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Validated method for the determination of ethylglucuronide and ethylsulfate in human urine

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Abstract

Detection of the alcohol metabolites ethylglucuronide (EtG) and ethylsulfate (EtS) has become routine in many forensic laboratories over the last few years. Most previously published methods using liquid chromatography coupled with electrospray tandem mass spectrometry require a post-chromatographic addition of solvent and/or extensive sample preparation prior to analysis. The aim of the study was to develop a simplified method. To 20 μL urine, internal standard containing EtG-d5 and EtS-d 5 was added and the mixture was treated with elution buffer internal standard. EtG and EtS were separated using a Shimadzu Prominence high performance liquid chromatography (HPLC) system with a C18 separation column (Restek Ultra Aqueous C18, 4.6 × 150 mm, 5 μm), using isocratic elution with a mobile phase consisting of 10 mM ammonium acetate buffer pH 7 (total run time, 6 min). The compounds were detected using an Applied Biosystems API 5000 liquid chromatography tandem mass spectrometry system (atmospheric pressure chemical ionization, multiple-reaction monitoring mode). The method was fully validated according to international guidelines. The assay was found to be selective for the compounds of interest. It was linear from 0.1 to 10 mg/L for all analytes (R 2 > 0.99). Matrix effects studies showed the presence of a slight but consistent ion enhancement (n = 10 different urine samples) at low concentrations and no effects at higher concentrations. Accuracy data were between 0.75% and 8.1% bias for EtG and between −5.0% and −11.3% bias for EtS. Precision data were between 4.3% and 6.9% relative standard deviations (RSD) for EtG and between 6.0% and 7.5% RSD for EtS. No instability was observed after repeated freezing and thawing. This fast, reliable, and accurate method enables the detection and quantification of alcohol metabolites in urine. The method is easier to use and more sensitive than previously published methods.

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Authors and Affiliations

  1. Department of Forensic Medicine, Victorian Institute of Forensic Medicine, Monash University, 57-83 Kavanagh St, Southbank, Melbourne, Victoria, 3006, Australia

    Jochen Beyer, Tu N. Vo, Dimitri Gerostamoulos & Olaf H. Drummer

  2. Department of Forensic Medicine, Monash University, Southbank, Victoria, 3006, Australia

    Jochen Beyer, Dimitri Gerostamoulos & Olaf H. Drummer

Authors
  1. Jochen Beyer
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  2. Tu N. Vo
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  3. Dimitri Gerostamoulos
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  4. Olaf H. Drummer
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Correspondence to Jochen Beyer.

Additional information

Published in the special issue Forensic Toxicology with Guest Editors Frank T. Peters, Hans H. Maurer, and Frank Musshoff.

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Beyer, J., Vo, T.N., Gerostamoulos, D. et al. Validated method for the determination of ethylglucuronide and ethylsulfate in human urine. Anal Bioanal Chem 400, 189–196 (2011). https://doi.org/10.1007/s00216-011-4667-2

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  • DOI: https://doi.org/10.1007/s00216-011-4667-2

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