Stable isotope labeling strategy for curcumin metabolite study in human liver microsomes by liquid chromatography-tandem mass spectrometry

Article abstract of DOI:10.1007/s13361-014-1064-z

The identification of drug metabolites is very important in drug development. Nowadays, the most widely used methods are isotopes and mass spectrometry. However, the commercial isotopic labeled reagents are usually very expensive, and the rapid and convenient identification of metabolites is still difficult. In this paper, an ¹⁸O isotope labeling strategy was developed and the isotopes were used as a tool to identify drug metabolites using mass spectrometry. Curcumin was selected as a model drug to evaluate the established method, and the ¹⁸O labeled curcumin was successfully synthesized. The non-labeled and ¹⁸O labeled curcumin were simultaneously metabolized in human liver microsomes (HLMs) and analyzed by liquid chromatography/mass spectrometry (LC-MS). The two groups of chromatograms obtained from metabolic reaction mixture with and without cofactors were compared and analyzed using Metabolynx software (Waters Corp.; Milford, MA, USA). The mass spectra of the newly appearing chromatographic peaks in the experimental sample were further analyzed to find the metabolite candidates. Their chemical structures were confirmed by tandem mass spectrometry. Three metabolites, including two reduction products and a glucuronide conjugate, were successfully detected under their specific HLMs metabolic conditions, which were in accordance with the literature reported results. The results demonstrated that the developed isotope labeling method, together with post-acquisition data processing using Metabolynx software, could be used for fast identification of new drug metabolites.

Full text of DOI:10.1007/s13361-014-1064-z

B American Society for Mass Spectrometry, 2015
J. Am. Soc. Mass Spectrom. (2015)
DOI: 10.1007/s13361-014-1064-z
RESEARCH ARTICLE
Stable Isotope Labeling Strategy for Curcumin
Metabolite Study in Human Liver Microsomes by Liquid
Chromatography-Tandem Mass Spectrometry
1
,2
1,2
5
1,2
3
5
Dan Gao, Xiaowu Chen, Xiaomei Yang, Qin Wu, Feng Jin, Hongliang Wen,
1,4
1,2
Yuyang Jiang, Hongxia Liu
1
State Key Laboratory Breeding Base-Shenzhen Key Laboratory of Chemical Biology, Graduate School at Shenzhen, Tsinghua
University, Shenzhen, 518055, China
Key Laboratory of Metabolomics at Shenzhen, Shenzhen, 518055, China
Neptunus Pharmaceutical Technology Center, Shenzhen, 518057, China
School of Medicine, Tsinghua University, Beijing, 100084, China
2
3
4
5
School of Chemical Engineering and Environment, Beijing Institute of Technology, Beijing, 100081, China
Abstract. The identification of drug metabolites is very important in drug develop-
ment. Nowadays, the most widely used methods are isotopes and mass spectrom-
etry. However, the commercial isotopic labeled reagents are usually very expensive,
and the rapid and convenient identification of metabolites is still difficult. In this paper,
18
an O isotope labeling strategy was developed and the isotopes were used as a tool
to identify drug metabolites using mass spectrometry. Curcumin was selected as a
18
model drug to evaluate the established method, and the O labeled curcumin was
18
successfully synthesized. The non-labeled and O labeled curcumin were simulta-
neously metabolized in human liver microsomes (HLMs) and analyzed by liquid
chromatography/mass spectrometry (LC-MS). The two groups of chromatograms
m/z
obtained from metabolic reaction mixture with and without cofactors were compared and analyzed using
Metabolynx software (Waters Corp., Milford, MA, USA). The mass spectra of the newly appearing chromato-
graphic peaks in the experimental sample were further analyzed to find the metabolite candidates. Their chemical
structures were confirmed by tandem mass spectrometry. Three metabolites, including two reduction products
and a glucuronide conjugate, were successfully detected under their specific HLMs metabolic conditions, which
were in accordance with the literature reported results. The results demonstrated that the developed isotope
labeling method, together with post-acquisition data processing using Metabolynx software, could be used for
fast identification of new drug metabolites.
