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Urine tested positive for ethyl glucuronide and ethyl sulphate after the consumption of “non-alcoholic” beer
Forensic Science International 202 (2010) 82–85
Contents lists available at ScienceDirect
Forensic Science International journal homepage: www.elsevier.com/locate/forsciint
Urine tested positive for ethyl glucuronide and ethyl sulphate after the consumption of ‘‘non-alcoholic’’ beer Annette Thierauf a,*, Heike Gnann a, Ariane Wohlfarth a, Volker Auwa¨rter a, Markus Große Perdekamp a, Klaus-Juergen Buttler b, Friedrich M. Wurst c, Wolfgang Weinmann d a
Institute of Forensic Medicine, Freiburg University Medical Centre, Albertstrasse 9, 79104 Freiburg, Germany Department of General Neurosurgery, Freiburg University Medical Centre, Freiburg, Germany Christian-Doppler-Klinik, University Clinic for Psychiatry II, Salzburg, Austria d Institute of Forensic Medicine, University of Bern, Bern, Switzerland b c
A R T I C L E I N F O
A B S T R A C T
Article history: Received 10 March 2010 Received in revised form 14 April 2010 Accepted 18 April 2010 Available online 8 May 2010
In abstinence maintenance programs, for reissuing the driving licence and in workplace monitoring programs abstinence from ethanol and its proof are demanded. Various monitoring programs that mainly use ethyl glucuronide (EtG) as alcohol consumption marker have been established. To abstain from ethanol, but not from the taste of alcoholic beverages, in particular non-alcoholic beer has become more and more popular. In Germany, these ‘‘alcohol-free’’ beverages may still have an ethanol content of up to 0.5 vol.% without the duty of declaration. Due to severe negative consequences resulting from positive EtG tests, a drinking experiment with 2.5 L of non-alcoholic beer per person was performed to address the question of measurable concentrations of the direct metabolites EtG and EtS (ethyl sulphate) in urine and blood. Both alcohol consumption markers – determined by LC–MS/MS – were found in high concentrations: maximum concentrations in urine found in three volunteers were EtG 0.30–0.87 mg/L and EtS 0.04– 0.07 mg/L, i.e., above the often applied cut-off value for the proof of abstinence of 0.1 mg EtG/L. In the urine samples of one further volunteer, EtG and EtS concentrations cumulated over-night and reached up to 14.1 mg/L EtG and 16.1 mg/L EtS in the next morning’s urine. Ethanol concentrations in blood and urine samples were negative (determined by HS-GC–FID and by an ADH-based method). ß 2010 Published by Elsevier Ireland Ltd.
Keywords: Ethanol Ethyl glucuronide Ethyl sulphate Non-alcoholic beer
1. Introduction Alcoholism is in many countries all around the world a common problem that extends into various areas of life, such as business and private life and the participation in the road traffic. Due to the incompatibility of ethanol consumption and work as well driving a vehicle, abstinence is demanded from people who suffer from alcoholism or have driven under the influence of high alcohol concentrations. To remain in employment or for reissuing a driver’s licence, workplace monitoring programs and Medical Psychological Assessments (MPA) for alcoholised drivers have been established [1]. Within these programs, abstinence must be proven for a certain period of time. Abstinence can for example be substantiated by the nondetectability of alcohol consumption markers like ethyl glucuronide (EtG) and ethyl sulphate (EtS) [2,3]. The proof of
* Corresponding author. Tel.: +49 761 203 6836; fax: +49 761 203 6858. E-mail address: [email protected] (A. Thierauf). 0379-0738/$ – see front matter ß 2010 Published by Elsevier Ireland Ltd. doi:10.1016/j.forsciint.2010.04.031
abstinence by the absence of EtG and EtS also takes place in withdrawal maintenance programs in psychiatric hospitals. In the organism, ethanol is not only metabolised in the liver by a two-stage oxidation process, first to acetaldehyde by alcohol dehydrogenase, further to acetic acid by aldehyde dehydrogenase, but also converted into conjugation products like EtG and EtS [4– 6]. They supply evidence for ethanol uptake with high sensitivity and specificity [7], and it was published that even very small amounts of ethanol can lead to detectable concentrations of EtG and EtS in urine [8–10]. In Germany, new regulations and guidelines regarding the driver’s licence and MPA programs stipulate that the monitored persons have to obtain six EtG negative urine test results per year or four negative results per half-year. The dates of sampling have to be unpredictable for the tested person, and the sampling has to happen within 1 day after the invitation to the test. For various purposes and in different countries, the recent cut-off value for these monitoring programs is 0.1 mg EtG/L urine [9]. To abstain from ethanol, but not from the taste of alcoholic beverages, in particular non-alcoholic beer is becoming more and
