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Introduction of sample tubes with sodium azide as a preservative for ethyl glucuronide in urine

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Abstract

Ethyl glucuronide (EtG) is a direct alcohol marker, which is widely used for clinical and forensic applications, mainly for abstinence control. However, the instability of EtG in urine against bacterial degradation or the post-collectional synthesis of EtG in contaminated samples may cause false interpretation of EtG results in urine samples. This study evaluates the potential of sodium azide in tubes used for urine collection to hinder degradation of ethyl glucuronide by bacterial metabolism taking place during growth of bacterial colonies. The tubes are part of a commercial oral fluid collection device. The sampling system was tested with different gram-positive and gram-negative bacterial species previously observed in urinary tract infections, such as Escherichia coli, Staphylococcus aureus, Enterecoccus faecalis, Staphylococcus epidermidis, Klebsiella pneumoniae, Enterobacter cloacae, and Pseudomonas aeruginosa. Inhibition of bacterial growth by sodium azide, resulting in lower numbers of colony forming units compared to control samples, was observed for all tested bacterial species. To test the prevention of EtG degradation by the predominant pathogen in urinary tract infection, sterile-filtered urine and deficient medium were spiked with EtG, and inoculated with E. coli prior to incubation for 4 days at 37 °C in tubes with and without sodium azide. Samples were collected every 24 hours, during four consecutive days, whereby the colony forming units (CFU) were counted on Columbia blood agar plates, and EtG was analyzed by LC-MS/MS. As expected, EtG degradation was observed when standard polypropylene tubes were used for the storage of contaminated samples. However, urine specimens collected in sodium azide tubes showed no or very limited bacterial growth and no EtG degradation. As a conclusion, sodium azide is useful to reduce bacterial growth of gram-negative and gram-positive bacteria. It inhibits the degradation of EtG by E. coli and can be used for the stabilization of EtG in urine samples.

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Acknowledgements

We would like to thank the Group for Oral Microbiology in Freiburg, Germany and the Team of Forensic Toxicology and Chemistry at the Institute of Forensic Medicine Bern, Switzerland. Furthermore, we would like to extend our gratitude to Michaela Brcak from Greiner Bio-One for providing the saliva transfer tubes from the saliva collection system.

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Authors and Affiliations

  1. Institute of Forensic Medicine, University of Bern, Bühlstrasse 20, 3012, Bern, Switzerland

    Marc Luginbühl & Wolfgang Weinmann

  2. Department of Operative Dentistry and Periodontology, University Medical Centre Freiburg, Hugstetterstrasse 55, 79106, Freiburg, Germany

    Ali Al-Ahmad

Authors
  1. Marc Luginbühl
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  2. Wolfgang Weinmann
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  3. Ali Al-Ahmad
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Corresponding author

Correspondence to Wolfgang Weinmann.

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Informend consent was given for obtaining the blank urine sample. All procedures performed were in accordance with the ethical standards of the institutional and national research committee and with the 1964 Helsinki declaration and its later amendments or comparable ethical standards.

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The authors declare that they have no conflict of interest.

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Luginbühl, M., Weinmann, W. & Al-Ahmad, A. Introduction of sample tubes with sodium azide as a preservative for ethyl glucuronide in urine. Int J Legal Med 131, 1283–1289 (2017). https://doi.org/10.1007/s00414-017-1633-3

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  • DOI: https://doi.org/10.1007/s00414-017-1633-3

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