Method development and validation for simultaneous analysis of four bile acids as CYP3A drug-drug interaction biomarkers.

AIM: To develop and validate a robust LC - MS/MS method for the simultaneous quantification of endogenous deoxycholic acid (DCA), 1β-hydroxydeoxycholic acid (1β-OH-DCA), 1β-hydroxydeoxycholic acid glycine conjugate (1β-OH-G-DCA), and 1β-hydroxydeoxycholic acid taurine conjugate (1β-OH-T-DCA) in human plasma as biomarkers for the assessment of CYP3A-mediated drug-drug interactions (DDI).

MATERIALS AND

METHODS

A surrogate matrix was employed for the preparation of calibration standards and quality control samples. Analytical quantification was achieved using protein precipitation extraction coupled with UHPLC - MS/MS analysis.

RESULTS

A multiplex LC - MS/MS assay was successfully developed and validated for the simultaneous quantification of DCA (4-2000 ng/mL), 1β-OH-DCA (0.05-100 ng/mL), 1β-OH-G-DCA (0.05-100 ng/mL), and 1β-OH-T-DCA (0.05-100 ng/mL) in human plasma. The method effectively addressed the analytical challenges associated with developing an integrated assay for endogenous analytes spanning markedly different concentration ranges and was compliant with the ICH M10 bioanalytical method validation guidance.

CONCLUSION

This is the first report describing the development and validation of a 4-in-1 LC - MS/MS method for the simultaneous quantification of DCA, 1β-OH-DCA, 1β-OH-G-DCA, and 1β-OH-T-DCA in human plasma. The assay provides a robust bioanalytical platform for evaluating CYP3A-mediated DDIs and offers a methodological framework applicable to other multiplexed methods for endogenous biomarkers.