Development and application of high-performance liquid chromatography for the study of ampelopsin...

Research Article

Development and application of high-performance liquid chromatography for thestudy of ampelopsin pharmacokinetics in ratplasma using cloud-point extraction

A simple, rapid and specific method based on cloud-point extraction (CPE) was developed

to determine ampelopsin in rat plasma after oral administration by reversed-phase high-

performance liquid chromatography. The non-ionic surfactant Genapol X-080 was chosen

as the extract solvent. Some important parameters affecting the CPE efficiency, such as

the nature and concentration of surfactant, extraction temperature and time, centrifuge

time and salt effect, were investigated and optimized. Separation was accomplished using

a C18 column by gradient elution with a acetonitrile–phosphate buffer solution as the

mobile phase. The detection wavelength was set at 290 nm. Under optimum conditions,

the linear range of ampelopsin in rat plasma was 20–2000 ng/mL (r2 5 0.9996). The limit

of detection was 6 ng/mL (S/N 5 3) with the limit of quantification being 20 ng/mL

(S/N 5 10). The proposed method has been successfully applied for pharmacokinetic

studies of ampelopsin from rat plasma after oral administration.

Keywords: Ampelopsin / Cloud-point extraction / PharmacokineticsDOI 10.1002/jssc.201000382

1 Introduction

Ampelopsis grossedentata (Hand Mazz) (Vitaceae) (Chinese

name: Rattan Tea) is widely distributed in South China [1].

In Chinese medicine, the dried whole herb of A. grosseden-tata was used to treat cold and tinea corporis [2]. It was

reported that A. grossedentata possessed many biological

activities including hypoglycemic [3], lipid-lowering [4], anti-

inflammatory, pain-relieving [5], anti-oxidative [6] and

hepatoprotective [7]. Ampelopsin is the main effective

constituent of A. grossedentata, which possesses a variety of

pharmacological and biological activities and is considered

to have therapeutic applications. Since the clinical use of

ampelopsin has greatly developed, it is essential to use a

specific and rapid method for the determination of

ampelopsin in plasma or serum.

Several methods such as thin-layer chromatography

(TLC) [8], high-performance liquid chromatography (HPLC)

[9] and liquid chromatography/mass spectrometry (LC-MS)

[10] have been reported for the analysis of ampelopsin in

biological samples by liquid–liquid extraction (LLE).

Although LLE is a common technique for the preconcen-

tration and clean-up prior to chromatographic or electro-

phoretic analysis, large organic solvent consumption,

tedious and analyte loss resulting from multi-stage opera-

tions cannot be neglected.

Recently, cloud-point extraction (CPE) has attracted

more attention as an alternative method to LLE. CPE has

some advantages, such as inexpensive, high concentration

efficiency, environmentally lower toxicity, simple procedure,

over the conventional LLE. Upon heating a surfactant

solution over a critical temperature, the solution was sepa-

rated into two distinct phases of a bulk aqueous phase and a

small surfactant-rich phase. A hydrophobic analyte can be

highly concentrated in the small volume of the surfactant-

rich phase [11–13], this enhanced the sensitivity of chro-

matographic analysis and allowed the analyte’s analysis and

quantification by techniques such as HPLC [14], capillary

electrophoresis [15] and LC-MS [16] without further sample

clean-up or evaporation steps.

All these indicated that CPE has great analytical

potential as an effective enrichment method. However, until

now no reports have been published about how to extract

ampelopsin from plasma. In this paper, the quantification

of ampelopsin in rat plasma by CPE preparation using

Genapol X-080 as the surfactant with HPLC was reported,

which demonstrated the feasibility of CPE in clinical and

preclinical pharmacokinetic studies.

Jun Zhou1�

Ping Zeng1

Hong Hai Tu2

Feng Qiao Wang3

1Department of Pharmacy,Urumqi General Hospital of PLA,Urumqi Xinjiang, P. R. China

2Institute for Drug andInstrument of Xinjing MiltaryCommand, Urumqi, Xinjiang,P. R. China

3Department of Chemistry,Fourth Military MedicalUniversity, Xi’an, Shanxi,P. R. China

Received June 3, 2010Revised November 4, 2010Accepted November 7, 2010

Abbreviations: CPE, cloud-point extraction; IS, internalstandard; LLE, liquid–liquid extraction; QC, quality control

