Quantitative analysis of methadone and two major metabolites in hair by positive chemical ionization ion trap mass spectrometry.

A sensitive and specific method for the quantitative determination of D,L-methadone (MD) and its metabolites, D,L-2-ethyl-1,5-dimethyl-3, 3-diphenylpyrrolinium (EDDP) and D,L-2-ethyl-5-methyl-3, 3-diphenyl-1-pyrroline (EMDP), in hair has been developed. Deuterated internal standards of MD, EMDP, and EDDP were added to 20-mg hair samples and digested overnight at room temperature with 1N sodium hydroxide. Calibration standards containing known concentrations of MD, EMDP, and EDDP dried onto human hair were also digested. Digest solutions were extracted by a liquid-liquid extraction procedure and analyzed with splitless injection on a Finnigan MagnumTM ion trap mass spectrometer. Chromatographic separation was achieved with helium carrier gas on a DB-5MS-30M-0.25-micron capillary column. Positive chemicaionization was used with acetone as the reagent gas. The assay was linear from 0.5 ng/mg (MD and EDDP) or 1.0 ng/mg (EMDP) to 50.0 ng/mg of human hair with correlation coefficients greater than 0.99. Intra-assay and interassay coefficients of variation were determined to be less than 20% for all three analytes at 2.0 and 10.0 ng/mg of hair. Recovery was estimated to be greater than 70% (MD and EDDP) and 53% (EMDP) at 2.0 and 10.0 ng/mg of hair. The method has been applied to the analysis of both human and rat hair. Male long-Evans rats were shaved prior to dosing to obtain their drug-free hair. Animals were then administered 15 mg/kg MD by intraperitoneal injection daily for five days. Fourteen days after the first dose, hair was collected and analyzed for MD, EMDP, and EDDP. The mean plus standard error of the mean (SEM; n = 3) concentrations of MD and EDDP in pigmented hair were 31.1 ng/mg +/- 9.6 ng/mg and 8.6 +/- 2.4 ng/mg, respectively. EMDP was detected in the hair of one of three rats. In another experiment, hair was collected from two human subjects who had received long-term methadone therapy for the treatment of heroin addiction. Subject A received 60 mg of methadone daily for at least six months; subject B received 80 mg of methadone daily for at least six months. The hair concentrations of MD were 10.1 ng/mg and 21.0 ng/mg for Subjects A and B, respectively. The hair concentrations of EDDP were 0.5 ng/mg and 2.6 ng/mg for Subjects A and B, respectively. EMDP was not detected in the hair of these two subjects. This method is being used to evaluate the incorporation of MD, EMDP, and EDDP in human and rat hair in dose-response studies.