High-throughput liquid chromatography-tandem mass spectrometric analysis of sirolimus in whole blood

Sirolimus appears as a new potent immunosuppressive agent taking advantage of therapeutic drug monitoring to optimize its use in organ transplantation. In the absence of any available commercial immunoassay it was mandatory to develop chromatographic assays. Some methods have already been proposed to quantify sirolimus in whole blood, based either on HPLC-UV, liquid chromatography-mass spectrometry (LC-MS) or liquid chromatography-tandem mass spectrometry (LC-MS/MS). We have developed a new faster and simpler LC-MS/MS method to quantify sirolimus in blood using ascomycin as an internal standard and multiple reaction monitoring (MRM) acquisition mode. This method displays a limit of detection of 0.3 microg/l, and the intra-assay reproducibility ranges from 4.1-7.9%. The pre-analytical preparation steps are quite similar to those required for semi-automated immunoassays. Ascomycin and sirolimus present retention times of 0.89 and 0.93 min, respectively, and the turnaround time for a result (2.5 min) is also similar to that observed using a clinical analyzer. The comparison performed between HPLC-UV and LC-MS/MS displays good correlation (r = 0.949). The LC-MS/MS method described above has been used routinely for more than 2000 patient blood specimens and may present several advantages over existing methods, e.g., specificity with sufficient sensitivity, rapidity, and small blood sampling (10 microl), making it particularly adapted for routine clinical use.