�Additional correspondence: Dr. Jun Zhou

E-mail: [email protected]

Correspondence: Dr. Feng Qiao Wang, Department of Chemis-try, Fourth Military Medical University, Xian, Shanxi 710032,P. R. ChinaE-mail: [email protected]: 186-29-84776945

& 2010 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim www.jss-journal.com

J. Sep. Sci. 2011, 34, 160–168160

2 Materials and methods

2.1 Chemicals, reagents and animals

Ampelopsin (MW 5 320, pKa 5 6.84, Log P 5 1.03) and the

internal standard (IS) (dihydroquercetin) were purchased

from Sigma (St. Louis, MO, USA). The structures of

ampelopsin and dihydroquercetin are shown in Fig. 1.

The non-ionic surfactant Genapol X-080 was also obtained

from Sigma and used without further purification. Various

concentrations (w/v) of aqueous surfactant solutions were

prepared by weighing appropriate amounts of the surfactant

and by directly dissolving the surfactant in distilled water.

Sodium chloride (5–10%) and phosphoric acid (0.1%)

(Beijing Chemical Factory, PR China) were prepared before

experiment. Acetonitrile was of HPLC grade and obtained

from Fisher (Leicestershire, UK). All other reagents

employed in this work were of analytical grade. Distilled

water (Millipore, Bedford, MA, USA) was obtained from

deionized water and used throughout the study.

Stock solutions of ampelopsin (10 mg/mL) and IS

(10 mg/mL) were prepared by dissolving suitable amounts of

each pure substance in methanol–water (50:50, v/v) and

kept stable for 2 months when stored at 41C in the refrig-

erator (assessed by HPLC).

Sprague–Dawley male rats (200720 g) were purchased

from the Experimental Animal Center of Fourth Military

Medical University. Rats were anesthesized by pentobarbital

sodium and blood was collected from abdominal artery in

clean heparinized glass tubes. The blank plasma was sepa-

rated by immediate centrifugation at 3000 rpm for 10 min

and stored at �201C until required.

2.2 Instrumentation and chromatographic condi-

tions

The chromatographic system was composed of a Dionex P680

HPLC pump, a thermostatted column compartment TCC-

100, a Dionex Chromatography Management System, a

Rheodyne 7225i injector and a PDA-100 photodiode array

detector (CA, USA). Separations were accomplished on an

Agilent Zorbax SB-C18 (150 mm� 4.6 mm id, 5 mm) column,

which was connected to an Agilent Zorbax Extend-C18 guard

column (12.5 mm� 2.1 mm id, 5 mm). The detector was

operated at 290 nm and the column temperature was

maintained at 251C. The mobile phase was a gradient elution

of A (0.1% phosphoric acid) and B (acetonitrile). The linear

gradient was as follows: 0–5% B over 0–4 min, 5–20% B over

4–8 min and returned to 5% B at 8 min immediately. The flow

rate was set at 1.0 mL/min. The injections were carried out

through a 20 mL loop. Retention data were recorded using the

above described chromatographic conditions. Column void

time was determined to be 1.16 min by the injection of

acetone. Retention behavior of the analyte was estimated by

retention factor (k) and calculated according to the equation,

k 5 (tR�t0)/ti0, where tR is the retention time of the analyte

and t0 is the elution time of the acetone (as a void marker) [17].

A thermostatic bath (HH-2, Guohua Medical Instru-

ment, Guangzhou, China) was used to implement CPE. To

accelerate the phase separation process, a high-speed centri-

fuge was employed to centrifuge the sample solutions (Anke

TCL-16G, Shanghai, China) in calibrated centrifugal tubes.

Vortex Genie Mixture was applied to the mixed sample

(CAY-1, Beijing Chang’an Instrumental Factory, China).

2.3 CPE procedure

About 200 mL of rat plasma sample and 40 mL of IS solution

(1.0 mg/mL) were added to a 1.5 mL capped centrifugal tube.

