Copyright © 2009 John Wiley & Sons, Ltd. Biomed. Chromatogr. 2010; 24: 550–555

Research Article

Received 20 February 2009, Revised 7 July 2009, Accepted 20 July 2009 Published online in Wiley Interscience: 30 September 2009

(www.interscience.wiley.com) DOI 10.1002/bmc.1325

Determination of asperosaponin VI in rat plasma by HPLC-ESI-MS and its application to preliminary pharmacokinetic studies

Kai Lia, Li Dingb, Zhong-Lin Yanga,*, E-Hu Liua, Lian-Wen Qia, Ping Lia and Yu-Zhu Hub

ABSTRACT: Asperosaponin VI (also named akebia saponin D) is a typical bioactive triterpenoid saponin isolated from the rhizome of Dipsacus asper Wall (Dipsacaceae). In this work, a sensitive high-performance liquid chromatography–electrospray ionization–mass spectrometry (HPLC-ESI-MS) assay has been established for determination of asperosaponin VI in rat plasma. With losartan as the internal standard (IS), plasma samples were prepared by protein precipitation with methanol. Chromatographic separation was performed on a C18 column with a mobile phase of 10 mM ammonium acetate buff er contain-ing 0.05% formic acid–methanol (32 : 68, v/v). The analysis was performed on an ESI in the selected ion monitoring mode using target ions at m/z 951.4 for asperosaponin VI and m/z 423.2 for the IS. The calibration curve was linear over the range 3–1000 ng/mL and the lower limit of quantifi cation was 3.0 ng/mL. The intra- and inter-assay variability values were less than 9.5 and 7.8%, respectively. The accuracies determined at the concentrations of 3.0, 100.0, 300.0 and 1000 ng/mL for aspero-saponin VI were within ±15.0%. The validated method was successfully applied to a pharmacokinetic study in rats after oral administration of asperosaponin VI. Copyright © 2009 John Wiley & Sons, Ltd.

Keywords: asperosaponin VI; HPLC-ESI-MS; pharmacokinetic

* Correspondence to: Zhing-Lin Yang, Key Laboratory of Modern Chinese

Medicines, China Pharmaceutical University, Ministry of Education, No. 24

Tongjiaxiang, Nanjing 210009, China. E-mail: likai--you@163.com

a Key Laboratory of Modern Chinese Medicines, China Pharmaceutical University, Ministry of Education, No. 24 Tongjiaxiang, Nanjing 210009, China

b Department of Pharmaceutical Analysis, China Pharmaceutical University, No. 24 Tongjiaxiang, Nanjing 210009, China

Abbreviations used: ME, matrix eff ect.

Introduction

Triterpenoid saponins possess a wide range of biological activi-

ties and are distributed in many important medicinal plants.

Asperosaponin VI (3-O-α-L-arabinopyranosyl hederagenin-28-β-

D-glucopyranoside-(1 → 6)-β-D-glucopyranoside, shown in Fig.

1), also named akebia saponin D, is a characteristic triterpenoid

saponin isolated from the rhizome of Dipsacus asper Wall

(Dipsacaceae). It has long been used as a tonic and anti-

infl ammatory agent in traditional Chinese medicine for the

therapy of low back pain, knee pain, rheumatic arthritis, trau-

matic hematoma, threatened abortion and bone fractures (Zhao

et al., 2006; Xiao, 2007). Studies showed that asperosaponins

could promote human osteoblast proliferation and diff erentia-

tion (Zheng, 2006), anti-oxidation (He and Qiu, 2005; Liu, 2007)

and promote agglutination of the bone injury (Ji et al., 1997).

Asperosaponin VI, the principal active component of aspero-

saponins, was reported to exert apoptosis-inducing activity via

induction of apoptosis through activation chiefl y via nitric oxide

and apoptosis-related p53 and Bax gene expression (Jeong et al.,

2008). Furthermore, it has been developed as a new drug to treat

osteoporosis and granted a Chinese patent by Zhejiang Dier

Pharmaceutical Co. Ltd (Yang, 2004).

