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

Document Type : Original Article

Authors

1 department of Quality Control, Shiraz Faculty of Pharmacy, Shiraz University of medical Sciences, Shiraz, Fars. IR

2 Department of Pharmacology, Faculty of Medicine, Shiraz University of Medical Sciences, Shiraz, Iran

3 Department of Quality Control, Faculty of Pharmacy, Shiraz University of Medical Sciences, Shiraz, Iran

10.30476/tips.2026.110754.1352

Abstract

Background: 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 (>800,000 patients), yet enantioselective therapeutic drug monitoring (TDM) is not available.
Objectives: 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.
Methods: Plasma samples from 20 clinically stable MMT patients (60–120 mg/day) were analyzed. Enantiomers were separated on a Chiral-AGP column (100 × 4 mm, 5 µm) using isocratic acetonitrile:0.01 M phosphate buffer (15:85, pH 6.8) at 1.2 mL/min with UV detection at 210 nm. Validation followed ICH Q2(R1) guidelines. Liquid–liquid extraction with n-hexane and imipramine as internal standard was applied.
Results: Calibration was linear (12.5–1000 ng/mL; r² ≥ 0.999). LOQ was 10 ng/mL. Intra- and inter-day precision were <3%, accuracy within ±3%, recovery 91–94%, and matrix effects 95–102% (CV <4.5%). Trough concentrations showed high variability: total methadone 452 ± 185 ng/mL; R- 226 ± 92 ng/mL; S- 181 ± 74 ng/mL; R:S ratio 0.6–1.9. Only 45% of patients were within the therapeutic R-methadone range (100–250 ng/mL), while 10% exhibited potentially elevated S-methadone levels.
Conclusion: This validated, low-cost (~$4/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

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