Comparison of ethyl glucuronide and fatty acid ethyl ester concentrations in hair of alcoholics, social drinkers and teetotallers

doi:10.1016/j.forsciint.2004.04.032

Forensic Science International

Volume 145, Issues 2–3, 29 October 2004, Pages 167-173

https://doi.org/10.1016/j.forsciint.2004.04.032 Get rights and content

Abstract

In previous investigations hair analysis for ethyl glucuronide (EtG) and fatty acid ethyl esters (FAEE) proved to be suitable for the detection of excessive alcohol consumption. The aim of this study was to compare EtG and FAEE concentrations in hair of alcoholics, social drinkers and teetotallers. Hair samples from 10 alcoholics in withdrawal treatment, 11 fatalities with documented excessive alcohol consumption, four moderate social drinkers who consumed up to 20 g ethanol per day, and three strict teetotallers were analysed. After external degreasing with n-heptane, extraction with a dimethyl sulfoxide/n-heptane mixture and headspace solid-phase microextraction of the extracts, four fatty acid ethyl esters (FAEEs) (ethyl myristate, ethyl palmitate, ethyl oleate and ethyl stearate) were analysed by gas chromatography–mass spectrometry (GC–MS) with deuterated internal standards. EtG was determined by GC–MS/NCI after ultrasonication of the samples with H₂O, cleanup by SPE with aminopropyl columns and PFP derivatisation.

The following concentrations were measured for the four groups: teetotallers EtG < 0.002 ng/mg, FAEE 0.05–0.37 ng/mg, moderate social drinkers EtG < 0.002 ng/mg, FAEE 0.26–0.50 ng/mg, alcoholic patients EtG 0.030–0.415 ng/mg, FAEE 0.65–20.50 ng/mg and the fatalities with alcohol history EtG 0.072–3.380 ng/mg, FAEE 1.30–30.60 ng/mg. The results confirm that by using a cut-off value of the sum of FAEE > 1 ng/mg and/or a positive EtG result in hair, excessive alcohol consumption can be identified using hair analysis. However, no significant correlation between the EtG and FAEE concentrations in the positive cases could be shown. Segmental analysis of some of the specimens did not reveal the same distribution for EtG compared to FAEE in hair, and no chronological accordance compared to the self-reported alcohol consumption could be observed for both parameters. These different results of both methods are discussed in terms of differences between EtG and FAEE in mechanism of formation and incorporation into hair and elimination from hair.

Introduction

Alcoholism is one of the most frequent addictions and is therefore of particular interest in forensic and clinical medicine. Hair analysis proved to be suitable for the detection of excessive alcohol consumption. Two major markers which are both products of the non-oxidative ethanol metabolism, are used: Fatty acid ethyl esters (FAEE), and ethyl glucuronide (EtG).

Detection of EtOH in body fluids is only possible during a relatively short time after alcohol consumption. About 90–95% of alcohol is eliminated by oxidation mainly in the liver whereas biotransformation of ethanol to ethyl glucuronide (ethyl-β-d-6 glucuronic acid, EtG) via conjugation with activated glucuronic acid represents only 0.5% of complete alcohol elimination. EtG can be used as a marker for alcohol consumption detectable even after complete elimination of ethanol from body fluids.

EtG is a non-volatile water-soluble substance, which was first detected in human urine by Jaakonmaki et al. [1] and Besserer and Schmidt [2]. In hair this minor metabolite of ethanol was determined by GC–MS–EI [3], [4], [5], by GC–MS/NCI [6] and by LC–MS/MS [7]. The more recent studies by GC–MS/NCI and LC–MS/MS showed that no EtG could be detected in hair of social drinkers and teetotallers, whereas in hair of alcoholics mainly positive EtG results were found. These studies showed also that a negative EtG hair result does not exclude alcohol consumption (EtG could be degraded by some hair treatments). However, if EtG is detected, chronically increased alcohol consumption has to be strongly assumed.

