CHAPTER TWO

Ethyl Glucuronide and Ethyl Sulfate Natalie E. Walsham*, Roy A. Sherwood†,1 *Department of Clinical Biochemistry, University Hospital Lewisham, London, United Kingdom † Department of Clinical Biochemistry, King’s College Hospital NHS Foundation Trust, London, United Kingdom 1 Corresponding author: e-mail address: [email protected]

Contents 1. 2. 3. 4. 5. 6.

Introduction Metabolism of Alcohol Stability Cutoff Values Detection Times Methods for Measurement of EtG and EtS 6.1 Hair EtG 7. Applications 7.1 Detoxification programs 7.2 Liver transplantation and liver disease 7.3 Fetal alcohol spectrum disorder 7.4 Postmortem 7.5 Sexual assault victims 7.6 Drink driving 8. Confounders Causing False-Positive or False-Negative Results 8.1 Urinary tract infection 8.2 Mouthwash 8.3 Hand sanitizers 8.4 Beverages and food 8.5 Drugs 8.6 Hair products 9. Conclusions Declarations References

48 49 51 51 52 52 54 55 56 57 58 59 60 60 60 60 61 61 62 62 63 63 64 64

Abstract Alcohol misuse is associated with significant morbidity and mortality. Although clinical history, examination, and the use of self-report questionnaires may identify subjects with harmful patterns of alcohol use, denial or under-reporting of alcohol intake is common. Existing biomarkers for detecting alcohol misuse include measurement of blood Advances in Clinical Chemistry, Volume 67 ISSN 0065-2423 http://dx.doi.org/10.1016/bs.acc.2014.09.006

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2014 Elsevier Inc. All rights reserved.

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Natalie E. Walsham and Roy A. Sherwood

or urine ethanol for acute alcohol consumption, and carbohydrate-deficient transferrin and gamma-glutamyl transferase for chronic alcohol misuse. There is a need for a biomarker that can detect excessive alcohol consumption in the timeframe between 1 day and several weeks. Ethyl glucuronide (EtG) is a direct metabolite of ethanol detectable in urine for up to 90 h and longer in hair. Because EtG has high specificity for excess alcohol intake, it has great potential for use in detecting “binge” drinking. Using urine or hair, this noninvasive marker has a role in a variety of clinical and forensic settings.

1. INTRODUCTION Alcohol misuse is associated with significant morbidity and mortality and is widely distributed throughout all socioeconomic groups worldwide. The 2011 Global Status Report on Alcohol from the World Health Organization estimated that an excess of 70 million people worldwide had recognizable alcohol misuse [1]. Alcohol abuse was responsible for 2.25 million deaths in the world each year (3.8% of the total). One-third of these were associated with accidents. In its publication “Statistics on Alcohol: England 2013,” the Health and Social Care Information Centre estimated that alcohol misuse costs the National Health Service £3.5 billion each year [2]. Although many subjects misusing alcohol can be identified by the clinical history and examination or by self-report questionnaires such as the Alcohol Use Disorders Identification Test (AUDIT) questionnaire, there are significant problems with deliberate under-reporting being common. A range of biomarkers for the detection of harmful alcohol consumption has been described [3]. These can be divided into direct and indirect markers. Direct markers include ethanol itself or its metabolites. Indirect markers are dependent on the action of alcohol at the molecular level or compounds released from organ damage associated with ethanol or metabolites. Ethanol measurements in breath or body fluids have high specificity for excessive alcohol intake, but relatively narrow timeframes for positivity after alcohol consumption (breath 4–6 h, blood 10–12 h, and urine 18–24 h). Other direct biomarkers of alcohol intake rely on alternative pathways of alcohol metabolism and include ethyl glucuronide (EtG), ethyl sulfate (EtS), and 5-hydroxytryptophol (5-HTOL). The most commonly used indirect biomarkers measured in blood are gamma-glutamyl transpeptidase (GGT), carbohydrate-deficient transferrin (CDT), and erythrocyte mean corpuscular volume (MCV) [4]. Both GGT and MCV require significant alcohol intake over a prolonged period of time (>1000 g over at least 2 weeks)

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49

to become abnormal. GGT is both induced by alcohol itself and released by hepatocytes damaged by alcohol or its metabolites, but it has poor specificity due to its increase in liver disease not associated with alcohol misuse. This particular problem is growing due to increased obesity in the developed world. For example, hepatic steatosis associated with obesity and diabetes mellitus causes increased GGT. In addition, MCV is increased in nutritional deficiencies, particularly folate and/or vitamin B12 deficiency, which may be present in those misusing alcohol with a chaotic lifestyle thus reducing specificity. Although CDT has good specificity for alcohol misuse, it is best used as a marker of chronic excessive alcohol consumption over 7–14 days. It will not test positive after a single session of heavy drinking. Excessive drinking in one session, “binge” drinking, appears to be an increasing problem in many areas of the world. Therefore, there is a need for a biomarker of alcohol misuse that can detect excessive consumption in the timeframe between those tests that are positive in the first 24 h only and CDT (which could be considered the HbA1c of alcohol intake).

2. METABOLISM OF ALCOHOL The main metabolic pathway of ingested alcohol takes place in the liver in a two-stage enzymatically catalyzed oxidation process. Alcohol is first converted to acetaldehyde by alcohol dehydrogenase and then further metabolized to acetate by aldehyde dehydrogenase. A small amount is excreted unchanged in urine, sweat, and expired air. EtG (ethyl β-D-6-glucuronide) is a direct metabolite of ethanol formed by the enzymatic conjugation of ethanol with glucuronic acid in the liver [5]. This phase II reaction is catalyzed by mitochondrial membrane-bound UDP-glucuronosyltransferase. Ethanol is also conjugated to sulfate by sulfotransferase to form EtS (Fig. 1). These are minor pathways with less than 1% of ethanol ingested entering these pathways and, being water-soluble, EtG and EtS are excreted in urine [6]. EtG and EtS are most commonly measured in urine as markers for alcohol intake, but can also be measured in whole blood, serum/plasma, and a range of other body fluids or tissues. Studies have shown that these minor metabolites are mainly distributed in the plasma compartment of blood rather than the cellular compartment with a median serum/plasma to whole blood ratio of 1.69 for EtG and 1.30 for EtS [7]. As these metabolites are formed in the liver, maximal plasma metabolite concentration occurs later than blood ethanol itself: approximately 2–3 h

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Natalie E. Walsham and Roy A. Sherwood

HOOC O

Sulfotransferase

C2H5O UDP-glucuronosyl transferase

CH3CH2OH Ethanol

C2H5O-SO3H Ethyl sulfate EtS

Alcohol dehydrogenase

OH OH

OH

Ethyl glucuronide EtG CH3CHO Acetaldehyde

Aldehyde dehydrogenase

CH3COOH Acetic acid

Figure 1 Metabolism of alcohol and formation of ethyl glucuronide and ethyl sulfate.

later for EtG and 1–2 h later for EtS [8,9]. In a study conducted with volunteers (n ¼ 18), the maximal concentration of EtG and EtS in serum was 4000 and 2000 μg/L, respectively, following consumption of 32 g alcohol, and 13,000 and 6000 μg/L, respectively, following consumption of 64 g alcohol. Peak concentrations were reached 1–3 h after alcohol ingestion [10]. There appear, however, to be wide interindividual variations in the maximum serum/plasma EtG and EtS concentration and there is a poor correlation between the metabolites and blood ethanol concentration [8]. Studies have found that metabolite elimination occurs exponentially with a median half-life of 2–4 h [9,11,12]. EtG can usually be detected in urine for 72–90 h. The elimination rate of EtG and EtS appears similar in healthy subjects and heavy drinkers during alcohol detoxification [11]. This study found decreased elimination rate and increased blood concentration in patients with renal disease which would delay excretion of these metabolites. Two small studies from one group have provided further evidence that renal impairment may cause increased EtG and EtS in urine and increased EtG in hair. In 14 subjects who each collected 10 urine samples after consuming 0.1–1.4 g of ethanol/kg body weight, detection times were found to be significantly longer in patients with decreased renal function versus healthy subjects (p < 0.01). Significantly increased hair EtG was found in 12 patients with renal disease versus 21 healthy volunteers (p ¼ 0.009) [13,14]. A study

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51

by Wurst et al.[15] found that EtG concentration was influenced by age, gender, cannabis use, kidney disease, and the amount of ethanol ingested in the previous month. Race, smoking, body mass index, liver cirrhosis, the age at which subjects began drinking regularly, and total body water had no significant influence on EtG concentration in urine [15]. In a way similar to some drug addicts, alcohol misusers sometimes attempt to lower the EtG and EtS urine concentration by drinking large volumes of water. Expressing EtG and EtS relative to urine creatinine can partly overcome this dilutional effect [6,16]. However, Helander et al.[17] reported that wide interindividual variation in EtG detection time was common despite normalization with creatinine. The interaction of alcohol with other metabolic pathways has resulted in several potential markers that have been compared to EtG. Fatty acid ethyl esters (FAEEs) are esterification products of ethanol and fatty acids that can be measured in blood and tissues as markers of alcohol intake [18]. Acute alcohol intake alters the normal metabolism of serotonin (5-hydroxytryptamine) to 5-hydroxyindole acetic acid (5-HIAA) resulting in the formation of 5-HTOL, albeit at 1% of the 5-HIAA concentration. The ratio of 5-HTOL to 5-HIAA in urine has been shown to be a more sensitive and specific marker of alcohol ingestion than urine or blood ethanol, remaining positive 6–15 h after the blood alcohol concentration (BAC) had returned to baseline [19].

