Journal of Pharmaceutical and Biomedical Analysis 117 (2016) 372379

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Journal of Pharmaceutical and Biomedical Analysis

journal homepage: www.elsevier.com/locate/jpba

Simultaneous determination of four furostanol glycosides in rat

plasma by UPLCMS/MS and its application to PK study after oral
administration of Dioscorea nipponica extracts
Min Liao, Cong Dai, Mengping Liu, Jiefeng Chen, Zuanguang Chen, Zhiyong Xie,
Meicun Yao
School of Pharmaceutical Sciences, Sun Yat-sen University, Guangzhou 510006, PR China

a r t i c l e

i n f o

Article history:
Received 26 July 2015
Received in revised form
15 September 2015
Accepted 16 September 2015
Available online 21 September 2015
Keywords:
Dioscorea nipponica
UPLCMS/MS
Furostanol glycosides
Lithium adduct
Rat plasma
Pharmacokinetics

a b s t r a c t
A novel, sensitive and rapid ultra-performance liquid chromatographytandem mass spectrometric
(UPLCMS/MS) method for simultaneous quantication of four furostanol glycosides in rat plasma was
established and validated. Ginsenoside Rb1 was used as an internal standard. Plasma samples were pretreated by liquidliquid extraction with n-butanol and chromatographed on a C18 column (2.1 50 mm
i.d., 2.6 m) using a gradient elution program consisting of acetonitrile and water (containing 0.03%
formic acid and 0.1 mM lithium acetate) at a ow rate of 0.4 mL/min. Lithium adduct ions were employed
to enhance the response of the analytes in electrospray positive ionization mode and multiple reaction monitoring transitions were performed for detection. All calibration curves exhibited good linearity
(r > 0.999) over the range of 1020,000 ng/mL for protodioscin and 24000 ng/mL for protogracillin, pseudoprotodioscin and pseudoprotogracillin. The recoveries of the whole analytes were more than 80.3% and
exhibited no severe matrix effect. Meanwhile, the intra- and inter-day precisions were all less than 10.7%
and accuracies were within the range of 8.112.9%. The four saponins showed rapid excretion and relative high plasma concentrations when the validated method was applied to the PK study of Dioscorea
nipponica extracts by intragastric administration at low, medium and high dose to rats. Moreover, the
T1/2 and AUC0t of each compound turned out to behave in a dose-dependent pattern by comparing them
at different dose levels.
2015 Elsevier B.V. All rights reserved.

1. Introduction
Dioscorea nipponica (DN), a folk medicine widely used in China
for thousands of years [1], as the major active ingredient of some
famous Chinese patent medicine, Diao Xinxuekang capsule [2],
ChuanShanLong Injection [3] and Guge Fengtong preparation
[4], is an important medicinal plant with high content of saponins
in its rhizome. Up to now, numerous bioactivities of DN have been
documented. For instance, DN showed therapeutic effect on gouty
arthritis based on the SDF-1/CXCR 4 and p38 MAPK pathway both
in vitro and in vivo [5]. Besides, it also exhibited cytotoxicity against
human oral cancer HSC-3 cells [6], human HepG2 cell [7], human
SH-SY5Y neuroblastoma cells [8], murine melanoma B16F10 cells
and human melanoma A2058 cells [9]. In addition, in a large amount

Corresponding author at: Laboratory of Pharmaceutical Analysis, School of Pharmaceutical Sciences, Sun Yat-sen University, Guangzhou Higher Education Meg
Center, Guangzhou 510006, PR China. Fax: +86 20 39943040.
E-mail address: yaomeicun@gmail.com (M. Yao).
http://dx.doi.org/10.1016/j.jpba.2015.09.021
0731-7085/ 2015 Elsevier B.V. All rights reserved.

