Validation of a Liquid Chromatography-Mass Spectrometric Assay for Tacrolimus in Peripheral Blood Mononuclear Cells

Abstract

As a potential alternative to whole-blood tacrolimus (TAC) monitoring, a sensitive and selective method was developed for quantifying this immunosuppressant in human peripheral blood mononuclear cell population (PBMCs). These cells, expected to be a more specific biological matrix than whole blood to reflect pharmacological efficacy, could be promising for TAC therapeutic drug monitoring (TDM). The assay was developed using liquid chromatography-tandem mass spectrometry (LC-MS/MS). PBMCs are isolated from 7 mL whole blood by centrifugation over Ficoll gradient density and washed twice with phosphate-buffered saline at 4 degrees C. Harvested cells were suspended within 1.5 mL of phosphate-buffered saline. Cell counts were performed to express and normalize TAC amount per 10 cells. TAC was extracted by a liquid-liquid extraction in basic medium (NH4OH) with 1-chlorobutane, and ascomycin was used as internal standard. After evaporation of the supernatant under nitrogen, the residue was reconstituted in methanol (MeOH). Compounds were eluted on a C18 column by a mixture of acetonitrile/water (90/10, vol/vol) containing 0.1% formic acid and 2 mmol/L of ammonium acetate. TAC and internal standard were monitored by detecting specific ammoniated product ions using multiple reaction monitoring acquisition mode in electrospray positive ionization. This method was fully validated in the range of 0.01-5 ng/mL. Limit of detection and of quantification are 0.005 and 0.01 ng/mL, respectively. Intra-assay and interassay recoveries ranged from 89.2% to 114.3% and 85.3% to 103.9%, respectively. Intra-assay and interassay imprecisions ranged from 9.3% to 12% and 10.7% to 12.2%, respectively, across the analytical range. Mean TAC extraction efficiency was 80.9% +/- 8.3%. Matrix effects were minimal with <8% ion suppression. This method is currently applied in clinical research protocols and allows the measurement of small intracellular amounts of TAC down to 0.006 ng per 10 PBMCs in kidney-transplanted recipients. This method could be a new potential tool for TAC TDM, providing new perspectives for optimizing immunosuppressive therapy. Further studies should be conducted to fully evaluate the benefit of intracellular TAC concentrations in refinement of TDM strategies for TAC to ensure optimal clinical outcomes.