Received: 10 Jul 2014 Revised and Accepted: 08 Aug 2014 ABSTRACT Objective: To develop method to measure both methadone enantiomers and its major metabolite 2-ethylidene-1, 5-dimethyl-3, 3- diphenylpyrrolidine (EDDP) in clinical samples Methods: Five hundredmicroliters plasma/serum was extracted using solid phase extraction (mixed mode SPE-C8/SCX). The eluent was evaporated, reconstituted in mobile phase (95:5, 0.003% formic acid in methanol: 20 mM* ammonium formate) and injected. Result: The recoveries of methadone enantiomers and EDDP were 97% and 89% respectively. Under this condition, methadone enantiomers were successfully separated at baseline but not EDPP. Precision of spiked plasma for intra-day and inter-day was less than five for both methadone enantiomers and less than 12 for EDDP at medium and high quality control samples. Linear relationship between peak area ratio and internal standard were obtained for methadone in the range 5-1000ng/ml, and for EDDP from 5-500ng/ml with correlation coefficients greater than 0.99. The limit of quantification was 5ng/ml. Conclusion: The assay was used to analyse serum samples obtained from patients enrolled in a methadone maintenance treatment program. Original Article VALIDATION FOR QUANTITATIVE OF METHADONE ENANTIOMERS AND ITS MAJOR METABOLITE USING VANCOMYCIN COLUMN COUPLED WITH MASS SPECTROMETRIC DETECTION AND ITS APPLICATION TO CLINICAL SAMPLES NURFADHLINA MUSA*, SIMHANN LIANG*, TAN SOOCHOON*, RUSLI ISMAIL** Pharmacogenomic& Novel Therapeutics Cluster, Institute for Research in Molecular Medicine (INFORMM), Universiti Sains Malaysia, 16150 KubangKerian, Kelantan, Malaysia, Centre of Excellence for Research in AIDS (CERiA), University of Malaya, Level 17, Wisma RND, Jalan Pantai Baharu, 59990, Kuala Lumpur, Malaysia Email: isrusliceria@gmail.com Keywords: Methadone, Vancomycin, LC/MS/MS, Enantiomers. INTRODUCTION Methadone was discovered in the 1930’s for use as an analgesic and has been used since 1960’s for the stabilization and maintenance of patients with addictive disorders [1]. With the advent of HIV/AIDS in the 1980’s, methadone became a widely used to substitute for illicit injectable opiates that propelled HIV spread. Over the past 10 years, interest in its use for pain treatment has also increased. Methadone however has complex pharmacokinetics and pharmacodynamics. This contribute to the poor relationship between dose, plasma levels and effects and the use of therapeutic concentrations. Earlier studies demonstrated that methadone doses from 60 to 100mg/day were effective; however it is now increasingly acknowledged that doses larger than 100mg/day may be required [2]. Thus, although 100mg/day is considered a maximum by many physicians, doses of more than 100mg/day are used in an increasing number of centres. On average, researchers have affirmed the benefit of a 150 to 600 ng/mL trough level to suppress opioid craving and a trough level at or above 400 ng/mL to provide opioid blockade during methadone maintenance[3, 4]. Methadone has an asymmetrical carbon atom in its structure. It exists as two enantiomers, having the same chemical composition but different spatial arrangements. It is marketed as a racemic mixture (50:50 mixture) of (R) or levo or l-methadone and (S) or dextro or d-methadone. R-methadone has 10-fold higher affinity than S-methadone for µ and δ opioid receptors [5]. It possesses up to 50 times the analgesic activity of S-methadone in human and in animal models[6]. R-methadone prevents opioid withdrawal but notS-methadone[7]. HoweverS-methadone blocks the potassium channel 3.5-fold more potently than R-methadone to cause prolonged QTC and sudden death [8]. It is proposed that the concentration of methadone enantiomers in serum of patients on MMT is higher than a certain maximum to cause prolonged QTC. It is also proposed that a therapeutic range exists for R-methadone for optimal effectiveness. Availability of a method to simultaneously measure methadone enantiomers would therefore be useful to test these hypotheses. The objective of this study was to develop method to measure both methadone enantiomers and its major metabolite 2-ethylidene-1, 5- dimethyl-3, 3-diphenylpyrrolidine (EDDP) in clinical samples.Methadone is mainly cleared via hepatic metabolism by cytochrome P450 (CYP) to the inactive metabolite EDDP through N- demethylation pathway. EDDP has no pharmacological activity and been reported to be of lower concentrations in plasma during therapeutic usage. However, measurement of EDDP was imperative in order to determine whether preferential metabolism of methadone had occurred. MATERIAL AND METHODS Chemicals and reagents S-Methadone, R-methadone, EDDPand deuterium-labeled (R,S)- [2H3]-Met, were purchased from Cerilliant (Austin, TX, USA). Methanol gradient grade for liquid chromatography LiChrosolv® Reag. Ph Eur and formic acid (98-100%) was purchased from Merck Millipore (Merck KGaA, Darmstadt, Germany),ammonium formate 99% was obtained fromAcros Organic (Geel, Belgium), and ultra- pure water wasobtained, using a Milli-Q water-purification system (Millipore, Milford, MA, USA). Pooled drug-free human plasmawas donated from Blood Bank, Hospital Universiti Sains Malaysia, Malaysia. For the SPE procedures, mixed mode Si-SCX/C8 columns (SampliQ) (100 mg sorbent mass, 1 ml column volume) were purchased from Agilent Technologies (USA). Phosphate buffer 0.01 M was prepared by dissolving disodium hydrogen phosphate (1.42 g) in water in a one litre volumetric flask. pH was adjusted to 6.0 (±0.1)either with sodium hydroxide 1.0 M or phosphoric acid. The acetic acidic solution 1 M, was prepared by an appropriate dilution of International Journal of Pharmacy and Pharmaceutical Sciences ISSN- 0975-1491 Vol 6, Issue 8, 2014 Innovare Academic Sciences