Chromatographia (2012) 75:103–109
DOI 10.1007/s10337-011-2165-5
ORIGINAL
Simultaneous Determination of Resibufogenin and Its Major
Metabolite 3-epi-Resibufogenin in Rat Plasma by HPLC
Coupled with Tandem Mass Spectrometry
Huirong Chen • Sa Deng • Peter R. Chang •
Changyuan Wang • Xiaochi Ma • Kexin Liu •
Yan Tian • Jihong Yao • Xiaoyu Guo
Received: 30 August 2011 / Revised: 5 November 2011 / Accepted: 10 November 2011 / Published online: 11 December 2011
Ó Springer-Verlag 2011
Abstract A rapid, sensitive and specific method for the
simultaneous quantification of resibufogenin (RBG) and
3-epi-resibufogenin (3-ERBG) in rat plasma was developed
by using a liquid–liquid extraction procedure and liquid
chromatography–electrospray ionization/tandem mass
spectrometric (LC–ESI–MS/MS) analysis. The separation
was performed by HPLC on a reversed phase C18 HPLC
column (150 9 2.1 mm, 3.5 lm) using a mobile phase of
acetonitrilel-0.1% formic acid aqueous solution (45:55,
v/v). The determination was performed by a triple-quad-
rupole mass spectrometer in the multiple reaction moni-
toring using positive mode of electrospray ionization (ESI).
The calibration curves were both linear (R [ 0.995) over
the concentration range of 3.0–5,000 ng mL-1, and the
lower limits of quantification were 3.0 ng mL-1 for both
RBG and 3-ERBG. The intra-day and inter-day precisions
(% RSD) were all less than 15%, and the accuracies (%RE)
were within the range of ±15%. The mean recoveries of
RBG, 3-ERBG and IS were over 82.7, 84.8 and 90.0%
H. Chen and S. Deng contributed equally to this work.
H. Chen
S. Deng
C. Wang
X. Ma (&)
K. Liu
Y. Tian

J. Yao
School of Pharmacy, Dalian Medical University,
Dalian 116044, China
e-mail: maxc1978@sohu.com
H. Chen
X. Guo (&)
Department of Natural Medicines, School of Pharmaceutical
Sciences, Peking University, Beijing 100191, China
e-mail: guoxy2006@bjmu.edu.cn
P. R. Chang
Bioproducts and Bioprocesses National Science Program,
Agriculture and Agri-Food Canada, 107 Science Place,
Saskatoon, SK S7N 0X2, Canada
(n = 6), respectively. The method was proved to be rapid,
sensitive and specific, and has been successfully applied to
determine RBG and its major metabolite 3-ERBG in rat
plasma after oral administration of RBG for pharmacoki-
netic study. Comparison of pharmacokinetic data with anti-
tumor activities of RBG and ERBG suggested that
3-ERBG, as a major metabolite of RBG in rats, was per-
haps also a bioactive form of RBG in vivo.
Keywords Resibufogenin
3-epi-Resibufogenin

