Generic method for the absolute quantification of glutathione S-conjugates: Application to the conjugates of acetaminophen, clozapine and diclofenac

Research output: Contribution to JournalArticleAcademicpeer-review

Abstract

Modification of cellular macromolecules by reactive drug metabolites is considered to play an important role in the initiation of tissue injury by many drugs. Detection and identification of reactive intermediates is often performed by analyzing the conjugates formed after trapping by glutathione (GSH). Although sensitivity of modern mass spectrometrical methods is extremely high, absolute quantification of GSH-conjugates is critically dependent on the availability of authentic references. Although 1H NMR is currently the method of choice for quantification of metabolites formed biosynthetically, its intrinsically low sensitivity can be a limiting factor in quantification of GSH-conjugates which generally are formed at low levels. In the present study, a simple but sensitive and generic method for absolute quantification of GSH-conjugates is presented. The method is based on quantitative alkaline hydrolysis of GSH-conjugates and subsequent quantification of glutamic acid and glycine by HPLC after precolumn derivatization with o-phthaldialdehyde/N-acetylcysteine (OPA/NAC). Because of the lower stability of the glycine OPA/NAC-derivate, quantification of the glutamic acid OPA/NAC-derivate appeared most suitable for quantification of GSH-conjugates. The novel method was used to quantify the concentrations of GSH-conjugates of diclofenac, clozapine and acetaminophen and quantification was consistent with 1H NMR, but with a more than 100-fold lower detection limit for absolute quantification.

Original languageEnglish
Pages (from-to)185-194
Number of pages10
JournalJournal of Chromatography B
Volume1046
DOIs
Publication statusPublished - 1 Mar 2017

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o-Phthalaldehyde
Diclofenac
Clozapine
Acetylcysteine
Acetaminophen
Glutathione
Metabolites
Glycine
Glutamic Acid
Nuclear magnetic resonance
Macromolecules
Pharmaceutical Preparations
Hydrolysis
Availability
Tissue
Limit of Detection
High Pressure Liquid Chromatography
Wounds and Injuries

Keywords

  • Acetaminophen
  • Alkaline hydrolysis
  • Clozapine
  • Diclofenac
  • Glutathione S-conjugates
  • O-phthaldialdehyde

Cite this

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title = "Generic method for the absolute quantification of glutathione S-conjugates: Application to the conjugates of acetaminophen, clozapine and diclofenac",
abstract = "Modification of cellular macromolecules by reactive drug metabolites is considered to play an important role in the initiation of tissue injury by many drugs. Detection and identification of reactive intermediates is often performed by analyzing the conjugates formed after trapping by glutathione (GSH). Although sensitivity of modern mass spectrometrical methods is extremely high, absolute quantification of GSH-conjugates is critically dependent on the availability of authentic references. Although 1H NMR is currently the method of choice for quantification of metabolites formed biosynthetically, its intrinsically low sensitivity can be a limiting factor in quantification of GSH-conjugates which generally are formed at low levels. In the present study, a simple but sensitive and generic method for absolute quantification of GSH-conjugates is presented. The method is based on quantitative alkaline hydrolysis of GSH-conjugates and subsequent quantification of glutamic acid and glycine by HPLC after precolumn derivatization with o-phthaldialdehyde/N-acetylcysteine (OPA/NAC). Because of the lower stability of the glycine OPA/NAC-derivate, quantification of the glutamic acid OPA/NAC-derivate appeared most suitable for quantification of GSH-conjugates. The novel method was used to quantify the concentrations of GSH-conjugates of diclofenac, clozapine and acetaminophen and quantification was consistent with 1H NMR, but with a more than 100-fold lower detection limit for absolute quantification.",
keywords = "Acetaminophen, Alkaline hydrolysis, Clozapine, Diclofenac, Glutathione S-conjugates, O-phthaldialdehyde",
author = "{den Braver}, {Michiel W.} and Vermeulen, {Nico P.E.} and Commandeur, {Jan N.M.}",
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T1 - Generic method for the absolute quantification of glutathione S-conjugates

T2 - Application to the conjugates of acetaminophen, clozapine and diclofenac

AU - den Braver, Michiel W.

AU - Vermeulen, Nico P.E.

AU - Commandeur, Jan N.M.

PY - 2017/3/1

Y1 - 2017/3/1

N2 - Modification of cellular macromolecules by reactive drug metabolites is considered to play an important role in the initiation of tissue injury by many drugs. Detection and identification of reactive intermediates is often performed by analyzing the conjugates formed after trapping by glutathione (GSH). Although sensitivity of modern mass spectrometrical methods is extremely high, absolute quantification of GSH-conjugates is critically dependent on the availability of authentic references. Although 1H NMR is currently the method of choice for quantification of metabolites formed biosynthetically, its intrinsically low sensitivity can be a limiting factor in quantification of GSH-conjugates which generally are formed at low levels. In the present study, a simple but sensitive and generic method for absolute quantification of GSH-conjugates is presented. The method is based on quantitative alkaline hydrolysis of GSH-conjugates and subsequent quantification of glutamic acid and glycine by HPLC after precolumn derivatization with o-phthaldialdehyde/N-acetylcysteine (OPA/NAC). Because of the lower stability of the glycine OPA/NAC-derivate, quantification of the glutamic acid OPA/NAC-derivate appeared most suitable for quantification of GSH-conjugates. The novel method was used to quantify the concentrations of GSH-conjugates of diclofenac, clozapine and acetaminophen and quantification was consistent with 1H NMR, but with a more than 100-fold lower detection limit for absolute quantification.

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