To these glass tubes, 1 mL of aqueous solution of Genapol

X-080 at a concentration of 5% w/v and 100 mL of 0.6 M

sodium chloride solutions were added. The contents were

mixed well with a Vortex Genie Mixture for 5 min, and then

incubated in the thermostatic bath at 551C for 20 min. After

that, the phase separation was then accelerated by centrifuga-

tion at 3500 rpm (1120� g) for 10 min. Followed with the

removal of the water phase, a surfactant-rich phase stuck to

the bottom of the tube was obtained (40 mL). Coextractants

such as hydrophobic proteins and most of the surfactants

were removed from the surfactant-rich phase by precipitation

with 200 mL of acetonitrile–water (30:70, v/v). Then, the

contents were vortex mixed and centrifuged at 16 000 rpm

(5120� g) for 5 min respectively. Most of the surfactants and

coextractants such as hydrophobic proteins were precipitated

at the bottom of the tube. Nearly 20 mL of the upper layer was

injected into the HPLC system for analysis.

2.4 LLE procedure

Forty microliters of IS stock solution (10 mg/mL) were added

to a 2.0 mL tube and the methanol was evaporated under the

reduced pressure at room temperature. Then, 1 mL of

plasma was added. After vortexing for 1 min, the plasma

Figure 1. Chemical structures of ampelopsin(A) and dihydroquercetin (IS) (B).

J. Sep. Sci. 2011, 34, 160–168 Liquid Chromatography 161


sample was adjusted to pH 4.0 with 10% phosphoric acid

and extracted with ethyl acetate (3 mL) three times. The

supernatants were transferred into a clean glass tube and

evaporated under nitrogen to dryness. The residue was

dissolved in 0.5 mL of methanol and filtered through a

0.45 mm filter. About 20 mL of the filter liquid was injected

into the HPLC system for analysis.

2.5 Method validation

2.5.1 Calibration curve

By spiking the appropriate stock solution containing the IS

at a constant concentration to 1.0 mL of blank plasma, six

effective concentrations were obtained separately 20, 50,

500, 1000, 1500 and 2000 ng/mL for ampelopsin. The

quality control (QC) samples were separately prepared in

the blank plasma at the concentrations of 20, 1000 and

2000 ng/mL containing the IS at a constant concentration,

respectively. The spiked plasma samples (standards and

QCs) were then treated as per the CPE procedure and

injected into the HPLC. The procedure was carried out in

triplicate for each concentration. The obtained analyte/IS

peak area ratios were plotted against the corresponding

concentrations of ampelopsin and the calibration curves

were set up by the least-squares method. The values of limit

of quantification (LOQ) and limit of detection (LOD) were

calculated, according to the Chinese Pharmacopeia [18]

guidelines, as the analyte concentrations gave rise to peaks

whose heights were ten and three times the baseline noise,

respectively.

2.5.2 Extraction recovery (absolute recovery)

By assaying the samples at three QC levels, absolute

recoveries of ampelopsin were determined. The analyte/IS

peak area ratios were compared to those obtained from the

direct injection of the compounds dissolved in the super-

natant of the processed blank plasma at the same theoretical

concentrations. The extraction yield values were calculated

as follows:

Absolute % recovery

¼ ðanalyte=IS peak area ratioÞ spiked blank

ðanalyte=IS peak area ratioÞ corresponding standard� 100%

2.5.3 Precision and accuracy

The precision, including intra-day and inter-day precisions

expressed as % RSD values, was assessed by assaying the

samples at three QC levels five times within the same day

and five different days. At the same time, the work was

accompanied by a standard calibration curve on each

analytical run. The accuracy was evaluated by the mean

recovery and expressed as (mean measured concentration)/

(spiked concentration)� 100% and % RSD values.

2.5.4 Selectivity

Blank plasma and drug plasma samples from rats were

injected into the HPLC. The resulting chromatograms were

checked for possible interference from endogenous substances

and metabolites of ampelopsin. The acceptance criterion was

no interfering peak in the place of an analyte peak.

2.5.5 Stability

To evaluate sample stability after freeze–thaw cycles and at

room temperature, five replicates of QC samples at each of

20, 1000 and 2000 ng/mL concentrations were subjected to

three freeze–thaw (from �20 to 251C) cycles or were stored

at room temperature (approximately 22–251C) for 4 h before

sample processing, respectively. Long-term stability was

studied by assaying samples that had been stored at �201C

for a certain period of time (15 days). Stability was assessed

by comparing the mean concentration of the stored QC

samples with the mean concentration of those prepared

freshly. Ampelopsin was considered stable under storage

conditions if the assay percent recovery was found to be

85–115% of the nominal initial concentration [19], (http://

www.fda.gov/cder/guidance/index.htm).

