Quantification of sirolimus by liquid chromatography-tandem mass spectrometry using on-line solid-phase extraction

Quantification of the new immunosuppressant sirolimus (syn.
rapamycin; Rapamune((R))) 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 mul) 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(R) 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((R)) 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 mug/l) and is
rugged and precise with monthly CVs