Use of 4β-hydroxycholesterol in animal and human plasma samples as a biomarker for CYP3A induction

Background: 4β-hydroxycholesterol (4βHC) has recently been proposed as a potential endogenous biomarker for CYP3A activity. Developing bioanalytical assays for 4βHC is challenging for several reasons, including endogenous background levels in plasma; the presence of free and ester forms; the inherent lack of MS sensitivity; and the presence of multiple positional isomers. Results: Bioanalytical assays in mouse, rat, dog and human plasma were adapted and modified from a previous published human plasma assay for 4βHC by using alkaline de-esterification, picolinic derivatization, a surrogate analyte (d7-4βHC) in authentic matrices and chromatographic conditions that showed good separation from isobaric, positional isomers. Conclusion: These assays were applied to multiple studies and demonstrated potential applications of 4βHC as a CYP3A biomarker across preclinical and clinical settings.