High-performance liquid chromatography of the adenosine A 1 agonist N 6-cyclopentyladenosine and the A 1 antagonist 8-cyclopentyltheophylline and its application in a pharmacokinetic study in rats

Abstract

This report describes a rapid and sensitive analysis for the simultaneous detection of the adenosine A 1 receptor ligands N 6-cyclopentyladenosine (CPA) and 8-cyclopentyltheophylline (CPT) in rat blood. The method involved alkaline extraction of the compounds and internal standard N 6-cyclohexyladenosine (CHA) with ethyl acetate, followed by isocratic reversed-phase high-performance liquid chromatography on a 3-μm MicroSphere C 18 column with UV detection at 269 nm. The mobile phase consisted of a mixture of 10 m M acetate buffer (pH 4.0)—methanol—acetonitrile (56:40:4, v/v/v) with a flow-rate of 0.50 ml/min. The total run time was ca. 19 min. For CPA and CPT extraction yields were greater than 77 and 66% in the concentration range of 0.010–0.75 μg/ml and 0.025–15 μg/ml, respectively, with intra- and inter-assay variations less than 9%. In 100 μl blood samples the corresponding limits of detection were 3.3 and 6.2 ng/ml (signal-to-noise ratio = 3). CPA was found to be degraded in rat blood in vitro with a half-life of 24 min at 37°C. The utility of the analytical method was established by analyzing blood samples from rats which had received an intravenous administration of 200 μg/kg CPA or 12 mg/kg CPT. Due to its rapidity and sensitivity this method is concluded to be particularly useful in pharmacokinetic studies with CPA and CPT.