Rapid and simultaneous determination of two immunosup-pressants in whole blood by high performance liquid chroma-tography

Abstract

Cyclosporine A and sirolimus are immunosuppressants that are widely used in many organ transplantation procedures. They exhibit some complementary mechanisms of action and interact synergistically when used together. However, they are critical⁃dose drugs and have a narrow therapeutic index. They provide the desired therapeutic effect with acceptable tolerability only within a specific range of blood concentrations. Therefore, the rapid and simultaneous detection of the concentrations of cyclosporine A and sirolimus in whole blood could provide valuable information on planning medicine administration after organ transplantations. In this study, firstly, the chromatographic behaviors of cyclosporine A and sirolimus on a biological liquid chromatography (BioLC)column and traditional liquid chromatography (TraLC)columns were investigated systematically under the same chromatographic conditions. The results suggested that the peak height and peak width of cyclosporine A and sirolimus on the BioLC column, ZORBAX 300SB C8 (250 mm X 4.6 mm, 5.0 μ m), were the highest and narrowest, respectively. The number of theoretical plates of cyclosporine A and sirolimus on the ZORBAX 300SB C8 column increased significantly when the volume ratio of acetonitrile in the mobile phases was greater than 70%. Their retention time on the BioLC and TraLC columns was negligibly affected by the use of formic acid and trifluoroacetic acid as the mobile phases. In the range of the experimental column temperature, the number of theoretical plates of cyclosporine A and sirolimus on the ZORBAX 300SB C8 column was significantly higher than that on the two TraLC columns. Furthermore, the relationship between the retention factor and column temperature of cyclosporine A on the ZORBAX 300SB C8 column was different from that on the two TraLC columns. A sample of whole blood with a volume of 50 μL was prepared by protein precipitation with 1 mol/L sodium hydroxide and then extracted into 500 μL of ether⁃methanol (95:5, v/v). After centrifugation at 14.000 r/min for 10 min, the organic layer was removed and evaporated under a stream of nitrogen at 50 ℃. The residue was then reconstituted in 200 μL of methanol for use. Cyclosporin A and sirolimus were separated through isocratic elution on the ZORBAX 300SB C8 column.