Ethyl Glucuronide in Alcoholic Beverages

Ethyl Glucuronide in Alcoholic Beverages Abstract Aims This study examines the biomarker ethyl glucuronide (EtG) in various alcoholic beverages. Short summary The biomarker EtG was consistently found to be a natural compound of wine, whereas it was not detected in any of the other tested alcoholic beverages, which included various distilled spirits, liqueurs and beer of different types and geographical origins. Methods Alcoholic beverages (n = 114) were analyzed by a validated liquid chromatography/tandem mass spectrometry assay. Beverages included samples from beer, wine, liqueurs and spirits from different manufacturers and geographical origins. Results EtG was not detected in any kind of distilled alcoholic beverages, regardless of the type of spirit (rum, gin, vodka, whiskey, fruit brandy, corn brandy, cordial) or liqueur (n = 52). EtG was also not detected in any of the analyzed samples of beer, which included pilsener, weissbier, lager beer and ale from different origins (n = 20). In contrast, EtG was detected in every of the analyzed samples of wine (n = 42) without exception. Highest amounts were found in red wine and ranged from 1425 to 3720 μg/l (n = 16). Significantly, lower concentrations of EtG were observed for white wine (347–1685 μg/l, n = 14) and sparkling wine (281–1447 μg/l, n = 10). Conclusions Wine is an external source of EtG. It has been shown that milligram amounts of the biomarker can be contained in a bottle of wine. This should be considered in biomarker testing, especially in EtG hair analysis, which is susceptible to external contamination. INTRODUCTION Ethyl glucuronide (EtG) is an applied state marker of alcohol consumption in clinical and forensic settings today. Due to the slower elimination kinetics compared to ethanol, the detection window of alcohol consumption is significantly improved by EtG urinalysis (Helander et al., 2009). EtG is also incorporated in keratinous matrices and hair analysis has become an important tool to evaluate alcohol consumption even over a period of several months (a comprehensive review of EtG hair analysis is given by Crunelle et al. (2014)). It is generally known that external contamination is one of the fundamental issues in hair analysis (Chatterton, 2015). There are manifold pathways both from the body (blood, sweat, sebum) and from the environment (external contamination) for the deposition of substances to the hair fiber. Therefore, external contamination of hair matrix with the target analyte should be carefully evaluated in hair testing. Only little evidence is presented in the literature regarding to external contamination in the context of EtG hair analysis today. Arndt et al. (2013) detected EtG in some herbal hair tonics (n = 7 out of 11 tested commercially available products) and Sporkert et al. (2012)—who also detected EtG in a herbal hair tonic—reported positive findings of EtG in hair of a person after exposition to this commercial hair tonic. However, there was only a single experiment on a single individuum. Further studies are necessary to investigate this aspect on a more representative scale and other potential sources of contamination—beside hair cosmetics—should be investigated. Since alcohol intake is commonly associated with the consumption and presence of alcoholic beverages, the beverages themselves are potential contaminants of body surfaces. The aim of this study was to investigate alcoholic beverages as external sources of EtG. MATERIALS AND METHODS Chemicals and reagents All solvents and reagents for sample preparation were of quality for analysis (p.a.) and were obtained from Merck Schuchard (Hohenbrunn, Germany). Solvents and chemicals for liquid chromatography/tandem mass spectrometry (LC/MS/MS) analysis (water, acetonitrile, formic acid) were of specified LC/MS grade (Chromasolv®) and purchased from Fluka (Munich, Germany). Stock solutions (1.0 mg/ml in methanol) of EtG and d5-EtG were obtained from Lipomed (Weil am Rhein, Germany). Calibrators and quality control samples for EtG in urine were purchased from Recipe (Munich, Germany). Alcoholic beverages and sample preparation Alcoholic beverages (n = 114) were collected from five different local stores; details reading the type and origin of the beverages are shown in Tables 1–3. Half of the samples (n = 57) were acquired in the originally sealed bottles (samples were marked individually in the data tables) and the other half (n = 57) of the samples were collected from original bottles, which had been opened for tastings before. Originally sealed bottles were opened on the day of analysis. Periods between opening and analysis of previously opened beverages (from tastings) could not be assessed, since information about the individual moment of bottle opening in the stores were not available. Samples were diluted prior to analysis: a 50-μl aliquot of each alcoholic beverage was added to a glass vial with 950 μl of an aqueous solution containing the internal standard 100 ng/ml d5-EtG. The samples were mixed thoroughly and directly analyzed by LC/MS/MS. Table 1. EtG in distilled alcoholic beverages Sample number Sample type Year Origin Alcohol by volume (%) EtG (μg/l) 1 Herbal Bitter 2005 Germany 49 n.d. 2 Herbal Bitter 2000 Germany 79 n.d. 3 Aquavit 2017 Germany 42 n.d. 4* Fruit Schnapps 2015 Mosel, Germany 40 n.d. 5 Kirschwasser 2015 Schwarzwald, Germany 40 n.d. 6* Quince Brandy 2017 Germany 40 n.d. 7 Fruit Brandy 2017 Heidelberg, Germany 40 n.d. 8* Mirabelle Brandy 2017 Oberrotweil, Germany 45 n.d. 9 Lambig 2017 Bretagne, France 40 n.d. 10* Pear Brandy 2015 Oberrotweil, Germany 42 n.d. 11 Apricot Brandy 2015 Merano, Italy 40 n.d. 12 Fruit Brandy 2017 Germany 56 n.d. 13* Dry Gin 2017 London, United Kingdom 40 n.d. 14 Gin 2017 Denmark 47 n.d. 15 Grappa 2017 Trentino, Italy 41 n.d. 16 Grappa 2015 Conegliano Veneto, Italy 39 n.d. 17 Grappa 2007 Piobesi d´Alba, Italy 40 n.d. 18* Korn 1995 Wilthen, Germany 32 n.d. 19 Korn 1995 Nordhausen, Germany 38 n.d. 20 Korn 1995 Hardenberg, Germany 38 n.d. 21 Rum 2017 Tholey, Germany 35 n.d. 22 Rum 2017 Cuba 40 n.d. 23* Rum 2017 Germany 54 n.d. 24 Rum 2017 La Réunion 37.5 n.d. 25 Rum, Sherry finish 2014 Mauritius 40 n.d. 26 Rum 2017 Chile 40 n.d. 27 Rum 2016 Havana, Cuba 40 n.d. 28* Rum 2013 Flensburg, Germany 54 n.d. 29 Rum 2015 Brasilia 40 n.d. 30 Tequila 2011 Guadalajara, Mexico 38 n.d. 31 Vodka 2012 Denmark 40 n.d. 32* Vodka 2016 Germany 40 n.d. 33 Vodka 2005 Russia 40 n.d. 34 Whiskey 2014 Kintyre, Scotland 46 n.d. 35 Whiskey 2014 India 42 n.d. 36 Bamboo spirit 2015 China 45 n.d. 37 Gin 2014 Jork, Germany 42 n.d. 38 Coffee Liqueur 2014 Mexiko 20 n.d. 39 Cranberry Liqueur 2016 Denmark 18 n.d. 40 Fruit Liqueur 2017 Germany 20 n.d. 41* Cordial 2017 Germany 30 n.d. 42* Cordial 2017 Germany 35 n.d. 43 Peach Liqueur 2017 Germany 18 n.d. 44 Apple Liqueur 2017 Germany 25 n.d. 45 Bitter Liqueur 2017 Italy 11 n.d. 46 Bitter Liqueur 2015 Milano, Italy 25 n.d. 47 Bitter Liqueur 2014 Amsterdam, The Netherlands 21 n.d. 48* Coffee Liqueur 2015 Brobergen, Germany 25 n.d. 49 Doppelbock Liqueur 2017 Germany 23 n.d. 50* Advocaat 2017 The Netherlands 15 n.d. 51* Brandy 2017 Eltville am Rhein, Germany 36 n.d. 52* Brandy 2017 Venetia, Italy 40 n.d. Sample number Sample type Year Origin Alcohol by volume (%) EtG (μg/l) 1 Herbal Bitter 2005 Germany 49 n.d. 2 Herbal Bitter 2000 Germany 79 n.d. 3 Aquavit 2017 Germany 42 n.d. 4* Fruit Schnapps 2015 Mosel, Germany 40 n.d. 5 Kirschwasser 2015 Schwarzwald, Germany 40 n.d. 6* Quince Brandy 2017 Germany 40 n.d. 7 Fruit Brandy 2017 Heidelberg, Germany 40 n.d. 8* Mirabelle Brandy 2017 Oberrotweil, Germany 45 n.d. 9 Lambig 2017 Bretagne, France 40 n.d. 10* Pear Brandy 2015 Oberrotweil, Germany 42 n.d. 11 Apricot Brandy 2015 Merano, Italy 40 n.d. 12 Fruit Brandy 2017 Germany 56 n.d. 13* Dry Gin 2017 London, United Kingdom 40 n.d. 14 Gin 2017 Denmark 47 n.d. 15 Grappa 2017 Trentino, Italy 41 n.d. 16 Grappa 2015 Conegliano Veneto, Italy 39 n.d. 17 Grappa 2007 Piobesi d´Alba, Italy 40 n.d. 18* Korn 1995 Wilthen, Germany 32 n.d. 19 Korn 1995 Nordhausen, Germany 38 n.d. 20 Korn 1995 Hardenberg, Germany 38 n.d. 21 Rum 2017 Tholey, Germany 35 n.d. 22 Rum 2017 Cuba 40 n.d. 23* Rum 2017 Germany 54 n.d. 24 Rum 2017 La Réunion 37.5 n.d. 25 Rum, Sherry finish 2014 Mauritius 40 n.d. 26 Rum 2017 Chile 40 n.d. 27 Rum 2016 Havana, Cuba 40 n.d. 28* Rum 2013 Flensburg, Germany 54 n.d. 29 Rum 2015 Brasilia 40 n.d. 30 Tequila 2011 Guadalajara, Mexico 38 n.d. 31 Vodka 2012 Denmark 40 n.d. 32* Vodka 2016 Germany 40 n.d. 33 Vodka 2005 Russia 40 n.d. 34 Whiskey 2014 Kintyre, Scotland 46 n.d. 35 Whiskey 2014 India 42 n.d. 36 Bamboo spirit 2015 China 45 n.d. 37 Gin 2014 Jork, Germany 42 n.d. 38 Coffee Liqueur 2014 Mexiko 20 n.d. 39 Cranberry Liqueur 2016 Denmark 18 n.d. 40 Fruit Liqueur 2017 Germany 20 n.d. 41* Cordial 2017 Germany 30 n.d. 42* Cordial 2017 Germany 35 n.d. 43 Peach Liqueur 2017 Germany 18 n.d. 44 Apple Liqueur 2017 Germany 25 n.d. 45 Bitter Liqueur 2017 Italy 11 n.d. 46 Bitter Liqueur 2015 Milano, Italy 25 n.d. 47 Bitter Liqueur 2014 Amsterdam, The Netherlands 21 n.d. 48* Coffee Liqueur 2015 Brobergen, Germany 25 n.d. 49 Doppelbock Liqueur 2017 Germany 23 n.d. 50* Advocaat 2017 The Netherlands 15 n.d. 51* Brandy 2017 Eltville am Rhein, Germany 36 n.d. 52* Brandy 2017 Venetia, Italy 40 n.d. Samples obtained from originally sealed bottles are marked by an asterisk. n.d., not detected. Table 1. EtG in distilled alcoholic beverages Sample number Sample type Year Origin Alcohol by volume (%) EtG (μg/l) 1 Herbal Bitter 2005 Germany 49 n.d. 2 Herbal Bitter 2000 Germany 79 n.d. 3 Aquavit 2017 Germany 42 n.d. 4* Fruit Schnapps 2015 Mosel, Germany 40 n.d. 5 Kirschwasser 2015 Schwarzwald, Germany 40 n.d. 6* Quince Brandy 2017 Germany 40 n.d. 7 Fruit Brandy 2017 Heidelberg, Germany 40 n.d. 8* Mirabelle Brandy 2017 Oberrotweil, Germany 45 n.d. 9 Lambig 2017 Bretagne, France 40 n.d. 10* Pear Brandy 2015 Oberrotweil, Germany 42 n.d. 11 Apricot Brandy 2015 Merano, Italy 40 n.d. 12 Fruit Brandy 2017 Germany 56 n.d. 13* Dry Gin 2017 London, United Kingdom 40 n.d. 14 Gin 2017 Denmark 47 n.d. 15 Grappa 2017 Trentino, Italy 41 n.d. 16 Grappa 2015 Conegliano Veneto, Italy 39 n.d. 17 Grappa 2007 Piobesi d´Alba, Italy 40 n.d. 18* Korn 1995 Wilthen, Germany 32 n.d. 19 Korn 1995 