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 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).
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 <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.
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.
Keywords: Calcineurin; Kidney transplantation; Pharmacodynamics; Tacrolimus; Transplantation; UHPLC-MS.
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