Determination of Icariin in Shenqi Bolikang Tablets by High Performance Liquid Chromatography - Science Definition

The ginseng Bolicom tablets are composed of ginseng, epimedium, astragalus, and rhizoma sinensis. They have the functions of nourishing qi and nourishing blood, nourishing yin and nourishing yang, and are used clinically to treat qi and blood deficiency, yin and yang deficiency, and body fatigue. , loss of appetite, palpitations, insomnia, weak waist and knees, night sweats and nocturnal emission [ll, has a good effect. The original drug standard is contained in the Chinese medicine prescription for the drug standard of the Ministry of Health. However, only the thin-layer chromatographic identification method of ginsenoside Re has no corresponding content determination index, and the inherent quality of the drug cannot be effectively controlled. To this end, this study used high performance liquid chromatography (HPLC) to determine the content of icariin in Shenqi Bolikang tablets [2-6], and rationally optimized the mobile phase, according to Epiglottis The properties of guanosine use the simplest methanol solution to extract icariin directly from the sample without complicated pretreatment. The method is fast, accurate and simple, and can control the intrinsic quality of the preparation. 1 Experiment 1.1 Instruments and reagents Waters 515 HPLC instrument (Waters, USA); KQ. 250B ultrasonic cleaner f Kunshan Ultrasonic Instrument Co., Ltd. 1. Icariin reference substance (China National Institute for the Control of Pharmaceutical and Biological Products, for determination of content); Shenqi Bolikang tablets and negative blank (homemade); acetonitrile (chromatographically pure); water is Wahaha pure water; other reagents are analyzed pure. 1.2 Chromatographic conditions Agilent ZORBAX SB·C18 column f250 mm x 4.6 mm, 5 m, Dalian Elite Analytical Instruments Co., Ltd.); Mobile phase: (acetonitrile): V (water) -30:70, flow rate: 1 .0 mL/min; detection wavelength 270 nm; column temperature: 40. C. 1.3 Preparation of the reference solution The icariin reference substance was accurately weighed, and methanol was added to make a reference solution of 0.158 mg/mL. 1.4 Preparation of sample solution Take appropriate amount of sample, grind finely, weigh about 1 g, place it in a 25 mL volumetric flask, add appropriate amount of methanol, remove it by sonication for 20 min, let cool to room temperature, and dilute to 25 mL with methanol. The supernatant was filtered through a 0.45 m microporous membrane, and the filtrate was used as a sample solution. 1.5 Preparation of negative blank sample solution According to the prescription of quality standard, it is formulated into group drug without epimedium, according to the process, it can be made into blank preparation of epimedium, and then made into negative according to the preparation method of sample solution. Blank sample solution. 2 Results 2.1 System Adaptability Test The above icariin reference solution, sample solution and negative blank sample solution were taken, and 10 L of each sample was injected according to the chromatographic conditions of 1.2, and the HPLC spectrum was measured. 2. The map shows no interference in the negative control solution chromatogram. The number of theoretical plates is more than 5,000 based on the peak of icariin, and the chromatographic resolution is greater than 1.5. 2.2 The relationship between linearity is accurately weighed the icariin reference substance, and methanol is added to make 0.063 mg/mL. The solution is accurately sucked into the above solution 1, 3, 5, 7, 9, l1, 13, 15, 17, 19, 20 L, and the peak area is determined according to the above chromatographic conditions, and the peak area value is the ordinate. The amount of glycosides (g) is a standard curve plotted on the abscissa, and the regression equation is: Y=3.920x10-X-1.098×10_. , r = 0.9999. The results showed that icariin had a good linear relationship in the range of 0.063~1.260 g. 2.3 Precision test Take the icariin reference solution, repeat the injection 8 times, 10 L each time under the same experimental conditions, and determine the peak area integral value. The relative standard deviation of the peak area was 0.79%, indicating that the precision of this method is good. 2.4 Stability test The test solution was injected at 10 L at 0, 0.5, 1, 2, 4, 6, 8, 10 h, and the peak area was recorded. The relative standard deviation of the peak area was 0. 63%, indicating that the test solution has good stability within 10 h. 2.5 Repeatability test Take 6 samples of Shenbo Bolicom tablets in parallel and operate according to the method of Section 1.4. Six sample solutions were determined in parallel. The results showed that the content of icariin was 0.273 and 0.275 respectively. 0.277, 0.276, 0.274, 0.271 mg/g, relative standard deviation 0.78% (n=6), indicating that the method has good repeatability. 2.6 Recovery test The sample sample recovery method was used to take 6 samples of Shenqi Bolikang tablets (content 0.273 mg/g), which were measured and accurately weighed, and accurately added to icariin. A certain amount of the reference solution, according to the method of 1.4, according to the law preparation, injection, record chromatogram, calculate the recovery rate, the results are shown in Table 1, the average recovery rate is 97.1%, the relative standard deviation is 0.83% . 2.7 Sample determination: 10 L of the reference substance and the sample solution were respectively accurately taken, and the content of icariin in the sample was calculated by the external standard method according to the above chromatographic conditions. The results are shown in Table 2. 3 Discussion on the reference substance of icariin The solution was scanned in the range of 200 to 400 nm. As a result, icariin had the maximum absorption at 270 nm, so it was selected as the measurement wavelength. Take appropriate amount of Shenqi Bolikang tablets, prepare according to the preparation method of sample solution, compare the ultrasonic time, respectively, ultrasonic 10, 20, 30, 40, 50 rain, and find the active ingredients in Shenli Bolicom tablets after 20 min. Extract completely. This product selects methanol as solvent, adopts ultrasonic vibration direct extraction method, compares with other organic solvent [ ] and extraction method f reflux extraction, etc. [10] test, the sample solution prepared by the above chromatographic conditions and methods has less impurity content. The interference is small, and the satisfactory separation effect can be achieved. The column efficiency is based on the main peak, and the number of theoretical plates is more than 7000. The operation is simple, the result is accurate, the repeatability is good, and the product quality can be effectively controlled.