Yegles 2004 Comparison EtG FSEE FSI

8/9/2019 Yegles 2004 Comparison EtG FSEE FSI

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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 [3–5], 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 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 haveproved 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 FAEEcan 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 mar-

kers with self reported data about thealcohol consumption of

the subjects.

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 thesespecimens 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.

2.2. Chemicals

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-D5 were purchased or prepared

as described in a previous study [9].

2.3. Instrumentation

Both FAEE and EtG studies were performed with a gas

chromatograph 6890 and a mass selective detector 5973

(Hewlett-Packard GmbH, Waldbronn, Germany).

Table 1

Patients in withdrawal treatment and their self-reported alcohol

consumption

Patient Self-reported alcohol consumption

before hair sampling

Segmental

lengths (cm)a

HV 20 Relapse with 0.7–1 L schnaps dailyafter a longer period of abstinence

3, 3, 4, 7

HV 21 During last 5 months 2–2.5 L beer

daily, only on weekends

7

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

weekends

6, 6, 6, 10

HV 25 During 6 months 0.5 L schnaps

(35%) daily

6, 5

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

else teetotal

6, 12

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

6

a Lengths of hair segments from proximal to distal.

168 M. Yegles et al. / Forensic Science International 145 (2004) 167 – 173


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For the FAEE study a multipurpose sampler MPS 2

(Gerstel, Mühlheim/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,

2 mL 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 theassay 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-d5 in

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 NH2columns (Isolute NH2, IST International, Sopachem, Bel-

gium) was done based on a method published by Janda and

Alt [7]. After conditioning with 3 mL methanol, 3 mL waterand 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 H2O/NH3 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-d5 (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 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 FAEEof social drinker being higher than those from teetotallers.

In each hair specimen of the patients in withdrawal

treatment EtG could be detected with concentrations rangingfrom 0.042 to 0.415 ng/mg (Table 4). CFAEE varied between

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 [10]. As a

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 FAEE

were higher than1 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

Table 2

Comparison between FAEE and EtG concentrations for teetotallers

Specimen C FAEE (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.

Table 3

Comparison between FAEE and EtG concentrations for social

drinkers


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


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substances are more ef ficiently incorporated than neutral or

acidic molecules [13].

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 coef ficient of

correlation R2

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 bemainly incorporated into hair from sebum after synthesis

from ethanol and fatty acids or lipids in the sebum glands

[10]. 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 [12]. It hasbeen shown that dying of hair with a commercial product

may decrease C FAEE by 65% in the hair matrix [12].

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 [14], a decrease of

78% was found after bleaching hair with a commercial

bleaching product.

In Fig. 2 the results of the segmental analysis of severalspecimens 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

Table 4

Comparison between FAEE and EtG concentrations for patients in

withdrawal treatment program


HV 20 1.23 0.030

HV 21a

0.70 0.044HV 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.a

The concentrations of this sample relate to the hair length 0–7 cm.

Table 5

Comparison between FAEE and EtG concentrations for fatalities

known to be alcoholics

Specimens C FAEE (ng/mg) EtG (ng/mg)

SN 550/01 11.70 0.531

SN 529/01 30.60 1.774SN 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.



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pattern may be the different incorporation mechanism and a

different removal by cosmetic treatment. It has been shown

in a previous paper [10] that for a person with a constant

alcohol consumption C FAEE 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

scale).



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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

suf ficiently reliable to allow an extended interpretation.

4. 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 con-

centrations in hair. Furthermore, for alcoholics, no clear

relationship could be determined between the EtG or FAEEconcentrations 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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Fig. 3. EtG and FAEE concentrations vs. monthly alcohol consumption for patient HV 27.



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