene (peak 6) as shown in Fig. 1.
The observation that peak 8 disappeared and one new peak
(peak 7 at 17.44 min) was shown in the LC chromatogram of
urine after treatment with b-glucuronidase and sulfatase
(Fig. 2(D)) also confirmed the presence of glucuronide
metabolites. Peak 7 had molecular ion m/z 271 [M–H]–, which
was 16 mass units higher than that of pterostilbene (m/z 255
[M-H]-), suggesting that it was mono-hydroxylated pter-
ostilbene. We also observed m/z 255 (271 – 16) as the major
product ion in the MS2 spectrum of peak 7. Moreover, the
MS3 spectrum of product ion m/z 255 [M–H]– of peak 7 (MS3:
m/z 255/271) was identical to the MS2 spectrum of authentic
pterostilbene (Figs. 5(C) and 7(A)). All these features
suggested that peak 7 was mono-hydroxylated pterostilbene.
The MS2 spectrum of peak 7 was identical to the MS3
spectrum of the product ion m/z 271 [M–H]– of peak 8 (MS3:
m/z 271/447) (Figs. 7(A) and 7(B)), which further confirmed
that peak 8 was the mono-glucuronide metabolite of mono-
hydroxylated pterostilbene. We observed only one glucur-
onide metabolite of mono-hydroxylated pterostilbene even
though it has two hydroxyl groups. The mono-glucuronide
metabolite can be generated from the glucuronidation of
mono-hydroxylated pterostilbene and/or the hydroxylation
of pterostilbene 40-glucuronide. Therefore, we tentatively
identify it as shown in Fig. 1.
Identification of the conjugated metabolites of
mono-hydroxylated pterostilbene
Since hydroxylation is considered to be one of the major phase
I biotransformations of resveratrol in human liver microsomes,
we also investigated the mono-hydroxylated pterostilbene and
its conjugated metabolites. Three conjugated metabolites were
identified in our study: mono-glucuronide, mono-sulfate, and
glucuronide sulfate metabolites of mono-hydroxylated pter-
ostilbene. Three major peaks were observed in the LC chroma-
togram under ESI-MS detection (Fig. 2(C)). They were marked
as peak 8 (RT: 12.32 min) with the molecular ion m/z 447 [M–
H]– (271 þ 176), peak 9 (RT: 31.36 min) with the molecular ion
m/z 351 [M–H]– (271 þ 80) and peak 10 (RT: 27.49 min) with the
molecular ion m/z 527 [M–H]– (271 þ 176 þ 80), suggesting that
they were mono-glucuronide, mono-sulfate, and glucuronide
and sulfate metabolites of mono-hydroxylated pterostilbene,
respectively.
The existence of the mono-glucuronide metabolite was
further confirmed by observing m/z 271 [448–176–H]– (loss of
one glucuronide moiety from m/z 448) and m/z 175 [M–H]–
(glucuronide moiety) as two of the major product ions in the
MS2 spectrum of molecular ion m/z 447 [M þ H]þ (Fig. 7(B)).