of animal experiment, DN presented anti-obesity [10], anti-diabetic

[11], anti-thrombotic [12], anti-arthritic [13], liver-protection [14]
bioactivities.
Meanwhile, furostane- and spirostane-type skeleton saponins
are two major active ingredients in DN [15]. However, spirostanetype, such as dioscin [16] and gracillin [17], showed an extremely
low bioavailability in rats. Therefore, in this study, the relative
higher bioavailability components, furostane-type saponins [3,18],
were the main ingredients we paid attention to. Among these
furostanol glycosides [1921], protodioscin (PD), protogracillin
(PG), pseudoprotodioscin (PPD), pseudoprotogracillin (PPG) were
the relative higher contents in DN and the chemical structures
of them are shown in Fig. 1. What is more, PD and PPD showed
anti-cancer [22,23] and anti-hyperlipidemic [24] effects as well. In
consideration of multiple bioactivities of DN above and mission of
the age to promote modernization of TCM, it is pressingly necessary
for us to gure out its pharmacokinetics (PK) behaviors.
To date, analytical methods for the quantication of the active
chemicals of DN included high performance liquid chromatography with ultraviolet (UV) [20] or evaporative light scattering

M. Liao et al. / Journal of Pharmaceutical and Biomedical Analysis 117 (2016) 372379

373

Fig. 1. The chemical structures of the four furostanol glycosides and IS.

detection (ELSD) [25]. Recently, an increasing number of

LC-ESIMS/MS methods were applied to characterization and
determination of DN [4,19,21]. Meanwhile, several researches had
previously investigated the metabolism and PK of DN and some
pure steroidal saponins in DN [3,18,19,26]. However, these methods were mainly focused on the quantication of total saponins
or one of them in plasma, and few of them were reported for the
simultaneous determination of multiple saponins in biological uids. Accordingly, as an explicit and effective herb medicine, it is
of great signicance to establish a sensitive and specic analytical method for simultaneous quantication of multiple saponins in
biological samples and evaluate its PK behaviors.
In our study, a newly developed UPLCMS/MS method was
established and validated for the simultaneous determination of
four furostanol glycosides in rat plasma. The rapid, sensitive and
specic method was successfully applied to evaluate the PKs of
four saponins after oral administration of DN extracts to rats.
2. Experimental
2.1. Plant materials
D. nipponica samples, cultivated with a growth period of 2
years and dried in the sun, were collected from Jilin Province,
China. The moisture content of DN sample was about 8.15%. The
plant materials were authenticated by Prof. Depo Yang, School
of Pharmaceutical Sciences, Sun Yat-sen University. Corresponding voucher specimens (voucher number: DN-JL-20150604) were
deposited in Lab of Pharmaceutical Analysis and Quality Assessment, Sun Yat-sen University.
2.2. Chemicals and reagents
The reference standards of PD, PG, PPD, PPG, ginsenoside Rb1
(internal standards, IS), with purity higher than 98% (checked
by NMR and HPLC-ELSD), were achieved from Beijing Xinrong

Technologies Co., Ltd. (Beijing, China). HPLC grade methanol and

acetonitrile were purchased from Honeywell Burdick & Jackson
(Muskegon, Michigan, USA). Formic acid was bought from Chengdu
Kelong Chemical Reagent Factory (Sichuan, China). Lithium acetate
was from Aladdin Industrial Corporation (Shanghai, China). Deionized water up to a resistivity of 18.2 M was prepared with a
Milli Q-purication system (Barnstead International Inc., Dubuque,
Iowa, USA).

2.3. Instrument and UPLCMS/MS conditions

Sample analysis was performed on an UPLCMS/MS system
(Waters Corporation, Milford, Connecticut, USA) composed of an
autosampler, a column oven (set at 30 C) and a binary solvent
delivery manager. A tandem mass spectrometry was connected
to the LC system through electrospray ionization (ESI) interface. LC separations were obtained on an Kinetex XB-C18 column
(50 mm 2.1 mm, 2.6 m; Phenomenex, Torrance, California, USA)
preceded by a SecurityGuard ULTRA Cartridges UHPLC precolumn.
The gradient elution consisted of acetonitrile (mobile phase A) and
ultrapure water contained 0.03% formic acid and 0.1 mM lithium
acetate (mobile phase B) was set as follows: starting at 20% A80%
B, increasing A to 50% in 1.5 min, then decreasing A to 20% in 0.1 min,
keeping constant for 0.5 min. The ow rate was 0.40 mL/min and the
sample injection volume was 10 L at room temperature. [M + Li]+
ions were used as the precursor for quantication in multiplereaction monitoring (MRM) mode. The optimized cone voltage and
collision energy for four saponins and IS are shown in Table 1. Other
parameters of MS analysis were as follows: capillary voltage, 3 kV;
ultra-high pure nitrogen and argon were used as desolvation gas
(900 L/h) and collision gas (0.21 mL/min), respectively. Cone gas
ow rate, 50 L/h; source temperature and desolvation temperature was 150 C and 400 C. MasslynxTM 4.1 software was used for
acquiring and processing data.

