Identification and quantification of metabolites of orally administered naringenin chalcone in rats

Article abstract of DOI:10.1021/jf901137x

Naringenin chalcone is the main active component of tomato skin extract, which has an antiallergic activity. In this study, naringenin chalcone was orally administered to rats, and the chemical structures and levels of the major metabolites in the plasma

Full text of DOI:10.1021/jf901137x

6432 J. Agric. Food Chem. 2009, 57, 6432–6437
DOI:10.1021/jf901137x
Identification and Quantification of Metabolites of Orally
Administered Naringenin Chalcone in Rats
MINEKA
Y
OSHIMURA,* ATSUSHI
SANO, JUN-ICHI
K
AMEI
,
AND
A
KIO
O
BATA
Research and Development Division, Kikkoman Corporation, 399 Noda, Noda City,
Chiba 278-0039, Japan
Naringenin chalcone is the main active component of tomato skin extract, which has an antiallergic
activity. In this study, naringenin chalcone was orally administered to rats, and the chemical
structures and levels of the major metabolites in the plasma and urine of rats were determined.
HPLC analysis indicated the presence of three major metabolites in the urine. LC-MS and NMR
analyses tentatively identified these as naringenin chalcone-20-O-β-
-glucuronide, and naringenin-40-O-β- -glucuronide. Naringenin chalcone-20-O-β-
the only metabolite detected in the plasma, and its peak plasma level was observed 1 h after
naringenin chalcone administration. Naringenin chalcone-20-O-β-
-glucuronide also inhibited hista-
D-glucuronide, naringenin-7-O-β-
D
D
D-glucuronide was
D
mine release from rat peritoneal mast cells stimulated with compound 48/80. This activity might
contribute to the antiallergic activity of naringenin chalcone in vivo. To the best of the authors’
knowledge, this study is the first to report determination of naringenin chalcone metabolites in rat
plasma and urine.
KEYWORDS: Naringenin chalcone; metabolite; naringenin chalcone glucuronide; antiallergy
INTRODUCTION
MATERIALS AND METHODS
Flavonoids are polyphenolic compounds that are present in
foods and beverages of plant origin. Epidemiological studies have
shown that the dietary intake of flavonoids is associated with
biological effects such as a lower incidence of cardiovascular
disease and cancer (1,2). Naringenin is a bioactive flavonoid, and
the inhibition effect of narigenin against hepatitis C virus was
reported recently (3). It is very important to evaluate the bioavail-
ability of flavonoids to determine whether the absorbed flavo-
noids have biological activity in vivo. There are some reports on
the pharmacokinetics of flavonoids, such as naringenin, hesper-
etin, and quercetin (4-8).
We have recently reported that a tomato skin extract, which
was prepared by extracting tomato skin with 60% (v/v) ethanol,
inhibited histamine release from rat peritoneal mast cells stimu-
lated with compound 48/80 and type I allergic reaction in mouse
model (9). Furthermore, in our previous experimental study, we
have confirmed that the main active component of tomato skin
extract is naringeninchalcone(Figure1) (9). Wealso reported that
tomato skin extract could relieve the symptoms of perennial
allergic rhinitis (10).
Chemicals and Reatents. Naringenin was purchased from Funakoshi
Co. (Tokyo, Japan). Compound 48/80 was purchased from Sigma
Chemical Co. (St. Louis, MO). Uridine-50-diphosphoglucuronic acid
was purchased from Nacalai Tesque Inc. (Kyoto, Japan). 2-[4-(2-Hydro-
xyethyl)-1-piperazinyl]ethanesulfonic acid (HEPES) was purchased from
Dojindo (Kumamoto, Japan). Rat liver microsomal protein (S-9) was
obtained from Kikkoman Co. (Chiba, Japan). Other chemicals and
solvents were purchased from Wako Pure Chemicals Co. (Osaka, Japan)
and used without further purification.
Animals. Male Sprague-Dawley rats (250-300 g) were purchased
from Japan SLC Co. (Shizuoka, Japan). These animals were acclimatized
in an environmentally controlled room (temperature, 23 ( 1 °C; humidity,
55 ( 5%; and illumination time, 7:00 a.m. to 7:00 p.m.) and were raised on
a type of MF standard diet (Oriental Yeast. Co., Tsukuba, Japan) and
water ad libitum for 1 week before the experiment. The experimental
protocols were approved by the animal research control committee of
Kikkoman Corp. and performed according to the guidelines for the care
and use of experimental animals of the Japanese Association for Labora-
tory Animals. The researchers who used the animals receive annual
instruction by the animal research control committee of Kikkoman Corp.
