Two rapid ultra performance liquid chromatography/tandem mass spectrometry (UPLC/MS/MS) methods with common sample pretreatment for therapeutic drug monitoring of immunosuppressants compared to immunoassay

Therapeutic drug monitoring of immunosuppressive agents is a critical and essential part of patient therapy after organ transplantation. We have developed high-throughput, robust, and rapid liquid chromatography–tandem mass spectrometry (UPLC/MS/MS) methods with common pretreatment procedures for simultaneous quantification of four immunosuppressive agents (everolimus, sirolimus, tacrolimus, and cyclosporin A) in whole blood and one immunosuppressant (mycophenolic acid) in plasma. The new approach used in this work is based on improved sample preparation procedures allowing the analysis of five immunosuppressive drugs. Whole blood was prepared by transferring 100 μL of blood into a 1.5-mL silanized conical test tube. Zinc sulfate solution (150 μL), containing deuterated internal standards, was added to perform hemolysis. The samples were vortexing for 10 s, followed by the addition of 250 μL acetonitrile, containing internal standard for cyclosporin A, to precipitate proteins. The mixture was vortexed for 1 min and centrifuged for 2 min at 14,000 rpm. The whole supernatant was transferred to a vial. To prepare blood plasma, the hemolysis step involving the addition of zinc sulfate was omitted and, instead of acetonitrile, methanol was used as the solvent for the internal standard (mycophenolic acid-d3). The volumes of chemicals used in this procedure were the same as those used in the procedure for immunosuppressants in whole blood. The basic validation parameters for the analytical methods were limits of detection (0.5 ng/mL for everolimus, sirolimus and tacrolimus, 25 ng/mL for cyclosporin A and 100 ng/mL for mycophenolic acid), precision (<15%), recovery (>84%), repeatability and reproducibility. Possible mutual ion suppression was eliminated in the presence of internal standards. The method developed for the quantitation of immunosuppressants in whole blood was used to analyze 276 patient samples containing tacrolimus and 55 samples containing cyclosporin A. The results from LC/MS/MS were compared to those obtained from immunoassays of the same samples. Immunoassays significantly overestimated the concentrations of immunosuppressants.