A. Thierauf et al. / Forensic Science International 202 (2010) 82–85
more popular. Non-alcoholic wine, non-alcoholic sparkling wine and other alcohol-free drinks are also available, but less common. In Germany, ethanol has to be declared in beverages above a content of 0.5 vol.%. Depending on the production process, low amounts of ethanol may be present in non-alcoholic beer. One way to produce a ‘‘non-alcoholic’’ beer is to extract ethanol by dialysis after the completed brewing process [11,12]. Another possibility is the use of complex maltose and lower temperatures during the fermentation and to stop the process of fermentation at low ethanol contents [13,14]. Recently the utilisation of mutated yeast further reduced the alcohol formation during fermentation [15,16]. Due to the aforementioned use of alcohol markers for the proof of abstinence, it is an important question, whether the consumption of non-alcoholic beer can lead to measurable blood alcohol concentrations and to detectable EtG concentrations in urine. This question was addressed in this investigation by a drinking experiment with non-alcoholic beer that is known to have a residual content of ethanol. 2. Materials and methods 2.1. Experimental set-up The drinking experiment was performed as follows: after at least 5 days of abstinence from alcoholic beverages and the collection of a void urine and blood sample, four volunteers (two males (30 y/91 kg, 23 y/54 kg), two females (30 y/ 67 kg, 27 y/59 kg), all of them classified as social drinkers) drank 2.5 l of nonalcoholic beer within 1 h about 4 h after a light lunch. One pretzel per person was eaten during this drinking period. This is in everyday life an uncommonly large drinking amount, but was chosen to gain as high ethanol concentrations as possible. For 20 h after the beginning of the beer consumption urine samples were collected and stored at 20 8C prior to analysis for EtG and EtS and at 4 8C prior to ethanol
83
determination. Urine samples were analysed for ethanol, EtG, EtS and creatinine. Blood samples were taken every 20–30 min up to 3.5 h after the beginning of the drinking experiment. The blood samples were analysed for ethanol, EtG and EtS. 2.2. Chemicals and instrumentation EtG and pentadeuterated EtG (D5-EtG) were obtained from Medichem (Steinenbronn, Germany). Sodium ethyl sulphate was purchased from ABCR (Karlsruhe, Germany). Deuterated ethyl sulphate (D5-EtS) was synthesised by an inhouse procedure with a purity of >99.9% detected by LC–MS/MS [17]. Acetonitrile and formic acid were obtained from Merck (Darmstadt, Germany). All solvents were of analytical or HPLC grade. Mass spectrometric analysis was performed by a liquid chromatography–tandem mass spectrometry (LC–MS/MS) system consisting of an API 365 triple quadrupole tandem mass spectrometer (Applied Biosystems, Darmstadt, Germany) with a Turbo IonSpray interface, an HPLC gradient system (Shimadzu 10AD, Shimazu, Duisburg, Germany) and a polar-endcapped phenylpropyl reversed phase column (Synergi Polar-RP 250 mm 2 mm, 4 mm) with a guard column (4 mm 2 mm, packing material identical; Phenomenex, Aschaffenburg, Germany). The ethanol concentrations in urine, blood and beer were measured using HSGC–FID (headspace gas chromatography/flame ionisation detector) using t-butanol as internal standard and additionally – as required by German forensic guidelines – with an ADH-based method (DRI alcohol dehydrogenase assay, Thermofisher Scientific, Dreieich, Germany). Ethanol determination was performed using linear calibration with 0.1, 0.2, 0.5, 1, 2, 3, 4, 5 g/L aqueous ethanol solutions for HS-GC/ FID. The lower limit of quantitation (LLOQ) was the lowest calibrator’s concentration (0.1 g/L or 0.08 g/kg for blood). Creatinine was determined by using the Jaffe´ reaction (DRI1 Creatinine-Detect1 Test) with a Konelab 30i analyzer (Thermofisher Scientific). The study had been approved by the ethics committee of the Freiburg University (285/09). 2.3. Sample preparation for EtG and EtS quantification by LC–MS/MS 0.1 mg D5-EtG and a constant amount of D5-EtS synthesised by an in-house procedure [17] were added as internal standards to 100 mL of urine or serum.