Nordhausen, Germany 38 n.d. 20 Korn 1995 Hardenberg, Germany 38 n.d. 21 Rum 2017 Tholey, Germany 35 n.d. 22 Rum 2017 Cuba 40 n.d. 23* Rum 2017 Germany 54 n.d. 24 Rum 2017 La Réunion 37.5 n.d. 25 Rum, Sherry finish 2014 Mauritius 40 n.d. 26 Rum 2017 Chile 40 n.d. 27 Rum 2016 Havana, Cuba 40 n.d. 28* Rum 2013 Flensburg, Germany 54 n.d. 29 Rum 2015 Brasilia 40 n.d. 30 Tequila 2011 Guadalajara, Mexico 38 n.d. 31 Vodka 2012 Denmark 40 n.d. 32* Vodka 2016 Germany 40 n.d. 33 Vodka 2005 Russia 40 n.d. 34 Whiskey 2014 Kintyre, Scotland 46 n.d. 35 Whiskey 2014 India 42 n.d. 36 Bamboo spirit 2015 China 45 n.d. 37 Gin 2014 Jork, Germany 42 n.d. 38 Coffee Liqueur 2014 Mexiko 20 n.d. 39 Cranberry Liqueur 2016 Denmark 18 n.d. 40 Fruit Liqueur 2017 Germany 20 n.d. 41* Cordial 2017 Germany 30 n.d. 42* Cordial 2017 Germany 35 n.d. 43 Peach Liqueur 2017 Germany 18 n.d. 44 Apple Liqueur 2017 Germany 25 n.d. 45 Bitter Liqueur 2017 Italy 11 n.d. 46 Bitter Liqueur 2015 Milano, Italy 25 n.d. 47 Bitter Liqueur 2014 Amsterdam, The Netherlands 21 n.d. 48* Coffee Liqueur 2015 Brobergen, Germany 25 n.d. 49 Doppelbock Liqueur 2017 Germany 23 n.d. 50* Advocaat 2017 The Netherlands 15 n.d. 51* Brandy 2017 Eltville am Rhein, Germany 36 n.d. 52* Brandy 2017 Venetia, Italy 40 n.d. Sample number Sample type Year Origin Alcohol by volume (%) EtG (μg/l) 1 Herbal Bitter 2005 Germany 49 n.d. 2 Herbal Bitter 2000 Germany 79 n.d. 3 Aquavit 2017 Germany 42 n.d. 4* Fruit Schnapps 2015 Mosel, Germany 40 n.d. 5 Kirschwasser 2015 Schwarzwald, Germany 40 n.d. 6* Quince Brandy 2017 Germany 40 n.d. 7 Fruit Brandy 2017 Heidelberg, Germany 40 n.d. 8* Mirabelle Brandy 2017 Oberrotweil, Germany 45 n.d. 9 Lambig 2017 Bretagne, France 40 n.d. 10* Pear Brandy 2015 Oberrotweil, Germany 42 n.d. 11 Apricot Brandy 2015 Merano, Italy 40 n.d. 12 Fruit Brandy 2017 Germany 56 n.d. 13* Dry Gin 2017 London, United Kingdom 40 n.d. 14 Gin 2017 Denmark 47 n.d. 15 Grappa 2017 Trentino, Italy 41 n.d. 16 Grappa 2015 Conegliano Veneto, Italy 39 n.d. 17 Grappa 2007 Piobesi d´Alba, Italy 40 n.d. 18* Korn 1995 Wilthen, Germany 32 n.d. 19 Korn 1995 Nordhausen, Germany 38 n.d. 20 Korn 1995 Hardenberg, Germany 38 n.d. 21 Rum 2017 Tholey, Germany 35 n.d. 22 Rum 2017 Cuba 40 n.d. 23* Rum 2017 Germany 54 n.d. 24 Rum 2017 La Réunion 37.5 n.d. 25 Rum, Sherry finish 2014 Mauritius 40 n.d. 26 Rum 2017 Chile 40 n.d. 27 Rum 2016 Havana, Cuba 40 n.d. 28* Rum 2013 Flensburg, Germany 54 n.d. 29 Rum 2015 Brasilia 40 n.d. 30 Tequila 2011 Guadalajara, Mexico 38 n.d. 31 Vodka 2012 Denmark 40 n.d. 32* Vodka 2016 Germany 40 n.d. 33 Vodka 2005 Russia 40 n.d. 34 Whiskey 2014 Kintyre, Scotland 46 n.d. 35 Whiskey 2014 India 42 n.d. 36 Bamboo spirit 2015 China 45 n.d. 37 Gin 2014 Jork, Germany 42 n.d. 38 Coffee Liqueur 2014 Mexiko 20 n.d. 39 Cranberry Liqueur 2016 Denmark 18 n.d. 40 Fruit Liqueur 2017 Germany 20 n.d. 41* Cordial 2017 Germany 30 n.d. 42* Cordial 2017 Germany 35 n.d. 43 Peach Liqueur 2017 Germany 18 n.d. 44 Apple Liqueur 2017 Germany 25 n.d. 45 Bitter Liqueur 2017 Italy 11 n.d. 46 Bitter Liqueur 2015 Milano, Italy 25 n.d. 47 Bitter Liqueur 2014 Amsterdam, The Netherlands 21 n.d. 48* Coffee Liqueur 2015 Brobergen, Germany 25 n.d. 49 Doppelbock Liqueur 2017 Germany 23 n.d. 50* Advocaat 2017 The Netherlands 15 n.d. 51* Brandy 2017 Eltville am Rhein, Germany 36 n.d. 52* Brandy 2017 Venetia, Italy 40 n.d. Samples obtained from originally sealed bottles are marked by an asterisk. n.d., not detected. Table 2. EtG in beer Sample no Sample type Year Origin Alcohol by volume (%) EtG (μg/l) 53* Kölsch 2017 Cologne, Germany 4.8 n.d. 54* Ale 2017 England 7.2 n.d. 55* Craft Beer 2017 Hamburg, Germany 6.3 n.d. 56* Helles 2017 München, Germany 5 n.d. 57* Lager Beer 2017 Budéjovice, Czech 5 n.d. 58* Lager Beer 2017 Poland 5.5 n.d. 59* Non-alcoholic Beer 2017 Krombach, Germany 0 n.d. 60* Pilsner 2017 Flensburg, Germany 4.8 n.d. 61* Pilsner 2017 Denmark 5 n.d. 62* Pilsner 2017 Bremen, Germany 4.9 n.d. 63* Pilsner 2017 Denmark 5 n.d. 64* Pilsner 2017 Pilzen, Czech 4.4 n.d. 65* Weissbier 2017 Erding, Germany 5.3 n.d. 66* Weissbier 2017 Munich, Germany 5.5 n.d. 67* Lager Beer 2017 Barcelona, Spain 5.4 n.d. 68* Weissbier 2017 Munich, Germany 5.5 n.d. 69* Beer 2017 Mexico 4.5 n.d. 70* Weissbier 2017 Bavaria, Germany 5.3 n.d. 71* Beer 2017 Vialonga, Portugal 5 n.d. 72* Non-alcoholic Beer 2017 Bremen, Germany 0 n.d. Sample no Sample type Year Origin Alcohol by volume (%) EtG (μg/l) 53* Kölsch 2017 Cologne, Germany 4.8 n.d. 54* Ale 2017 England 7.2 n.d. 55* Craft Beer 2017 Hamburg, Germany 6.3 n.d. 56* Helles 2017 München, Germany 5 n.d. 57* Lager Beer 2017 Budéjovice, Czech 5 n.d. 58* Lager Beer 2017 Poland 5.5 n.d. 59* Non-alcoholic Beer 2017 Krombach, Germany 0 n.d. 60* Pilsner 2017 Flensburg, Germany 4.8 n.d. 61* Pilsner 2017 Denmark 5 n.d. 62* Pilsner 2017 Bremen, Germany 4.9 n.d. 63* Pilsner 2017 Denmark 5 n.d. 64* Pilsner 2017 Pilzen, Czech 4.4 n.d. 65* Weissbier 2017 Erding, Germany 5.3 n.d. 66* Weissbier 2017 Munich, Germany 5.5 n.d. 67* Lager Beer 2017 Barcelona, Spain 5.4 n.d. 68* Weissbier 2017 Munich, Germany 5.5 n.d. 69* Beer 2017 Mexico 4.5 n.d. 70* Weissbier 2017 Bavaria, Germany 5.3 n.d. 71* Beer 2017 Vialonga, Portugal 5 n.d. 72* Non-alcoholic Beer 2017 Bremen, Germany 0 n.d. Samples obtained from originally sealed bottles are marked by an asterisk. n.d., not detected. Table 2. EtG in beer Sample no Sample type Year Origin Alcohol by volume (%) EtG (μg/l) 53* Kölsch 2017 Cologne, Germany 4.8 n.d. 54* Ale 2017 England 7.2 n.d. 55* Craft Beer 2017 Hamburg, Germany 6.3 n.d. 56* Helles 2017 München, Germany 5 n.d. 57* Lager Beer 2017 Budéjovice, Czech 5 n.d. 58* Lager Beer 2017 Poland 5.5 n.d. 59* Non-alcoholic Beer 2017 Krombach, Germany 0 n.d. 60* Pilsner 2017 Flensburg, Germany 4.8 n.d. 61* Pilsner 2017 Denmark 5 n.d. 62* Pilsner 2017 Bremen, Germany 4.9 n.d. 63* Pilsner 2017 Denmark 5 n.d. 64* Pilsner 2017 Pilzen, Czech 4.4 n.d. 65* Weissbier 2017 Erding, Germany 5.3 n.d. 66* Weissbier 2017 Munich, Germany 5.5 n.d. 67* Lager Beer 2017 Barcelona, Spain 5.4 n.d. 68* Weissbier 2017 Munich, Germany 5.5 n.d. 69* Beer 2017 Mexico 4.5 n.d. 70* Weissbier 2017 Bavaria, Germany 5.3 n.d. 71* Beer 2017 Vialonga, Portugal 5 n.d. 72* Non-alcoholic Beer 2017 Bremen, Germany 0 n.d. Sample no Sample type Year Origin Alcohol by volume (%) EtG (μg/l) 53* Kölsch 2017 Cologne, Germany 4.8 n.d. 54* Ale 2017 England 7.2 n.d. 55* Craft Beer 2017 Hamburg, Germany 6.3 n.d. 56* Helles 2017 München, Germany 5 n.d. 57* Lager Beer 2017 Budéjovice, Czech 5 n.d. 58* Lager Beer 2017 Poland 5.5 n.d. 59* Non-alcoholic Beer 2017 Krombach, Germany 0 n.d. 60* Pilsner 2017 Flensburg, Germany 4.8 n.d. 61* Pilsner 2017 Denmark 5 n.d. 62* Pilsner 2017 Bremen, Germany 4.9 n.d. 63* Pilsner 2017 Denmark 5 n.d. 64* Pilsner 2017 Pilzen, Czech 4.4 n.d. 65* Weissbier 2017 Erding, Germany 5.3 n.d. 66* Weissbier 2017 Munich, Germany 5.5 n.d. 67* Lager Beer 2017 Barcelona, Spain 5.4 n.d. 68* Weissbier 2017 Munich, Germany 5.5 n.d. 69* Beer 2017 Mexico 4.5 n.d. 70* Weissbier 2017 Bavaria, Germany 5.3 n.d. 71* Beer 2017 Vialonga, Portugal 5 n.d. 72* Non-alcoholic Beer 2017 Bremen, Germany 0 n.d. Samples obtained from originally sealed bottles are marked by an asterisk. n.d., not detected. Table 3. EtG in wine Sample no Sample type Year Origin Alcohol by volume (%) EtG (μg/l) 73 Red Wine, Spätburgunder 2016 Pfalz, Germany 13 3070 74 Red Wine, Canti Primitivo 2015 Puglia, Italy 12 1425 75 Red Wine, Bobos 2015 Spain 13.5 3617 76 Red Wine 2012 Bordeaux, France 14 3001 77 Red Wine 2014 Pfalz, Germany 13 2120 78 Red Wine, Primitivo 2015 Italy 14 2201 79* Red Wine, Chianti Classico 2015 Toscana, Italy 13.5 3176 80 Red Wine, Chianti 2016 Italy 12 1525 81* Red Wine, Merlot 2016 Sicilia, Italy 13 3720 82 Red Wine, Merlot 2005 Chile 13.5 3614 83 Red Wine 2016 Alentejo, Portugal 13.5 3409 84* Red Wine, Montepulciano d´Abruzzo 2015 Montepulciano, Italy 14.5 3335 85 Red Wine (Glühwein) 2017 Hamburg, Germany 12 3040 86* Red Wine, Heroldrebe 2015 Rotenberg, Germany 13 3463 87* Red Wine, Non-alcoholic 2017 Trier, Germany 0 1881 88* Red Wine, Non-alcoholic 2016 Rüdesheim, Germany 0 2714 89* Dornfelder Rosé 2015 Dornfeld, Germany 10 672 90 Tempranillo Rosado 2016 Ribera del Duero, Spain 13.5 1508 91* Sparkling Wine 2017 Germany 12.5 281 92* Sparkling Wine 2017 Freyburg, Germany 11 869 93* Sparkling Wine 2017 Trier, Germany 5 771 94* Sparkling Wine 2017 Italy 10 738 95* Sparkling Wine 2017 Maxhütte-Heudorf, Germany 11 826 96* Sparkling Wine 2017 Germany 11 307 97* Sparkling Wine 2017 Paris, France 11 1447 98* Sparkling Wine, Prosecco 2017 Venetia, Italy 10.5 463 99* Sparkling Wine, Prosecco 2017 Venetia, Italy 11 532 100* Sparkling Wine, Non-alcoholic 2017 Trier, Germany 0 1328 101 White Wine, Gutedel 2015 Freiburg, Germany 12 514 102* White Wine, Sauvignon Blanc 2017 Western Cape, South Africa 12.5 1207 103 White Wine 2015 France 14 646 104 White Wine, Grecanico 2016 Sicilia, Italy 13 1685 105 White Wine, Sauvignon 2015 Centre-val de Loire, France 12.5 665 106 White Wine, Grauer Burgunder 2016 Bad Dürkheim, Germany 13 1081 107 White Wine, Pinot Grigio 2016 Italy 12.5 1028 108* White Wine, Sauvignon Blanc 2017 South Africa 12,5 546 109 White Wine, Chardonnay 2017 Petersbach, France 12.5 1452 110 White Wine, Riesling 2004 Mosel-Saar, Germany 11.5 1500 111 White Wine, Pinot Grigio 2003 Veneto, Italy 11.5 347 112* White Wine, Chardonnay 2015 South Africa 12 1071 113* White Wine, Riesling 2016 Rheingau, Germany 12 1057 114* White Wine, Riesling Cabinet 2011 Flein-Talheim, Germany 11 1265 Sample no Sample type Year Origin Alcohol by volume (%) EtG (μg/l) 73 Red Wine, Spätburgunder 2016 Pfalz, Germany 13 3070 74 Red Wine, Canti Primitivo 2015 Puglia, Italy 12 1425 75 Red Wine, Bobos 2015 Spain 13.5 3617 76 Red Wine 2012 Bordeaux, France 14 3001 77 Red Wine 2014 Pfalz, Germany 13 2120 78 Red Wine, Primitivo 2015 Italy 14 2201 79* Red Wine, Chianti Classico 2015 Toscana, Italy 13.5 3176 80 Red Wine, Chianti 2016 Italy 12 1525 81* Red Wine, Merlot 2016 Sicilia, Italy 13 3720 82 Red Wine, Merlot 2005 Chile 13.5 3614 83 Red Wine 2016 Alentejo, Portugal 13.5 3409 84* Red Wine, Montepulciano d´Abruzzo 2015 Montepulciano, Italy 14.5 3335 85 Red Wine (Glühwein) 2017 Hamburg, Germany 12 3040 86* Red Wine, Heroldrebe 2015 Rotenberg, Germany 13 3463 87* Red Wine, Non-alcoholic 2017 