Preparation of Naringenin Chalcone from Tomato Skin Extract.
Tomato (Lycopersicon esculentum Miller) skin extract containing poly-
phenols was prepared by incubating a mixture of the skin with 60% (v/v)
ethanol at 60 °C for 2 h, followed by lyophilization. Preparation of
naringenin chalcone was based on the identification method of naringenin
chalcone, which was reported (9). Naringenin chalcone was separated
from the tomato skin extract by HPLC. The column used was a 250 mm ꢀ
15 mm i.d. Capcell Pak C18 column (Shiseido, Tokyo, Japan). Elution was
carried out with acetonitrile/water (33:67, v/v) containing 0.1% trifluor-
oacetic acid at a flow rate of 5 mL/min at room temperature and
monitored at 350 nm. The peak fraction was collected, concentrated under
reduced pressure to remove the organic solvents, and then freeze-dried.
There are many reports on the bioavailability of naringenin (5-7),
which is the cyclized structure of naringenin chalcone, but none
on that of naringenin chalcone itself. In this study, to estimate the
metabolism of naringenin chalcone, we examined the levels of its
metabolites in plasma and urine of rats by using HPLC, LC-MS,
and NMR analyses.
*Corresponding author (telephone þ81-4-7123-5540; fax þ81-4-
7123-5540; e-mail myoshimura@mail.kikkoman.co.jp).
pubs.acs.org/JAFC
Published on Web 06/26/2009
© 2009 American Chemical Society

Article
J. Agric. Food Chem., Vol. 57, No. 14, 2009 6433
Figure 1. Chemical structures of naringenin chalcone and its metabolites.
(Advantec), and injected into the HPLC system. The column used was a
250 ꢀ 10 mm i.d. YMC-Pack Pro C18 column (YMC, Kyoto, Japan).
Elution was carried out with acetonitrile/water (25:75, v/v) containing
0.1% trifluoroacetic acid at a flow rate of 5 mL/min at room temperature
and monitored between 250 and 400 nm. Three major fractions were
collected, concentrated under reduced pressure to removethe solvents, and
then freeze-dried. They were analyzed by LC-MS and NMR.
Table 1. HPLC-MS Data for Metabolites of Naringenin
mass ion (ESI)
retention time (min)
UV max (nm)
[M þ H]þ
major fragments
M1
M2
M3
16.9
10.4
11.4
365
283
283
449
449
449
273
273
273
HPLC System. HPLC analysis used a JASCO HPLC system con-
sisting of a pump PU-2080 Plus, an autoinjectorAS-2057 Plus, and a
multiwavelength detector MD-2010 Plus (JASCO, Tokyo, Japan).
The fraction was identified as naringenin chalcone by LC-MS (98%
purity).
LC-MS Analysis. LC-MS analysis used a system of JMS-BU30
(JEOL, Tokyo, Japan). The column used was a 150 ꢀ 4.6 mm i.d. Capcell
Pak C18-UG120 (Shiseido) for calculating the molecular weight. The LC-
MS conditions were as follows: atmospheric pressure chemical ionization
(APCI) in the positive ion mode; desolving plate, 220 °C; orifice 1, 70 °C;
and lens ring voltage, 50 V. Elution was carried out with acetonitrile/water
(20:80, v/v) containing 0.1% acetic acid at a flow rate of 1 mL/min at room
temperature. MS was performed in the selected ion monitoring mode
(SIM).
In Vivo Study. The rats were orally administered 0.5% carboxymethyl
cellulose sodium salt solution (5 mL/kg of body weight) with or without
naringenin chalcone (20 mg/kg). They were anesthetized with diethyl ether
at 0, 0.5, 1, 2, 4, and 24 h after sample administration. Blood samples from
the jugular vein were collected into heparinized tubes. They were centri-
fuged at 2000g for 5 min at 4 °C, and the supernatants were collected.
Urine samples were collected hourly from the 0-10 and 10-24 h periods in
test tubes. Each plasma and urine sample was mixed with an equal volume
of methanol and centrifuged at 2500g for 10 min at 4 °C. The supernatant
of each sample was collected in a test tube and also stored at -20 °C until
further analysis.
