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Methadone is often invoked for detoxification and maintenance of the opioid addict. We have developed and validated a sensitive and specific method for the analysis of methadone and its metabolites, 2-ethylidene-1,5-dimethyl-3, 3-diphenylpyrrolidine (EDDP) and 2-ethyl-5-methyl-3,3-diphenylpyrroline (EMDP), in human plasma, urine, and liver microsomes. This assay uses a solid-phase extraction. Separation and analysis of the analytes are performed by capillary gas chromatography-positive ion chemical ionization-mass spectrometry. The protonated molecules (MH+) are monitored at m/z 264 and 267 for EMDP-d0 and -d3, m/z 278 and 281 for EDDP-d0 and -d3, and m/z 310 and 313 for methadone-d0 and -d3. The recovery of methadone and its metabolites exceeded 85% in the different matrices analyzed. Linear standard curves in plasma and in urine were obtained over the concentration range of 10-600 ng/mL (coefficients of determination: methadone, &gt; or = 0.995; EMDP, &gt; or = 0.994; and EDDP, &gt; or = 0.996). With plasma and urine fortified at 25, 100, and 300 ng/mL, the assay was precise (intra-assay coefficients of variation [CVs], 2-12%; interassay CVs, 1-15%) and accurate (intra-assay percent of target, 85-107; interassay percent of target, 88-105) for all three analytes. Stability studies indicated that methadone and its metabolites are stable at room temperature in plasma and in urine for at least 1 week and in liver microsomes for at least 24 h. This method has now been shown to be useful for quantitation of methadone, EDDP, and EMDP in human urine and plasma and is also useful for quantitation of the amount of EDDP produced in human liver microsomes incubated with methadone. It provides an accurate and precise analytical tool for further studies on the metabolism of methadone.

Determination of Methadone and its N-Demethylation Metabolites in Biological Specimens by GC-PICI-MS