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

Abstract

ABSTRACT: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 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, with phenolic metabolites being further substantiated by selective liquid-liquid extraction after adjustments to the pH. Two glucuronide conjugates of hydroxynorketamine were also identified. Analysis by LCcoupled 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 after oral ketamine administration, as facilitated by the addition of deuterated metabolites generated from the in vitro biosynthesis. To our knowledge, phenolic metabolites of ketamine, including an intact glucuronide conjugate, are here reported for the first time. The use of biologically synthesized deuterated material as an internal chromatographic and mass spectrometric marker is a viable approach to aid in the identification of metabolites. Metabolites that have particular diagnostic value can be selected as candidates for chemical synthesis of standards.

Drug Pharmacokinetics Determined by Real‐Time Analysis of Mouse Breath

“…Furthermore,f ragment ions formed from the [M + H] + ion of Ket (Figure 1c) matched those previously reported, [11] and fragments obtained from Ket standard (Figure S2 a). Not only Ket, but also its main metabolites could all be detected with our setup.…”

Glucuronide Production by Whole-Cell Biotransformation Using Genetically Engineered Fission YeastSchizosaccharomyces pombe

“…In the first approach, a labeled aglycon served as a substrate, as in a recent study where pooled human liver microsomes were used for a similar biotransformation (Turfus et al, 2009). In this case, 4-fold deuterated T was successfully glucuronidated by strain DB53 (Fig.…”

A data-independent acquisition workflow for qualitative screening of new psychoactive substances in biological samples

“…However, it has also been used as a new psychoactive substance since the 1960s [25,26] and several intoxication cases have been reported [25,27]. Some research groups have studied the in vitro metabolism of KET [28,27] and identified two major metabolites: norketamine (NK) and dehydronorketamine (DHNK). Additionally, these studies [28,27] found several isomers of hydroxyketamine (HK) and hydroxynorketamine (HNK).…”

“…Some research groups have studied the in vitro metabolism of KET [28,27] and identified two major metabolites: norketamine (NK) and dehydronorketamine (DHNK). Additionally, these studies [28,27] found several isomers of hydroxyketamine (HK) and hydroxynorketamine (HNK). However, the confirmation of these metabolites was not done by AMMS.…”

“…At this point, reference standards for the isomers of HNK were not commercially available; however by including chemical formulae of metabolites from the literature into our library we were able to tentatively identify six isomers of HNK (Table 2) in one of the serum samples. The tentative identification of the three detected isomers HNK 1, 2 and 3 ( Fig SI-1 and Fig SI-2) was facilitated by the elucidation of fragmentation pathways of the qualified product ions, by the possible isomeric structures sourced from the literature [28,29] and by in silico predictions from Nexus software.…”