Trier, Germany 0 1881 88* Red Wine, Non-alcoholic 2016 Rüdesheim, Germany 0 2714 89* Dornfelder Rosé 2015 Dornfeld, Germany 10 672 90 Tempranillo Rosado 2016 Ribera del Duero, Spain 13.5 1508 91* Sparkling Wine 2017 Germany 12.5 281 92* Sparkling Wine 2017 Freyburg, Germany 11 869 93* Sparkling Wine 2017 Trier, Germany 5 771 94* Sparkling Wine 2017 Italy 10 738 95* Sparkling Wine 2017 Maxhütte-Heudorf, Germany 11 826 96* Sparkling Wine 2017 Germany 11 307 97* Sparkling Wine 2017 Paris, France 11 1447 98* Sparkling Wine, Prosecco 2017 Venetia, Italy 10.5 463 99* Sparkling Wine, Prosecco 2017 Venetia, Italy 11 532 100* Sparkling Wine, Non-alcoholic 2017 Trier, Germany 0 1328 101 White Wine, Gutedel 2015 Freiburg, Germany 12 514 102* White Wine, Sauvignon Blanc 2017 Western Cape, South Africa 12.5 1207 103 White Wine 2015 France 14 646 104 White Wine, Grecanico 2016 Sicilia, Italy 13 1685 105 White Wine, Sauvignon 2015 Centre-val de Loire, France 12.5 665 106 White Wine, Grauer Burgunder 2016 Bad Dürkheim, Germany 13 1081 107 White Wine, Pinot Grigio 2016 Italy 12.5 1028 108* White Wine, Sauvignon Blanc 2017 South Africa 12,5 546 109 White Wine, Chardonnay 2017 Petersbach, France 12.5 1452 110 White Wine, Riesling 2004 Mosel-Saar, Germany 11.5 1500 111 White Wine, Pinot Grigio 2003 Veneto, Italy 11.5 347 112* White Wine, Chardonnay 2015 South Africa 12 1071 113* White Wine, Riesling 2016 Rheingau, Germany 12 1057 114* White Wine, Riesling Cabinet 2011 Flein-Talheim, Germany 11 1265 Samples obtained from originally sealed bottles are marked by an asterisk. Table 3. EtG in wine Sample no Sample type Year Origin Alcohol by volume (%) EtG (μg/l) 73 Red Wine, Spätburgunder 2016 Pfalz, Germany 13 3070 74 Red Wine, Canti Primitivo 2015 Puglia, Italy 12 1425 75 Red Wine, Bobos 2015 Spain 13.5 3617 76 Red Wine 2012 Bordeaux, France 14 3001 77 Red Wine 2014 Pfalz, Germany 13 2120 78 Red Wine, Primitivo 2015 Italy 14 2201 79* Red Wine, Chianti Classico 2015 Toscana, Italy 13.5 3176 80 Red Wine, Chianti 2016 Italy 12 1525 81* Red Wine, Merlot 2016 Sicilia, Italy 13 3720 82 Red Wine, Merlot 2005 Chile 13.5 3614 83 Red Wine 2016 Alentejo, Portugal 13.5 3409 84* Red Wine, Montepulciano d´Abruzzo 2015 Montepulciano, Italy 14.5 3335 85 Red Wine (Glühwein) 2017 Hamburg, Germany 12 3040 86* Red Wine, Heroldrebe 2015 Rotenberg, Germany 13 3463 87* Red Wine, Non-alcoholic 2017 Trier, Germany 0 1881 88* Red Wine, Non-alcoholic 2016 Rüdesheim, Germany 0 2714 89* Dornfelder Rosé 2015 Dornfeld, Germany 10 672 90 Tempranillo Rosado 2016 Ribera del Duero, Spain 13.5 1508 91* Sparkling Wine 2017 Germany 12.5 281 92* Sparkling Wine 2017 Freyburg, Germany 11 869 93* Sparkling Wine 2017 Trier, Germany 5 771 94* Sparkling Wine 2017 Italy 10 738 95* Sparkling Wine 2017 Maxhütte-Heudorf, Germany 11 826 96* Sparkling Wine 2017 Germany 11 307 97* Sparkling Wine 2017 Paris, France 11 1447 98* Sparkling Wine, Prosecco 2017 Venetia, Italy 10.5 463 99* Sparkling Wine, Prosecco 2017 Venetia, Italy 11 532 100* Sparkling Wine, Non-alcoholic 2017 Trier, Germany 0 1328 101 White Wine, Gutedel 2015 Freiburg, Germany 12 514 102* White Wine, Sauvignon Blanc 2017 Western Cape, South Africa 12.5 1207 103 White Wine 2015 France 14 646 104 White Wine, Grecanico 2016 Sicilia, Italy 13 1685 105 White Wine, Sauvignon 2015 Centre-val de Loire, France 12.5 665 106 White Wine, Grauer Burgunder 2016 Bad Dürkheim, Germany 13 1081 107 White Wine, Pinot Grigio 2016 Italy 12.5 1028 108* White Wine, Sauvignon Blanc 2017 South Africa 12,5 546 109 White Wine, Chardonnay 2017 Petersbach, France 12.5 1452 110 White Wine, Riesling 2004 Mosel-Saar, Germany 11.5 1500 111 White Wine, Pinot Grigio 2003 Veneto, Italy 11.5 347 112* White Wine, Chardonnay 2015 South Africa 12 1071 113* White Wine, Riesling 2016 Rheingau, Germany 12 1057 114* White Wine, Riesling Cabinet 2011 Flein-Talheim, Germany 11 1265 Sample no Sample type Year Origin Alcohol by volume (%) EtG (μg/l) 73 Red Wine, Spätburgunder 2016 Pfalz, Germany 13 3070 74 Red Wine, Canti Primitivo 2015 Puglia, Italy 12 1425 75 Red Wine, Bobos 2015 Spain 13.5 3617 76 Red Wine 2012 Bordeaux, France 14 3001 77 Red Wine 2014 Pfalz, Germany 13 2120 78 Red Wine, Primitivo 2015 Italy 14 2201 79* Red Wine, Chianti Classico 2015 Toscana, Italy 13.5 3176 80 Red Wine, Chianti 2016 Italy 12 1525 81* Red Wine, Merlot 2016 Sicilia, Italy 13 3720 82 Red Wine, Merlot 2005 Chile 13.5 3614 83 Red Wine 2016 Alentejo, Portugal 13.5 3409 84* Red Wine, Montepulciano d´Abruzzo 2015 Montepulciano, Italy 14.5 3335 85 Red Wine (Glühwein) 2017 Hamburg, Germany 12 3040 86* Red Wine, Heroldrebe 2015 Rotenberg, Germany 13 3463 87* Red Wine, Non-alcoholic 2017 Trier, Germany 0 1881 88* Red Wine, Non-alcoholic 2016 Rüdesheim, Germany 0 2714 89* Dornfelder Rosé 2015 Dornfeld, Germany 10 672 90 Tempranillo Rosado 2016 Ribera del Duero, Spain 13.5 1508 91* Sparkling Wine 2017 Germany 12.5 281 92* Sparkling Wine 2017 Freyburg, Germany 11 869 93* Sparkling Wine 2017 Trier, Germany 5 771 94* Sparkling Wine 2017 Italy 10 738 95* Sparkling Wine 2017 Maxhütte-Heudorf, Germany 11 826 96* Sparkling Wine 2017 Germany 11 307 97* Sparkling Wine 2017 Paris, France 11 1447 98* Sparkling Wine, Prosecco 2017 Venetia, Italy 10.5 463 99* Sparkling Wine, Prosecco 2017 Venetia, Italy 11 532 100* Sparkling Wine, Non-alcoholic 2017 Trier, Germany 0 1328 101 White Wine, Gutedel 2015 Freiburg, Germany 12 514 102* White Wine, Sauvignon Blanc 2017 Western Cape, South Africa 12.5 1207 103 White Wine 2015 France 14 646 104 White Wine, Grecanico 2016 Sicilia, Italy 13 1685 105 White Wine, Sauvignon 2015 Centre-val de Loire, France 12.5 665 106 White Wine, Grauer Burgunder 2016 Bad Dürkheim, Germany 13 1081 107 White Wine, Pinot Grigio 2016 Italy 12.5 1028 108* White Wine, Sauvignon Blanc 2017 South Africa 12,5 546 109 White Wine, Chardonnay 2017 Petersbach, France 12.5 1452 110 White Wine, Riesling 2004 Mosel-Saar, Germany 11.5 1500 111 White Wine, Pinot Grigio 2003 Veneto, Italy 11.5 347 112* White Wine, Chardonnay 2015 South Africa 12 1071 113* White Wine, Riesling 2016 Rheingau, Germany 12 1057 114* White Wine, Riesling Cabinet 2011 Flein-Talheim, Germany 11 1265 Samples obtained from originally sealed bottles are marked by an asterisk. Liquid chromatography/tandem mass spectrometry A well-established, validated and accredited LC/MS/MS approach, which has been used for routine EtG urinalysis for more than 5 years, was applied for the analysis of beverages with slight modifications. Chromatography was performed by Waters Acquity® UPLC with a C18 Acquity® HSS T3, 1.7 μm, 2.1 × 150 mm column at 40°C. The injection volume of the samples was 5 μl, the flow rate at 0.3 ml/min and the binary gradient as follows: 0–3.8 min: 2–30% B, 4.0–6.9 min: 2% B. Mobile phase A consisted of water with 0.1% formic acid and mobile phase B was acetonitrile with 0.1% formic acid. A Waters XEVO®TQ-S triple quadrupole mass analyzer operating in multiple reaction monitoring was used for detection, five transitions were monitored: EtG: m/z 221 > 75 (quantifier), m/z 221 > 85, m/z 221 > 57, m/z 221 > 113 (qualifiers), d5-EtG (internal standard I.S.) m/z 226 > 85. The assay was routinely calibrated using six commercially available standards of spiked urine (80, 191, 582, 1464, 4873 and 9621 ng/ml) and there were also commercially available quality control samples (111, 518 and 2052 pg/mg) included in each analytical series. For the analysis of beverages, additionally aqueous calibrators of EtG (50, 100, 200, 500, 1000, 2000 and 5000 ng/ml) were prepared in house from an independent source and analyzed by the LC/MS/MS procedure. Calibration derived from those aqueous standards was congruent with the routinely used calibration from urinary calibrators. Validation parameters including accuracy, interferences, linearity of calibration, matrix effects and in-process stability complied with the international standards. Matrix effects in alcoholic beverages were assessed at two concentration levels (corresponding to QC1 and QC3) by the procedure described by Matuszewski et al. (2003). Comparable absolute signal areas and signal variabilities (difference <15%) were observed in different EtG-spiked beverages (n = 10) and EtG-spiked aqueous solutions. Matrix effects in wine could not be assessed by this procedure (EtG was present in every sample), but analysis did not raise suspicion to serious matrix effects: (a) predilution of the samples did not affect quantitative results (in case of strong ion suppression, non-linear decrease of analyte signal and impact on quantitative result is commonly observed by dilution of the sample) and (b) signal areas of internal standard d5-EtG (which elutes very closely to EtG) were also unchanged in samples from wine. The limit of detection of the analytical method was 5 ng/ml and the lower limit of quantification was 17 ng/ml. Accuracy was assessed at three concentration levels (corresponding to QC1-3) on different days. Intraday coefficient of variation was 5% (QC1), 3% (QC2) and 3%(QC3), interday coefficient of variation was 8% (QC1), 4% (QC2) and 4% (QC3). Regular external quality control is performed by periodical proficiency testing. RESULTS EtG was not detected in distilled alcoholic beverages (Table 1). This was consistent for all analyzed samples of rum (n = 9), whisky (n = 2), grappa (n = 3), korn (n = 3), brandy (n = 2), fruit brandy (n = 9), gin (n = 3), vodka (n = 3), herbal bitters (n = 3), tequila (n = 1), bamboo spirit (n = 1) and liqueurs (n = 13). EtG was also not detected in any of the analyzed beers (n = 20, Table 2), regardless to type (e.g. pilsner, weissbeer, ale) and origin (e.g. Mexico, Czech, Denmark, Spain). In contrast, EtG was detected in every of the analyzed samples of wine (n = 42) without exception. EtG concentrations in wine ranged from 281 to 3720 μg/l and different quantitative results were observed for the different types of wine. The results are box plotted in Fig. 1 and individual EtG concentrations are listed in Table 3. EtG concentrations in red wine were significantly higher (P < 0.001) than in white wine and sparkling wine. As shown in Fig. 2, this was also observed for the EtG/alcohol ratio of wine, differences in EtG concentrations were not compensated by differences in alcohol content (red wine: average 13.2%, range 12–14.5%; white