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 microl) 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 microg/l) and is rugged and precise with monthly CVs <7% at a sirolimus concentration of 13.1 microg/l in routine use; the instrumentation proved to be reliable and required minimal maintenance. Clin Chem
Vogeser
Michael
M
Institute of Clinical Chemistry, Ludwig-Maximilians-Universität München, Klinikum Grosshadern, Munich, Germany. mvogeser@klch.med.uni-muenchen.de
Fleischer
Claudia
C
Meiser
Bruno
B
Groetzner
Jan
J
Spöhrer
Ute
U
Seidel
Dietrich
D
D002853:Chromatography, Liquid / Q000379:methods*; D006801:Humans; D007166:Immunosuppressive Agents / Q000097:blood*; D013058:Mass Spectrometry / Q000379:methods*; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity; D020123:Sirolimus / Q000097:blood*
D007166:Immunosuppressive Agents; D020123:Sirolimus