Determination of a renin inhibitor in plasma by solid-phase extraction using acetone as protein binding displacer...

H 218/54 is a potent inhibitor of human renin activity (pIC50 = 8.3 at pH 6) and is therefore a potential agent for blood pressure reduction. This lipophilic compound is highly bound to plasma proteins, e.g. 99.7% in rats and 99.6% in humans. For pharmacokinetic studies, a quantitative assay for 3H-H 218/54 in plasma has been developed. On top of an AASP phenyl solid-phase cartridge 70 microliters of rat plasma or 1 ml of cynomolgus plasma was mixed with 200 microliters of water containing 20% acetone. The acetone displaced the substance from plasma proteins without precipitation of the sample and clogging of the extraction column. The mixture was passed through the cartridge, which adsorbed 3H-H 218/54. The cartridge was placed in an AASP autosampler connected to a reversed-phase LC system, with a Vydac C-18 column and CH3CN-H2O-TFA (60:40:0.1, v/v/v) as mobile phase. The effluent from the separation column was collected in fractions for radioactivity counting. Recovery, as measured after adding various amounts of tritium-labelled H 218/54 to blank plasma followed by repeated analysis of the samples, was close to 100% with relative standard deviations between 1.4 and 3.0%. At the lowest level tested, 200 dpm per sample, the recovery was 120% with a relative standard deviation of only 10%. The sensitivity of the method will depend on the specific radioactivity of the dose given.