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 beta-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 C(18) column (250 mm x 4.6 mm, 5 microm) and kept at 30 degrees 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 10h 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-infinity) and was found to be within the range, which indicated that the two products were bioequivalence.
Li
Weiyong
W
Institute of Clinic Pharmacy, Union Hospital Affiliated to Tongji Medical College, Huazhong University of Science & Technology, Wuhan 430022, PR China. lweiyong2002@yahoo.com
Deng
Jungang
J
Qiao
Jian
J
Li
Qian
Q
Zhang
Ying
Y
D000781:Anethole Trithione / Q000378:metabolism*; D019540:Area Under Curve; D002851:Chromatography, High Pressure Liquid / Q000379:methods*; D004355:Drug Stability; D005966:Glucuronidase / Q000378:metabolism; D006801:Humans; D006868:Hydrolysis; D013810:Therapeutic Equivalency
D005966:Glucuronidase; D000781:Anethole Trithione