Hasler F, Krapf R, Brenneisen R, Bourquin D, Krähenbühl S
Institute of Pharmacy, University of Berne, Switzerland.
J Chromatogr. 1993 Oct 22;620(1):73-82. doi: 10.1016/0378-4347(93)80053-7.
Methods have been developed and characterized allowing rapid isolation and quantification of 18 beta-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 beta-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 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 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.
已开发并表征了一些方法，可快速分离和定量人及大鼠生物体液中的18β-甘草次酸（GRA）。样品制备包括用尿素-甲醇提取血浆样品，以及用固相萃取（SPE）处理尿液和胆汁样品。通过用β-葡萄糖醛酸酶处理来进行尿液和胆汁中GRA葡萄糖醛酸苷的水解。合成了GRA的3-O-甲基衍生物MGRA作为抗水解的内标。使用甲醇-水-乙酸（83:16.8:0.2，v/v/v）作为溶剂，在Lichrocart RP-18柱上于30℃进行等度系统的高效液相色谱（HPLC），并进行紫外检测。这些方法能够从所有测试的生物体液中基线分离GRA和MGRA，检测限为0.15mg/l。方法的验证包括测定人及大鼠血浆、胆汁和尿液中的回收率、准确度和精密度。通过研究GRA在正常大鼠和胆瘘大鼠中的药代动力学进一步评估了这些方法。静脉注射10mg/kg剂量后，GRA的血浆浓度-时间曲线在对照大鼠和胆瘘大鼠中均符合一室模型。对照大鼠和胆瘘大鼠的消除半衰期平均分别为15.0±2.2分钟和16.8±2.4分钟（差异不显著）。在给予GRA后90分钟内，两组中GRA及其葡萄糖醛酸苷的尿排泄均小于1%，而胆汁排泄平均为51.3±3.1%。结果表明，所开发的方法可用于人及大鼠中GRA的药代动力学研究。
