Development and validation of a rapid and sensitive UPLC-MS/MS assay for the quantification of tofacitinib in human...

A highly sensitive, selective and rapid ultra-performance liquid chromatography-tandem mass spectrometry method has been developed for the quantification of a Janus kinase (JAK) inhibitor, tofacitinib (TOF). The assay employed liquid-liquid extraction with methyl-tert butyl ether to extract tofacitinib and tofacitinib-13C3 15 N (as internal standard) from human plasma. The samples were analyzed on a UPLC BEH C₁₈ (50 × 2.1 mm, 1.7 μm) column using acetonitrile and 10.0 mm ammonium acetate, pH 4.5 (75:25, v/v) as the mobile phase within 1.4 min. The precursor/product ion transitions were monitored at m/z 313.3/149.2 and 317.4/149.2 for tofacitinib and tofacitinib-13C3 15 N, respectively, in the positive electrospray ionization mode. The calibration curves were linear (r²  ≥ 0.9978) across the concentration range of 0.05-100 ng/mL. The mean extraction recovery of tofacitinib across quality controls was 98.6%. The intra- and inter-batch precision (CV) and accuracy ranged from 2.1-5.1 and 96.2-103.1%, respectively. All validation results complied well with the current guidelines. The method is amenable to high sample throughput and was applied to determine TOF plasma concentration in a pharmacokinetic study with 12 healthy Indian subjects after oral administration of 5 mg tablets.