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M. Liao et al. / Journal of Pharmaceutical and Biomedical Analysis 117 (2016) 372379

Table 1
MS/MS parameters for detection of four saponins and IS.
Compound

Parent ion (m/z)

Cone voltage (v)

Collision energy [eV]

Daughter ion (m/z)

PD

1056

1038
892b

60

65

PG

1072

1054a
908b

60

70

PPD

1038

892a
566b

60

65

PPG

1054

908a
349b

60

65

IS

1116

349a
774b

95

55

a
b

Transition used for quantication.

Transition used for qualitatively analysis.

2.4. Animals
Fifteen SPF-grade healthy male Wistar rats (225 25 g) (Certicate no. SCXK2011-0029) were obtained from the Laboratory
Animal Center in Sun Yat-sen University. Animals were kept
in an environmentally controlled breeding room (temperature:
2326 C, relative humidity: 4560%) for 3 days before starting the
experiments and free accessible to food and water. Diet was prohibited for 12 h before the experiment, while water was taken ad
libitum. The studies were approved by the Animal Ethics Committee of Sun Yat-sen University and followed the Guide for Care and
Use of Laboratory Animals published by the US National Institute
of Health [27].
2.5. Preparation of DN extracts
DN (50 g) was chopped into 60 mesh pieces and extracted three
times by ultrasonic (120 w, 40 kHz) with methanol (1:10, w/v) for
30 min each time. The combined ltrate was concentrated to dryness and then suspended with water to gain DN extracts (DNE) at a
concentration equivalent to 1 g/mL of the raw DN material before
it is used. The stability data of extract DN in water is listed in Supplementary Table 2. To gure out the dosage of administration, the
amount of four saponins in the extract solution were quantied
with UPLCMS/MS. The quantities of PD, PG, PPD and PPG in the
extract were 11.849, 0.991, 1.195 and 0.160 mg/mL, respectively.
In order to picture the global chemical prole of DN, an HPLC-UV
method was established. The method was as follows: chromatographic separation was performed at 35 C and detected at 203 nm
on an Apollo C18 column (250 4.60 mm; 5 m; Alltech, Illinois,
Deereld, USA); the mobile phase consisted of acetonitrilewater
through gradient elution at a ow rate of 0.8 mL/min; the injection
volume was 10 l. According to area normalization methods, the
amounts of the four analytes came to more than 50% (Fig. 4).
2.6. Preparation of standard and quality control (QC) samples
The stock solutions of PD, PG, PPD, PPG and IS were prepared with methanol at concentration of 2 mg/mL, respectively. The
mixed standard solutions of PD, PG, PPD and PPG for the preparation
of calibration curves were serially diluted with methanol to give
nal concentrations of 20, 50, 200, 800, 4000, 20,000, 30,000 and
40,000 ng/mL for PD and 4, 10, 40, 160, 800, 4000, 6000, 8000 ng/mL
for the others, respectively. The working solutions at low, medium
and high concentration were prepared in 50, 4000, 30,000 ng/mL for
PD and 10, 800, 6000 ng/mL for the rest by diluting the initial stock
solution with methanol. A 200 ng/mL solution of the IS was also
obtained by dilution of the IS stock solution with methanol. Both
stock and working solutions were stored at 4 C and the stability of

