VALIDATION FOR QUANTITATIVE OF METHADONE ENANTIOMERS AND ITS MAJOR METABOLITE USING VANCOMYCIN COLUMN COUPLED WITH MASS SPECTROMETRIC DETECTION AND ITS APPLICATION TO CLINICAL SAMPLES
Keywords:Methadone, Vancomycin, LCMSMS, Enantiomers
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.
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