Detection Of Ethyl Glucuronide Research Articles

Dried blood spot (DBS) for ethyl glucuronide (EtG) and ethyl sulfate (EtS) determination in blood with a new design of “volumetric” DBS filter paper card

Determination of EtG in blood can be performed from capillary blood with high sensitivity and detection limits of down to 1 ng/mL for EtG–and in addition to phosphatidyl ethanol (PEth) determination, EtG concentrations can be used for interpretation of consumption habits of patients in maintenance therapy. Sampling of capillary blood is a crucial preanalytical step–and different methods have been suggested for collection of a defined volume (VAMS, Capitainer, HemaXis, and capillaries for collection of samples). We used and validated a new volumetric capillary blood sampling card (DBSv cards, Protzek, Lörrach/Germany) which can be used either for self-sampling of dried blood spots (up to 4 times 20 microliter of blood) on filter paper with convenient handling by the patients or also in laboratory work. In contrast to standard DBS cards with printed circles on a filter paper, a newly designed card with a paper comb with four teeth per card, which allows a volumetric collection of 20 microlitres of blood per tooth, was used. Venous blood was pipetted on the teeth of DBSv cards, dried, and one paper tooth was ripped off and extracted by adding 490 microliter of methanol and 10 microliter of ISTD (400 ng/ml D5-EtG and 400 ng/ml D5-EtS). The samples were then vortexed for 5 minutes and centrifuged for 10 minutes. After transferring them into vials, the solutions were evaporated under a stream of nitrogen and reconstituted in 80 microliter of a water/0.1% formic acid. The samples were shaken and 10 μL were injected onto the LC column. A previously published LC-MS/MS method was adapted for a QTrap 5500 triple-quadrupole ion trap mass spectrometer (Sciex, Toronto, Canada) with an Acquity C18 HSS T3 column (Waters, Wexford, Ireland) and detection of EtG, D5-EtG, EtS and D5-EtS was performed according to forensic guidelines using one precursor and two product ions. Lower limits of quantification were 10 ng/mL for EtG and 20 ng/mL for EtS–with linear ranges of 10 to 5000 ng/mL for EtG and (50 to 2500 ng/mL for EtS. Traces of EtS were found in HPLC grade solvent, and a solvent selection between products of different manufacturers had to be performed. One brand methanol showed the lowest baseline level of EtS, which was in the range of 20 ng/mL. Thus, for determination of EtS, a lower limit of quantitation of 50 ng/mL was defined, whereas EtG could be quantified at concentrations above 10 ng/mL. A validation with spiked blood samples was performed. Samples from forensic cases and from drinking studies, where DBS were collected for the analysis of alcohol biomarkers (PEth and EtG/EtS) have been analyzed. The dried blood spot sample collection in laboratory or by self-sampling (of capillary blood) can be used to detect EtG and EtS at very low levels. The results show the potential of DBS analysis for alcohol markers EtG and EtS–which can be used for abstinence monitoring by very low limits of detection, especially for EtG, or for the detection of a recent alcohol consumption, when ethanol has already been eliminated from the body. A contamination of pure HPLC-grade solvent methanol was found for EtS determination, this has to be kept in mind, and needs further optimization for detection of EtS at lower concentrations in biological samples.

Testing venous carbohydrate-deficient transferrin or capillary phosphatidylethanol with concurrent ethyl glucuronide and ethyl palmitate hair tests to assess historical and recent alcohol use.

Hair biomarkers, ethyl glucuronide (EtG) and ethyl palmitate (EtPa), together with blood biomarker tests, carbohydrate-deficient transferrin (CDT) or phosphatidylethanol (PEth), are commonly used in identifying patterns of alcohol consumption as they possess different windows of detection. The detection of EtG in hair samples is mainly used in combination with EtPa when hair cosmetic treatments such as hair colouring and bleaching affect EtG levels. The main purpose of our study was to investigate the differences in frequency distribution of positive CDT and PEth results indicating alcohol had been used, when EtG and EtPa were not detected, where evidence of abstinence is paramount. Of the total 602 cases, for 179 (29.7%), neither EtG nor EtPa markers were detected. Of these, 0.5% of the cases produced positive CDT. However, 18.6% produced positive PEth, a significantly higher proportion. A similar pattern emerges when results are evaluated according to whether hair had been either cosmetically treated or untreated. When hair was untreated, one case produced positive CDT, and 19.3% were positive for PEth (median of 51 ng/ml). No cases of positive CDT results, but 20.8% of PEth were positive (median of 106.5 ng/ml) when hair samples had been cosmetically treated. Whether EtG or EtPa markers were detected or not, significantly higher proportions of PEth than CDT were seen. The results of this study substantiate the case for using hair EtG in conjunction with a PEth test, rather than CDT test, for efficient monitoring of recent and historical alcohol consumption.

