(M1130-06-33) A Derivatization-Free LCMS Assay for 4β-Hydroxycholesterol

Purpose: In clinical studies, 4β-hydroxycholesterol is an important biomarker for assessing the activity of CYP3A. Numerous quantitative methods have been reported for analyzing the total amount of 4β-hydroxycholesterol (free and conjugated) after saponification and derivatization. Derivatization is often required to achieve clinically relevant assay sensitivity when utilizing LCMS for detection, but this additional step can be complex and time-consuming. We have developed and validated a method for 4β-hydroxycholesterol that doesn't require derivatization, simplifying the extraction process and achieving clinically relevant detection levels. Removal of the derivation step also increased the robustness of the method with regard to UPLC retention time and over-pressuring issues.

Methods: Total 4β-hydroxycholesterol (free and conjugated) is extracted from human plasma using a liquid-liquid extraction methodology, following alkaline hydrolysis. After the analyte is recovered with hexane, the samples are evaporated and then reconstituted with 1% acetic acid in 80% methanol and water. The resulting extracts are analyzed using LC-MS/MS detection. The analyte is separated from potential interferences using a reverse phase column and gradient conditions. A Sciex 6500+ mass spectrometer operating in positive APCI mode is used to detect 4β-Hydroxycholesterol and the isotopically labeled internal standard. Detection was accomplished by multiple-reaction-monitoring (MRM) that monitored a precursor ion formed by the loss of water from 4β-Hydroxycholesterol, 385 m/z, and a product ion of 109 m/z. Because 4β-Hydroxycholesterol is an endogenous compound, calibrators and quality control samples are prepared in saline with 5% bovine serum albumin. The analytical range of the assay is 4.00 to 400 ng/mL.

Results: Removal of the derivatization step improved the overall robustness of the method compared to prior validated assays that utilized this process. A surrogate matrix was used to prepare calibrators and quality control samples. Stability assessments used endogenous QCs and included freeze/thaw cycles and short-term and long-term storage conditions. A dilution of endogenous QCs with the surrogate matrix was performed to demonstrate parallelism. The validation experiments included three accuracy and precision runs, selectivity and specificity, parallelism, and hemolysis effect. Additionally, analyte stability assessments for long-term, short-term, freeze-thaw, and whole blood were performed. All experiments met a priori acceptance criteria and demonstrated that the assay is suitable for supporting regulated clinical studies.

Conclusion: A method of analysis for 4β-Hydroxycholesterol in human plasma was developed and rigorously validated to support regulated clinical studies. This method was validated following the principles outlined in the FDA M10 bioanalytical method validation guidance and from the AAPS “Biomarker Assay Validation by Mass Spectrometry” white paper. The validation process included comprehensive experiments to measure accuracy and precision in an authentic and surrogate matrix, parallelism, and stability. This method, which does not require derivatization of the analyte, has significantly increased the ruggedness and robustness of the assay for supporting clinical studies, providing a reliable tool for biomarker analysis.