title: A simple, fast, sensitive and robust LC-MS/MS bioanalytical assay for evaluating 7α-hydroxy-4-cholesten-3-one biomarker in a clinical program.
abstract: OBJECTIVE
  Serum 7α-hydroxy-cholesten-3-one (C4) has been reported as a biomarker to assess CYP7A1 enzyme activity and bile acid synthesis. To support a clinical program, a sensitive and reliable assay without derivatization was required for the analysis of C4 in human serum. Methodology & results: A systematic approach was used to optimize mass spectrometry, LC and sample extraction conditions, therefore, significantly improved assay sensitivity, and achieved the required quantification limit without derivatization. A surrogate matrix approach was used to overcome the interference from endogenous C4. A stable isotope-labeled C4 was used as internal standard. The samples were extracted using a simple protein precipitation method with 2% formic acid in acetonitrile.
  
  
  CONCLUSIONS
  A simple, fast, sensitive and robust UHPLC-MS/MS method for the quantification of 0.50 ng/ml C4 in 100 µl human serum was developed and fit for purpose validated. The method was successfully applied to the bioanalysis of C4 in a clinical study.
authors.affiliation: Bioanalytical Sciences, Bristol-Myers Squibb Co., Princeton, NJ 08543, USA.
authors.forename: Long
authors.initials: L
authors.lastname: Yuan
authors.affiliation: Clinical Translational Technologies & Operations, Bristol-Myers Squibb Co., Princeton, NJ 08543, USA.
authors.forename: Yi
authors.initials: Y
authors.lastname: Luo
authors.affiliation: Bioanalytical Sciences, Bristol-Myers Squibb Co., Princeton, NJ 08543, USA.
authors.forename: Hamza
authors.initials: H
authors.lastname: Kandoussi
authors.affiliation: Bioanalytical Sciences, Bristol-Myers Squibb Co., Princeton, NJ 08543, USA.
authors.forename: Qin C
authors.initials: QC
authors.lastname: Ji
mesh_terms: D015415:Biomarkers / Q000097:blood*; D001774:Blood Chemical Analysis / Q000295:instrumentation / Q000379:methods*; D002112:Calcifediol / Q000737:chemistry; D002783:Cholestenones / Q000097:blood* / Q000592:standards; D002790:Cholesterol 7-alpha-Hydroxylase / Q000378:metabolism; D002851:Chromatography, High Pressure Liquid / Q000592:standards; D006801:Humans; D007553:Isotope Labeling; D011786:Quality Control; D012015:Reference Standards; D053719:Tandem Mass Spectrometry / Q000592:standards
chemical_list: D015415:Biomarkers; D002783:Cholestenones; C002656:7 alpha-hydroxy-4-cholesten-3-one; C553219:CYP7A1 protein, human; D002790:Cholesterol 7-alpha-Hydroxylase; D002112:Calcifediol
keywords: LC-MS/MS; biomarker; surrogate matrix