Determination of ethyl glucuronide (EtG) in blood samples using partial least squares discriminant analysis applied to surface-enhanced Raman spectroscopy

Consumption of alcohol and problems related to alcohol vary widely around the world. Especially illegally produced and distributed alcohols have a great risk for human health, since they include high quantity of toxic substances. Ethanol measurements have high specificity for excessive alcohol intake. Ethyl Glucuronide (EtG) is considered as a direct biomarker of recent ethanol consumption.The study aims to identify EtG in blood samples by using Raman Spectroscopy. Silver colloidal nanoparticles were synthesized to obtain SERS (Surface-Enhanced Raman Spectroscopy) spectra of blood samples and EtG. PLS-DA (Partial Least Squares Discriminant Analysis) models were built on the collected SERS spectra. It is observed that EtG and the blood samples taken from people using alcohol are in the same group and the blood samples taken from no alcohol users are in a separate cluster from EtG. The sources of data variability are modelled by the so-called Latent Variables (LVs). The percentage of LVs were found to be 29.53% and 8.34%, respectively.In the study, it has been observed that the level of EtG changes according to the level of ethanol in blood samples. However, there is no study on the detection of EtG in blood with Raman spectroscopy. Outcomes of the present study encourage the feasibility of the application of Raman Spectroscopy for determination of EtG in blood samples.

A Real-Time Wearable Sensor to Monitor Alcohol Consumption through Ethyl Glucuronide Detection

Here we demonstrate for the first time a wearable sensor that can differentiate Ethyl Glucuronide (EtG) concentrations in synthetic human sweat correlating to the predicted alcohol consumption profiles of moderate drinkers. Alcohol consumption monitoring devices typically report blood alcohol content (BAC) through correlation with breath alcohol content (BrAC). However, the rapid decay of expressed ethanol can limit the application space of these devices. Alcohol is metabolized and excreted from the body by different processes or pathways, ethyl Glucuronide (EtG) is one of them and is produced in the liver. EtG is readily expressed and detectable in transdermal human sweat making it a promising candidate for wearable alcohol consumption monitoring. A wearable biosensor device capable of reporting EtG levels in sweat both continuously via low power impedance spectroscopy is reported. The device was not only able to detect and respond to EtG present in the samples, but also able to differentiate each test scenario from one another. The three distinct EtG concentrations was designed as simulated drinking scenarios estimated to be 1, 2, and 3 standard U.S. drinks consumed over a duration of 60 minutes. It shows the ability for this device to act as an accurate reporter of EtG levels in low volume sweat (less than 5 microliter). The device will serve as a candidate tool for researchers to better understand and characterize the relationship between sweat EtG and consumed alcohol in real-time.

AWARE: A Wearable Awareness with Real-time Exposure, for monitoring alcohol consumption impact through ethyl glucuronide detection.

Here we demonstrate for the first time a dynamic monitoring of the ethanol metabolite ethyl glucuronide (EtG) for a more robust evaluation of alcohol consumption, compared to conventional methods. A wearable biosensor device capable of reporting EtG levels in sweat continuously via low power impedance spectroscopy is reported. The custom hardware was compared against a conventional benchtop potentiostat, and demonstrated comparable results in the application of EtG detection in low volume sweat. The device successfully differentiated three distinct EtG concentrations correlating to simulated drinking scenarios estimated to be 1, 2, and 3 standard U.S. drinks consumed over a duration of 60min, with p<0.0001. This device has the potential to enable moderate drinkers to engage in guided decision-making, based on objective data, to address the needs of alcohol-sensitive populations. The device also will serve as a tool for researchers to better understand and characterize the relationship between sweat EtG and consumed alcohol.

