Quantification of Efavirenz Hydroxymetabolites in Human Plasma Using LC-HRMS/MS

Area of Science:

• Pharmacology
• Analytical Chemistry
• Pediatric Medicine

Background:

• Efavirenz (EFV) is a key HIV treatment, but its efficacy and safety depend on plasma concentrations.
• Suboptimal EFV levels lead to poor viral suppression, while high levels cause neuropsychiatric side effects.
• The metabolism of EFV in children and its impact on treatment outcomes remain understudied.

Purpose of the Study:

• To develop and validate a sensitive method for quantifying EFV and its metabolites in pediatric plasma.
• To investigate the presence and levels of EFV metabolites, including a newly discovered one, in children.
• To establish a tool for future studies on EFV pharmacokinetics and treatment efficacy in pediatric populations.

Main Methods:

• Quantification of EFV, 7-hydroxyefavirenz (7-OH-EFV), 8-hydroxyefavirenz (8-OH-EFV), and a novel metabolite "EFAdeg" in plasma.
• Sample preparation involved protein precipitation and hydrolysis for phase II metabolite analysis.
• Analysis was performed using Liquid Chromatography coupled with High-Resolution Orbitrap Mass Spectrometry (LC-HRMS/MS).

Main Results:

• A validated LC-HRMS/MS method with a wide measuring range for EFV and its metabolites was established.
• The method met European Medicines Agency guidelines for precision, accuracy, and selectivity.
• The newly discovered metabolite "EFAdeg" was confirmed in pediatric plasma samples, with carryover noted for 8-OH-EFV.

Conclusions:

• A robust LC-HRMS/MS method for quantifying EFV and its phase I/II metabolites in children was successfully developed and validated.
• This method is suitable for analyzing pediatric plasma samples and aids in understanding EFV metabolism.
• The identification of "EFAdeg" offers new insights into factors influencing 8-OH-EFV concentrations and potentially treatment outcomes.