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

BackgroundTherapeutic 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. MethodsThis methodology is based on monitoring the Ca2+-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). ResultsOur 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 <4.3% and 10.3%, respectively. The mean recovery for peptide was 91.6 ± 4.0%. Matrix effect study displayed ion suppression, and no carry-over and interferences were observed. There were no differences in CNA between healthy and TAC-treated patients. Furthermore, CNA showed maximum inhibition at 1 h after drug intake when TAC reached the highest blood concentration. ConclusionsThis 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.