Determination of Polydatin in Rat Serum and Its Changes by UPLC Method

Haisheng ZENG, Zhiqiang WANG, Meiyuan LU, Chao ZENG*, Mengxia YANG, Guilin YANG

1. The Affiliated Hospital of Youjiang Medical University for Nationalities, Baise 533000, China; 2. The First Affiliated Hospital of Guangxi University of Chinese Medicine, Nanning 530023, China; 3. Guangxi Food and Drug Evaluation & Inspection Center, Nanning 530029, China

Abstract [Objectives] To establish a UPLC-UV method for the determination of polydatin in the serum of Wistar rats. [Methods] Acquity UPLC BEH shield RP18 column (1.7 μm, 2.1 mm × 100 mm, Waters Corporation, USA) was used as the analytical column, acetonitrile-water (55∶45) was used as the mobile phase, the flow rate was 0.5 mL/min, and the column temperature was 30 ℃ and the detection wavelength was 306 nm. [Results] The linear range of the established serum sample analysis method was 1.0-20.0 μg/mL, and the correlation coefficient was r=0.999 4; the intraday and interday RSD of Wistar rat serum was less than 3.0%, and the accuracy was higher than 90%. [Conclusions] This method is sensitive, accurate, and rapid. It is suitable for monitoring the concentration of polydatin in serum after intragastric administration, and can also be used for pharmacokinetics and bioavailability studies.

Key words Polydatin, Drug concentration in serum, Content determination, Ultra Performance Liquid Chromatography (UPLC)

1 Introduction

Polydatin is a monomer extracted from the dried rhizomes of the traditional Chinese medicine Polygoni Cuspidati Rhizoma Et Radix, which has the functions of resolving dampness and reduce jaundice, clearing heat and detoxification, dispelling blood stasis and relieving pain, relieving cough and resolving phlegm, and improving the body’s immunity[1]. Polygoni Cuspidati Rhizoma Et Radix is widely used in the treatment of cardiovascular diseases, and research on its key components will provide a new idea for the development of new traditional Chinese medicines. Polydatin is the main effective component of Polygoni Cuspidati Rhizoma Et Radix. It is the product of the combination of resveratrol and glucose. It has proven that polydatin has effects of improving myocardial damage, protecting blood vessels, reducing blood lipids, resisting lipid peroxidation, resisting pulmonary hypertension, resisting shock, resisting vascular injury, and resisting thrombus,etc[2-4].

To play its effect, the drug must enter the blood circulation from the drug site. And only the components absorbed by the blood can have an effect. The nature and concentration of the chemical components that directly affect the body determine the efficacy and toxicology of the drug. In recent years, Ultra Performance Liquid Chromatography (UPLC) has the advantages of high sensitivity and strong specificity, and has been widely used in drug analysis and determination[5-6]. In this study, using the UPLC method, we determined the polydatin in serum and analyzed the relationship between different dosages and drug concentration in blood. This method is convenient to operate, has high sensitivity and good selectivity, and will provide a theoretical basis for the study of the absorption and metabolism of polydatin and the rational clinical use of polydatin.

2 Materials

2.1 InstrumentsWaters Ultra-Performance Liquid Chromatograph (American Waters Corporation, thermostatic autosampler, diode array detector); Empower 3 workstation; MTN-2800D nitrogen blowing instrument (Tianjin Automatic Science Instrument Co., Ltd.); GL124-1SCN electric balance (Sartorius Scientific Instruments (Beijing) Co., Ltd.); TGL-16G refrigerated centrifuge (Shanghai Anting Scientific Instrument Factory); KQ-300VDE ultrasonic cleaner (Kunshan Ultrasonic Instruments Co., Ltd.).

2.2 Drugs and reagentsPolydatin reference substance (batch No.111575-200502, China Food and Drug Control Research Institute); acetonitrile (chromatographically pure, TEDIA, USA); water was ultrapure water; other reagents were analytical pure.

2.3 Experimental animalsMale SD rats weighing (180±20) g were purchased from the Laboratory Animal Center of Guangxi Medical University, license number: SCXK (Gui) 2014-0002. Before the experiment, rats were placed in our laboratory for 4 d.

3 Methods and results

3.1 Preparation of reference solutionPrecisely weighed 20 mg of polydatin, added methanol to dissolve and diluted to the mark, shook well, prepared a 0.8 mg/mL reference substance stock solution, and stored in a refrigerator at 4 ℃. Before the determination, diluted with methanol to obtain different concentrations of reference solution for the plotting of the standard curve.

3.2 Preparation of administration samples and administration methodsUsing 0.5% CMC-Na, prepared polydatin into different concentrations of suspensions. The rats were randomly divided into a blank group and 5 administration groups. The intragastric administration was performed at doses of 30, 50, 100, 200, 500 mg/kg, once a day for 3 consecutive days. Blood was taken at 0.5, 1.0, 2.0, 2.5, 3.0, 4.0, 6.0, 8.0, 12 h after the last administration. Placed for 30 min, centrifuged at 3 000 rpm for 15 min, and took the supernatant. Stored in the refrigerator at -80 ℃ for later use.