wine: average 12.3%, range 11–14%; sparkling wine: average 10.3%, range 5–12.5%). EtG was also present in ‘non-alcoholic’ wine. EtG concentrations in wine in relation to vintages (except to sparkling wine (2017 n = 10), samples of wine derived from different vintages (2017 n = 5, 2016 n = 10, 2015 n = 11, 2014 n = 1, 2012 n = 1, 2011 n = 1, 2005 n = 1, 2004 n = 1 and 2003 n = 1)) are presented in Fig. 3. There was no obvious correlation between vintages and EtG concentrations and no extraordinary findings in very aged wine (2005 n = 1, 2004 n = 1 and 2003 n = 1). Differences in EtG concentrations of wine from previously opened and originally sealed bottles were not observed (Fig. 4). Fig. 1. View largeDownload slide Boxplots of EtG concentrations in wine. Fig. 1. View largeDownload slide Boxplots of EtG concentrations in wine. Fig. 2. View largeDownload slide Boxplots of EtG/alcohol by volume ratios in wine. Fig. 2. View largeDownload slide Boxplots of EtG/alcohol by volume ratios in wine. Fig. 3. View largeDownload slide Comparison of EtG concentrations and vintages of wine. Triangles: red wine, dots: white wine and diamonds: sparkling wine. Fig. 3. View largeDownload slide Comparison of EtG concentrations and vintages of wine. Triangles: red wine, dots: white wine and diamonds: sparkling wine. Fig. 4. View largeDownload slide Comparison of EtG concentrations found in samples from originally sealed and previously opened bottles. Triangles: red wine, dots: white wine and diamonds: sparkling wine. Fig. 4. View largeDownload slide Comparison of EtG concentrations found in samples from originally sealed and previously opened bottles. Triangles: red wine, dots: white wine and diamonds: sparkling wine. DISCUSSION Since its detection more than 50 years ago, a substantial amount of scientific studies regarding the formation and kinetics of EtG in the human body has been published in the literature. Formation of EtG is characterized by the conjugation of uridine-5`-diphospho (UDP)-glucuronic acid to ethanol. This is catalyzed by the superfamily of UDP-glucuronosyltransferases (UGTs), and it was found that multiple isoforms of UGT (predominantly UGT1A1 and UGT2B7) are responsible for the formation of EtG in human liver microsomes (Foti and Fisher, 2005). However, only marginal data are available regarding exogenous sources of EtG from outside the human body. Helander et al. (2007) reported possible post-collection synthesis of EtG in urine samples contaminated by bacteria, which was observed as well as bacterial degradation of EtG. Politi et al. (2005) reported positive testings of EtG in Italian wine (n = 8). Sporkert et al. (2012) detected EtG (2.7 mg/l) in an alcoholic (44% ethanol by volume) plant extract, which was used as a hair tonic and finally caused a positive finding of EtG in hair of an individuum (n = 1) after external application. Arndt et al. (2013) identified EtG (0.07–1.06 mg/l) in herbal hair tonics, which also contained ethanol (21–39% by volume). Sporkert et al. (2014) examined different ethanolic extracts from several different plants (n = 17), EtG was confirmed by mass spectrometry in four ethanolic plant extracts (thyme, birch, oak and plantain). Baumgartner et al. (2013) discussed a nucleophilic reaction during hydro-ethanolic plant extraction as the principle mechanism of EtG formation. However, ethanolic herbal preparations (e.g. cordial liqueurs, bitters) were tested negative for EtG in the presented study. In contrast, EtG was consistently found in wine, which is finally derived from the preparation of fruit. This is in accordance with the findings of Politi et al. (2005), who also found EtG in Italian wine (n = 8), but not in samples from beer (n = 4). The underlying biochemical mechanism is still unclear and will be examined in further studies, but the results of the presented work suggest that EtG formation rather takes place in early steps of wine production (disintegration, mashing and fermentation of grapes) than during wine storage. This could probably explain the obvious differences in EtG concentrations of white and red wine and is additionally supported by the following observations: (a) there were comparable EtG concentrations in wine of different ages, (b) differences in EtG concentrations were not compensated by differences in alcohol concentrations and (c) EtG was also present in non-alcoholic wine (which is fermented before the reduction of ethanol and the subsequent storage). Preliminary results from the analysis of grapes, grape juices and other products derived from grapes (data not presented) suggest that EtG is not an ingredient of the grapes themselves. Microbial activity of yeast or bacteria during fermentation even does not seem to be the only crucial step of EtG formation, since EtG was not detected in any of the analyzed beer samples. Further studies are necessary to investigate if there is any impact of the results on EtG biomarker analysis. Hoiseth et al. (2010) did not observe positive EtG testings in urine of 12 subjects after the consumption of 0.75 l non-alcoholic wine, which contained 3 mg/l of EtG, so the impact of externally supplied EtG on urinary EtG seems to be of minor importance. Otherwise, the investigations of Sporkert et al. (2012) strongly point out that EtG hair testings can be affected by external exposition to EtG: the application of a herbal hair tonic containing 2.7 mg/l EtG lead to an significant increase of hair EtG concentration of an individuum (n = 1) and a possible misinterpretation of the drinking behavior. Although it has not yet been tested, it can be easily assumed that external application of wine (containing EtG comparable to the described herbal hair tonic) to hair could falsify hair EtG results in a similar manner. Although this is a rather uncommon exposition setting (beside the famous ‘champagne shower’, red wine is used as a hair colorant in combination with henna and as an ingredient in natural hair cosmetics), the impact of incidental and unintended external exposures should be considered. This is addressed to a future study of a population of abstinent cooks and wine stewards. It is the authors’ own experience and also reported by others (Kintz and Nicholson, 2014) that unexpected positive results of hair EtG analysis are sometimes observed, which are in contradiction to all other relevant factors of a case and, therefore, raise reasonable suspicion to external contamination. Since an application of herbal hair tonics or other cosmetic hair treatments are frequently negated in those cases, further investigations of external contamination in EtG hair analysis are necessary. CONFLICT OF INTEREST STATEMENT None declared. REFERENCES Arndt T , Schröfel S , Stemmerich K . ( 2013 ) Ethyl glucuronide identified in commercial hair tonics . Forensic Sci Int 231 : 195 – 8 . Google Scholar CrossRef Search ADS PubMed Baumgartner MR , Binz TM , Schwanninger A , et al. . ( 2013 ) Ethyl glucuronide in hair tonics: formation of EtG during hydro-ethanolic extraction of plant material for cosmetic products, TIAFT 51st Meeting, Madeira, Abstract book OM3, pp. 63–4. Chatterton G . ( 2015 ) External contamination: still a debate? In Kintz P , Salomone A , Vincenti M (eds) . Hair Analysis in Clinical and Forensic Toxicology . New York : Academic Press , 47 – 70 . Google Scholar CrossRef Search ADS Crunelle CL , Yegles M , van Nuijs ALN , et al. . ( 2014 ) Hair ethyl glucuronide levels as a marker for alcohol use and abuse: a review of the current state of the art . Drug Alcohol Depend 134 : 1 – 11 . Google Scholar CrossRef Search ADS PubMed Foti RS , Fisher MB . ( 2005 ) Assessment of UDP-glucuronosyltransferase catalyzed formation of ethyl glucuronide in human liver microsomes and recombinant UGTs . Forensic Sci Int 153 : 109 – 16 . Google Scholar CrossRef Search ADS PubMed Helander A , Böttcher M , Fehr C , et al. . ( 2009 ) Detection times for urinary ethyl glucuronide and ethyl sulfate in heavy drinkers during alcohol detoxification . Alcohol Alcohol 44 : 55 – 61 . Google Scholar CrossRef Search ADS PubMed Helander A , Olsson I , Dahl H . ( 2007 ) Postcollection synthesis of ethyl glucuronide by bacteria in urine may cause false identification of alcohol consumption . Clin Chem 53 : 1855 – 7 . Google Scholar CrossRef Search ADS PubMed Hoiseth G , Yttredal B , Karinen R , et al. . ( 2010 ) Levels of ethyl glucuronide and ethyl sulfate in oral fluid, blood, and urine after use of mouthwash and ingestion of nonalcoholic wine . J Anal Toxicol 34 : 84 – 8 . Google Scholar CrossRef Search ADS PubMed Kintz P , Nicholson D . ( 2014 ) Testing for ethanol markers in hair: discrepancies after simultaneous quantification of ethyl glucuronide and fatty acid ethyl esters . Forensic Sci Int 243 : 44 – 6 . Google Scholar CrossRef Search ADS PubMed Matuszewski BK , Constanzer ML , Chavez-Eng CM . ( 2003 ) Strategies for the assessment of matrix effect in quantitative bioanalytical methods based on HPLC-MS/MS . Anal Chem 75 : 3019 – 30 . Google Scholar CrossRef Search ADS PubMed Politi L , Morini L , Groppi A , et al. . ( 2005 ) Direct determination of the ethanol metabolites ethyl glucuronide and ethyl sulfate in urine by liquid chromatography/electrospray tandem mass spectrometry . Rapid Commun Mass Spectrom 19 : 1321 – 31 . Google Scholar CrossRef Search ADS PubMed Sporkert F , Kharbouche H , Augsburger MP , et al. . ( 2012 ) Positive EtG findings in hair as a result of a cosmetic treatment . Forensic Sci Int 218 : 97 – 100 . Google Scholar CrossRef Search ADS PubMed Sporkert F , Sizun A , Giroud C . ( 2014 ) Ethyl glucuronide in plant extracts . Toxicol Anal Clin 26 : 11 . © The Author(s) 2018. Medical Council on Alcohol and Oxford University Press. All rights reserved. This article is published and distributed under the terms of the Oxford University Press, Standard Journals Publication Model (https://academic.oup.com/journals/pages/about_us/legal/notices) http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png Alcohol and Alcoholism Oxford University Press

Ethyl Glucuronide in Alcoholic Beverages

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Oxford University Press
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© The Author(s) 2018. Medical Council on Alcohol and Oxford University Press. All rights reserved.