2.6 Application to pharmacokinetic study

Sprague–Dawley male rats (200720 g) were specifically

pathogen free and kept in an environmentally controlled

breeding room (temperature maintained at 25711C and

with a 12:12 h light–dark cycle) for at least 1 wk before

starting the experiment. Before oral administration of

ampelopsin at the dose of 100 mg/kg body weight, the rats

were fasted for 24 h, maintained with physiological saline.

All procedures involving animals were in accordance with

the Regulations of Experimental Animal Administration

issued by the State Committee of Science and Technology of

People’s Republic of China. Five rats were anesthesized by

pentobarbital sodium and blood was collected from

abdominal artery in clean heparinized glass tubes predose

and 5, 15, 30, 45, 60, 75, 90, 120, 150 and 180 min postdose.

Plasma was separated by centrifugation at 3500 rpm for

10 min. The plasma obtained was stored frozen at �201C

until analysis.

Data from these samples were used to construct phar-

macokinetic profiles by plotting drug concentration versustime. All data were subsequently processed by the DAS 2.0

statistical software (Pharmacology Institute of China).

3 Results and discussion

3.1 Optimization of the chromatographic conditions

The maximum absorption wavelength of ampelopsin and

dihydroquercetin is 290 and 276 nm, respectively. A value of

J. Sep. Sci. 2011, 34, 160–168162 J. Zhou et al.


290 nm was used to detect two analytes with a good

sensitivity. In order to obtain good HPLC chromatograms

with a baseline separation of compounds in plasma, various

HPLC columns were investigated, and the results showed

that the Zorbax SB-C18 column was suitable for the analysis.

Since there existed interference ingredients in the

plasma, several kinds of mobile phase systems were inves-

tigated. Finally, gradient eluent system (acetonitrile �0.1%

phosphoric acid; Table 1) was chosen as well as run time

was 10 min. No interference was observed under the assay

conditions. The peaks of the analytes in the plasma were

identified by comparing their retention times with those of

the standards and further confirmed by their online UV–Vis

spectra. The retention times were 5.45 and 7.32 min for

ampelopsin and IS, respectively.

Under these optimum chromatographic conditions, the

peaks were neat, symmetric and well separated (Fig. 2).

3.2 Optimization of the CPE procedure

In order to find suitable extraction method, SPE, LLE and

CPE were evaluated for the sample preparation. However,

SPE and LLE methods need to use relatively high volumes

of organic solvents and a long time for the extraction, which

are harmful to analysts and the environment. Therefore,

CPE was chosen.

3.2.1 Selection of the surfactant

At the beginning of this study, Triton X-100, Triton X-114,

Triton X-45 and Genapol X-080 were all tried as extraction

solvents. However, the Triton X series showed high UV

absorbance and gave very broad peaks in the HPLC

chromatogram, which interfered severely with the

determination of ampelopsin and IS. Genapol X-080 is a

polyoxyethylene glycol mono ether-type surfactant that has

eight oxyethylene units and tridecyl alkyl moieties (critical

micellar concentration 5 0.05 mmol/L (0.028%, w/v), cloud-

point 421C (in pure water)). Several research groups have

successfully used Genapol X-080 in the extraction proce-

dures [20, 21]. Because it possesses no aromatic moiety,

Genapol X-080 does not absorb above 210 nm, thus it will

not interfere with the determination of ampelopsin and IS.

So, Genapol X-080 was chosen as the CPE surfactant in this

study.

3.2.2 Effect of surfactant concentration

The effect of the concentration of surfactant was examined

in our study and the result is shown in Fig. 3. From Fig. 3,

the extraction efficiency of ampelopsin in rat plasma (five

independently samples) can be seen increased when the

surfactant concentration increases from 0.5 to 5% w/v. It

tends to remain fairly constant in the surfactant concentra-

tion range of 5–10%. It is known that ampelopsin is a

Table 1. HPLC mobile phase gradient conditions for the analy-

sis of ampelopsin

Time (min) Flow rate (mL/min) Acetonitrile (v/v) (%)

0–4 1.0 0–5

4–8 1.0 5–20

8 1.0 20–5

Figure 2. Typical HPLC chromatograms of a cloud-point extractof plasma samples: (A) a blank plasma sample; (B) a blankplasma sample spiked with ampelopsin and dihydroquercetin;(C) plasma sample 0.5 h after oral administration. Peak identifi-cation: 1, ampelopsin; 2, dihydroquercetin.



