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The authors describe an isocratic liquid chromatographic/electrospray single quadrupole mass spectrometric assay suitable for routine therapeutic monitoring of the antirejection drugs tacrolimus, sirolimus, and cyclosporine in blood. The drugs and added internal standards, ascomycin, desmethoxysirolimus, and cyclosporine G, are extracted from a protein-free supernatant of patient blood on a Strata SDB-L styrene-divinylbenzene polymeric extraction cartridge (Phenomenex, Torrance, CA). A Gilson Aspec XL-4 (Gilson Instruments, Middleton, WI) is programmed to perform the extraction and transfer the extract to autosampler vials. The extract is automatically injected onto a C18 column held at 75°C. Mobile phase, acetonitrile/water 90/10 (vol/vol), is pumped at 0.5 mL/min through a silica saturating column positioned in the column oven before the injector valve. Detection is by selected ion monitoring of the sodium adduct of each analyte, [M + 23]+. A linear response is achieved for tacrolimus and sirolimus from limit of quantitation (LOQ) 1.0 μg/L to at least 80.0 μg/L. Cyclosporine was linear from LOQ 25 μg/L to at least 2000 μg/L. Interferences and ionization suppression are minimal. Analytic recovery for cyclosporine is 99.9 ± 6.2% over a range of 104-1162 μg/L; sirolimus 107.7 ± 9.3% over a range of 3.2-29.2 μg/L; and tacrolimus 101.9 ± 2.5% over a range of 2.9-38.1 μg/L. Between-run relative standard deviations are 3.0% to 5.1% over a range of 9.8 μg/L to 30.7 μg/L for tacrolimus; 5.5% to 7.6% over a range of 13.5 μg/L to 35.2 μg/L for sirolimus; and 2.6% to 4.3% over a range of 186.0 μg/L to 1428.7 μg/L for cyclosporine. In the authors' laboratory, the method is robust and shows improved selectivity, precision, detection limits, and lower cost per test over available immunoassays. The authors find the method suitable for routine monitoring and therapy management of these drugs with over 50,000 samples analyzed over the course of 1 year.