Abstract: Objective To establish a method for the determination of berberine hydrochloride in Shaoyao Tang. Methods High performance liquid chromatography was used. The column: AgilentXDBC18 (4.6mm × 250mm, 5μm); mobile phase: acetonitrile-0.05mol / L potassium dihydrogen phosphate aqueous solution-triethylamine (30: 70: 0.14); flow rate: 1.0 mL / min; column temperature: 25 ℃; detection wavelength: 345nm.
Shaoyao Decoction comes from "Su Wen Qi Qi Qi Yi Bao Ming Ji" [1], which is composed of 9 Chinese herbs such as Paeonia, Angelica, Coptis, Scutellaria, Betel Nut, Woody, Licorice, Rhubarb, Cinnamon, etc. The efficacy of humid diarrhea. Shaoyao Decoction is a commonly used prescription for the treatment of ulcerative colitis [2], but there are few studies on its effective ingredients and quality standards, and only the content of paeoniflorin contained in it has been reported [3]. Coptis chinensis and Radix Scutellariae are the Chinese medicine of Fang Zhong [1]. The active ingredient of Coptis chinensis is berberine. Therefore, in this study, berberine hydrochloride was used as the main active ingredient of the compound. The method of berberine content. The results show that the method is simple, accurate and reproducible, which provides a basis for the quality control of Shaoyao Tang.
1 Instruments and reagents
High performance liquid chromatograph: AgilentHPLC1100 chromatograph (G1311A quaternary pump, G1313A autosampler, G1316A column thermostat, G1315BDAD detector, HP ChemStation); SartoriusCP225D electronic balance.
Berberine hydrochloride standard (provided by China National Institute for the Control of Pharmaceutical and Biological Products, batch number 110713-200910, for content determination); all the medicinal materials used in Fangzhong were purchased from Beijing Fengtai Jinyuan Pharmaceutical Co., Ltd., and were researched by the Chinese Academy of Chinese Medical Sciences. The researcher Hu Shilin appraised all the authentic products included in the 2005 edition of the Pharmacopoeia of the People's Republic of China (Part 1). Acetonitrile (chromatographically pure, Fisher); other reagents are analytically pure; water is Wahaha purified water.
2 Methods and results
2.1 Chromatographic conditions
Chromatography column: AgilentXDBC18 (4.6mm × 250mm, 5μm); mobile phase: acetonitrile-0.05mol / L potassium dihydrogen phosphate aqueous solution-triethylamine (30: 70: 0.14); flow rate: 1.0mL / min; column temperature: 25 ℃; detection wavelength: 345nm.
2.2 Extraction of prescription drugs
Weigh each flavor of Chinese medicine according to the prescription, put it in a decoction, extract it twice using traditional water decoction method, add water amount of 6, 4 times, decoction for 30min each time, combine decoction liquid, concentrate, vacuum dry to dry Paste powder, set aside.
2.3 Preparation of reference solution
Precisely weigh the appropriate amount of berberine hydrochloride reference substance, add hydrochloric acid-methanol solution (1: 100) to make a solution containing 70μg per 1mL, that is, get.
2.4 Preparation of test solution
Take Shaoyao Tang dry paste powder (through No. 4 sieve) about 50mg, weigh accurately, add 30mL of hydrochloric acid-methanol solution (1: 100) precisely, weigh it, ultrasonic extraction (power 250W, frequency 40kHz) 30min, let cool, Weigh it again, make up the lost weight with hydrochloric acid-methanol solution, shake well and filter. After the filtrate is concentrated, it is transferred to a 10mL volumetric flask, diluted to the mark with hydrochloric acid-methanol solution, shaken, and filtered with a microporous filter membrane (0.45μm), and the continuous filtrate is taken.
2.5 Investigation of linear relationship
Precisely draw the above reference solution 1, 3, 5, 7, 9, 11, 20μL injection analysis, determine the peak area. The integral value of the peak area was regressed by the injection volume, and the regression equation was obtained: Y = 2513.5X-32.56, r = 0.9999, berberine hydrochloride showed a good linear relationship between 0.07 ～ 1.40μg.
