Development and Validation of an Enantioselective HPLC–UV Method for Stereospecific Therapeutic Drug Monitoring of Methadone Enantiomers in Iranian MMT Patients: A Pilot Study

Methadone used in maintenance therapy is a racemic mixture of R- and S-enantiomers with distinct pharmacological and safety profiles. R-methadone mediates therapeutic effects via μ-opioid receptor activation, whereas S-methadone inhibits hERG potassium channels and may contribute to QT prolongation. Iran hosts the largest national methadone maintenance therapy (MMT) population (>۸۰۰,۰۰۰ patients), yet enantioselective therapeutic drug monitoring (TDM) is not available. To develop, validate, and clinically apply a simple and cost-effective HPLC–UV method for enantioselective quantification of methadone in plasma of Iranian MMT patients. Plasma samples from ۲۰ clinically stable MMT patients (۶۰-۱۲۰ mg/day) were analyzed. Enantiomers were separated on a Chiral-AGP column (۱۰۰ × ۴ mm, ۵ µm) using isocratic acetonitrile:۰.۰۱ M phosphate buffer (۱۵:۸۵, pH ۶.۸) at ۱.۲ mL/min with UV detection at ۲۱۰ nm. Validation followed ICH Q۲(R۱) guidelines. Liquid–liquid extraction with n-hexane and imipramine as internal standard was applied. Calibration was linear (۱۲.۵-۱۰۰۰ ng/mL; r² ≥ ۰.۹۹۹). LOQ was ۱۰ ng/mL. Intra- and inter-day precision were <۳%, accuracy within ±۳%, recovery ۹۱–۹۴%, and matrix effects ۹۵–۱۰۲% (CV <۴.۵%). Trough concentrations showed high variability: total methadone ۴۵۲±۱۸۵ ng/mL; R- ۲۲۶±۹۲ ng/mL; S- ۱۸۱±۷۴ ng/mL; R:S ratio ۰.۶-۱.۹. Only ۴۵% of patients were within the therapeutic R-methadone range (۱۰۰-۲۵۰ ng/mL), while ۱۰% exhibited potentially elevated S-methadone levels. This validated, low-cost (~۴/sample) method enables stereospecific methadone monitoring. Marked interindividual variability highlights the clinical value of enantioselective TDM to optimize efficacy and minimize cardiovascular risk in MMT programs.

کلیدواژه ها:

Methadone enantiomers ، HPLC–UV ، therapeutic drug monitoring ، chiral analysis ، methadone maintenance therapy ، stereoselective pharmacokinetics