Systematic optimisation of ethyl glucuronide extraction conditions from scalp hair by design of experiments and its potential effect on cut-off values appraisal.

The quantitative determination of ethyl glucuronide (EtG) in hair samples is consistently used throughout the world to assess chronic excessive alcohol consumption. For administrative and legal purposes, the analytical results are compared with cut-off values recognised by regulatory authorities and scientific societies. However, it has been recently recognised that the analytical results depend on the hair sample pretreatment procedures, including the crumbling and extraction conditions. A systematic evaluation of the EtG extraction conditions from pulverised scalp hair was conducted by Design of Experiments (DoE) considering the extraction time, temperature, pH, and solvent composition as potential influencing factors. It was concluded that an overnight extraction at 60°C with pure water at neutral pH represents the most effective conditions to achieve high extraction yields. The absence of differential degradation of the internal standard (isotopically-labelled EtG) under such conditions was confirmed and the overall analytical method was validated according to SGWTOX and ISO17025 criteria. Twenty real hair samples with different EtG content were analysed with 3 commonly accepted procedures: (a) hair manually cut in snippets and extracted at room temperature; (b) pulverised hair extracted at room temperature; (c) hair treated with the optimised method. Average increments of EtG concentration around 69% (from a to c) and 29% (from b to c) were recorded. In light of these results, the authors urge the scientific community to undertake an inter-laboratory study with the aim of defining more in detail the optimal hair EtG detection method and verifying the corresponding cut-off level for legal enforcements.

Sonochemically Synthesized Zinc Oxide Nanoflakes Based Electrochemical Immunosensor for Ethyl Glucuronide (EtG) Detection

Continuous monitoring of alcohol detection is imperative for the prevention of accidents, point of care monitoring, and personal safety. The cutting-edge technologies are mostly focused to detect alcohol from the breath (e.g. breathalyzer), which is not compatible for the real-time monitoring. The relationship between the blood and breath alcohol concentrations and the improvement of the detection techniques of alcohol biomarkers from the sweat render an opportunity for point of care monitoring. Due to its prolonged existence in sweat, Ethylglucuronide (EtG) is considered as one of the most promising biomarkers for alcohol detection. We report on label-free highly sensitive electrochemical immunosensor for the detection and the quantification of EtG based on sonochemically synthesized two-dimensional (2D) zinc oxide (ZnO) nanoflakes (NFs) on flexible Au-coated polyethylene terephthalate (PET) substrate. Highly sensitive detection of EtG using cyclic voltammetry (CV) is achieved by immobilizing EtG antibody on the as-synthesized sensing electrodes of ZnO NFs.

Sonochemically Synthesized Zinc Oxide Nanoflakes Based Electrochemical Immunosensor for Ethyl Glucuronide (EtG) Detection

