Comparison of ethyl glucuronide and fatty acid ethyl ester concentrations in hair of alcoholics, social drinkers and teetotallers
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.
Comparison of ethyl glucuronide and fatty acid ethyl ester
concentrations in hair of alcoholics, social drinkers
, A. Labarthe
, V. Auwa
, S. Hartwig
, H. Vater
, F. Pragst
Laboratory National de Sante
, Toxicologie, CRP-Sante
du Luxembourg, Campus Limbertsberg,
162a, Av. de la Faı
encerie, L-1511 Luxembourg
Institute of Legal Medicine, Humboldt-University, Hannoversche Straße 6, D-10115 Berlin, Germany
Available online 24 June 2004
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 H
O, 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.
# 2004 Elsevier Ireland Ltd. All rights reserved.
Alcoholism is one of the most frequent addictions and is
therefore of particular interest in forensic and clinical med-
icine. 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 glucur-
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
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.
Forensic Science International 145 (2004) 167–173
Corresponding author. Tel.: þ352 466644 480;
fax: þ352 221331.
E-mail address: firstname.lastname@example.org (M. Yegles).
0379-0738/$ – see front matter # 2004 Elsevier Ireland Ltd. All rights reserved.
EtG is a non-volatile water-soluble substance, which was
ﬁrst detected in human urine by Jaakonmaki et al.  and
Besserer and Schmidt . In hair this minor metabolite of
ethanol was determined by GC–MS–EI [3–5],byGC–MS/
NCI  and by LC–MS/MS . 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 consump-
tion 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 [8–12]. The sum of the concentrations of ethyl myr-
istate, ethyl palmitate, ethyl oleate and ethyl stearate C
can be used as a criterion for interpretation. Thus, in general,
excessive alcohol consumption can be assumed, if C
1 ng/mg hair, whereas for teetotallers and weak social
< 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 mar-
kers with self reported data about the alcohol consumption of
2. Material and methods
2.1. 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 docu-
mented excessive alcohol consumption (Table 1). For these
specimens segmental hair analysis was performed. Eleven
specimens were from fatalities that were postmortem exam-
ined at the Institute of Legal Medicine of the Humboldt-
University, Berlin. The history of heavy alcohol consump-
tion was known from the police reports and the typical
pathological ﬁndings found during autopsy. Four specimens
were collected from social drinkers (colleagues and friends
of the authors) together with the data about their drinking
behaviour and habits of hair care on a questionnaire. Their
consumption was up to 20 g ethanol per day. Three speci-
mens were from strict teetotallers (children or adults who did
not drink any alcoholic beverages). For the samples from the
fatalities, social drinkers and teetotallers only the proximal
hair segment 0–6 cm was analysed. The postmortem sam-
ples were washed with deionised water in order to remove
water soluble impurities and dried. Afterwards, they were
washed twice with n-heptane for removal of the external
lipids. The specimens from the living volunteers were only
washed with n-heptane.
All reagents were of analytical grade purity. EtG and
deuterated EtG were purchased from Medichem (Stuttgart,
Germany). The FAEE, ethyl myristate, ethyl palmitate, ethyl
oleate and ethyl stearate, as well as the corresponding
deuterated standards FAEE-D
were purchased or prepared
as described in a previous study .
Both FAEE and EtG studies were performed with a gas
chromatograph 6890 and a mass selective detector 5973
(Hewlett-Packard GmbH, Waldbronn, Germany).
Patients in withdrawal treatment and their self-reported alcohol
Patient Self-reported alcohol consumption
before hair sampling
HV 20 Relapse with 0.7–1 L schnaps daily
after a longer period of abstinence
3, 3, 4, 7
HV 21 During last 5 months 2–2.5 L beer
daily, only on weekends
HV 22 4 L beer daily for a long time 6, 4
HV 23 2.5–3 L beer þ 0.5 L liqueur daily
for a long time
3, 3, 8
HV 24 1 month teetotal, before that during
3 weeks 0.7 L brandy þ 2 L beer,
before 2–3 L beer daily only on
6, 6, 6, 10
HV 25 During 6 months 0.5 L schnaps
HV 26 During 6 weeks 1.5 L schnaps
(35%) daily, before 2 months teetotal,
before that 1.5 L schnaps daily
3, 3, 5, 5, 8
HV 27 6 days teetotal, before during 2
months 2 L wine daily, before
that 4 months teetotal, before
during 3 weeks 2 L wine daily
3, 3, 6
HV 28 Every 2–3 months during 1 week
2 L beer and 0.8 L schnaps daily
HV 29 1 week 1.5–2.5 L beer þ 0.25
schnaps daily, before it 2 months
abstinent, before it short
consumption periods else teetotal
Lengths of hair segments from proximal to distal.
