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

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 Cmax, AUC0-t and AUC0-∞ and was found to be within the range, which indicated that the two products were bioequivalence.