NMR Analysis. To determine the structure of naringenin chalcone
metabolites, one- and two-dimensional (1D- and 2D) NMR spectra,
1
1
including H-1H correlation spectroscopy (COSY), H-13C heteronuc-
lear multiple quantum coherence (HMQC), and 1H-13C heteronuclear
multiple bond connectivity (HMBC), were recorded in CD3OD at 300 K
using a Bruker Avance 500 spectrometer (Bruker Biospin, Tsukuba,
Japan), operated at 500.1 MHz for 1H and at 125.7 MHz for 13C. A
summary of 1H and 13C resonance of isolated metabolites is given in
Tables 2 and 3.
Isolation of Metabolites from Plasma and Urine. Each sample was
filtered through a cellulose acetate DISMIC filter (Advantec, Dublin, CA)
and analyzed by HPLC. The column used was a 150 ꢀ 4.6 mm i.d. Capcell
Pak C18-UG120 (Shiseido). Elution was carried out with acetonitrile/
water (20:80, v/v) containing 0.1% trifluoroacetic acid at a flow rate of
1 mL/min at room temperature and monitored between 250 and 400 nm.
Retention times of major metabolites, M1, M2, and M3, are given in
Table 1. The limit of metabolite detection was 0.3 μM.
Enzymatic Synthesis of Related Glucuronides. We synthesized the
corresponding glucuronides of naringenin chalcone enzymatically. Nar-
ingenin chalcone was dissolved in 50% (v/v) ethanol (20 mM, 2.5 mL) and
mixed with 22 mL of a reaction buffer solution [0.5 mM HEPES (pH 5.0),
3 mM uridine-50-diphosphoglucuronic acid, 0.1 mM MgCl2, and 25 mL of
rat liver microsomal protein solution]. The mixture was incubated at 25°C.
After 3 h, it was mixed with an equal volume of methanol and centrifuged
at 2000g for 10 min at 4 °C. The supernatant was concentrated under
vacuum, dissolved in dimethyl sulfoxide, filtered through a DISMIC filter
Assay of the Inhibition of Histamine Release from Rat Peritoneal
Mast Cells. Preparation of the mast cells and assay of the inhibition of
histamine release from rat peritoneal mast cells were based on reported
methods (11). Rat peritoneal mast cells were collected from the peritoneal
cavity after the injection of a buffer solution [0.15 M NaCl, 3.7 mM KCl,
3.0 mM Na2HPO4, 3.5 mM KH2PO4, 0.9 mM CaCl2, 5.6 mM glucose, and
0.1% (w/v) gelatin] and then partially purified by centrifugation. Test
samples, which were dissolved in the buffer solution containing 0.5% (v/v)
dimethyl sulfoxide, were added to the cell suspension (2.5 ꢀ 105 cells/mL in
80 μL) and subsequently incubated at 37 °C for 10 min. The cells were then

6434 J. Agric. Food Chem., Vol. 57, No. 14, 2009
Yoshimura et al.
Table 2. 1H and 13C NMR Spectroscopic Data for Naringenin Chalcone and
M1 in CD3OD
RESULTS AND DISCUSSION
Detection of Naringenin Chalcone Metabolites in Rat Urine. In
the present study, we identified the three major metabolites of
naringenin chalcone and determined their structures by LC-MS
and NMR analyses. Figure 2 shows representative chromato-
grams of rat urine samples 1 h after the administration of vehicle
(Figure 2A) or naringenin chalcone (Figure 2B), respectively.
Table 1 shows the mass data of these metabolites. The positive
APCI-MS of M1 showed molecular ion peaks ([M þ H]þ) at m/z
273 and 449 corresponding to a naringenin chalcone and a
naringenin chalcone monoglucuronide, respectively. The UV
absorption maximum of M1 was the same as that of naringenin
chalcone (Figure 3A). M2 and M3 showed the same [M þ H]þ
peaks of M1, but their UV spectra were the same as that of
naringenin (Figure 3B,C). M2 and M3 appear to be two different
types of naringenin monoglucuronide, considering that their
retention times were inconsistent.