FAEE are also direct alcohol markers containing the unchanged ethyl group of ethanol. After ethanol consumption they are enzymatically formed in a side route of the ethanol metabolism in almost all tissues from free fatty acids or lipids. FAEE are detectable in blood up to 24 h after the end of drinking and accumulate in fat tissues. They have proved to be an interesting marker of alcohol consumption in hair [8], [9], [10], [11], [12]. The sum of the concentrations of ethyl myristate, ethyl palmitate, ethyl oleate and ethyl stearate C_FAEE can be used as a criterion for interpretation. Thus, in general, excessive alcohol consumption can be assumed, if C_FAEE > 1 ng/mg hair, whereas for teetotallers and weak social drinkers C_FAEE < 0.4 ng/mg hair was found.

The aim of the present study was to determine EtG and FAEE in the same hair specimens of alcoholics, social drinkers and teetotallers and to compare both alcohol markers with self reported data about the alcohol consumption of the subjects.

Section snippets

Hair specimens

The scalp hair samples were collected in the usual way, by fixing a strand of hair in the vertex posterior region and cutting it as close as possible to the skin. Ten specimens together with self-reported data about alcohol consumption were obtained from patients of a psychiatric clinic in Berlin who were in a withdrawal treatment program after documented excessive alcohol consumption (Table 1). For these specimens segmental hair analysis was performed. Eleven specimens were from fatalities

Results and discussion

In Table 2 the results of the FAEE and EtG determinations are shown for the three teetotallers. No EtG could be detected, whereas C_FAEE varied between 0.05 and 0.37 ng/mg. In the hair of social drinkers, the EtG results were all negative, too, whereas C_FAEE varied between 0.26 and 0.50 ng/mg hair (Table 3). C_FAEE of teetotallers and social drinkers were all below the cut-off of 1 ng/mg, C_FAEE of social drinker being higher than those from teetotallers.

In each hair specimen of the patients in

Conclusions

From the results it follows that EtG and FAEE are suitable qualitative hair markers of chronically excessive alcohol consumption: A positive EtG result and/or a value of the sum of FAEE above the cut-off of 1 ng/mg hair can be taken as strong evidence for excessive drinking behaviour.

However, the data shows that there is no significant quantitative correlation between the EtG and FAEE concentrations in hair. Furthermore, for alcoholics, no clear relationship could be determined between the EtG

References (14)

P. Jaakonmaki et al.
The characterization by gas–liquid chromatography of ethyl β-d-glucuronic acid as metabolite of ethanol in rat and man
Eur. J. Clin. Pharmacol
(1967)
I. Janda et al.
Improvement of ethyl glucuronide determination in human urine and serum samples by solid-phase extraction
J. Chromatogr. B Biomed. Sci. Appl
(2001)
F. Pragst et al.
Are there possibilities for the detection of chronically elevated alcohol consumption by hair analysis? A report about the state of investigation
Forensic Sci. Int
(2000)
F. Pragst et al.
Analysis of fatty acid ethyl esters in hair as possible markers of chronically elevated alcohol consumption by headspace solid-phase microextraction and gas chromatography-mass spectrometry (GC–MS)
Forensic Sci. Int
(2001)
S. Hartwig et al.
Effect of hair care and hair cosmetics on the concentrations of fatty acid ethyl esters in hair as markers of chronically elevated alcohol consumption
Forensic Sci. Int
(2003)
G.L. Henderson
Mechanisms of drug incorporation into hair
Forensic Sci. Int
(1993)
K. Besserer et al.
Ein Beitrag zur renalen Ausscheidung von Äthlylglucuronid nach oraler Alkoholaufnahme
Zentrabl Rechtsmed
(1983)

There are more references available in the full text version of this article.

