A Simple and Sensitive Method for Quantifying Sirolimus in Whole Blood Using Liquid Chromatography/Electrospray Ionization Tandem Mass Spectrometry

Abstract

We describe a rapid and sensitive method for the quantitative analysis of the immunosuppressant sirolimus in whole blood using liquid chromatography/electrospray tandem mass spectrometry (LC/ESI-MS/MS). Ascomycin was first added to samples as the internal standard and then they were purified by precipitating blood protein with a zinc sulfate/methanol solution. Next, chromatographic separation was conducted using a Capcell Pak UG 120 (1.5 x 150 mm, 5μm) column with the mobile phase consisting of methanol/10 mM ammonium acetate (70/30, v/v, pH 7.0) at a flow-rate of 0.1 mL/min. Selected reaction monitoring (SRM) of the above compounds (m/z 932>m/z 865 for sirolimus and m/z 809>m/z 765 for ascomycin, respectively) was performed using ESI in the positive ion mode.
In whole blood samples from healthy subjects and a patient receiving sirolimus, no interfering peaks were observed. The assay produced a linear dynamic range of 1-100 ng/mL (r<0.999) for sirolimus in whole blood and the lower limit of detection was 0.5 ng/mL (S/N>3). The method of analysis we studied provides acceptable intra-and inter-assay accuracy and precision in the expected therapeutic range.
In conclusion, the simplicity and sensitivity of our method make it suitable for the therapeutic drug monitoring of sirolimus.