Although literature on the pharmacological eff ects of aspero-

saponins is extensive and some analytical methods for determin-

ing the asperosaponin VI have been developed to control the

quality of Dipacus asperoides (Yang et al., 2000; Tan et al., 2006;

Ma et al., 2007), to the best of our knowledge, little information

is available about its pharmacokinetics. LC-MS methods are pre-

ferred for the study of the pharmacokinetics of many active com-

ponents (Wu et al., 2005; Liu et al., 2005; Paek et al., 2006; Xu

et al., 2007; Xie et al., 2008) due to their high sensitivity and selec-

tivity. However, no previous method has been described for the

determination of asperosaponin VI in biological samples. Herein

is described a sensitive high-performance liquid chromatogra-

phy (HPLC)–electrospray ionization (ESI)–MS method for the

determination of asperosaponin VI in plasma with the lower limit

of quantifi cation (LLOQ) of 3.0 ng/mL using losartan as the inter-

nal standard (IS). The developed method was then applied to its

pharmacokinetic study, which helped in taking a limited view of

the pharmacokinetic profi les and designing rational dosage

regimens.

Experimental

Chemicals and Reagents

Asperosaponin VI was isolated from Dipsacus asperoides at the author’s

laboratory. Its structure was elucidated and unequivocally identifi ed by 5

50

Determination of asperosaponin VI in rat plasma

Biomed. Chromatogr. 2010; 24: 550–555 Copyright © 2009 John Wiley & Sons, Ltd. www.interscience.wiley.com/journal/bmc

spectroscopic methods (UV, IR, MS, 1H-NMR and 13C-NMR). The IS losartan

shown in Fig. 1 was purchased from Sigma-Aldrich Trading Co. Ltd

(Shanghai, China). The purity of the two reference compounds was deter-

mined to be higher than 98% by normalization of the peak area detected

by HPLC/MS.

Methanol of HPLC grade was purchased from Merck KGaA (Darmstadt,

German). Ammonium acetate and formic acid were of analytical grade

and purchased from Nanjing Chemical Reagent Co. Ltd (Nanjing, China).

Water was prepared with double distillation.

Instrumental and Conditions

HPLC-ESI-MS analysis was performed using an Agilent 1100 Series

LC-MSD SL system (Agilent Technologies, Palo Alto, CA, USA) with a

SecurityGuard-C18 (4 × 2.0 mm, 5 μm, Phenomenex) and Zorbax SB-C18

column (2.1 × 150 mm, 5 μm, Agilent). The mobile phase was 10 mM

ammonium acetate buff er containing 0.05% formic acid–methanol

(32 : 68, v/v) at a fl ow rate of 0.25 mL/min. The column temperature was

maintained at 35°C. HP ChemStation software (10.02 A) supplied by

Agilent controlled the LC-MS system. HPLC-ESI-MS in positive ion mode

was carried out using nitrogen to assist nebulization. A quadrupole MS

equipped with an ESI source was set with a drying gas (N2) fl ow at 10 L/

min, nebulizer pressure at 40 psi, drying gas temperature at 350°C, capil-

lary voltage at 4.0 kV and fragmentor voltage at 180 V. HPLC-ESI-MS was

performed in the selected-ion monitoring (SIM) mode using target ions

at m/z 951.4 for asperosaponin VI and m/z 423.2 for the IS.

Preparation of Standard Solutions

The stock solution of asperosaponin VI with a concentration of 1 mg/mL

was accurately prepared in methanol and stored at −20°C. It was diluted

consecutively with methanol to prepare a series of working solutions of

10 μg/mL, 1.0 μg/mL, 100 ng/mL and 10 ng/mL. A solution of IS of 10 μg/

mL was also prepared. All the solutions were stored in refrigerator at −20°C.

Sample Preparation

All frozen standards and samples were allowed to thaw at room tempera-

ture and homogenized by vortexing. Aliquots of 50 μL of spiked sample

standards, quality control (QC) samples and unknown plasma samples

were placed into a 1 mL centrifuge tube, and added with 20 μL of IS

(10 μg/mL), respectively. For protein precipitation, 200 μL of methanol

was added to the mixture and vortexed for approximate 3 min, then

allowed to stand for 30 min. The precipitate was removed by centrifuga-

tion at 16,000 rpm for 10 min. Finally, a 3 μL aliquot was injected into the

LC-ESI-MS system for analysis.

