title: Development and validation of a liquid chromatography-mass spectrometric assay for simultaneous determination of tacrolimus and 13-O-desmethyl tacrolimus in rat kidney tissue.
abstract: A sensitive and robust LC-MS/MS method has been developed and validated to determine the concentrations of tacrolimus and its major metabolite 13-O-desmethyl tacrolimus (13-ODMT) in kidney tissue from rats who received tacrolimus intra-peritoneally at doses of 0.5mg/kg and 2mg/kg. The samples were prepared by a liquid-liquid extraction procedure using ethyl ether as the extraction solvent and ascomycin as the internal standard. Chromatographic separation was achieved using Phenomenex Kinetex column (2.6μm C18 100Å, 100×2.1mm, Phenomenex, Torrance CA) and a gradient mobile phase of water and methanol-acetonitrile (50:50, v/v) both containing 0.1% formic acid. The limit of quantification was 0.25ng/ml and the calibration curves covered a concentration range from 0.25 to 50ng/ml. Intra-and inter-assay precision and accuracy for both tacrolimus and 13-ODMT were all within FDA guidelines for bioanalysis. Extraction efficiency for tacrolimus ranged from 67.00 to 74.90% and from 66.70 to 78.40% for 13-ODMT. Several challenges interfering with the performance of the method such as phospholipid build-up have also been addressed. Kidney tissue samples from six rats receiving either 0.5 or 2mg/kg dose were analyzed and resulted in a median concentration of 11.54 and 0.72ng/ml for tacrolimus and 13-ODMT, respectively, for the lower dose level, and a median concentration of 8.89ng/ml and 1.50ng/ml for tacrolimus and 13-ODMT, respectively, at the higher dose level.
authors.affiliation: College of Pharmacy, University of Iowa, Iowa City, IA, United States.
authors.forename: Tatian
authors.initials: T
authors.lastname: Kirresh
authors.affiliation: College of Pharmacy, University of Iowa, Iowa City, IA, United States.
authors.forename: Sony
authors.initials: S
authors.lastname: Tuteja
authors.affiliation: College of Medicine, University of Iowa, Iowa City, IA, United States.
authors.forename: D
authors.initials: D
authors.lastname: Russo
authors.affiliation: College of Medicine, University of Iowa, Iowa City, IA, United States.
authors.forename: P D
authors.initials: PD
authors.lastname: Brophy
authors.affiliation: College of Pharmacy, University of Iowa, Iowa City, IA, United States. Electronic address: dj.murry@unmc.edu.
authors.forename: D J
authors.initials: DJ
authors.lastname: Murry
mesh_terms: D000818:Animals; D002853:Chromatography, Liquid / Q000379:methods*; D004305:Dose-Response Relationship, Drug; D007166:Immunosuppressive Agents / Q000008:administration & dosage / Q000032:analysis / Q000378:metabolism / Q000493:pharmacokinetics*; D007274:Injections, Intraperitoneal; D007668:Kidney / Q000737:chemistry / Q000378:metabolism*; D057230:Limit of Detection; D008297:Male; D013058:Mass Spectrometry / Q000379:methods*; D051381:Rats; D017207:Rats, Sprague-Dawley; D015203:Reproducibility of Results; D016559:Tacrolimus / Q000008:administration & dosage / Q000031:analogs & derivatives* / Q000032:analysis / Q000378:metabolism / Q000493:pharmacokinetics
chemical_list: C503135:13-O-demethyl tacrolimus; D007166:Immunosuppressive Agents; D016559:Tacrolimus
keywords: 13-O-desmethyl tacrolimus; IT-TOF; Mass spectroscopy; Nephrotoxicity; Organ rejection; Tacrolimus