A liquid chromatography-tandem mass spectrometry method was developed for the quantification of circulating levels of multiple immunosuppressant drugs including cyclosporine (CsA), tacrolimus, methotrexate (Mtx), prednisone, prednisolone, methylprednisone, total and free mycophenolic acid (MPA), as well as MPA phenolic (MPAG) and acyl (AcMPAG) glucuronide metabolites. Linearity, precision and accuracy were validated within the typical therapeutic range of concentrations for each compound. The assay was linear over 0.125-25ng/mL for tacrolimus, 1-500ng/mL for prednisone/methylprednisone, 2-400ng/mL for Mtx, 2-1000ng/mL for prednisolone and from 7.5 to 1500ng/mL for CsA with the lowest limit of quantification (LLOQ) being 0.125, 1.00, 2.00, 2.00 and 7.5ng/mL, respectively. The calibration curve concentrations for MPA and MPAG ranged from 50 to 50,000ng/mL (LLOQ: 50ng/mL) and 10 to 10,000ng/mL (LLOQ: 10ng/mL) for AcMPAG. Mean recoveries in blood and plasma were 84%±5.7%. The method could measure individual drugs with high sensitivity, accuracy (bias≤14%), and reproducibility (CV≤12.8%). Its clinical application was validated by measuring levels of these drugs in samples obtained from hematopoietic stem cell transplant recipients treated with combined immunosuppressive drug therapy. Our results indicate that this approach is suitable for simultaneous determination of in vivo levels of immunosuppressive drugs commonly used in combined therapies.
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