ISSN
0735-0414
eISSN
1464-3502
D.O.I.
10.1093/alcalc/agy033
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Abstract

Abstract Aims This study examines the biomarker ethyl glucuronide (EtG) in various alcoholic beverages. Short summary The biomarker EtG was consistently found to be a natural compound of wine, whereas it was not detected in any of the other tested alcoholic beverages, which included various distilled spirits, liqueurs and beer of different types and geographical origins. Methods Alcoholic beverages (n = 114) were analyzed by a validated liquid chromatography/tandem mass spectrometry assay. Beverages included samples from beer, wine, liqueurs and spirits from different manufacturers and geographical origins. Results EtG was not detected in any kind of distilled alcoholic beverages, regardless of the type of spirit (rum, gin, vodka, whiskey, fruit brandy, corn brandy, cordial) or liqueur (n = 52). EtG was also not detected in any of the analyzed samples of beer, which included pilsener, weissbier, lager beer and ale from different origins (n = 20). In contrast, EtG was detected in every of the analyzed samples of wine (n = 42) without exception. Highest amounts were found in red wine and ranged from 1425 to 3720 μg/l (n = 16). Significantly, lower concentrations of EtG were observed for white wine (347–1685 μg/l, n = 14) and sparkling wine (281–1447 μg/l, n = 10). Conclusions Wine is an external source of EtG. It has been shown that milligram amounts of the biomarker can be contained in a bottle of wine. This should be considered in biomarker testing, especially in EtG hair analysis, which is susceptible to external contamination. INTRODUCTION Ethyl glucuronide (EtG) is an applied state marker of alcohol consumption in clinical and forensic settings today. Due to the slower elimination kinetics compared to ethanol, the detection window of alcohol consumption is significantly improved by EtG urinalysis (Helander et al., 2009). EtG is also incorporated in keratinous matrices and hair analysis has become an important tool to evaluate alcohol consumption even over a period of several months (a comprehensive review of EtG hair analysis is given by Crunelle et al. (2014)). It is generally known that external contamination is one of the fundamental issues in hair analysis (Chatterton, 2015). There are manifold pathways both from the body (blood, sweat, sebum) and from the environment (external contamination) for the deposition of substances to the hair fiber. Therefore, external contamination of hair matrix with the target analyte should be carefully evaluated in hair testing. Only little evidence is presented in the literature regarding to external contamination in the context of EtG hair analysis today. Arndt et al. (2013) detected EtG in some herbal hair tonics (n = 7 out of 11 tested commercially available products) and Sporkert et al. (2012)—who also detected EtG in a herbal hair tonic—reported positive findings of EtG in hair of a person after exposition to this commercial hair tonic. However, there was only a single experiment on a single individuum. Further studies are necessary to investigate this aspect on a more representative scale and other potential sources of contamination—beside hair cosmetics—should be investigated. Since alcohol intake is commonly associated with the consumption and presence of alcoholic beverages, the beverages themselves are potential contaminants of body surfaces. The aim of this study was to investigate alcoholic beverages as external sources of EtG. MATERIALS AND METHODS Chemicals and reagents All solvents and reagents for sample preparation were of quality for analysis (p.a.) and were obtained from Merck Schuchard (Hohenbrunn, Germany). Solvents and chemicals for liquid chromatography/tandem mass spectrometry (LC/MS/MS) analysis (water, acetonitrile, formic acid) were of specified LC/MS grade (Chromasolv®) and purchased from Fluka (Munich, Germany). Stock solutions (1.0 mg/ml in methanol) of EtG and d5-EtG were obtained from Lipomed (Weil am Rhein, Germany). Calibrators and quality control samples for EtG in urine were purchased from Recipe (Munich, Germany). Alcoholic beverages and sample preparation Alcoholic beverages (n = 114) were collected from five different local stores; details reading the type and origin of the beverages are shown in Tables 1–3. Half of the samples (n = 57) were acquired in the originally sealed bottles (samples were marked individually in the data tables) and the other half (n = 57) of the samples were collected from original bottles, which had been opened for tastings before. Originally sealed bottles were opened on the day of analysis. Periods between opening and analysis of previously opened beverages (from tastings) could not be assessed, since information about the individual moment of bottle opening in the stores were not available. Samples were diluted prior to analysis: a 50-μl aliquot of each alcoholic beverage was added to a glass vial with 950 μl of an aqueous solution containing the internal standard 100 ng/ml d5-EtG. The samples were mixed thoroughly and directly analyzed by LC/MS/MS. Table 1. EtG in distilled alcoholic beverages Sample number Sample type Year Origin Alcohol by volume (%) EtG (μg/l) 1 Herbal Bitter 2005 Germany 49 n.d. 2 Herbal Bitter 2000 Germany 79 n.d. 3 Aquavit 2017 Germany 42 n.d. 4* Fruit Schnapps 2015 Mosel, Germany 40 n.d. 5 Kirschwasser 2015 Schwarzwald, Germany 40 n.d. 6* Quince Brandy 2017 Germany 40 n.d. 7 Fruit Brandy 2017 Heidelberg, Germany 40 n.d. 8* Mirabelle Brandy 2017 Oberrotweil, Germany 45 n.d. 9 Lambig 2017 Bretagne, France 40 n.d. 10* Pear Brandy 2015 Oberrotweil, Germany 42 n.d. 11 Apricot Brandy 2015 Merano, Italy 40 n.d. 12 Fruit Brandy 2017 Germany 56 n.d. 13* Dry Gin 2017 London, United Kingdom 40 n.d. 14 Gin 2017 Denmark 47 n.d. 15 Grappa 2017 Trentino, Italy 41 n.d. 16 Grappa 2015 Conegliano Veneto, Italy 39 n.d. 17 Grappa 2007 Piobesi d´Alba, Italy 40 n.d. 18* Korn 1995 Wilthen, Germany 32 n.d. 19 Korn 1995 Nordhausen, Germany 38 n.d. 20 Korn 1995 Hardenberg, Germany 38 n.d. 21 Rum 2017 Tholey, Germany 35 n.d. 22 Rum 2017 Cuba 40 n.d. 23* Rum 2017 Germany 54 n.d. 24 Rum 2017 La Réunion 37.5 n.d. 25 Rum, Sherry finish 2014 Mauritius 40 n.d. 26 Rum 2017 Chile 40 n.d. 27 Rum 2016 Havana, Cuba 40 n.d. 28* Rum 2013 Flensburg, Germany 54 n.d. 29 Rum 2015 Brasilia 40 n.d. 30 Tequila 2011 Guadalajara, Mexico 38 n.d. 31 Vodka 2012 Denmark 40 n.d. 32* Vodka 2016 Germany 40 n.d. 33 Vodka 2005 Russia 40 n.d. 34 Whiskey 2014 Kintyre, Scotland 46 n.d. 35 Whiskey 2014 India 42 n.d. 36 Bamboo spirit 2015 China 45 n.d. 37 Gin 2014 Jork, Germany 42 n.d. 38 Coffee Liqueur 2014 Mexiko 20 n.d. 39 Cranberry Liqueur 2016 Denmark 18 n.d. 40 Fruit Liqueur 2017 Germany 20 n.d. 41* Cordial 2017 Germany 30 n.d. 42* Cordial 2017 Germany 35 n.d. 43 Peach Liqueur 2017 Germany 18 n.d. 44 Apple Liqueur 2017 Germany 25 n.d. 45 Bitter Liqueur 2017 Italy 11 n.d. 46 Bitter Liqueur 2015 Milano, Italy 25 n.d. 47 Bitter Liqueur 2014 Amsterdam, The Netherlands 21 n.d. 48* Coffee Liqueur 2015 Brobergen, Germany 25 n.d. 49 Doppelbock Liqueur 2017 Germany 23 n.d. 50* Advocaat 2017 The Netherlands 15 n.d. 51* Brandy 2017 Eltville am Rhein, Germany 36 n.d. 52* Brandy 2017 Venetia, Italy 40 n.d. Sample number Sample type Year Origin Alcohol by volume (%) EtG (μg/l) 1 Herbal Bitter 2005 Germany 49 n.d. 2 Herbal Bitter 2000 Germany 79 n.d. 3 Aquavit 2017 Germany 42 n.d. 4* Fruit Schnapps 2015 Mosel, Germany 40 n.d. 5 Kirschwasser 2015 Schwarzwald, Germany 40 n.d. 6* Quince Brandy 2017 Germany 40 n.d. 7 Fruit Brandy 2017 Heidelberg, Germany 40 n.d. 8* Mirabelle Brandy 2017 Oberrotweil, Germany 45 n.d. 9 Lambig 2017 Bretagne, France 40 n.d. 10* Pear Brandy 2015 Oberrotweil, Germany 42 n.d. 11 Apricot Brandy 2015 Merano, Italy 40 n.d. 12 Fruit Brandy 2017 Germany 56 n.d. 13* Dry Gin 2017 London, United Kingdom 40 n.d. 14 Gin 2017 Denmark 47 n.d. 15 Grappa 2017 Trentino, Italy 41 n.d. 16 Grappa 2015 Conegliano Veneto, Italy 39 n.d. 17 Grappa 2007 Piobesi d´Alba, Italy 40 n.d. 18* Korn 1995 Wilthen, Germany 32 n.d. 19 Korn 1995 Nordhausen, Germany 38 n.d. 20 Korn 1995 Hardenberg, Germany 38 n.d. 21 Rum 2017 Tholey, Germany 35 n.d. 22 Rum 2017 Cuba 40 n.d. 23* Rum 2017 Germany 54 n.d. 24 Rum 2017 La Réunion 37.5 n.d. 25 Rum, Sherry finish 2014 Mauritius 40 n.d. 26 Rum 2017 Chile 40 n.d. 27 Rum 2016 Havana, Cuba 40 n.d. 28* Rum 2013 Flensburg, Germany 54 n.d. 29 Rum 2015 Brasilia 40 n.d. 30 Tequila 2011 Guadalajara, Mexico 38 n.d. 31 Vodka 2012 Denmark 40 n.d. 32* Vodka 2016 Germany 40 n.d. 33 Vodka 2005 Russia 40 n.d. 34 Whiskey 2014 Kintyre, Scotland 46 n.d. 35 Whiskey 2014 India 42 n.d. 36 Bamboo spirit 2015 China 45 n.d. 37 Gin 2014 Jork, Germany 42 n.d. 38 Coffee Liqueur 2014 Mexiko 20 n.d. 39 Cranberry Liqueur 2016 Denmark 18 n.d. 40 Fruit Liqueur 2017 Germany 20 n.d. 41* Cordial 2017 Germany 30 n.d. 42* Cordial 2017 Germany 35 n.d. 43 Peach Liqueur 2017 Germany 18 n.d. 44 Apple Liqueur 2017 Germany 25 n.d. 45 Bitter Liqueur 2017 Italy 11 n.d. 46 Bitter Liqueur 2015 Milano, Italy 25 n.d. 47 Bitter Liqueur 2014 Amsterdam, The Netherlands 21 n.d. 48* Coffee Liqueur 2015 Brobergen, Germany 25 n.d. 49 Doppelbock Liqueur 2017 Germany 23 n.d. 50* Advocaat 2017 The Netherlands 15 n.d. 51* Brandy 2017 Eltville am Rhein, Germany 36 n.d. 52* Brandy 2017 Venetia, Italy 40 n.d. Samples obtained from originally sealed bottles are marked by an asterisk. n.d., not detected. Table 1. EtG in distilled alcoholic beverages Sample number Sample type Year Origin Alcohol by volume (%) EtG (μg/l) 1 Herbal Bitter 2005 Germany 49 n.d. 2 Herbal Bitter 2000 Germany 79 n.d. 3 Aquavit 2017 Germany 42 n.d. 4* Fruit Schnapps 2015 Mosel, Germany 40 n.d. 5 Kirschwasser 2015 Schwarzwald, Germany 40 n.d. 6* Quince Brandy 2017 Germany 40 n.d. 7 Fruit Brandy 2017 Heidelberg, Germany 40 n.d. 8* Mirabelle Brandy 2017 Oberrotweil, Germany 45 n.d. 9 Lambig 2017 Bretagne, France 40 n.d. 10* Pear Brandy 2015 Oberrotweil, Germany 42 n.d. 11 Apricot Brandy 2015 Merano, Italy 40 n.d. 12 Fruit Brandy 2017 Germany 56 n.d. 13* Dry Gin 2017 London, United Kingdom 40 n.d. 14 Gin 2017 Denmark 47 n.d. 15 Grappa 2017 Trentino, Italy 41 n.d. 16 Grappa 2015 Conegliano Veneto, Italy 39 n.d. 17 Grappa 