Liquid chromatographic-tandem mass spectrometric assay for simultaneous quantitation of tofacitinib, cabozantinib and afatinib in human plasma and urine

Purpose: To develop a simple, adequately sensitive, and practical liquid chromatographic-mass spectrometric method to simultaneously quantify three tyrosine kinase inhibitors, viz, tofacitinib (TOF), cabozantinib (CBZ) and afatinib (AFB) after their extraction from both human plasma and urine. Methods: Blood and urine samples were obtained from healthy volunteers who admitted to not being on any medications. The investigated analytes were chromatographically separated on a C18 column (Luna (R)-PFP 100 angstrom column, 50 mm x 2.0 mm i. d., 3.0 mu m) with the aid of a mobile phase containing A; acetonitrile (ACN) and B; 0.01 M ammonium formate buffer (pH 4.1) pumped at a rate of 0.3 mL.min(-1) in the ratio A: B, 50: 50 v/v. Analyte monitoring was achieved by tandem mass spectrometry interfaced with an electrospray ionization source with the aid of multiple reaction monitoring (MRM) mode for analytes quantification. Results: The proposed method permitted a specific and sensitive determination of the investigated TKIs in the linear range of 1.0 - 100 ng mL-1 with correlation coefficient (r(2)) of 0.9991, 0.9997, and 0.9998 for TOF, CBZ and AFB, respectively. The method was validated with regard to its limits of quantification (ranging from 0.91 to 1.24 ng mL-1 for the 3 analytes), intra-and inter assay accuracy (in the range -1.85 to 1.22 %) and precision (0.71 - 5.12 %). The method was also validated in terms of recovery from both studied matrices, robustness and matrix effect. Conclusion: The results obtained reveal that the developed method is simple, specific and highly efficient for routine determination of the studied analytes in human plasma and urine. It can be reliably applied for high throughput analysis of clinical samples containing the investigated analytes.