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, School of Pharmacy, Shiraz University of Medical Sciences, Shiraz, Iran

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

10.30476/tips.2026.110754.1352

Abstract

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. 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 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. 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. 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.

Highlights

Marzieh Faraz (Google Scholar)

Hashem Montaseri (Google Scholar)

Keywords

References

1.    De Rosa F, Giannatiempo B, Charlier B, Coglianese A, Mensitieri F, Gaudino G, et al. Pharmacological Treatments and Therapeutic Drug Monitoring in Patients with Chronic Pain. Pharmaceutics. 2023;15(8):2088. doi: 10.3390/pharmaceutics15082088. 
2.    Eap CB, Buclin T, Baumann P. Interindividual variability of the clinical pharmacokinetics of methadone: implications for the treatment of opioid dependence. Clin Pharmacokinet. 2002;41(14):1153-93. doi: 10.2165/00003088-200241140-00003. 
3.    Dyer KR, White JM. Patterns of symptom complaints in methadone maintenance patients. Addiction. 1997;92(11):1445-55. 
4.    Kristensen K, Christensen CB, Christrup LL. The mu1, mu2, delta, kappa opioid receptor binding profiles of methadone stereoisomers and morphine. Life Sci. 1995;56(2):PL45-50. doi: 10.1016/0024-3205(94)00426-s. 
5.    Eap CB, Crettol S, Rougier JS, Schläpfer J, Sintra Grilo L, Déglon JJ, et al. Stereoselective block of hERG channel by (S)-methadone and QT interval prolongation in CYP2B6 slow metabolizers. Clin Pharmacol Ther. 2007;81(5):719-28. doi: 10.1038/sj.clpt.6100120. 
6.    Roden DM. Drug-induced prolongation of the QT interval. N Engl J Med. 2004;350(10):1013-22. doi: 10.1056/NEJMra032426. 
7.    Foster DJ, Somogyi AA, Bochner F. Methadone N-demethylation in human liver microsomes: lack of stereoselectivity and involvement of CYP3A4. Br J Clin Pharmacol. 1999;47(4):403-12. doi: 10.1046/j.1365-2125.1999.00921.x.
8.    Totah RA, Sheffels P, Roberts T, Whittington D, Thummel K, Kharasch ED. Role of CYP2B6 in stereoselective human methadone metabolism. Anesthesiology. 2008;108(3):363-74. doi: 10.1097/ALN.0b013e3181642938. 
9.    Crettol S, Déglon JJ, Besson J, Croquette-Krokar M, Hämmig R, Gothuey I, et al. ABCB1 and cytochrome P450 genotypes and phenotypes: influence on methadone plasma levels and response to treatment. Clin Pharmacol Ther. 2006;80(6):668-81. doi: 10.1016/j.clpt.2006.09.012. 
10.    Oz MD, Ozdemir F, Suzen HS. The Influence of Genetic Variations and Drug Interactions Based on Metabolism of Antidepressants and Anticonvulsants. Curr Drug Metab. 2021;22(8):596-627. doi: 10.2174/1389200221999201125211058. PMID: 33243113.
11.    Di Salvo C, Valdiserra G, Balestrieri S, Beucci G, Paciulli G, Luculli GI, et al. The Role of Clinical Pharmacogenetics and Opioid Interactions in Pain Management: Current Evidence and Future Perspectives. Pharmaceutics. 2026;18(1):59.
12.    Fonseca Casals F. Pharmacogenomic study of oppioid addicts in methadone treatment: Universitat Pompeu Fabra; 2010.
13.    Ramli FF. Pharmacogenomics biomarkers for personalized methadone maintenance treatment: The mechanism and its potential use. Bosn J Basic Med Sci. 2021;21(2):145-154. doi: 10.17305/bjbms.2020.4897. 
14.    Ranjbari E, Golbabanezhad-Azizi AA, Hadjmohammadi MR. Preconcentration of trace amounts of methadone in human urine, plasma, saliva and sweat samples using dispersive liquid-liquid microextraction followed by high performance liquid chromatography. Talanta. 2012;94:116-22. doi: 10.1016/j.talanta.2012.03.004. 
15.    Nakahara Y. Hair analysis for abused and therapeutic drugs. J Chromatogr B Biomed Sci Appl. 1999;733(1-2):161-80. doi: 10.1016/s0378-4347(99)00059-6. 
16.    MD F, Karch SB. Postmortem toxicology of abused drugs: CRC Press; 2007.
17.    Cruces-Blanco C, García-Campaña A. Capillary electrophoresis for the analysis of drugs of abuse in biological specimens of forensic interest. TrAC Trends  Anal Chem. 2012;31:85-95.
18.    Health UDo, Services H. FDA. Bioanalytical Method Validation Guidance for Industry. 2018.
19.    ICH I, editor Q2 (R1): Validation of analytical procedures: text and methodology. International conference on harmonization, Geneva; 2005.
20.    Valentová J, Horáková R, Pechová I, Okruhlica L, Devínsky F. Stereoselective determination of methadone and its main metabolite in serum and urine from methadone maintenance patients. Neuro Endocrinol Lett. 2006;27 Suppl 2:130-3. 
21.    Gumustas M, Ozkan SA, Chankvetadze B. Analytical and Preparative Scale Separation of Enantiomers of Chiral Drugs by Chromatography and Related Methods. Curr Med Chem. 2018;25(33):4152-88. doi: 10.2174/0929867325666180129094955.