3. STABILITY EtG and EtS have been shown to be stable markers in vitro. Urine samples stored at 4  C for 5 weeks were found to have no change in EtG concentration [20]. When stored at room temperature in ventilated vials, the concentration of EtG was found to increase due to water evaporation. During this study, there was no evidence of analyte decomposition. EtGpositive tissue material allowed to slowly decompose at room temperature exhibited decreased EtG concentration over time. No postmortem formation was found.

4. CUTOFF VALUES Studies in healthy volunteers who ingested alcohol (0.1–0.8 g/kg body weight) have consistently shown that the best cutoff value in urine is 100–200 μg/L for EtG and 100–110 μg/L for EtS [17]. For clinical use,

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cutoffs as high as 500 μg/L have been used to reduce the risk of false-positive results [17]. In a volunteer study, a maximal plasma EtG concentration of 360 μg/L (range 280–410 μg/L) was found in samples taken 1.5–24 h after a single alcohol dose (0.5 g/kg) [21]. Cutoffs of 500 μg/L for EtG and 50–100 μg/L for EtS in urine were supported in a preliminary study in healthy volunteers [22]. Meta-analysis of 15 studies found that mean hair EtG concentration in social drinkers, heavy drinkers, and deceased subjects with a known history of chronic alcohol misuse was 7.5, 142.7, and 586.1 ng/g, respectively. A cutoff of 30 ng/g for EtG in hair was proposed to limit false negatives and better distinguish social and heavy drinkers [23].

5. DETECTION TIMES There have been a number of studies characterizing the timeframe during which EtG and EtS remain detectable in urine following alcohol intake in healthy volunteers [6,8,9,24–27]. Although these studies involved a range of alcohol doses (0.1–0.85 g/kg body weight), the detection timeframe was relatively consistent (24–48 h) for both EtG and EtS. One study, using a larger dose of alcohol (>1 g/kg), found that EtG remained above the limit of detection (100 μg/L) for 39–102 h [28]. This longer timeframe was in agreement with two studies conducted in alcohol-intoxicated subjects (40–130 h) [17,29].

6. METHODS FOR MEASUREMENT OF EtG AND EtS Various methods for measuring EtG and EtS have been published over the past 10 years. The most commonly used methods are based on liquid chromatography–mass spectrometry (LC-MS) because it is highly sensitive, specific, and is able to simultaneously measure EtG and EtS [30–33]. This technology has been used to determine EtG in urine, whole blood, serum, meconium [34], saliva [35], hair [36], nails [37], and dried blood spots [38,39]. LC-MS detection of EtG can be carried out using selected ion monitoring of the precursor ion (m/z 221) and the principal daughter ion (m/z 75) with penta-deuterated EtG (ETG-D5, m/z 226) as the internal standard [30].

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53

The alternative transition m/z 221 ! 85 has also been used [31,35]. Corresponding transitions for EtS are m/z 125 ! 97 and m/z 125 ! 80 [40]. Most LC-MS methods have a limit of quantitation (LOC) of 50–100 μg/L for EtG and EtS. In some applications, urine can be injected without extraction following centrifugation and dilution with water and supplementation with an internal standard. Serum/plasma samples can be analyzed after deproteinization with methanol or acetonitrile, centrifugation, and addition to an aliquot of the mobile phase. A comparison of five LC-MS methods for measurement of urinary EtG and EtS recommended that solid-phase extraction followed by LC-MS-MS should be adopted as the reference method because of its high selectivity and sensitivity [41]. Other methods include reversed-phase liquid chromatography with pulsed electrochemical detection [42], microwave-assisted extraction followed by gas chromatography–mass spectrometry (GC-MS) [43,44], GC-MS with solid-phase extraction for sweat samples [45] and GC-MS of silylated derivatives [46]; capillary electrophoresis [47], capillary zone electrophoresis–mass spectrometry [48], capillary isotachophoresis, and zone electrophoresis [49]; and an ELISA based on polyclonal antibodies [50] (Table 1). An LC-MS/MS method for urine EtG/EtS has been validated using forensic guidelines [51]. A monoclonal antibody-based enzyme immunoassay (EIA) is commercially available for EtG analysis in urine (DRI Ethyl Glucuronide Enzyme Immunoassay, Thermo Fisher Scientific Diagnostics, Hemel Hempstead, UK). Comparison with an established LC-MS method showed good agreement (r2 ¼ 0.931), indicating a low cross-reactivity of the EtG antibody to other urinary constituents [52]. The method evaluation showed the EIA is sensitive, specific, and offers a low but clinically relevant measuring range (0–500 μg/L). Higher results may be obtained by dilution (detection limit of 100 μg/L). Although correlation to LC-MS was good, the EIA method is considered a screening test. EtG-positive samples should always be confirmed by LC-MS/MS with EtS measurement to rule out false positives (see Section 8). To ascertain if EtG crosses the human placenta to the fetus, a method for the measurement of EtG in placental perfusate and tissue was developed using headspace solid-phase microextraction coupled with GC-MS. This was used in an ex vivo placental perfusion model to show that EtG could be detected in the fetal circulation within 20 min [53]. EtG has also been measured in dental tissue by LC-MS/MS and correlated well with the Michigan Alcohol Screening Test (r ¼ 0.914) [54].

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Table 1 Characteristics of selected methods for the measurement of EtG Method

Extraction

Sample matrix

LoD

Ref.

Electrospray LC-MS

Direct injection

Urine

100 μg/L

[30]

Electrospray LC-MS

Direct injection

Urine

–

[31]

Anion exchange LC-MS/MS

Direct injection

Urine

100 μg/L

[32]

Electrospray LC-MS/MS

Protein precipitation

Serum

0.2 μmol/L [33]

LC-MS/MS

Solid-phase

Meconium

5 ng/g

[34]

UPLC-MS/MS

Solid-phase

Oral fluid

4.4 μg/L

[35]

GC-MS/MS

Solid-phase

Hair

8.4 ng/g

[36]

LC-MS/MS

Water

Nails

10 ng/g

[37]

LC-MS/MS

Methanol

Blood spots

0.1 mg/L

[38]

Electrospray LC-MS

Direct injection

Urine

50 μg/L

[40]

LC-pulsed ECD

Liquid–liquid

Urine

10 μg/L

[42]

GC-MS

Microwave assisted Urine

100 μg/L

[43]

GC-MS

Microwave assisted Hair

0.3 ng/mg

[44]

GC-MS

Solid phase

Sweat

1 μg/L

[45]

GC-MS

Silylated derivatives

Hair

–

[46]

Capillary electrophoresis

Direct injection

Serum

100 μg/L

[47]

Capillary isotachophoresis + CZE

Water dilution

Serum

0.01 μmol/ [49] L

ELISA

Direct sample

Serum

300 μg/L

[50]

EIA

Direct sample

Urine

100 μg/L

[51]

LC, liquid chromatography; MS, mass spectrometry; GC, gas chromatography; LoD, limit of detection; UPLC, ultra-performance liquid chromatography; ECD, electrochemical detection; CZE, capillary zone electrophoresis; ELISA, enzyme linked immunosorbent assay; EIA, enzyme immunoassay.

6.1. Hair EtG Analysis of drugs of abuse in hair samples has long been used to identify chronic use over extended timeframes (weeks to months). As such, there has been considerable interest in testing hair EtG to extend the detection period beyond traditional markers such as CDT. Hair EtG, as a marker for detection of alcohol intake, has been recently reviewed [55].

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Initial methods using GC-MS for hair EtG were hampered by LOCs (0.5–2.0 μg/g) [46,56,57]. Over the past decade, LC-MS methods developed for urine EtG have contributed to substantially improve the LOC in hair so that it is now typically in the range of 2-10 ng/g. [58–65]. An EtG cutoff value of 4–30 ng/g in hair has been proposed to distinguish social (40 g ethanol/day). This approach has yielded good sensitivity and specificity (90–95%). In subjects with low to moderate alcohol intake, i.e., daily consumption of 16–32 g alcohol over a 3-month period, the maximum hair EtG concentration was 11 ng/g [66]. This study proposed an abstinence threshold of 30 ng/g. The latter cutoff produced a higher positivity rate versus CDT in a fitness-to-drive following previous alcohol problems program [67]. Because of the false positivity concerns in cases with legal implications (fitness-to-drive, workplace testing, child custody, etc.), several groups have recommended a combination of hair EtG and FAEE measurement [68,69]. Hair analysis for EtG requires extraction prior to analysis by any of the methods detailed earlier. Washing the hair sample with dichloromethane and methanol followed by sonication (30 min) extracts more than 50% of the EtG [70]. An alternative approach is to use micropulverization [71]. Body site is independent, i.e., chest, arm, and leg hair samples provide equivalent EtG values when compared to scalp hair [72]. Recent methods for hair EtG analysis include hydrophilic interaction liquid chromatography–tandem mass spectrometry (HILIC-MS/MS) with liquid–liquid extraction that has a lower LOC of 0.18 ng/g [73] and UHPLC-MS/MS with an LOC of 1.0 ng/g [74,75]. Hair and nail EtG was measured in 606 undergraduate students by LC-MS/MS [76]. Nail EtG demonstrated better sensitivity versus hair EtG for detecting any weekly alcohol use (p ¼ 0.02).