Calibration Curves

The calibration standards were prepared as follows: the proper amounts

of each asperosaponin VI working solution were separately added to

seven 1 mL centrifuge tubes and evaporated to dryness under a gentle

fl ow of nitrogen at room temperature, respectively. And then a 50 μL

aliquot of blank plasma was added to each centrifuge tube and vortexed

to form calibration concentrations of 3.0, 10.0, 30.0, 100.0, 300.0, 800.0

and 1000 ng/mL for the calibration curve. The calibration curve was pre-

pared and assayed along with QC samples and each run of unknown

plasma samples.

Preparation of QC Samples

Quality control samples at concentration levels of 3.0, 100.0, 300.0 and

1000 ng/mL were prepared using the same method of the calibration

standards. QC samples were analyzed with processed test samples at

intervals in each run. The results of the QC samples provided the basis of

accepting or rejecting the run.

Method Validation

Linearity, Limit of Detection and Lower Limit of

Quantifi cation

Calibration standards of asperosaponin VI concentration levels at

3.0, 10.0, 30.0, 100.0, 300.0, 800.0 and 1000 ng/mL were prepared

and assayed. The calibration curve was constructed by plotting

the peak-area ratios of asperosaponin VI to the IS vs the concen-

trations of asperosaponin VI, using weighted least squares linear

regression (weighting factor was 1/C). The limit of detection

(LOD) was considered as the fi nal concentration producing a

signal to noise ratio of 3. The lower limit of quantitation (LLOQ)

was defi ned as the lowest concentration on the calibration curve

at which precision was within 20% and accuracy was within

±20% (Guidance for Industry, Bioanalytical Method Validation,

2001), and it was established using fi ve samples independent of

standards.

Precision and Accuracy

Validation samples were prepared and analyzed on three sepa-

rate runs to evaluate the accuracy, intra-run and inter-run preci-

sions of the analytical method. The accuracy, intra-run and

inter-run precisions of the method were determined by analyz-

ing four replicates at 3.0, 100.0, 300.0 and 1000 ng/mL of aspero-

saponin VI along with one standard curve on each of three runs.

Assay precision was calculated using the relative standard devia-

tion (RSD%). The accuracy is the degree of closeness of the deter-

mined value to the nominal or known true value under prescribed

conditions. Accuracy is defi ned as the relative deviation in the

calculated value (E) of a standard from that of its true value (T)

expressed as a percentage (RE%). It was calculated using the

Figure 1. Chemical structures of asperosaponin VI and losartan.

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K. Li et al.

www.interscience.wiley.com/journal/bmc Copyright © 2009 John Wiley & Sons, Ltd. Biomed. Chromatogr. 2010; 24: 550–555

formula: RE% = (E − T)/T × 100. The accuracy of the assay was

checked by preparation of QC samples at the start of the phar-

macokinetic study. These QC samples were assayed along with

unknown samples in each run to monitor the performance of the

assay and to assess the integrity and validity of the result of the

unknown plasma samples.

Extraction Recovery

The extraction recoveries of asperosaponin VI were evaluated by

analyzing fi ve replicates of asperosaponin VI plasma samples at

each concentration level of 3.0, 100.0, 300.0 and 1000 ng/mL.

Recovery was calculated by comparing the peak areas of aspero-

saponin VI prepared in plasma with those obtained from direct

injection of standards dissolved in the supernatant of the pro-

cessed blank plasma.

Stability

Stability tests were performed to verify the stability of aspero-

saponin VI during handling procedures. Samples were assayed at

the two QC concentrations of 3.0 and 1000 ng/mL for aspero-

saponin VI in triplicate. The long-term stability was performed at

−20°C in refrigerator for 4 weeks. The stability at ambient tem-

perature was tested for 12 h. The samples were subjected to the

three freeze–thaw cycles with each freeze cycle lasting at least

24 h. The post-preparative stability of processed samples under

autosampler conditions was evaluated, where samples were pre-

pared and placed in injection vials for 24 h at room temperature

before injecting into the LC-MS system. The stability of the stock

solutions of asperosaponin VI and IS were determined by placing

the stock solution at −20°C for 1 month. The results were com-

pared with the solutions freshly prepared.