Fig. 1. (a–d) EtG100 and EtS100 concentrations of the four participants.
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A. Thierauf et al. / Forensic Science International 202 (2010) 82–85
250 mL of methanol was added for protein precipitation and the samples were centrifuged for 10 min at 13000 rpm. 270 mL of the supernatant was separated and evaporated in a vacuum centrifuge. The dried extracts were reconstituted with 140 mL of 0.1% aqueous formic acid, and 10 mL aliquots were injected into the LC– MS/MS system. HPLC separation was performed at 40 8C with isocratic elution (0.1% formic acid) with a flow rate of 0.2 mL/min. To increase the volatility of the eluent, acetonitrile was added post-column at a flow rate of 0.2 mL/min via a T-union. EtG and EtS were analysed in one run by a validated MS/MS-method with electrospray ionisation (ESI) [18,19]. The method was calibrated from 0.05 to 50 mg/L for EtG and 0.04 to 50 mg/L for EtS. The lowest calibration level corresponds to the lowest limit of quantitation (LLOQ) in urine. Values below the LLOQ were reported as negative. The following MS/MS transitions with precursor ion/product ion were used: EtG: m/z 221/75 as quantifier, m/z 221/85 as first qualifier, and 221/203, 221/113 as additional qualifiers, m/z 226/75 for the deuterated internal standard. EtS: m/z 125/ 97 as quantifier, m/z 125/80 as qualifier and m/z 130/98 for the deuterated internal standard. Criteria for compound identification were based on forensic guidelines which require a relative ion-intensity ratio of qualifier-to-quantifier area intensity of 20% compared to a reference compound [17,18]. The concentrations were normalised to 100 mg/dL creatinine.
The ethanol content of the beer was determined in a repeated determination for each of the four lots of beer and was 3.1 and 3.2 g/kg (equal to 0.41 and 0.42 vol.%). The ingested amount of ethanol after drinking 2.5 L of ‘‘non-alcoholic’’ beer is about 8.3 g of ethanol and equals about one shot of hard liquor or 26 mL of a 40 vol.% spirit. Ethanol concentrations in urine and blood were below LLOQ (<0.08 g/kg).
the legal limit for alcohol-free beverages, but not a negligible amount of ethanol. One litre of such a beer contains approximately 3.3 g ethanol. In former studies, the ethanol amount of 1 g – contained in less than one bottle of ‘‘non-alcoholic’’ beer – already led to positive EtG and EtS findings in urine for a few hours after drinking [10]. Also in this setting with a common and usual beverage, the ethanol metabolites EtG and EtS could be detected in concentrations that were clearly positive and above the presently applied cut-off value of 0.1 mg/L in urine, which may have negative consequences for monitored persons. As a consequence, individuals that are under any kind of monitoring for alcohol markers have to be educated about the possible presence of ethanol in some food products, beverages and other products, and in particularly of those beverages that are declared as ‘‘non-alcoholic’’, in order to avoid negative implications. Interestingly, one participant explicitly exceeded the EtG and EtS concentrations of the other volunteers; the unexpectedly high concentrations were detected about 17 h after the drinking period, in the next morning’s urine sample. Accumulation of EtG and EtS in this urine sample is one possible explanation, since the period of time between the previous sample and the morning sample is 14 h. Another plausible explanation for this finding would be further ethanol uptake after the experimental set-up. This was denied in a detailed interview. Not only were the measured concentrations remarkable in this participant, but also the ratio between EtG and EtS with higher concentrations of EtS than EtG. The authors lack an explanation for this occurrence; further examination of the metabolism of this participant will be performed.