3.3 Serum sample treatment methodTook 500 μL of rat serum, added 4 times the amount of acetonitrile, vortex mixed for 1 min, centrifuged at 10 000 rpm for 15 min, took the supernatant, blow dried with nitrogen, added 250 μL of acetonitrile to the residue, vortex dissolved for 30 s, passed through a 0.22 μm microporous membrane, and performed the UPLC analysis.

3.4 Chromatographic conditions and analysis conditionsChro-matographic column: Acquity UPLC BEH shield RP18(2.1 mm×100 mm, 1.7 μm, Waters Corporation, USA); mobile phase: acetonitrile∶water (55∶45); detection wavelength: 306 nm; flow rate: 0.5 mL/min; injection volume: 3 μL; chromatographic column temperature: 30 ℃.

3.5 Methodology examination

3.5.1Method specificity. Took the rat blank serum, the blank serum added with the reference substance of polydatin, and serum after intragastric administration of polydatin in rats, operated according to the serum sample processing method, and took 3 μL of each sample into the ultra performance liquid chromatograph to obtain the UPLC chromatogram. From the chromatogram, it can be seen that the retention time of polydatin is 4.85 min, the chromatographic separation of the administered serum sample is good, and the endogenous substances in the serum do not interfere with the determination of polydatin.

3.5.2Plotting of standard curve. Took 500 μL of blank serum and added 50 μL of polydatin standard series solution to prepare serum samples with concentrations of polydatin of 1.0, 3.0, 5.0, 10.0, 15.0, and 20.0 μg/mL. In accordance with the "serum sample processing" operation, took 3 μL sample and added into the ultra high performance liquid chromatograph, and established a standard curve to obtain the UPLC chromatogram. Took the concentration of polydatin as the abscissa and the peak area of polydatin as the ordinate, the regression operation was performed by the weighted least square method, and obtained the linear equationy=75.12x-36.29 (r=0.999 4), showing that the concentration of polydatin in the serum has a good linear relationship in the range of 1.0-20.0 μg/mL.

3.5.3Precision and accuracy. Took 500 μL of blank serum, added appropriate amount of polydatin standard solution to obtain 5 quality control samples of low, medium, and high concentrations (1.0, 5.0, 10.0 μg/mL), and determined the intra-day and inter-day precision. Continuously measured the precision for 5 consecutive days once a day (the results are shown in Table 1). Experimental data show that the intra-day and inter-day precision (RSD) are both lower than 3.0%, indicating that the detection method has good precision and accuracy.

Table 1 Inter-day and intra-day precision and accuracy determination of polydatin in rat serum (n=5)

3.5.4Stability. Prepared the rat serum samples of polydatin with low, medium, and high concentrations, and injected and analyzed the samples every 2 h at room temperature to investigate the stability of polydatin in the process of sample determination. For each concentration, 3 samples were analyzed. The results show that the samples are stable after placing 24 h at room temperature.

3.5.5Recovery rate. Took 500 μL of rat blank serum, prepared low, medium, and high concentrations (1.0, 5.0, 10.0 μg/mL) quality control samples in accordance with method in "plotting of standard curve", and analyzed 5 samples for each concentration. Replaced acetonitrile with serum, processed in accordance with the serum processing method, prepared samples of corresponding concentrations. For each concentration, analyzed 5 samples to obtain the corresponding peak area. The results indicate that the recovery rates are all greater than 90%, which is relatively stable (Table 2).

Table 2 Determination of recovery rate of polydatin in rat serum (n=5)

Absolute recovery rate=Measured sample concentration (Cmeasurement)/Theoretical sample concentration (Ctheory)

4 Determination of samples

Wistar rats were divided into 6 groups, namely, blank group, 30, 50, 100, 200, 500 mg/kg groups, and the rats were intragastrically administered for 3 d. After the last administration, took the blood at 0.5, 1.0, 2.0, 2.5, 3.0, 4.0, 6.0, 8.0 and 12 h. After taking the blood, placed for 30 min, centrifuged at 3 000 rpm/min for 16 min, and took the supernatant. Processed in accordance with the method in Section3.3to obtain a chromato-gram of the serum sample and determine the concentration of polydatin in the serum. The drug concentration-time curve is shown in Fig.1.

Fig.1 Drug concentration-time curve of rats intragastrically administered with different doses of polydatin

With the increase of the intragastric administration dose, the concentration of polydatin in the blood also tends to increase. There are two different situations in the five doses with 100 mg/kg as the limit; for doses below 100 mg/kg (i.e. 50 and 30 mg/kg), the time for the first appearance of the maximum drug concentration is 1 h, and the time for the second appearance of the maximum drug concentration is 3 h; at doses above 100 mg/kg (i.e, 500 and 200 mg/kg), the first time the maximum drug concentration appears is 0.5 h.

5 Conclusions

The above experimental results show that the UPLC-UV method used in this study has high sensitivity, good selectivity, simple pretreatment method, stable recovery rate, and short analysis time. It is a method suitable for rapid qualitative and quantitative determination of polydatin in serum. The drug concentration-time curve shows that the concentration in the rat serum gradually increases with the increase of the dose. Considering individual differences in rats, after intragastric administration to Wistar rats, as time goes by, it generally takes on a declining trend in the form of a curve.