Pharmacokinetic Study

Sprague–Dawley rats (male, n = 6, 200–250 g, Experimental

Animal Center of Suzhou University, Suzhou, China), were housed

with unlimited access to food and water except for fasting 12 h

before the experiment. The animals were maintained on a 12 h

light/12 h dark cycle (light on at 8:00) at ambient temperature

(22–25°C) and at 60% relative humidity. Asperosaponin VI was

dissolved in freshly prepared normal saline and was administered

to the rats by oral gavage at a dose of 100.0 mg/kg. Blood samples

(0.2 mL) were collected from the oculi chorioidea vein of the rats

at 0.33, 0.67, 1, 1.33, 2, 3, 4, 6, 10, 15 and 20 h postdose. Plasma

was separated by centrifugation and stored at −20°C until analy-

sis. Aliquots of 50 μL plasma samples were processed and ana-

lyzed for asperosaponin VI concentration. The pharmacokinetic

parameters of asperosaponin VI were obtained from the plasma

concentration–time data. The maximum plasma concentration

(Cmax) and the time to reach it (tmax) were noted directly. The elimi-

nation rate constant (ke) was calculated by linear regression of

the terminal points of the semi-log plot of plasma concentration

against time. The elimination half-life (t1/2) was calculated by the

formula t1/2 = 0.693/ke. The area under the plasma concentration–

time curve from zero to the last measurable plasma concentra-

tion (AUC0–t) was calculated by the linear trapezoidal method. The

area under the plasma concentration–time curve to time infi nity

(AUC0–∞) was calculated as follows: AUC0–∞ = AUC0–t + Ct/ke, where

Ct is the last measurable plasma concentration and ke is the elimi-

nation rate constant.

Results and Discussion

Conditions for ESI-MS

The positive and negative ion detection modes were investi-

gated during the preliminary assay. The results showed that the

positive ion detection mode could off er greater sensitivity for the

analytes than the negative ion detection mode. Therefore, ESI in

positive ion mode was adopted for the assay of asperosaponin

VI. Figure 2 shows a typical full-scan ESI mass spectrum of aspero-

saponin VI. ESI produced abundant target ions of [M + Na]+ at m/z

951.4 for asperosaponin VI in the scan mode. By monitoring this

ion, a highly sensitive assay for asperosaponin VI was developed.

The intensity of the ion of asperosaponin VI at m/z 951.4 was

compared using fragmentor voltages of 30, 60, 90, 120, 150, 180

and 210 V to determine the optimal collision energy. The result

showed that the highest sensitivity was obtained at the fragmen-

tor voltage of 180 V. Therefore, a fragmentor voltage of 180 V was

used to carry out LC-ESI-MS in the scan mode. At this collision

energy the most intensive ion of IS was the molecular ion [M +

H]+ at m/z 423.2 (Fig. 2). Therefore, the positive molecular ion [M

+ H]+ at m/z 423.2 was selected as the target ion of IS in the SIM.

Chromatographic Conditions

Selection of mobile phase components is also a critical factor.

Since phosphate buff er cannot be used with MS, ammonium

acetate was employed to supply the ionic strength. Increasing

the percentage of buff er in the mobile phase could enhance

analyte peak symmetry and resolution. Using a 10 mM concentra-

tion of ammonium acetate buff er, the chromatographic peaks

became sharp and symmetrical. The ionization of samples at the

LC-MS interface is also aff ected by the mobile phase. Hence, a

mobile phase containing volatile acid or salt is used frequently.

In this case, the responses were measured using 0.2, 0.5, 1 and

2% formic acid in aqueous phase. The response to asperosaponin

VI was maximal by addition of 0.5% formic acid to the mobile

phase. Finally, the acceptable retention and separation of aspero-

saponin VI were obtained by using an elution system of 10 mM

ammonium acetate buff er containing 0.05% formic acid–metha-

nol (32 : 68, v/v) as the mobile phase. Under the present chro-

matographic conditions, the run time of each sample was 5.0 min.

The retention times were 3.6 and 2.4 min for asperosaponin VI

and IS, respectively. Representative SIM are shown in Fig. 3.

Preparation of Plasma Samples

Sample preparation is a critical step for accurate and reliable

LC-MS assays. The biological sample preparation methodologies

which are the most widely employed include liquid–liquid

extraction, protein precipitation and solid-phase extraction. The

recoveries of asperosaponin VI using these three methods were

investigated and compared. The results showed that protein pre-

cipitation provided higher recovery compared with the other

two methods. Thus, the asperosaponin VI plasma samples were

prepared by protein precipitation procedure. Three kinds of pre-

cipitation reagents (methanol, acetonitrile and ethanol) were

investigated during the experiment. In the case of acetonitrile

and ethanol as precipitation reagents, when the supernatant of

prepared samples was directly injected into the LC-MS system,

the results showed peak fronting of the asperosaponin VI and IS.

