Received for publication December 22, 2008.
Revised May 6, 2009.
Accepted for publication May 15, 2009.

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

Abstract

In vitro biosynthesis using pooled human liver microsomes was applied to help identify in vivo metabolites of ketamine by LC-MS/MS. Microsomal synthesis produced dehydronorketamine, seven structural isomers of hydroxynorketamine, and at least five structural isomers of hydroxyketamine. To aid identification, stable isotopes of the metabolites 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 spectra indicating the presence of a phenolic group, phenolic metabolites being further substantiated by selective liquid-liquid extraction following adjustments to the pH. Two glucuronide conjugates of hydroxynorketamine were also identified. Analysis by LC-coupled ion cyclotron resonance mass spectrometry gave unique masses in accordance with the predicted elemental composition. The metabolites, including the phenols, were subsequently confirmed to be present in urine of subjects following oral ketamine administration, as facilitated by the addition of deuterated metabolites generated from the in vitro biosynthesis. To our knowledge, phenolic metabolites of ketamine are here reported for the first time, including an intact glucuronide conjugate. The use of biologically-synthesized deuterated material for use as an internal chromatographic and mass spectrometric marker is a viable approach to aid the identification of metabolites. Those metabolites that are particularly of diagnostic value can be selected as candidates for chemical synthesis of standards.

Key words: drug analysis, mass spectrometry, metabolite identification, microsomes