18
Keywords: Curcumin, O isotope labeling, Metabolite, HLMs, HPLC/QqQ-MS
Received: 27 September 2014/Revised: 18 November 2014/Accepted: 9 December 2014
derived from drug metabolites are weaker than the signals from
Introduction
complex matrix [2]. Isotopic tracers in which a specific atom or
atoms replaced with a different isotope have been used as a key
technique to analyze the metabolic pathway [3]. The isotopic
tracers can be either radioactive or stable; however, the major
disadvantage of radiolabeling is the risk of exposure in humans.
Therefore, stable isotope labeling has become the mostly wide-
ly used method for the structural identification of metabolites in
drug discovery, especially for new drugs [4]. Stable isotopes
such as carbon-13 [5, 6], nitrogen-15 [7, 8], and deuterium [9–
he investigation of drug metabolites in vitro and in vivo is
T
one of the essential studies in the early stages of drug
development [1]. Meanwhile, more drug safety evaluation is
needed because metabolites generated from phase I biotrans-
formations are more likely to be chemical reactive, pharmaco-
logically active, or toxic. However, the identification of drug
metabolites is very difficult because the analytical signals
12] could be incorporated strategically into drug molecules to
assist the structure elucidation of their metabolites using LC/
MS and NMR. Numerous studies have reported using the
Correspondence to: Hongliang Wen; e-mail: wen.hongliang816@gmail.com,
Hongxia Liu; e-mail: liuhx@sz.tsinghua.edu.cn

D. Gao et al.: Stable Isotope Labeling for Metabolite Study
mixtures of equal amount of non-labeled and isotope-labeled
compounds to study in vitro and in vivo metabolic disposition
and compounds [13–15]. Because the labeled and unlabeled
analogues retained almost identical physicochemical proper-
ties, the separation of the parent compound or its metabolites to
form a twin ion pair pattern by mass spectrometry could help
detect and identify the unknown drug metabolites. Moreover,
the incorporation of stable isotopes into the parent compound
also provides an opportunity to explore the possible metabolic
pathways or the enzymes responsible for the formation of the
metabolites. Another example is the use of oxygen-18 labeled
superfamily is a large and diverse group of enzymes that is
involved in drug metabolism and bioactivation, and 75% of the
total number of different metabolic reactions are catalyzed by
them [37]. Human liver microsomes (HLMs) are commonly
used as the carrier of P450 and have been widely used for
in vitro drug metabolism studies because of advantages includ-
ing low cost, simplicity of use, and fewer ethical objections
[38, 39].
Because of the importance of a drug metabolism study, we
1
8
developed an O isotope labeling strategy to analyze the
metabolites of curcumin in HLMs by LC-MS with
Metabolynx analyzer (Waters Corp., Milford, MA, USA).
Owing to the clear metabolic pathways of curcumin, it was
selected as a model drug to evaluate the reliability of the
1
8
18
oxygen molecules ( O ) and water (H O) in metabolism
2
2
studies to understand the metabolic pathways or metabolites
structures [16, 17]. However, the incorporation of oxygen-18
into the compounds for drug metabolism research has not been
reported. Currently, liquid chromatography-tandem mass spec-
trometry (LC-MS/MS) has become an important and reliable
analytical technique for metabolites identification because of
its high separation ability and the ability to provide structural
information. Furthermore, tandem mass spectrometry (MS/
MS) can be used to confirm chemical structures of metabolites
through their mass-to-charge ratio (m/z) and MS/MS fragmen-
tations with high resolution [18].
1
8
established method. The O labeled curcumin was synthe-
sized, and the reaction products mixture, including curcumin,
1
8
18
[ O]-curcumin, and [ O ]-curcumin were then simultaneous-
2
ly metabolized by HLMs—three major metabolites including
two reduction products and a glucuronide conjugate, in accor-
dance with the literature reported results. Our developed meth-
od with the advantages of minimal operator intervention and
reduced data interpretation time is expected to be applied for
the metabolism study of other compounds by the stable isotope
labeling strategy.