168 M. Yegles et al. / Forensic Science International 145 (2004) 167–173
For the FAEE study a multipurpose sampler MPS 2
hlheim/Ruhr, Germany) was used. All the steps
of the HS-SPME experiments could be programmed and
automatically carried out with the MPS 2. The SPME
experiments were performed with a 65 mm polydimethylsi-
loxane/divinylbenzene ﬁber (PDMS/DVB) from Supelco
(Deisenhofen, Germany) ﬁtting to the MPS 2. The liquid
extraction of the hair samples was carried out using a mixer
5433 with a rack for 12 samples (Eppendorf, Hamburg,
Germany): the solvents were removed by an evaporator
(Liebisch, Bielefeld, Germany).
For the EtG study, the gas chromatograph was equipped
with a HP-Ultra 2 capillary column (12 m 0.2 mm
0.33 mm ﬁlm thickness). The temperature was programmed
from 70 8C (2 min hold) to 280 8C with 20 8C/min. The
injector temperature was 260 8C; the helium carrier gas ﬂow
rate was 1 mL/min. The mass spectrometer was operated in
the NCI mode with methane as the carrier gas (ﬂow of 40%).
2.4. Sample preparation and determination by GC/MS
2.4.1. FAEE determination
The methods for extraction of the hair samples were
previously described in detail [9,10]. Brieﬂy, 30 mg of the
washed and dried hair sample were cut into pieces of about
1 mm length and weighed. Then, 0.5 mL dimethylsulfoxide,
2mL n-heptane and 20 ng of each of the four deuterated
internal standards in 10 mL chloroform were added. The
mixture was shaken during 20 h at 25 8C. Then, the n-
heptane layer was collected and evaporated for the determi-
nation by HS-SPME and GC/MS in the selected ion mon-
itoring (SIM) mode. The detection limits of the FAEE were
between 0.01 and 0.04 ng/mg and the assay repeatability was
between 3.5 and 16% depending on the concentration.
2.4.2. EtG determination
The washed and dried hair was pulverised in a ball mill
(Retsch, Haan Germany). 2 mL water and 5 ng EtG-d
10 mL water were added to about 30 mg of the pulverised
sample and were ultra-sonicated for 2 h. After centrifuga-
tion, a solid phase extraction using Aminopropyl NH
columns (Isolute NH
, IST International, Sopachem, Bel-
gium) was done based on a method published by Janda and
Alt . After conditioning with 3 mL methanol, 3 mL water
and 3 mL acetonitrile, the sample was applied to the car-
tridge. The columns were washed with n-hexane and dried
during 15 min by a strong vacuum. The elution was done
using 2 mL of H
2%. The residue obtained was
derivatised with pentaﬂuoropropionic anhydride (PFPA)/
pentaﬂuoropropanol (PFPOH) (100 ml/70 ml) for 30 min at
90 8C. The mixture was again dried under nitrogen at 40 8C
and reconstituted in 50 mL of ethyl acetate. Two microlitre of
the sample were injected into the GC/MS system which was
operated in the selected ion monitoring (SIM) mode for the
detection of EtG (m/z ¼ 496, 347) with EtG-d
(m/z ¼ 501,
352) used as internal standard. The recovery was 65% and
the LOD and LOQ were 2 and 4 pg/mg hair, respectively.
The calibration curve was linear from 4 to 6000 pg/mg hair,
whereas the accuracy was 2.5%. The intra-day repeatability
was 5.7% (n ¼ 10), whereas the inter-day repeatability was
2.8% (5 days, n ¼ 6).
3. 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
varied between 0.05 and
0.37 ng/mg. In the hair of social drinkers, the EtG results
were all negative, too, whereas C
varied between 0.26
and 0.50 ng/mg hair (Table 3). C
of teetotallers and
social drinkers were all below the cut-off of 1 ng/mg, C
of social drinker being higher than those from teetotallers.
In each hair specimen of the patients in withdrawal
treatment EtG could be detected with concentrations ranging
from 0.042 to 0.415 ng/mg (Table 4). C
0.65 and 20.48 ng/mg, only 6 of the 10 specimens showed
concentrations higher than 1 ng/mg. In previous investiga-
tions a cut-off as high as 1 ng/mg was chosen in order to
exclude false positive results as far as possible .Asa
consequence a certain portion of false negative cases are
found, particularly between socially integrated alcoholics
with regular hair care.
Regarding the results of the 11 specimens from fatalities
known to be alcoholics, EtG could be detected, in every
specimen, with concentrations varying from 0.072 to
3.380 ng/mg (Table 5). All the C
were higher than
1 ng/mg hair with concentrations varying from 1.3 to
30.6 ng/mg hair.
The low concentrations of the acidic compound EtG in
hair could be explained by the fact that in general basic
Comparison between FAEE and EtG concentrations for teetotallers
(ng/mg) EtG (ng/mg)
Children hair pool 0.24 N
Child 1 0.37 N
SH 031 0.05 N
Investigated hair length 0–6 cm. N: negative, LOD of 2 pg/mg.
Comparison between FAEE and EtG concentrations for social
(ng/mg) EtG (ng/mg)
SH 002 0.26 N
SH 016 0.50 N
SH 044 0.50 N
SH 051 0.37 N
Investigated hair length 0–6 cm. N: negative, LOD of 2 pg/mg.
M. Yegles et al. / Forensic Science International 145 (2004) 167–173 169
substances are more efﬁciently incorporated than neutral or
acidic molecules .