Cited by (164)

Unlocking the potential of forensic traces: Analytical approaches to generate investigative leads
2022, Science and Justice
Forensic investigation involves gathering the information necessary to understand the criminal events as well as linking objects or individuals to an item, location or other individual(s) for investigative purposes. For years techniques such as presumptive chemical tests, DNA profiling or fingermark analysis have been of great value to this process. However, these techniques have their limitations, whether it is a lack of confidence in the results obtained due to cross-reactivity, subjectivity and low sensitivity; or because they are dependent on holding reference samples in a pre-existing database. There is currently a need to devise new ways to gather as much information as possible from a single trace, particularly from biological traces commonly encountered in forensic casework. This review outlines the most recent advancements in the forensic analysis of biological fluids, fingermarks and hair. Special emphasis is placed on analytical methods that can expand the information obtained from the trace beyond what is achieved in the usual practices. Special attention is paid to those methods that accurately determine the nature of the sample, as well as how long it has been at the crime scene, along with individualising information regarding the donor source of the trace.
Metabolites of Ethanol
2022, Encyclopedia of Forensic Sciences: Volume 1-4, Third Edition
Nonoxidative metabolites of ethanol (EtOH) have been the subject of much attention in recent years due to their diagnostic value. Due to the frequency of alcohol abuse throughout the world, forensic toxicologists are frequently confronted with the need to determine EtOH consumption. This article describes EtOH metabolites, specifically ethyl glucuronide, ethyl sulfate, ethyl phosphate, fatty acid ethyl esters, and phosphatidylethanol in humans. Methods of detection of the metabolites, interpretations of the measured EtOH metabolites in specimens such as plasma/blood, urine, hair, and vitreous humor, as well as information on the relative advantages of various specimens are described.
Applications of gas chromatography in forensic science
2021, Gas Chromatography
Gas chromatography (GC) is now a mature and indispensable analytical instrument in investigational forensic science. High sensitivity, selectivity, resolution and speed, good accuracy and precision, wide dynamic concentration range, simple and robust instrument design, and its ability to easily interface with many established and emerging detection systems have made GC the instrument of choice in many facets of investigational forensic science. A continuous influx of new column chemistries, development of high-precision thermal and pneumatic controlling systems, introduction to programmable temperature vaporizer, advancement in control electronics, and the large number of available detection systems have positioned GC in a unique place in forensic analytical laboratories. Although the applications of GC are limited to volatile and semivolatile organic compounds, rapid development in derivatization chemistry, technology, and high-temperature capabilities have made hundreds of new organic compounds with forensic significance amenable to GC and thus extended its horizon.
The introduction of multidimensional GC (GC × GC), GC–isotope ratio mass spectrometry, and fast GC has contributed significantly in expanding forensic applications by their unmatched quality of analytical separation; high data quality and relatively low operational cost have also added additional advantages to its users.
This chapter introduces major application of GC in different subdisciplinary areas within the vast domain of forensic science and the current trend in GC with forensic implications.
Improved measurement of ethyl glucuronide concentrations in hair using UPLCMS/MS for the evaluation of chronic ethanol consumption
2020, Forensic Science International
The presence of Ethyl glucuronide (EtG) in hair provides a strong indication of ethanol consumption and its investigation is of interest in both clinical and forensic contexts because of the wide window of detection. However, due to the possibility of false negative results in cases of small ethanol intake or excessive hair washing, the combined measurement of ethyl palmitate (EtP) with EtG could be useful. In this study, a sensitive UHPLC-MS/MS procedure for the measurement of EtG in hair was developed and validated, using optimized sample preparation and chromatographic separation. Milled hair was extracted with water for 24 h at room temperature, followed by clean-up of the extract by ion-exchange solid phase extraction (SPE). Extraction was highly efficient, with yield of 96.93–101.06%. Chromatographic separation was performed with a Fluoro-Phenyl stationary phase. The assay was linear from 4 to 500 pg mg⁻¹, with accuracy in the range of 100.30–106.16%. Matrix effects (−0.87 to 5.89%) were adequately compensated by the use of deuterated EtG as internal standard. EtG was measured in hair samples of 46 volunteers, and results were compared with hair concentrations of ethyl palmitate (EtP) and the score in the AUDITC questionnaire. EtG hair concentrations were significantly correlated to the AUDIT-C classification (r_s = 0.365, p < 0.05), but not to EtP hair levels. The diagnostic performance of EtG hair concentrations to identify excessive or moderate ethanol use was similar to the capability of AUDIT-C to identify severe and high health risk (Kappa, p = 0.013). The developed assay is suitable for clinical use, providing a useful tool to evaluate chronic ethanol consumption.
