Simultaneous Determination of Cyclosporine, Sirolimus, and Tacrolimus in Whole Blood Using Liquid ChromatographyTandem Mass Spectrometry

A multiple reaction monitoring, positive ionization electrospray, liquid chromatography–tandem mass spectrometry (LC-MS/MS) method is described for the simultaneous quantification of cyclosporine, sirolimus, and tacrolimus in human whole blood. Proteins in the samples are precipitated with a mixture of methanol and zinc sulfate. The supernatant is injected into the LC-MS/MS for analysis. Chromatography involves the use of a C18 column and ammonium acetate/water/methanol-containing mobile phases. The MS/MS is operated in positive ion electrospray mode. Quantification is achieved by comparing peak area ratios of MRMs of analytes and internal standards with that of calibrators. Calibration curves are constructed from peak area ratios of MRMs of calibrators and internal standards versus concentrations. MRMs used are ascomycin (m /z 809.5 → 756.5), cyclosporine A (m /z 1,219.9 → 1,203.1), cyclosporine D (m/z 1,234.0 → 1,217.0), sirolimus (m /z 931.6 → 864.5), and tacrolimus (m/z 821.5 → 768.4).