SOFT 2004 Meeting Abstracts - Society of Forensic Toxicologists

A60
THE DETERMINATION OF ETHYL GLUCURONIDE IN URINE USING REVERSED-PHASE
HPLC AND PULSED ELECTROCHEMICAL DETECTION
Romina Kaushik*, Barry Levine and William R. LaCourse
University ofMaryland, Baltimore, Maryland, USA
There is a need for a method to distinguish between ethanol levels detected in biological matrices due to
alcohol consumption versus ethanol production after death as a result of decomposition. Ethyl glucuronide
(EtG) is a non-volatile, water-soluble, direct metabolite of ethanol that can serve as a biological marker of
alcohol consumption. This metabolite can be a marker of acute alcohol consumption even at low levels
unlike traditional biomarkers. It is an intermediate marker of alcohol consumption, bridging the gap
between long-term (COT, MCV & GGT) and very short-term (ethanol & HTOL) biomarkers. EtG is a
highly sensitive and specific alcohol consumption marker that can be detected for up to 80 hours after
complete alcohol elimination from the body. This has very important clinical and forensic applications.
Clinical applications for this biological marker would include monitoring patients in treatment for alcohol
abuse. This marker could improve therapy outcome and quality of life in patients by preventing relapse
episodes. Furthermore monitoring would increase safety in the workplace and prevent traffic accidents.
Finally, fetal alcohol syndrome could be avoided by detecting and monitoring EtG in the mother which
would reduce costs by making therapy more effective. In addition, the metabolite exhibits very high storage
stability which is important as forensic samples are often stored for extended periods of time. Sometimes
samples are not analyzed until the case goes to court which could take months or years. In previous studies
there have been various methods used to detect EtG. These include gas chromatography (GC) coupled with
mass spectrometry (MS), and liquid chromatography (LC) coupled with MS. GC/MS is available at almost
all forensic facilities at a moderate cost. However, GC·MS detection of this metabolite requires prior
derivatization. LC/MS is advantageous because it doesn't require derivatization nonetheless it is an
expensive technique. For widespread use of EtG as a marker, simpler and less expensive methods are
necessary.
The current study involved developing a method for the detection of EtG in postmortem urine samples
using reversed-phase liquid chromatography with pulsed electrochemical detection. Methyl glucuronide
served as the internal standard. The mobile phase consisted of 1% acetic acid/water and acetonitrile (98:2),
with a 600mM sodium hydroxide post-column system attached to enable pulsed electrochemical detection
(PED). This amperometric detection technique applies alternated positive and negative potential pulses at a
noble metal electrode. The analyte is oxidized followed by oxidative and reductive cleaning steps. The
analyte concentration is determined by measuring the electric current resulting from the molecule gaining
or losing electrons. In order to separate EtG from the biological matrix a solid-phase extraction (SPE) was
used using aminopropyl columns. EtG was found to have a retention time of 5.3 minutes with LOQ and
LOD values of 0.4 and 0.1 uglmL respectively. The extraction recovery following SPE was approximately
50%. This method is specific, reproducible and sensitive. Reversed-phase chromatography enabled a
simple separation of the analyte without requiring the ion-pairing reagents typically associated with ion
chromatography. PED is a direct (no derivatization) and affordable detection method. This method is a
potential tool to clinical and forensic toxicologists for determining alcohol consumption in live and
deceased individuals.
Keywords: Alcohol, Biomarker, Direct and affordable analysis
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