积雪草酸代谢物的水解及其RP-HPLC测定

Hydrolysis of the metabolites of Asiatic acid and its determination by RP-HPLC

建立了积雪草酸代谢物的水解方法及其定量测定方法.中药积雪草酸在体内的主要代谢物为积雪草酸葡萄糖醛酸结合物和硫酸酯结合物,实验以积雪草苷为代谢物模型药物,1 mol/L氢氧化钠乙醇（70%）水溶液为水解液,水解时间3 h,温度60℃.水解液药物浓度通过RP-HPLC方法进行检测,色谱条件为：色谱柱Diamonsil C18柱（250 mm×4.6 mm,5μm）、流动相乙腈：0.3%磷酸溶液（50∶50）、流速1.0 mL/min、柱温25℃、检测波长200 nm.测定方法的回收率为（100.76±1.47）%,积雪草酸在0.5~20μg/mL浓度范围内线性关系良好,回归方程为y=10 418x-1371.8,R=0.999 9,定量限为（0.5±0.045）μg/mL（S/N=10）.所建立的水解方法用于水解大鼠粪便样品,方便可行,高效液相色谱法能准确地测定积雪草酸代谢产物的含量.

The hydrolytic method of the metabolites of Asiatic acid and a RP-HPLC method for the quantitative determination of the Asiatic acid were developed. Asiatic acid can be metabolized into its glucuronic acid conjugations and sulfuric acid conjugations in vivo, asiaticoide was selected as model drug. Hydrolyzation needed 3 hours at 60 ~C with the ethanol （70~） solution of 1 mol/L Sodium hydroxide. Asiatic acid in the hydrolysate was detected by RP-HPLC. Chromatographic separation was performed on an Diamonsil C18（5μm, 4.6 mm×250 mm）; the mobile phase was acetonitrile： 0. 3% phosphoric acid solution （50 ： 50）, with a flow rate of 1. 0 mL/min; the temperature of the column was 25 ℃ and was detected at 200 nm. The average recovery is （100.76±1. 47）%; A calibration curve ranging from 0.5 to 20μg/mL was shown to be linear, and the regression equation is y = 10 418x- 1 371. 8, R = 0. 999 9, the lowest limit of quantification was （0.5±0. 045）μg/mL（S/N： 10）. The hydrolysis method is convenient and stable for rat fecal samples, and the RP-HPLC method is a very accurate method, for the quantitative determination of the metabolite of Asiatic acid.