We describe a multiple reaction monitoring positive ion HPLC/tandem mass spectrometric method for quantification of tacrolimus in human whole blood with online extraction and cleanup. Included in this procedure: API 2000 triple quadrupole mass spectrometer with turbo-ion spray source (Applied Biosystems, Foster City, CA); 10-port diverter/switching valve (Valco, Houston, TX); HPLC system (Agilent Technologies series 1100, Wilmington, DE); 10mm (C18 ) guard cartridge (Perkin Elmer, Norwalk, CT) used as an extraction column; a Nova-Pak C18 analytical column (2.1 � 150mm I.D., 4μm, Waters Corp, Milford, MA); washing solution, methanol: 30mM ammonium acetate pH 5.1 (80:20); eluting solution, methanol:30mM ammonium acetate pH 5.1 (97:3); flow rate 0.8 mL/min; and a run-time of 2.8min. The first and third quadrupoles were set to detect the ammonium adduct ion and a high mass fragment of tacrolimus (m/z821.5→768.3), and of an internal standard (ascomycin) (m/z901.8→834.4). The lower limit of quantification of this method is 3.75mg/L. The concentration of drug is determined by comparing peak-area ratios for tacrolimus and internal standard to a standard curve constructed using non-weighted linear through zero regression.