Previous studies indicated that metabolites of naringenin were
found in plasma and urine as glucuronides (13, 14). In our study,
naringenin chalcone might be extensively metabolized by glucu-
tonidases. The amount of the three isolated metabolites of
naringenin chalcone in the urine sample was too little to be
analyzed by NMR. We prepared the glucronides of naringenin
chalcone by incubating naringenin chalcone with rat liver micro-
somes and uridine-50-diphosphoglucuronic acid. Three major
peaks were detected from the reaction solution by HPLC (data
not shown). These were purified and analyzed by HPLC and
LC-MS. They were found to be identical to the same compounds
of the three metabolites of naringenin chalcone in urine, deter-
mined from retention times, their spectra, and molecular weights
(data not shown). These enzymatically synthesized metabolites
were used for NMR analysis.
naringenin chalcone
M1
position
δH
δC
δH
δC
194.5
chalcone CdO
R
194.2
125.6
143.6
8.06
7.69
7.98
7.66
125.9
144.1
β
1
128.5
131.3
116.8
161.0
128.5
131.8
116.9
161.1
2, 6
3, 5
4
7.48
6.81
7.62
6.83
10
20
30
40
50
60
105.9
166.0
96.0
107.8
161.6
95.7
5.84
5.84
6.19
6.01
166.2
96.0
165.7
98.7
166.0
165.7
100
200
300
400
500
600
5.22
102.0
3.59-3.52
3.59-3.52
3.66
77.7-72.9
77.7-72.9
77.7-72.9
77.7-72.9
171.9
4.02
Table 3. 1H and 13C NMR Spectroscopic Data for M2 and M3 in CD3OD
M2 M3
position
δH
δC
δH
δC
Identification of Metabolites of Naringenin Chalcone. For
detailed structural analysis, M1 was analyzed by NMR (Table 2).
The 1H NMR spectrum of naringenin chalcone glucuronide
showed nonequivalence of the two aromatic protons (H-30 and
H-50) and downfield shifts to δH 6.19 and 6.01, respectively, in
comparison with those of naringenin chalcone at δH 5.84 (2H,
br s, H-30 and H-50). Comparison of the 13C NMR spectrum of
naringeninchalconeglucuronide withthat ofnaringeninchalcone
revealed that two equivalent signals of naringenin chalcone at δC
166.0 (C-20 and C-60) and δC 96.0 (C-30 and C-50) shifted to
nonequivalent signals of naringenin chalcone glucuronide at δC
165.7 (C-60), 161.6 (C-20), 98.7 (C-50), and 95.7 (C-30) as deter-
mined by HMQC and HMBC data. In addition, on comparison
of the HMBC signals of naringenin chalcone glucuronide and
naringenin chalcone, the upfield shifted carbon of naringenin
chalcone glucuronide at δC 161.6 (C-20) correlated with the
proton signal at δH 5.22 (H-100). The glycoside linkage was
assigned as β on the basis of the anomeric proton signal at
2
5.39
3.17, 2.75
80.7
44.1
5.40
3.09, 2.73
80.1
44.1
3
4
198.6
105.1
197.5
103.4
4a
5, 7, 8a
166.6-164.6
97.9
96.9
168.4-164.7
97.1
96.2
6
8
6.18
6.18
5.89
5.89
10
130.8
129.1
116.3
159.1
134.6
128.8
118.0
159.0
20, 60
30, 50
40
7.32
6.81
7.43
7.12
100
200
300
400
500
600
5.07
3.48
3.48
3.59
4.02
101.0
74.3
77.1
72.8
76.5
172.0
5.01
3.50
3.50
3.62
3.99
102.3
74.6
77.3
73.0
76.6
172.1
δ
H 5.22 (d, J=7.3 Hz, H-100). These results indicate that the C-20
position of the naringenin chalcone skeleton is modified and
bonded glycosidically with glucuronic acid in the β form
(Figure 1). M1 was tentatively identified as naringenin chal-
stimulated with compound 48/80 (final concentration = 0.5 μg/mL) for
10 min. After centrifugation of the cell suspension at 1500g for 5 min at
4 °C, each supernatant was mixed with the same volume of HCl (0.1 N) to
stop histamine release from the cells. The amount of histamine in each
solution was analyzed by HPLC using the on-column derivatization
method with o-phthalaldehyde (12).
cone-20-O-β-
D-glucuronide.
In addition, M2 and M3 were identified as naringenin-7-O-β-
D
-glucuronide and naringenin-40-O-β-
D-glucuronide, respectively,
1
by specific chemical shifts in the H NMR spectra and correla-
tions in the HMBC spectra (Table 3) (13). The proton signals in
the conjugated aromatic ring were shifted downfield in compar-
ison with those of naringenin. In M2, the proton signal at δH 6.18
(2H, m, H-6, 8) in the conjugated aromatic ring was equivalently
shifted downfield in comparison with those of naringenin at δH
5.89 (2H, H-6, 8). In M3, the carbon signal at δC 159.0 (C-40) was
Statistical Analysis. Each data value is expressed as the mean value
and its standard error (SEM). Statistical analysis was performed by
nonparametric Mann-Whitney’s U test for comparisons between the
groupsadministered vehicle ornaringeninchalcone. All statistical analyses
were two-tailed, and statistical significance was established at p<0.05.
