Methodology for the identification of the urinary metabolites of the analgesic-narcotic antagonist, codorphone, by gas chromatography mass spectrometry

Methodology is presented for the identification of codorphone and its metabolites in urine samples using gas chromatography mass spectrometry. The procedure focuses on the clean-up of biological samples and a derivatization technique suitable for these samples. Sep-Pak C-18 cartridges were employed in the clean-up procedure permitting the biological sample to be derivatized in a relatively small volume of reagents. The derivatization procedure incorporated a one-step trimethylsilyloxime reaction to prevent enol formation while simultaneously derivatizing free hydroxyl groups with the excess trimethylsilylimidazole present in the reaction mixture. This was followed by the addition of BSTFA directly to this reaction mixture to complete derivatization of any metabolites possessing dealkylation of the nitrogen. Using this derivatization scheme, synthetic metabolites were analyzed by gas chromatography mass spectrometry, and their mass spectra were characterized emphasizing the diagnostic fragment ions observed in the spectra. To illustrate the usefulness of this methodology, a urine sample obtained from a dog that had been dosed with codorphone was analyzed by gas chromatography mass spectrometry, and the metabolites were identified by comparison to the mass spectra of the synthetic derivatives.