HPLC-QAMS同时测定益肾养元颗粒中9种成分的含量

目的 建立一测多评法（quantitative analysis of multi-components by single-marker，QAMS）同时测定益肾养元颗粒中2，3，5，4’-四羟基二苯乙烯-2-O-β-D-葡萄糖苷、虎杖苷、大黄素、大黄素甲醚、薯蓣皂苷元、齐墩果酸、乌索酸、蔷薇酸和坡模酸的含量。方法 采用Spursil C₁₈色谱柱（4.6 mm×250 mm，5 μm）进行HPLC测定，柱温为30℃；甲醇-0.1%甲酸溶液为流动相，梯度洗脱，流速为1.0 mL·min^-1；检测波长分别为280 nm （检测2，3，5，4’-四羟基二苯乙烯-2-O-β-D-葡萄糖苷、虎杖苷、大黄素和大黄素甲醚）和210 nm （检测薯蓣皂苷元、齐墩果酸、乌索酸、蔷薇酸和坡模酸）。以薯蓣皂苷元为内参物，建立其与其他组分的相对校正因子，计算各成分含量，同时将外标法实测值与QAMS计算值进行比较，验证QAMS的准确性和可靠性。结果 2，3，5，4’-四羟基二苯乙烯-2-O-β-D-葡萄糖苷、虎杖苷、大黄素、大黄素甲醚、薯蓣皂苷元、齐墩果酸、乌索酸、蔷薇酸和坡模酸的线性范围分别为23.89~477.80，8.67~173.40，4.26~85.20，2.99~59.80，9.86~197.20，0.69~13.80，2.17~43.40，1.28~25.60，1.66~33.20 μg·mL^-1，r为0.999 4~0.999 9；平均回收率分别为99.45%，98.49%，97.94%，98.91%，100.03%，96.90%，99.02%，97.86%和98.35%，RSD分别为1.01%，0.87%，1.37%，1.19%，0.70%，0.98%，1.03%，0.82%和1.53%（n=9）；经独立样本T-检验，2，3，5，4’-四羟基二苯乙烯-2-O-β-D-葡萄糖苷、虎杖苷、大黄素、大黄素甲醚、薯蓣皂苷元、齐墩果酸、乌索酸、蔷薇酸和坡模酸含量的计算值和实测值之间无明显差异。结论 所建立的QAMS操作便捷、结果准确，可同时测定益肾养元颗粒中9种成分含量。

Simultaneous Determination of Nine Components in Yishen Yangyuan Granules by HPLC-QAMS

OBJECTIVE To establish a quantitative analysis of multi-components by single-marker(QAMS) method for simultaneous determination of 2,3,5,4'-tetrahydroxystilbene-2-O-β-D-glucoside, polydatin, emodin, physcion, diosgenin, oleanolic acid, ursolic acid, euscaphic acid and pomolic acid in Yishen Yangyuan granules. METHODS HPLC analysis was performed on an Spursil C₁₈(4.6 mm×250 mm, 5 μm) with a mobile phase consisted of methanol-0.1% formic acid solution in gradient elution manner at a flow rate of 1.0 mL·min^-1. The column temperature was maintained at 30℃. The detection wavelength were 280 nm for 2,3,5,4'-tetrahydroxystilbene-2-O-β-D-glucoside, polydatin, emodin, physcion, and 210 nm for diosgenin, oleanolic acid, ursolic acid, euscaphic acid, pomolic acid. Diosgenin was used as the internal reference substance, its relative correction factor with other components was established and its content was calculated, at the same time, the method was evaluated by comparison of the quantitative results between external standard method and QAMS method. RESULTS 2,3,5,4'-Tetrahydroxystilbene-2-O-β-D-glucoside, polydatin, emodin, physcion, diosgenin, oleanolic acid, ursolic acid, euscaphic acid and pomolic acid had good relations within the range of 23.89-477.80, 8.67-173.40, 4.26-85.20, 2.99-59.80, 9.86-197.20, 0.69-13.80, 2.17-43.40, 1.28-25.60, 1.66-33.20 μg·mL^-1(r=0.999 4-0.999 9), The average recovery were 99.45%, 98.49%, 97.94%, 98.91%, 100.03%, 96.90%, 99.02%, 97.86% and 98.35%, RSDs were 1.01%, 0.87%, 1.37%, 1.19%, 0.70%, 0.98%, 1.03%, 0.82% and 1.53%(n=9), respectively. There was no significant difference between the calculated and measured values of 2,3,5,4'-tetrahydroxystilbene-2-O-β-D-glucoside, polydatin, emodin, physcion, diosgenin, oleanolic acid, ursolic acid, euscaphic acid and pomolic acid according to T-test analysis. CONCLUSION The QAMS method can simultaneously determine the content of nine components in Yishen Yangyuan granules, the method is simple and accurate.