Automated quantitative determination of the new renin inhibitor CGP 60536 by high-performance liquid chromatography

A fully automated high-performance liquid chromatography method with fluorescence detection for the determination of the renin inhibitor CGP 60536 in animal and human plasma and urine has been developed and validated. After addition of an internal standard, the compounds were automatically extracted from 400 μl of plasma or urine with methyl alcohol–acetic acid (99:1, v/v) on 100-mg Bond-Elut CN cartridges using the Gilson ASPEC system. The on-line chromatographic separation was performed on a LiChrospher 100 RP8 5-μm particle size packed analytical column (25×0.4 cm I.D.). The mobile phase consisted of acetonitrile–0.01 M potassium dihydrogenphosphate (65:35, v/v) at a flow-rate of 0.8 ml/min. The analytes were detected using a fluorescence detector at excitation and emission wavelengths of 280 and 330 nm, respectively. The limit of quantitation was established at 4.5 ng/ml in plasma (accuracy 106% and precision 1%), and 9.0 ng/ml in urine (accuracy 101% and precision 13%). The method was applied to the investigation of the pharmacokinetics of CGP 60536.