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Determination of asperosaponin VI in rat plasma

Biomed. Chromatogr. 2010; 24: 550–555 Copyright © 2009 John Wiley & Sons, Ltd. www.interscience.wiley.com/journal/bmc

Figure 2. Positive mass spectrum of asperosaponin VI (A) and losartan (B).

Using methanol as a precipitation reagent, sharp peak shape and

higher extraction recovery of asperosaponin VI were obtained.

Method Validation

Selectivity. Selectivity was assessed by comparing the chro-

matograms of six diff erent batches of blank rat plasma with the

corresponding spiked plasma. Figure 3 shows the typical chro-

matograms of a blank, a spiked plasma sample with asperosapo-

nin VI and IS, and a plasma sample from a rat at 0.67 h after an

oral administration of asperosaponin VI. Interferences from the

matrices at the expected retention times of the target ions were

not observed and typical retention times for asperosaponin VI

and IS were 3.6 and 2.4 min, respectively. Therefore an accept-

able selectivity was obtained by this method.

Matrix eff ect. To evaluate the matrix eff ect (ME), i.e. the poten-

tial ion suppression or enhancement due to co-eluting matrix

components, six diff erent batches of blank plasma were precipi-

tated by methanol and then spiked with the analyte at four con-

centrations of 3.0, 100.0, 300.0 and 1000 ng/mL. The corresponding

peak areas of the analytes in spiked plasma post-extraction (A)

were then compared with those of the standards in mobile phase

(B) at equivalent concentrations. The ratio (A/B × 100) was defi ned

as the ME. The ME data at the four concentrations of asperosapo-

nin VI in six diff erent batches of rat plasma are presented in Table

1. The results of ME in the range of 92.5–99.3% showed that there

was no signifi cant ME in this study.

Linearity of calibration curves and LLOQ. Visual inspection of

the plotted duplicate calibration curves and correlation coeffi -

cients >0.999 confi rmed that the calibration curve was linear over

the concentration ranges of 3.0–1000 ng/mL for asperosaponin

VI. A typical standard curve was f = 3.12 × 10−4C + 2.45 × 10−3,

where f represents the peak area ratio of asperosaponin VI to IS

and C represents the plasma concentration of asperosaponin VI.

The LOD was determined to be 1 ng/mL with a signal-to-noise

ratio of 3. The present LC-ESI-MS method off ered a LLOQ of

3.0 ng/mL when using 50 μL plasma samples. Under the present

LLOQ of 3.0 ng/mL, the asperosaponin VI concentration could be

determined in plasma samples until 20 h after a single oral

administration of 100.0 mg/kg asperosaponin VI. This was sensi-

tive enough to investigate the pharmacokinetic behaviors of

asperosaponin VI.

Precision and accuracy. Asperosaponin VI plasma samples at

four concentration levels of 3.0, 100.0, 300.0 and 1000 ng/mL

were analyzed for accuracy and precision. The data obtained for

asperosaponin VI is shown in Table 1. The precision was calcu-

lated by one-way-ANOVA. For the four concentration levels of

asperosaponin VI, the intra-run precision was less than 9.5%, the

inter-run precision was less than 7.8% and the accuracy was

within ±15.0%. The data obtained for asperosaponin VI was

within the acceptable limits to meet the guidelines for bioanalyti-

cal methods.

Recovery. The mean extraction recoveries were measured at

four diff erent concentration levels for asperosaponin VI (3.0,

100.0, 300.0 and 1000 ng/mL) by comparing the peak areas of

asperosaponin VI prepared in plasma with those obtained from

direct injection of standards dissolved in the supernatant of the

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K. Li et al.

www.interscience.wiley.com/journal/bmc Copyright © 2009 John Wiley & Sons, Ltd. Biomed. Chromatogr. 2010; 24: 550–555

VI plasma samples. The accuracy values of low (3.0 ng/mL) and

high (1000 ng/mL) concentration of asperosaponin VI in rat

plasma were 101.4 and 98.8% after three freeze–thaw cycles, and

99.6 and 98.5% at −20°C for 4 weeks. The post-preparative stabil-

ity test showed that the prepared samples under autosampler

conditions were stable for 24 h at least before injection into the

LC-MS system. The stock solutions of asperosaponin VI and IS in

methanol were shown to remain stable for 1 month at −20°C.