7. APPLICATIONS The measurement of EtG has been carried out in a variety of clinical and forensic settings. Alcohol misuse can be implicated in a significant proportion of subjects admitted to a hospital emergency department with gastrointestinal symptoms or following minor injury. Self-report of alcohol intake using the AUDIT questionnaire tends to be unreliable due to underestimation of alcohol consumption by the subjects. Two studies on the use of

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EtG measurements in the emergency room setting have been carried out using urine [18] and plasma [77]. Most subjects either tested negative for blood ethanol or had low BAC in the range 0.01–0.07 g/L. Interestingly, a substantial percentage (25–38%) tested positive for EtG irrespective of positive (8 points) or negative AUDIT score.

7.1. Detoxification programs Monitoring abstinence in subjects undergoing alcohol detoxification programs is important. Blood or urine ethanol measurement is problematic due to the relatively short timeframe for these markers following alcohol ingestion. In a group of 139 detoxified alcohol-dependent patients followed up for 12 weeks after discharge from in-patient treatment, 28% of subjects denying relapse tested positive for EtG and EtS by LC-MS/MS [78]. Similarly 4 out of 30 patients, in whom neither clinical assessment nor routine laboratory testing suggested relapse, tested positive for urine EtG at concentrations from 4200 to 196,600 μg/L [19]. It should be noted, however, that the subject with the highest urine EtG concentration had detectable serum EtG. A double-blind placebo-controlled oral acamprosate study was conducted in 56 alcohol-dependent subjects (30 males) [79]. Urine was obtained at baseline and weekly for EtG and EtS. On the first day, 72% of subjects tested positive. This number decreased to 31% after 3 weeks with no difference between the acamprosate and placebo groups. Significantly, 28% of samples from subjects who denied alcohol consumption in the day prior to testing were positive for EtG and EtS. In a similar study of 24 out-patients undergoing treatment for alcohol or drug dependency, urine EtG and EtS were compared to self-reporting [80]. This study found high concordance (87%) for self-report and EtG/EtS results. A single patient specimen was positive for EtS only. Subjects undergoing opioid maintenance therapy often abuse alcohol, but often deny it with negative AUDIT scores. Urine and hair EtG measurement identified cases of excess alcohol intake in subjects on a methadone maintenance program [81–83]. Many of these would have been missed using self-report alone. In health-care professionals recovering from substancerelated disorders, complete abstinence from drugs, including alcohol, is required before they can return to work. Random urine testing is usually incorporated into such programs. In one study, 100 urines were collected and tested for alcohol use [84]. Although none tested positive for alcohol, seven tested positive for EtG (0.5–196 mg/L).

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Oral fluid EtG has been measured in a Norwegian employee recruitment exercise [85]. In this nondetoxification study, about 2.1% tested positive for EtG (>2.2 μg/L). Hair testing for EtG and FAEE has potential application in workplace testing for employees in high-risk occupations [86].

7.2. Liver transplantation and liver disease Orthotopic liver transplantation (OLT) for treatment of end-stage liver disease resulting from alcohol misuse remains controversial because a substantial percentage of subjects (20–25%) return to harmful drinking. As such, most transplant programs require a period of abstinence to remain on the waiting list. Detection of alcohol misuse in these patients represents a challenge because GGT is typically increased due to hepatic fibrosis and CDT may be increased secondary to reduced clearance from the circulation into bile [87]. In addition, patients refrain from drinking in the 24–36-h period prior to breath, blood, or urine alcohol testing. Another study was conducted in 18 OLT candidates who denied alcohol consumption [88]. This report found that almost half (49%) of urine specimens were positive for EtG, whereas only 1 of 127 breath alcohol tests was positive. A cross-sectional anonymous study of adult OLT candidates (n ¼ 109) found that 20% of subjects were positive for urine EtG and EtS versus 4% by self-reported questionnaire [89]. A large study of OLT candidates (n ¼ 141) reported a positive predictive value (PPV) of 89.3% and negative predictive value of 98.9% for urine EtG in detecting alcohol misuse [90]. This German report demonstrated that EtG was clearly superior to CDT, MCV, or GGT. These findings have been confirmed in two recent studies including one that used hair EtG. In a study of 121 OLT candidates/recipients, urine EtG was compared to serum and urine ethanol, CDT, and the AUDIT-c questionnaire [91]. Alcohol consumption was defined as a positive AUDIT-c or by patient confirmation when challenged with the test results. Receiver Operator Characteristics analysis found that urine EtG was the best predictor of alcohol consumption (AUC 0.94) versus CDT (AUC 0.63). Urine EtG combined with the AUDIT-c increased the AUC to 0.98. In 63 OLT candidates, hair EtG was compared to urine EtG, blood ethanol, and CDT [92]. Although 19 patients (30%) admitted alcohol consumption in the previous 6 months, 39 patients (62%) tested positive for at least one marker. In the 44 patients claiming abstinence, 52% had one positive marker with hair EtG above the cutoff (30 ng/g) in 83% of cases providing a specificity of

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98% and a PPV of 92%. Interestingly, the authors claimed that renal and liver function had no effect on hair EtG concentration. Others evaluated urine EtG and EtS in patients with liver disease (n ¼ 120) and hair EtG in patients with liver disease (n ¼ 200) [93,94]. Urine EtG (cutoff 100 μg/L) had a sensitivity of 76% and specificity of 93%. Urine EtS (cutoff 25 μg/L) had a sensitivity of 82% and specificity of 86%. Hair EtG (cutoff 8 ng/g) demonstrated an AUC of 0.93 for detecting ethanol ingestion (average 28 g of ethanol a day over a 3-month period).

7.3. Fetal alcohol spectrum disorder Fetal alcohol syndrome (FAS) and fetal alcohol spectrum disorder (FASD) are recognized as a cause of congenital abnormalities, cognitive dysfunction, and developmental delay. It is estimated that FAS affects 2/1000 and FASD 9/1000 live births in the developed world. Diagnosis after birth, however, is difficult. EtG and EtS have been measured by LC-MS/MS in meconium samples from the infants of 177 randomly selected women from Italy and Spain [95]. EtG was detectable in over 80% of samples while EtS was only found in 50%. A cutoff of 2 nmol/g was found to have 100% sensitivity and specificity to distinguish heavy maternal ethanol consumption during pregnancy from occasional or no use (defined by questionnaire and meconium FAEE measurement). This cutoff was validated in a study from the same group using a subset of mothers who self-reported alcohol consumption during pregnancy [96]. This study showed that neonatal hair EtG was a poor predictor of maternal alcohol intake. A similar study of 602 meconium samples from a maternal health evaluation in Germany found only 97 (16.3%) of cases had detectable EtG [97]. In none of the 602 cases did the mothers report serious alcohol consumption and no evidence of FAS or FASD were found in the newborn infants. When EtG was compared to FAEE, a cutoff of 274 ng/g provided the best agreement between the two markers. Two outliers (EtG 10,200 and 82,000 ng/g) suggested heavy alcohol consumption that was not reported. The authors concluded that combined EtG and FAEE in meconium minimized both false-positive and false-negative results. An LC-MS/MS method has been developed for the simultaneous measurement of FAEE, EtG, and EtS [98]. An ELISA has been developed and validated for the measurement of EtG in meconium [99]. An EtG cutoff of 0.9 nmol/g provided excellent sensitivity (100%) and good specificity (78%) when compared to LC-MS/MS confirmation.

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59

Urine and hair EtG and EtS measurement during pregnancy has been reported [100]. In this Swedish study, women (n ¼ 103) provided urine and hair for EtG, EtS, and FAEE measurement and completed the AUDIT questionnaire. Although 26 women (25.2%) were identified as possible alcohol consumers and 7 women had hair EtG or FAEE concentrations highly suspicious of heavy drinking, only 1 was positive by self-reported AUDIT questionnaire. An Italian group compared the performance of FAEE and EtG in meconium with maternal hair and nail EtG in predicting fetal exposure to alcohol [101]. Similar results to other groups were obtained for FAEE and EtG in meconium. None, however, tested positive for hair or nail EtG despite confirmed alcohol consumption in 18 of 151 cases.

