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

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-1 ZnSO4. Chromatographic separation was achieved on a Phenyl-Hexyl column by a gradient elution using 20 mmol.L-1 ammonium formate/0.1% (v/v) formic acid in water (mobile phase A) and 20 mmol.L-1 ammonium formate/0.1% (v/v) formic acid in methanol (mobile phase B) with flow rate 1 mL.min-1. 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-1 for Tacrolimus, Sirolimus and Everolimus and 5 μg.L-1 for Cyclosporine A. The method demonstrated adequate accuracy and precision with sufficient linearity range.