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

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). PBMCs were collected using Cell-Preparation-Tubes; cells number and MCV were evaluated by an automatic cell counter. TAC and EVE were quantified using UHPLC-MS/MS coupled with an automated on-line SPE platform. Chromatographic run was performed on an Acquity UPLC® BEH C18 1,7 µm (2,1 x 50 mm) column at 45 °C, for 6 minutes 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 (1x10mm) SPE cartridges were used and the internal standard was ascomycin. Following FDA guidelines, method validation resulted in high sensitivity and specificity. Calibration curves were linear (r(2)  = 0.998) and intra- 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 5 pediatric patients in co-treatment resulted within the calibration range (0.039-5 ng). Concentrations from each patient were standardized using their evaluated MCV: 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.