We report on label free highly sensitive electrochemical immunosensor for monitoring alcohol consumption through the detection and quantification of ethyl glucuronide (EtG) -a metabolite of ethanol-based on two-dimensional (2D) zinc oxide (ZnO) nanoflakes (NFs), which were synthesized on flexible Au-coated polyethylene terephthalate (PET) substrate using simple one step sonochemical approach. Highly sensitive detection of EtG using cyclic voltammetry (CV) is achieved by immobilizing EtG antibody on the as-synthesized sensing electrodes of ZnO NFs. Continuous monitoring of alcohol detection is imperative for the prevention of accidents, point of care monitoring, and personal safety. The cutting-edge technologies are mostly focused to detect alcohol from the breath (e.g. breathalyzer), which is not compatible for the real-time monitoring. The relationship between the blood and breath alcohol concentrations and the improvement of the detection techniques of alcohol biomarkers from the sweat render an opportunity for point of care monitoring. Among different biomarkers, Ethylglucuronide (EtG) has been found as a promising indicator of alcohol due toit’s prolonged existence in sweat (24 hours for one or two “drinks” or 2-4 days for binge drinking), which prevents relapsing. PET has gained popularity as substrate for wearables because of its intrinsic elasticity, thermal stability, hydrophobicity, excellent dielectric properties, low coefficient of thermal expansion, structural resiliency against repeated bending forces and compatibility with roll-to-roll fabrication processes for low cost and scalable manufacturing. ZnO, a semiconducting material with piezoelectricity,high catalytic efficiency, biocompatibility, chemical stability in physiological environments, low toxicity and a high isoelectric point (IEP) of about 9.5, has been proposed for biosensing applications. ZnO nanostructures (NSs) with different morphologies such as nanorods, nanowalls, nanobelts and quantum dots have been used as immobilizing matrix in fabrication of biosensors for the detection of physiologically relevant biomarkers such as cholesterol, galactose, glucose. Considering their promising properties and potential applications, many techniques have been developed to synthesize various ZnO NSs. In comparison to the more conventional approaches such as hydrothermal method, sonochemical synthesis method is not only significantly faster, inexpensive and performed at ambient conditions but also highly stable and reproducible with the advantages of more uniform size distribution, faster reaction time and a higher surface area, which are essential to design sensing platform withimproved performance. Compared to bulk materials, the 2D ZnO NFs provide unique sensing advantages with polarized (0001) plane orientation and high surface charge density, which could enhance EtG antibody loading and thus improve sensing performance. High isoelectric point allows immobilization of most biomolecules without any additional biding layer. This provides a direct, stable pathway for rapid electron transport when an analyte is immobilized on NFs and improves electron transfer rate greater than 1D ZnO nanorods, high isoelectric point,that results in change of the resistance.Taking advantage of these unique properties, we immobilizedEtG antibody on the synthesized ZnO NFs. When an analyte binds the antibody, there is a change in the current due to increase in the charge transfer resistance. The cyclic voltammetry (CV) measurements were taken in the potential range of -0.1V-0.8V at a scan rate of 50mVs-1. For the EtGconcentrationsof1ng/mL-100µg/mLusing PET/Au/ZnO NFs as the working electrode. The minimum detection limit was found to be much lower than 1ng/mL which covers the physiological range. The sensitivity of the sensor was found to be 18.48 mA/ng/mL/cm-2.

Ethyl glucuronide in keratinous matrices as biomarker of alcohol use: A correlation study between hair and nails

To quantify alcohol use, objective, specific and sensitive long-term alcohol markers are necessary. Ethyl glucuronide (EtG), a direct metabolite of alcohol, accumulates in keratinous matrices such as hair and nails, and is a specific and sensitive long-term biomarker for the detection of chronic alcohol consumption. So far, research has primarily focused on the detection of EtG in hair, and studies on its measurement in nails are scarce. In this article, we assessed EtG concentrations in hair, finger- and toenails from the same individuals in order to evaluate the direct correlation between the matrices. To this end, a total amount of 45 hair, 41 fingernail, and 13 toenail samples were collected from patients treated for alcohol use disorders at two psychiatric centers in Belgium. Samples were analyzed by gas chromatography-tandem mass spectrometry. Hair EtG concentrations ranged from <LLOQ to 1149pg/mg (median=164pg/mg, IQR [42; 283]). Fingernail EtG concentrations ranged from <LLOQ to 4090pg/mg (median=250pg/mg, IQR [74; 645]). Toenail EtG concentrations ranged from 127 to 3792pg/mg (median=687pg/mg, IQR [379; 1370]). EtG levels in hair and nails were significantly and positively correlated (p-values<0.001, r=0.70 and r=0.62, respectively). Higher concentrations were present in finger- and toenails compared to hair, which might be attributed to the slower growth rate of nails, resulting in increased accumulation of EtG. Hence, nail analysis may be interesting when low concentrations of EtG are expected, e.g. to discriminate between teetotalers and social drinkers. In addition, the current study proposes preliminary cut-off values for EtG concentrations in fingernails: >123pg/mg for chronic excessive alcohol consumption, 59–123pg/mg for moderate alcohol consumption, and <59pg/mg for alcohol abstinence. In light of these results, nails may be a useful alternative to hair samples for monitoring of long-term alcohol consumption, e.g., in cases where hair is not available. Further studies are needed to establish cut-off values for EtG levels in nails.

Alcohol Consumption during Pregnancy: Analysis of Two Direct Metabolites of Ethanol in Meconium.