hydrophobic compound and cannot be extracted by water. It

was demonstrated in our experiment that ampelopsin can

be extracted by surfactant solution at a specific concentra-

tion. The ability of the aqueous non-ionic Genapol X-080

solution in extracting ampelopsin may be related to the

solubility-enhancement effect of the surfactant micelles. In

this experiment, when the concentration of surfactant is

below 5%, it suspends in the bulk solution and is very

difficult to be separated into two phases. Simultaneously,

when the surfactant concentration rises to 10%, the

extraction efficiency of ampelopsin increases. But the

solution becomes too sticky to handle. According to these

experimental results, 5.0% Genapol X-080 was selected for

obtaining best response signals and highest extraction

efficiency.

3.2.3 Effect of sodium chloride concentration

The addition of salt to the solution can influence the

extraction process. For most non-ionic surfactant, the

presence of salts may facilitate phase separation since they

increase the density of the aqueous phase [22]. In this paper,

sodium chloride was employed as the modifier because it is

both cost effective and environment friendly. The study of

the influence of the ionic strength on the extraction

efficiency was carried out by varying the concentration of

sodium chloride between 0.1 and 1.0 M. The result shows

that the addition of sodium chloride facilitates the separa-

tion between the surfactant-rich phase and the aqueous

phase. With the increase in the salt concentration, the

micelle size and the aggregation number are increased and

the critical micellar concentration remains constant. In

addition, analytes may become less soluble in the solution at

higher salt concentrations and thus contribute to higher

extraction efficiency. That is to say, the inert salt increases

the extraction efficiency by decreasing the solubility of the

organic species in the aqueous phase. The result obtained in

Fig. 4 indicates that the CPE at a salt concentration of 0.6 M

gives the optimum extraction efficiency. When the concen-

tration is higher than 0.6 M, the surfactant-rich phase will

be on the surface of the solution, which will make it more

difficult to separate the extraction solvent into two phases

and the accuracy and reproducibility probably were not

satisfactory. The extraction effect is best when the concen-

tration of sodium chloride is 0.6 M.

3.2.4 Effect of the equilibrium temperature and time

Theoretically, the optimal equilibration temperature for the

extraction occurs when the temperature is 15–201C higher

than the cloud point of surfactant [23]. So, the influence of

temperature on the extraction efficiency was examined in

the range of 45–701C. As can be seen from Fig. 5, the

highest extraction efficiency occurred when the equilibrium

temperature reached 551C. Higher temperatures only led to

the more difficult separation of phases due to the increasing

rate of molecular thermodynamic movement.

The effect of incubation time on the extraction effi-

ciency was studied by varying the incubation time from 5 to

55 min. The results indicated that the extraction recovery of

ampelopsin increased with the increase in the extraction

time. Figure 6 shows the best extraction effect was reached

when extracted for 20 min. When the extraction time was

longer than 20 min, the extraction efficiency of ampelopsin

remained constant. Therefore, 20 min was selected for the

extraction time.

3.2.5 Effect of centrifugation time

In general, centrifugation time only slightly affects micelle

formation but accelerates phase separation in the same

sense as a conventional separation of a precipitate from its

original aqueous environment. The effect of centrifugation

time upon extraction efficiency was studied at 3500 rpm

(1120� g) in the range of 5–20 min. The complete phase

separation was achieved after 5 min. Centrifugation time of

10 min was chosen as optimal, with good efficiency for

separating both phases and experimental convenience.

Figure 3. Effect of concentration of Genapol X-080 (%) on theextraction efficiency. Other extraction conditions: equilibriumtemperature: 551C, equilibrium time: 20 min, concentration ofsodium chloride solution: 0.6 M.

Figure 4. Effect of the ionic strength on the extraction efficiency.Other extraction conditions: equilibrium temperature: 551C,equilibrium time: 20 min, concentration of Genapol X-080 (%):5%.



3.3 Comparison with LLE

To prove the validity of the method the results obtained by

use of CPE were compared with those obtained by the use of

LLE. Compared with LLE, CPE has higher extraction

efficiency under identical experimental conditions. The

preconcentration effect of CPE is clearly demonstrated in

Fig. 7.

