Inhibition of bacterial degradation of EtG by collection as dried urine spots (DUS) |
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Authors: | Redondo Ana Hernández Körber Christiane König Stefan Längin Andreas Al-Ahmad Ali Weinmann Wolfgang |
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Institution: | (1) Institute of Forensic Medicine, Faculty of Medicine, University of Bern, B?hlstrasse 20, Bern, 3012, Switzerland;(2) Department of Operative Dentistry and Periodontology, University Medical Centre Freiburg, Hugstetterstrasse 55, 79106 Freiburg, Germany |
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Abstract: | Ethyl glucuronide (EtG) and ethyl sulfate (EtS) are direct alcohol consumption markers widely used nowadays for clinical and
forensic applications. They are detectable in blood and urine even after consumption of trace amounts of ethanol and for a
longer time frame, being detectable even when no more ethanol is present. The instability of EtG against bacterial degradation
in contaminated urine samples and/or the possible postcollection synthesis of this metabolite in samples containing, e.g.,
Escherichia coli and ethanol, may cause false identification of alcohol uptake. Therefore, it is of paramount importance to constrict these
error sources by inhibition of any bacterial growth causing hydrolization or synthesis of EtG. This study evaluates a new
method of collecting urine samples on filter paper, dried urine spots (DUS), for simultaneous detection of EtG, EtS and creatinine,
having the great advantage of inhibiting bacterial activity. In addition, a method validation for the determination of EtG
and EtS in DUS was performed according to the FDA guidelines. Sterile-filtered urine was spiked with EtG and EtS, inoculated
with E. coli and incubated. Liquid and dried urine samples were collected after various time intervals up to 96 h. Liquid samples were
frozen immediately after collection, whereas aliquots for DUS were pipetted onto filter paper, allowed to dry and stored at
RT until analysis 1 week after. The specimens were analyzed by LC–ESI–MS/MS. As expected, degradation of EtG, but not of EtS,
was observed in contaminated liquid urine samples. However, the specimens collected on filter paper and stored at RT showed
no degradation during storage. Therefore, collecting urine samples on filter paper for EtG and EtS analysis turns out to be
a reliable method to avoid bacterial degradation of EtG and EtS, and consequently, stabilization of these ethanol metabolites
is achieved. In addition, simultaneous measurement of creatinine content as an indicator of urine dilution helps to interpret
the results. Method validation for EtG and EtS in DUS was satisfactory, showing the linearity of the calibration curves in
the studied concentration range, good precision, accuracy and selectivity. |
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