LC–ESI–MS/MS
Pharmacokinetics
Metabolite
Introduction
ChanSu, also called toad venom, is one of important tra-
ditional Chinese medicines (TCM), originated from the
skin secretions of Bufo bufo gargarizans Cantor and
B. melsanostictus Schneider. Chansu and its related prep-
arations, such as Liu-Shen pills and Niu-Huang-Xiao-Yan
tablets, are widely used for pain relief and anticancer in
China, Japan and other Asian countries [1, 2]. Resibufog-
enin (RBG) is a major bufadienolide with the content of
1–6% in Chansu, and it has strong cytotoxic activities
against human myeloid leukemia, prostate cancer and
human hepatoma cells with IC50 values of 1–10 nmol L-1
[3–5]. For some cancer cells, its activities were mediated
by induction of cell apoptosis and cell differentiation,
stronger than that of Taxol. However, as a cardioactive
steroid with a-pyrone ring, RBG can adversely affect the
myocardium to produce the most life-threatening toxicity
including ventricular ectopy and hyperkalemia with a
narrow therapeutic index [6, 7]. So it is necessary to
investigate the pharmacokinetic behaviors of RBG and its
metabolites in vivo to define the pharmacokinetic and
123
104
safety profiles, and to establish optimal doses for maximal
efficacy as preclinical and clinical trials.
To our knowledge, only some literature on pharmacoki-
netics of Chansu in animal plasma has been published
[8–10]. In our previous report, the 3-epi-resibufogenin
(3-ERBG) as a major metabolite was detected in the plasma
of rats after oral administration of RBG [11, 12]. The
metabolites of cinobufagin (CB), as a derivative of RBG
from Chansu, were investigated. And a series of CB
metabolites excreted from bile were isolated and identified as
the mono- or dihydroxylated derivatives of 3-epi-cinobufa-
gin (3-ECB) at C-1 and C-5 by 1H, 13C and 2D NMR tech-
nologies [13], all of which suggest that the isomerization of
bufadienolides such as RBG and CB was the major metabolic
reaction in rats. So, 3-ERBG would be an important meta-
bolic intermediate, and perhaps play a vital role in the
pharmacologic activities and toxicity. But compared with the
extensive literature on phytochemical and pharmacological
investigations, no investigation in simultaneously deter-
mining pharmacokinetics of RBG and its metabolite by LC–
ESI–MS/MS method was found. Due to its strong bioactiv-
ities and toxicities, it was very necessary to simultaneously
determine RBG and its metabolite in vivo, and to clarify the
relationships between pharmacologic activities and phar-
macokinetics of RBG and its metabolite.
In the present paper, a LC–ESI–MS/MS method was
developed and applied successfully for simultaneous deter-
mination of RBG and its major metabolite (3-ERBG) in rats
after oral administration of RBG. In addition, according to
the pharmacokinetics and cytotoxicity profiles, our results
suggested that 3-epi-resibufogenin (3-ERBG), as a major
metabolite, should be one of bioactive form in vivo.
Experiment
Chemicals and Materials
Reference standards of RBG and bufalin (Internal standard,
IS) were isolated from Chansu in our laboratory. Chan Su
was purchased from Ping-An Traditional Chinese Medicine
Trade (Qingdao, Shandong, China). Reference standard of
3-ERBG was prepared by microbial transformation from
Fusarium solani AS 3.1829 [11]. The chemical structures
of RBG, 3-ERBG and bufalin were identified on the basis
Table 1 Optimized multiple reaction monitoring (MRM) parameters for resibufogenin, 3-epi-resibufogenin and bufalin (IS)
Analyte Precursor ion Product ion Declustering potential (eV)
RBG 385.2 253.1 45
3-ERBG 385.2 253.1 45
Bufalin 387.4 351.3 40
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H. Chen et al.
of NMR and MS spectral analysis. Their purities were
[99.5% as determined by HPLC analysis. Deionized water
was prepared from a Milli-Q Reagent Water System
(Millipore, MA, USA). Acetonitrile and methanol (Tedia,
USA) were of HPLC grade. Other reagents were of ana-
lytical grade, and purchased from Tianjin Kermel Chemi-
cal Reagent (Tianjing, China).
Apparatus and Chromatographic Conditions
Agilent 1200 HPLC system consisted of a quaternary
delivery system, a degasser, an auto-sampler and UV-
detector. The chromatograph was equipped with an Elite
Kromasil C18 (150 9 2.1 mm, 3.5 lm) analytical column.
