A new HPLC-MS/MS method for simultaneous determination of Cyclosporine A, Tacrolimus, Sirolimus and Everolimus for routine therapeutic drug monitoring.

Abstract

A rapid, simple and robust HPLC-MS/MS method for simultaneous determination of immunosuppressants Cyclosporine A, Tacrolimus, Sirolimus and Everolimus has been developed and validated. Sample of whole blood with volume of 50 μL was prepared by a protein precipitation with methanol and 0.5 mol. L ZnSO. Chromatographic separation was achieved on a Phenyl-Hexyl column by a gradient elution using 20 mmol.L ammonium formate/0.1% (v/v) formic acid in water (mobile phase A) and 20 mmol.L ammonium formate/0.1% (v/v) formic acid in methanol (mobile phase B) with flow rate 1 mL.min. The run time was 3.5 min. Electrospray ionization and multiple reaction monitoring was used. The lower limit of quantification (LLOQ) was set at 0.5 μg.L for Tacrolimus, Sirolimus and Everolimus and 5 μg.L for Cyclosporine A. The method demonstrated adequate accuracy and precision with sufficient linearity range.