Liquid chromatographic-electrospray mass spectrometric determination of cyclosporin A in human plasma

A rapid, sensitive and selective liquid chromatography-mass spectrometry (LC-MS) assay has been developed for determination of cyclosporin A (CyA) in human plasma; cyclosporin B (CyB) was used as internal standard (IS). The method utilized a combination of a column-switching valve and a reversed-phase symmetry column. The mobile phase was a 25: 75 (v/v) mixture of 10% aqueous glacial acetic acid and acetonitrile. Running time per single run was less than 10 min. Sample preparation included C-8 SPE of human plasma spiked with the analyte and internal standard, evaporation of the eluate to dryness at 50 degrees C under N-2 gas, and finally reconstitution in the mobile phase. Detection of cyclosporin A and the IS was performed in selected ion-monitoring mode at m/z 601.3 and 594.4 Da for CyA and IS, respectively. Quantitation was achieved by use of the regression equation of relative peak area of cyclosporin to IS against concentration of cyclosporin. The method was validated according to FDA guideline requirements. The linearity of the assay in the range 5.0-400.0 ng mL(-1) was verified as characterized by the least-squares regression line Y = (0.00268 +/- 1.9 x 10(-4)) X + (0.00078 +/- 1.8 x 10(-3)), correlation coefficient, r = 0.9986 +/- 1.1 x 10(-3) (n = 48). Intra and inter-day quality-control measurements in the range 5.0-350.0 ng mL(-1) revealed almost 100% accuracy and <= 9% CV for precision. The mean absolute recovery of CyA was found to be 84.01 +/- 9.9% and the respective relative recovery was 100.3 +/- 9.19. The limit of quantitation (LOQ) achieved was 5 ng mL(-1). Eventually, stability testing of the analyte and IS in plasma or stock solution revealed that both chemicals were very stable when stored for long or short periods of time at room temperature or -20 degrees C.