Application of high-performance liquid chromatography/chemical reaction interface mass spectrometry for the analysis of conjugated metabolites: a demonstration using deuterated acetaminophen.

The combination of a universal high-performance liquid chromatography/mass spectrometry (HPLC/MS) interface (UI) and the element and isotope-selective capabilities of the chemical reaction interface (CRI) has potential as a comprehensive analysis system for drug conjugates. In this work, we found equal sensitivity for model compounds as their sulfate or glucuronide conjugates. We examined urine and bile samples from Syrian golden hamsters after dosing with (2H4)acetaminophen (D4-APAP), with particular emphasis on the rich range of conjugated metabolites that are known to be produced. Seventeen metabolites were quantified from a single chromatogram of urine; 14 were conjugates. With a combination of authentic standards, selective hydrolysis, and sulfur-selective CRIMS detection, at least partial identification of most of these metabolites was accomplished. The glutathione conjugate of APAP appears the dominant metabolite in bile. The quantitative pattern of APAP metabolism found here is consistent with literature values. It does appear that this HPLC/UI/CRIMS combination has substantial ability to carry out comprehensive metabolite determinations, especially for conjugated species.