Alcohol consumption in young women is a widespread habit that may continue during pregnancy and induce alterations in the fetus. We aimed to characterize prevalence of alcohol consumption in parturient women and to assess fetal ethanol exposure in their newborns by analyzing two direct metabolites of ethanol in meconium. This is a cross-sectional study performed in September 2011 and March 2012 in a series of women admitted to an obstetric unit following childbirth. During admission, socio-demographic and substance use (alcohol, tobacco, cannabis, cocaine, and opiates) during pregnancy were assessed using a structured questionnaire and clinical charts. We also recorded the characteristics of pregnancy, childbirth, and neonates. The meconium analysis was performed by liquid chromatography—tandem mass spectrometry (LC-MS/MS) to detect the presence of ethyl glucuronide (EtG) and ethyl sulfate (EtS). Fifty-one parturient and 52 neonates were included and 48 meconium samples were suitable for EtG and EtS detection. The median age of women was 30 years (interquartile range (IQR): 26–34 years); EtG was present in all meconium samples and median concentration of EtG was 67.9 ng/g (IQR: 36.0–110.6 ng/g). With respect to EtS, it was undetectable (<0.01 ng/g) in the majority of samples (79.1%). Only three (6%) women reported alcohol consumption during pregnancy in face-to-face interviews. However, prevalence of fetal exposure to alcohol through the detection of EtG and EtS was 4.2% and 16.7%, respectively. Prevention of alcohol consumption during pregnancy and the detection of substance use with markers of fetal exposure are essential components of maternal and child health.

A wearable biochemical sensor for monitoring alcohol consumption lifestyle through Ethyl glucuronide (EtG) detection in human sweat.

We demonstrate for the first time a wearable biochemical sensor for monitoring alcohol consumption through the detection and quantification of a metabolite of ethanol, ethyl glucuronide (EtG). We designed and fabricated two co-planar sensors with gold and zinc oxide as sensing electrodes. We also designed a LED based reporting for the presence of EtG in the human sweat samples. The sensor functions on affinity based immunoassay principles whereby monoclonal antibodies for EtG were immobilized on the electrodes using thiol based chemistry. Detection of EtG from human sweat was achieved through chemiresistive sensing mechanism. In this method, an AC voltage was applied across the two coplanar electrodes and the impedance across the sensor electrodes was measured and calibrated for physiologically relevant doses of EtG in human sweat. EtG detection over a dose concentration of 0.001–100 μg/L was demonstrated on both glass and polyimide substrates. Detection sensitivity was lower at 1 μg/L with gold electrodes as compared to ZnO, which had detection sensitivity of 0.001 μg/L. Based on the detection range the wearable sensor has the ability to detect alcohol consumption of up to 11 standard drinks in the US over a period of 4 to 9 hours.

Ethyl glucuronide in nails: method validation, influence of decontamination and pulverization, and particle size evaluation

Ethyl glucuronide (EtG), a minor metabolite of alcohol, is present in hair and nails, and thus can be used as a maker to estimate previous alcohol consumption. Due to the low concentrations of EtG in keratinized matrices (pg/mg range), sensitive analytical methods and optimized sample preparation procedures are essential. While several methods for the detection of EtG in hair have been published, its detection in nails has been scarcely investigated. In this study, an analytical method for the determination of EtG in nails using gas chromatography coupled with tandem mass spectrometry (GC–MS/MS) was developed and validated. In addition, the influence of sample preparation on EtG concentrations was evaluated. The calibration curve was linear from 2 to 100 pg/mg, and the lower limit of quantification (LLOQ) was 2 pg/mg. The method was applied to finger- and toenails of five regular alcohol consumers, with concentrations ranging from <LLOQ to 123 pg/mg. No significant difference in EtG concentrations in washing solutions was found with the use of different decontamination procedures, including hexane, dichloromethane, methanol, acetone, and water as solvents. More intense pulverization of the nail samples increased EtG concentrations, in parallel with a decrease in particle size, but reaching a plateau. In conclusion, we present a sensitive, validated GC–MS/MS technique for the determination of EtG in nails which were pulverized to obtain a high extraction surface area.