Curcumin, [1,7-bis(4-hydroxy-3-methoxyphenyl)-1,6-
heptadiene-3,5-dione], is a potent antioxidant extracted from
the powered rhizomes of turmeric (Curcuma longa). Turmeric
is widely used as a food coloring and flavoring agent. Many Experimental
studies have shown that curcumin possessed a wide range of
Chemicals and Reagents
biological and pharmacological activities including anti-in-
flammatory, anti-viral, anti-oxidant, anti-carcinogenic, and
cancer chemoprevention activities [19–23]. The studies have
indicated that it has no serious toxicity on animal models or
humans even at very high doses. Its wonderful biological
activities and pharmacological safety make it as a potential
therapeutic reagent for the treatment of several human diseases
1
2
8
H
O was purchased from Shanghai Engineering Research
1
8
Center of Stable Isotope (Shanghai, China). K OH was pre-
1
2
8
pared by the reaction of H
O with t-BuOK in dry tetrahydro-
furan and dried under vacuum before use. All other chemicals
were of analytical grade. All solvents used in this study were
HPLC-grade. Acetonitrile was obtained from Merck
(Darmstadt, Germany). Uridine 5′-diphosphoglucuronic acid
(UDPGA), nicotinamide adenine dinucleotide phosphate
(NADPH), D-saccharic acid-1,4-lactone, HLMs, magnesium
[24, 25]. However, there are several inherent drawbacks that
prevent its entering the market as a drug so far. The major
obstacles are the poor aqueous solubility and absorption, rapid
metabolism, chemical instability, and low oral bioavailability
chloride (MgCl ), and ammonium acetate were purchased from
2
[
26–28]. In addition, several researches have implied that
Sigma-Aldrich Chemical Co. (St. Louis, MO, USA).
curcumin metabolites might regulate some of the biological
effects of curcumin [29–31]. The metabolic pathways have
been widely studied. In vivo and in vitro studies have demon-
strated that curcumin undergoes both phase I and phase II
metabolism [32–34]. Phase I biotransformation of curcumin
generates serial reduction products by reduction of the olefinic
double bonds, including tetrahydro-curcumin, hexahydro-
curcumin, and octahydro-curcumin. Phase II metabolism of
curcumin is the conjunction of curcumin and its reductive
metabolites with glucuronic and sulfate. Glucuronidation is
the major phase II conjugation reaction of curcumin in humans
1
8
Synthesis of O Labeled Curcumin
18
As shown in Scheme 1, [ O]-2-methoxy-4-methylphenol was
obtained via hydroxylation of 1-iodo-2-methoxy-4-methyl-
18
18
benzene with K OH in t-BuOH-DMSO-H O catalyzed with
2
CuI/8-hydroxyquinoline. The oxidation of the methyl group at
C-4 of [ O]-2-methoxy-4-methylphenol with 2,3-dichloro-
18
5
,6-dicyano-1,4-benzoquinone (DDQ) in CH Cl at room tem-
2 2
18
perature gave [ O]-vanillin. Finally, condensation of vanillin
18
and [ O]-vanillin with pentane-2,4-dione gave curcumin,
18
18
[
O]-curcumin and [ O ]-curcumin.
2
[35]. Therefore, reduction and conjugation are regarded as the
common metabolic pathways of curcumin that take place in
hepatic and intestinal tissues [32, 36]. In mammals, the liver is
the major organ involved in the metabolism of various endog-
enous compounds and drugs. The cytochrome P450 (CYP)
Incubations with HLMs for Curcumin Metabolism
Metabolites of curcumin were generated by incubation with
HLMs in the presence of appropriate cofactors according to the

D. Gao et al.: Stable Isotope Labeling for Metabolite Study
(
a)
18
18
2
Scheme 1. Reagents and conditions: (i) CuI, 8-hydroxyquinoline, K OH, t-BuOH-DMSO-H O, 100°C, 48 h, 87.5%; (ii) DDQ,
2 2 2 3
CH Cl , rt, 4 h, 75.2%; (iii) pentane-2, 4-dione, B O , 1, 2, 3, 4-tetrahedroquinoline, acetic acid, 110°C, 24 h; (iv) aq acetic acid (20%),
20°C, 24 h, 80.5% (two steps)
reported procedures [40]. Briefly, HLMs were pre-incubated
on ice for 30 min. Curcumin and O isotope-labeled curcumin
temperature was set at 25°C and the injection volume was
10 μL.