stock solutions were validated and related data was appended in

Supplementary Table 1.
2.7. Samples preparation
Rat plasma samples were stored at 20 C and thawed to
room temperature when used. To every 100 L plasma sample,
50 L IS working solution (200 ng/mL), 50 L methanol and 500 L
n-butanol were added. After vortex for 3 min, the mixture was centrifuged at 16,000 g for 5 min at 4 C, and then the organic layer
was transferred to another blank Eppendorf tube and evaporated
to dryness at 45 C. The residue was reconstituted with 100 L
acetonitrilewater (20:80, v/v), a 10 L aliquot was injected into
the UPLCMS/MS for analysis after centrifugation at 16,000 g for
5 min at 4 C.
2.8. Method validation
The establishment of validation method was performed in
accordance with the USA Food and Drug Administration Bioanalytical Method Validation Guidance [28].
Calibration standards were prepared in triplicate and analyzed
in duplicate in three independent runs. Linear regression method at
least squares principle was applied to determine the slope, intercept and square regression coefcient (r) of the linear regression
equation. The lower limit of quantitation (LLOQ) was determined
as the lowest concentration with values for precision and accuracy
within 20% and the analyte response at the LLOQ was more than
ve times the response compared to blank samples at the same
retention time. To evaluate specicity, chromatograms of the analytes at the LLOQ level were compared to those of blank plasma
samples in quintuplicate.
Intra- and inter-day precisions and accuracies of the method
were determined by testing ve replicates of each of the QC samples at low, middle and high concentration (25 ng/mL, 2000 ng/mL,
15,000 ng/mL for PD and 5 ng/mL, 400 ng/mL, 3000 ng/mL for the
other three) in three separate analytical runs. Precision is expressed
as the relative standard deviation (RSD) and accuracy as the relative
error (RE), respectively.
To assess potential interferences by matrix, matrix effect was
carried out by comparing the responses achieved from postextraction blank rat plasma and mobile phase spiked with low,
middle and high concentrations of the analyte, separately.
The absolute recovery of the analytes, after liquidliquid extraction, was determined by comparing the responses of the analytes
from QC samples at low, middle and high concentrations with
analytes spiked in post-extracted blank rat plasma at equivalent
concentrations.

M. Liao et al. / Journal of Pharmaceutical and Biomedical Analysis 117 (2016) 372379

375

Fig. 2. Product ion mass spectra, proposed collision-induced fragmentations of precursor ions and monitored transitions of the four analytes and IS. (A) PG, (B) PD, (C) PPD,
(D) PPG, (E) IS.

Carryover was determined by measuring the peak area for the

analytes by injecting two processed blank matrix samples sequentially after injecting an upper limit of quantitation (ULOQ) sample.
The response in the rst blank matrix at the retention times of each
analyte should be less than 20% of the response of its LLOQ sample,
correspondingly.
The stabilities of the target chemicals in rat plasma were evaluated by analyzing ve aliquots of low and high QC samples tested for
pre-treatment, post-treatment and three cycles of freezethaws.
The results were compared with those for freshly prepared QC
samples and the percentage concentration deviation was calculated. Accuracy should be within 85115% of the nominal values
and precision within 15% RSD.
2.9. Pharmacokinetic study
Fifteen rats were randomly divided into low (5 g/kg DNE),
medium (10 g/kg DNE), high (20 g/kg DNE) dose group, equally.
All groups were treated with intragastric administration. Blood
samples (200 L) were acquired with heparinized tubes by puncture of the retro-orbital sinus at 0 (predose), 5, 10, 30, 60, 90,
150, 210, 270, 360, 480, 600 min after dosing. Plasma samples
(100 L) were gained by centrifuging at 16,000 g for 1 min and
then samples were stored at 20 C until analysis. Pharmacokinetic parameters for the analytes were calculated with Winnonlin
5.0.1 (Pharsight, USA) by non-compartmental analysis. Data was
expressed as mean SD.