P8: The influence of cleansing shampoos on ethyl glucuronide concentrations in hair analyzed with an optimized and validated LC-MS/MS method

Introduction Ethyl glucuronide (EtG) has been established as a routine biomarker for monitoring alcohol abuse or abstinence. Recently, social networks and blogs have become a platform for sharing information on how to avoid EtG detection through bleaching, dyeing, shaving or straightening the hair. In this study, we investigated the influence of four special cleansing shampoos that promise to ‘detox’ and thereby reduce ethyl glucuronide concentrations in hair. Methods An optimized LC-MS/MS method was developed using a hypercarb ® porous graphitic carbon (PGC) column and validated according to the guidelines of the German Society of Toxicological and Forensic Chemistry. For the comparative analysis of the present study, 25 real case hair samples were analyzed in total with 21 positive hair samples covering a concentration range of EtG ( c ETG ) from 7-288 pg/mg and 4 negative hair samples with c ETG Results EtG concentrations in hair did not show any significant differences after a single application of the various cleansing shampoos. EtG was still detectable in all hair samples at equal concentration levels, respectively. These results clearly demonstrated that a single application of the tested cleansing shampoos did not remove or reduce EtG from hair and therefore had no influence on EtG concentrations. Conclusion The reported LC-MS/MS method for the analysis of EtG in hair has been used to study the influence of so-called “detox” shampoos on EtG concentration in hair. This study showed that none of the four selected shampoos influenced the detected concentration of EtG in hair. These results demonstrate that these shampoos do not remove EtG from hair after a single application.

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

Ethyl glucuronide (EtG) measurement in neonatal meconium has emerged as a reliable marker to objectively assess prenatal exposure to maternal ethanol complementary to fatty acid ethyl ester (FAEEs) measurement. The detection of EtG in meconium is currently a lengthy, difficult and expensive process using liquid chromatography tandem mass spectrometry (LC-MS/MS) as the analytical procedure. An enzyme-linked immunosorbent assay (ELISA) for the identification of EtG in meconium was developed, validated and applied to authentic meconium specimens from newborns collected in Europe. The ELISA procedure was calibrated using 0.45, 0.9, 1.35 and 1.8 nmol/g (100, 200 300 and 400 ng/g) standards. Meconium (0.25 g) was mixed thoroughly, with extraction buffer (pH 7.3; 0.5 mL). The tube was capped, sonicated, centrifuged and the supernatant was decanted. An aliquot of the extract (50 μL) was placed in the well of the microplate followed by enzyme conjugate (150 μL). The plate was incubated for 1 h, washed with deionized water, dried and substrate (200 μL) was added. After 30 min incubation, stop solution was added and the plate was read at 450 nm and 650 nm. Samples were also analyzed for EtG and FAEEs by validated LC-MS/MS assays. Using an EtG cut-off of 0.9 nmol/g for both ELISA screening test and confirmatory LC-MS/MS, immunoassay sensitivity was 100%; specificity 78%; positive-predictive value (PPV) 29% and negative-predictive value (NPV) 100%. The assay is proposed as a preliminary screening test for the meconium of newborns suspected of being born to mothers drinking alcohol during pregnancy.

Interpretation of a highly positive ethyl glucuronide result together with negative fatty acid ethyl esters result in hair and negative blood results