1
8
dissolved in DMSO with the final concentration of 20 μg, 10
The MS system consisted of an ESI interface and a triple
quadrupole (QqQ) mass analyzer. The MS parameters were
as follows: capillary voltage was set at 3.0 kV and cone
voltage was maintained at 25 V in negative ion mode.
Source temperature was kept at 120°C whereas the
desolvation temperature was 250°C. N2 was used for
mM MgCl , and 0.15 mg HLMs were added to 100 mM
2
phosphate buffer solution (pH 7.4) to incubate for 5 min at
3
7°C. Subsequently, NADPH with the final concentration of 1
mM was added to start the reactions and incubated at 37°C in
shaking incubator block to generate reduction metabolites.
After 90 min, reactions were terminated by adding 150 μL of
ice-cold acetonitrile. For glucuronidation of curcumin by
HLMs, 20 μg curcumin, and O-18 isotope-labeled curcumin
dissolved in DMSO, 0.15 mg HLMs in 100 mM phosphate
–
1
desolvation gas with the flow rate of 500 L h . For MS/
MS analysis, the collision energy was set at 15 eV whereas
–
1
the flow rate of the collision gas was 0.15 mL min . The
samples were analyzed in full-scan and selected ion record-
ing (SIR) mode. All data collected in centroid mode were
processed using a Waters Metabolynx (v 4.1) program.
buffer solution (pH 7.4), 10 mM MgCl , 10 mM D-saccharic
2
acid 1,4-lactone, and 2 mM UDPGA were incubated at 37°C
for 1.5 h. Control incubations were conducted with HLMs in
the absence of NADPH or UDPGA. All incubations were
carried out in triplicate. Samples were vortexed and centrifuged
at 10,000g for 5 min at 4°C. The supernatants were filtered and
analyzed by HPLC-MS.
Data Analysis and Metabolite Identification
The chromatograms of the in vitro metabolic reaction
1
8
mixture from the non-labeled and the
O isotope-
labeled curcumin with or without cofactors were collect-
ed by separate LC-MS measurements. The reaction mix-
ture without addition of cofactors was regarded as con-
trol. By comparison of the chromatograms corresponding
to experimental and control samples, several new chro-
matographic peaks could be easily found in the chro-
matogram of the experimental sample using Metabolynx
software. Metabolynx is a post-acquisition data process-
ing software that employs an extensive list of potential
biotransformation reactions consisting of hydrogenation
and glucuronidation, in combination with the elemental
compositions of the substrate molecules, to generate a
series of extracted ion chromatograms (EICs). These
EICs are compared between the control and experimental
sample to eliminate those chromatographic peaks in the
LC-MS Analysis
LC/MS analysis was performed on a system equipped with an
Alliance HPLC Waters 2695 system (Waters) and a Quattro
Premier XE mass spectrometer (Waters). The HPLC system
was comprised of a quaternary pump, an online degasser, a
column heater, and an autosampler. HPLC separation of
curcumin and its metabolites was carried out on a X Bridge
C8 column (2.1 mm × 100 mm i.d., 3.5 μm, Waters) with the
following solvent system: A = 5 mM ammonium in water, B =
5
mM ammonium in acetonitrile in negative ion mode at a flow
–
1
rate of 0.3 mL min . Elution gradient was linearly increased
from 10% to 90% B within 30 min, followed by return to 10%
B. Total running time was 35 min per separation. Column

D. Gao et al.: Stable Isotope Labeling for Metabolite Study
sample that also appear in the control. Only those peaks
with the area ratio of experimental to control sample
above 5 were considered to be the compound-related
metabolites. The mass spectra of these chromatographic
peaks should be further analyzed to validate whether it
was the compound-related metabolites. Finally, the chem-
ical structures of the metabolite candidates were further
confirmed by tandem mass spectrometry (MS/MS).