3. Results and discussion

3.1. Optimization for UPLCMS/MS conditions
For purpose of exploring more sensitive ionization mode, both
positive and negative modes were compared using the abundance
of four reference compounds and IS as index. As a result, the
response of the analytes in positive ionization mode was much
higher than in negative. Ultimately, [M + Li]+ was chosen as the precursor ion for both target compounds and IS in positive ionization
mode. The advantages of [M + Li]+ over [M + Na]+ and [M + H]+ for
such kind of chemicals were claried in our previous study [18]. The
optimized both precursor ions and daughter ions for the analytes
are listed in Table 1. Meanwhile, the proposed collision-induced
fragmentations of precursor ions of the analytes are shown in Fig. 2.
The chromatographic conditions were optimized to achieve
symmetrical peak shapes, enhance the signal response and obtain
shorter chromatographic cycle times for simultaneous analysis of
four components and IS. In the rst place, compared with Waters
UPLC BEH C18 column (50 mm 2.1 mm, 1.7 m) and Phenomenex
Kinetex XB-C18 column (50 mm 2.1 mm, 2.6 m), the latter was
our nal choice for better peek shapes and a relative shorter retention time (detailed in Supplementary Fig. 1). Meanwhile, methanol
was without our consideration as mobile phase for hydroxyl of
furostanols at position C-22 could be transformed into methyl
ethers in methanol under the high temperature of ion source
[29]. Furthermore, water phase with 0.03% (v/v) FA could suppress

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M. Liao et al. / Journal of Pharmaceutical and Biomedical Analysis 117 (2016) 372379

Table 2
The regression equations, linear range and LLOQs of the four saponins.
Analyte

Range (ng/mL)

Calibration curves

Correlation coefcient (r)

LLOQ (ng/mL)

PG
PD
PPD
PPG

24000
1020000
24000
24000

Y = 0.0055X 0.0824
Y = 0.0045X 0.3055
Y = 1.1321X 12.896
Y = 1.1207X 22.879

0.9998
0.9994
0.9999
0.9996

2
10
2
2

signal noise and increase the response of analytes. Besides, 0.1 mM

lithium acetate was applied to assist the formation of [M + Li]+ .
Eventually, the mobile phases composed of (A) acetonitrile and
(B) aqueous formic acid (0.03%, v/v) and 0.1 mM lithium acetate
with a gradient elution at a ow rate of 0.4 mL/min were used.
In this study, ginsenoside Rb1 was selected as the internal standard because of its similarity of chemical structure, retention and
extraction behavior with the four saponins.
3.2. Sample pretreatment
On account of the differences among the analytes in the physiochemical properties, it is important to explore an appropriate
method to prepare the samples with a high recovery and without endogenous interferences for all these analytes. To begin with,
protein precipitation (PPT) was evaluated. However, PPT was left
out of account because of the severe matrix effect for PD and PG.
Then, a conventional liquidliquid extraction (LLE) approach was
used and ethyl acetate, n-butanol and acetone were all tested as
extraction solvent. Finally, n-butanol was adopted because of its
higher extraction efciency (>80%) for all these saponins without
obvious endogenous interference.
3.3. Validation results
3.3.1. Specicity and selectivity
As shown in Fig. 3, no severe endogenous interferences were
observed in the retention times for four saponins and IS by comparing chromatograms of blank plasma of ve different rats with
chromatograms of plasma spiked with IS and the target compound standards at LLOQ level. Representative chromatograms of
rat plasma are also presented at 1 h after intragastric administration
of DNE at low, medium and high dose in Fig. 3.

3.3.2. Linearity of calibration curves and LLOQ

As depicted in Table 2, all calibration curves were found to be
adequate for the subsequent analysis of rat plasma samples and
exhibited good linearity over the concentration of 1020,000 ng/mL
for PD and 24000 ng/mL for the others with correlation coefcient
(r) within the range of 0.99940.9999. The LLOQs were all suitable
for the determination of analytes in the pharmacokinetic studies.
Moreover, according to Table 3, the accuracies and precisions of the
analytes at LLOQ levels were all below 10% which were acceptable.
3.3.3. Accuracy and precision
The UPLCMS/MS method provided high within-run and
between-run precision as well as accuracy of quantication of
four saponins in rat plasma. As described in Table 3, the intraday and inter-day accuracies of the four analytes were 8.112.0%
and 7.112.9%, respectively. Meanwhile, precisions represented
as RSD no more than 10.7% for both within-run and between-run. In
a word, the results above suggested that the method was accurate,
reliable and reproducible.
3.3.4. Carryover
Conspicuous carryover was observed after injection of the rst
blank plasma samples, it was approximately 30% of the LLOQ for
PD and no more than 5% for the other three and IS. Nevertheless,
carryovers of the whole analytes were less than 5% of the LLOQ after
injection of the second blank plasma samples which were within
the acceptable range. Therefore, in our succeeding sample analysis,
all samples were tested from low to high concentration. In addition,
a blank plasma sample would be injected if the former sample was
close to ULOQ level.
3.3.5. Matrix effect and recovery
The average extraction recoveries and matrix effects of the QC
samples are summarized in Table 4. Recoveries of the four saponins

