Quantification and stability of everolimus (SDZ RAD) in human blood by high-performance liquid chromatography–electrospray tandem mass spectrometry

We report here a validated method for the quantification of a new immunosuppressant drug, everolimus (SDZ RAD), using HPLC–tandem mass spectrometry. Whole blood samples (500 μl) were prepared by protein precipitation, followed by C18 solid-phase extraction. Mass spectrometric detection was by selected reaction monitoring with an electrospray interface operating in positive ionization mode. The assay was linear from 0.5 to 100 μg/l (r2>0.996, n=9). The analytical recovery and inter-day imprecision, determined using whole blood quality control samples (n=5) at 0.5, 1.2, 20.0, and 75.0 μg/l, was 100.3–105.4% and ≤7.6%, respectively. The assay had a mean relative recovery of 94.8±3.8%. Extracted samples were stable for up to 24 h. Fortified everolimus blood samples were stable at −80 °C for at least 8 months and everolimus was found to be stable in blood when taken through at least three freeze–thaw cycles. The reported method provides accurate, precise and specific measurement of everolimus in blood over a wide analytical range and is currently supporting phase II and III clinical trials.