Determination of naringin content by HPLC - Master's thesis - Dissertation

The bone capsule prescription is a capsule prepared by extracting eight kinds of herbs such as sclerotium, epimedium and myrrh, and has the effects of strengthening kidney and strengthening bone, continuation and pain relief. In this prescription, sclerotherapy is a medicinal herb, and its flavonoid naringin is an indicator component. The method for determining the content of naringin is reported in the literature. I1], we refer to the method for determination of naringin in the Chinese Pharmacopoeia by high performance liquid chromatography [5], and the naringin contained in the preparation of the granules in the preparation. The method validation test and the determination of the content of multiple batches of samples show that the method is simple and easy to use and can be used for quality control of the preparation. 1 Instruments and reagents 1.1 Instrument high performance liquid chromatography, SPD-20AVP UV detector (Shimadzu, Japan); LC_2OATVP infusion pump (Shimadzu, Japan), Zhejiang University N200O chromatography workstation; electronic balance FA2004 (Shanghai Precision Science Instrument Co., Ltd.). 1.2 Test substance Naringin reference substance is provided by China National Institute for the Control of Pharmaceutical and Biological Products (for content determination, batch number 110722-2OO309). Zhuanggu Capsule (homemade). The reagent was analytically pure, the water was double distilled water, and the methanol was chromatographically pure. 2 Methods and Results 2.1 Chromatographic conditions Column: Phenomenex ODS column (250 mmX 4.6 mm, 5, urn); mobile phase: methanol-acetic acid-water (37:2: 61); detection wavelength: 283 nm; flow rate : 1.2 mL·rain_. Column temperature: room temperature. The number of theoretical plates is not less than 3,000 according to the naringin peak. The chromatogram is shown in Figure 1. 2.2 Preparation of the reference solution The appropriate amount of naringin reference substance dried at 110 ° C to constant weight is accurately weighed and added. Methanol is made into a solution containing 0.08 mg per 1 mL. 2.3 Preparation of the test solution Take the contents of 10 capsules, grind finely and mix well; accurately weigh 1.0 g, place in a triangular flask, add methanol 30 mL, place in ultrasonic oscillator, ultrasonic extraction After 30 min, let it cool, filter and evaporate. The residue was dissolved in methanol and transferred to a 10 mL measuring flask, diluted with methanol to the mark, shaken, filtered, and the filtrate was taken. 2.4 Methodological investigation 2.4.1 Linear relationship experiment Precision extraction of prepared naringin reference solution (mass concentration of 0.092 g·L) 2,4,6,8 and 1O belly L, respectively injected High-performance liquid chromatograph, the peak area was determined according to the above-mentioned chromatographic conditions, and the integral value (y) of the peak area was used to perform regression analysis on the mass concentration (X) of the reference substance, and the regression equation was obtained as Y-172 1] 5 X + 43 318,r a 0.999 5 The experimental results show that the naringin reference substance has a good linear relationship with the peak area in the mass concentration range of 0.184~0.920 g·L-. . 2.4.2 Precision experiment Accurately absorb 1O L of the naringin reference solution, and inject it 6 times according to the chromatographic conditions outlined above. The RSD value of the peak area integral value of the naringin reference substance was 0.9. 2.4.3 Stability test Take the same batch of Zhuanggu Capsules, prepare sample solution, and inject the sample once every 2 h according to the above-mentioned chromatographic conditions for 4 times. The RSD value of the peak area of ​​naringin is measured. 1.2, the experimental results show that the prepared sample solution has good stability within 8 h. 2.4.4 Reproducibility experiment Take the same batch of Zhuanggu Capsules, prepare 6 sample solutions according to the preparation method of the prepared test solution, and measure them according to the above-mentioned chromatographic conditions. The RSD is 1.6. This indicates that the method is reproducible. 2.4.5 Recovery rate The precision is weighed 0.5 g of the same batch of the known sample, a total of 6 parts, each of which is precisely added to the naringin reference solution, according to the proposed preparation method of the test solution A total of 6 samples of the test solution were prepared, and the naringin content of the sample was determined according to the chromatographic conditions outlined above, and the recovery was calculated respectively. The average recovery was 99.1 and the RSD was 1.9. 2.5 Sample determination Take different batches of samples, prepare the test solution according to the proposed preparation method of the test solution, determine the peak area integral value according to the chromatographic conditions as defined above, and calculate the content of naringin in the 6 batches. They were 0.36, 0.39, 0.32, 0.41, 0.40 and 0.38 mg·particles, respectively. 3 Discussion Ethanol, volume fraction of 70 ethanol and methanol were used as extraction solvents. Three methods of cold dip extraction, reflux extraction and ultrasonic extraction were compared. The results showed that the extraction efficiency of methanol was better than that of ethanol, because the preparation was concentrated and concentrated. After the capsule, the naringin can be completely extracted in the preparation by simple sonication. Through the examination of the ultrasonic extraction time, the results showed that after the sample was extracted by ultrasonic for 30 min, the naringin was completely extracted. Therefore, the sample was treated with methanol as solvent and sonicated for 30 min to prepare the test solution.