sitivity detection method and new strategies to solve
1
8
these problems. In this study, an O isotope labeling
strategy was applied for the discovery of metabolite
1
8
candidates. O isotope-labeled curcumin was synthesized
and was used as a model to evaluate the feasibility of
1
8
the method. Briefly, 20 μM of non-labeled and
O
isotope-labeled curcumin mixture were incubated in
HLMs with and without cofactors (corresponding to ex-
perimental sample and control sample), and the reaction
mixtures were then analyzed by LC-MS. The discovery
of metabolite candidates is to compare the chromato-
grams corresponding to experimental and control samples
using Metabolynx software and to find those chromato-
graphic peaks only present in the experimental sample.
Only those peaks with the area ratio of experimental to
control sample above 5 were considered to be the poten-
tial metabolites of curcumin. The representative chro-
matographic results of the two groups obtained from
the experimental group and control group about the
glucuronidation and reduction system are shown in
Figure 2. During the data processing using Metabolynx
software, a large number of peaks were generated be-
cause of the existence of unrelated peaks. Therefore, we
must pick up some peaks that are likely to be curcumin-
related metabolites. For glucuronidation of curcumin,
four additional chromatographic peaks with high signal
intensity were found present in the experimental sample,
which are indicated by vertical red lines in Figure 2a and
b. Therefore, they were selected for further analysis to
find the potential glucuronide metabolites. Taking the
results from the Metabolynx software together with the
MS data containing three m/z values with specific mass-
shift of 2.00 ± 0.02 Da, it was indicated that only the
mass spectrum of the chromatographic peak at the reten-
tion time of 9.28 had the matched mass-shift, which was
suspected to the glucuronide metabolite, and the three
ions had the m/z values of 542.78, 544.77, and 546.79,
respectively, as shown in Figure 3. Besides, the relative
mass spectrometry signal intensities of the three ions
were similar with the labeled and non-labeled parent
compounds. From the corresponding m/z values obtained
from the glucuronidation reaction system, we deduced
that the most probable metabolite was the glucuronide
conjugate of curcumin. The results of the curcumin re-
duction in HLMs are shown in Figure 2c and d. From
the results, we could see that several chromatographic
peaks were found at the retention time of 16.65 min and
from 24.71 to 30.69 min in the experimental sample, but
there were no three paired ions with the mass difference
of 2.00 ± 0.02 Da from the retention time of 24.71 to
30.69 min. However, the mass spectrum of the chromato-
graphic peak at the retention time of 16.65 min had
several groups of ions with m/z values of 367.05,
Results and Discussion
Characterization of the Synthesized
18
O Isotope-Labeled Curcumin
1
8
O isotope-labeled curcumin were synthesized according to
the procedures described in Scheme 1. The chemical structures
of synthesized labeled and unlabeled curcumin were character-
ized by ESI-QqQ-MS/MS. Mass spectrometry results were
obtained in the mass range m/z 50–1000 in the negative ion
–
mode. As shown in Figure 1, curcumin with [M – H] =
1
8
–
18
3
66.94, [ O]-curcumin with [M – H] = 368.92, and [ O ]-
2
–
curcumin with [M – H] = 370.94 could be detected. The ratio
of their relative mass signal intensities was about 2:1:1. The
result indicated that the amounts of the reaction-generated
1
8
18
[
O]-curcumin and [ O ]-curcumin were nearly the same
2
because of the similar chemical properties and similar ioniza-
tion capacity.
