[Abstract] Objective To determine the content of adenosine in Digu Jiangtang capsules. Method using liquid chromatography. The chromatographic column is a Kromasil C18 column (250mm × 4.6m, 5μm); phosphate buffer (pH 6.8 takes 250ml of 0.2mol / L potassium dihydrogen phosphate solution. Add 118ml of 0.2mol / L sodium hydroxide solution and dilute to 100ml with water , That is) tetrahydrofuran (99: 1) is the mobile phase, the flow rate: 1.0ml / min; the detection wavelength is 20Onm. Results Adenosine had a good linear relationship in the range of 0.05-0.5 μg, with an average recovery of 96.87% and RSD = 1.65%. Conclusion This method can be used for the determination of adenosine in Digu Jiangtang capsules.
ã€Key words】 Digu Jiangtang capsule; Cordyceps sinensis; Adenosine; High-performance liquid chromatography Jiangtang capsule is composed of six flavors such as turmeric, digu skin, and Cordyceps sinensis. It is used to treat thirst and type â…¡ diabetes caused by Yin deficiency and blood stasis. The main ingredient of Cordyceps sinensis in the prescription is adenosine, and its active ingredient is adenosine. We determined the content of adenosine in Jiangtang capsules by HPLC. This method is simple, accurate, and highly sensitive.
1 Instrument and reagent LC-2010A high-performance liquid chromatograph, PDA-lOAvp detector (Shimazu Corporation of Japan), SB5200 ultrasonic cleaner (Shanghai Branson Corporation); adenosine reference substance (China National Institute for the Control of Pharmaceutical and Biological Products for content determination Use); Digu Jiangtang capsule and negative blank (Qinhai Tibetan Medicine Factory in Qinghai Province); tetrahydrofuran (chromatographically pure); water is ultrapure water; the remaining reagents are analytically pure.
2 Chromatographic conditions Kromasil C18 liquid chromatography column (250mmx4.6mm, 5μm); mobile phase: phosphate buffer (pH6.8 take 250ml of 0.2mol / L potassium dihydrogen phosphate solution, add 118ml of 0.2mol / L sodium hydroxide solution , Diluted with water to 100ml, that is) tetrahydrofuran (99: 1), flow rate: 1.0ml / min; detection wavelength 26Onm.
3 Experimental methods and results 3.1 Preparation of the sample solution Take the contents of this product (approximately equivalent to 4 capsules), weigh accurately, add 90ml methanol 10ml, weigh accurately, sonicate for 20min, let cool, add 90% methanol Make up for the lost weight, filter, and take the filtrate. Filter with 0.45μm microporous membrane as the test solution. In addition, a negative control product containing no Cordyceps sinensis was prepared, and a negative control solution was prepared by the same method.
3.2 Preparation of reference substance solution Weigh accurately adenosine reference substance, add 90% methanol to make 0.015mg / ml reference substance solution.
3.3 System adaptability test Take the test sample, negative blank sample and reference substance solution, according to two chromatographic conditions, each injection lOμl, measured HPLC spectrum. The spectrum indicates that the negative control chromatogram is free of interference. The theoretical plate number is not less than 3,000 based on the adenosine peak calculation.
3.4 Examination of linear relationship Weigh accurately adenosine reference substance, add 90% methanol to make a solution of 0.05mg / ml, precisely draw the above solutions 1, 2, 4, 6, 8, 10ml and add 90 to a 10ml volumetric flask Dilute to the mark with% methanol, shake well, inject 1Oμl each, determine the peak area according to the above chromatographic conditions, draw the standard curve with the peak area value as the ordinate, and the adenosine amount (μg) as the abscissa. = 3806623X + 6617r = 0.9998. It shows that adenosine has a good linear relationship in the range of 0.05 ～ 0.5μg.
3.5 Precision test Take the reference solution, inject 10μl, repeat the injection 6 times, the RSD of the peak area is 1.82%.
3.6 Stability test The prepared reference solution was injected into 10 μl at 0, 1, 2, 4, 6, and 8 h, and the peak area was recorded. The RSD of the peak area was 1.94%. It can be seen that the test solution is stable within 8h.
3.7 Reproducibility test Take hypoglycemic capsules (batch number 20010710), 6 samples in parallel, according to the sample determination method, the results contain adenosine 0.042mg, 0.04mg, 0.04mg, 0.043mg, 0.042 mg, 0.040mg (RSD = 2.52%, n = 6) shows that this method has good reproducibility.
3.8 Recovery rate test: Add sample recovery method, take 6 samples of hypoglycemic capsules with a known content (batch number 20010710 content is 0.041 mg / capsule), accurately weigh them, add the appropriate amount of adenosine, and prepare according to the sample solution Under the item, prepare, inject, record chromatograms and calculate the recovery rate according to the law.
3.9 Determination of the sample: Accurately draw 10 μl each of the reference substance and the sample solution into the liquid chromatograph, measure according to the above chromatographic conditions, record the chromatogram, and calculate the content of adenosine in the sample using the external standard method.
4 Discussion 4.1 The determination methods of adenosine in preparations mainly include thin layer scanning method, spectrophotometry, high performance liquid chromatography, capillary electrophoresis, etc. In this paper, HPLC method is used to determine the content of adenosine in Digu Jiangtang capsules. The method is simple, accurate, and highly sensitive, which can control the internal quality of the preparation.
4.2 Digu Jiangtang capsule is a compound preparation of traditional Chinese medicine with complex ingredients. In the experiment, 15% tetrahydrofuran phosphate buffer solution was tried; methanol-0.05mol / L potassium dihydrogen phosphate solution (13:87), 6% acetonitrile, Phosphate buffer solution (pH6.8 take 250mol of 0.2mol / L potassium dihydrogen phosphate solution, add 18mol of 0.2mol / L sodium hydroxide solution, dilute with water to 1000ml, then get) -tetrahydrofuran (99: 1) and other solvents The system was separated, and it was determined to use phosphate buffer (pn6.8 to take 250ml of 0.2mol / L potassium dihydrogen phosphate solution, add 18ml of 0.2mol sodium hydroxide solution, and dilute to 1000ml with water)-tetrahydrofuran (99: 1) As mobile phase, adenosine is well separated, and the negative control has no interference.
4.3 Preparation of the sample solution. In the experiment, three extraction methods of reflux, cold immersion and ultrasonic extraction were investigated with 90% methanol. The results show that the extraction effect of the three is basically the same. Because the ultrasonic extraction method is simple and fast, the ultrasonic extraction method is selected to prepare the sample solution.
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[4] Qi Yanfei. HPLC determination of adenosine in compound monkey head capsules [J]. Chinese patent medicine, 2O02, 24 (3): 181
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