Direct Quantification of Ethyl Glucuronide in Clinical Urine Samples by Liquid Chromatography–Mass Spectrometry

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Abstract
Ethyl glucuronide is a minor metabolite of ethanol, and its presence in urine can be used as a laboratory test to detect recent alcohol intake, even for some time after the ethanol is no longer measurable. A simple analytical procedure was developed based on direct injection of urine diluted with a deuterated internal standard into an electrospray liquid chromatographic–mass spectrometric (LC-MS) system. A novel LC system using a porous graphite column (Hypercarb) enabled an isocratic elution with retention times of 5–6 minutes. The intra- and inter-assay coefficients of variation were 2–12%, and the measuring range was 0.1–1,500 mg/L (0.45–6,750 μmol/L). Ethyl glucuronide was found to be stable in urine for more than 4 days at room temperature, and no artifactual formation was observed on storage of urine samples fortified with 1% ethanol. Ethyl glucuronide was not detected in urine samples collected after abstinence from alcohol. Intake of a very low amount (7 g) of ethanol produced ethyl glucuronide values up to 8.4 mg/L after 4 hours and was still detectable at 6 hours. When the method was applied for routine screening of 252 clinical urine samples (range, 0–1,240 mg/L), it fulfilled the need for a simple and reliable assay to be used in the evaluation of urinary ethyl glucuronide as a routine test of recent alcohol intake.