Strategy for Metabolite Discovery
and the Identification of Its Metabolites
The detection of drug metabolites is very important but it
is very difficult because most of the metabolites in real
biological samples are at very low concentrations. In
addition, the interference signals from the background
will also be serious. Therefore, it requires a higher sen-
3
66.94
100
3
68.92 370.94
0
3
52 356 360 364 368 372 376 380 384
m/z
18
Figure 1. Mass spectrum of curcumin and O isotope labeled
3
69.03, 371.08, 373.04, and 375.06, as shown in the
curcumin. Spectrum was obtained in the negative ion mode

D. Gao et al.: Stable Isotope Labeling for Metabolite Study
(a)
1
00 0.96
Control
1
.06
7
.37
7
.97
.41
1
5.26
16.49
8
20.6923.01
25.83
27.60 30.46
34.33
1
0.88
0
5.00
10.00
15.00
20.00
25.00
30.00
35.00
40.00
0
.87
(b)
Glucuronidation
1
00
1
.08
.11
.24
1
2
7.65
1
9
.32
28.31
7
.95
11.36
20.68
26.46
33.34
34.02
7
.47
16.40
2
3.00
0
5.00
10.00
15.00
20.00
25.00
30.00
35.00
40.00
Time
(c)
1
00 0.96
Control
1
.06
7
.37
7
.97
.41
1
5.26
16.49
8
20.6923.01
25.83
27.60 30.46
34.33
1
0.88
0
5
.00
10.00
15.00
20.00
25.00
30.00
35.00
40.00
3
67.05
(d)
0.84
1
00
100
369.03
3
71.08
Reduction
1
.16
3
72.08
73.04
3
3
68.05
1
.23
370.03
3
75.06
0
3
54 358 362 366 370 374 378 382
m/z
2
7.75
7.81 30.20
2
0.80
2
1
6.65
24.62
26.16
1
9.97
32.33
2
4.71
0
5.00
10.00
15.00
20.00
25.00
30.00
35.00
40.00
Time
18
Figure 2. Analytical ion chromatograms of the mixtures of curcumin and O isotope labeled curcumin metabolized in HLMs
obtained in the negative ion mode. The metabolic reaction mixture without addition of cofactors was as control. (a, b)
Glucuronidation of the curcumin mixture; (c, d) reduction of the curcumin mixture. The insert mass spectrum was obtained from
the chromatographic peak at the retention time of 9.28 min. The vertical red lines or rectangle indicate the different chromatographic
peaks between the experimental and control samples
1
8
inset mass spectrum of Figure 2b. These ions could
match with the mass-shift of 2.00 ± 0.02 Da, but they
could not be confirmed as the reduction metabolites
because of the same molecular weight between reduction
metabolites and O-18-labeled curcumin. For example,
and tetrahydro-curcumin, dihydro-[ O ]-curcumin and
2
1
8
tetrahydro-[ O]-curcumin have the same molecular
weight. The major reason might be their overlapped
chromatographic peaks between the curcumin and its
reduction metabolites because of their similar chemical
structures and properties. Besides, the concentration of
1
8
18
[
O]-curcumin and dihydro-curcumin, [ O ]-curcumin
2

D. Gao et al.: Stable Isotope Labeling for Metabolite Study
5
42.78
100
hexahydro-curcumin were the major products among the re-
duction products of curcumin. Of those, tetrahydro-curcumin
was one of the major metabolites to exhibit the same physio-
logical and pharmacologic activities as curcumin [22, 41].
Therefore, the protonated ions at m/z 367, 369, 371, and 373
were simultaneously monitored by a split SIR descriptor. The
total ion chromatogram for the four ions monitored by SIR
HPLC-MS for the curcumin reduction system is shown in
Figure 4. It was observed that four chromatographic peaks
were completely separated within less than 15 min under the
gradient elution condition. However, the monitored ion at m/z
365 was not detected under the above conditions. This might be
caused by its extremely low trace level, which did not reach the
detection sensitivity of ESI-MS. The chromatographic peak at
the retention time of 0.79 min was the solvent peak. The
corresponding mass spectra of chromatographic peaks at the
retention time of 7.07, 8.62, and 12.72 min are shown in Figure
5
46.79
5
47.87
5
12.81
13.52
5
5
48.83
49.71
5
0
4
20
460
500
540
580
620
660
m/z
Figure 3. The corresponding mass spectrum of the chromato-
graphic peak at the retention time of 9.28 min. The spectrum
was obtained in the negative ion mode
5
. As shown in Figure 5a, c, and e, three paired ions with the
mass difference of 2.00 Da (m/z 371, 373, and 375; m/z 369,
71, and 373; m/z 367, 369, and 371) were, and their relative
reduction metabolites of curcumin might be at a very
low level. Therefore, a new analytical strategy or new
separation condition should be adopted to further confirm
the above detection result.