The mobile phase was consisted of acetonitrile-0.1% for-
mic acid aqueous solution (45:55, v/v) at a flow rate of
0.7 mL min-1. The run time was 4.0 min for each injec-
tion, and the column temperature was controlled at 30 °C.
An Applied Biosystems MDS Sciex API 3200 Triple
Quadrapole mass spectrometer (MS/MS) equipped with an
electrospray ionization (ESI) source was used, and the
system was operated in positive mode. Optimization of MS
conditions was carried out using standard solutions con-
taining 500 ng mL-1 of RBG, 3-ERBG and IS, delivered
via a Harvard syringe pump at a constant flow rate of
8 lL min-1. The optimized ionspray voltage and temper-
ature were set at 5,000 V and 600 °C, respectively. The
curtain gas (CUR) was set at 8 L min-1; gas 1 and gas 2
(nitrogen) were set at 55 and 60 psi, respectively, and the
dwell times were 200 ms. Nitrogen was used as the curtain
gas and collision gas, which were controlled at 13 and
6 psi, respectively. The quantification assay was performed
using multiple reaction monitoring (MRM). The optimized
MRM parameters and product ions of detected analytes and
IS were listed in Table 1.
Preparation of Stock and Working Solutions
The standard stock solutions of RBG, 3-ERBG and bufalin
were prepared in acetonitrile with final concentrations of
5 mg mL-1. The stock solutions were further diluted with
acetonitrile together to achieve standard working solutions
with concentrations of 15, 25, 100, 250, 2,500, 5,000, and
25,000 ng mL-1 for RBG and 3-ERBG, respectively. Each
working standard solution contains both of the two analytes
Entrance potential (eV) Collision energy (eV)
9 27
9 27
6 25
Simultaneous Determination of Resibufogenin
with the same concentration. Internal standard working
solution (1,500 ng mL-1) was prepared by diluting the
stock solution of bufalin with acetonitrile. All the stock and
working solutions were kept at -20 °C.
Calibration Curves
Calibration curves were prepared by spiking 20 lL of the
above-mentioned working standard solutions of RBG and
3-ERBG into 100 lL of blank plasma to obtain the series
of standard solutions (3.0, 5.0, 20, 50, 250, 1,000, and
5,000 ng mL-1) for calibration curves.
Sample preparation
A 100-lL aliquot of plasma sample was combined with
20 lL IS, then vortexed for 30 s. Samples were then
extracted with 400 lL ethyl acetate by vortex-mixing for
5.0 min. After centrifugation at 10,000g for 5 min, the
above extraction was repeated twice. The upper organic
layer was collected and evaporated to dryness at 35 °C
under a gentle stream of nitrogen gas. The residue was
dissolved in 100 lL mobile phase and a 10-lL aliquot was
injected into the LC–MS/MS system.
Animal Handling and Sample Collection
Male Sprague–Dawley rats, weighing approximately
230–250 g, were provided by the SPF animal experiment
center of Dalian Medical University. The animal experi-
mentation was approved by Dalian Medical University. Six
rats received an oral administration of 10 mg kg-1 RBG.
The drug was suspended in aqueous solution containing
10% propylene glycol. Blood samples (300 lL) were col-
lected in heparinized tubes from each rat at 0, 2, 5, 10, 15,
30, 45, 60, 120, 240, 480, and 720 min after administration.
Blood samples were immediately centrifuged and then the
plasma samples were stored at -20 °C before analysis.
Plasma collected from six vehicle-administrated rats served
as blank samples.
Determination of the Pharmacokinetic Parameters
Pharmacokinetic parameters including the elimination rate
constant (Ke), maximal concentration (Cmax), time for
maximal concentration (tmax), half-life (t1/2Ka and t1/2Ke),
the area under the concentration–time curve from zero to
the time of the final measurable sample (AUC0–t), the area
under the plasma concentration–time curve from zero to
infinity (AUC0–?), the area under the first moment curve
(AUMC), and the mean residence times (MRT) were cal-
culated with 3P97 software (Chinese Society of Mathe-
matical Pharmacology).
105
Bioassay
HeLa, HL-60, and Bel-7402 cells were maintained in
RPMI1640 medium (GIBCO/BRL; MD, USA) supple-
mented with 10% (v/v) fetal bovine serum and culture in
96-well microtiter plates for the assay. Appropriate dilu-
tions of both analytes were added to the cultures. And then
cells were cultured at 37 °C, 5% CO2 for 72 h. The sur-
vival rates of the cancer cells were evaluated by the MTT
