High performance liquid chromatographic analysis of a novel aminoalkylpyridine anticonvulsant 2-(4-chlorophenyl)amino-2-(4-pyridyl)ethane

Abstract

A simple, rapid and specific high performance liquid chromatographic (HPLC) method for the quantitation of 2-(4-chlorophenyl)amino-2-(4-pyridyl)ethane (AAP-Cl) and identification of its putative metabolite, 2-(4-chlorophenyl)amino-2-(4-pyridyl)ethanol ( β-AA) in rat blood and urine has been developed. AAP-Cl, β-AA and an appropriate internal standard were extracted from rat biofluids by a solid phase extraction technique using C18 cartridges prior to the HPLC analysis. The extractibility was 92% for AAP-Cl and 98% for β-AA. The HPLC analysis employed a symmetrical or standard reversed-phase HPLC column (Apex ODS, 5 μm, 25 cm×0.46 cm) for blood or urine analysis, a mobile phase of water–methanol–acetonitrile (40:30:30) containing 20 μl 100 ml −1 diethylamine at a flow rate of 1 ml min −1, and UV detection at 254 nm. The limit of detection was 100 ng ml −1 for both analytes in both blood and urine. The calibration curves for AAP-Cl in rat biofluids were shown to be linear in both low and high concentration ranges (blood: 0–1 and 1–10 μg ml −1; urine: 0–10 and 10–100 μg ml −1) with intra- and inter-day coefficients of variation of no more than 18% for blood and 14% for urine. The method developed was successfully applied to a preliminary analysis of intact AAP-Cl in both blood and urine obtained from rats dosed with AAP-Cl.