3.2. Ethyl glucuronide/ethyl sulphate
5. Conclusions
In urine, the non-normalised peak concentrations of EtG of the participants ranged from 0.30 to 14.1 mg/L and after normalisation from 0.49 to 10.37 mg/L. The maximum EtS concentrations were from below the lowest limit of quantitation (0.04 mg/L) up to 16.1 mg/L non-normalised and up to 11.84 mg/L after normalisation to 100 mg/dL creatinine. The highest concentrations in the participants were measured between 3.3 and 17 h after the start of drinking. In two of the volunteers EtG and/or EtS were still detectable in the last collected urine samples 20 h after the start of the drinking experiment, and the concentrations were higher in the male participants. The EtG and EtS concentrations of one of the participants showed a late determined maximum concentration 17 h after the start of drinking, i.e., in the morning of the day after the experiment, and very much exceeded the values of the other participants. Fig. 1(a)–(d) shows the EtG100- and EtS100 concentrations of the four participants in urine over the time. In serum samples collected from the start of drinking until 2.5 h after the end of drinking, neither EtG nor EtS was detectable.
The ethanol metabolites EtG and EtS can be found in urine after drinking ‘‘non-alcoholic’’ beer, and therefore negative consequences may arise for monitored persons. Education of these persons is essential to avoid negative implications.
3. Results 3.1. Ethanol
3.3. Creatinine During and after the drinking period the creatinine concentrations decreased to 6 mg/dL and showed low concentrations for about 3.5 h after the start of drinking. The creatinine concentrations in the urine samples of the volunteer who excreted EtG and EtS in high concentrations were 136 mg/dL in the next morning’s urine sample (17 h after the start of drinking) and 138 mg/dL 1.25 h later. 4. Discussion The ‘‘non-alcoholic’’ beer that was used for this experiment contained 0.41 or 0.42 vol.% ethanol, respectively. This is within
Acknowledgement We thank the ‘‘Bund gegen Alkohol und Drogen im Straßenverkehr e.V’’. for financial support of this study. References [1] G.E. Skipper, W. Weinmann, A. Thierauf, P. Schaefer, G. Wiesbeck, J.P. Allen, M. Miller, F.M. Wurst, Ethyl glucuronide: a biomarker to identify alcohol use by health professionals recovering from substance use disorders, Alcohol Alcohol. 39 (2004) 445–449. [2] F.M. Wurst, G.E. Skipper, W. Weinmann, Ethyl glucuronide—the direct ethanol metabolite on the threshold from science to routine use, Addiction 98 (2003) 51–61. [3] F.M. Wurst, M. Yegles, C. Alling, S. Aradottir, J. Dierkes, G.A. Wiesbeck, C.C. Halter, F. Pragst, V. Auwaerter, Measurement of direct ethanol metabolites in a case of former driving under the influence (DUI) of alcohol offender, now claiming abstinence, Int. J. Legal Med. 122 (2008) 235–239. [4] H. Schneider, H. Glatt, Sulpho-conjugation of ethanol in humans in vivo and by individual sulphotransferase forms in vitro, Biochem. J. 383 (2004) 543– 549. [5] R.S. Foti, M.B. Fisher, Assessment of UDP-glucuronosyl-transferase catalyzed formation of ethyl glucuronide in human liver microsomes and recombinant UGTs, Forensic Sci. Int. 153 (2005) 109–116. [6] C.C. Halter, S. Dresen, V. Auwaerter, F.M. Wurst, W. Weinmann, Kinetics in serum and urinary excretion of ethyl sulphate and ethyl glucuronide after medium dose ethanol intake, Int. J. Legal Med. 122 (2008) 123–128. [7] S. Baranowski, A. Serr, A. Thierauf, W. Weinmann, M. Große Perdekamp, F.M. Wurst, C.C. Halter, In vitro study of bacterial degradation of ethyl glucuronide and ethyl sulphate, Int. J. Legal Med. 122 (2008) 389–393. [8] A. Costantino, E.J. Digregorio, W. Korn, S. Spayd, F. Rieders, The effect of the use of mouthwash on ethylglucuronide concentration in urine, J. Anal. Toxicol. 30 (2006) 659–662. [9] T.P. Rohrig, C. Huber, L. Goodson, W. Ross, Detection of ethylglucuronide in urine following the application of Germ-X, J. Anal. Toxicol. 30 (2006) 703–704.
