HPLC determination of 4-hydroxy-anethole trithione in plasma via enzymatic hydrolysis and its application to bioequivalence study

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• 作者：Weiyong Li ; Jungang Deng ; Jian Qiao ; Qian Li ; Ying Zhang
• 关键词：Anethole trithione ; 4-Hydroxy-anethole trithione ; β ; -Glucuronidase ; HPLC ; Bioequivalence
• 刊名：Journal of Pharmaceutical and Biomedical Analysis
• 出版年：2008
• 期刊代码：65_07317085
• 类别：ch
• 出版时间：15 July 2008
• 卷：47
• 期：3
• 页码：612-617
• 文件大小：345 K

摘要

A simple, selective and reproducible high-performance liquid chromatographic (HPLC) method via enzymatic hydrolysis of glucuronide conjugates of 4-hydroxy-anethole trithione (ATX) was established for simultaneous determination of ATX. Human plasma samples were hydrolyzed by β-glucuronidase and followed by subsequent extraction with cyclohexane–isopropanol (95:5, v/v) using mifepristone as the internal standard. Chromatography was carried out on a reverse phase C₁₈ column (250 mm × 4.6 mm, 5 μm) and kept at 30 °C, with UV detection set at 346 nm. The mobile phase consisted of a mixture of methanol and water (75:25, v/v), at a flow rate of 1 ml/min. It was validated and proved to be linear in the range of 20–1500 ng/ml, with the regression equation Y = 0.0016C − 0.0069, r = 0.9992. And the limit of quantification (LOQ) concentration in plasma was 20 ng/ml. The absolute recoveries of ATX at three concentrations were 32.04, 38.95 and 44.06%and the relative recoveries were 104.80, 102.53 and 107.04%, which showed that the analytical method was sensible, accurate and reproducible. The method was utilized on a double-blind, randomized, single dose, two period, and crossover bioequivalence study of ATT tablets produced by different companies in 20 healthy male Chinese subjects, with a washout between every two periods. Blood samples were collected over each period of 10 h and various pharmacokinetic parameters were determined. Natural log-transformed values were compared by analysis of variance followed by classical 90%confidence interval for C_max, AUC_0–t and AUC_0–∞ and was found to be within the range, which indicated that the two products were bioequivalence.