Microwave assisted extraction for the determination of ethyl glucuronide in urine by gas chromatography‐mass spectrometry

Abstract

Alcohol is the most frequently abused 'addictive substance' that causes serious social problems throughout the world; thus alcoholism is of particular interest in clinical and forensic medicine. Ethyl glucuronide (EtG) is a marker of recent alcohol consumption that detects alcohol use reliably over a definite time period. The present paper describes a new method for the determination of EtG in urine. It was based both on microwave assisted extraction (MAE) to extract the analyte from urine samples, and gas chromatography-mass spectrometry (GC-MS) to identify and quantify the EtG in selected ion monitoring (SIM) mode. The method was applied to 33 urine samples from alcohol users, obtaining positive results in all cases. It was fully validated including a linear range (0.1-100 microg ml(-1)) and the main precision parameters. In summary, the use of microwave assisted extraction turned out to be a substantially simpler, faster and more sensitive procedure than any other conventional sample preparations.

Comparison of analytical approaches for liquid chromatography/mass spectrometry determination of the alcohol biomarker ethyl glucuronide in urine

“…Quantitation of EtG is currently performed by a number of methods showing variable analytical sensitivity and specificity,29–31, 34, 41–43 but there is no consensus regarding a common cutoff (i.e., lower quantitation) limit for application in forensic or clinical decision making. In addition to analytical uncertainty in measurement (method imprecision), there is a risk for false‐positive identifications of alcohol ingestion attributable to unintentional ethanol exposure,24, 26–28, 44 if the EtG analysis focuses on maximum sensitivity (i.e., uses a very low LOQ).…”

Ethyl glucuronide

“…A comparison of five LC-MS methods for measurement of urinary EtG and EtS recommended that solid-phase extraction followed by liquid chromatography-tandem mass spectrometry (LC-MS/MS) be adopted as the reference method as it had the best selectivity and sensitivity. 41 Other methods developed include reversed-phase liquid chromatography with pulsed electrochemical detection, 42 microwave-assisted extraction followed by gas chromatography-mass spectrometry (GC-MS), 43,44 GC-MS with solidphase extraction for sweat samples 45 and GC-MS of silylated derivatives, 46 capillary electrophoresis, 47 capillary zone electrophoresis-mass spectrometry, 48 capillary isotachophoresis and zone electrophoresis 49 and an enzyme-linked immunosorbent assay based on polyclonal antibodies. 50 Recently, a commercially available enzyme immunoassay (EIA) method based on a new monoclonal antibody (DRI Ethyl Glucuronide Enzyme Immunoassay; Thermo Fisher Scientific Diagnostics, Hemel Hempstead, UK) came onto the market for the analysis of EtG in urine.…”

“…Other methods developed include reversed-phase liquid chromatography with pulsed electrochemical detection, 42 microwave-assisted extraction followed by gas chromatography-mass spectrometry (GC-MS), 43,44 GC-MS with solid-phase extraction for sweat samples 45 and GC-MS of silylated derivatives, 46 capillary electrophoresis, 47 capillary zone electrophoresis-mass spectrometry, 48 capillary isotachophoresis and zone electrophoresis 49 and an enzyme-linked immunosorbent assay based on polyclonal antibodies. 50…”

A High-Performance Liquid Chromatographic-Tandem Mass Spectrometric Method for the Determination of Ethyl Glucuronide and Ethyl Sulfate in Urine Validated According to Forensic Guidelines

“…Several methods exist to determine EtG and EtS in urine, blood, hair, and other species. For the analyses of urine and serum, the following analytical methods are used: gas chromatography-mass spectrometry (GC-MS) (13,14,16,31), liquid chromatography-mass spectrometry (LC -MS) (15,18,24,35,36), LC-multiple mass spectrometry (MS n ) (14, 19 -21, 25, 27, 30), LC with pulsed electrochemical detection (22,26), capillary zone electrophoresis (23,29,33,34), and immunochemical tests (17,28,32).…”

“…Major differences exist with regard to the applied extraction procedures. Besides simple dilutions or precipitations with methanol or acetonitrile for protein precipitation (13 -15, 17-21, 23 -25, 27, 29, 30, 32, 34), solid-phase extraction procedures (16,22,26,33,35,36) and extractions by microwaves (31) are used for the analysis of urine samples. Basic data for methods for the determination of EtG/EtS in urine and serum are summarized in Table I (13 -36).…”