Determination of N-(trans-4-isopropylcyclohexylcarbonyl)-D-phenylalanine in human plasma by solid-phase extraction and column-switching high-performance liquid chromatography with ultraviolet detection

A column-switching high-performance liquid chromatography method with ultraviolet detection at 210 nm has been developed for the determination of N-(trans-4-isopropylcyclohexylcarbonyl)-D-phenylalanine (AY4166, I) in human plasma. Plasma samples were prepared by solid-phase extraction with Sep-Pak Light tC(18), followed by HPLC. The calibration graph for I was linear in the range 0.1-20 mu g/ml. The limit of quantitation of I, in plasma, was 0.05 mu g/ml. The recovery of spiked I (0.5 mu g/ml) to drug-free plasma was over 92% and the relative standard deviation of spiked I (0.5 mu g/ml) compared to drug-free plasma was 4.3% (n = 8).