A. Thierauf et al. / Forensic Science International 202 (2010) 82–85 [10] A. Thierauf, C.C. Halter, S. Rana, V. Auwaerter, F.M. Wurst, W. Weinmann, Urine tested positive for ethyl glucuronide after trace amounts of ethanol, Addiction 104 (2009) 2007–2012. [11] W. Attenborough, Evaluation of processes for the production of low- and nonalcohol beer, Fermentation 1 (1988) 40–44. [12] I. Leskosek, M. Mitrovic, V. Nedovic, Factors influencing alcohol and extract separation in beer dialysis, World J. Microbiol. Biotechnol. 11 (1995) 512–514. [13] M.F.M. van Iersel, E. Meersman, W. Swinkels, T. Abee, F.M. Rombouts, Continuous production of non-alcohol beer by immobilized yeast at low temperature, J. Ind. Microbiol. Biotechnol. 14 (1995) 495–501. [14] R. Olmi, V.V. Meriakri, A. Ignesti, S. Priori, C. Riminesi, Monitoring alcoholic fermentation by microwave dielectric spectroscopy, J. Microw. Power Electromagn. Energy 41 (2007) 37–49. [15] E. Nevoigt, R. Pilger, E. Mast-Gerlach, U. Schmidt, S. Freihammer, M. Eschenbrenner, L. Garbe, U. Stahl, Genetic engineering of brewing yeast to reduce the content of ethanol in beer, FEMS Yeast Res. 2 (2002) 225–232.
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[16] M. Navratil, Z. Do¨meny, E. Sturdik, D. Smogrovicova, P. Gemeiner, Production of non-alcoholic beer using free and immobilized cells of Saccharomyces cerevisiae deficient in the tricarboxylic acid cycle, Biotechnol. Appl. Biochem. 35 (2002) 133–140. [17] S. Dresen, W. Weinmann, F.M. Wurst, Forensic confirmatory analysis of ethyl sulfate – a new marker for alcohol consumption – by liquid-chromatography/ electrospray ionization/tandem mass spectrometry, J. Am. Soc. Mass Spectrom. 15 (2004) 1644–1648. [18] W. Weinmann, P. Schaefer, A. Thierauf, A. Schreiber, F.M. Wurst, Confirmatory analysis of ethyl glucuronide in urine by liquid-chromatography/electrospray ionization/tandem mass spectrometry according to forensic guidelines, J. Am. Soc. Mass Spectrom. 15 (2004) 188–193. [19] L. Politi, L. Morini, A. Groppi, V. Poloni, F. Pozzi, A. Polettini, Direct determination of the ethanol metabolites ethyl glucuronide and ethyl sulphate in urine by liquid chromatography/electrospray tandem mass spectrometry, Rapid Commun. Mass Spectrom. 19 (2005) 1321–1331.
Contents lists available at ScienceDirect
Forensic Science International journal homepage: www.elsevier.com/locate/forsciint
Urine tested positive for ethyl glucuronide and ethyl sulphate after the consumption of ‘‘non-alcoholic’’ beer Annette Thierauf a,*, Heike Gnann a, Ariane Wohlfarth a, Volker Auwa¨rter a, Markus Große Perdekamp a, Klaus-Juergen Buttler b, Friedrich M. Wurst c, Wolfgang Weinmann d a
Institute of Forensic Medicine, Freiburg University Medical Centre, Albertstrasse 9, 79104 Freiburg, Germany Department of General Neurosurgery, Freiburg University Medical Centre, Freiburg, Germany Christian-Doppler-Klinik, University Clinic for Psychiatry II, Salzburg, Austria d Institute of Forensic Medicine, University of Bern, Bern, Switzerland b c
A R T I C L E I N F O
A B S T R A C T
Article history: Received 10 March 2010 Received in revised form 14 April 2010 Accepted 18 April 2010 Available online 8 May 2010
In abstinence maintenance programs, for reissuing the driving licence and in workplace monitoring programs abstinence from ethanol and its proof are demanded. Various monitoring programs that mainly use ethyl glucuronide (EtG) as alcohol consumption marker have been established. To abstain from ethanol, but not from the taste of alcoholic beverages, in particular non-alcoholic beer has become more and more popular. In Germany, these ‘‘alcohol-free’’ beverages may still have an ethanol content of up to 0.5 vol.% without the duty of declaration. Due to severe negative consequences resulting from positive EtG tests, a drinking experiment with 2.5 L of non-alcoholic beer per person was performed to address the question of measurable concentrations of the direct metabolites EtG and EtS (ethyl sulphate) in urine and blood. Both alcohol consumption markers – determined by LC–MS/MS – were found in high concentrations: maximum concentrations in urine found in three volunteers were EtG 0.30–0.87 mg/L and EtS 0.04– 0.07 mg/L, i.e., above the often applied cut-off value for the proof of abstinence of 0.1 mg EtG/L. In the urine samples of one further volunteer, EtG and EtS concentrations cumulated over-night and reached up to 14.1 mg/L EtG and 16.1 mg/L EtS in the next morning’s urine. Ethanol concentrations in blood and urine samples were negative (determined by HS-GC–FID and by an ADH-based method). ß 2010 Published by Elsevier Ireland Ltd.
