The Importance Of Sample Clean Up Demonstrated By The Revalidation Of The Biomarker 4β-hydroxycholesterol For Assay Robustness

4β-hydroxycholesterol (4β-OHC) is a key endogenous biomarker for CYP3A enzyme activity, with plasma levels shifting in response to CYP3A inhibitors and inducers, making it critical for evaluating drug interactions. Although a previously validated method for measuring 4β-OHC in human plasma had passed initial performance checks, recurring issues, such as systemic contamination, UPLC overpressure, inconsistent derivatization, and retention time drift, raised concerns about the assay’s robustness.

A thorough investigation pinpointed two key problem areas: the derivatization step and final sample cleanup. Rather than overhauling the method entirely, our team focused on targeted improvements that preserved cross-validation compatibility while significantly enhancing reliability. Through a systematic, stepwise approach, we traced contamination to derivatization conditions and linked LC issues to extract components.

Discover how this refined method strengthens assay performance without sacrificing continuity, and what it reveals about best practices in biomarker quantification, by exploring the full poster.