Statistical analyses were performed using SPSS Systems (SPSS Inc.,
Chicago, IL).

Article
J. Agric. Food Chem., Vol. 57, No. 14, 2009 6435
Figure 2. Representative HPLC chromatograms of naringenin chalcone metabolites in urine at 310 nm. The urine sample was collected 1 h after the
administration of vehicle (A) or 20 mg/kg of naringenin chalcone (B).
observed in correlation with the proton signal at δH 5.01 (1H,
H-100) with HMBC analysis, as well as downfield shifts of the
proton signals at δH 7.30 (2H, H-20,60) and δH 6.81 (2H, H-30,50)
in naringenin to δH 7.43 (2H, H-20,60) and δH 7.12 (2H, H-30,50).
The anomeric proton of glucuronic acid (J > 7.0 Hz) exhibited
HMBC correlations to an A-ring carbon (C-7) and a B-ring
carbon (C-40), indicating β positions of naringenin-7-O-β-
quantitative standards. When naringenin chalcone was orally
administered to rats, only M1 was detected in plasma (Figure 6).
The peak was not detected in the plasma of rats administered
vehicle. Two types of naringenin glucuronide were not detected in
the plasma samples. The peak plasma level for M1 was observed 1
h after naringenin chalcone administration, and its concentration
was 5.0 ( 1.0 μM (Figure 4). M1 had a half-life of 5.5 ( 1.7 h. The
level of M1 in the plasma decreased to nearly undetectable levels
at 24 h after administration. It had been reported that the
glucuronidation of naringenin occurs in the intestinal epithe-
lium (20). Furthermore, naringenin chalcone might be glucuro-
nized in the intestinal epithelium and transferred to the plasma
shortly after oral administration. The absence of naringenin
glucuronides in the plasma might indicate that once these have
been released into the bloodstream, they are rapidly removed by
excretion via the kidney. Such pharmacokinetics had been
reported for quercetin metabolites (8). Further investigation is
required to determine the pathway through which naringenin
glucuronides are synthesized from naringenin chalcone in vivo.
Figure 5 shows the cumulative excretion of the three metabo-
lites of naringenin chalcone in the urine. The three metabolites
were observed 1 h after oral administration. These peaks were not
detected in the plasma of rat administered vehicle. M1 was
excreted more rapidly than two types of naringenin glucuronide
in the urine. The level of M1 reached a peak between 2 and 4 h and
increased throughout a 24 h period, whereas those of two types of
naringenin glucuronide, M2 and M3, reached a peak between
3 and 6 h. The three metabolites were not detected 48 h after
administration (data not shown). The recovery rates of M1, M2,
and M3 in urine were 7.5 ( 0.4, 5.4 ( 0.4, and 7.9 ( 0.1% of
intake, respectively. The total cumulative amount of the three
metabolites after 24 h was 20.9 ( 1.6% of the dosage. Because the
recovery of the metabolites in the urine was only 21%, a large
amount of the ingested dose was unaccounted for. It has been
reported that naringenin (21) and phloretin (22), naringenin
D
-glucuronide and naringenin-40-O-β-
(Figure 1).
LC-MS and NMR analyses showed that the major metabolites
in urine were naringenin chalcone-20-O-β-
-glucuronide, narin-
genin-7-O-β- -glucuro-
-glucuronide, and naringenin-40-O-β-
nide. This study reports for the first time the presence of
naringenin chalcone-20-O-β-
-glucuronide in rat plasma and
D-glucuronide, respectively
D
D
D
D
urine after naringenin chalcone administration. It has been
reported that phloretin (20,40,60,4-tetrahydroxydihydrochalcone),
which has a chalcone form like naringenin chalcone, is metabo-
lized to a phloretin glucuronide that has a chalcone form (15).
Therefore, it is reasonable to assume that naringenin chalcone
was metabolized to naringenin chalcone glucuronide, keeping a
chalcone form. Naringenin chalcone is metabolized to naringenin
by chalcone isomerase. Some chalcone isomerase has been iso-
lated from human fecal bacteria (16). Several studies have noted
that naringenin is metabolized to naringenin glucuro-
nides (13, 14, 17). The glucuronosyltransferase, which is involved
in the synthesis of glucuronide metabolites of flavonoids, has
been found in the intestines and livers of rat and humans (18,19).