2007 Piobesi d´Alba, Italy 40 n.d. 18* Korn 1995 Wilthen, Germany 32 n.d. 19 Korn 1995 Nordhausen, Germany 38 n.d. 20 Korn 1995 Hardenberg, Germany 38 n.d. 21 Rum 2017 Tholey, Germany 35 n.d. 22 Rum 2017 Cuba 40 n.d. 23* Rum 2017 Germany 54 n.d. 24 Rum 2017 La Réunion 37.5 n.d. 25 Rum, Sherry finish 2014 Mauritius 40 n.d. 26 Rum 2017 Chile 40 n.d. 27 Rum 2016 Havana, Cuba 40 n.d. 28* Rum 2013 Flensburg, Germany 54 n.d. 29 Rum 2015 Brasilia 40 n.d. 30 Tequila 2011 Guadalajara, Mexico 38 n.d. 31 Vodka 2012 Denmark 40 n.d. 32* Vodka 2016 Germany 40 n.d. 33 Vodka 2005 Russia 40 n.d. 34 Whiskey 2014 Kintyre, Scotland 46 n.d. 35 Whiskey 2014 India 42 n.d. 36 Bamboo spirit 2015 China 45 n.d. 37 Gin 2014 Jork, Germany 42 n.d. 38 Coffee Liqueur 2014 Mexiko 20 n.d. 39 Cranberry Liqueur 2016 Denmark 18 n.d. 40 Fruit Liqueur 2017 Germany 20 n.d. 41* Cordial 2017 Germany 30 n.d. 42* Cordial 2017 Germany 35 n.d. 43 Peach Liqueur 2017 Germany 18 n.d. 44 Apple Liqueur 2017 Germany 25 n.d. 45 Bitter Liqueur 2017 Italy 11 n.d. 46 Bitter Liqueur 2015 Milano, Italy 25 n.d. 47 Bitter Liqueur 2014 Amsterdam, The Netherlands 21 n.d. 48* Coffee Liqueur 2015 Brobergen, Germany 25 n.d. 49 Doppelbock Liqueur 2017 Germany 23 n.d. 50* Advocaat 2017 The Netherlands 15 n.d. 51* Brandy 2017 Eltville am Rhein, Germany 36 n.d. 52* Brandy 2017 Venetia, Italy 40 n.d. Sample number Sample type Year Origin Alcohol by volume (%) EtG (μg/l) 1 Herbal Bitter 2005 Germany 49 n.d. 2 Herbal Bitter 2000 Germany 79 n.d. 3 Aquavit 2017 Germany 42 n.d. 4* Fruit Schnapps 2015 Mosel, Germany 40 n.d. 5 Kirschwasser 2015 Schwarzwald, Germany 40 n.d. 6* Quince Brandy 2017 Germany 40 n.d. 7 Fruit Brandy 2017 Heidelberg, Germany 40 n.d. 8* Mirabelle Brandy 2017 Oberrotweil, Germany 45 n.d. 9 Lambig 2017 Bretagne, France 40 n.d. 10* Pear Brandy 2015 Oberrotweil, Germany 42 n.d. 11 Apricot Brandy 2015 Merano, Italy 40 n.d. 12 Fruit Brandy 2017 Germany 56 n.d. 13* Dry Gin 2017 London, United Kingdom 40 n.d. 14 Gin 2017 Denmark 47 n.d. 15 Grappa 2017 Trentino, Italy 41 n.d. 16 Grappa 2015 Conegliano Veneto, Italy 39 n.d. 17 Grappa 2007 Piobesi d´Alba, Italy 40 n.d. 18* Korn 1995 Wilthen, Germany 32 n.d. 19 Korn 1995 Nordhausen, Germany 38 n.d. 20 Korn 1995 Hardenberg, Germany 38 n.d. 21 Rum 2017 Tholey, Germany 35 n.d. 22 Rum 2017 Cuba 40 n.d. 23* Rum 2017 Germany 54 n.d. 24 Rum 2017 La Réunion 37.5 n.d. 25 Rum, Sherry finish 2014 Mauritius 40 n.d. 26 Rum 2017 Chile 40 n.d. 27 Rum 2016 Havana, Cuba 40 n.d. 28* Rum 2013 Flensburg, Germany 54 n.d. 29 Rum 2015 Brasilia 40 n.d. 30 Tequila 2011 Guadalajara, Mexico 38 n.d. 31 Vodka 2012 Denmark 40 n.d. 32* Vodka 2016 Germany 40 n.d. 33 Vodka 2005 Russia 40 n.d. 34 Whiskey 2014 Kintyre, Scotland 46 n.d. 35 Whiskey 2014 India 42 n.d. 36 Bamboo spirit 2015 China 45 n.d. 37 Gin 2014 Jork, Germany 42 n.d. 38 Coffee Liqueur 2014 Mexiko 20 n.d. 39 Cranberry Liqueur 2016 Denmark 18 n.d. 40 Fruit Liqueur 2017 Germany 20 n.d. 41* Cordial 2017 Germany 30 n.d. 42* Cordial 2017 Germany 35 n.d. 43 Peach Liqueur 2017 Germany 18 n.d. 44 Apple Liqueur 2017 Germany 25 n.d. 45 Bitter Liqueur 2017 Italy 11 n.d. 46 Bitter Liqueur 2015 Milano, Italy 25 n.d. 47 Bitter Liqueur 2014 Amsterdam, The Netherlands 21 n.d. 48* Coffee Liqueur 2015 Brobergen, Germany 25 n.d. 49 Doppelbock Liqueur 2017 Germany 23 n.d. 50* Advocaat 2017 The Netherlands 15 n.d. 51* Brandy 2017 Eltville am Rhein, Germany 36 n.d. 52* Brandy 2017 Venetia, Italy 40 n.d. Samples obtained from originally sealed bottles are marked by an asterisk. n.d., not detected. Table 2. EtG in beer Sample no Sample type Year Origin Alcohol by volume (%) EtG (μg/l) 53* Kölsch 2017 Cologne, Germany 4.8 n.d. 54* Ale 2017 England 7.2 n.d. 55* Craft Beer 2017 Hamburg, Germany 6.3 n.d. 56* Helles 2017 München, Germany 5 n.d. 57* Lager Beer 2017 Budéjovice, Czech 5 n.d. 58* Lager Beer 2017 Poland 5.5 n.d. 59* Non-alcoholic Beer 2017 Krombach, Germany 0 n.d. 60* Pilsner 2017 Flensburg, Germany 4.8 n.d. 61* Pilsner 2017 Denmark 5 n.d. 62* Pilsner 2017 Bremen, Germany 4.9 n.d. 63* Pilsner 2017 Denmark 5 n.d. 64* Pilsner 2017 Pilzen, Czech 4.4 n.d. 65* Weissbier 2017 Erding, Germany 5.3 n.d. 66* Weissbier 2017 Munich, Germany 5.5 n.d. 67* Lager Beer 2017 Barcelona, Spain 5.4 n.d. 68* Weissbier 2017 Munich, Germany 5.5 n.d. 69* Beer 2017 Mexico 4.5 n.d. 70* Weissbier 2017 Bavaria, Germany 5.3 n.d. 71* Beer 2017 Vialonga, Portugal 5 n.d. 72* Non-alcoholic Beer 2017 Bremen, Germany 0 n.d. Sample no Sample type Year Origin Alcohol by volume (%) EtG (μg/l) 53* Kölsch 2017 Cologne, Germany 4.8 n.d. 54* Ale 2017 England 7.2 n.d. 55* Craft Beer 2017 Hamburg, Germany 6.3 n.d. 56* Helles 2017 München, Germany 5 n.d. 57* Lager Beer 2017 Budéjovice, Czech 5 n.d. 58* Lager Beer 2017 Poland 5.5 n.d. 59* Non-alcoholic Beer 2017 Krombach, Germany 0 n.d. 60* Pilsner 2017 Flensburg, Germany 4.8 n.d. 61* Pilsner 2017 Denmark 5 n.d. 62* Pilsner 2017 Bremen, Germany 4.9 n.d. 63* Pilsner 2017 Denmark 5 n.d. 64* Pilsner 2017 Pilzen, Czech 4.4 n.d. 65* Weissbier 2017 Erding, Germany 5.3 n.d. 66* Weissbier 2017 Munich, Germany 5.5 n.d. 67* Lager Beer 2017 Barcelona, Spain 5.4 n.d. 68* Weissbier 2017 Munich, Germany 5.5 n.d. 69* Beer 2017 Mexico 4.5 n.d. 70* Weissbier 2017 Bavaria, Germany 5.3 n.d. 71* Beer 2017 Vialonga, Portugal 5 n.d. 72* Non-alcoholic Beer 2017 Bremen, Germany 0 n.d. Samples obtained from originally sealed bottles are marked by an asterisk. n.d., not detected. Table 2. EtG in beer Sample no Sample type Year Origin Alcohol by volume (%) EtG (μg/l) 53* Kölsch 2017 Cologne, Germany 4.8 n.d. 54* Ale 2017 England 7.2 n.d. 55* Craft Beer 2017 Hamburg, Germany 6.3 n.d. 56* Helles 2017 München, Germany 5 n.d. 57* Lager Beer 2017 Budéjovice, Czech 5 n.d. 58* Lager Beer 2017 Poland 5.5 n.d. 59* Non-alcoholic Beer 2017 Krombach, Germany 0 n.d. 60* Pilsner 2017 Flensburg, Germany 4.8 n.d. 61* Pilsner 2017 Denmark 5 n.d. 62* Pilsner 2017 Bremen, Germany 4.9 n.d. 63* Pilsner 2017 Denmark 5 n.d. 64* Pilsner 2017 Pilzen, Czech 4.4 n.d. 65* Weissbier 2017 Erding, Germany 5.3 n.d. 66* Weissbier 2017 Munich, Germany 5.5 n.d. 67* Lager Beer 2017 Barcelona, Spain 5.4 n.d. 68* Weissbier 2017 Munich, Germany 5.5 n.d. 69* Beer 2017 Mexico 4.5 n.d. 70* Weissbier 2017 Bavaria, Germany 5.3 n.d. 71* Beer 2017 Vialonga, Portugal 5 n.d. 72* Non-alcoholic Beer 2017 Bremen, Germany 0 n.d. Sample no Sample type Year Origin Alcohol by volume (%) EtG (μg/l) 53* Kölsch 2017 Cologne, Germany 4.8 n.d. 54* Ale 2017 England 7.2 n.d. 55* Craft Beer 2017 Hamburg, Germany 6.3 n.d. 56* Helles 2017 München, Germany 5 n.d. 57* Lager Beer 2017 Budéjovice, Czech 5 n.d. 58* Lager Beer 2017 Poland 5.5 n.d. 59* Non-alcoholic Beer 2017 Krombach, Germany 0 n.d. 60* Pilsner 2017 Flensburg, Germany 4.8 n.d. 61* Pilsner 2017 Denmark 5 n.d. 62* Pilsner 2017 Bremen, Germany 4.9 n.d. 63* Pilsner 2017 Denmark 5 n.d. 64* Pilsner 2017 Pilzen, Czech 4.4 n.d. 65* Weissbier 2017 Erding, Germany 5.3 n.d. 66* Weissbier 2017 Munich, Germany 5.5 n.d. 67* Lager Beer 2017 Barcelona, Spain 5.4 n.d. 68* Weissbier 2017 Munich, Germany 5.5 n.d. 69* Beer 2017 Mexico 4.5 n.d. 70* Weissbier 2017 Bavaria, Germany 5.3 n.d. 71* Beer 2017 Vialonga, Portugal 5 n.d. 72* Non-alcoholic Beer 2017 Bremen, Germany 0 n.d. Samples obtained from originally sealed bottles are marked by an asterisk. n.d., not detected. Table 3. EtG in wine Sample no Sample type Year Origin Alcohol by volume (%) EtG (μg/l) 73 Red Wine, Spätburgunder 2016 Pfalz, Germany 13 3070 74 Red Wine, Canti Primitivo 2015 Puglia, Italy 12 1425 75 Red Wine, Bobos 2015 Spain 13.5 3617 76 Red Wine 2012 Bordeaux, France 14 3001 77 Red Wine 2014 Pfalz, Germany 13 2120 78 Red Wine, Primitivo 2015 Italy 14 2201 79* Red Wine, Chianti Classico 2015 Toscana, Italy 13.5 3176 80 Red Wine, Chianti 2016 Italy 12 1525 81* Red Wine, Merlot 2016 Sicilia, Italy 13 3720 82 Red Wine, Merlot 2005 Chile 13.5 3614 83 Red Wine 2016 Alentejo, Portugal 13.5 3409 84* Red Wine, Montepulciano d´Abruzzo 2015 Montepulciano, Italy 14.5 3335 85 Red Wine (Glühwein) 2017 Hamburg, Germany 12 3040 86* Red Wine, Heroldrebe 2015 Rotenberg, Germany 13 3463 87* Red Wine, Non-alcoholic 2017 Trier, Germany 0 1881 88* Red Wine, Non-alcoholic 2016 Rüdesheim, Germany 0 2714 89* Dornfelder Rosé 2015 Dornfeld, Germany 10 672 90 Tempranillo Rosado 2016 Ribera del Duero, Spain 13.5 1508 91* Sparkling Wine 2017 Germany 12.5 281 92* Sparkling Wine 2017 Freyburg, Germany 11 869 93* Sparkling Wine 2017 Trier, Germany 5 771 94* Sparkling Wine 2017 Italy 10 738 95* Sparkling Wine 2017 Maxhütte-Heudorf, Germany 11 826 96* Sparkling Wine 2017 Germany 11 307 97* Sparkling Wine 2017 Paris, France 11 1447 98* Sparkling Wine, Prosecco 2017 Venetia, Italy 10.5 463 99* Sparkling Wine, Prosecco 2017 Venetia, Italy 11 532 100* Sparkling Wine, Non-alcoholic 2017 Trier, Germany 0 1328 101 White Wine, Gutedel 2015 Freiburg, Germany 12 514 102* White Wine, Sauvignon Blanc 2017 Western Cape, South Africa 12.5 1207 103 White Wine 2015 France 14 646 104 White Wine, Grecanico 2016 Sicilia, Italy 13 1685 105 White Wine, Sauvignon 2015 Centre-val de Loire, France 12.5 665 106 White Wine, Grauer Burgunder 2016 Bad Dürkheim, Germany 13 1081 107 White Wine, Pinot Grigio 2016 Italy 12.5 1028 108* White Wine, Sauvignon Blanc 2017 South Africa 12,5 546 109 White Wine, Chardonnay 2017 Petersbach, France 12.5 1452 110 White Wine, Riesling 2004 Mosel-Saar, Germany 11.5 1500 111 White Wine, Pinot Grigio 2003 Veneto, Italy 11.5 347 112* White Wine, Chardonnay 2015 South Africa 12 1071 113* White Wine, Riesling 2016 Rheingau, Germany 12 1057 114* White Wine, Riesling Cabinet 2011 Flein-Talheim, Germany 11 1265 Sample no Sample type Year Origin Alcohol by volume (%) EtG (μg/l) 73 Red Wine, Spätburgunder 2016 Pfalz, Germany 13 3070 74 Red Wine, Canti Primitivo 2015 Puglia, Italy 12 1425 75 Red Wine, Bobos 2015 Spain 13.5 3617 76 Red Wine 2012 Bordeaux, France 14 3001 77 Red Wine 2014 Pfalz, Germany 13 2120 78 Red Wine, Primitivo 2015 Italy 14 2201 79* Red Wine, Chianti Classico 2015 Toscana, Italy 13.5 3176 80 Red Wine, Chianti 2016 Italy 12 1525 81* Red Wine, Merlot 2016 Sicilia, Italy 13 3720 82 Red Wine, Merlot 2005 Chile 13.5 3614 83 Red Wine 2016 Alentejo, Portugal 13.5 3409 84* Red Wine, Montepulciano d´Abruzzo 2015 Montepulciano, Italy 14.5 3335 