Measurement of calcineurin activity in peripheral blood mononuclear cells by ultra-high performance liquid chromatography-tandem mass spectrometry. Renal transplant recipients application (pharmacodynamic monitoring)

Abstract Background Therapeutic drug monitoring of calcineurin inhibitor, tacrolimus (TAC), is routinely used in post-transplantation. Currently, measurement of calcineurin activity has been proposed as a promising clinical tool to evaluate efficacy and to optimize drug dosing. The main aim of our study was to develop a method to measure phosphatase calcineurin activity (CNA) in peripheral blood mononuclear cells (PBMCs) by ultra-high performance liquid chromatography-tandem mass spectrometry and to validate it following FDA and EMA guidelines. Methods This methodology is based on monitoring the Ca 2+ -dependent dephosphorylation of a phosphopeptide substrate. CNA was evaluated in 5 healthy volunteers and in 5 renal transplant patients receiving twice-daily formulation of TAC before drug intake. Moreover, we studied pharmacodynamic effect of TAC and blood concentrations of TAC in different drug dose intervals (0, 1, 3, 6 and 12 h). Results Our results showed linearity in the range 0.04–2.00 μM with a lower limit of quantification of 0.04 μM. Coefficients of variation and absolute relative biases were Conclusions This method improves the extraction phase of PBMCs and achieves faster determination compared to other techniques, bringing us closer to be applied in daily laboratory practice.