DETERMINATION OF 18-BETA-GLYCYRRHETINIC ACID IN BIOLOGICAL-FLUIDS FROM HUMANS AND RATS BY SOLID-PHASE EXTRACTION AND HIGH-PERFORMANCE LIQUID-CHROMATOGRAPHY

Methods have been developed and characterized allowing rapid isolation and quantification of 18beta-glycyrrhetinic acid (GRA) in biological fluids from both humans and rats. Sample preparation includes extracti on with urea-methanol for plasma samples, and solid-phase extraction ( SPE) for urine and bile samples. Hydrolysis of GRA glucuronides in uri ne and bile was performed by treatment with beta-glucuronidase. MGRA, the 3-0-methyl derivative of GRA was synthesized as an internal standa rd resistant to hydrolysis. High-performance liquid chromatography (HP LC) was performed with an isocratic system using methanol-water-acetic acid (83:16.8:0.2, v/v/v) as solvent on a Lichrocart RP-18 column at 30-degrees-C with ultraviolet detection. The methods allowed base line separation of GRA and MGRA from all biological fluids tested, with a detection limit of 0. 15 mg/l. Validation of the methods included dete rmination of recovery, accuracy and precision in plasma, bile and urin e from humans and rats. The methods were further evaluated by investig ating the pharmacokinetics of GRA in normal rats and in rats with a bi le fistula. Following an intravenous dose of 10 mg/kg, the plasma conc entration-time curve of GRA could be fitted to a one compartment model both in control and bile fistula rats. The elimination half life aver aged 15.0 +/- 2.2 versus 16.8 +/- 2.4 min in control and bile fistula rats (difference not significant). Within 90 min following administrat ion of GRA, urinary elimination of GRA and GRA glucuronides was less t han 1 % in both groups whereas biliary elimination averaged 51.3 +/- 3 .1 %. The results show that the methods developed allow pharmacokineti c studies of GRA in humans and rats.