method. The activity was shown as the IC50 value, which is
the concentration of test compound given 50% inhibition of
cell growth. Results were expressed as the mean value of
triplicate determinations.
Results and Discussion
Chromatography
Different ionization methods (including positive and neg-
ative modes) were tested to obtain good specificities and
sensitivities for RBG and 3-ERBG. The positive ionization
mode was more appropriate for both analytes detection
than the negative mode. The positive ion electrospray mass
spectra of RBG, 3-ERBG and IS in full-scan mode showed
quasi-molecular ions [M ? H]? as the base peaks, at m/z
385.2 for RBG and 3-ERBG, and m/z 387.4 for IS,
respectively. Fragmentation patterns of the quasi-molecular
ions were observed by increasing the collision energy. The
product ion mass spectra of RBG and 3-ERBG are shown
in Fig. 1. The most intense product ions were observed at
m/z 253.1 for RBG and 3-ERBG, and m/z 351.3 for IS,
respectively.
The different organic solvents as mobile phase were
tested. The results suggested that acetonitrile could sig-
nificantly increase the response signals of both analytes.
Formic acid was added to the mobile phase for helping to
form the protonated molecule ion [M ? H]?. After opti-
mization, a proper concentration of formic acid in the
mobile phase was chosen. In order to achieve a short run
time for the simultaneous analysis, the rate of acetonitrile
in mobile phase was increased, and all analytes were eluted
rapidly within 4.0 min with the satisfactory separation
(Fig. 2). The retention times of RBG, 3-ERBG and IS were
approximately 2.8, 3.4 and 1.8 min, respectively.
Method Validation
Assay Selectivity and Matrix Effect
The LC–MS/MS method has high selectivity because only
selected ions produced from selected precursor ions are
monitored. Comparison of the blank and the spiked rat
123
106
Fig. 1 Full-scan product ion
spectra of [M ? H]? ions and
fragmentation pathways for
(a) resibufogenin, (b) 3-epi-
resibufogenin, and c IS (bufalin)
plasma indicated no significant interference at the retention
times of the analytes and IS.
The possibility of a matrix effect caused by ionization
competition occurring between the analytes and endoge-
nous co-eluents was evaluated at three concentrations
(n = 3). The matrix effect experiments were carried out by
comparing the mass peak responses of the post-extraction
spiked samples with that of pure standards in mobile phase.
Our results suggested that the matrix effect occurred was
negligible in this method. The rates of the peaks response
were acceptable in the ranges from 91.5 to 101.0%.
Calibration Curves
The calibration curves for spiked rat plasma containing each
analyte were good linear in the range 3.0–5,000 ng mL-1
with a correlation coefficient r [ 0.9990. The regression
equation of the target curves and their correlation coeffi-
cients were calculated as follows: resibufogenin,
y = 7.82 9 10-3x ? 1.63 9 10-2 (r = 0.9992); 3-epi-
resibufogenin, y = 1.56 9 10-2x ? 2.37 9 10-2 (r =
0.9991). Signals 10 times higher than the peak noise height
was regarded as the lower limit of quantification (LLOQ).
The lower limits of quantification for determination of each
123
H. Chen et al.
analyte in plasma were both 3.0 ng mL-1. These LLOQs are
sufficient for the pharmacokinetic studies of two analytes
after an oral administration of RBG to rats.
Precision and Accuracy
The accuracy of the method was determined by calculating
the percentage deviation observed in the analysis of QCs.
Intra- and inter-day precision was assessed from the results
of QC samples by using a one-way analysis of variance
(ANOVA). The mean values and RSD for QC samples at
three concentration levels were calculated in triplicate.
Table 2 summarized the intra- and interday precision and
accuracy for two analytes from the QC samples. All intra-
and inter-day precision and accuracy were acceptable.
Extraction Recovery
Spiked plasma samples were prepared at concentrations of
5, 100, and 2,000 ng mL-1, and assayed as described
above. Extraction recovery was calculated by comparing
the peak area obtained from the extracted sample with that
of standard solution containing the same concentration of
analytes and IS in the blank plasma. For both analytes at
Simultaneous Determination of Resibufogenin 107