Keywords: Ethanol Ethyl glucuronide Ethyl sulphate Non-alcoholic beer
1. Introduction Alcoholism is in many countries all around the world a common problem that extends into various areas of life, such as business and private life and the participation in the road traffic. Due to the incompatibility of ethanol consumption and work as well driving a vehicle, abstinence is demanded from people who suffer from alcoholism or have driven under the influence of high alcohol concentrations. To remain in employment or for reissuing a driver’s licence, workplace monitoring programs and Medical Psychological Assessments (MPA) for alcoholised drivers have been established [1]. Within these programs, abstinence must be proven for a certain period of time. Abstinence can for example be substantiated by the nondetectability of alcohol consumption markers like ethyl glucuronide (EtG) and ethyl sulphate (EtS) [2,3]. The proof of
* Corresponding author. Tel.: +49 761 203 6836; fax: +49 761 203 6858. E-mail address: [email protected] (A. Thierauf). 0379-0738/$ – see front matter ß 2010 Published by Elsevier Ireland Ltd. doi:10.1016/j.forsciint.2010.04.031
abstinence by the absence of EtG and EtS also takes place in withdrawal maintenance programs in psychiatric hospitals. In the organism, ethanol is not only metabolised in the liver by a two-stage oxidation process, first to acetaldehyde by alcohol dehydrogenase, further to acetic acid by aldehyde dehydrogenase, but also converted into conjugation products like EtG and EtS [4– 6]. They supply evidence for ethanol uptake with high sensitivity and specificity [7], and it was published that even very small amounts of ethanol can lead to detectable concentrations of EtG and EtS in urine [8–10]. In Germany, new regulations and guidelines regarding the driver’s licence and MPA programs stipulate that the monitored persons have to obtain six EtG negative urine test results per year or four negative results per half-year. The dates of sampling have to be unpredictable for the tested person, and the sampling has to happen within 1 day after the invitation to the test. For various purposes and in different countries, the recent cut-off value for these monitoring programs is 0.1 mg EtG/L urine [9]. To abstain from ethanol, but not from the taste of alcoholic beverages, in particular non-alcoholic beer is becoming more and
A. Thierauf et al. / Forensic Science International 202 (2010) 82–85
more popular. Non-alcoholic wine, non-alcoholic sparkling wine and other alcohol-free drinks are also available, but less common. In Germany, ethanol has to be declared in beverages above a content of 0.5 vol.%. Depending on the production process, low amounts of ethanol may be present in non-alcoholic beer. One way to produce a ‘‘non-alcoholic’’ beer is to extract ethanol by dialysis after the completed brewing process [11,12]. Another possibility is the use of complex maltose and lower temperatures during the fermentation and to stop the process of fermentation at low ethanol contents [13,14]. Recently the utilisation of mutated yeast further reduced the alcohol formation during fermentation [15,16]. Due to the aforementioned use of alcohol markers for the proof of abstinence, it is an important question, whether the consumption of non-alcoholic beer can lead to measurable blood alcohol concentrations and to detectable EtG concentrations in urine. This question was addressed in this investigation by a drinking experiment with non-alcoholic beer that is known to have a residual content of ethanol. 2. Materials and methods 2.1. Experimental set-up The drinking experiment was performed as follows: after at least 5 days of abstinence from alcoholic beverages and the collection of a void urine and blood sample, four volunteers (two males (30 y/91 kg, 23 y/54 kg), two females (30 y/ 67 kg, 27 y/59 kg), all of them classified as social drinkers) drank 2.5 l of nonalcoholic beer within 1 h about 4 h after a light lunch. One pretzel per person was eaten during this drinking period. This is in everyday life an uncommonly large drinking amount, but was chosen to gain as high ethanol concentrations as possible. For 20 h after the beginning of the beer consumption urine samples were collected and stored at 20 8C prior to analysis for EtG and EtS and at 4 8C prior to ethanol