Therefore, some of the naringenin chalcone might be converted to
naringenin by chalcome isomerase of fecal bacteria and metabo-
lized to naringenin glucuronides.
Time Course Analysis of Naringenin Chalcone Metabolites in
Plasma and Urine. The time-course characteristics for changes in
the concentrations of M1, M2, and M3 in the plasma and urine
after naringenin chalcone administration are shown in Figures 4
and 5. The enzymatically synthesized glucuronides were used as

6436 J. Agric. Food Chem., Vol. 57, No. 14, 2009
Yoshimura et al.
Figure 5. Cumulative urinary excretion curves of naringenin chalcone
metabolites of rats after oral administration of naringenin chalcone. Each
value represents the mean ( SEM (n = 10).
Figure 6. Representative HPLC chromatograms of naringenin chalcone
metabolites in plasma at 310 nm. The plasma sample was collected
1 h after the administration of vehicle (A) or 20 mg/kg of naringenin
chalcone (B).
Figure 3. UV spectra of M1 (A), M2 (B), and M3 (C) in the urine of rat
administered naringenin chalcone.
liver microsomes (23, 24) and the intestinal microflora (25, 26)
were capable of effecting flavonoid ring-fission and conversion of
flavonoids to related phenolic acids and phloroglucinol. Narin-
genin chalcone might be converted to a phenolic acid that was not
identified in this study. Further investigation is required to detect
the ring-fission metabolites of naringenin chalcone.
Effect of Metabolites on Histamine Release from Rat Peritoneal
Mast Cells. Recently, several studies have reported that flavonoid
metabolites, such as (þ)-catechin and quercetin metabolites, have
biological activities (5, 27, 28). To determine the antiallergic
properties of the three metabolites of naringenin chalcone, we
studied histamine release assay with rat peritoneal mast cells. All
of the metabolites exhibited dose-dependent inhibition (data not
shown). The IC50 values of M1, M2, and M3 were 510.3 μM,
3.2 mM, and 2.8 mM, respectively. M1 showed stronger anti-
allergic activity than M2 and M3. The activity of M1 might be
lower than that of naringenin chalcone (IC50=33.1 μM) in this
study. Recently, it was reported that the metabolites of poly-
phenols can be deconjugated during inflammation and contribute
to its aglycone bioactivity (29). M1 might be deconjugated and
contribute to the antiallergic activity of naringenin chalcone in
vivo. Further investigation is required to detect the active form in
the inflammatory site.
Figure 4. Plasma concentration of naringenin chalcone-20-O-β-
D-glucur-
onide in rats after oral administration of naringenin chalcone. Each value
represents the mean ( SEM (n = 7).
dihydrochalcone, is metabolized to the corresponding glucuro-
nide and ring-fission metabolites such as p-hydroxyphenylpro-
pionic acid. There is some evidence to suggest that the enzyme in

Article
In conclusion, the major metabolites of naringenin chalcone
were tentatively identified as naringenin chalcone-20-O-β-
-glucuronide, naringenin-7-O-β- -glucuronide, and naringen-
in-40-O-β-
-glucuronide. These three metabolites were detected in
the urine, but only naringenin chalcone-20-O-β-
-glucuronide
was detected in the plasma. Naringenin chalcone-20-O-β-
-glucuronide inhibited histamine release, and this might con-
J. Agric. Food Chem., Vol. 57, No. 14, 2009 6437
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D
D
D
D
D
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ABBREVIATIONS USED
HEPES, 2-[4-(2-hydroxyethyl)-1-piperazinyl]ethanesulfonic
acid; HPLC, high-performance liquid chromatography; LC-
MS, liquid chromatography-mass spectrometry; NMR, nuclear
magnetic resonance; APCI, atmospheric pressure chemical ioni-
zation; SIM, selected ion monitoring mode; COSY, 1H-1H
correlation spectroscopy; HMQC, 1H-13C heteronuclear multi-
ple quantum coherence; HMBC, 1H-13C heteronuclear multiple
bond connectivity; SEM, standard error of the mean.
ACKNOWLEDGMENT
We acknowledge the advice of Dr. Taichi Yamamoto, Chil-
dren’s Hospital Oakland Research Institute, for this study. We
are grateful for the assistance of our laboratory staffs.
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Received April 6, 2009. Revised manuscript received June 11, 2009.
Accepted June 15, 2009.