Simultaneous Quantification of Sirolimus, Everolimus, Tacrolimus and Cyclosporine by Liquid Chromatography-Mass Spectrometry (LC-MS)

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Abstract
We developed a universal liquid chromatography-mass spectrometry (LC-MS) assay with automated online extraction to quantify simultaneously the immunosuppressants sirolimus, everolimus, tacrolimus, and cyclosporine. Whole blood (300 microl) plus 6 microl 32-desmethoxyrapamycin (1 ng/microl) as internal standard was treated with 600 microl methanol/0.2 M ZnSO4 (80/20 v/v). After vortexing (30 s) and centrifugation (20000 g, 5 min) 50 microl 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 column-switching valve, were back-flushed by 0.25 ml/min methanol/ water (90/10 v/v) onto a C18 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 degrees C. 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 microg/l and for cyclosporine from 5 to 1000 microg/l (all r2>0.999). Recovery of all immunosuppressants and the internal standard was >90% and in general, inter-day and intraday precision was <10%. The simultaneous quantification of blood levels by LC-MS seems to be the method of choice in transplanted patients receiving multiple immunosuppressants.