7.4. Postmortem Confirming a role for alcohol as a contributor to cause of death has been difficult due to the inherent instability of peptide markers such as CDT postmortem. EtG was compared to CDT in serum, urine, cerebrospinal fluid (CSF), and vitreous humor in postmortem forensic cases with a positive (n ¼ 38) and negative (n ¼ 22) history of alcohol misuse [102]. EtG (mean  SD) in urine (339,000  389,000 μg/L; p < 0.001), vitreous humor (4200  4800 μg/L; p < 0.001), serum (6900  8900 μg/L; p < 0.01), and CSF (1700  2.7 μg/L; p < 0.01) were significantly higher in the alcohol-positive group, whereas CDT was only increased in CSF. The same group demonstrated that the commercially available immunoassay (Thermo Scientific) could also be applied to vitreous humor samples and correlated well with LC-MS/MS (r ¼ 0.94) [103]. An immunoassay cutoff of 300 μg/L for vitreous humor EtG provided high sensitivity (92%). In contrast, blood alcohol (cutoff 100 mg/L) was positive in only 68% of cases. Postmortem urine (n ¼ 800) was tested for EtG by immunoassay and LC-MS/MS [104]. The LC-MS/MS method had a statistically significant proportional bias (p < 0.0001). An LC-MS/MS cutoff of 100 μg/L provided the best sensitivity and specificity that equated to a 92 μg/L immunoassay cutoff. EtG has also been detected in postmortem hair samples together with tissue samples (gluteal and abdominal fat, liver, and brain) from intoxicated subjects [105]. It is unclear, however, why EtG was not detected in the liver or gluteal fat of one subject who died intoxicated.

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7.5. Sexual assault victims Delay in testing for alcohol intake is common in cases of sexual assault due to the late presentation of many victims. Urine from 59 female victims of sexual assault in Norway was tested for EtG and EtS by UPLC-MS/MS [106]. EtG and EtS were positive in 45 of 48 cases with self-reported alcohol intake, whereas ethanol was only detected in 20 cases (sensitivity: EtG 94%; ethanol 42%).

7.6. Drink driving In a number of European countries, drink-driving offenders have to prove abstinence for a period of time to regain their driving licenses. Recently, the United Kingdom replaced GGT and MCV with CDT. In Germany, a program that encompasses both urine alcohol and drug tests is in place. In a Canadian study, drivers regaining their licenses were required to install ignition interlock devices that prevented the vehicle being driven if BAC limit was exceeded. Urine EtG/EtS and hair EtG were compared with conventional biomarkers and the ignition interlock BAC [107,108]. The authors concluded that testing for EtG in either urine or hair improved the detection rate for problem drinkers. Similar conclusions for hair EtG were reached in Switzerland [67] and Germany [109].

8. CONFOUNDERS CAUSING FALSE-POSITIVE OR FALSE-NEGATIVE RESULTS 8.1. Urinary tract infection As EtG measurement becomes commonplace in clinical and medico-legal settings, i.e., the use of urine EtG in Germany for return of driving licenses after conviction of driving under the influence of alcohol, it has become increasingly important to understand the potential causes of false-positive or false-negative results. False-positive and false-negative results for urine EtG have been reported when bacteria are present. Glucuronide and sulfate conjugates are cleaved by β-glucuronidase and sulfatase enzymes, respectively. Studies have shown that EtG, but not EtS, was sensitive to bacterial hydrolysis when exposed to Escherichia coli and Clostridium sordellii [110,111]. As E. coli is the most common pathogen in urinary tract infections (UTIs), falsely decreased EtG may occur in its presence. Under these conditions, EtG should be combined with EtS LC-MS analysis because EtS appears unaffected by

Ethyl Glucuronide and Ethyl Sulfate

61

bacterial contamination. Preservatives such as fluoride and immediately freezing specimens may prevent or mitigate bacterial growth [110]. Other urine preservatives such as boric acid have not been investigated with respect to EtG. Interestingly, EtG may be formed postcollection in samples infected with E. coli in the presence of ethanol via fermentative processes [112]. This risk is increased in diabetic subjects if glycosuria is also present. Formation of EtG postcollection may not always be prevented by fluoride preservatives or by storage at 4  C [112], and therefore, caution is advised when interpreting results. Formation of EtS in these bacterially contaminated samples did not occur, supporting the recommendation that EtS should accompany or be used to verify EtG results.

8.2. Mouthwash It is important to determine if sources of ethanol other than overt consumption can be responsible for the presence of EtG or EtS. Although ethanol absorbed into the body from alcohol-based mouthwash may result in the presence of EtG in the urine, normal routine use did not generate high urine values [113]. Routine alcohol-based mouthwash use, i.e., three times a day after meals, resulted in 29% of subjects having urine EtG >50 μg/L. In two smaller studies (n ¼ 14 subjects), only one person was positive for urine EtG, whereas seven had detectable EtS (maximum concentration 104 μg/L) [114,115].

8.3. Hand sanitizers It has also been demonstrated that EtG was detected in urine when alcoholcontaining hand sanitizer gels are used frequently [116]. These products typically contain 60–65% ethanol by weight. When used eight times over an 8-h period, urine EtG and EtS up to 103 and 51 μg/L respectively, were reported. A study on intensive use of hand sanitizers was conducted in volunteers (n ¼ 11) who cleansed their hands with an alcohol-based sanitizer (62% ethanol) every 5 min for 10 h on three consecutive days [117]. Urine EtG and EtS could be detected (maximum concentration 2001 and 84 μg/L, respectively) at the end of the study day. Only two specimens had detectable EtG the next morning (96 and 139 μg/L) and only one had detectable EtS (64 μg/L). A more recent study suggested that inhalation of ethanol vapor not transdermal absorption caused the increase in EtG [118]. Using LC-MS/ MS, 2-propyl glucuronide, a metabolite of 2-propanol (a compound

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frequently used in disinfectants), was found in urine. As such, the presence of this metabolite could potentially be used to identify false-positive EtG results. The methodology employed for urine EtG measurement is highly important with respect to false positivity. Positive results after the use of a hand sanitizer in one case could not be confirmed by LC-MS/MS [119]. 1-Propylglucuronide and 2-propylglucuronide were detected, i.e., in vivo metabolites of 1-propanol and 2-propanol, respectively. Interestingly, the two parent compounds accounted for 75% by weight of the sanitizer solution.

8.4. Beverages and food Low alcohol or “alcohol-free” beers have become popular in many parts of the world. Despite having up to 0.5% alcohol, these are still deemed nonalcoholic. Four volunteers who consumed 2.5 L of these nonalcoholic beers had urine EtG concentrations ranging from 300 to 14,100 μg/L the next morning [120]. Positive EtG results were also found in another study 13 h after consumption of nonalcoholic beers [121]. In the same study, the authors showed that consuming foodstuffs that contain alcohol caused positive urine EtG results including samples taken 5 h after eating sauerkraut and 3.5 h after consuming matured bananas [121]. Similarly, in vivo fermentation of baker’s yeast to ethanol with subsequent formation of EtG and EtS has been reported [122]. Paradoxically, ingestion of brewer’s yeast did not result in any positive EtG or EtS results.

8.5. Drugs The case below highlights the importance of method selection for measuring urine EtG. In this report, the patient was taking a number of medications including levetiracetam, gabapentin, clomethiazol, and chloral hydrate [123]. Despite confirmed alcohol abstinence, the patient had urine EtG (up to 8000 μg/L) as determined by commercial immunoassay. Further investigation by LC-MS/MS revealed no urine EtG or EtS. To validate these findings, urine was collected from a control subject who ingested 500 mg chloral hydrate. The control urine was found to have an EtG concentration of 280 μg/L. Trichloroethyl glucuronide was proposed as the most likely cross-reacting compound. Unfortunately, this premise could not be confirmed due to the lack of a pure standard.

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8.6. Hair products Interest in quantifying hair EtG has led a number of groups to investigate the impact of various hair treatments on false-positive and false-negative results. LC-MS/MS demonstrated the presence of EtG (64%) and EtS (27%) in 11 herbal hair tonics [124]. EtG concentration ranged from 70 to 1060 μg/L. A case report of an individual with a hair EtG concentration of 910 ng/g, but normal CDT and GGT, who regularly used a hair tonic was investigated as a potential false-positive [125]. Overnight incubation of EtG-free hair in the lotion resulted in a hair EtG of 140 ng/g. Another report, however, found no increase in hair EtG in seven volunteers using a hair tonic for up to 1 month despite the tonic containing 44% (v/v) ethanol [126]. Although coloring does not to affect hair EtG content, bleaching and perming caused decreased hair EtG (mean decrease 73.5% and 95.7%, respectively) [127]. In vitro experiments using hydrogen peroxide to simulate bleaching and ammonium thioglycate to simulate perming showed similar decreases in hair EtG suggesting chemical degradation of EtG.