3.4 Calibration and validation

3.4.1 Linearity, LOD and LOQ

The calibration curves were constructed by calculating the

peak area ratios (Y) of ampelopsin to IS against ampelopsin

standard concentrations. The calibration curve was

Y 5�0.0225410.00856X with a correlation coefficient above

0.9996. The mean of five calibration curves was made over a

period of 5 days, each calibration curve originating from a

new set of extractions. Calibration curves were linear in the

concentration range investigated with coefficients of correla-

tion (r)Z0.9990. Table 2 shows inter-day precision in the

slope, intercept and correlation coefficient of standard

curves (r 5 0.9995–0.9998) made over a period of 5 days.

The coefficient of variation (CV) (%) (n 5 5) of the slope

calculated with calibration curve data was 1.93%, showing

good repeatability. Further evaluations such as residual

plots examination and lack-of-fit test were carried out to

check the model’s adequacy. No significant lack of fit was

observed in any of the calibration curves. The correlation

coefficient using linear regression model of calibration

curve is acceptable (r 5 0.9996). The limit of LOQ for

ampelopsin in plasma was 20 ng/mL and the limit of LOD

was 6 ng/mL.

3.4.2 Accuracy and precision

The intra-day and inter-day accuracy and precision values of

the assay method are shown in Table 3. All intra-day RSD

(%) for ampelopsin were below 6.5%. All inter-day RSD (%)

were below 5.6%. The accuracies were determined by

comparing the mean calculated concentration with

the spiked target concentration of the QC samples. The

Figure 5. Effect of the equilibrium temperature on the extractionefficiency. Other extraction conditions: equilibrium time: 20 min,concentration of sodium chloride: 0.6 M, concentration ofGenapol X-080 (%): 5%.

Figure 6. Effect of the equilibrium time on the extractionefficiency. Other extraction conditions: equilibrium temperature:551C, concentration of sodium chloride: 0.6 M, concentration ofGenapol X-080 (%): 5%.

Figure 7. Typical chromatograms of determination of ampelop-sin and dihydroquercetin in plasma samples; (A) plasma sample0.5 h after oral administration with LLE; (B) plasma sample 0.5 hafter oral administration with CPE. Peak identification: 1,ampelopsin; 2, dihydroquercetin.



intra-day and inter-day accuracies for ampelopsin were

found to be within 93.5 and 97.9%.

3.4.3 Extraction recovery

To determine the recovery of ampelopsin in rat plasma, a

blank rat plasma was spiked with ampelopsin to achieve a

final concentration of 20, 1000 and 2000 ng/mL. The plasma

samples were subjected to the CPE procedure and injected

into the HPLC. Six samples were analyzed for each

concentration. The analysis was performed for three

replicates at the abovementioned concentration levels. The

mean recoveries of ampelopsin from rat plasma at

concentrations of 20, 1000 and 2000 ng/mL were 91.8,

93.9 and 96.0%. Using the same method, the recovery of IS

in rat plasma was obtained, which was 93.7%.

3.4.4 Selectivity and stability

Selectivity was evaluated by comparing the chromatograms

of blank plasma and drug plasma samples, which were

subjected to the CPE procedure and injected into the HPLC.

Figure 2 shows the typical chromatograms of a blank

plasma sample, of a spiked plasma sample with ampelopsin

(1000 ng/mL) and IS, and of a plasma sample from 0.5 h

after an oral administration. It also shows no significant

interference from endogenous substances and metabolites

of ampelopsin observed in the place of the analytes.

Ampelopsin in rat plasma was shown to be stable for at

least 15 days stored at �201C. The RE % of ampelopsin in

rat plasma between the initial concentrations and the

concentrations of the following three freeze–thaw cycles

ranged from 2.45 to 5.48%, which indicated that ampelopsin

Table 2. Inter-day precision in the slope, intercept and correla-

tion coefficient (r) of standard curves

(r 5 0.9995–0.9998)

Day Slope Intercept r

1 �0.02287 0.00851 0.9995

2 �0.02211 0.00864 0.9998

3 �0.02282 0.00867 0.9996

4 �0.02201 0.0086 0.9998

5 �0.02289 0.00837 0.9995

Mean7S.D. �0.0225470.000436 0.0085670.000121 0.999670.00015

CV (%) 1.93 14.1 0.03

CV 5 coefficient of variation.

