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Xingnao Reconstruction Capsules are contained in the fourth volume of the "Medical Standards for Traditional Chinese Medicine and Chinese Medicine Preparations", which has the effect of revitalizing the brain and stimulating the wind. It is used for recovery and sequelae of unconsciousness, language sputum, sputum sputum, kidney deficiency, muscle aches and pains, hand and foot restraint, hemiplegia and cerebral thrombosis. According to the determination method of the content of astragaloside in the 2005 edition of the Pharmacopoeia of the People's Republic of China (Part I), this experiment established high performance liquid chromatography-evaporative light scattering detection (HPLC-ELSD) to determine the content of astragaloside in Xingnao Reconstruction Capsules. content. The method is accurate, high in sensitivity and good in reproducibility, and can be used for quality control of Xingnao Reconstruction Capsule.
1. Instruments and reagents: Japan Shimadzu LC-10AT VP high performance liquid chromatography, Hitachi ALLTECH ELSD2000 evaporative light scattering detector, SPD-10AT VP type autosampler. The reference substance of astragaloside IV is provided by China National Institute for the Control of Pharmaceutical and Biological Products (batch number 110781-200512; specification: 20 mg for content determination). Xingnao Reconstruction Capsule is provided by Jilin Huaqiao Pharmaceutical Co., Ltd. (batch numbers 20060903, 20061010, 20061221). Acetonitrile is chromatographically pure, water is purified water, and other reagents are of analytical grade.
2, methods and results
2.1 Chromatographic conditions: Column HPLC vials: Phenomenex Gemini C18 (250 mm × 4.60 mm, 5 μm), mobile phase: acetonitrile-water (36:64); flow rate: 1.0 mL/min; column temperature: 40 °C; Hitachi ALLTECH ELSD2000 Evaporative light scattering detector, drift tube temperature: 105 °C; carrier gas flow rate: 2.7 L / min; theoretical plate number should be no less than 4 000 with the astragaloside peak, resolution (R) > 1.5.
2.2 Preparation of reference solution: Accurately weigh the appropriate amount of astragaloside reference substance, add methanol to make 0.1 mg solution per 1 mL as the reference solution.
2.3 Preparation of the test solution: Mix the product under the difference of the amount of the preparation, mix and grind, take about 0.7 g, place the Soxhlet extractor, add the appropriate amount of methanol, cold soak overnight, add methanol, heat Reflux for 4 h. The extract was recovered with methanol and concentrated to dryness. 10 mL of water was added to the residue, and the mixture was dissolved with a little heat. The mixture was extracted with water-saturated n-butanol for 4 times, 40 mL each time HPLC vials. The n-butanol extract was combined and washed twice with ammonia test solution for 40 mL each time. Ammonia is discarded, n-butanol solution is evaporated to dryness, and 10 mL of water is added to the residue to dissolve and let cool. D101 type macroporous adsorption resin column (inner diameter 1.5 cm, length 12 cm), eluted with 50 mL of water, discard the aqueous solution, and then elute with 40 mL of 40% ethanol, discard the eluate, and use 70%. Evaporate 80 mL of ethanol, collect the eluate, evaporate to dryness, dissolve in methanol and transfer to a 5 mL volumetric flask. Add methanol to the mark and shake well.
2.4 Linear relationship investigation: Accurately weigh the methanol solution (0.097 2 mg/mL) of the reference substance of astragaloside, and accurately measure the 5, 10, 15, 20, 25 μL of the reference, and take the natural logarithm of the reference quality as the abscissa. The standard curve is drawn by taking the natural logarithm of the peak area as the ordinate. The results showed that the linear relationship of astragaloside IV in the range of 0.486～2.43 μg was good, HPLC vials and its linear logarithmic equation was: Y=1.952 6X+13.628 6 (r=0.999 7).
2.5 Stability test: Take the Xingnao Reconstruction Capsule (batch No. 20060903), prepare the test solution according to the method of “2.3”, and accurately absorb the sample solution 20 μL at 0, 2, 4, 8, 12, 24 h after preparation. Inject into a liquid chromatograph to determine the peak area. The results showed that astragaloside IV was basically stable within 24 h, and RSD was 1.0%.
2.6 Precision test: 20 μL of the test solution was accurately aspirated and injected into the liquid chromatograph. The injection was repeated 6 times, and the result was RSD=0.6%.
2.7 Repeatability test: Take the same batch of 6 samples, HPLC vials and prepare the test solution according to the method of “2.3”. The content of the test solution is 0.203 7 mg/particle, and the result is RSD=1.1%.
2.8 Sample recovery test: accurately weigh 6 parts of the same batch of the test sample (batch number 20060903, content 0.203 7 mg / grain), and accurately add the reference solution (0.101 6 mg / mL) 2.0 mL, respectively. According to the law, the average recovery was calculated to be 100.5%, and RSD=0.3%.
2.9 Sample determination
3. Discussion: During the selection of chromatographic conditions, a series of comparisons were made between acetonitrile-water (32:68), acetonitrile-water (34:66), and acetonitrile-water (36:64) mobile phases. The results showed that Using acetonitrile-water (36:64) as the mobile phase system, the separation effect is good, the peak shape is symmetrical, the retention time is appropriate, the impurity peak reaches the baseline separation, and the negative is non-interfering. Therefore, the experiment uses acetonitrile-water (36:64) as the flow. phase.
Different samples require different conditions HPLC vials. The carrier gas flow rate and drift tube temperature of the evaporative light scattering detector have a great influence on the results, which is an important parameter affecting the test results. The conditions selected in this test can obtain the ideal signal-to-noise ratio and baseline. .
This is the end of the introduction of Determination of Astragaloside IV in Xingnao Reconstruction Capsules by HPLC-ELSD. I hope it can help you.