9. CONCLUSIONS EtG was first described as a metabolite of ethanol in 1967 [128]; however, the increased use of mass spectrometry over the past decade has resulted in the development of accurate and reliable methods for EtG and EtS in biologic samples. Although most methods were initially developed for urine, there has been renewed interest in testing hair to increase the timeframe for detecting alcohol misuse. Published data suggest that EtG has potential as a marker of high sensitivity and specificity for the detection of alcohol misuse in a variety of settings in both clinical and forensic medicine. As a noninvasive marker, EtG in urine or hair could have a role in screening, diagnosis, and monitoring treatment in selected groups of subjects or in general population studies. Urine EtG remains positive for periods of up to 48–72 h following heavy alcohol consumption. As such, EtG has potential use in the intermediate timeframes, i.e., between those times in which ethanol and GGT/CDT measurements are performed. This approach has been successfully applied to establish abstinence in patients on liver transplant waiting lists and in alcohol detoxification programs. Whether EtG will be adopted in workplace monitoring or regranting driving licenses requires further work.

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The availability of an immunoassay for EtG that can be performed on general clinical chemistry analyzers will make it easier to conduct larger studies. Because bacterial UTIs cause both false-positive and false-negative EtG results, mass spectrometry-based methods that measure both EtG and EtS may be preferable. Nearly all methods for hair measurement of EtG and EtS use mass spectrometry which allows for identification of other alcohols that could interfere with immunoassay-based methods. Further work is clearly required before the full potential of these direct ethanol biomarkers can be realized and incorporated into the armamentarium of alcohol biomarkers in general.

DECLARATIONS Conflicts of interest None. Funding No funding applicable to this review. Ethical approval Ethical approval not required with regard to the content of this review. Guarantor R. A. S. Contributorship The authors contributed equally to the work.

REFERENCES [1] WHO Global Status Report on Alcohol 2011. www.who.int/substance_abuse/ publications/global_alcohol_report/en/ (accessed 31.03.14). [2] Health and Social Care Information Centre, Statistics on Alcohol: England 2013. www.hscic.gov.uk/catalogue/PUB10932/alc-eng-2013-rep.pdf (accessed 31.03.14). [3] G.B. Ingall, Alcohol biomarkers, Clin. Lab. Med. 32 (2012) 391–406. [4] M.L. Hannuksela, M.K. Liisanantti, A.E. Nissinen, M.J. Savolainen, Biochemical markers of alcoholism, Clin. Chem. Lab. Med. 45 (2007) 953–961. [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] H. Dahl, N. Stephanson, O. Beck, A. Helander, Comparison of urinary excretion characteristics of ethanol and ethyl glucuronide, J. Anal. Toxicol. 26 (2002) 2001–2004. [7] G. Høiseth, L. Morini, A. Polettini, A.S. Christophersen, L. Johnsen, R. Karinen, et al., Serum/whole blood concentration ratio for ethyl glucuronide and ethyl sulphate, J. Anal. Toxicol. 22 (2009) 208–211. [8] 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. Leg. Med. 122 (2008) 123–128. [9] G. Høiseth, J.P. Bernard, R. Karinen, L. Johnsen, A. Helander, A.S. Christophersen, et al., A pharmacokinetic study of ethyl glucuronide in blood and urine: applications to forensic toxicology, Forensic Sci. Int. 172 (2007) 119–124. [10] A.M. Lostia, J.L. Vicente, D.A. Cowan, Measurement of ethyl glucuronide, ethyl sulphate and their ratio in the urine and serum of healthy volunteers after two doses of alcohol, Alcohol Alcohol. 48 (2013) 74–82.

Ethyl Glucuronide and Ethyl Sulfate

65

[11] G. Høiseth, L. Morini, A. Polettini, A. Christophersen, J. Mørland, Blood kinetics of ethyl glucuronide and ethyl sulphate in heavy drinkers during alcohol detoxification, Forensic Sci. Int. 188 (2009) 52–56. [12] P. Droenner, G. Schmitt, R. Aderjan, H. Zimmer, A kinetic model describing the pharmacokinetics of ethyl glucuronide in humans, Forensic Sci. Int. 126 (2002) 24–29. [13] G. Høiseth, K. Nordal, E. Pettersen, J. Mørland, Prolonged urinary detection times of EtG and EtS in patients with decreased renal function, Alcohol. Clin. Exp. Res. 36 (2012) 1148–1151. [14] G. Høiseth, L. Morini, R. Ganss, K. Nordal, J. Mørland, Higher levels of hair ethyl glucuronide in patients with decreased kidney function, Alcohol. Clin. Exp. Res. 37 (Suppl. 1) (2013) E14–E16. [15] F.M. Wurst, G.A. Wiesbeck, J.W. Metzger, W. Weinmann, On sensitivity, specificity, and the influence of various parameters on ethyl glucuronide levels in urine— results from the WHO/ISBRA study, Alcohol. Clin. Exp. Res. 28 (2004) 1220–1228. [16] M. Goll, G. Schmitt, B. Ganssmann, R.E. Aderjan, Excretion profiles of ethyl glucuronide in human urine after internal dilution, J. Anal. Toxicol. 26 (2002) 262–266. [17] A. Helander, M. B€ ottcher, C. Fehr, N. Dahmen, O. Beck, Detection times for urinary ethyl glucuronide and ethyl sulphate in heavy drinkers during alcohol detoxification, Alcohol Alcohol. 44 (2009) 55–61. [18] M.J. Kip, C.D. Spies, T. Neumann, Y. Nachbar, C. Alling, S. Aradottir, et al., The usefulness of direct ethanol metabolites in assessing alcohol intake in nonintoxicated male patients in an emergency room setting, Alcohol. Clin. Exp. Res. 32 (2008) 1284–1291. [19] F.M. Wurst, C. Kempter, S. Seidl, A. Alt, Ethyl glucuronide—a marker of alcohol consumption and a relapse marker with clinical and forensic implications, Alcohol Alcohol. 34 (1999) 71–77. [20] H. Schloegl, S. Dresen, K. Spaczynski, M. Stoertzel, F.M. Wurst, W. Weinmann, Stability of ethyl glucuronide in urine, post mortem tissue and blood samples, Int. J. Leg. Med. 120 (2006) 83–88. [21] G. Høiseth, B. Yttredal, R. Karinen, H. Gjerde, J. Mørland, A. Christopherson, Ethyl glucuronide concentrations in oral fluid, blood, and urine after volunteers drank 0.5 and 1.0 g/kg doses of ethanol, J. Anal. Toxicol. 34 (2010) 319–324. [22] M.E. Albermann, F. Musshoff, E. Doberentz, P. Heese, M. Banger, B. Madea, Preliminary investigations on ethyl glucuronide and ethyl sulfate cutoffs for detecting alcohol consumption on the basis of an ingestion experiment and on data from withdrawal treatment, Int. J. Leg. Med. 126 (2012) 757–764. [23] R. Boscolo-Berto, G. Viel, M. Montisci, C. Terranova, D. Favretto, S.D. Ferrara, Ethyl glucuronide concentrations in hair for detecting heavy drinking and/or abstinence: a meta-analysis, Int. J. Leg. Med. 127 (2013) 611–619. [24] A. Helander, O. Beck, Mass spectrometric identification of ethyl sulfate as an ethanol metabolite in humans, Clin. Chem. 50 (2004) 936–937. [25] A. Helander, O. Beck, Ethyl sulfate: a metabolite of ethanol in humans and a potential biomarker of acute alcohol intake, J. Anal. Toxicol. 29 (2005) 270–274. [26] M.H. Wojcik, J.S. Hawthorne, Sensitivity of commercial ethyl glucuronide (ETG) testing in screening for alcohol abstinence, Alcohol Alcohol. 42 (2007) 317–320. [27] F.M. Wurst, S. Dresen, J.P. Allen, G. Weisbeck, M. Graf, W. Weinmann, Ethyl sulphate: a direct ethanol metabolite reflecting recent alcohol consumption, Addiction 101 (2006) 204–211. [28] K. Borucki, R. Schreiner, J. Dierkes, K. Jachau, D. Krause, S. Westphal, et al., Detection of recent ethanol intake with new markers: comparison of fatty acid esters in serum and of ethyl glucuronide and the ratio of 5-hydroxytryptophol to 5-hydroxyindole acetic acid in urine, Alcohol. Clin. Exp. Res. 29 (2005) 781–787.