Fig. 2 Representative MRM

chromatograms: (a) a blank rat
plasma sample; (b) a blank rat
plasma sample spiked with
resibufogenin at the LLQ
(3.0 ng mL-1), 3-epi-
resibufogenin at the LLOQ
(3.0 ng mL-1) and IS (bufalin,
150 ng mL-1); (c) a plasma
sample from a rat at 2 min after
oral administration of
resibufogenin at a dose of
10 mg kg-1. I resibufogenin
(m/z 385.2 ? 253.1) and
3-epi-resibufogenin
(m/z 385.2 ? 253.1); II IS
(m/z 387.4 ? 351.3)

Stability
Table 2 Precision and accuracy of resibufogenin and 3-epi-resibuf-
ogenin in rat plasma determined by LC–ESI–MS/MS assay (n = 6)
The stability of both analytes in plasma sample was
Analyte Added C Found C Intra-day Inter-day Accuracy
assessed by spiking the standard solution of RBG and
(ng mL-1) (ng mL-1) RSD RSD (%)
(%) (%) 3-ERBG in blank plasma at three different concentrations.
Both analytes were found to be stable under a variety of
RBG 5 5.1 5.2 8.6 102.3 storage and process conditions, the ranges of the accuracy
100 102.8 3.6 5.7 102.8 for the analytes were 86.4–113.8% (Table 4).
2,000 2,169.4 2.8 11.5 108.5
3-ERBG 5 5.0 4.3 9.6 100.7 Application of the Analytical
100 103.1 2.4 13.7 103.1 Method to Pharmacokinetic Studies
2,000 2,122.8 2.1 12.1 106.1
The present method was successfully applied to the phar-
macokinetic study of RBG and 3-ERBG after an oral
administration of 10 mg kg-1 RBG to six male rats. The
the plasma concentrations of 5, 100 and 2,000 ng mL-1, mean plasma concentration–time profiles of RBG and
the mean recoveries of RBG, 3-ERBG and IS were over 3-ERBG are presented in Fig. 3. It can be seen that two
82.7, 84.8 and 90.0% (n = 6), respectively, shown in analytes were rapidly eliminated from rat plasma. The
Table 3, which suggesting that a single step of liquid– concentration–time curves of two analytes in rat plasma
liquid extraction with EtOAc should be simple and fitted a one-compartment model. The main pharmacoki-
successful. netic parameters were listed in Table 5.

123
108 H. Chen et al.

To our knowledge, the transformation of RBG to 3-ERBG is 128 times that of RBG, which suggests that the
3-ERBG should use the 3-ketone-RBG as an intermediate, amount of 3-ERBG in vivo was obviously higher than that
which may include the involvement of CPY450 and other of RBG, and that RBG could be transformed to 3-ERBG in
enzymes [14–16]. In addition, small amounts of 3-ERBG rats. Comparison of pharmacokinetic data of RBG and
were detected in the intestinal contents of rats, and the ERBG with anti-tumor activities suggested that 3-ERBG,
plasma concentration of 3-ERBG could be detected in large
amounts in a short time of administration. These results
implied that RBG could be metabolized rapidly in vivo and
the formation of 3-ERBG maybe carried out in liver and
intestinal tract at the same time. The metabolic enzyme
systems and transporters of RBG should be investigated in
the future.
The in vitro cytotoxicities of RBG and 3-ERBG against
human cancer cell lines (HeLa, HL-60 and Bel-7402) were
also investigated. 3-ERBG showed cytotoxic activities with
IC50 values of 1.05 lM (Hela), 1.48 lM (HL-60) and
0.58 lM (Bel-7402), respectively, while the IC50 values
of RBG were 7.9 9 10-2 lM (Hela), 9.6 9 10-2 lM
(HL-60) and 6.8 9 10-2 lM (Bel-7402), respectively. Fig. 3 Mean plasma concentration–time profiles of resibufogenin
This evidence suggested that both RBG and 3-ERBG had and its metabolite after a single oral dose of resibufogenin to rats,
cytotoxic activities, but the activities of 3-ERBG against each point represents mean ± SD (n = 6)
three cancer cell lines were weaker than those of RBG.
From the pharmacokinetic data of RBG and 3-ERBG
(a major metabolite), it can be concluded that tmax of Table 5 Pharmacokinetic parameters of RBG and its metabolite
RBG is obviously shorter than that of 3-ERBG, Cmax 3-ERBG after oral administration of 10 mg kg-1 RBG in rats
(mean ± SD, n = 6)
of 3-ERBG is about 60 times that of RBG, and AUC0–? of
Parameter RBG 3-ERBG

