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

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

Research output: Contribution to journalArticle

36 Citations (Scopus)

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.

LanguageEnglish
Pages1769-1778
Number of pages10
JournalDrug Metabolism and Disposition
Volume37
Issue number8
DOIs
Publication statusPublished - 1 Aug 2009
Externally publishedYes

Fingerprint

Ketamine
Microsomes
Mass Spectrometry
Glucuronides
Liquid Chromatography
Isotopes
Ions
Cyclotrons
Liquid-Liquid Extraction
Phenols
Liver Microsomes
Tandem Mass Spectrometry
Oral Administration
Urine
norketamine
In Vitro Techniques

Cite this

Turfus, Sophie C. ; Parkin, Mark C. ; Cowan, David A. ; Halket, John M. ; Smith, Norman W. ; Braithwaite, Robin A. ; Elliot, Simon P. ; Steventon, Glyn B. ; Kicman, Andrew T. / Use of human microsomes and deuterated substrates : An alternative approach for the identification of novel metabolites of ketamine by mass spectrometry. In: Drug Metabolism and Disposition. 2009 ; Vol. 37, No. 8. pp. 1769-1778.
@article{75d06f13b2e94bfa97eb84f570ccbf80,
title = "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 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.",
author = "Turfus, {Sophie C.} and Parkin, {Mark C.} and Cowan, {David A.} and Halket, {John M.} and Smith, {Norman W.} and Braithwaite, {Robin A.} and Elliot, {Simon P.} and Steventon, {Glyn B.} and Kicman, {Andrew T.}",
year = "2009",
month = "8",
day = "1",
doi = "10.1124/dmd.108.026328",
language = "English",
volume = "37",
pages = "1769--1778",
journal = "Drug Metabolism and Disposition",
issn = "0090-9556",
publisher = "American Society for Pharmacology and Experimental Therapeutics",
number = "8",

}

Use of human microsomes and deuterated substrates : An alternative approach for the identification of novel metabolites of ketamine by mass spectrometry. / Turfus, Sophie C.; Parkin, Mark C.; Cowan, David A.; Halket, John M.; Smith, Norman W.; Braithwaite, Robin A.; Elliot, Simon P.; Steventon, Glyn B.; Kicman, Andrew T.

In: Drug Metabolism and Disposition, Vol. 37, No. 8, 01.08.2009, p. 1769-1778.

Research output: Contribution to journalArticle

TY - JOUR

T1 - Use of human microsomes and deuterated substrates

T2 - Drug Metabolism and Disposition

AU - Turfus, Sophie C.

AU - Parkin, Mark C.

AU - Cowan, David A.

AU - Halket, John M.

AU - Smith, Norman W.

AU - Braithwaite, Robin A.

AU - Elliot, Simon P.

AU - Steventon, Glyn B.

AU - Kicman, Andrew T.

PY - 2009/8/1

Y1 - 2009/8/1

N2 - 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.

AB - 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.

UR - http://www.scopus.com/inward/record.url?scp=67650783182&partnerID=8YFLogxK

U2 - 10.1124/dmd.108.026328

DO - 10.1124/dmd.108.026328

M3 - Article

VL - 37

SP - 1769

EP - 1778

JO - Drug Metabolism and Disposition

JF - Drug Metabolism and Disposition

SN - 0090-9556

IS - 8

ER -