Determination of Paeoniflorin in Lupu Lingling Granules by High Performance Liquid Chromatography - Science Definition

Lupusling granules are mainly composed of five kinds of traditional Chinese medicines such as red peony, peony bark, rehmannia root and honeysuckle vine. They have the effects of cooling blood, dispersing phlegm and clearing away heat and detoxification. Among them, paeoniflorin is the main active ingredient, and paeoniflorin has concentration and function. Dependent two-way immunomodulation j, clinically used to treat systemic lupus erythematosus. HPLC method was used to determine the content of paeoniflorin in Radix Paeoniae Alba. The HPLC method for determination of paeoniflorin was established. It can be used for quality control of the preparation. 1 Instruments and reagents Agilent 1 100 high performance liquid chromatography, Mettler Toledo AG 135 electronic analytical balance (Mettler, Switzerland), SCQ-250 ultrasonic extractor (Shanghai Shenbo Ultrasonic). Paeoniflorin reference substance (provided by China National Institute for the Control of Pharmaceutical and Biological Products, batch number: 110736.200732); acetonitrile is chromatographically pure, water is pure water, and other reagents are of analytical grade. 2 Methods and Results 2.1 Chromatographic conditions: Column: Hypersil ODS-2 (4.6 mm × 150 mm, 5.01xm, Elite); Mobile phase: B; 1~-o. 4% aqueous phosphoric acid solution (10:90); detection wavelength: 230 nm; flow rate 1.0 ml/min; column temperature: 25 ° C; injection volume: lOlxl. 2.2 Preparation of solution 2.2.1 Preparation of reference solution: Accurately weigh 3.04 mg of paeoniflorin reference substance, place it in a 10 ml volumetric flask, add methanol to dissolve and dilute to volume, shake well, then obtain control Solution (1 ml containing paeoniflorin 0.304 mg). 2.2.2 Preparation of the test solution: Weigh about 0.50 g of the test sample, place it in a 50 ml volumetric flask, add 30 ml of methanol, place it for 4 h, and sonicate for 20 min. Cool to room temperature. Make up to volume with methanol, shake well, filter, and get. 2.2.3 Preparation of Negative Control Solution: A negative sample preparation lacking red peony was prepared according to the preparation process of lupus granules, and then prepared according to the method of 2.2.2 to prepare a negative control solution. 2.3 Linear relationship investigation: Take the above reference solution, and automatically inject 2 l, 4 txl, 6 txl, 8 l, 10 l. The integral value of the paeoniflorin peak area is plotted on the ordinate (Y), and the injection amount (g) of the paeoniflorin reference substance is plotted on the abscissa (X). The regression equation is: Y=1427.68X+5.96, r=0.9999. The results show that the linear relationship is good in the range of 0.608~3.041xg. 2.4 Specificity test: precision draw reference solution, test solution, negative control solution each lOla, 1, and determined according to the above chromatographic conditions. The results showed no interference peaks at the peak of paeoniflorin (see Figures 1, 2, and 3). 2.5 Precision test: precision draw the reference solution 101xl, repeat the injection 5 times, the peak area integral value was measured, and the RSD of paeoniflorin was calculated to be 1.19% (n=5). The results showed that the precision was good. 2.6 Stability test: The test solution (lot number) of the same batch number is accurately taken and measured according to the chromatographic conditions at 0, 4, 8, 12, 16 and 24 h after preparation. The peak area was recorded and the RSD of paeoniflorin was calculated to be 1.30% (n=5), indicating that the paeoniflorin content was stable within 24 h. 2.7 Repeatability test: Precisely weigh 5 samples of the same batch number (batch number), prepare according to the preparation method of the test solution, and test according to the above chromatographic conditions, the average content of paeoniflorin is 9.73 mg/g, RSD. It is 1.24% (n=5). 2.8 Sample recovery test: accurately weigh the known content of lupus granules to 0.10 g, precisely add a certain amount of paeoniflorin reference substance, prepared according to the preparation method of the test solution, and determined according to the above chromatographic conditions. The results are shown in Table 1. The average recovery was 97.77% and the RSD was 0.62%. 2.9 Sample determination: Weigh accurately about 0.5g of different batches of Lupu Lingling particles, prepared according to the preparation method of the test solution, and accurately draw the reference solution and the test solution for each 10/. Zl, injected into a high performance liquid chromatograph, tested according to the above chromatographic conditions. The results are shown in Table 2. According to the experimental results, the source of the medicinal materials, and the production and storage of the preparations, the batches of each sample are tentatively prepared with the content of paeoniflorin as no more than 9.7 mg per 1 g of granules. 3 Discussion In this experiment, the mobile phase used in the determination of paeoniflorin in the Chinese version of the Chinese Pharmacopoeia in 2005 was used: methanol-0.05mol/L potassium dihydrogen phosphate solution (40:65), the main peak separation was not good, and there was interference. After analysis and comparison, it is finally determined that acetonitrile-0.4% phosphoric acid aqueous solution (10:90) is the mobile phase, and the peaks are concentrated, which can make the paeoniflorin get better separation. In this experiment, HPLC method was used to determine the content of paeoniflorin in Lupu Lingling Granule. The extraction method was simple, the analysis was fast, the precision was high, the reproducibility was good, and the negative was non-interference, which could better control the quality of this product.