First UHPLC-MS/MS method coupled with automated online SPE for quantification both of tacrolimus and everolimus in peripheral blood mononuclear cells and its application on samples from co-treated pediatric patients.

Abstract

Tacrolimus (TAC, FK-506) and everolimus (EVE, RAD001) are immunosuppressors used to treat pediatric patients undergoing liver transplantation. Their hematic TDM by liquid chromatography became standard practice. However, it does not always reflect concentrations at their active site. Our aim was to develop and validate a new method for the simultaneous TAC and EVE quantification into target cells: peripheral blood mononuclear cells (PBMCs). Peripheral blood mononuclear cells were collected using cell preparation tubes; cells number and mean cell volume were evaluated by an automatic cell counter. TAC and EVE were quantified using UHPLC-MS/MS coupled with an automated online solid-phase extraction platform. Chromatographic run was performed on an Acquity UPLC® BEH C18 1.7 μm (2.1 × 50 mm) column at 45 °C, for 6 min at 0.5 ml/min. Mobile phases were water and methanol, both with 2 mm ammonium acetate and 1 ml/l formic acid). XBridge® C8 10 μm (1 × 10 mm) SPE cartridges were used, and the internal standard was ascomycin. Following Food and Drug Administration guidelines, method validation resulted in high sensitivity and specificity. Calibration curves were linear (r = 0.998) and intra-day and inter-day imprecision and inaccuracy were <15%. A reproducible matrix effect was observed, with a good recovery for all compounds. Drug amounts in 15 'real' PBMCs samples from five pediatric patients in co-treatment resulted within the calibration range (0.039-5 ng). Concentrations from each patient were standardized using their evaluated mean cell volume: intra-PBMCs concentration was meanly 19.23 and 218.61 times higher than the hematic one for TAC and EVE, respectively. This method might be useful in clinical routine, giving reliable data on drugs concentration at the active site. Copyright © 2017 John Wiley & Sons, Ltd.

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

“…One can raise the hypothesis that it might be due to the normalization of TAC intra-PBMC concentration in quantity per number of cells instead of volumetric unit. Indeed, as suggested by Pensi et al, it would be more accurate to normalize intracellular concentration by the mean cell volume of each patient [7,36]. Unfortunately, measurement of cells volume was not available on the instrument use in our study and we could not check whether the underestimated concentrations were due to extreme values in PBMC volumes in these individuals.…”

Monitoring the tacrolimus concentration in peripheral blood mononuclear cells of kidney transplant recipients

“…In liver transplant recipients, [Tac] cells significantly correlated with both the development and the severity of rejection 8 . Moreover, previous studies reported poor to moderate correlations between [Tac] cells and [Tac] blood , indicating that [Tac] blood does not always reflect the concentration at the target site 8,11,13–17 …”

“…8 Moreover, previous studies reported poor to moderate correlations between [Tac] cells and [Tac]blood , indicating that [Tac] blood does not always reflect the concentration at the target site. 8,11,[13][14][15][16][17] This poor correlation may reflect the activity of the efflux transporter protein ABCB1 in mononuclear cell membranes. Differences in ABCB1 activity may result in differences in intralymphocytic tacrolimus accumulation and tacrolimus pharmacodynamics.…”

Barriers and opportunities for the clinical implementation of therapeutic drug monitoring in oncology

“…30 Also, as these samples contain high and variable amounts of cells and phospholipids that may affect the analysis, evaluation of the matrix effect is critical. 31,32 In contrast to the above-described alternative sample matrices, CSF requires highly invasive sampling from lumbar puncture. For several drugs, similar to PBMCs, the concentration in CSF directly reflects the concentration at the target site.…”

“…However, intracellular TDM is not (yet) routinely performed, due to a lack of standardised collection/isolation and cell count procedures for the processing of PBMCs, both of which influence the accuracy of the quantitative measurements 30 . Also, as these samples contain high and variable amounts of cells and phospholipids that may affect the analysis, evaluation of the matrix effect is critical 31,32 …”