Stereoselective Quantification of Methadone and a d6-labeled Isotopomer Using High Performance Liquid Chromatography-Atmospheric Pressure Chemical Ionization Mass-Spectrometry: Application to a Pharmacokinetic Study in a Methadone Maintained Subject

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There is evidence that the apparent oral clearance of rac-methadone is induced during the early phase of methadone maintenance treatment. However, it is not known if this is due to changes in bioavailability or if this phenomenon is stereoselective. This knowledge can be obtained by administering a dose of stable-labeled methadone at selected times during ongoing treatment. Therefore, the authors developed a stereoselective high performance liquid chromatography-atmospheric pressure chemical ionization mass-spectrometry assay for the quantification of the enantiomers of methadone and a d6-labeled isotopomer. The compounds were quantified in a single assay after liquid-liquid extraction and stereoselective high performance liquid chromatograph with atmospheric pressure chemical ionization-mass spectrometry detection. The following ions were monitored: m/z 310.15 for unlabeled methadone; m/z 316.15 for methadone-d6; and m/z 313.15 for the methadone-d3 (internal standard). Calibration curves ranged from 0.5 to 75 ng/mL for each compound. Extraction recovery was approximately 80% for all analytes, without evidence of differences between the unlabeled and stable-labeled compounds or concentration dependency. Minor ion promotion was observed (<15%) but this was identical for all analytes including the d3-labeled internal standard, with peak area ratios in extracted samples identical to control injections. The isotopomers did not alter each others’ ionisation, even at 10:1 concentration ratios, and 10-fold diluted samples were within 10% of the nominal concentration. Assay performance was acceptable, with interassay and intra-assay bias and precision <10% for all compounds, including the upper and lower limits of quantitation. In conclusion, the assay was successfully applied to quantify the concentration of the methadone enantiomers of both orally administered unlabeled methadone and an intravenous 5 mg dose of methadone-d6 in a patient receiving chronic oral methadone maintenance therapy.

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