MICROWAVE ASSISTED EXTRACTION OF ETHYL GLUCURONIDE 773 Copyright © 2008 John Wiley & Sons, Ltd. J. Appl. Toxicol. 2008; 28: 773–778 DOI: 10.1002/jat JOURNAL OF APPLIED TOXICOLOGY J. Appl. Toxicol. 2008; 28: 773–778 Published online 14 March 2008 in Wiley InterScience (www.interscience.wiley.com) DOI: 10.1002/jat.1338 Microwave assisted extraction for the determination of ethyl glucuronide in urine by gas chromatography-mass spectrometry Iván Álvarez Freire, Ana María Bermejo Barrera,* Purificación Cid Silva, María Jesús Tabernero Duque, Purificación Fernández Gómez and Patricia López Eijo Institute of Legal Medicine, Forensic Toxicology Service, Faculty of Medicine, C/ San Francisco s/n, 15782 Santiago de Compostela, Spain Received 19 November 2007; Revised 17 December 2007; Accepted 4 January 2008 ABSTRACT: Alcohol is the most frequently abused ‘addictive substance’ that causes serious social problems through- out 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 quan- tify 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 μg ml -1 ) and the main pre- cision 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. Copyright © 2008 John Wiley & Sons, Ltd. KEY WORDS: ethyl glucuronide; microwave assisted extraction; gas chromatography-mass spectrometry * Correspondence to: Ana María Bermejo Barrera, Institute of Legal Medi- cine, Forensic Toxicology Service, Faculty of Medicine, C/ San Francisco s/n, 15782 Santiago de Compostela, Spain. E-mail: apimlana@usc.es identify high risk groups; (b) evaluate current treatment programs; (c) develop more effective treatment strategies; (d) disclose recent drinking in social drinkers in inappro- priate and risky situations; and (e) elucidate the role of neuropsychological impairment following alcohol intake (i.e. the hangover state). Detection of ethanol in body fluids is only possible during a relatively short time after alcohol consumption. About 90–95% of alcohol is eliminated by oxidation mainly in the liver, whereas biotransformation of ethanol to ethyl glucuronide (ethyl-β-D-6 glucuronic acid) (EtG), via conjugation with activated glucuronic acid, represents only 0.02–0.06% of complete alcohol elimination (Yegles et al., 2004; Wurst et al., 2003). Although the percentage of alcohol metabolized by this latter pathway is small, it represents a useful tool as EtG becomes detectable up to 4 days after complete elimination of alcohol from the body. With its specific time frame of detection, interme- diate between short- and long-term markers, and a high sensitivity and specificity (i.e. EtG is not detectable unless alcohol has been consumed), EtG is a promising marker of alcohol consumption in general and a marker for relapse detection (Wurst et al., 2002a; 2002b). Another advantage of EtG detection in urine is that obtaining a urine sample is far less invasive than taking a blood sample, and larger amounts can be available. Generally, some extraction methods have been used in forensic toxicology, such as liquid–liquid extraction, solid phase extraction or solid phase microextraction and so on Introduction Alcohol has traditionally been considered a serious pub- lic health problem because of several physical, mental and social problems associated with its abuse, as it exerts an enormous toll on the lives and communities of many nations (Politi et al., 2006). In fact, the Spanish 2005 National Report, presented to the European Monitoring Centre for Drugs and Drug Addiction, has shown that alcohol is by far the most widely consumed psychoactive substance of abuse by the Spanish population. In 2003, 48.2% had consumed alcoholic beverages at least once a week in the 12 months prior to the survey, 75% had had a drink on more than eight of the preceding 30 days and 14.1% on a daily basis in the last month. With regard to indicators of more problematic drinking, 21.2% had been drunk at least once in the 12 months prior to the survey, and 5.3% were ‘drinkers at risk’ (European Monitoring Centre for Drugs and Drug Addiction, 2006). To prevent and control health and social problems related to alcohol use for the individual and society, bio- logical state markers and marker combinations capable of monitoring alcohol consumption with a high sensitivity and specificity are needed. Such markers may help to (a)