Determination of 18β-glycyrrhetinic acid in biological fluids from humans and rats by solid-phase extraction and high-performance liquid chromatography

Abstract

Methods have been developed and characterized allowing rapid isolation and quantification of 18β-glycyrrhetinic acid (GRA) in biological fluids from both humans and rats. Sample preparation includes extraction with urea—methanol for plasma samples, and solid-phase extraction (SPE) for urine and bile samples. Hydrolysis of GRA glucuronides in urine and bile was performed by treatment with β-glucuronidase. MGRA, the 3-O-methyl derivative of GRA was synthesized as an internal standard resistant to hydrolysis. High-performance liquid chromatography (HPLC) 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°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 determination of recovery, accuracy and precision in plasma, bile and urine from humans and rats. The methods were further evaluated by investigating the pharmacokinetics of GRA in normal rats and in rats with a bile fistula. Following an intravenous dose of 10 mg/kg, the plasma concentration—time curve of GRA could be fitted to a one compartment model both in control and bile fistula rats. The elimination half life averaged 15.0 ± 2.2 versus 16.8 ± 2.4 min in control and bile fistula rats (difference not significant). Within 90 min following administration of GRA, urinary elimination of GRA and GRA glucuronides was less than 1% in both groups whereas biliary elimination averaged 51.3 ± 3.1%. The results show that the methods developed allow pharmacokinetic studies of GRA in humans and rats.