title: Quantitation of P450 3A4 endogenous biomarker - 4β-hydroxycholesterol - in human plasma using LC/ESI-MS/MS.
abstract: 4β-Hydroxycholesterol (4β-HC) has been proposed as a new endogenous biomarker for cytochrome P450 3A4/5 activity. Therefore, it is important to have a robust method for its accurate determination in human plasma. Here a liquid chromatography-tandem mass spectrometry with electrospray ionization (LC/ESI-MS/MS) assay for the quantitation of 4β-HC in human plasma is described. While the calibration standards were prepared in a surrogate matrix for human plasma, the quality control samples were prepared in human plasma to mimic the incurred study samples. In order to achieve accurate determination of 4β-HC, the chromatographic separation of 4β-HC from its isomers, especially 4α-hydroxycholesterol (4α-HC), was crucial. In the absence of an authentic 4α-HC standard at the time of this study, an alternative selectivity test strategy was developed to confirm the separation. After being alkalized with potassium hydroxide, the human plasma sample (50 μL) was extracted with hexane, derivatized into picolinyl esters using picolinic acid, extracted again with hexane, and then analyzed by LC/ESI-MS/MS. The calibration curve range was 5-500 ng/mL and the chromatographic separation was achieved on a 50 × 2.1 mm Thermal Hypersil Gold column with a gradient elution. The assay accuracy, precision, linearity, selectivity and analyte stability throughout the analysis were established. The validated assay was successfully applied to a Phase I clinical study for the measurement of 4β-HC in human plasma.
authors.affiliation: Janssen Research and Development LLC, Pharmaceutical Companies of Johnson & Johnson, 1400 McKean Road, Spring House, PA, 19477, USA.
authors.forename: Mike-Qingtao
authors.initials: MQ
authors.lastname: Huang
authors.forename: Weisheng
authors.initials: W
authors.lastname: Lin
authors.forename: Weimin
authors.initials: W
authors.lastname: Wang
authors.forename: Wei
authors.initials: W
authors.lastname: Zhang
authors.forename: Zhongping John
authors.initials: ZJ
authors.lastname: Lin
authors.forename: Naidong
authors.initials: N
authors.lastname: Weng
mesh_terms: D015415:Biomarkers / Q000097:blood; D002851:Chromatography, High Pressure Liquid / Q000379:methods*; D051544:Cytochrome P-450 CYP3A / Q000378:metabolism*; D006801:Humans; D006888:Hydroxycholesterols / Q000097:blood*; D021241:Spectrometry, Mass, Electrospray Ionization / Q000379:methods; D053719:Tandem Mass Spectrometry / Q000379:methods*
chemical_list: D015415:Biomarkers; D006888:Hydroxycholesterols; C099828:cholest-5-ene-3,4-diol; D051544:Cytochrome P-450 CYP3A; C510163:CYP3A4 protein, human
keywords: 4β-hydroxycholesterol; CYP3A4/5; LC/ESI-MS/MS; endogenous biomarker