Fig. 3. Extracted ion current chromatograms of the analytes and the IS (100 ng/mL) acquired during the analysis of plasma samples: (A) blank rat sample; (B) blank plasma
spiked with the analytes at LLOQ and IS, i.e., 10 ng/mL for PD and 2 ng/mL for the rest; (C), (D), (E) incurred rat plasma obtained at 30 min after intragastric administration of
low (5 g/kg), medium (10 g/kg), high (20 g/kg) dose of DNE to rats, respectively. 1: IS, 2: PG, 3: PD, 4: PPD, 5: PPG.

M. Liao et al. / Journal of Pharmaceutical and Biomedical Analysis 117 (2016) 372379

377

Table 3
Intra-day and inter-day precisions and accuracies for the determination of the four saponins from the assay samples (mean SD).
Analytes

Nominal conc. (ng/mL)

Intra-day (n = 5)
Observed
concentration
(ng/mL)

PG

PD

2.0
5.0
400.0
3000.0
10.0
25.0
2000.0
15000.0

2.1
4.6
417.7
2963.7
10.6

0.1
0.3
11.6
71.6
0.4

25.4 0.8
2240.3 22.5
14359.6 308.0

Inter-day (n = 15)
Precision (RSD, %)

Accuracy (RE, %)

5.3
7.1
2.8
2.4
4.2

4.0
7.2
4.4
1.2
6.2

3.3
1.0
2.1

1.6
12.0
4.3

Observed
concentration
(ng/mL)
2.1
4.7
422.0
2965.6
10.7

0.1
0.2
9.5
77.4
0.6

26.4 1.3
2257.6 30.3
14639.8 428.7

Precision (RSD, %)

Accuracy (RE, %)

5.6
5.3
2.3
2.6
5.3

3.3
6.6
5.5
1.1
6.7

4.9
1.3
2.9

5.5
12.9
2.4

PPD

2.0
5.0
400.0
3000.0

2.2
4.8
387.1
2775.1

0.2
0.2
6.2
138.8

7.7
3.7
1.6
5.0

8.0
4.4
3.2
7.5

2.2
4.8
386.7
2893.6

0.2
0.3
11.0
163.6

10.7
5.9
2.8
5.7

8.3
4.2
3.3
3.5

PPG

2.0
5.0
400.0
3000.0

2.0
4.8
367.7
2938.3

0.2
0.3
25.8
190.3

10.6
5.7
7.0
6.5

2.0
4.0
8.1
2.1

2.0
4.8
371.7
3072.9

0.2
0.2
19.4
208.8

7.8
4.9
5.2
6.8

1.7
3.6
7.1
2.4

at all levels were higher than 80.3% and IS was about 81.5%, which
was adequate and acceptable for the PK studies. Meanwhile, the
matrix effects of IS and QC samples of the four target compounds
at three concentrations were observed to be within 97.3% and the
range of 86.9107.4%, respectively. Data above suggested that ion
suppression or enhancement from plasma matrix is negligible for
this method.

3.3.6. Stability tests

The results of stability evaluation under different conditions
were illustrated in Table 5. The deviations of the measured concentrations from the standard ones for the whole analytes in
the stability tests were within the 15% assay variability limit.
Namely, the four saponins were stable enough in our anticipant
conditions.