Ethyl glucuronide hair testing: A review
2019, Forensic Science International
Ethyl glucuronide (EtG) is a minor, non-oxidative ethanol metabolite that can be detected in several matrices (e.g. blood, urine, hair, meconium) for variable periods of time. Quantification of EtG in hair (hEtG) has established itself, over recent years, as one of the most reliable biomarkers of long-term alcohol consumption habits, with the Society of Hair Testing (SoHT) offering cut-off values for assessment of both abstinence and heavy drinking (>60 g/day). Despite its high diagnostic performance, however, issues concerning inter- and intra-laboratory variability as well as data interpretation are still being investigated and represent the ultimate barrier to widespread acceptance of hEtG in the forensic context. The aim of this review is to summarize currently available analytical methods of hEtG testing, provide a framework to understand current hEtG cut-offs and their possible upcoming changes (in particular, a lower abstinence cut-off has been proposed for the 2019 revision of the SoHT consensus), and offer a schematic but exhaustive overview of the pitfalls in result reproducibility and interpretation that may limit applications of hEtG testing in the forensic context. Ultimately, the purpose of the authors is not to undermine the reliability of hEtG as an alcohol use marker, but rather to enhance it by promoting familiarization with all aspects related to it, from ethanol pharmacokinetics and EtG incorporation into hair, to sample preparation and analytical methods, to specific cases warranting close attention and additional tests for correct interpretation of hEtG results.
Alcohol consumption assessment in a student population through combined hair analysis for ethyl glucuronide and fatty acid ethyl esters
2019, Forensic Science International
Citation Excerpt :
One way to minimize these situations is by the combined measurement of EtG and FAEEs, which has been recommended to improve discrimination between abstinence, moderate and excessive drinking [7,9,11,12]. The combined analysis of both markers was performed in several studies [7–9,11,12,18,22], however no studies have so far been made on student population, where excessive alcohol consumption is common [23–25]. We have previously reported results from a study on a university student population based on the analysis of EtG [25].
This study aimed to assess alcohol consumption in a university student population though the combined analysis of the alcohol biomarkers ethyl glucuronide (EtG) and fatty acid ethyl esters (FAEEs) in hair samples. A total of 975 hair samples were analysed for EtG and FAEEs using liquid chromatography coupled to mass spectrometry (LC–MS/MS) and gas chromatography coupled to mass spectrometry (GC–MS/MS), respectively. The results were analysed using the cut-offs proposed by the Society of Hair Testing and receiver operating characteristic (ROC) curve analysis was performed to verify the adequacy of the proposed values for the study population. Good sensitivity and specificity were obtained for both biomarkers, especially for EtG, and a correlation was found with the self-reported alcohol consumption habit. In 56.3% of the abstinent, 65.8% of the moderate and 80.0% of the excessive drinking cases, self-reported alcohol consumption could be confirmed by combined alcohol biomarker analysis. Combined analysis of EtG and FAEEs in hair samples proved to be a valuable tool for the monitoring of alcohol consumption in a student population. For a feasible result interpretation, it is very important to document the use of hair products, cosmetic treatments and washing frequency, and for these to be considered during interpretation. Overall the participants were aware of their consumption pattern, however for doubtful cases and to account for academic calendars, repeated analysis of samples collected at different time frames would be advisable.

View all citing articles on Scopus

View full text

Comparison of ethyl glucuronide and fatty acid ethyl ester concentrations in hair of alcoholics, social drinkers and teetotallers

Abstract

Introduction

Section snippets

Hair specimens

Results and discussion

Conclusions

Eur. J. Clin. Pharmacol

J. Chromatogr. B Biomed. Sci. Appl

Forensic Sci. Int

Forensic Sci. Int

Forensic Sci. Int

Forensic Sci. Int

Ein Beitrag zur renalen Ausscheidung von Äthlylglucuronid nach oraler Alkoholaufnahme

Zentrabl Rechtsmed