Determination of cyclosporin A in human and mouse plasma by reversed-phase high-performance liquid chromatography

Abstract

An isocratic reversed-phase high-performance liquid chromatographic method with ultraviolet detection at 227 nm has been validated for the determination of cyclosporin A in human and mouse plasma. The cyclosporin D analog PSC 833 was used as internal standard. Plasma samples were pretreated by liquid–liquid extraction with diethyl ether. A good chromatographic separation between cyclosporin A, the internal standard and two potentially interfering endogenous peaks was achieved using a stainless steel column packed with 5 μm Nova-Pak phenyl material operated at 72°C, and a mobile phase consisting of acetonitrile–methanol–water (20:52:28, v/v/v). The calibration curve for cyclosporin A in human plasma was linear over the tested concentration range of 0.11 to 5.34 μ M. Murine plasma samples (200 μl) were diluted up to a total volume of 500 μl with blank human plasma and the concentrations were read from the calibration curve prepared in human plasma. The lower limit of quantitation was 0.11 μ M using 500 μl of human plasma and 0.28 μ M using 200 μl of mouse plasma. The validation data showed that the assay is sensitive, selective and reproducible for determination of cyclosporin A. The applicability was demonstrated in a pharmacokinetic experiment where mice received oral cyclosporin A.