Fig. 4. (A) HPLC-UV (203 nm) prole of DNE; 1: PD, 2: PG, 3: PPD, 4: PPG, 5: dioscin and gracillin. (A1), (A2), (A3), (A4) Mean rat plasma concentrationtime proles of PD,
PG, PPD and PPG after oral administration of DNE at low (), medium () and high () dose to rats. The gures in top right corner of (A1)(A3) were the amplied ones of
low dose level; the plasma concentration of PPG at low dose level was under LLOQ. Vertical bars represent SD. The comparison of AUC0t (B) and T1/2 (C) of the four saponins
at each does level (mean SD, n = 5). *Values are signicantly different from each other (P < 0.05), **Values are signicantly different from each other (P < 0.01).

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M. Liao et al. / Journal of Pharmaceutical and Biomedical Analysis 117 (2016) 372379

Table 4
The absolute recovery and matrix effect of four saponins and IS in rat plasma (n = 5).
Analytes

Spiked concentration
(ng/mL)

Recovery (%)
(mean SD)

RSD (%)

Matrix effect (%)

(mean SD)

RSD (%)

PG

5.0
400.0
3000.0

103.3 6.0
89.8 0.7
86.0 5.2

5.8
0.7
6.0

107.4 6.7
91.5 1.9
101.8 6.4

6.2
2.1
6.3

PD

25.0
2000.0
15000.0

96.8 1.5
91.2 9.6
80.4 6.6

1.5
10.5
8.2

106.0 2.9
86.9 4.7
88.8 1.3

2.7
5.4
1.4

100.6 8.1
89.6 5.2
91.9 4.5

8.0
5.8
4.9

PPD

5.0
400.0
3000.0

84.0 9.0
90.0 6.1
83.3 10.8

10.7
6.8
13.0

PPG

5.0
400.0
3000.0

92.6 8.3
91.3 9.4
80.3 4.6

8.9
10.3
5.7

91.1 12.4
91.5 6.6
93.6 2.8

13.6
7.2
2.9

100.0

81.5 7.8

9.6

97.3 12.6

13.0

IS

Table 5
The stability of the four saponins in rat plasma under different storage conditions (n = 5).
Analytes

Nominal conc. (ng/mL)

25 C for 4 h
Precision
(RSD, %)

Accuracy
(RE, %)

Frozen for 15 days

Three-freezethaw cycles

4 C for 24 h

Precision
(RSD, %)

Precision
(RSD, %)

Precision
(RSD, %)

Accuracy
(RE, %)

Accuracy
(RE, %)

Accuracy
(RE, %)

PG

5.0
3000.0

5.4
3.8

4.4
2.7

5.6
7.4

2.8
4.9

4.7
1.7

2.4
4.3

10.2
2.6

5.2
5.5

PD

25.0
15000.0

7.4
1.1

8.5
0.1

4.7
0.8

4.0
9.5

2.9
1.2

1.9
0.9

3.2
2.2

7.4
1.3

PPD

5.0
3000.0

3.4
6.6

6.0
10.3

7.2
7.2

6.0
8.2

11.2
3.4

8.0
9.5

11.6
4.5

11.6
4.3

PPG

5.0
3000.0

7.9
3.4

6.4
5.2

11.4
4.0

7.6
0.5

12.1
1.3

3.2
1.5

8.0
8.6

6.0
5.4

Table 6
Pharmacokinetic parameters of four saponins after intragastric administration of DNE at doses of 5 g/kg (n = 5), 10 g/kg (n = 5), and 20 g/kg (n = 5) to rats.
Analytes

