title: Simultaneous determination of itraconazole, hydroxy itraconazole, keto itraconazole and N-desalkyl itraconazole concentration in human plasma using liquid chromatography with tandem mass spectrometry.
abstract: A high-performance liquid chromatography tandem mass spectrometry (LC-MS/MS) assay was developed and validated for simultaneous determination of itraconazole (ITZ), hydroxy-itraconazole (OH-ITZ), keto-itraconazole (keto-ITZ) and N-desalkyl itraconazole (ND-ITZ) concentration in human plasma. One hundred and fifty microliters of human plasma were extracted using a solid-supported liquid extraction (SLE) method and the final extracts were analyzed using reverse-phase chromatography and positive electrospray ionization mass spectrometry. The standard curve range is 5-2500 ng/mL for ITZ and OH-ITZ and 0.4-200 ng/mL for keto-ITZ and ND-ITZ. The curve was fitted to a 1/x(2) weighted linear regression model for all analytes. The precision and accuracy of the LC-MS/MS assay based on the five analytical quality control (QC) levels were well within the acceptance criteria from both FDA and EMA guidance for bioanalytical method validation. Average extraction recovery was 97.4% for ITZ, 112.9% for OH-ITZ, 103.4% for keto-ITZ, and 102.3% for ND-ITZ across their respective curve range. Matrix factor was close to 1.0 at both high and low QC levels of all 4 analytes, which indicates minimal ion suppression or enhancement in our validated assay. Itraconazole and all three metabolites are stable in human plasma for 145 days stored at -70 °C freezers. The validated assay was successfully applied to a clinical study, which has a drug-drug interaction (DDI) arm using ITZ as a cytochrome P450, family 3, subfamily A (CYP3A) inhibitor.
authors.affiliation: Drug Metabolism and Pharmacokinetics, Genentech Inc., South San Francisco, CA 94080, United States. Electronic address: Liang.Xiaorong@gene.com.
authors.forename: Xiaorong
authors.initials: X
authors.lastname: Liang
authors.affiliation: Bioanalytical Department, Covance Laboratories Inc., Madison, WI 53704, United States.
authors.forename: Michael
authors.initials: M
authors.lastname: Van Parys
authors.affiliation: Drug Metabolism and Pharmacokinetics, Genentech Inc., South San Francisco, CA 94080, United States.
authors.forename: Xiao
authors.initials: X
authors.lastname: Ding
authors.affiliation: Covance Pharmaceutical Research and Development Co. Ltd., Shanghai 201203, PR China.
authors.forename: Ning
authors.initials: N
authors.lastname: Zeng
authors.affiliation: Covance Pharmaceutical Research and Development Co. Ltd., Shanghai 201203, PR China.
authors.forename: Luke
authors.initials: L
authors.lastname: Bi
authors.affiliation: Bioanalytical Department, Covance Laboratories Inc., Madison, WI 53704, United States.
authors.forename: Drew
authors.initials: D
authors.lastname: Dorshort
authors.affiliation: Bioanalytical Department, Covance Laboratories Inc., Madison, WI 53704, United States.
authors.forename: Janine
authors.initials: J
authors.lastname: McKnight
authors.affiliation: Bioanalytical Department, Covance Laboratories Inc., Madison, WI 53704, United States.
authors.forename: Dennis
authors.initials: D
authors.lastname: Milanowski
authors.affiliation: Drug Metabolism and Pharmacokinetics, Genentech Inc., South San Francisco, CA 94080, United States.
authors.forename: Jialin
authors.initials: J
authors.lastname: Mao
authors.affiliation: Drug Metabolism and Pharmacokinetics, Genentech Inc., South San Francisco, CA 94080, United States.
authors.forename: Yuan
authors.initials: Y
authors.lastname: Chen
authors.affiliation: Department of Clinical Pharmacology, Genentech Inc., South San Francisco, CA 94080 United States.
authors.forename: Joseph A
authors.initials: JA
authors.lastname: Ware
authors.affiliation: Drug Metabolism and Pharmacokinetics, Genentech Inc., South San Francisco, CA 94080, United States.
authors.forename: Brian
authors.initials: B
authors.lastname: Dean
authors.affiliation: Drug Metabolism and Pharmacokinetics, Genentech Inc., South San Francisco, CA 94080, United States.
authors.forename: Cornelis E C A
authors.initials: CE
authors.lastname: Hop
authors.affiliation: Drug Metabolism and Pharmacokinetics, Genentech Inc., South San Francisco, CA 94080, United States.
authors.forename: Yuzhong
authors.initials: Y
authors.lastname: Deng
mesh_terms: D002853:Chromatography, Liquid / Q000379:methods*; D056148:Chromatography, Reverse-Phase; D004347:Drug Interactions; D004355:Drug Stability; D006801:Humans; D017964:Itraconazole / Q000097:blood*; D016014:Linear Models; D053719:Tandem Mass Spectrometry / Q000379:methods*
chemical_list: D017964:Itraconazole
keywords: Drug-drug interaction; Human plasma; Hydroxy itraconazole; Itraconazole; Keto itraconazole; LC–MS; N-desalkyl itraconazole