3
mass spectrometry signal intensities were similar to their parent
compounds. We considered that m/z 371 (367 + 4H), m/z 369
(
367 + 2H), and m/z 367 were tetrahydrocurcumin,
A new separation condition and a new data acquisition
mode were applied in the following experiment. The optimized
elution gradient condition was that A was linearly decreased
from 80% to 40% within 15 min. It was well known that
selected ion recording (SIR) mode had the advantage of en-
hanced sensitivity and reduced measurement time for the mon-
itored ions. In order to provide confirmation at the higher
resolution and to determine if identification was possible at
the lower resolution, mass spectra acquisition was made in SIR
mode, with a dwell time of 100 ms. Several researches have
shown that dihydro-curcumin, tetrahydro-curcumin, and
dihydrocurcumin, and curcumin, separately. Their structures
were further confirmed by MS/MS. An extra energy of 25 eV
was applied to the extracted ions m/z 371, 369, and 367 to get
the corresponding fragments. As shown in Figure 5b, the
fragment peaks of m/z 148.68 and 217.05 indicated the exis-
tence of curcumin. The fragment peaks of m/z 148.77 and
2
5
18.88 indicated the existence of dihydrocurcumin (Figure
d). The fragment peaks of m/z 150.85 and 218.92 indicated
the existence of tetrahydrocurcumin (Figure 5f).
Based on the data we obtained, we propose that major
metabolic pathways for curcumin were reduced to
dihydrocurcumin and tetrahydrocurcumin by an endogenous
reductase system in HLMs, and glucuronidated by UDP-
glucuronosyl transferases, as shown in Figure 6. Several
researches have reported that curcumin could be metabo-
lized via conjugation (glucuronidation and sulfation) and
reduction [42]. However, only glucuronidation and reduc-
tion products were detected in our experiment. The sulfate
conjugates were not detected, which was caused by the
absence of sulfatase enzyme in the HLMs metabolic reac-
tion system. Therefore, the metabolic results were nearly in
accordance with the literature reported under our experi-
mental conditions. The results of the study show that the
stable isotopic labeling strategy is an efficient approach to
find metabolite candidates by direct detecting signals de-
rived from labeled and non-labeled parent compounds with
a specified mass-shift, and it is nearly not influenced by the
metabolic complexity.
1
2.72
1
00
0
.79
8.62
8
.88
7.07
0
2.00
6.00
10.00
Time
14.00
18.00
Figure 4. A typical SIR chromatogram of four monitored ions
at m/z 367, 369, 371, and 375 in curcumin reduction system

D. Gao et al.: Stable Isotope Labeling for Metabolite Study
O
O
O
O-
H CO
3
H CO
3
OCH3
H CO
3
-
-O
HO
O
HO
3
67.00
(
a) 100
(b) 100
148.68
m/z 367
2
17.05
3
69.00
1
72.84
3
71.00
3
73.00
367.01
350
1
33.77
2
01.09
3
75.00
376
0
0
3
64
368
372
380
0
50
100
150
200
m/z
250
300
400
m/z
OH
O
H CO
3
H3CO
HO
OCH3
-
-O
O
3
69.00
148.77
(
c) 100
(d)100
m/z 369
O-
3
71.00
O
H3CO
HO
3
67.00
218.88
3
73.00
1
74.87
16.90
2
3
69.01
3
75.00
190.89
2
19.41
250
0
0
3
62
366
370
374
378
0
50
100
150
200
m/z
300
350
400
m/z
OH
O
H CO
3
H CO
3
OCH3
m/z 371
-O
-
HO
O
3
71.00
150.85
(
e) 100
(f) 100
O
O-
2
18.92 H CO
3
3
73.00
1
74.77
HO
3
75.00
1
35.77
220.98
3
71.03
2
17.60
3
69.00
266.15
3
67.00
0
0
3
66
368
370
372
374
376
0
50
100
150
200
m/z
250 300
350
400
m/z
Figure 5. The corresponding mass spectra of the chromatographic peaks at the retention times of 7.07, 8.62, and 12.72 min, and