22.    Vyas R, Bhushan R, Nagar H, Sharma A. Reversed-phase-HPLC enantioseparation and control of enantiomeric purity of duloxetine using a new chiral reagent and recovery of enantiomers. Biomed Chromatogr. 2021;35(12):e5228. doi: 10.1002/bmc.5228. 
23.    Narayana ChL, Suresh T, Rao SM, Dubey PK, Babu JM. A validated chiral HPLC method for the enantiomeric separation of Linezolid on amylose based stationary phase. J Pharm Biomed Anal. 2003;32(1):21-8. doi: 10.1016/s0731-7085(03)00031-1. 
24.    Bouquié R, Hélène Hernando, Guillaume Deslandes, Mostefa Daho AB, Renaud C, Grall-Bronnec M, et al. Chiral on-line solid phase extraction coupled to liquid chromatography-tandem mass spectrometry assay for quantification of (R) and (S) enantiomers of methadone and its main metabolite in plasma. Talanta. 2015;134:373-378. doi: 10.1016/j.talanta.2014.11.052.
25.    Ward LF, Enders JR, Bell DS, Cramer HM, Wallace FN, McIntire GL. Improved Chiral Separation of Methamphetamine Enantiomers Using CSP-LC-MS-MS. J Anal Toxicol. 2016;40(4):255-63. doi: 10.1093/jat/bkw005. 
26.    Mueller F, Losacco GL, Nicoli R, Guillarme D, Thomas A, Grata E. Enantiomeric methadone quantitation on real post-mortem dried matrix spots samples: Comparison of liquid chromatography and supercritical fluid chromatography coupled to mass spectrometry. J Chromatogr B Analyt Technol Biomed Life Sci. 2021 Jul 1;1177:122755. doi: 10.1016/j.jchromb.2021.122755. 
27.    Aebi B, Bernhard W. Modern Gas Chromatography–Mass Spectrometry in Human Forensic Toxicology. Biological Concepts and Techniques in Toxicology An Integrated Approach New York: Editorial Taylos & Francis. 2006:365-75.
28.    Leek H, Andersson S. Preparative Scale Resolution of Enantiomers Enables Accelerated Drug Discovery and Development. Molecules. 2017;22(1):158. doi: 10.3390/molecules22010158. 
29.    Recarey-Rama S, Gómez-Trigo J, Gil-Rodriguez A, Dominguez E, Sánchez-Martínez I, Riveiro-Recimil Á, et al. The Relevance of Pharmacokinetic Biomarkers in Response to Methadone Treatment: A Systematic Review. Pharmaceuticals (Basel). 2025;18(5):623. doi: 10.3390/ph18050623. 
30.    Crettol S, Déglon JJ, Besson J, Croquette-Krokkar M, Gothuey I, Hämmig R, et al. Methadone enantiomer plasma levels, CYP2B6, CYP2C19, and CYP2C9 genotypes, and response to treatment. Clin Pharmacol Ther. 2005 Dec;78(6):593-604. doi: 10.1016/j.clpt.2005.08.011. 
31.    Packiasabapathy S, Aruldhas BW, Horn N, Overholser BR, Quinney SK, Renschler JS, et al. Pharmacogenomics of methadone: a narrative review of the literature. Pharmacogenomics. 2020 Aug;21(12):871-887. doi: 10.2217/pgs-2020-0040. 
32.    Sadat Aghabozorg Afjeh S, Boshehri B, Hamednia S, Amini A, Mashayekhi P, Omrani MD. Investigating the CYP2B6 rs3745274 and rs3211371 polymorphisms in Methadone-Responder and Non-Responder Addicts in Iran. Iran Biomed J. 2021;25(3):220-5. doi: 10.29252/ibj.25.3.220. 
33.    Vallamkonda B, Sethi S, Satti P, Das DK, Yadav S, Vashistha VK. Enantiomeric Analysis of Chiral Drugs Using Mass Spectrometric Methods: A Comprehensive Review. Chirality. 2024;36(8):e23705. doi: 10.1002/chir.23705. 
34.    Valladares Chávez EY, Moreno-Rocha LA, Ortega Cabello L, García-Gutiérrez P, Miranda-Calderón JE. Evolution, Validation and Current Challenges in Bioanalytical Methods for Praziquantel: From Fluorometry to LC–MS/MS. Scientia Pharmaceutica. 2025;94(1):4.
35.    Ansermot N, Rudaz S, Brawand-Amey M, Fleury-Souverain S, Veuthey J-L, Eap CB. Validation and long-term evaluation of a modified on-line chiral analytical method for therapeutic drug monitoring of (R, S)-methadone in clinical samples. Journal of Chromatography B. 2009;877(23):2301-7.
36.    Meini M, Moncini M, Daini L, Giarratana T, Scaramelli D, Chericoni S, et al. Relationship between plasma concentrations of the l-enantiomer of methadone and response to methadone maintenance treatment. Eur J Pharmacol. 2015;760:1-6. doi: 10.1016/j.ejphar.2015.03.081. 
37.    Foster DJ, Somogyi AA, Dyer KR, White JM, Bochner F. Steady-state pharmacokinetics of (R)- and (S)-methadone in methadone maintenance patients. Br J Clin Pharmacol. 2000;50(5):427-40. doi: 10.1046/j.1365-2125.2000.00272.x. 
38.    Hendrix CW, Wakeford J, Wire MB, Lou Y, Bigelow GE, Martinez E, et al. Pharmacokinetics and pharmacodynamics of methadone enantiomers after coadministration with amprenavir in opioid-dependent subjects. Pharmacotherapy. 2004;24(9):1110-21. doi: 10.1592/phco.24.13.1110.38091. 
Trends in Pharmaceutical Sciences and Technologies
Volume 12, Issue 1
March 2026
Pages 67-80

Files

Share

How to cite

Statistics