Table 3. Accuracy and precision for the assay of ampelopsin in rat plasma (n 5 5)

Theoretical concentration (ng/mL) Assayed concentration (ng/mL) (mean7SD) Accuracy (%) Precision (RSD %)

Intra-day

20 18.773.42 93.5 6.5

1000 954.6728.85 95.5 4.5

2000 1943.5760.51 97.2 2.8

Inter-day

20 19.173.83 95.5 5.6

1000 968.3733.22 96.8 4.6

2000 1958.4758.45 97.9 3.9

RSD 5 relative standard deviation.

Table 4. Summary of stability of ampelopsin in rat plasma (n 5 5)

Concentration found (mg/mL) (mean7SD) Concentration added (ng/mL) (mean7SD)

20 1000 2000

Freeze and thaw stability

At the beginning 20.771.35 1013.6750.26 2009.4775.04

After three freeze–thaw cycle 21.972.11 1067.4749.18 2062.5780.63

Bias (RE %) 5.48 5.04 2.57

Short-term room temperature stability

At the beginning 20.271.21 1005.8752.73 2000.4774.47

After 4 h at room temperature 21.471.19 1047.6751.23 2048.5775.18

Biasa) (RE%) 5.61 3.99 2.35

Long-term cold storage stability

At the beginning 20.471.32 1010.8749.62 2003.6781.23

After 15 days at �20%1C 21.772.07 1072.1753.33 2079.7778.93

Biasa) (RE %) 5.99 5.72 3.66

a) Bias (RE %) 5 (Cactual�Ccalculated)/Cactual (%).



was stable during the three freeze–thaw cycles. Processed

samples were also found to be stable for at least 4 h at room

temperature. The above stability data are summarized in

Table 4. The result shows that no significant deterioration of

the analytes was observed under any of these conditions.

3.5 Pharmacokinetic study of ampelopsin in rat

plasma

After a single oral administration of ampelopsin to rats, the

plasma concentrations of ampelopsin were determined by

the developed method. The mean plasma concentration–

time profile is shown in Fig. 8; the pharmacokinetic

parameters were calculated and summarized in Table 3.

The results indicated that ampelopsin was absorbed rapidly

with Tmax at 32.2 min, and eliminated with mean residence

time (MRT) lasting 81.4 min in rats. Plasma concentrations

were below the LOQ of 20 ng/mL after 180 min. The

ampelopsin concentration in plasma was in conformation

with a two-compartment model with first-order absorption.

Other pharmacokinetic parameters in this study are shown

in Table 5. This method could be applied to pharmacoki-

netic studies after oral administration of ampelopsin.

4 Concluding remarks

The CPE technique has been successfully applied for the

first time as an effective method for the extraction and

preconcentration of ampelopsin from rat plasma samples.

The proposed CPE procedure is less polluting and time

consuming than the LLE procedures. It was also shown that

this method was applied successfully used to assay

ampelopsin in plasma and to study in vitro pharmacoki-

netics of ampelopsin for the first time. Doubtlessly, the

chromatographic condition and sample preparation proce-

dure in this paper will likely facilitate the development and

validation of other methods to analyze ampelopsin in other

biological matrixes such as urine and tissue homogenates in

our future work.

This work was financially supported by the NationalNatural Science Foundation of China (NSFC. NO. 20842007).

The authors have declared no conflict of interest.

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Figure 8. Plasma concentration–time curve of ampelopsin afteroral administration. Each point and bar represent the mean7SD(n 5 5).

Table 5. Pharmacokinetic data of ampelopsin in rat plasma

(n 5 5)

Parameter Estimate (mean7SD)

T1/2a (min) 12.773.13

T1/2b (min) 17.974.85

T1/2a (min) 13.273.32

Tmax (min) 32.278.48

AUC0�t (ng h mL�1) 1556.67522.5

AUC0�N (ng h mL�1) 1768.27557.2

CL (mL kg min�1) 45.6716.2

Vd (L/kg) 345.5771.3

Cmax (ng/mL) 156.1710.1

Note: Vd, volume of distribution; AUC0�N, the area under the

concentration–time curve; T1/2a, distribution half-life; T1/2b,

elimination half-life; MRT, mean residence time; CL, the

elimination clearance; Tmax, time to maximum plasma concen-

tration; Cmax, maximum plasma concentration.



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Development and application of high-performance liquid chromatography for the study of ampelopsin...

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