Table 3 Recoveries of resibufogenin and 3-epi-resibufogenin in rat Ke (1 min-1) 0.025 ± 0.013 0.0097 ± 0.0029
plasma (mean ± SD, n = 6) t1/2(Ka) (min) 1.67 ± 1.52 4.15 ± 2.18
Analyte Added concentration Extraction RSD t1/2(Ke) (min) 37.9 ± 25.10 76.8 ± 22.01
(ng mL-1) recovery ± SD (%) (%) Tmax (min) 6.94 ± 5.63 17.5 ± 6.55
RBG 5 84.9 ± 3.8 4.5 Cmax (ng mL-1) 25.4 ± 13.02 1,457 ± 842
100 82.7 ± 11.9 14.4 CL/F 0.027 ± 0.048 0.000074 ± 0.000044
(mg kg-1 min-1)
2,000 83.9 ± 3.5 4.2
AUC0–? [(ng mL-1) 1,648 ± 948 2.106 9 105 ± 1.071 9 105
3-ERBG 5 93.2 ± 9.3 10.0 min]
100 84.8 ± 10.2 12.0 AUC0–t [(ng mL-1) 1,322 ± 1023 2.083 9 105 ± 1.085 9 105
2,000 86.4 ± 3.1 3.6 min]
IS 150 90.0 ± 2.4 2.7 MRT0-? (min) 56.8 ± 32.65 124 ± 37.12

Table 4 Stability of the resibufogenin and 3-epi-resibufogenin in rat plasma (n = 6)

Analyte Added (ng mL-1) Recovery (%, mean ± SD)
Storage at room Storage at 4 °C Storage at -20 °C 3 freeze–thaw
temperature for 8 h for 24 h for 30 days cycles

RBG 5 86.4 ± 2.90 113.8 ± 9.20 101.4 ± 8.00 96.2 ± 7.10

100 93.0 ± 4.80 102.6 ± 3.40 104.8 ± 6.40 108.2 ± 2.70
2,000 85.7 ± 2.20 103.4 ± 3.50 100.6 ± 3.40 109.2 ± 4.10
3-ERBG 5 93.8 ± 5.90 113.6 ± 8.40 100.1 ± 6.10 97.0 ± 7.00
100 92.3 ± 3.00 95.7 ± 3.50 109.2 ± 7.70 103.8 ± 6.00
2,000 91.6 ± 3.80 96.1 ± 4.10 107.6 ± 4.80 100.7 ± 4.90

123
Simultaneous Determination of Resibufogenin
as a major metabolite of RBG in rats, was also a bioactive
form of RBG in vivo. The pharmacological function and
toxicities of 3-ERBG will be evaluated in the future.
Conclusion
In this paper, a rapid, sensitive and specific LC–ESI–MS/
MS method was developed and validated for simultaneous
quantification of RBG and its metabolite (3-ERBG) in rat
plasma. Due to the side effect of RBG, the present LC–
ESI–MS/MS method could be used for preclinical and
clinical pharmacokinetic evaluation. In addition, our
research also indicated that 3-ERBG, as the major metab-
olite of RBG, was perhaps also the bioactive form after oral
administration of RBG.
Acknowledgments We thank Education Department of Liaoning
Province (LS2010059), Program for Liaoning Excellent Talents in
University (LNET) and National Natural Science Foundation of
China (30701088, 81073013 and 81160559) for financial support.
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