66

Natalie E. Walsham and Roy A. Sherwood

[29] A. Alt, F.M. Wurst, S. Seidl, Evaluation of the ethylglucuronide in urine samples with the internal standard d5-ethylglucuronide, Blutalkohol 34 (1997) 360–365. [30] N. Stephanson, H. Dahl, A. Helander, O. Beck, Direct quantification of ethyl glucuronide in clinical urine samples by liquid chromatography-mass spectrometry, Ther. Drug Monit. 24 (2002) 645–651. [31] W. Weinmann, P. Schaefer, A. Thierauf, Confirmatory analysis of ethyl glucuronide in urine by liquid chromatography/electrospray ionisation/tandem mass spectrometry according to forensic guidelines, J. Am. Soc. Mass Spectrom. 15 (2004) 188–193. [32] W. Bicker, M. La¨mmerhofer, T. Keller, R. Schuhmacher, R. Krska, W. Lindner, Validated method for the determination of the ethanol consumption markers ethyl glucuronide, ethyl phosphate and ethyl sulphate in human urine by reversed phase/weak anion exchange liquid chromatography-tandem mass spectrometry, Anal. Chem. 78 (2006) 5884–5892. [33] L. Morini, L. Politi, A. Zucchella, A. Polettini, Ethyl glucuronide and ethyl sulphate determination in serum by liquid chromatography-electrospray tandem mass spectrometry, Clin. Chim. Acta 376 (2007) 213–219. [34] L. Morini, E. Marchei, M. Pellegrini, A. Groppi, C. Stramesi, F. Vagnarelli, et al., Liquid chromatography with tandem mass spectrometric detection for the measurement of ethyl glucuronide and ethyl sulphate in meconium: new biomarkers of gestational ethanol exposure? Ther. Drug Monit. 30 (2008) 725–732. [35] S. Hegstad, L. Johnsen, J. Mørland, A.S. Christophersen, Determination of ethylglucuronide in oral fluid by ultra-performance liquid chromatography-tandem mass spectrometry, J. Anal. Toxicol. 33 (2009) 204–207. [36] H. Kharbouche, F. Sporkert, S. Troxler, M. Augsburger, P. Mangin, S. Staub, Development and validation of a gas chromatography-negative chemical ionisation tandem mass spectrometry method for the determination of ethyl glucuronide in hair and its application to forensic science, J. Chromatogr. B Analyt. Technol. Biomed. Life Sci. 877 (2009) 2337–2343. [37] L. Morini, M. Colucci, M.G. Roberto, A. Groppi, Determination of ethyl glucuronide in nails by liquid chromatography tandem mass spectrometry as a potential new biomarker for chronic alcohol abuse and binge drinking behaviour, Anal. Bioanal. Chem. 402 (2012) 1865–1870. [38] A. Hernandez Redondo, A. Schroeck, B. Kneubuehl, W. Weinmann, Determination of ethyl glucuronide and ethyl sulfate from dried blood spots, Int. J. Leg. Med. 127 (2013) 769–775. [39] M. Winkler, G. Skopp, A. Alt, E. Miltner, T. Jochum, C. Daenhardt, et al., Comparison of direct and indirect alcohol markers with PEth in blood and urine in alcohol dependent inpatients during detoxification, Int. J. Leg. Med. 127 (2013) 761–768. [40] L. Politi, L. Morini, A. Groppi, V. Poloni, F. Pozzi, A. Polettini, 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 (2005) 1321–1331. [41] A. Helander, N. Kenan, O. Beck, Comparison of analytical approaches for liquid chromatography/mass spectrometry determination of the alcohol biomarker ethyl glucuronide in urine, Rapid Commun. Mass Spectrom. 24 (2010) 1737–1743. [42] R. Shah, W.R. Lacourse, An improved method to detect ethyl glucuronide in urine using reversed-phase liquid chromatography and pulsed electrochemical detection, Anal. Chim. Acta 576 (2006) 239–245. [43] I.A. Freire, A.M. Barrera, P.C. Silva, M.J. Duque, P.F. Go´mez, P.L. Eijo, Microwave assisted extraction for the determination of ethyl glucuronide in urine by gas chromatography–mass spectrometry, J. Appl. Toxicol. 28 (2008) 773–778.

Ethyl Glucuronide and Ethyl Sulfate

67

[44] I. Alvarez, A.M. Bermajo, M.J. Tabernero, P. Ferna´ndez, P. Carbarcos, P. Lo´pez, Microwave-assisted extraction: a simpler and faster method for the determination of ethyl glucuronide in hair by gas chromatography–mass spectrometry, Anal. Bioanal. Chem. 393 (2009) 1345–1350. [45] C. Schummer, B.M. Appenzeller, R. Wennig, Quantitative determination of ethyl glucuronide in sweat, Ther. Drug Monit. 30 (2008) 536–539. [46] G. Skopp, G. Schmitt, L. P€ otsch, P. Dr€ onner, R. Aderjan, R. Mattern, Ethyl glucuronide in human hair, Alcohol Alcohol. 35 (2000) 283–285. [47] L. Kriva´nkova´, J. Caslavska, H. Mala´skova´, P. Gebauer, W. Thormann, Analysis of ethyl glucuronide in human serum by capillary electrophoresis with sample selfstacking and indirect detection, J. Chromatogr. A 1081 (2005) 2–8. [48] J. Caslavska, B. Jung, W. Thormann, Confirmation analysis of ethyl glucuronide and ethyl sulfate in human serum and urine by CZE-ESI-MS(n) after intake of alcoholic beverages, Electrophoresis 32 (2011) 1760–1764. [49] M. Nova´kova´, L. Kriva´nkova´, Determination of ethyl glucuronide in human serum by hyphenation of capillary isotachophoresis and zone electrophoresis, Electrophoresis 29 (2008) 1694–1700. [50] H. Zimmer, G. Schmitt, R. Aderjan, Preliminary immunochemical test for the determination of ethyl glucuronide in serum and urine: comparison of screening method results with gas chromatography–mass spectrometry, J. Anal. Toxicol. 26 (2002) 11–16. [51] M.E. Albermann, F. Musshoff, B. Madea, A high-performance liquid chromatographic-tandem mass spectrometric method for the determination of ethyl glucuronide and ethyl sulfate in urine validated according to forensic guidelines, J. Chromatogr. Sci. 50 (2012) 51–56. [52] M. B€ ottcher, O. Beck, A. Helander, Evaluation of a new immunoassay for urinary ethyl glucuronide testing, Alcohol Alcohol. 43 (2008) 46–48. [53] J.N. Matlow, K. Aleksa, A. Lubetsky, G. Koren, The detection and quantification of ethyl glucuronide in placental tissue and placental pefusate by headspace solid-phase microextraction coupled with gas chromatography–mass spectrometry, J. Popul. Ther. Clin. Pharmacol. 19 (2012) e473–e482. [54] C. Zeren, A. Keten, S. C ¸ elik, I. Damlar, N. Daglioglu, A. C ¸ eliker, et al., Demonstration of ethyl glucuronide in dental tissue samples by liquid chromatography/electrospray tandem mass spectrometry, J. Forensic Leg. Med. 20 (2013) 706–710. [55] C.L. Crunelle, M. Yegles, A.L. Nuijs, A. Covaci, M. De Doncker, K.E. Maudens, et al., 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 (2014) 1–11. [56] A. Alt, I. Janda, S. Seidl, F.M. Wurst, Determination of ethyl glucuronide in hair samples, Alcohol Alcohol. 35 (2000) 313–314. [57] C. Jurado, T. Soriano, M.P. Gime´nez, M. Mene´ndez, Diagnosis of chronic alcohol consumption. Hair analysis for ethyl-glucuronide, Forensic Sci. Int. 145 (2004) 167–173. [58] L. Politi, L. Morini, F. Leone, A. Polettini, Ethyl glucuronide in hair: is it a reliable marker of chronic high levels of alcohol consumption, Addiction 101 (2006) 1408–1412. [59] F. Lamoureux, J.M. Gaulier, F.L. Sauvage, M. Mercerolle, C. Vallejo, G. Lach^atre, Determination of ethyl-glucuronide in hair for heavy drinking detection using liquid chromatography-tandem mass spectrometry following solid-phase extraction, Anal. Bioanal. Chem. 394 (2009) 1895–1901. [60] L. Morini, L. Politi, S. Acito, A. Groppi, A. Polettini, Comparison of ethyl glucuronide in hair with carbohydrate-deficient transferrin in serum as markers of chronic high levels of alcohol consumption, Forensic Sci. Int. 188 (2009) 140–143.