85 Red Wine (Glühwein) 2017 Hamburg, Germany 12 3040 86* Red Wine, Heroldrebe 2015 Rotenberg, Germany 13 3463 87* Red Wine, Non-alcoholic 2017 Trier, Germany 0 1881 88* Red Wine, Non-alcoholic 2016 Rüdesheim, Germany 0 2714 89* Dornfelder Rosé 2015 Dornfeld, Germany 10 672 90 Tempranillo Rosado 2016 Ribera del Duero, Spain 13.5 1508 91* Sparkling Wine 2017 Germany 12.5 281 92* Sparkling Wine 2017 Freyburg, Germany 11 869 93* Sparkling Wine 2017 Trier, Germany 5 771 94* Sparkling Wine 2017 Italy 10 738 95* Sparkling Wine 2017 Maxhütte-Heudorf, Germany 11 826 96* Sparkling Wine 2017 Germany 11 307 97* Sparkling Wine 2017 Paris, France 11 1447 98* Sparkling Wine, Prosecco 2017 Venetia, Italy 10.5 463 99* Sparkling Wine, Prosecco 2017 Venetia, Italy 11 532 100* Sparkling Wine, Non-alcoholic 2017 Trier, Germany 0 1328 101 White Wine, Gutedel 2015 Freiburg, Germany 12 514 102* White Wine, Sauvignon Blanc 2017 Western Cape, South Africa 12.5 1207 103 White Wine 2015 France 14 646 104 White Wine, Grecanico 2016 Sicilia, Italy 13 1685 105 White Wine, Sauvignon 2015 Centre-val de Loire, France 12.5 665 106 White Wine, Grauer Burgunder 2016 Bad Dürkheim, Germany 13 1081 107 White Wine, Pinot Grigio 2016 Italy 12.5 1028 108* White Wine, Sauvignon Blanc 2017 South Africa 12,5 546 109 White Wine, Chardonnay 2017 Petersbach, France 12.5 1452 110 White Wine, Riesling 2004 Mosel-Saar, Germany 11.5 1500 111 White Wine, Pinot Grigio 2003 Veneto, Italy 11.5 347 112* White Wine, Chardonnay 2015 South Africa 12 1071 113* White Wine, Riesling 2016 Rheingau, Germany 12 1057 114* White Wine, Riesling Cabinet 2011 Flein-Talheim, Germany 11 1265 Samples obtained from originally sealed bottles are marked by an asterisk. Table 3. EtG in wine Sample no Sample type Year Origin Alcohol by volume (%) EtG (μg/l) 73 Red Wine, Spätburgunder 2016 Pfalz, Germany 13 3070 74 Red Wine, Canti Primitivo 2015 Puglia, Italy 12 1425 75 Red Wine, Bobos 2015 Spain 13.5 3617 76 Red Wine 2012 Bordeaux, France 14 3001 77 Red Wine 2014 Pfalz, Germany 13 2120 78 Red Wine, Primitivo 2015 Italy 14 2201 79* Red Wine, Chianti Classico 2015 Toscana, Italy 13.5 3176 80 Red Wine, Chianti 2016 Italy 12 1525 81* Red Wine, Merlot 2016 Sicilia, Italy 13 3720 82 Red Wine, Merlot 2005 Chile 13.5 3614 83 Red Wine 2016 Alentejo, Portugal 13.5 3409 84* Red Wine, Montepulciano d´Abruzzo 2015 Montepulciano, Italy 14.5 3335 85 Red Wine (Glühwein) 2017 Hamburg, Germany 12 3040 86* Red Wine, Heroldrebe 2015 Rotenberg, Germany 13 3463 87* Red Wine, Non-alcoholic 2017 Trier, Germany 0 1881 88* Red Wine, Non-alcoholic 2016 Rüdesheim, Germany 0 2714 89* Dornfelder Rosé 2015 Dornfeld, Germany 10 672 90 Tempranillo Rosado 2016 Ribera del Duero, Spain 13.5 1508 91* Sparkling Wine 2017 Germany 12.5 281 92* Sparkling Wine 2017 Freyburg, Germany 11 869 93* Sparkling Wine 2017 Trier, Germany 5 771 94* Sparkling Wine 2017 Italy 10 738 95* Sparkling Wine 2017 Maxhütte-Heudorf, Germany 11 826 96* Sparkling Wine 2017 Germany 11 307 97* Sparkling Wine 2017 Paris, France 11 1447 98* Sparkling Wine, Prosecco 2017 Venetia, Italy 10.5 463 99* Sparkling Wine, Prosecco 2017 Venetia, Italy 11 532 100* Sparkling Wine, Non-alcoholic 2017 Trier, Germany 0 1328 101 White Wine, Gutedel 2015 Freiburg, Germany 12 514 102* White Wine, Sauvignon Blanc 2017 Western Cape, South Africa 12.5 1207 103 White Wine 2015 France 14 646 104 White Wine, Grecanico 2016 Sicilia, Italy 13 1685 105 White Wine, Sauvignon 2015 Centre-val de Loire, France 12.5 665 106 White Wine, Grauer Burgunder 2016 Bad Dürkheim, Germany 13 1081 107 White Wine, Pinot Grigio 2016 Italy 12.5 1028 108* White Wine, Sauvignon Blanc 2017 South Africa 12,5 546 109 White Wine, Chardonnay 2017 Petersbach, France 12.5 1452 110 White Wine, Riesling 2004 Mosel-Saar, Germany 11.5 1500 111 White Wine, Pinot Grigio 2003 Veneto, Italy 11.5 347 112* White Wine, Chardonnay 2015 South Africa 12 1071 113* White Wine, Riesling 2016 Rheingau, Germany 12 1057 114* White Wine, Riesling Cabinet 2011 Flein-Talheim, Germany 11 1265 Sample no Sample type Year Origin Alcohol by volume (%) EtG (μg/l) 73 Red Wine, Spätburgunder 2016 Pfalz, Germany 13 3070 74 Red Wine, Canti Primitivo 2015 Puglia, Italy 12 1425 75 Red Wine, Bobos 2015 Spain 13.5 3617 76 Red Wine 2012 Bordeaux, France 14 3001 77 Red Wine 2014 Pfalz, Germany 13 2120 78 Red Wine, Primitivo 2015 Italy 14 2201 79* Red Wine, Chianti Classico 2015 Toscana, Italy 13.5 3176 80 Red Wine, Chianti 2016 Italy 12 1525 81* Red Wine, Merlot 2016 Sicilia, Italy 13 3720 82 Red Wine, Merlot 2005 Chile 13.5 3614 83 Red Wine 2016 Alentejo, Portugal 13.5 3409 84* Red Wine, Montepulciano d´Abruzzo 2015 Montepulciano, Italy 14.5 3335 85 Red Wine (Glühwein) 2017 Hamburg, Germany 12 3040 86* Red Wine, Heroldrebe 2015 Rotenberg, Germany 13 3463 87* Red Wine, Non-alcoholic 2017 Trier, Germany 0 1881 88* Red Wine, Non-alcoholic 2016 Rüdesheim, Germany 0 2714 89* Dornfelder Rosé 2015 Dornfeld, Germany 10 672 90 Tempranillo Rosado 2016 Ribera del Duero, Spain 13.5 1508 91* Sparkling Wine 2017 Germany 12.5 281 92* Sparkling Wine 2017 Freyburg, Germany 11 869 93* Sparkling Wine 2017 Trier, Germany 5 771 94* Sparkling Wine 2017 Italy 10 738 95* Sparkling Wine 2017 Maxhütte-Heudorf, Germany 11 826 96* Sparkling Wine 2017 Germany 11 307 97* Sparkling Wine 2017 Paris, France 11 1447 98* Sparkling Wine, Prosecco 2017 Venetia, Italy 10.5 463 99* Sparkling Wine, Prosecco 2017 Venetia, Italy 11 532 100* Sparkling Wine, Non-alcoholic 2017 Trier, Germany 0 1328 101 White Wine, Gutedel 2015 Freiburg, Germany 12 514 102* White Wine, Sauvignon Blanc 2017 Western Cape, South Africa 12.5 1207 103 White Wine 2015 France 14 646 104 White Wine, Grecanico 2016 Sicilia, Italy 13 1685 105 White Wine, Sauvignon 2015 Centre-val de Loire, France 12.5 665 106 White Wine, Grauer Burgunder 2016 Bad Dürkheim, Germany 13 1081 107 White Wine, Pinot Grigio 2016 Italy 12.5 1028 108* White Wine, Sauvignon Blanc 2017 South Africa 12,5 546 109 White Wine, Chardonnay 2017 Petersbach, France 12.5 1452 110 White Wine, Riesling 2004 Mosel-Saar, Germany 11.5 1500 111 White Wine, Pinot Grigio 2003 Veneto, Italy 11.5 347 112* White Wine, Chardonnay 2015 South Africa 12 1071 113* White Wine, Riesling 2016 Rheingau, Germany 12 1057 114* White Wine, Riesling Cabinet 2011 Flein-Talheim, Germany 11 1265 Samples obtained from originally sealed bottles are marked by an asterisk. Liquid chromatography/tandem mass spectrometry A well-established, validated and accredited LC/MS/MS approach, which has been used for routine EtG urinalysis for more than 5 years, was applied for the analysis of beverages with slight modifications. Chromatography was performed by Waters Acquity® UPLC with a C18 Acquity® HSS T3, 1.7 μm, 2.1 × 150 mm column at 40°C. The injection volume of the samples was 5 μl, the flow rate at 0.3 ml/min and the binary gradient as follows: 0–3.8 min: 2–30% B, 4.0–6.9 min: 2% B. Mobile phase A consisted of water with 0.1% formic acid and mobile phase B was acetonitrile with 0.1% formic acid. A Waters XEVO®TQ-S triple quadrupole mass analyzer operating in multiple reaction monitoring was used for detection, five transitions were monitored: EtG: m/z 221 > 75 (quantifier), m/z 221 > 85, m/z 221 > 57, m/z 221 > 113 (qualifiers), d5-EtG (internal standard I.S.) m/z 226 > 85. The assay was routinely calibrated using six commercially available standards of spiked urine (80, 191, 582, 1464, 4873 and 9621 ng/ml) and there were also commercially available quality control samples (111, 518 and 2052 pg/mg) included in each analytical series. For the analysis of beverages, additionally aqueous calibrators of EtG (50, 100, 200, 500, 1000, 2000 and 5000 ng/ml) were prepared in house from an independent source and analyzed by the LC/MS/MS procedure. Calibration derived from those aqueous standards was congruent with the routinely used calibration from urinary calibrators. Validation parameters including accuracy, interferences, linearity of calibration, matrix effects and in-process stability complied with the international standards. Matrix effects in alcoholic beverages were assessed at two concentration levels (corresponding to QC1 and QC3) by the procedure described by Matuszewski et al. (2003). Comparable absolute signal areas and signal variabilities (difference <15%) were observed in different EtG-spiked beverages (n = 10) and EtG-spiked aqueous solutions. Matrix effects in wine could not be assessed by this procedure (EtG was present in every sample), but analysis did not raise suspicion to serious matrix effects: (a) predilution of the samples did not affect quantitative results (in case of strong ion suppression, non-linear decrease of analyte signal and impact on quantitative result is commonly observed by dilution of the sample) and (b) signal areas of internal standard d5-EtG (which elutes very closely to EtG) were also unchanged in samples from wine. The limit of detection of the analytical method was 5 ng/ml and the lower limit of quantification was 17 ng/ml. Accuracy was assessed at three concentration levels (corresponding to QC1-3) on different days. Intraday coefficient of variation was 5% (QC1), 3% (QC2) and 3%(QC3), interday coefficient of variation was 8% (QC1), 4% (QC2) and 4% (QC3). Regular external quality control is performed by periodical proficiency testing. RESULTS EtG was not detected in distilled alcoholic beverages (Table 1). This was consistent for all analyzed samples of rum (n = 9), whisky (n = 2), grappa (n = 3), korn (n = 3), brandy (n = 2), fruit brandy (n = 9), gin (n = 3), vodka (n = 3), herbal bitters (n = 3), tequila (n = 1), bamboo spirit (n = 1) and liqueurs (n = 13). EtG was also not detected in any of the analyzed beers (n = 20, Table 2), regardless to type (e.g. pilsner, weissbeer, ale) and origin (e.g. Mexico, Czech, Denmark, Spain). In contrast, EtG was detected in every of the analyzed samples of wine (n = 42) without exception. EtG concentrations in wine ranged from 281 to 3720 μg/l and different quantitative results were observed for the different types of wine. The results are box plotted in Fig. 1 and individual EtG concentrations are listed in Table 3. EtG concentrations in red wine were significantly higher (P < 0.001) than in white wine and sparkling wine. As shown in Fig. 