Skip to main page content
U.S. flag

An official website of the United States government

Dot gov

The .gov means it’s official.
Federal government websites often end in .gov or .mil. Before sharing sensitive information, make sure you’re on a federal government site.

Https

The site is secure.
The https:// ensures that you are connecting to the official website and that any information you provide is encrypted and transmitted securely.

Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
Clinical Trial
. 2009 Aug;37(8):1769-78.
doi: 10.1124/dmd.108.026328. Epub 2009 May 15.

Use of human microsomes and deuterated substrates: an alternative approach for the identification of novel metabolites of ketamine by mass spectrometry

Sophie C Turfus  1 , Mark C Parkin, David A Cowan, John M Halket, Norman W Smith, Robin A Braithwaite, Simon P Elliot, Glyn B Steventon, Andrew T Kicman
Affiliations
Clinical Trial

Use of human microsomes and deuterated substrates: an alternative approach for the identification of novel metabolites of ketamine by mass spectrometry

Sophie C Turfus et al. Drug Metab Dispos. 2009 Aug.

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 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 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.

PubMed Disclaimer

Figures

F<sc>ig</sc>. 1.
Fig. 1.
Extracted ion chromatograms of selected metabolites obtained from microsomal incubates: ketamine (m/z 238), norketamine (m/z 224), hydroxynorketamine (m/z 240), tetradeuterated hydroxynorketamine (m/z 244), trideuterated hydroxynorketamine (m/z 243), hydroxyketamine (m/z 254), tetradeuterated hydroxyketamine (m/z 258), trideuterated hydroxyketamine (m/z 257), and dehydronorketamine (m/z 222). Dotted lines are drawn through the peaks discussed in the main text.
F<sc>ig</sc>. 2.
Fig. 2.
MS/MS spectra obtained from fragmenting alcoholic or phenolic hydroxynorketamine. 35Cl and 37Cl isotopes were targeted and used to help elucidate metabolite structure. a and b, typical spectra produced, respectively, for alcoholic and phenolic metabolites from 35Cl isotopes. c and d, fragmentation of the 37Cl isotopes. Prominent fragments greater than 5% of the intensity of the base peak are labeled or ions that become dominant at other collision energies.
F<sc>ig</sc>. 3.
Fig. 3.
Postulated fragments for the MS/MS spectra shown in Fig. 2. a, possible structures of fragments of alcoholic isomers of the hydroxylated metabolites. b, possible structures for phenolic metabolites. Of importance is m/z 125 (alcohols) versus m/z 141 (phenols) and m/z 222 (alcohols) compared with m/z 223 (phenols) as shown. Note that the benzyl group is drawn as a tropylium in this figure and elsewhere although no evidence of a structure has been determined (McLafferty and Winkler, 1974).
F<sc>ig</sc>. 4.
Fig. 4.
Chromatograms of a urine sample spiked with deuterated metabolites before extraction, showing isomers of hydroxynorketamine. a, superimposed chromatograms for the transitions m/z 240/125 and 244/129 for alcoholic hydroxynorketamine and hydroxynorketamine-d4 showing overlap at peaks 1 to 3 and 7. b, superimposed chromatograms for the transitions m/z 240/141 and 243/144 for phenolic hydroxynorketamine and hydroxynorketamine-d3 showing overlap at peaks 4 to 6.
See this image and copyright information in PMC

References

    1. Adamowicz P and Kala M (2005) Urinary excretion rates of ketamine and norketaine following therapeutic ketamine administration: method and detection window consideration. J Anal Toxicol 29 376–382. - PubMed
    1. Adams JD Jr, Baillie TA, Trevor AJ, and Castagnoli N Jr (1981) Studies on the biotransformation of ketamine. 1–Identification of metabolites produced in vitro from rat liver microsomal preparations. Biomed Mass Spectrom 8 527–538. - PubMed
    1. Anari MR, Burton RW, Gopaul S, and Abbott FS (2000) Metabolic profiling of valproic acid by cDNA-expressed human cytochrome P450 enzymes using negative-ion chemical ionization gas chromatography-mass spectrometry. J Chromatogr B Biomed Sci Appl 742 217–227. - PubMed
    1. Apollonio LG, Pianca DJ, Whittall IR, Kyd JM, and Maher WA (2006) A comparison of atmospheric pressure chemical ionization and electrospray ionization in testing for amphetamine-type substances and ketamine using ultra-performance liquid chromatography/mass spectrometry. Rapid Commun Mass Spectrom 20 2777–2780. - PubMed
    1. Arnell R, Johannisson R, Lindholm J, Fornstedt T, Ersson B, Ballagi A, and Caldwell K (2007) Biotechnological approach to the synthesis of 9α-hydroxylated steroids. Prep Biochem Biotechnol 37 309–321. - PubMed

Publication types

MeSH terms

LinkOut - more resources