Dose
mg/kg

T1/2
min

Tmax
min

Cmax
ng/mL

AUC0t
ng min/mL

AUC0
ng min/mL

MRT0
min

PG

4.96
9.91
19.82

195.9 26.2
150.7 26.3
116.4 34.6

52.5 15.0
52.5 15.0
67.5 15.0

37.5 12.6
486.3 303.7
2011.4 426.2

10231.9 3162.7
59249.1 31,887.2
238463.5 61210.8

11597.1 3270.5
61963.0 31,488.8
242221.1 61,137.3

287.6 39.5
165.7 41.5
129.1 21.2

PD

59.25
118.49
236.98

194.8 27.0
142.9 32.2
95.9 13.2

35.0 28.9
37.5 15.0
82.5 15.0

293.3 120.5
4557.7 2638.5
15575.1 3115.0

71135.7 23397.3
482572.4 246825.0
1869125.1 412759.7

79507.9 23,160.5
503093.0 245815.1
1890521.7 4064858.0

271.6 49.4
151.8 33.9
120.8 25.4

PPD

5.98
11.95
23.90

120.1 38.6
103.7 37.4
87.0 15.4

90.0 42.4
52.5 15.0
82.5 15.0

25.5 7.9
436.1 282.0
1903.0 626.9

5927.4 2667.4
50868.0 28654.3
259101.6 63653.1

6254.5 2793.4
51547.3 28429.8
261328.4 64499.0

211.5 47.1
126.2 27.2
131.5 21.5

PPG

1.60
3.20

165.5 15.1
153.9 39.9

50.0 17.3
67.5 15.0

37.9 12.0
86.7 52.2

4154.1 1088.3
12887.3 7547.3

4411.9 1019.0
13823.7 7950.6

167.7 42.6
206.2 88.0

3.4. Application of the assay method

The established method depicted above was successfully
applied to the PK study of four furostanol glycosides in rat plasma
after oral administration of DNE at low, medium and high dose. The
mean plasma concentrationtime proles and the comparison of
AUC0600 min and T1/2 of the different dose groups for four saponins
in rats are described in Fig. 4. Furthermore, non-compartmental
mode was applied to calculate all of the pharmacokinetic parameters by using Winnonlin 5.0.1 software and the results are listed in
Table 6.
As illustrated in Table 6, the pharmacokinetic parameters
including half-time (T1/2 ), time to reach the maximum concentrations (Tmax ), maximum plasma concentration (Cmax ), area under
concentrationtime curve (AUC0t and AUC0 ), mean residence

time (MRT0 ) are listed as mean SD. Signicant differences of

T1/2 were observed between low and high dose groups for PG
and PD. What is more, AUC0600 min between low and high dose
groups, medium and high dose groups was statistical signicance
for PG, PD and PPD as well. Generally, four furostanol glycosides
exhibited rapid excretion and relative high plasma concentrations in rats. In our preliminary studies and reported in literatures
[20,21], spirostanol saponins, such as dioscin and gracillin, also
accounted a high amount in DN. However, it is difcult to simultaneously quantify them with furostanol glycosides in a PK study
for the extremely low plasma concentrations and relative longer
half-time [17]. Meanwhile, as a well-known effective component
treating with cardiovascular disease [2], it may be reasonable
for us to monitor its ingredients working quickly and effectively
in vivo.

M. Liao et al. / Journal of Pharmaceutical and Biomedical Analysis 117 (2016) 372379

In addition, it is interesting to nd that Cmax and AUC of PPD

after intragastric administration of DN were much higher than a
single oral administration of PPD in rats compared with our previous study [18]. It is supposed that the other ingredients in DN
may enhance the absorption of PPD and improve its bioavailability.
As a consequence, this ubiquitous phenomenon in TCM may well
explain the reason why most of TCMs work effectively despite the
appearance of possessing simply trace chemical constituents.
4. Conclusion
In summary, a specic, sensitive and rapid UPLCMS/MS
method was the rst time established and validated to simultaneously quantify the concentration of four furostanol glycosides in
rat plasma with a LLOQ value of 10 ng/mL for PD and 2 ng/mL for
the others. In addition, the method was successfully applied to the
pharmacokinetic study of the four saponins following intragastric
administration at low, medium and high dose of DNE to rats. The
four furostanol glycosides showed rapid excretion and relative high
plasma concentrations in rats. Moreover, the T1/2 and AUC0t of each
compound turned out to behave in a dose-dependent manner by
comparing them at three dose levels.
Acknowledgments
This work was funded by the Guangzhou Science and Technology Program (No. 2014J4100171), the National Key Technology
R&D Program during the Twelfth Five-Year Plan Period of Peoples
Republic of China (No. 2013BAD10B04-2).
Appendix A. Supplementary data
Supplementary data associated with this article can be found, in
the online version, at http://dx.doi.org/10.1016/j.jpba.2015.09.021.
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