83
determination. Urine samples were analysed for ethanol, EtG, EtS and creatinine. Blood samples were taken every 20–30 min up to 3.5 h after the beginning of the drinking experiment. The blood samples were analysed for ethanol, EtG and EtS. 2.2. Chemicals and instrumentation EtG and pentadeuterated EtG (D5-EtG) were obtained from Medichem (Steinenbronn, Germany). Sodium ethyl sulphate was purchased from ABCR (Karlsruhe, Germany). Deuterated ethyl sulphate (D5-EtS) was synthesised by an inhouse procedure with a purity of >99.9% detected by LC–MS/MS [17]. Acetonitrile and formic acid were obtained from Merck (Darmstadt, Germany). All solvents were of analytical or HPLC grade. Mass spectrometric analysis was performed by a liquid chromatography–tandem mass spectrometry (LC–MS/MS) system consisting of an API 365 triple quadrupole tandem mass spectrometer (Applied Biosystems, Darmstadt, Germany) with a Turbo IonSpray interface, an HPLC gradient system (Shimadzu 10AD, Shimazu, Duisburg, Germany) and a polar-endcapped phenylpropyl reversed phase column (Synergi Polar-RP 250 mm 2 mm, 4 mm) with a guard column (4 mm 2 mm, packing material identical; Phenomenex, Aschaffenburg, Germany). The ethanol concentrations in urine, blood and beer were measured using HSGC–FID (headspace gas chromatography/flame ionisation detector) using t-butanol as internal standard and additionally – as required by German forensic guidelines – with an ADH-based method (DRI alcohol dehydrogenase assay, Thermofisher Scientific, Dreieich, Germany). Ethanol determination was performed using linear calibration with 0.1, 0.2, 0.5, 1, 2, 3, 4, 5 g/L aqueous ethanol solutions for HS-GC/ FID. The lower limit of quantitation (LLOQ) was the lowest calibrator’s concentration (0.1 g/L or 0.08 g/kg for blood). Creatinine was determined by using the Jaffe´ reaction (DRI1 Creatinine-Detect1 Test) with a Konelab 30i analyzer (Thermofisher Scientific). The study had been approved by the ethics committee of the Freiburg University (285/09). 2.3. Sample preparation for EtG and EtS quantification by LC–MS/MS 0.1 mg D5-EtG and a constant amount of D5-EtS synthesised by an in-house procedure [17] were added as internal standards to 100 mL of urine or serum.
Fig. 1. (a–d) EtG100 and EtS100 concentrations of the four participants.
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A. Thierauf et al. / Forensic Science International 202 (2010) 82–85
250 mL of methanol was added for protein precipitation and the samples were centrifuged for 10 min at 13000 rpm. 270 mL of the supernatant was separated and evaporated in a vacuum centrifuge. The dried extracts were reconstituted with 140 mL of 0.1% aqueous formic acid, and 10 mL aliquots were injected into the LC– MS/MS system. HPLC separation was performed at 40 8C with isocratic elution (0.1% formic acid) with a flow rate of 0.2 mL/min. To increase the volatility of the eluent, acetonitrile was added post-column at a flow rate of 0.2 mL/min via a T-union. EtG and EtS were analysed in one run by a validated MS/MS-method with electrospray ionisation (ESI) [18,19]. The method was calibrated from 0.05 to 50 mg/L for EtG and 0.04 to 50 mg/L for EtS. The lowest calibration level corresponds to the lowest limit of quantitation (LLOQ) in urine. Values below the LLOQ were reported as negative. The following MS/MS transitions with precursor ion/product ion were used: EtG: m/z 221/75 as quantifier, m/z 221/85 as first qualifier, and 221/203, 221/113 as additional qualifiers, m/z 226/75 for the deuterated internal standard. EtS: m/z 125/ 97 as quantifier, m/z 125/80 as qualifier and m/z 130/98 for the deuterated internal standard. Criteria for compound identification were based on forensic guidelines which require a relative ion-intensity ratio of qualifier-to-quantifier area intensity of 20% compared to a reference compound [17,18]. The concentrations were normalised to 100 mg/dL creatinine.
