Highly sensitive and rapid determination of tacrolimus in peripheral blood mononuclear cells by liquid chromatography–tandem mass spectrometry

Abstract

After solid organ transplantation, tacrolimus is given to prevent rejection. Therapeutic drug monitoring is used to reach target concentrations of tacrolimus in whole blood. Because the site of action of tacrolimus is the lymphocyte, and tacrolimus binds ~80% to erythrocytes, the intracellular tacrolimus concentration in lymphocytes is possibly more relevant. For this purpose, we aimed to develop, improve and validate a UPLC–MS/MS method to measure tacrolimus concentrations in isolated peripheral blood mononuclear cells (PBMCs). PBMCs were isolated using a Ficoll separation technique, followed by a washing step using red blood cell lysis. A cell suspension of 50 μL containing 1 million PBMCs was used in combination with MagSiMUS‐TDMPREP. To each sample we added 30 μL lysis buffer, 20 μL reconstitution buffer containing 13C2H4‐tacrolimus as internal standard, 40 μL MagSiMUS‐TDMPREP Type I Particle Mix and 175 μL Organic Precipitation Reagent VI for methanol‐based protein precipitation. A 10 μL aliquot of the supernatant was injected into the UPLC–MS/MS system. The method was validated, resulting in high sensitivity and specificity. The method was linear (r2 = 0.997) over the range 5.0–1250 pg/1 × 106 PBMCs. The inaccuracy was <5% and the imprecision was <15%. The washing steps following Ficoll isolation could be performed at either room temperature or on ice, with no effect of the temperature on the results. A method for the analysis of tacrolimus concentrations in PBMCs was developed and successfully validated. Further research will be performed to investigate the correlation between concentrations in PBMCs and clinical outcome.

Immunomonitoring of Tacrolimus in Healthy Volunteers: The First Step from PK- to PD-Based Therapeutic Drug Monitoring?

“…There was no correlation between tacrolimus concentrations in whole blood and PBMCs at 1.5 h post-dose. Evidence supporting this finding is scarce and conflicting; some papers do report a correlation in transplantation patients [19,20], whereas other papers do not [21,22]. Tacrolimus levels in T cells, on the other hand, did correlate significantly with whole blood concentrations.…”

“…Whole blood and intracellular tacrolimus concentrations were measured using a Waters Acquity UPLC–MS/MS system by the Department of Hospital Pharmacy, Erasmus Medical Center, as described previously [19].…”

Pharmacogenetic—Whole blood and intracellular pharmacokinetic—Pharmacodynamic (PG-PK2-PD) relationship of tacrolimus in liver transplant recipients

“…The authors reported a good correlation between intrahepatic or intra-PBMC concentrations of TAC, and an histological rejection score determined at day-7 posttransplantation whereas no association was found with whole blood TAC concentrations. Besides, the relationship between TAC whole blood concentrations and TAC concentrations in PBMC have been studied in various organ transplant patients [5][6][7][8][9][10][11]. These works were concordant to report a poor correlation between trough whole blood and trough intracellular concentrations, meaning that monitoring TAC in its target cell could be a better surrogate marker of its pharmacological effect.…”

Usage of Tacrolimus and Mycophenolic Acid During Conception, Pregnancy, and Lactation, and Its Implications for Therapeutic Drug Monitoring: A Systematic Critical Review

“…Tac concentration in lymphocytes could be an appropriate matrix to monitor Tac treatment. [175][176][177][178] In a study by Capron et al, 179 intracellular Tac concentration was correlated with both the development and severity of rejection after liver transplantation. However, the authors of this review think that at present, there is insufficient evidence to make recommendations regarding the optimal matrix for TDM of Tac during pregnancy.…”