2.6 Precision test
Accurately draw 5μL of the same test solution, analyze according to the above chromatographic conditions, repeat the injection 6 times, the peak area RSD = 0.33%, indicating that the instrument precision is good.
2.7 Stability test
Take 1 sample solution, put it at room temperature, and inject 5μL at 0, 1, 2, 3, 4, 5, 6h, respectively, and analyze according to the above chromatographic conditions. Its peak area RSD = 0.61%, indicating that the sample solution The stability is good within 6h.
2.8 Repeatability test
Prepare 6 parts of the test product solution according to the method under "2.4", draw 5μL respectively, analyze according to the above chromatographic conditions, determine the peak area and calculate the content. As a result, the average content of berberine hydrochloride in the test product is 1.55%, RSD = 1.14%. Shows good repeatability.
2.9 Sample recovery test
Adopt sample recovery method. Take about 50mg of Shaoyao Tang dry ointment powder with known content (about 0.775mg of berberine hydrochloride), weigh accurately, take a total of 6 parts, and add 4mL of berberine hydrochloride reference solution (0.194mg / mL) respectively, Prepare the test solution according to the method under "2.4", draw 5μL respectively, analyze according to the above chromatographic conditions, determine the peak area and calculate the content. The average recovery rate is 98.63%, RSD = 1.21%. See Table 1. Table 1 Sample recovery test results (omitted)
2.10 Sample determination
Accurately draw 10 μL each of the reference solution and the test solution, and then inject it into the liquid chromatograph to measure the peak area and calculate the content of berberine hydrochloride in the test product. The chromatogram is shown in Figure 1; the content determination results are shown in Table 2. Table 2 Sample measurement results (omitted)
3 Discussion
In this study, the preparation method of the paeoniae soup sample was optimized. Three methods were used to prepare the samples: the water decoction, the water decoction evaporated methanol to dissolve, and the water decoction was made into dry paste powder methanol. The results showed that the water The percentage of berberine hydrochloride in the test solution prepared by the ultrasonic extraction method of the dry paste powder made from the decoction powder was significantly higher than that of other methods.
This study optimized the separation conditions of the instrument. A full-wavelength ultraviolet scan of berberine hydrochloride was found to have three absorption peaks around 250, 280, and 340 nm. 254, 280, 340, 345, and 350 nm were selected for detection. Both are greater than other wavelengths, so 345nm is selected as the detection wavelength. In the selection of chromatographic columns, we selected AgilentSBC18 (4.6mm × 250mm, 5μm), AgilentXDBC18 (4.6mm × 250mm, 5μm), AgilentExtendC18 (4.6mm × 250mm, 5μm) for the determination. , 5μm) chromatographic column is superior to other chromatographic columns in terms of peak shape, plate number, retention time, etc. In this study, the mobile phase and column temperature were also screened. The mobile phase was selected from acetonitrile-0.2mol / L potassium dihydrogen phosphate aqueous solution (25:75) and acetonitrile-0.033mol / L potassium dihydrogen phosphate aqueous solution (30:70). , Acetonitrile-0.05mol / L potassium dihydrogen phosphate aqueous solution (30:70), acetonitrile-0.05mol / L potassium dihydrogen phosphate aqueous solution-triethylamine (30: 70: 0.14), acetonitrile-0.05mol / L dihydrogen phosphate The potassium aqueous solution (SDS1.7g per 1000mL aqueous solution) (30:70) was tested. The results showed that the separation effect of acetonitrile-0.05mol / L potassium dihydrogen phosphate aqueous solution-triethylamine (30: 70: 0.14) was the best. On the column temperature, the utility selected 25 and 35 ℃ for comparison, and finally selected the column temperature at 25 ℃ for separation.
We screened the extraction solvent and compared water, 75% ethanol, 95% ethanol, methanol, hydrochloric acid-methanol (1: 100), and the results showed that the content of berberine hydrochloride was significantly higher when hydrochloric acid-methanol (1: 100) was used as the solvent. Used in other solvents. In the screening of the amount of extraction solvent, extraction method and extraction time, cold immersion, reflux, and ultrasonic method were used. After optimization, hydrochloric acid-methanol (1: 100) 30mL ultrasonic extraction was used for 0.5h as the extraction condition of the sample.
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