Use of Human Microsomes and Deuterated Substrates: An Alternative Approach for the Identification of Novel Metabolites of Ketamine by Mass Spectrometry

In vitro biosynthesis using pooled human liver microsomes was applied to help identify in vivo metabolites of ketamine by liquid chromatography (LC)-tandem mass spectrometry. Microsomal synthesis produced dehydronorketamine, seven structural iso-mers of hydroxynorketamine, and at least five structural isomers of hydroxyketamine. To aid identification, stable isotopes of the me-tabolites were also produced from tetra-deuterated isotopes of ketamine or norketamine as substrates. Five metabolites (three hydroxynorketamine and two hydroxyketamine isomers) gave chromatographically resolved components with product ion spec-tra indicating the presence of a phenolic group, with phenolic metabolites being further substantiated by selective liquid-liquid extraction after adjustments to the pH. Two glucuronide conju-gates of hydroxynorketamine were also identified. Analysis by LC-coupled ion cyclotron resonance mass spectrometry gave uniqu