Abstract: Paeoniflorin is hygroscopic amorphous tan powder (90% is off-white powder), [α] 16D-12.8. (C = 4.6, methanol), tetraacetate is colorless needle crystal, melting point: 196 ℃. Paeoniflorin is stable in acidic environment (pH 2 ～ 6), and unstable in alkaline environment.
Objective To establish a method for determining the content of paeoniflorin in Langshouling granules. Methods High performance liquid chromatography was used. Column: HypersilODS-2 (150mm × 4.6mm, 5.0μm); mobile phase: acetonitrile-0.4% phosphoric acid aqueous solution (10:90); detection wavelength: 230nm; flow rate: 1.0mL / min, column temperature: 25 ° C. Results Paeoniflorin showed a good linear relationship (r = 0.9999) in the range of 0.608 ～ 3.04μg, the recovery rate was 97.77%, and the RSD = 0.62% (n = 5). Conclusion The method is fast, feasible, simple, accurate, reproducible, and reliable. It can be used to control the quality of Lupuling Granules.
Lupuling granules are mainly compound preparations of hospital composed of traditional Chinese medicines such as red peony root, peony bark, radix rehmanniae, honeysuckle, etc. They have the effects of cooling blood, removing blood stasis, clearing heat and detoxifying. Paeoniflorin in the prescription is the main active ingredient and has a concentration- and function-dependent two-way immunomodulatory effect. It is mainly used clinically to treat systemic lupus erythematosus. In this study, high performance liquid chromatography (HPLC) method was used to determine the content of paeoniflorin in Langshouling granules, and an HPLC method for determining the content of paeoniflorin was established [1-2].
1 Instruments and reagents
Agilent1100 high-performance liquid chromatograph, MettlerToledo AG135 electronic analytical balance (Switzerland Mettler company), SCQ-250 ultrasonic extractor (Shanghai Shenbo Ultrasonic Company). Paeoniflorin reference substance (provided by China National Institute for the Control of Pharmaceutical and Biological Products, batch number 110736-200732); lupushenling granules (provided by the Affiliated Hospital of Chengdu University of Traditional Chinese Medicine, batch number 100315, 100318, 100322, 100329, specifications 15g / bag). Acetonitrile is chromatographically pure, water is pure water, and other reagents are analytically pure.
2 Methods and results
2.1 Chromatographic conditions
Chromatography column: HypersilODS-2 (4.6mm × 150mm, 5.0μm, Elite); mobile phase: acetonitrile-0.4% phosphoric acid aqueous solution (10:90); detection wavelength: 230nm; flow rate 1.0mL / min; column temperature: 25 ℃; injection volume: 10μL.
2.2 Solution preparation
2.2.1 Preparation of reference substance solution Precisely weigh the paeoniflorin reference substance 3.04mg, place it in a 10mL volumetric flask, add methanol to dissolve and dilute to the mark, shake to obtain the reference substance solution (1mL containing paeoniflorin 0.304mg).
2.2.2 Preparation of the solution for the test product Take 0.5g of this product, accurately weigh it, put it in a conical flask with a stopper, add 50mL of methanol precisely, shake well, weigh it, ultrasonic treatment (power 300W, frequency 40kHz) 20min, Let it cool to room temperature [3], weigh it again, make up the lost weight with methanol, shake it up, filter it, and take the filtrate to get it.
2.2.3 Preparation of negative reference solution The negative sample preparation lacking red peony was prepared according to the preparation process of lupushenling granules, and then the negative reference solution was prepared according to the method under "2.2.2".
2.3 Investigation of linear relationship
Take the above reference solution and automatically inject 2, 4, 6, 8, and 10 μL respectively. A standard curve was drawn with the integral value of the peak area of ​​paeoniflorin as the ordinate, and the injection volume (μg) of the paeoniflorin reference substance as the abscissa. The regression equation is: Y = 1427.68X + 5.96, r = 0.9999. The results show that the injection volume is in a good linear relationship in the range of 0.608 ～ 3.04μg.
2.4 Specific test
Accurately draw 10μL each of the reference solution, test solution and negative reference solution, according to the above chromatographic conditions.
2.5 Precision test
Accurately draw 10μL of the reference solution and repeat the injection 5 times. The peak area integral value was measured and calculated. The RSD of paeoniflorin was 1.19% (n = 5), indicating good precision.
2.6 Stability test
Accurately draw the test solution of the same batch (batch number 100318), and measure at 0, 4, 8, 12, 16, 24h after preparation according to the chromatographic conditions. The peak area was recorded, and the RSD of paeoniflorin was calculated to be 1.30% (n = 5), indicating that the content of paeoniflorin was stable within 24h.
2.7 Repeatability test
Precisely weigh 5 samples of the same batch number (lot number 100318), prepare according to the preparation method of the test sample solution, and determine according to the above chromatographic conditions, the average content of paeoniflorin measured is 9.73mg / g, RSD = 1.24% (n = 5) .
2.8 Sample recovery test
Precisely weigh 0.10g of lupusling granules with known content, add a certain amount of paeoniflorin reference substance accurately, prepare according to the preparation method of test solution, and determine according to the above chromatographic conditions. Results The average recovery rate was 97.77%, RSD = 0.62%. See Table 1.
2.9 Sample determination
Precisely weigh about 0.5g of Lupuling granules from different batches, prepare according to the preparation method of the test solution, and test according to the above chromatographic conditions. The results are shown in Table 2. According to the test results, taking into account the origin of the medicinal materials, as well as the production and storage of the preparation, etc., it is tentatively planned that each batch of samples should be based on the content of paeoniflorin, and the particle size should not be less than 9.7mg per 1g.
Table 1 Sample recovery test results
Sample weight
Amount (g) The sample contains
Amount (mg) Amount added
(mg) Measured amount
(mg) Recovery rate
(%) Average recycling
Rate (%) RSD
(%)
0.10190.9911.0011.96797.43
0.10240.9960.9751.95598.35
0.10170.9890.9871.94696.9297.770.62
0.10150.9870.9901.96198.29
0.10230.9950.9841.95897.84
Table 2 Determination results of paeoniflorin in samples (mg / g)
Batch number of paeoniflorin content average content
123
1003159.789.719.739.74
1003229.749.709.719.72
1003299.729.789.789.76
3 Discussion
In this experiment, three methods of water bath reflux extraction, ultrasonic extraction, and Soxhlet extraction were investigated, and the ultrasonic extraction method was superior to the reflux method and Soxhlet extraction method; three kinds of extraction solvents, such as methanol, 70% ethanol, and 50% ethanol, were investigated. The effect of glycoside extraction results showed that the methanol extraction effect was significantly better than 70% ethanol and 50% ethanol; the effect of extraction time on the extraction effect of paeoniflorin was investigated. The result was that methanol was used as the extraction solvent, and ultrasonic extraction for 20 minutes was better.
In this experiment, the mobile phase used in the determination of paeoniflorin in Radix Paeoniae Rubra (part one) of the 2005 edition of the People's Republic of China: methanol-0.05mol / L potassium dihydrogen phosphate solution (40:65). There is interference. After analysis and comparison, it is determined that acetonitrile-0.4% phosphoric acid aqueous solution (10:90) is the mobile phase, and the concentration of hetero peaks can make the paeoniflorin get better separation.
The extraction method of this method is simple, rapid analysis, high precision, good repeatability, negative interference-free, and can better control the quality of this product.
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