Quantification of Sirolimus by Liquid Chromatography-Tandem Mass Spectrometry Using On-Line Solid-Phase Extraction

Quantification of the new immunosuppressant sirolimus (syn. rapamycin; Rapamune®) in whole blood by chromatography is essential for its clinical use since no immunoassay is available although monitoring is mandatory. Here we report on a rapid and convenient liquid chromatography (LC)-tandem mass spectrometry method and describe our practical experience with its routine use. Whole blood samples were hemolyzed and deproteinized using an equal volume (150 μl) of a mixture of methanol/zinc sulfate solution containing the internal standard desmethoxy-rapamycin. After centrifugation, the clear supernatants were submitted to an on-line solid-phase extraction procedure using the polymeric Waters Oasis HLB® material, with elution of the extracts onto the analytical column in the back-flush mode by column switching. For analytical chromatography a RP-C18 column was used with 90/10 methanol/2 mM ammonium acetate as the mobile phase. A 1:10 split was used for the transfer to the mass spectrometer, a Micromass Quattro LC-tandem mass spectrometry system equipped with a Z-spray® source and used in the positive electrospray ionization mode. The following transitions were recorded: sirolimus, 931>864 m/z, and desmethoxy-rapamycin (I.S.), 901>834 m/z. The analytical running time was 5 min, including on-line extraction. The method has a linear calibration curve (r>0.99; range 1.6–50 μg/l) and is rugged and precise with monthly CVs