UPLC–MS/MS assay validation for tacrolimus quantitative determination in peripheral blood T CD4+ and B CD19+ lymphocytes

    loading  Checking for direct PDF access through Ovid


Monitoring tacrolimus (Tac) exposure in cell matrices enriched with lymphocytes can improve Tac therapeutic drug monitoring (TDM) in solid organ transplant recipients. An UPLC–MS/MS based assay for Tac quantification in peripheral blood T CD4+ and B CD19+ lymphocytes was developed. Peripheral blood mononuclear cells (PBMC) were obtained by density gradient centrifugation and highly purified (purity >90%) T CD4+ and B CD19+ cell suspensions were acquired by magnetic negative selection from whole blood of 6 healthy volunteers. The purity of lymphocyte suspensions was checked by flow cytometry. Tac extraction was performed in a liquid–liquid zinc sulfate, methanol and acetonitrile based medium. Ascomycin was used as internal standard. The equipment used was a Waters® Acquity™ UPLC system (Waters Corporation, Milford, MA, USA). The chromatographic run was performed on a Waters® MassTrak TDM C18 (2.1 × 10 mm) column (Waters Corporation, Milford, MA, USA). at a flow rate of 0.4 mL/min. The instrument was set in electrospray positive ionization mode. The method was validated according to Clinical Laboratory Standard Institute (CLSI) guidelines and showed a high sensitivity and specificity over a range of 0–5.2 ng/mL in PBMC, 0–5.0 ng/mL in T CD4+ Lymphocytes and 0–5.3 ng/mL in B CD19+ lymphocytes. Precision was appropriate with CV of intra-assay quantifications ranging from 4.9 to 7.4%, and of inter-assay quantifications from 7.2 to 13.9%. Limit of detection and quantification were 0.100 and 0.115 ng/mL in PBMC, 0.058 and 0.109 ng/mL in T CD4+ and 0.017 and 0.150 ng/mL in B CD19+ cells. Matrix effect was not significant among all the studied matrices. Samples showed stability for Tac quantification over a period of 90 days either at room temperature or at −30 °C storage conditions. The method was applied to clinical samples of 20 kidney transplant recipients. Concentrations ranged from 2.200 to 11.900 ng/mL in whole blood, from 0.005 to 0.570 ng/106 cells in PBMC, from 0.081 to 1.432 ng/106 cells in T CD4+, and from 0.197 to 1.564 ng/106 cells in B CD19+ cell matrices. The method has potential applicability for Tac TDM in solid organ transplant recipients.

Related Topics

    loading  Loading Related Articles