68

Natalie E. Walsham and Roy A. Sherwood

[61] L. Morini, L. Politi, A. Polettini, Ethyl glucuronide in hair: a sensitive and specific marker of chronic heavy drinking, Addiction 104 (2009) 915–920. [62] G. Høiseth, L. Morini, A. Polettini, A. Christopherson, J. Mørland, Ethyl glucuronide in hair compared with traditional alcohol biomarkers—a pilot study of heavy drinkers referred to an alcohol detoxification unit, Alcohol. Clin. Exp. Res. 33 (2009) 812–816. [63] P. Bendroth, R. Kronstrand, A. Helander, J. Greby, N. Stephanson, P. Krantz, Comparison of ethyl glucuronide in hair with phosphatidylethanol in whole blood as postmortem markers of alcohol abuse, Forensic Sci. Int. 176 (2008) 76–81. [64] R. Paul, R. Kingston, L. Tsanaclis, A. Berry, A. Guwy, Do drug users use less alcohol than non-drug users? A comparison of ethyl glucuronide concentrations in hair between the two groups in medico-legal cases, Forensic Sci. Int. 176 (2008) 82–86. [65] I. Janda, W. Weinmann, T. Kuehnle, M. Lahode, A. Alt, Determination of ethyl glucuronide in human hair by SPE and LC-MS/MS, Forensic Sci. Int. 128 (2002) 59–65. [66] R. Kronstrand, L. Brinkhagen, F.H. Nystr€ om, Ethyl glucuronide in human hair after daily consumption of 16 or 32 g of ethanol for 3 months, Forensic Sci. Int. 215 (2011) 51–55. [67] B. Liniger, A. Nguyen, A. Friedrich-Koch, M. Yegles, Abstinence monitoring of suspected drinking drivers: ethyl glucuronide in hair versus CDT, Traffic Inj. Prev. 11 (2010) 123–126. [68] F. Pragst, M. Rothe, B. Moench, M. Hastedt, S. Herre, D. Simmert, Combined use of fatty acid ethyl esters and ethyl glucuronide in hair for diagnosis of alcohol abuse: interpretation and advantages, Forensic Sci. Int. 196 (2010) 101–110. [69] M.E. Albermann, F. Musshoff, B. Madea, Comparison of ethyl glucuronide (EtG) and fatty acid ethyl esters (FAEEs) concentrations in hair for testing abstinence, Anal. Bioanal. Chem. 400 (2011) 175–181. [70] L.C. Bossers, R. Paul, A.J. Berry, R. Kingston, C. Middendorp, A.J. Guwy, An evaluation of washing and extraction techniques in the analysis of ethyl glucuronide and fatty acid ethyl esters from hair samples, J. Chromatogr. B Analyt. Technol. Biomed. Life Sci. 15 (2014) 953–954. [71] B. M€ onch, R. Becker, I. Nehis, Determination of ethyl glucuronide in hair: a rapid sample pretreatment involving simultaneous milling and extraction, Int. J. Leg. Med. 128 (2014) 69–72. [72] A. Pianta, B. Liniger, M.R. Baumgartner, ethyl glucuronide in scalp and non-head hair: an intra-individual comparison, Alcohol Alcohol. 48 (2013) 295–302. [73] F. Yaldiz, N. Daglioglu, A. Hilal, A. Keten, M.K. Gu¨lmen, Determination of ethyl glucuronide in human hair by hydrophilic interaction liquid chromatography-tandem mass spectrometry, J. Forensic Leg. Med. 20 (2013) 799–802. [74] V. Pirro, D. Di Corcia, F. Seganti, A. Salomone, M. Vincenti, Determination of ethyl glucuronide levels in hair for the assessment of alcohol abstinence, Forensic Sci. Int. 232 (2013) 229–236. [75] P. Cabarcos, H.M. Hassan, M.J. Tabernero, K.S. Scott, Analysis of ethyl glucuronide in hair samples by liquid chromatography–electrospray ionization-tandem mass spectrometry, J. Appl. Toxicol. 33 (2013) 638–643. [76] L. Berger, M. Fendrich, J. Jones, D. Fuhrmann, C. Plate, D. Lewis, Ethyl glucuronide in hair and fingernails as a long-term alcohol biomarker, Addiction 109 (2014) 425–431. [77] T. Neumann, A. Helander, H. Dahl, T. Holzmann, B. Neuner, E. Weiss-Gerlach, et al., Value of ethyl glucuronide in plasma as a biomarker for recent alcohol consumption in the emergency room, Alcohol Alcohol. 43 (2008) 431–435. [78] K. Junghanns, I. Graf, J. Pflu¨ger, G. Wetterling, C. Ziems, D. Ehrenthal, et al., Urinary ethyl glucuronide (EtG) and ethyl sulphate (EtS) assessment: valuable tools to improve verification of abstention in alcohol-dependent patients during in-patient treatment and at follow-ups, Addiction 104 (2009) 921–926.

Ethyl Glucuronide and Ethyl Sulfate

69

[79] H. Dahl, A. Hammarberg, J. Franck, A. Helander, Urinary ethyl glucuronide and ethyl sulfate testing for recent drinking in alcohol-dependent outpatients treated with acamprosate or placebo, Alcohol Alcohol. 46 (2011) 553–557. [80] H. Dahl, A. Voltaire Carlsson, K. Hillgren, A. Helander, Urinary ethyl glucuronide and ethyl sulfate testing for detection of recent drinking in an outpatient treatment program for alcohol and drug dependence, Alcohol Alcohol. 46 (2011) 278–282. [81] F.M. Wurst, K.M. Du¨rsteler-MacFarland, V. Auwaerter, S. Ergovic, N. Thon, M. Yegles, et al., Assessment of alcohol use among methadone maintenance patients by direct ethanol metabolites and self-reports, Alcohol. Clin. Exp. Res. 32 (2008) 1552–1557. [82] F.M. Wurst, P.S. Haber, G. Wiesbeck, B. Watson, C. Wallace, J.B. Whitfield, et al., Assessment of alcohol consumption among hepatitis C-positive people receiving opioid maintenance treatment using direct ethanol metabolites and self-report: a pilot study, Addict. Biol. 13 (2008) 416–422. [83] F.M. Wurst, N. Thon, M. Yegles, C. Halter, W. Weinmann, B. Laskowska, et al., Optimizing heroin-assisted treatment (HAT): assessment of the contribution of direct alcohol metabolites in identifying hazardous and harmful alcohol use, Drug Alcohol Depend. 115 (2011) 57–61. [84] G.E. Skipper, W. Weinmann, A. Thierauf, P. Schaefer, G. Wiesbeck, J.P. Allen, et al., Ethyl glucuronide: a biomarker to identify alcohol use by health professionals recovering from substance use disorders, Alcohol Alcohol. 39 (2004) 445–449. [85] H. Gjerde, A.S. Christopherson, I.S. Moan, B. Yttredal, J.M. Walsh, P.T. Normann, et al., Use of alcohol and drugs by Norwegian employees: a pilot study using questionnaires and analysis of oral fluid, J. Occup. Med. Toxicol. 5 (2010) 13–21. [86] M. Halstedt, S. Herre, F. Pragst, M. Rothe, S. Hartwig, Workplace alcohol testing program by combined use of ethyl glucuronide and fatty acid ethyl esters in hair, Alcohol Alcohol. 47 (2012) 127–132. [87] J.P. Allen, F.M. Wurst, N. Thon, R.Z. Litten, Assessing the drinking status of liver transplant patients with alcoholic liver disease, Liver Transpl. 19 (2013) 369–376. [88] Y. Erim, M. B€ ottcher, U. Dahmen, O. Beck, C.E. Broelsch, A. Helander, Urinary ethyl glucuronide testing detects alcohol consumption in alcoholic liver disease patients awaiting liver transplantation, Liver Transpl. 13 (2007) 757–761. [89] I. Webzell, D. Ball, J. Bell, R.A. Sherwood, A. Marsh, J.G. O’Grady, et al., Substance use in liver transplant candidates: an anonymous urinalysis study, Liver Transpl. 17 (2011) 757–761. [90] K. Staufer, H. Andresen, E. Vettorazzi, N. Tobias, B. Nashan, M. Sterneck, Urinary ethyl glucuronide as a novel screening tool in patients pre-liver and post-liver transplantation improves detection of alcohol consumption, Hepatology 54 (2011) 1640–1649. [91] S. Piano, L. Marchioro, E. Gola, S. Rosi, F. Morando, M. Cavallin, et al., Assessment of alcohol consumption in liver transplant candidates and recipients: the best combination among the tools available, Liver Transpl. 20 (2014) 815–822. [92] M. Sterneck, M. Yegles, G. von Rothkirch, K. Staufer, E. Vewttorazzi, K.H. Scheultz, et al., Determination of ethyl glucuronide in hair improves evaluation of long-term alcohol abstinence in liver transplant candidates, Liver Int. 34 (2013) 469–476. [93] S.H. Stewart, D.G. Koch, I.R. Wilner, P.K. Randall, A. Reuben, Hair ethyl glucuronide is highly sensitive and specific for detecting moderate-to-heavy drinking in patients with liver disease, Alcohol Alcohol. 48 (2013) 83–87. [94] S.H. Stewart, D.G. Koch, D.M. Burgess, I.R. Wilner, A. Reuben, Sensitivity and specificity of urinary ethyl glucuronide and ethyl sulfate in liver disease patients, Alcohol. Clin. Exp. Res. 37 (2013) 150–155. [95] S. Pichini, L. Morini, E. Marchei, I. Palmi, M.C. Rotolo, F. Vagnarelli, et al., Ethylglucuronide and ethylsulfate in meconium to assess gestational ethanol exposure:

70

[96] [97]

[98]

[99]

[100]

[101]

[102]

[103] [104] [105] [106] [107] [108] [109] [110]

