title: Therapeutic monitoring of tacrolimus concentrations in blood: semi-automated extraction and liquid chromatography-electrospray ionization mass spectrometry.
abstract: The authors describe a highly selective liquid chromatographic/mass spectrometric (LC/MS) method for measurement of the immunosuppressive drug tacrolimus. A protein-free supernatant of a patient's whole-blood sample (500 microL) is applied to an Empore styrene-divinylbenzene (SDB-XC) disk cartridge (Varian Sample Preparation Products; Harbor City, CA) to isolate tacrolimus and the internal standard, ascomycin. The entire extraction is automated on the Gilson ASPEC XL4 (Gilson; Middleton, WI). Separation takes place on an octyldecyl (C18) high-performance liquid chromatographic (HPLC) column maintained at 75 degrees C with a simple mobile phase of acetonitrile/water (90/10, v/v). An atmospheric pressure ionization (API)-electrospray ionization(ESI)-mass spectrometer (MS) is used for analysis. Detection is by selected ion monitoring (SIM) of the positively charged sodium adduct of tacrolimus and ascomycin, m/z 826.5 and 814.5, respectively. Collision-induced dissociation (CID) fragment ions of tacrolimus and ascomycin (m/z 616.4 and 604.4, respectively) are also monitored to enhance overall selectivity. Total run time is 1 minute per injection. A plot of chromatographic peak height ratio of m/z 826.5 to m/z 814.5 vs tacrolimus blood concentration is linear from the lowest limit of quantitation (0.3 microg/L) to at least 120.0 microg/L. Metabolites of tacrolimus and other immunosuppressive and commonly prescribed drugs do not interfere. Analytical recovery is 94% to 113% over a range of 4.4 to 41.0 microg/L. Between-run precision coefficients of variation (CV) are 3.6% to 4.2% over a range of 8.1 to 32.4 microg/L. Comparison data (n = 156) of the LC/MS method versus a commercial immunoassay (Abbott Tacrolimus IMx MEIA II) (Abbott; Chicago, IL) demonstrated that the tacrolimus concentration as measured by LC/MS = (0.912 x MEIA concentration) - 0.049; r2 = 0.968. This validated LC/MS method demonstrates significant advantages over conventional immunoassays and improves on the lower limit of detection, sensitivity, accuracy, and range of linearity. In the authors' laboratory, this method is approximately 60% less costly to perform than the most commonly implemented immunoassay. Together, the authors' daily clinical experience during 1 year and participation in a proficiency testing program has demonstrated that the method is robust and suitable for routine therapeutic monitoring of tacrolimus.
authors.affiliation: University of Wisconsin Hospitals & Clinics, Division of Laboratory Medicine, Toxicology Laboratory, Madison, Wisconsin, USA. gl.lensmeyer@hosp.wisc.edu
authors.forename: G L
authors.initials: GL
authors.lastname: Lensmeyer
authors.forename: M A
authors.initials: MA
authors.lastname: Poquette
mesh_terms: D002853:Chromatography, Liquid; D016903:Drug Monitoring; D006801:Humans; D007166:Immunosuppressive Agents / Q000097:blood*; D013058:Mass Spectrometry; D016559:Tacrolimus / Q000097:blood*
chemical_list: D007166:Immunosuppressive Agents; D016559:Tacrolimus