2, this was also observed for the EtG/alcohol ratio of wine, differences in EtG concentrations were not compensated by differences in alcohol content (red wine: average 13.2%, range 12–14.5%; white wine: average 12.3%, range 11–14%; sparkling wine: average 10.3%, range 5–12.5%). EtG was also present in ‘non-alcoholic’ wine. EtG concentrations in wine in relation to vintages (except to sparkling wine (2017 n = 10), samples of wine derived from different vintages (2017 n = 5, 2016 n = 10, 2015 n = 11, 2014 n = 1, 2012 n = 1, 2011 n = 1, 2005 n = 1, 2004 n = 1 and 2003 n = 1)) are presented in Fig. 3. There was no obvious correlation between vintages and EtG concentrations and no extraordinary findings in very aged wine (2005 n = 1, 2004 n = 1 and 2003 n = 1). Differences in EtG concentrations of wine from previously opened and originally sealed bottles were not observed (Fig. 4). Fig. 1. View largeDownload slide Boxplots of EtG concentrations in wine. Fig. 1. View largeDownload slide Boxplots of EtG concentrations in wine. Fig. 2. View largeDownload slide Boxplots of EtG/alcohol by volume ratios in wine. Fig. 2. View largeDownload slide Boxplots of EtG/alcohol by volume ratios in wine. Fig. 3. View largeDownload slide Comparison of EtG concentrations and vintages of wine. Triangles: red wine, dots: white wine and diamonds: sparkling wine. Fig. 3. View largeDownload slide Comparison of EtG concentrations and vintages of wine. Triangles: red wine, dots: white wine and diamonds: sparkling wine. Fig. 4. View largeDownload slide Comparison of EtG concentrations found in samples from originally sealed and previously opened bottles. Triangles: red wine, dots: white wine and diamonds: sparkling wine. Fig. 4. View largeDownload slide Comparison of EtG concentrations found in samples from originally sealed and previously opened bottles. Triangles: red wine, dots: white wine and diamonds: sparkling wine. DISCUSSION Since its detection more than 50 years ago, a substantial amount of scientific studies regarding the formation and kinetics of EtG in the human body has been published in the literature. Formation of EtG is characterized by the conjugation of uridine-5`-diphospho (UDP)-glucuronic acid to ethanol. This is catalyzed by the superfamily of UDP-glucuronosyltransferases (UGTs), and it was found that multiple isoforms of UGT (predominantly UGT1A1 and UGT2B7) are responsible for the formation of EtG in human liver microsomes (Foti and Fisher, 2005). However, only marginal data are available regarding exogenous sources of EtG from outside the human body. Helander et al. (2007) reported possible post-collection synthesis of EtG in urine samples contaminated by bacteria, which was observed as well as bacterial degradation of EtG. Politi et al. (2005) reported positive testings of EtG in Italian wine (n = 8). Sporkert et al. (2012) detected EtG (2.7 mg/l) in an alcoholic (44% ethanol by volume) plant extract, which was used as a hair tonic and finally caused a positive finding of EtG in hair of an individuum (n = 1) after external application. Arndt et al. (2013) identified EtG (0.07–1.06 mg/l) in herbal hair tonics, which also contained ethanol (21–39% by volume). Sporkert et al. (2014) examined different ethanolic extracts from several different plants (n = 17), EtG was confirmed by mass spectrometry in four ethanolic plant extracts (thyme, birch, oak and plantain). Baumgartner et al. (2013) discussed a nucleophilic reaction during hydro-ethanolic plant extraction as the principle mechanism of EtG formation. However, ethanolic herbal preparations (e.g. cordial liqueurs, bitters) were tested negative for EtG in the presented study. In contrast, EtG was consistently found in wine, which is finally derived from the preparation of fruit. This is in accordance with the findings of Politi et al. (2005), who also found EtG in Italian wine (n = 8), but not in samples from beer (n = 4). The underlying biochemical mechanism is still unclear and will be examined in further studies, but the results of the presented work suggest that EtG formation rather takes place in early steps of wine production (disintegration, mashing and fermentation of grapes) than during wine storage. This could probably explain the obvious differences in EtG concentrations of white and red wine and is additionally supported by the following observations: (a) there were comparable EtG concentrations in wine of different ages, (b) differences in EtG concentrations were not compensated by differences in alcohol concentrations and (c) EtG was also present in non-alcoholic wine (which is fermented before the reduction of ethanol and the subsequent storage). Preliminary results from the analysis of grapes, grape juices and other products derived from grapes (data not presented) suggest that EtG is not an ingredient of the grapes themselves. Microbial activity of yeast or bacteria during fermentation even does not seem to be the only crucial step of EtG formation, since EtG was not detected in any of the analyzed beer samples. Further studies are necessary to investigate if there is any impact of the results on EtG biomarker analysis. Hoiseth et al. (2010) did not observe positive EtG testings in urine of 12 subjects after the consumption of 0.75 l non-alcoholic wine, which contained 3 mg/l of EtG, so the impact of externally supplied EtG on urinary EtG seems to be of minor importance. Otherwise, the investigations of Sporkert et al. (2012) strongly point out that EtG hair testings can be affected by external exposition to EtG: the application of a herbal hair tonic containing 2.7 mg/l EtG lead to an significant increase of hair EtG concentration of an individuum (n = 1) and a possible misinterpretation of the drinking behavior. Although it has not yet been tested, it can be easily assumed that external application of wine (containing EtG comparable to the described herbal hair tonic) to hair could falsify hair EtG results in a similar manner. Although this is a rather uncommon exposition setting (beside the famous ‘champagne shower’, red wine is used as a hair colorant in combination with henna and as an ingredient in natural hair cosmetics), the impact of incidental and unintended external exposures should be considered. This is addressed to a future study of a population of abstinent cooks and wine stewards. It is the authors’ own experience and also reported by others (Kintz and Nicholson, 2014) that unexpected positive results of hair EtG analysis are sometimes observed, which are in contradiction to all other relevant factors of a case and, therefore, raise reasonable suspicion to external contamination. Since an application of herbal hair tonics or other cosmetic hair treatments are frequently negated in those cases, further investigations of external contamination in EtG hair analysis are necessary. CONFLICT OF INTEREST STATEMENT None declared. REFERENCES Arndt T , Schröfel S , Stemmerich K . ( 2013 ) Ethyl glucuronide identified in commercial hair tonics . Forensic Sci Int 231 : 195 – 8 . Google Scholar CrossRef Search ADS PubMed Baumgartner MR , Binz TM , Schwanninger A , et al. . ( 2013 ) Ethyl glucuronide in hair tonics: formation of EtG during hydro-ethanolic extraction of plant material for cosmetic products, TIAFT 51st Meeting, Madeira, Abstract book OM3, pp. 63–4. Chatterton G . ( 2015 ) External contamination: still a debate? In Kintz P , Salomone A , Vincenti M (eds) . Hair Analysis in Clinical and Forensic Toxicology . New York : Academic Press , 47 – 70 . Google Scholar CrossRef Search ADS Crunelle CL , Yegles M , van Nuijs ALN , et al. . ( 2014 ) Hair ethyl glucuronide levels as a marker for alcohol use and abuse: a review of the current state of the art . Drug Alcohol Depend 134 : 1 – 11 . Google Scholar CrossRef Search ADS PubMed Foti RS , Fisher MB . ( 2005 ) Assessment of UDP-glucuronosyltransferase catalyzed formation of ethyl glucuronide in human liver microsomes and recombinant UGTs . Forensic Sci Int 153 : 109 – 16 . Google Scholar CrossRef Search ADS PubMed Helander A , Böttcher M , Fehr C , et al. . ( 2009 ) Detection times for urinary ethyl glucuronide and ethyl sulfate in heavy drinkers during alcohol detoxification . Alcohol Alcohol 44 : 55 – 61 . Google Scholar CrossRef Search ADS PubMed Helander A , Olsson I , Dahl H . ( 2007 ) Postcollection synthesis of ethyl glucuronide by bacteria in urine may cause false identification of alcohol consumption . Clin Chem 53 : 1855 – 7 . Google Scholar CrossRef Search ADS PubMed Hoiseth G , Yttredal B , Karinen R , et al. . ( 2010 ) Levels of ethyl glucuronide and ethyl sulfate in oral fluid, blood, and urine after use of mouthwash and ingestion of nonalcoholic wine . J Anal Toxicol 34 : 84 – 8 . Google Scholar CrossRef Search ADS PubMed Kintz P , Nicholson D . ( 2014 ) Testing for ethanol markers in hair: discrepancies after simultaneous quantification of ethyl glucuronide and fatty acid ethyl esters . Forensic Sci Int 243 : 44 – 6 . Google Scholar CrossRef Search ADS PubMed Matuszewski BK , Constanzer ML , Chavez-Eng CM . ( 2003 ) Strategies for the assessment of matrix effect in quantitative bioanalytical methods based on HPLC-MS/MS . Anal Chem 75 : 3019 – 30 . Google Scholar CrossRef Search ADS PubMed Politi L , Morini L , Groppi A , et al. . ( 2005 ) Direct determination of the ethanol metabolites ethyl glucuronide and ethyl sulfate in urine by liquid chromatography/electrospray tandem mass spectrometry . Rapid Commun Mass Spectrom 19 : 1321 – 31 . Google Scholar CrossRef Search ADS PubMed Sporkert F , Kharbouche H , Augsburger MP , et al. . ( 2012 ) Positive EtG findings in hair as a result of a cosmetic treatment . Forensic Sci Int 218 : 97 – 100 . Google Scholar CrossRef Search ADS PubMed Sporkert F , Sizun A , Giroud C . ( 2014 ) Ethyl glucuronide in plant extracts . Toxicol Anal Clin 26 : 11 . © The Author(s) 2018. Medical Council on Alcohol and Oxford University Press. All rights reserved. This article is published and distributed under the terms of the Oxford University Press, Standard Journals Publication Model (https://academic.oup.com/journals/pages/about_us/legal/notices)

Journal

Alcohol and AlcoholismOxford University Press

Published: May 26, 2018

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