Synthesis and applications of deuterated methadone and metabolites to biotransformation and disposition studies
Creators: name: Kang, Gun-Il
Publisher: name: University of British Columbia
Year: 2010

description: Deuterium labeled methadone and deuterium labeled metabolites were synthesized to use in gas chromatography mass spectrometry (GCMS) studies of the metabolic pathways of metha-done in rats. These compounds were also useful to develop sensitive and selective analytical methods to study the pharmacokinetics and disposition of methadone. Synthesis of the deuterium labeled compounds was mainly achieved by using known procedures with special treatments required to provide label enrichment. Using the labeled and unlabeled derivatives, mass fragmentation processes that are common to methadone and its metabolites were defined. Aryl ring migration was observed in a fragmentation process for 2-ethylidene-l,5-dimethyl-3,3-diphenyl pyrrolidine (EDDP). This aryl ring migration was not a favorable process for ring substituted EDDP analogs. Various aspects regarding the optimization of the selected ion monitoring (SIM) analysis of methadone and its metabolites in biological fluids are described. The SIM analysis using deuterium labeled compounds as internal standards generally proved to be selective but not as sensitive as expected using electron impact ionization (EI) conditions of GCMS. One advantage of using SIM over GC analysis was described in terms of ratio analysis. Quantitation of methadone in human plasma and saliva using SIM gave a lower limit of sensitivity of 20 ng/0.5 ml of sample by monitoring the base peak, m/e 72. The mean methadone ratios of saliva to plasma for two patients were 0.55 ± 0.15 (standard deviation) and 0.48 ± 0.10 (standard deviation). Methadone metabolism studies emphasized the detection of minor metabolites using special extraction methods for rat bile and using labeled and unlabeled compounds. Comparison of the mass spectra from total ion current (TIC) profiles of metabolites from unlabeled compounds with those from labeled compounds run as separate experiments gave GCMS evidence for methadone nitrone (N-methylene-l-methyl-3,3-diphenyl-4-oxo-hexanamine-oxide). Possibilities for the metabolic formation of N-hydroxynormethadone and the pharmacological significance of the detection of methadone nitrone were described. A proposal for metabolic studies to examine the potential formation of other methadone metabolites resulting from metabolic oxidation of nitrogen was presented. Structural evidence for the methadone nitrone molecule was obtained indirectly by chemical oxidation studies of metha done metabolites. m-Chloroperbenzoic acid treatment of EDDP perchlorate gave three products: methadone nitrone, 4,4-diphen yl-2,5-heptanedione (diketone), and 2-acetyl-5-methyl-3,3-di-phenyl-l-pyrroline. These compounds were identified from their IR, NMR and mass spectral data. Mass fragmentation processes were defined for the methadone nitrone. Possible mechanisms for the formation of methadone nitrone and diketone from chemical oxidation of EDDP are proposed. Since diazepam is a drug widely abused by methadone maintenance patients, methadone-diazepam interaction studies were designed to analyze metabolites using deuterium labeled authentic compounds as internal standards. Metabolites in the conjugated fraction of rat bile were analyzed using deuterium labeled biosynthetic internal standards. Diazepam (5 mg/kg) was given to rats through a cannulated jugular vein and a subcutaneous dose of methadone (10 mg/kg) was given. Bile was collected through the cannulated bile duct over a period of 24 hours. The deuterium label was found to be stable even under severe conditions of incubation temperature and time. SIM analysis of bile sample extracts showed that concomitant administration of diazepam with methadone did not affect biliary excretion of EDDP nor the conjugated metabolites. This indicates that diazepam does not interact with methadone at the hepatic metabolism level and with transport of the metabo-: lites by the biliary excretion route. Application of the use of a biosynthetic internal standard to drug metabolism and pharmacokinetic studies by means of ratio analysis was described with examples.