In Fig. 1 a comparison of the concentrations of the two
alcohol abuse markers in hair is shown for the fatalities and
the patients in withdrawal treatment. From the coefﬁcient of
of 0.0914 it follows that there is no signiﬁcant
correlation between the concentrations of EtG and FAEE in
hair. This lack of correlation may result from the fact that
both alcohol markers are formed at different body sites and
are deposited in a different way in hair. FAEE seem to be
mainly incorporated into hair from sebum after synthesis
from ethanol and fatty acids or lipids in the sebum glands
. Thus, the incorporation rate may depend more on the
activity of the sebum glands which highly varies depending
on sex, age and season. Furthermore, the intensity and
frequency of cosmetic treatment of hair may play a role,
removing the sebum layer to a different degree . It has
been shown that dying of hair with a commercial product
may decrease C
by 65% in the hair matrix .
Concerning EtG, due to its hydrophilic properties the
dominant way of incorporation seems to be by sweat.
Furthermore, EtG may be washed out due to its polarity.
EtG has only poor binding properties to the keratinized
matrix implying easy removal by washing. Moreover,
bleaching also causes a decrease of EtG concentrations.
As was shown in a previous study , a decrease of
78% was found after bleaching hair with a commercial
In Fig. 2 the results of the segmental analysis of several
specimens from patients of the withdrawal treatment pro-
gram are shown in logarithmic scale. It is seen that EtG and
FAEE are differently distributed along the hair length. EtG
decreases in most cases from proximal to distal, whereas for
FAEE mainly an increase from proximal to distal and then a
decrease is seen. Reasons for this different distribution
Comparison between FAEE and EtG concentrations for patients in
withdrawal treatment program
(ng/mg) EtG (ng/mg)
HV 20 1.23 0.030
HV 22 0.71 0.304
HV 23 4.16 0.415
HV 24 0.65 0.046
HV 25 3.38 0.140
HV 26 0.85 0.305
HV 27 1.15 0.193
HV 28 20.48 0.042
HV 29 2.83 0.076
Investigated hair length 0–6 cm. The samples were analysed in one
to ﬁve segments (cf. Table 1). The concentrations given in this table
are the mean values of the segmental concentrations of the hair
length 0–6 cm.
The concentrations of this sample relate to the hair length 0–
Comparison between FAEE and EtG concentrations for fatalities
known to be alcoholics
(ng/mg) EtG (ng/mg)
SN 550/01 11.70 0.531
SN 529/01 30.60 1.774
SN 562/01 9.80 3.380
SN 547/01 3.50 0.214
SN 538/01 10.20 0.327
SN 530/01 7.30 0.166
SN 012/02 3.30 0.361
SN 226/02 13.20 0.255
SN 063/01 11.10 2.043
SN 537/01 1.30 0.072
SN 555/01 1.30 0.887
Investigated hair length 0–6 cm.
Fig. 1. Comparison of EtG and FAEE concentrations in hair.
170 M. Yegles et al. / Forensic Science International 145 (2004) 167–173
pattern may be the different incorporation mechanism and a
different removal by cosmetic treatment. It has been shown
in a previous paper  that for a person with a constant
alcohol consumption C
increases up to a hair length of
5–10 cm, and after that decreases in distal direction. This
was explained by an accumulation of FAEE from sebum in
the proximal hair shaft, whereas in the distal part the removal
of FAEE by hair washing prevails. Finally, the amount of
alcohol consumed every month before cutting hair was
calculated for each patient in the treatment program from
the self-reported drinking amounts. These data were com-
pared with the concentrations of EtG and FAEE based on the
segmental analysis results. In Fig. 3 the data for the patient
HV 27 are shown. In this case, but also in the other cases
(data not shown), no clear relationship between EtG/FAEE
concentrations and documented alcohol consumption could
be determined. These results indicate that FAEE and EtG
cannot be interpreted as a timetable of alcohol consumption.
Fig. 2. Segmental analysis of FAEE (&) and EtG (&) in hair from four patients in the treatment program (histograms are in a logarithmic
M. Yegles et al. / Forensic Science International 145 (2004) 167–173 171
This lack of correlation with the drinking history should at
least in part be caused by the incorporation of both EtG and
FAEE outside the hair root leading to a positive result in hair
segments which grew during periods of abstinence. The poor
correlation between the EtG and the FAEE concentrations can
be understood by means of the different paths of formation
and incorporation on the one hand and the different removal
by cosmetic treatment due to the different chemical and
physical properties of the substances, on the other hand.
In addition, as information on alcohol consumption is
based mainly on personal statements, this data may not be
sufﬁciently reliable to allow an extended interpretation.
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 con-
centrations in hair. Furthermore, for alcoholics, no clear
relationship could be determined between the EtG or FAEE
concentrations and the self-reported alcohol consumption.
Also the segmental analysis of the specimens did not reveal
the same distribution for EtG compared to FAEE in hair.
Therefore, the time-resolved drinking history of the subjects
cannot be ascertained by the corresponding segmental con-
centrations of EtG or FAEE.
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