Simultaneous quantification of sirolimus, everolimus, tacrolimus and cyclosporine by liquid chromatography-mass spectrometry (LC-MS)

We developed a universal liquid chromatographymass spectrometry (LCMS) assay with automated online extraction to quantify simultaneously the immunosuppressants sirolimus, everolimus, tacrolimus, and cyclosporine. Whole blood (300 mul) plus 6 mul 32-desmethoxyrapamycin (1 ng/mul) as internal standard was treated with 600 mul methanol/0.2 M ZnSO4 (80/20 v/v). After vortexing (30 s) and centrifugation (20000 g, 5 min) 50 mul of the supernatant were loaded on an extraction column, were washed by 0.35 ml/min water for 3 min and, after activation of a columnswitching valve, were backflushed by 0.25 ml/min methanol/water (90/10 v/v) onto a C-18 analytical column. After 22 min the extraction column was washed for 2 min with methanol and for 3 min with water before starting the next run. Column temperatures were kept at 33 degreesC. Sodium adduct ions [M+Na](+) ions were detected in the selected ion mode. For sirolimus, everolimus and tacrolimus the assay was linear from 0.3 to 200 mug/l and for cyclosporine from 5 to 1000 mug/l (all r(2)>0.999). Recovery of all immunosuppressants and the internal standard was >90% and in general, interday and intraday precision was <10%. The simultaneous quantification of blood levels by LCMS seems to be the method of choice in transplanted patients receiving multiple immunosuppressants.