Simultaneous quantification of sirolimus, everolimus, tacrolimus and cyclosporine by liquid chromatography-mass spectrometry (LC-MS).
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.
Deters
Michael
M
Institute of Pharmacology, Medical School Hannover, Germany. Deters.Michael@MH-Hannover.de
Kirchner
Gabriele
G
Resch
Klaus
K
Kaever
Volkhard
V
D001774:Blood Chemical Analysis / Q000379:methods*; D002138:Calibration; D002853:Chromatography, Liquid / Q000379:methods*; D016572:Cyclosporine / Q000032:analysis*; D000068338:Everolimus; D007166:Immunosuppressive Agents / Q000032:analysis*; D013058:Mass Spectrometry / Q000379:methods*; D020123:Sirolimus / Q000031:analogs & derivatives* / Q000032:analysis*; D016559:Tacrolimus / Q000032:analysis*; D013696:Temperature; D013997:Time Factors
D007166:Immunosuppressive Agents; D016572:Cyclosporine; D000068338:Everolimus; D020123:Sirolimus; D016559:Tacrolimus