A novel UPLC–MS/MS method for the estimation of an immunosuppressant in human whole blood

Abstract

To facilitate estimation of Ciclosporine (CsA), an immunosuppressant, in whole blood sample, ultra-performance liquid chromatography–mass spectrometric method employing positive electrospray ionization was developed for the determination of CsA concentration in human whole blood. CsA together with the internal standard (Ciclosporine D) was extracted from 250µL of human whole blood by protein precipitation followed by solid phase extraction. The chromatography was performed using C18, sub 2µ column with a mobile phase consisting of methanol and 0.1% ammonia solution. Multiple reaction monitoring mode was used for the quantification of CsA in whole blood. The validation of the method including sensitivity, linearity, reproducibility and stability was examined. The lower limit of quantification (LLOQ) of the developed assay method for CsA was 10ng/mL and the linear calibration curve was acquired with r > 0.99 between 10 and 2000 ng/ml. The intra-day and inter-day variation of the current assay was evaluated and the coefficient of variation were found within 6.4% at LLOQ and 6.8% for other quality control samples, whereas the mean accuracy ranged from 92.3% to 108.6%. The present method provides a robust, rapid and sensitive analytical tool for Ciclosporine in human whole blood.