Determination of Paeoniflorin in Xiaoyao Pills by Saturated Flow-out Solid Phase Extraction

Abstract: Objective To prepare Xiaoyao Pill test solution by SPEHPLC method and determine the content of paeoniflorin by high performance liquid chromatography. Methods ZorbaxSBC18 column (4.6 mm × 250 mm, 5 μm), using acetonitrile water (20:80) as mobile phase, flow rate of 1 ml / min, detection wavelength of 230 nm. Results The measurement of paeoniflorin had a good linear relationship in the range of 0.123 2 ～ 1.232 μg, r = 0.999 9, the average recovery rate was 99.16%, and the RSD was 1.6%. Conclusion The method is simple, rapid, highly sensitive and effective to remove the protective matrix of the drug matrix. It can be used for the determination of paeoniflorin in Xiaoyao pills.

Keywords: Xiaoyao Pill; Paeoniflorin; Solid Phase Extraction Device; High Performance Liquid Chromatography

Content Determination of Peoniflorin in XiaoYao Pill bySaturated Flowthrough SPEHPLC

Abstract: ObjectiveTo develop saturated flowthrough SPEHPLCmethod for the assay of Peoniflorin in XiaoYao pill. Methods Separation was performed on a ZorbaxSBC18 column (4.6 mm × 250mm, 5 μm). The mobile phase consisted of acetonitrile-water (20:80, v / v). The flow rate was 1 ml / min. The UVdetection was set at 230 nm. Results Peoniflorin had a good linear relationship in the range of 0.123 2 ～ 1.232 μg, the average recovery of XiaoYao pill was 99.16% and the RSD was 1.6%. Conclusion Themethod is simple, sensitive and rapid, and reduce the damage ofbase material to the chromatographic column, it can be applied tothe determination of peoniflorin in XiaoYao Pill．

Key words: XiaoYao Pill; Peoniflorin; SPE; HPLC

Xiaoyao pill (water pill) is a water pill made from the mixture of Bupleurum, Angelica, white peony root, Atractylodes macrocephala, Poria, licorice root, mint seven flavor traditional Chinese medicine powder and ginger decoction. Applicable to the depression caused by liver depression and spleen deficiency, chest pain, dizziness, dizziness, loss of appetite, irregular menstruation [1]. High-performance liquid chromatography has the advantages of strong specificity and high precision, but it has high requirements for the purification of the test solution. This product is directly used as a drug powder, and a large amount of matrix will damage the chromatography column. Saturated effluent solid phase extraction Chinese medicine sample purification method has the advantages of simple and rapid, less loss of components to be tested, good reproducibility and effective protection of the chromatographic column [2], this article reports the application of this pretreatment method to prepare the test solution, HPLC method for determination of paeoniflorin in Xiaoyao pills. The results are reported below.

1 Instruments and reagents

1.1 Instrument American Waters600 high-performance liquid chromatograph, Waters600E quaternary pump, 717 automatic sampler, 2487 UV-visible spectrum detector, online degassing device, Empower chromatography workstation, Agilent ZorbaxSBC18 column, solid phase extraction cartridge (self-made) , Germany SartoriusBP221S (one ten thousandth), BT25S (one hundred thousandth) electronic balance.

1.2 Test drug Xiaoyao Pill (Shuiwan, Guangzhou Jingxiutang Pharmaceutical Co., Ltd., batch number: C11018), paeoniflorin reference substance (provided by China National Institute for the Control of Pharmaceutical and Biological Products, 110736200423), alumina (Shanghai May Fourth Chemical Reagent Factory 100200 mesh) ), Acetonitrile is chromatographically pure, water is ultrapure water, and the remaining reagents are analytically pure.

2 Methods and results

2.1 Chromatographic conditions [3] Column: Agilent: Zorbax SBC18 column (4.6 mm × 250 mm, 5 μm); mobile phase: acetonitrile water (20:80); flow rate: 1 ml / min; detection wavelength 230 nm; column temperature 30 ℃, inlet Sample volume 5μl. Under the above chromatographic conditions, the retention time of paeoniflorin was 6.09 min. The results are shown in Figure 1.

2.2 Preparation of reference substance solution Precisely weigh 1.85 mg of the paeoniflorin reference substance of phosphorus pentoxide and dry to constant weight, place in a 10ml volumetric flask, add dilute ethanol to dissolve, and dilute to the mark as a stock solution. Precisely transfer 5 ml of the stock solution into a 25 ml volumetric flask, then add 10 ml of dilute ethanol to dilute it, shake well, and obtain a reference solution with a mass concentration of 61.6 μg / ml.

2.3 Preparation of test solution Take a hollow injection needle barrel of appropriate size, place a stopper plate at the bottom, add 5.0g of neutral alumina to obtain a self-made solid phase extraction cartridge [4], use 3 Activate by pre-washing twice the volume of water. Take an appropriate amount of Xiaoyao pill, grind it finely, take about 0.8g, accurately weigh it, place it in a conical flask with a stopper, add 50 ml of dilute ethanol precisely, close the plug, weigh it, ultrasonic extraction (power 250W, frequency 33kHz) 40min, Let it cool, weigh it again, make up the lost weight with dilute ethanol, shake it up, filter it, and continue the filtrate through the above small column, the flow rate is controlled at 0.4 ~ 0.6ml / min, collect the 8 ~ 10 ml effluent as Test solution.