The sulfate metabolite of mono-hydroxylated pterostil-
bene was confirmed by observing m/z 271 [352–80–H]– (loss
255.09
240.06
17.69
100
100
MS2: 271[M-H]-
MS3: 255/271
100
SIM: m/z 271[M-H]-
(A)
OH
80 RT: 17.69
80 RT: 17.69
80
Mono-hydroxylated Pt
OH
255
Peak-7
60
40
20
0
60
40
20
0
60
40
20
0
H3CO
241.26
250
OCH3
150
100
200
300
100
150
200
250
300
0
5
10
15
20
25
30
35
Time (min)
m/z
m/z
12.32
100
80
60
40
20
0
SIM: m/z 447[M-H]-
Mono-hydroxylated Pt
glucuronide
271.08
255.06
100
80
60
40
20
0
100
80
60
40
20
0
MS2: 447[M-H]-
RT: 12.32
MS3: 271/447
RT: 12.32
(B)
Peak-8
175.00
256.11
250
241.17
250
m/z
150
200
300
m/z
350
400
450
500
0
5
10
15
20
25
30
35
100
150
200
300
350
400
Time (min)
31.36
271.11
255.06
100
SIM: m/z 351[M-H]-
100
100
80
60
40
20
0
MS2: 351[M-H]-
RT: 31.36
MS3: 271/351
(C)
80 Mono-hydroxylated Pt sulfate
80 RT: 31.36
Peak-9
60
60
40
20
0
40
20
0
241.17
250
m/z
0
5
10
15
20
25
30
35
100
150
200
250
m/z
300
350
400
100
150
200
300
350
400
Time (min)
27.49
271.14
351.01
271.27
100
80
SIM: m/z 527[M-H]-
Mono-hydroxylated Pt
glucuronide-sulfate
Peak-10
MS3: 447/527
RT: 27.49
100
100
100
80
60
40
20
0
MS2: 527[M-H]-
MS3: 351/527
RT: 27.49
80 RT: 27.49
80
60
40
20
0
60
40
20
0
60
40
20
0
(D)
175.20
271.24
175.07
446.99
0
5
10
15
20
25
30
35
100 150 200 250 300 350 400 450 500 550
m/z
150 200 250 300 350 400 450 500 550
150 200 250 300 350 400 450 500 550
Time (min)
m/z
m/z
Figure 7. LC/ESI-MS2 and -MS3 (negative ion) spectra of (A) mono-hydroxylated pterostilbene glucuronide (peak 7 in
Fig. 2(D)), (B) mono-hydroxylated pterostilbene glucuronide (peak 8 in Fig. 2(C)), (C) mono-hydroxylated pterostilbene sulfate
(peak 9 in Fig. 2(C)), and (D) mono-hydroxylated pterostilbene glucuronide sulfate (peak 10 in Fig. 2(C)).
Copyright # 2010 John Wiley & Sons, Ltd.
Rapid Commun. Mass Spectrom. 2010; 24: 1770–1778
DOI: 10.1002/rcm

1778 X. Shao et al.
of one sulfate moiety from m/z 352) as one of the major
product ions in the MS2 spectrum of molecular ion m/z 351
[M–H]– (Fig. 7(C)). Even our chromatography method did
not give us good separation of the sulfate metabolites of
pterostilbene and mono-hydroxylated pterostilbene. The
disappearance of peak 9 was observed in the LC chromato-
gram of urine after treatment with b-glucuronidase and
sulfatase (Fig. 2(D)). In addition, the MS3 spectrum of
product ion m/z 271 [M–H]– of peak 9 (MS3: m/z 271/351) was
identical to the MS2 spectrum of peak 7 (Figs. 7(A) and 7(C)),
which confirmed that peak 9 was the mono-sulfate meta-
bolite of mono-hydroxylated pterostilbene. Similar to the
mono-glucuronide metabolite, we observed only one sulfate
metabolite of the mono-hydroxylated pterostilbene. The
mono-sulfate metabolite can be generated from the sulfation
of mono-hydroxylated pterostilbene and/or the hydroxyl-
ation of pterostilbene 40-sulfate. Therefore, we tentatively
identify it as shown in Fig. 1.
oral gavage (Fig. 1 and Table 1). To our knowledge, this is the
first study to establish the mouse urinary metabolic profile of
pterostilbene using multi-stage tandem mass spectrometry.
Our results clearly indicate that pterostilbene can be
metabolized in mice to generate the mono-demethylated
and mono-hydroxylated metabolites. Pterostilbene and its
mono-demethylated and mono-hydroxylated metabolites
are good substrates for glucuronidation and sulfation to
form related phase II metabolites.
It has been reported that resveratrol is extensively meta-
bolized in vivo to generate the glucuronide and sulfate
metabolites which are predominantly circulated in plasma
and distributed to various tissues. Similarly, we found that
glucuronide and sulfate metabolites are also the major
metabolites of pterostilbene in mice. A possible metabolism
pathway is established based on our observations and the
literature reports on the biotransformation of resveratrol
(Fig. 1). It is worthwhile to further study the pharmacoki-
netics of pterostilbene and the tissue distribution of ptero-
stilbene and its major metabolites. Whether the newly
identified mono-demethylated, mono-hydroxylated, glucur-
onide, and sulfate metabolites of pterostilbene are bioactive
remains to be determined. The results would help us assess
the relative contribution of pterostilbene metabolites to the
disease preventive effects of pterostilbene.
