HIGH-PERFORMANCE LIQUID-CHROMATOGRAPHIC DETERMINATION OF A NEW ORAL THROMBIN INHIBITOR IN THE BLOOD OF RATS AND DOGS

A reliable reversed-phase high-performance liquid chromatographic meth od has been developed for the determination of a new oral thrombin inh ibitor (compound I) in the blood of rats and dogs. The analyte was dep roteinized with a 1.5 volume of methanol and a 0.5 volume of 10% zinc sulfate, and the supernatant was injected into a 5-mu m Capcell Pak C- 18 column (150x4.6 mm I.D.). The mobile phase was a mixture of acetoni trile and 0.2% triethylamine of pH 2.3 (31:69, v/v) with a flow-rate o f 1.0 ml/min at UV 231 nm. The retention time of compound I was approx imately 9.3 min. The calibration curve was linear over the concentrati on range of 0.05-100 mg/l for rat blood (r(2)>0.9995, n=6) and dog blo od (r(2)>0.9993, n=6). The limit of quantitation was 0.05 mg/l for bot h bloods using a 100-mu l sample. For the 5 concentrations (0.05, 0.1, 1, 10, and 100 mg/l), the within-day recovery (n=4) and precision (n= 4) were 98.1-104.1% and 1.5-6.8% for rat blood and 95.4-105.7% and 1.4 -5.3% for dog blood, respectively. The between-day recovery (n=6) and precision (n=6) were 99.8-105.3% and 3.7-12.6% for rat blood and 87.5- 107.1% and 2.9-15.3% for dog blood, respectively. The absolute recover ies were 82.4-93.3%. No interferences from endogenous substances were observed. In conclusion, the presented simple, sensitive, and reproduc ible HPLC method proved and was used successfully for the determinatio n of compound I in the preclinical pharmacokinetics. (C) 1998 Elsevier Science B.V. All rights reserved.