Considering the widespread nature of alcohol-related problems, the diagnosis of excessive alcohol consumption is an important task from medical and legal viewpoints. Alcohol abuse can be documented by usual blood [e.g., carbohydrate deficient transferrin (CDT)] and liver function [e.g., γ-GT or mean corpuscular volume (MCV)] tests, and, over a long-term basis, by hair analysis. Major markers of ethanol consumption in hair are ethyl glucuronide (EtG) and fatty acid ethyl esters (FAEEs). Detection of EtG in hair is said to be associated with excessive alcohol consumption, whereas a negative result does not unambiguously exclude alcohol abuse. Investigations on FAEEs can also be used to monitor excessive alcohol consumption. Four FAEEs (ethyl myristate, ethyl palmitate, ethyl oleate, and ethyl stearate) are the most suitable markers for the detection of heavy alcohol consumption and show different concentrations in hair of children, adult teetotalers, and social drinkers in comparison with FAEEs concentrations found in the hair of alcoholics. The Society of Hair Testing has provided guidelines for hair testing for chronic excessive alcohol consumption, and states positive cutoffs for a 0–3 cm hair segment as 30 pg/mg and 0.5 ng/mg for EtG and FAEEs, respectively. This study reviews the difficulties in interpreting blood and hair results of a 41-year-old woman involved in a child custody legal dispute. Her EtG and FAEEs concentrations in a 0–3 cm segment were 203 pg/mg and 0.29 ng/mg, respectively, and blood parameters were in the normal range (0.6 %, 93 fl, 14 IU/l for CDT, MCV, and γ-GT, respectively). Although the high EtG concentration suggested excessive drinking behavior, the second hair marker, FAEEs, and the three bloods tests were inconspicuous and in accordance with the claim of abstinence from alcohol by the subject. The woman declared having dyed her hair more than 6 months prior to sampling, use of weight-loss medication, and consumption of about four energy drinks per day. A hair lotion based on ethanolic plant extracts was regularly used by the subject, and could have been a source of contamination. Therefore, we recommend that possible external sources of hair contamination always be taken into consideration, especially if contradictory biological results are obtained and if the subject denies any alcohol intake.

Inhibition of bacterial degradation of EtG by collection as dried urine spots (DUS)

Ethyl glucuronide (EtG) and ethyl sulfate (EtS) are direct alcohol consumption markers widely used nowadays for clinical and forensic applications. They are detectable in blood and urine even after consumption of trace amounts of ethanol and for a longer time frame, being detectable even when no more ethanol is present. The instability of EtG against bacterial degradation in contaminated urine samples and/or the possible postcollection synthesis of this metabolite in samples containing, e.g., Escherichia coli and ethanol, may cause false identification of alcohol uptake. Therefore, it is of paramount importance to constrict these error sources by inhibition of any bacterial growth causing hydrolization or synthesis of EtG. This study evaluates a new method of collecting urine samples on filter paper, dried urine spots (DUS), for simultaneous detection of EtG, EtS and creatinine, having the great advantage of inhibiting bacterial activity. In addition, a method validation for the determination of EtG and EtS in DUS was performed according to the FDA guidelines. Sterile-filtered urine was spiked with EtG and EtS, inoculated with E. coli and incubated. Liquid and dried urine samples were collected after various time intervals up to 96 h. Liquid samples were frozen immediately after collection, whereas aliquots for DUS were pipetted onto filter paper, allowed to dry and stored at RT until analysis 1 week after. The specimens were analyzed by LC-ESI-MS/MS. As expected, degradation of EtG, but not of EtS, was observed in contaminated liquid urine samples. However, the specimens collected on filter paper and stored at RT showed no degradation during storage. Therefore, collecting urine samples on filter paper for EtG and EtS analysis turns out to be a reliable method to avoid bacterial degradation of EtG and EtS, and consequently, stabilization of these ethanol metabolites is achieved. In addition, simultaneous measurement of creatinine content as an indicator of urine dilution helps to interpret the results. Method validation for EtG and EtS in DUS was satisfactory, showing the linearity of the calibration curves in the studied concentration range, good precision, accuracy and selectivity.

Detection of ethyl glucuronide in blood spotted on different surfaces

This study aims to show that sensitive detection of ethyl glucuronide in dried blood spotted onto various surfaces after a period of 24h is feasible. At present, there is insufficient information how tightly ethyl glucuronide (EtG) binds to various materials and how easily it can be eluted. 4ml aliquots of blood samples obtained from seven volunteers after consumption of alcoholic beverages were applied to six different surfaces. After drying and a 24h-storage at 20±2°C the samples were re-dissolved in water, and EtG was subsequently analyzed by a LC-MS Paul-type ion trap. A comparison was made between dried and corresponding fluid samples. EtG was detectable in all subjects' samples following consumption of alcohol. EtG was also detectable after a storage time of four weeks at 4°C in whole blood that had been preserved with EDTA. EtG was detectable in all samples dried on different surfaces and its concentration remained relatively constant irrespective of the particular condition of the material. Detection of EtG in blood spots from the scene may indicate recent alcohol consumption in cases where collection of blood remained undone or could not be performed.