The ethanol content of the beer was determined in a repeated determination for each of the four lots of beer and was 3.1 and 3.2 g/kg (equal to 0.41 and 0.42 vol.%). The ingested amount of ethanol after drinking 2.5 L of ‘‘non-alcoholic’’ beer is about 8.3 g of ethanol and equals about one shot of hard liquor or 26 mL of a 40 vol.% spirit. Ethanol concentrations in urine and blood were below LLOQ (<0.08 g/kg).
the legal limit for alcohol-free beverages, but not a negligible amount of ethanol. One litre of such a beer contains approximately 3.3 g ethanol. In former studies, the ethanol amount of 1 g – contained in less than one bottle of ‘‘non-alcoholic’’ beer – already led to positive EtG and EtS findings in urine for a few hours after drinking [10]. Also in this setting with a common and usual beverage, the ethanol metabolites EtG and EtS could be detected in concentrations that were clearly positive and above the presently applied cut-off value of 0.1 mg/L in urine, which may have negative consequences for monitored persons. As a consequence, individuals that are under any kind of monitoring for alcohol markers have to be educated about the possible presence of ethanol in some food products, beverages and other products, and in particularly of those beverages that are declared as ‘‘non-alcoholic’’, in order to avoid negative implications. Interestingly, one participant explicitly exceeded the EtG and EtS concentrations of the other volunteers; the unexpectedly high concentrations were detected about 17 h after the drinking period, in the next morning’s urine sample. Accumulation of EtG and EtS in this urine sample is one possible explanation, since the period of time between the previous sample and the morning sample is 14 h. Another plausible explanation for this finding would be further ethanol uptake after the experimental set-up. This was denied in a detailed interview. Not only were the measured concentrations remarkable in this participant, but also the ratio between EtG and EtS with higher concentrations of EtS than EtG. The authors lack an explanation for this occurrence; further examination of the metabolism of this participant will be performed.
3.2. Ethyl glucuronide/ethyl sulphate
5. Conclusions
In urine, the non-normalised peak concentrations of EtG of the participants ranged from 0.30 to 14.1 mg/L and after normalisation from 0.49 to 10.37 mg/L. The maximum EtS concentrations were from below the lowest limit of quantitation (0.04 mg/L) up to 16.1 mg/L non-normalised and up to 11.84 mg/L after normalisation to 100 mg/dL creatinine. The highest concentrations in the participants were measured between 3.3 and 17 h after the start of drinking. In two of the volunteers EtG and/or EtS were still detectable in the last collected urine samples 20 h after the start of the drinking experiment, and the concentrations were higher in the male participants. The EtG and EtS concentrations of one of the participants showed a late determined maximum concentration 17 h after the start of drinking, i.e., in the morning of the day after the experiment, and very much exceeded the values of the other participants. Fig. 1(a)–(d) shows the EtG100- and EtS100 concentrations of the four participants in urine over the time. In serum samples collected from the start of drinking until 2.5 h after the end of drinking, neither EtG nor EtS was detectable.
The ethanol metabolites EtG and EtS can be found in urine after drinking ‘‘non-alcoholic’’ beer, and therefore negative consequences may arise for monitored persons. Education of these persons is essential to avoid negative implications.
3. Results 3.1. Ethanol
3.3. Creatinine During and after the drinking period the creatinine concentrations decreased to 6 mg/dL and showed low concentrations for about 3.5 h after the start of drinking. The creatinine concentrations in the urine samples of the volunteer who excreted EtG and EtS in high concentrations were 136 mg/dL in the next morning’s urine sample (17 h after the start of drinking) and 138 mg/dL 1.25 h later. 4. Discussion The ‘‘non-alcoholic’’ beer that was used for this experiment contained 0.41 or 0.42 vol.% ethanol, respectively. This is within
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