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

摘要

Introduction Tacrolimus is a potent immunosuppressant drug that is well established for primary immunosuppression in kidney transplantation. A simple, rapid, and sensitive LC-MS/MS method has been developed and validated for the quantitation of tacrolimus in peripheral blood mononuclear cells (PBMCs). Methods PBMCs are isolated from 7 mL whole blood by centrifugation over Ficoll gradient density and washed twice with phosphate-buffered saline at 4°C. Harvested cells were suspended within 1.2 mL of phosphate-buffered saline. Cell counts were performed to express and normalize tacrolimus amount per 10~6 cells. Ascomycin was used as an internal standard(IS). Tacrolimus was extracted by a liquid-liquid extraction in methyl tert-butyl ether. The separation of the analyte was achieved on a Venusil XBP C18 column (50 × 2.1 mm, 5 μm; Agela, USA) with a mobile phase consisting of 2 mM ammonium acetate buffer and methanol (5:95, v/v) containing 0.1% formic acid. Result The proposed method has been validated with linear range of 0.01-5 ng/mL. The within- and between-run precision and accuracy of the QC samples were 1.333-18.142% (at LLOQ level) and 86.80-114.5%, respectively. Tacrolimus was stable at room temperature for 17 h and also during three freeze-thaw cycles. Conclusion This method is currently used for the measurement of small intracellular amounts of tacrolimus down to 0.0051 ng per 10~6 PBMCs in kidney-transplanted recipients.