Liquid chromatography–tandem mass spectrometry method for simultaneous determination of cyclosporine A and its three metabolites AM1, AM9 and AM4N in whole blood and isolated lymphocytes in renal transplant patients

Abstract A LC‐MS/MS method was developed and validated for the determination of cyclosporine A (CsA) and its three phase 1 metabolites AM1, AM9, and AM4N in whole blood and lymphocytes isolated on the Histopaque gradient. 200 μL of whole blood was precipitated with 10 mol/L zinc sulfate in acetonitrile/methanol (40:60, v/v) and lymphocytes isolated from 1.5 mL blood were extracted with acetonitrile/methanol (40:60, v/v). The analytes and internal standard cyclosporine D were separated on RP column BEH C18, 2.1×50 mm, 1.7 μm using gradient LC‐MS/MS analysis in positive electrospray mode. Time of analysis was 5 min. Linearity in blood was 5–2000 μg/L for CsA, AM1, and AM9; 2–500 μg/L for AM4N; and 2–500 μg/L for all substances in lymphocytes. Coefficient of variations was 1.8–9.8% and recovery was 92.0–110.0%. The method was used in early and chronic renal transplant patients for therapeutic drug monitoring of CsA to compare either its share in lymphocytes as target organ or binding to one lymphocyte. The same parameters were calculated for all metabolites tested.