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

This report describes a rapid and sensitive analysis for the simultane ous detection of the adenosine A1 receptor ligands N6-cyclopentyladeno sine (CPA) and 8-cyclopentyltheophylline (CPT) in rat blood. The metho d involved alkaline extraction of the compounds and internal standard N6-cyclohexyladenosine (CHA) with ethyl acetate, followed by isocratic reversed-phase high-performance liquid chromatography on a 3-mum Micr oSphere C18 column with UV detection at 269 nm. The mobile phase consi sted of a mixture of 10 mM acetate buffer (pH 4.0)-methanol-acetonitri le (56:40:4, v/v/v) with a flow-rate of 0.50 ml/min. The total run tim e 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 mug/ml and 0.025-1 5 mug/ml, respectively, with intra- and inter-assay variations less th an 9%. In 100 mul blood samples the corresponding limits of detection were 3.3 and 6.2 ng/ml (signal-to-noise ratio = 3). CPA was found to b e degraded in rat blood in vitro with a half-life of 24 min at 37-degr ees-C. The utility of the analytical method was established by analyzi ng blood samples from rats which had received an intravenous administr ation of 200 mug/kg CPA or 12 mg/kg CPT. Due to its rapidity and sensi tivity this method is concluded to be particularly useful in pharmacok inetic studies with CPA and CPT.