Natalie E. Walsham and Roy A. Sherwood

preliminary results in two Mediterranean cohorts, Can. J. Clin. Pharmacol. 16 (2009) 370–375. L. Morini, E. Marchei, F. Vagnarelli, O. Garcia Algar, A. Groppi, L. Mastrobattista, et al., Ethyl glucuronide and ethyl sulfate in meconium and hair—potential biomarkers of intrauterine exposure to ethanol, Forensic Sci. Int. 196 (2010) 74–77. A. Bakdash, P. Burger, T.W. Goecke, P.A. Fasching, U. Reulbach, S. Bleich, et al., Quantification of fatty acid ethyl esters (FAEE) and ethyl glucuronide (EtG) in meconium from newborns for detection of alcohol abuse in a maternal health evaluation study, Anal. Bioanal. Chem. 396 (2010) 2469–2477. S.K. Himes, M. Concheiro, K.B. Scheidweiler, M.A. Huestis, Validation of a novel method to identify in utero ethanol exposure: simultaneous meconium extraction of fatty acid esters, ethyl glucuronide, and ethyl sulfate followed by LC-MS/MS quantification, Anal. Bioanal. Chem. 406 (2014) 1945–1955. S. Pichini, L. Morini, R. Pacifici, J. Tuyay, W. Rodrigues, R. Solimini, et al., Development of a new immunoassay for the detection of ethyl glucuronide (EtG) in meconium: validation with authentic specimens analyzed using LC-MS/MS. Preliminary results, Clin. Chem. Lab. Med. 52 (2014) 1179–1185. F.M. Wurst, E. Kelso, W. Weinmann, F. Pragst, M. Yegles, I. Sondstr€ om Poromaa, Measurement of direct ethanol metabolites suggests a higher rate of alcohol use among pregnant women than found with the AUDIT—a pilot study in a population-based sample of Swedish women, Am. J. Obstet. Gynecol. 198 (2008) 407, e1–e5. L. Morini, E. Marchai, L. Tarini, M. Trivelli, G. Rapisardi, M.R. Elicio, et al., Testing ethyl glucuronide in maternal hair and nails for the assessment of fetal exposure to alcohol: comparison with meconium testing, Ther. Drug Monit. 35 (2013) 402–407. J. Rainio, S. Ahola, P. Kangastupa, J. Kultti, H. Tuomi, P.J. Karhunen, et al., Comparison of ethyl glucuronide and carbohydrate-deficient transferrin in different body fluids for post-mortem identification of alcohol use, Alcohol Alcohol. 49 (2014) 55–59. J. Rainio, J. Kultti, P. Kangastupa, H. Tuoni, S. Haola, P.J. Karhunen, et al., Immunoassay for ethyl glucuronide in vitreous humor: a new tool for post-mortem diagnostics of alcohol use, Forensic Sci. Int. 226 (2013) 261–265. S.C. Turfus, T. Vo, N. Niehaus, D. Gerostamoulos, J. Beyer, An evaluation of the DRI-ETG EIA method for the determination of ethyl glucuronide concentrations in clinical and post-mortem urine, Drug Test. Anal. 5 (2013) 439–445. F.M. Wurst, R. Schu¨ttler, C. Kempter, S. Siedl, T. Glig, K. Jachau, et al., Can ethyl glucuronide be determined in post-mortem body fluids and tissues, Alcohol Alcohol. 34 (1999) 262–263. S. Hegstadt, A. Helland, C. Hagelmann, L. Michelsen, O. Sprigset, EtG/EtS in urine from sexual assault victims determined by UPLC-MS-MS, J. Anal. Toxicol. 37 (2013) 227–232. P.R. Marques, A.S. Tippetts, M. Yegles, Ethylglucoronide in hair is a top predictor of impaired driving recidivism, alcohol dependence, and a key marker of the highest BAC interlock tests, Traffic Inj. Prev. 15 (2014) 361–369. P. Marques, S. Tippetts, J. Allen, M. Javors, C. Alling, M. Yegles, et al., Estimating driver risk using alcohol biomarkers, interlock blood alcohol concentration tests and psychometric assessments: initial descriptives, Addiction 105 (2010) 226–239. R. Aguis, T. Nadulski, H.G. Kahl, B. Dufaux, Ethyl glucuronide in hair—a highly effective test for the monitoring of alcohol consumption, Forensic Sci. Int. 218 (2012) 10–14. A. Helander, H. Dahl, Urinary tract infection: a risk factor for false-negative urinary ethyl glucuronide but not ethyl sulphate in the detection of recent alcohol consumption, Clin. Chem. 51 (2005) 1728–1730.

Ethyl Glucuronide and Ethyl Sulfate

71

[111] S. Baranowski, A. Serr, A. Thierauf, W. Weinmann, M. Grosse Perdekamp, F.M. Wurst, et al., In vitro study of bacterial degradation of ethyl glucuronide and ethyl sulphate, Int. J. Leg. Med. 122 (2008) 389–393. [112] A. Helander, I. Olsson, H. Dahl, Postcollection synthesis of ethyl glucuronide by bacteria in urine may cause false identification of alcohol consumption, Clin. Chem. 53 (2007) 1855–1857. [113] A. Costantino, E.J. DiGregorio, W. Korn, S. Spayd, F. Rieders, The effect of the use of mouthwash on ethyl glucuronide concentrations in urine, J. Anal. Toxicol. 30 (2006) 659–662. [114] G. Høiseth, B. Yttredal, R. Karinen, H. Gjerde, A. Christopherson, Levels of ethyl glucuronide and ethyl sulfate in oral fluid, blood and urine after use of mouthwash and ingestion of non-alcoholic wine, J. Anal. Toxicol. 34 (2010) 84–88. [115] G.M. Reisfield, B.A. Goldberger, A.J. Pesce, B.O. Crews, G.R. Wilson, S.A. Teitelbaum, et al., Ethyl glucuronide, ethyl sulfate, and ethanol in urine after intensive exposure to high ethanol content mouthwash, J. Anal. Toxicol. 35 (2011) 264–268. [116] J.T. Jones, M.R. Jones, C.A. Plate, D. Lewis, Ethyl glucuronide and ethyl sulphate concentrations following use of ethanol containing hand sanitizer, USDTL Research Monograph 2006.02. [117] G.M. Reisfield, B.A. Goldberger, B.O. Crews, A.J. Pesce, G.R. Wilson, S.A. Teitelbaum, et al., Ethyl glucuronide, ethyl sulfate, and ethanol in urine after sustained exposure to an ethanol-based hand sanitizer, J. Anal. Toxicol. 35 (2011) 85–91. [118] T. Arndt, S. Schr€ ofel, B. Gu¨ssregen, K. Stemmerich, Inhalation but not transdermal resorption of hand sanitizer ethanol causes positive ethyl glucuronide findings in urine, Forensic Sci. Int. 237 (2014) 126–130. [119] T. Arndt, J. Gru¨ner, S. Schr€ ofel, K. Stemmerich, False-positive ethyl glucuronide immunoassay screening caused by a propyl alcohol-based hand sanitizer, Forensic Sci. Int. 223 (2012) 359–363. [120] A. Thierauf, H. Gnann, A. Wohlfarth, V. Auwa¨rter, M.G. Pederkamp, K.J. Buttler, et al., Urine tested positive for ethyl glucuronide and ethyl sulphate after the consumption of “non-alcoholic” beer, Forensic Sci. Int. 202 (2010) 82–85. [121] F. Musshoff, E. Albermann, B. Madea, Ethyl glucuronide and ethyl sulfate in urine after consumption of various beverages and foods—misleading results? Int. J. Leg. Med. 124 (2010) 623–630. [122] A. Thierauf, A. Wohlfarth, V. Auwa¨rter, M.G. Pederkamp, F.M. Wurst, W. Weinmann, Urine tested positive for ethyl glucuronide and ethyl sulfate after the consumption of yeast and sugar, Forensic Sci. Int. 202 (2010) e45–e47. [123] T. Arndt, B. Girten, B. Gussregen, A. Werle, J. Gruner, False-positive ethyl glucuronide immunoassay screening associated with chloral hydrate medication as confirmed by LC-MS/MS and self-medication, Forensic Sci. Int. 184 (2009) e27–e29. [124] T. Arndt, S. Schr€ ofel, K. Stemmerich, Ethyl glucuronide identified in commercial hair tonics, Forensic Sci. Int. 231 (2013) 195–198. [125] F. Sporkert, H. Kharbouche, M.P. Augsburger, C. Klemm, M.R. Baumgartner, Positive EtG findings in hair as a result of a cosmetic treatment, Forensic Sci. Int. 218 (2012) 97–100. [126] L. Martins Ferreira, T. Binz, M. Yegles, The influence of ethanol containing cosmetics on ethyl glucuronide concentration in hair, Forensic Sci. Int. 218 (2012) 123–125. [127] I. Kerekes, M. Yegles, Coloring, bleaching, and perming: influence on EtG content in hair, Ther. Drug Monit. 35 (2013) 527–529. [128] P.I. Jaakonmaki, K.L. Knox, E.C. Horning, M.G. Horning, The characterization by gas–liquid chromatography of ethyl-β-D-glucosiduronic-acid as a metabolite of ethanol in rat and man, Eur. J. Pharmacol. 1 (1967) 63–70.