Pharmacokinetic studies. The described method was success-

fully applied to a pharmacokinetic study in rats. The mean plasma

concentration–time curve of asperosaponin VI was shown in Fig.

4. The main pharmacokinetic parameters of asperosaponin VI

were calculated. After oral administration of 100.0 mg/kg aspero-

saponin VI, the mean values of Tmax and Cmax were 1.2 h (range

0.8–1.9 h) and 306.5 ng/mL (range 243.2–419.4 ng/mL), respec-

tively. The elimination half-life of asperosaponin VI was 2.8 ±

0.7 h. The AUC0–t and AUC0–∞ values obtained were 52.2 ± 27.4

and 53.1 ± 27.8 μg h/mL, respectively. Additionally, it was inter-

esting to note that double peaks were observed in both indi-

vidual and mean plasma–concentration curves of asperosaponin

VI. This phenomenon was also found in the pharmacokinetics of

timosaponin B-II (Cai et al., 2008), macranthoidin B, macranthoi-

din A and dipsacodise B (Chen et al., 2008) in rat plasma after

oral administration. It might be involved in enterohepatic

recirculation.

Conclusion

A sensitive LC-ESI-MS method for the quantifi cation of aspero-

saponin VI in rat plasma was fi rst established for pharmacokinetic

study of asperosaponin VI after oral administration. The pharma-

cokinetic parameters of oral administration of asperosaponin VI

in rats were obtained. The results of validation have shown that

the method is rapid, sensitive and accurate. This LC-ESI-MS

method required only 50 μL of plasma with a LLOQ of 3.0 ng/mL

for asperosaponin VI and the determination of one plasma

sample needed only 5 min. These results indicated that it was

suitable for routine analysis of large number of biological

samples. The metabolite-related identifi cation of asperosaponin

Figure 3. Typical SIM mass chromatograms of blank plasma (A), plasma

spiked with asperosaponin VI (10 ng/mL) and IS (B), and plasma obtained

from a rat at 40 min after oral administration of 100.0 mg/kg asperosapo-

nin VI, the plasma concentration of asperosaponin VI was estimated to

be 121.7 ng/mL (C).

Table 1. Matrix eff ect data, precision and accuracy, and recovery data of asperosaponin VI in rat plasma

3.0 ng/mL 100.0 ng/mL 300.0 ng/mL 1000.0 ng/mL

Matrix eff ect (%)a

ME (mean ± SD, %) 92.5 ± 8.6 97.8 ± 4.7 98.6 ± 4.1 99.3 ± 3.2

Precision and accuracyb

Mean found concentration (ng/mL) 2.814 97.46 293.0 2978RSD (%) intra-day 9.5 2.1 1.6 1.2RSD (%) inter-day 7.8 3.4 2.5 2.4RE (%) −6.2 −2.5 −2.3 −0.7

Recovery (%)c

Mean found concentration (ng/mL) 2.769 90.73 279.0 925.3RSD (%) 7.4 3.6 2.4 2.1Extraction recovery (%) 92.3 90.7 93.0 92.5

a Matrix eff ect data for asperosaponin VI in rat plasma (n = 6).b Precision and accuracy of asperosaponin VI in plasma (n = 3 runs, fi ve replicates per run).c Recovery data of asperosaponin VI in rat plasma (n = 5).

processed blank plasma. As shown in Table 1, recoveries fell in

the range 90.7–93.0% when spiking diff erent concentrations,

demonstrating the acceptable accuracy of this method.

Stability. The results of stability experiments showed that no

signifi cant degradation occurred at ambient temperature for

12 h and during the three freeze-thaw cycles for asperosaponin

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Determination of asperosaponin VI in rat plasma

Biomed. Chromatogr. 2010; 24: 550–555 Copyright © 2009 John Wiley & Sons, Ltd. www.interscience.wiley.com/journal/bmc

Figure 4. Mean plasma concentration-time profi le of asperosaponin VI after oral administration of asperosaponin VI 100.0 mg/kg in rats.

VI in plasma, urine and other biological matrix, as well as mecha-

nism of action will be further reported in the future.

Acknowledgements

The authors greatly appreciate the fi nancial support from the Key

Project of the National Natural Science Foundation of China (no.

30730113).

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