Confirmation analysis of ethyl glucuronide and ethyl sulfate in human serum and urine by CZE‐ESI‐MSn after intake of alcoholic beverages

CZE coupled to sheath liquid-based electrospray ionization (ESI) and multiple-stage ion trap mass spectrometry (MS(n) ) was used for the confirmation analysis of ethyl glucuronide (EtG) and ethyl sulfate (EtS) in human serum and urine collected after intake of alcoholic beverages. Electrophoretic separations were performed in uncoated fused-silica capillaries using a pH 9.5 ammonium acetate background electrolyte and normal polarity. MS detection of EtG and EtS occurred after negative ionization using a spray liquid containing 0.5% v/v ammonia in isopropanol/water (60:40%, v/v). CZE-MS and CZE-MS² results obtained after injection of solid-phase extracts for EtG and EtS and of diluted urine confirmed the presence of EtG and EtS in samples whose levels were previously determined by CZE with indirect UV detection. Detection limits of each compound were estimated to be around 2.0 (injection of diluted urine) and 0.2 μg/mL (extracts).

Population Baseline of Meconium Ethyl Glucuronide and Ethyl Sulfate Concentrations in Newborns of Nondrinking Women in 2 Mediterranean Cohorts

The detection of ethyl glucuronide (EtG) and ethyl sulfate (EtS) in meconium has been investigated recently as an alternative to meconium fatty acid ethyl esters (FAEEs) measurement as an objective estimate of prenatal alcohol exposure, independent of maternal self-reporting. We report the results of the first study conducted to investigate the concentrations of EtG and EtS in meconium from newborns with and without intrauterine exposure to ethanol, defined by questionnaire and meconium FAEEs concentration. FAEEs, EtG, and EtS were quantified by liquid chromatography tandem mass spectrometry in meconium samples obtained from the Arcispedale Santa Maria Nuova, Reggio Emilia, Italy (n = 80) and from the Hospital del Mar in Barcelona, Spain (n = 105). Median EtG and EtS values in meconium from newborns without intrauterine exposure to ethanol varied between 0.100 and 0.140 nmol/g and 0.010 and 0.020 nmol/g in Reggio Emilia and Barcelona samples, respectively. In meconium from newborns with uncertain prenatal ethanol exposure, the EtG median value was 0.160 nmol/g in the Italian cohort and 0.250 nmol/g in the Spanish one. The median EtS concentration was 0.020 in both cohorts. EtG and EtS median values in 5 meconium samples from newborns of heavily drinking mothers were 7.240 nmol/g and 0.033 nmol/g, respectively. A positive cutoff of 2.0 nmol/g for EtG yielded the best sensitivity and specificity (100%) to discriminate for true prenatal exposure to ethanol. It was not possible to establish a proper cutoff for EtS because of the low number of positive samples. Based on our results, meconium EtG can be proposed as an alternate biomarker for intrauterine alcohol exposure. In contrast to the 7 FAEEs, EtG is just one molecule that could be screened in meconium samples from all newborns by a simple, low-cost, easy-to-perform immunoassay, which can be routinely applied in neonatology wards for the early diagnosis of prenatal exposure to ethanol.

L'éthylglucuronide: un marquer de la consommation d'alcool

Excessive alcohol consumption represents a major risk factor for morbidity and mortality. It is therefore indispensable to be able to detect at-risk drinking. Ethyl glucuronide (EtG) is a specific marker of alcohol consumption. The determination of ethyl glucuronide in urine or blood can be used to prove recent driving under the influence of alcohol, even if ethanol is no longer detectable. The commercialization of an EtG specific immunological assay now allows to obtain preliminary results rapidly and easily with satisfying sensitivity. Moreover, the detection of ethyl glucuronide in hair offers the opportunity to evaluate an alcohol consumption over a long period. The EtG concentration in hair is in correlation with the amount of ingested alcohol. Thus, the analysis of ethyl glucuronide can be used to monitor abstinence, to detect alcohol relapse and to identify at-risk drinkers. However, a cut off allowing to detect chronic alcohol abuser reliably still does not exist. Therefore, it is recommended to perform the analysis of ethyl glucuronide in complement to the existing blood markers. A study financed by the Swiss Foundation for Alcohol Research is actually conducted by the West Switzerland University Center of Legal Medicine in order to establish an objective cut-off.