the corresponding tandem mass spectrometry. Spectra were obtained in the negative ion mode

D. Gao et al.: Stable Isotope Labeling for Metabolite Study
O
O
H3CO
HO
OCH3
curcumin
OH
Reductase
UDP-glucuronosyl transferase
O
O
O
O
H3CO
HO
OCH3
OH
H3CO
OCH3
OH
HOOC
O
HO
HO
O
Dihydrocurcumin
OH
Curcumin-glucuronoside
Reductase
O
O
H3CO
HO
OCH3
OH
Tetrahydrocurcumin
Figure 6. Proposed biotransformation and metabolites of curcumin in HLMs containing NADPH and UDPGA cofactors
4
. Chen, Q., Wu, J., Zhang, Y., Lin, J.M.: Qualitative and quantitative analysis
of tumor cell metabolism via stable isotope labeling assisted microfluidic
chip electrospray ionization mass spectrometry. Anal. Chem. 84, 1695–
Conclusions
We developed an isotopic labeling strategy for the dis-
covery metabolites with Metabolynx software and the
stable isotopic mass-shift at the similar retention time.
The method is efficient and fast in detecting metabolite
candidates even under a complex matrix. Curcumin was
selected as a model compound to validate the feasibility
of our approach. Three metabolites, including two reduc-
tion products and one glucuronide conjugate, were suc-
cessfully detected under their specific HLMs metabolic
conditions. The results indicate that the isotopic labeling
approach together with the specific isotopic mass-shift
and automatic data analysis has the ability to identify
drug metabolites, which is very useful in drug discovery.
In addition, if using high resolution MS for detection,
the approach might become more accurate to filter a
specific mass-shift.
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Y.H., Khan, S.I., Adelli, V.R., Sahu, R., Elsohly, M.A., McChesney, J.D.,
Khan, I.A., Walker, L.A.: Profiling primaquine metabolites in primary
human hepatocytes using UHPLC-QTOF-MS with 13C stable isotope
labeling. J. Mass Spectrom. 48, 276–285 (2013)
. Liu, J., Tang, M.H., Lai, H.J., Dong, Y.F., Xie, C.F., Ye, H.Y., Ma, L., Qiu,
N., Li, Y.F., Cai, L.L., Chen, L.J.: Identification of metabolites of honokiol
in rat urine using C-13 stable isotope labeling and liquid chromatography
coupled with quadrupole time-of-flight tandem mass spectrometry. J.
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Acknowledgments
The authors acknowledge support for this work by the National
Youth Science Foundation (no. 21305074), the National Nat-
ural Science Foundation of China (no. 21172129 and no.
1
0. Lu, W.C., Sheen, J.F., Hwang, L.S., Wei, G.J.: Identification of
5,7,3',4'-tetramethoxyflavone metabolites in rat urine by the isotope-
labeling method and ultrahigh-performance liquid chromatography-
electrospray ionization-mass spectrometry. J. Agric. Food Chem.
2
1475073), the National High Technology Research and De-
6
0, 8123–8128 (2012)
velopment Program of China (no. 2013AA092902), and China
Postdoctoral Science Foundation (no. 2014M552238).
11. Wei, G.J., Sheen, J.F., Lu, W.C., Hwang, L.S., Ho, C.T., Lin, C.I.: Iden-
tification of sinensetin metabolites in rat urine by an isotope-labeling
method and ultrahigh-performance liquid chromatography-electrospray
ionization mass spectrometry. J. Agric. Food Chem. 61, 5016–5021 (2013)
1
2. Ruseva, S., Lozanov, V., Markova, P., Girchev, R., Mitev, V.: In vivo
investigation of homocysteine metabolism to polyamines by high-
resolution accurate mass spectrometry and stable isotope labeling. Anal.
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