Evaluation of a cyano stationary phase for the determination of tacrolimus, sirolimus and cyclosporin A in whole blood by high-performance liquid chromatography–tandem mass spectrometry

The potential of a cyano HPLC column for the analysis of three immunosuppressants is investigated. Tacrolimus, sirolimus and cyclosporin A, were used to probe differences in the retention and efficiency of a cyano column compared to the more widely used C18 column. The cyano column showed comparable retention for all three compounds, whereas the C18 column showed stronger retention, especially for cyclosporin A. Furthermore, the efficiencies at 50 °C were up to 12 times higher on the cyano column. As a result, a baseline separation was achieved in less than three minutes with the cyano column, using an isocratic mobile phase of 52/48 (v/v) acetonitrile/water at 0.45 mL/min. The analysis of immunosuppressant drugs in human whole blood was performed with the cyano column using a selected reaction monitoring (SRM) method for each analyte with negative ion mode electrospray ionization on a triple quadrupole mass spectrometer. Detection limits were 0.05 ng/mL for sirolimus, 0.1 ng/mL for cyclosporin A and 0.2 ng/mL for tacrolimus. Calibration curves were linear over three orders of magnitude. Good agreement was obtained with the actual levels of immunosuppressant drugs in patient samples with an average error of less than 10%.