Quantitation of Sirolimus Using Liquid Chromatography-Tandem Mass Spectrometry (LC-MS-MS)

A multiple reaction monitoring positive ion HPLC method with tandem mass spectrometric detection (MS-MS) for determination of sirolimus in human blood samples is described. This method utilizes an online cleanup step that provides simple and rapid sample preparation with a switching valve technique. This procedure includes: instrumentation, API 3000 triple quadrupole with turbo-ion spray (Applied Biosystems, Foster City, CA); HPLC system (Agilent Technologies series 1100, Wilmington, DE); two position switching valve (Valco, Houston, TX); 10 mm guard cartridge (C₁₈ ) used as an extraction column (Perkin Elmer, Norwalk, CT); analytical column (Nova-Pak C₁₈ column, 2.1 � 150 mm I.D., 4 μm, Waters Corp, Milford, MA) maintained at 65�C; extraction solution, ammonium acetate (30 mM, pH 5.2), flow rate 1.0 mL/min; eluting solution, methanol:30 mM ammonium acetate buffer (pH 5.2, 97:3 v/v), flow rate 0.8 mL/min with ˜ 1/3 of the flow split post-column into the MS-MS; total run-time 3.5 min. Sample preparation is based on simple protein precipitation with a mixture of methanol and zinc sulfate (7:3, v/v) followed by online sample cleanup. This procedure provides a decreased sample preparation time by a factor of four compared to a method that uses an SPE column. The first and third quadrupoles were set to detect the ammonium adduct ion and a high mass fragment of sirolimus (m/z 931.8→864.6), and of an internal standard (ascomycin) (m/z 809.5→756.5). The lower limit of quantification of this method is 2.5 μg/L. The quantification of drug is made from standard curve using peak-area ratio of analyte vs. internal standard. Calibration curve is constructed using non-weighted linear through zero regression.