Rapid and sensitive UPLC-MS-MS determination of tacrolimus in Wistar rats and human blood.

A simple, sensitive and high throughput ultra-high performance liquid chromatography tandem mass spectrometry (UPLC-MS-MS) method was developed for the determination of tacrolimus in the whole blood of Wistar rats and humans. Sample preparation involved protein precipitation of the analyte, using sirolimus as the internal standard with ZnSO4 from 50 µL of rat blood/human blood, followed by solid-phase extraction. Chromatographic analysis was conducted on a Waters Acquity UPLC BEH C18 column (50 × 2.1 mm, 1.7 µm) using 10 mM ammonium acetate (pH 6.0) and methanol (5:95, v/v) under isocratic conditions and detection by MS-MS. Quantitation of the analytes was achieved by multiple reaction monitoring under positive ionization mode. The method was validated over a dynamic concentration range of 0.200-200 ng/mL and had a chromatographic run time of 1.2 min. The extraction recovery for tacrolimus was >96% across three quality control levels. Matrix effect was assessed by the precision (coefficient of variation) values for the calculated slopes of calibration curves from six lots of blood. The method was applied to a pre-clinical study in 25 rats and to a bioequivalence study in 20 healthy Indian subjects. The reproducibility of the assay was successfully demonstrated by the reanalysis of 80 subject samples.