UHPLC-MS/MS method for simultaneous determination of tacrolimus, Cyclosporine A, sirolimus and Everolimus in human blood and clinical application

The whole blood concentrations of Tacrolimus, Cyclosporine A, Sirolimus, and Everolimus are critical for managing organ transplant rejection, hypersensitivity reactions, and autoimmune diseases. Routine monitoring of these immunosuppressive drugs aids in dose adjustment and toxicity prevention. An ultra-high performance liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS) method for simultaneous determination of whole blood Tacrolimus, Cyclosporine A, Sirolimus, and Everolimus was developed, validated, and applied in clinical samples. The ion transitions were m/z 821.5 > 768.4 for Tacrolimus, m/z 1219.7 > 1208.8 for Cyclosporine A, m/z 931.5 > 864.5 for Sirolimus, and m/z 975.6 > 908.4 for Everolimus. The flow rate was 0.6 mL/min with a run time of 3.5 min. The calibration ranges were 1.25–42.9 ng/mL for Tacrolimus, 21.7–1270 ng/mL for Cyclosporine A, 1.38–45.6 ng/mL for Sirolimus, and 1.22–44.6 ng/mL for Everolimus, respectively. The intra-day and inter-day inaccuracy and imprecision were within ±15 % for all analytes. Consistent internal standard -normalized recovery and minimal matrix effects were observed at all quality control levels. The UHPLC-MS/MS method offers significant advantages over traditional immunoassays and HPLC methods, including higher specificity, sensitivity, and the ability to detect multiple drugs simultaneously. It simplifies therapeutic drug monitoring (TDM) procedures, increases detection throughput, and supports optimizing immunosuppressive therapy and improving patient outcomes.