title: Determination of 7α-OH cholesterol by LC-MS/MS: Application in assessing the activity of CYP7A1 in cholestatic minipigs.
abstract: An LC-MS/MS method was developed and validated to determine 7α-OH cholesterol in liver microsome. This method was convenient and fast with high specificity and sensitivity. Briefly, a gradient elution was performed on a Synergi polar-C18 column (50×4.6mm i.d., 3μm). The mobile phase (consisting of 0.1% HCOOH solution and acetonitrile) eluted in gradient at a flow rate of 1ml/min. MS detection was operated on APCI (+) mode; the MRM transitions for 7α-OH cholesterol and D7-cholesterol (I.S.) were 385.1≥159.1 and 376.4≥266.3, respectively. The linear response range of 7α-OH cholesterol was covered from 1.563 to 100.0ng/ml. All of the validation items meet the requirement of FDA guidance for bioanalytical method validation. This method was applied to enzymatic studies for determination of cholesterol 7alpha-hydroxylation activity catalyzed by CYP7A1 in the cholestatic minipigs liver microsomes.
authors.affiliation: Department of Pharmaceutics, University of Houston, 1441 Moursund St. Houston, TX 77030, United States.
authors.forename: Changhong
authors.initials: C
authors.lastname: Yun
authors.affiliation: Department of Pharmaceutics, University of Houston, 1441 Moursund St. Houston, TX 77030, United States.
authors.forename: Taijun
authors.initials: T
authors.lastname: Yin
authors.affiliation: Department of Pharmaceutics, University of Houston, 1441 Moursund St. Houston, TX 77030, United States.
authors.forename: Katherine
authors.initials: K
authors.lastname: Shatzer
authors.affiliation: USDA Children's Nutrition Research Center, Department of Pediatrics, Baylor College of Medicine, 1100 Bates St. Houston, TX 77030, United States.
authors.forename: Douglas G
authors.initials: DG
authors.lastname: Burrin
authors.affiliation: USDA Children's Nutrition Research Center, Department of Pediatrics, Baylor College of Medicine, 1100 Bates St. Houston, TX 77030, United States.
authors.forename: Liwei
authors.initials: L
authors.lastname: Cui
authors.affiliation: Department of Pharmaceutics, University of Houston, 1441 Moursund St. Houston, TX 77030, United States.
authors.forename: Yifan
authors.initials: Y
authors.lastname: Tu
authors.affiliation: Department of Pharmaceutics, University of Houston, 1441 Moursund St. Houston, TX 77030, United States. Electronic address: mhu@uh.edu.
authors.forename: Ming
authors.initials: M
authors.lastname: Hu
mesh_terms: D000818:Animals; D002779:Cholestasis / Q000097:blood* / Q000201:enzymology / Q000378:metabolism; D002790:Cholesterol 7-alpha-Hydroxylase / Q000097:blood / Q000378:metabolism*; D002853:Chromatography, Liquid / Q000379:methods*; D016014:Linear Models; D008862:Microsomes, Liver / Q000378:metabolism; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity; D013552:Swine; D013556:Swine, Miniature; D053719:Tandem Mass Spectrometry / Q000379:methods*
chemical_list: D002790:Cholesterol 7-alpha-Hydroxylase
keywords: 7α-OH cholesterol; CYP7A1; Cholesterol 7α-hydroxylase activity; LC–MS/MS; Liver microsome