Development of a UPLC-MS/MS Method for Determination of Tacrolimus and Cyclosporine A in Human Whole Blood

An accurate and validated liquid chromatography method and a triple quadrupole mass spectrometry method were developed and validated for determination of tacrolimus and cyclosporine A in human whole blood. Whole blood samples were prepared by precipitating protein with acetonitrile after adding ZnSO4. The analytes were separated using a reversed-phase BEH C18 column (2.1 x 50 mm, 1.7 mu m, Waters, USA) maintained at 60 degrees C. The mobile phase consisted of acetonitrile and water (both containing 10 mM ammonium acetate by adding 0.1% formic acid) with a gradient elution pumped at a flow humane of 0.4 mL/min. The analytes were detected with positive electrospray ionization in multiple reaction monitoring (MRM) mode for target fragment ions m/z 822.36 -> 769.37 for tacrolimus, m/z 1220.95 -> 1203.74 for cyclosporine A and m/z 285.1 -> 193.1 for diazepam (IS). Good linearity was achieved to quantify the concentration ranges of 0.5-10 ng/mL for tacrolimus and 10-500 ng/mL for cyclosporine A in human whole blood. The mean recoveries of tacrolimus and cyclosporine A from the whole blood exceeded 75.58%. The intra-run and inter-run assay precisions of tacrolimus and cyclosporine A were both less than 8.9%. The validated method proved stability of tacrolimus and cyclosporine A in human whole blood and the correlation study showed the capability of the method to be used as an alternative for whole blood analysis in therapeutic drug monitoring.