2.4 Preparation of the standard curve Use the autosampler to draw the reference solution 2, 4, 5, 10, 15, and 20 μl into the sample respectively, determine the peak area according to the above chromatographic conditions, and compare the peak area (A) with the injection mass (μg) Perform a linear regression to find the regression equation: Y = 79 546X-37610, r = 0.999 9, indicating that paeoniflorin has a good linear relationship in the range of 0.123 2 ~ 1.232 μg.

2.5 Precision experiment: Draw 5μl of the reference solution accurately and inject 6 times continuously. The RSD of the peak area response value is 0.4%, indicating that the system precision is good.

2.6 Stability experiment Take the test solution prepared from the same batch of samples, inject 5μl each at 0, 2, 4, 6, and 8 h, record the chromatogram, and the measured peak area response value RSD = 0.3%, visible The test product is stable within 8 h.

2.7 Repeatability experiment The same batch of samples were weighed in parallel with exactly 6 parts, each about 0.8 g, and the test solution was prepared according to the method 2.3, and then each was injected with 5 μl, and the average content measured was 4.298 mg / g, RSD = 1.6% , Indicating that the method has good repeatability.

2.8 Sample recovery rate experiment Weigh 6 samples of known content (C11018) accurately, put them in conical flasks with stoppers, add 1ml of reference stock solution (0.185 mg / ml) to each precision, and prepare for The sample solution is measured by injection. The results are shown in Table 1.

a Chromatogram of paeoniflorin reference substance b Chromatogram of solution with pretreatment c Chromatogram of solution without pretreatment

3 Discussion

3.1 Sample pretreatment and preparation of elution curve This product uses dilute ethanol as a solvent, a large number of highly polar macromolecular compounds are precipitated, and the solution is directly exposed to the high-performance liquid phase [3], there are too many interfering components, and the peak of the measured component reaches Before the baseline separation, a large amount of matrix damages the analytical column; when the solution passes through the solid phase extraction column, a large amount of matrix is ​​adsorbed by the polar alumina packing, thereby effectively protecting the analytical column, and the components to be tested have reached a good Baseline separation effect. Due to the strong adsorption of alumina on polar compounds, the polar substances adsorbed on the solid phase extraction cartridge are not easily eluted by the solvent. As the solution continues to be injected, the solid phase extraction cartridge will detect the When the adsorption reaches saturation, the concentration of the component to be tested in the effluent is the same as the concentration of the component to be tested before being injected into the cartridge. Collect the effluent of the extraction cartridge 11ml in units of 1ml, number the samples and measure them in sequence, with the effluent volume as the abscissa, the percentage of the peak area of ​​paeoniflorin in each effluent and the average peak area of ​​the paeoniflorin in the saturated effluent section is the ordinate , Draw the outflow curve. The results are shown in Figure 2.

From the 8th tube (ie, the 8th milliliter), the adsorption of paeoniflorin by the solid phase column has reached saturation. To ensure the accuracy of the experiment, the 8-10 milliliters of effluent is collected as the test solution.

3.2 The choice of packing The three kinds of alumina, acidic, basic and neutral, were used as the column packing. It was found that there was no difference in the content of paeoniflorin in the effluent, but the content of paeoniflorin was different when the adsorption saturation was reached. Aluminum is the best, so it is used as a small column packing in this experiment.

3.3 The relationship between the solution concentration and the amount of filler during the experiment, we found that the solution concentration is directly proportional to the amount of filler, that is, the solution concentration is low and the filler is low, and the concentration is high and the filler is high. However, the concentration of the solution is too high, and the components to be tested are not completely extracted; if it is too low, the saturation section of the solution over-extraction cartridge is too narrow, making it difficult to collect an accurate test solution. In this experiment, the composition of solution concentration 0.016g / ml and 5.0g neutral alumina filler was selected.

references:

[1] National Pharmacopoeia Commission. Chinese Pharmacopoeia [S]. Beijing: Chemical Industry Press, 2005: 564.

[2] Zhu Binghui, Liang Yiying, Mo Jinyuan. SepPakC18 Solid Phase Extraction High Performance Liquid Chromatography Determination of Icariin in Haima Baji Pills [J]. Chinese Patent Medicine, 1999, 21 (10): 500.

[3] Zhuang Qiuhong. Determination of Paeoniflorin in Xiaoyao Pills by High Performance Liquid Chromatography [J]. Strait Pharmaceuticals, 2004, 16 (2): 50.

[4] Gao Liqin. Study on the extraction recovery rate of tenoxicam plasma samples with different solid-phase small columns [J]. Chinese Journal of Pharmacy, 2002, 37 (10): 774.

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