In SIM mode with negative ESI-MS detection, we observed
one peak corresponding to molecular ion m/z 527 [M–H]–
(271 þ 176 þ 80) (peak 10 at 27.49 min, Fig. 7(D)). The MS2
spectrum of peak 10 had product ions m/z 447 (loss of one
sulfate moiety from m/z 527), m/z 351 (loss of one glucuronide
moiety from m/z 527), m/z 271 (loss of one glucuronide moiety
and one sulfate moiety from m/z 527), and m/z 175
(glucuronide moiety). All these features suggested that peak
10 was the glucuronide sulfate metabolite of mono-hydro-
xylated pterostilbene. In addition, the MS3 spectrum of the
product ion m/z 447 [M–H]– (MS3: m/z 447/527) was almost
identical to the MS2 spectrum of mono-hydroxylated
pterostilbene glucuronide (peak 8: m/z 447 [M–H]–) and
the MS3 spectrum of product ion m/z 351 [M–H]– (MS3: m/z
351/527) was almost identical to the MS2 spectrum of mono-
hydroxylated pterostilbene sulfate (peak 9: m/z 351 [M–H]–)
(Fig. 7(D)). All of these features suggested that that peak 10
was the glucuronide sulfate metabolite of mono-hydroxyl-
ated pterostilbene. The disappearance of peak 10 was
observed in the LC chromatogram of urine after treatment
with b-glucuronidase and sulfatase (Fig. 2(D)) further sup-
ported our conclusion that peak 10 was the glucuronide
sulfate metabolite of mono-hydroxylated pterostilbene. This
metabolite can be generated by further sulfation of the mono-
hydroxylated pterostilbene glucuronide (peak 8) or by
further glucuronidation of the mono-hydroxylated pteros-
tilbene sulfate (peak 9) (Fig. 1). Since we only observed one
peak, we tentatively identify it as shown in Fig. 1.
SUPPORTING INFORMATION
Additional supporting information may be found in the
online version of this article.
REFERENCES
1. Langcake P, Cornford CA, Pryce RJ. Phytochemistry 1979; 18:
1027.
2. Maurya R, Ray AB, Duah FK, Slatkin DJ, Schiff PL. J. Nat.
Prod. 1984; 47: 179.
3. Sehadri R. Phytochemistry. 1972; 11: 881.
4. Paul B, Masih I, Deopujari J, Charpentier C. J Ethnopharmacol.
1999; 68: 71.
5. Cichocki M, Paluszczak J, Szaefer H, Piechowiak A,
Rimando AM, Baer-Dubowska W. Mol. Nutr. Food Res.
2008; 52 (Suppl 1): S62.
6. Suh N, Paul S, Hao X, Simi B, Xiao H, Rimando AM, Reddy
BS. Clin. Cancer Res. 2007; 13: 350.
7. Pari L, Satheesh MA. Life Sci. 2006; 79: 641.
8. Rimando AM, Cuendet M, Desmarchelier C, Mehta RG,
Pezzuto JM, Duke SO. J. Agric. Food Chem. 2002; 50: 3453.
9. Manickam M, Ramanathan M, Jahromi MA, Chansouria JP,
Ray AB. J. Nat. Prod. 1997; 60: 609.
10. Remsberg CM, Yanez JA, Ohgami Y, Vega-Villa KR,
Rimando AM, Davies NM. Phytother. Res. 2008; 22: 169.
11. Lambert JD, Sang S, Hong J, Kwon SJ, Lee MJ, Ho CT, Yang
CS. Drug Metab. Dispos. 2006; 34: 2111.
12. Baur JA, Sinclair DA. Nat. Rev. Drug Discov. 2006; 5: 493.
13. Li S, Wang Z, Sang S, Huang MT, Ho CT. Mol. Nutr. Food Res.
2006; 50: 291.
CONCLUSIONS
In this study, using both LC/APCI-MS/MS and LC/ESI-
MS/MS analysis we successfully identified nine novel meta-
bolites of pterostilbene from mouse urine samples collected
24 h after administration of 200 mg/kg pterostilbene through
14. Steenwyk RC, Tan B. Xenobiotica 2010; 40: 62.
Copyright # 2010 John Wiley & Sons, Ltd.
Rapid Commun. Mass Spectrom. 2010; 24: 1770–1778
DOI: 10.1002/rcm