Rapid Liquid Chromatography–Tandem Mass Spectrometry Routine Method for Simultaneous Determination of Sirolimus, Everolimus, Tacrolimus, and Cyclosporin A in Whole Blood

With the introduction of the novel immunosuppressive agents sirolimus and everolimus, new, potentially more effective immunosuppressive regimens are undergoing clinical evaluation. Whereas the calcineurin inhibitors cyclosporin A (CsA) and tacrolimus suppress early activation of T lymphocytes through inhibition of cytokines such as interleukin 2, the primary target of sirolimus and everolimus is mammalian target of rapamycin (mTOR), a specific cell-cycle regulatory protein. The inhibition of mTOR leads to suppression of cytokine-driven T-lymphocyte proliferation (1). Because of their distinct modes of action, a calcineurin inhibitor and an mTOR inhibitor act synergistically to block acute allograft rejection (2)(3)(4). Current evidence (5) suggests that drug monitoring is necessary, not only for the calcineurin inhibitors, but also for the mTOR inhibitors. In contrast to the calcineurin inhibitors, however, there are currently no commercially available immunoassays for the latter two drugs. There is a need, therefore, for assays that can simultaneously quantify both the calcineurin and the mTOR inhibitors. Furthermore, a major shortcoming of the commercial immunoassays for CsA and tacrolimus is their cross-reactivity with unpredictable amounts of both active and inactive metabolites of the parent drugs in patient samples. In the case of CsA, no immunoassay has completely fulfilled the criteria recommended by a consensus panel (6). New developments in immunosuppression protocols require that assays for calcineurin inhibitors provide broader dynamic ranges. On the one hand, C2 monitoring has been proposed for optimizing dosage of the CsA microemulsion (7), thereby necessitating quantification of CsA at concentrations up to 2500 μg/L: the lack of validated dilution protocols can be a problem for C2 monitoring (8). On the other hand, lower target ranges for CsA (5) and tacrolimus (9) are being investigated for long-term maintenance therapy to minimize the adverse side effects, in particular nephrotoxicity, of these drugs. Current immunoassays exhibit …