A simple HPLC method for the determination of cyclosporin A in human whole blood

A simple high performance liquid chromatography method for the determination of cyclosporin A in human whole blood was developed. Human blood samples were deproteinated with a zinc sulfate saturated acetonitrile-water (1:1) solution, and centrifuged. The supernatant was transferred to clean tubes, evaporated, and reconstituted for an HPLC analysis. Cyclosporin D was used as an internal standard. Chromatography was carried out using XTerra® C ₁₈ 150 x 4.6 mm 5 μm column (Waters, Watford, UK) maintained at 80°C, with a mobile phase consisting of acetonitrile-water-t-butyl methyl ether-phosphoric acid (55:40:5:1, v/v/v/v). The flow rate of the mobile phase was adjusted to 1 mL/min. The detection wavelength was set at 210 nm. Under these conditions, cyclosporin A and cyclosporin D were cleanly separated with no interfering endogenous peaks present. The calibration curve for cyclosporin A in human blood was linear over the concentration range examined (50 - 3000 ng/mL), with a correlation coefficient greater than 0.999. The lower limit of quantification (LOQ) approached 50 ng/mL. The intra- and inter-day variations were in the ranges of 0.48 - 13.33% for precision and 98.30 - 103.74% for accuracy, respectively. The applicability of this method was demonstrated in a pharmacokinetic study of cyclosporin A in human volunteers.