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Abstract
Fatty acid ethyl esters (FAEE) are products of the nonoxidative ethanol metabolism, which are known to be detectable in
blood only about 24 h after the last alcohol intake. After deposition in hair they should be suitable long-term markers of
chronically elevated alcohol consumption. Therefore, a method for the analysis of ethyl myristate, ethyl palmitate, ethyl oleate
and ethyl stearate from hair was developed based on the extraction of the hair sample by a dimethylsulphoxide (DMSO)/nhexane mixture, separation and evaporation of the n-hexane phase and application of headspace solid-phase microextraction
(HS-SPME) in combination with gas chromatography-mass spectrometry (GC-MS) to the extract. For use as internal
standards, the corresponding D5-ethyl esters were prepared. The HS-SPME/GC-MS measurements were automatically
performed using a multi-purpose sampler. The detection limits of the FAEE were between 0.01 and 0.04 ng/mg and the
reproducibility was between 3.5 and 16%. By application of the method to hair samples of 21 fatalities with known heavy
alcohol abuse 0.0452.4 ng/mg ethyl myristate, 0.3513.5 ng/mg ethyl palmitate, 0.257.7 ng/mg ethyl oleate and 0.05
3.85 ng/mg ethyl stearate were measured. For social drinkers (3060 g ethanol per week), the concentrations were about one
order of magnitude smaller. For 10 teetotalers negative results or traces of ethyl palmitate were found. It was shown by
supplementary investigations in single cases that FAEE are also present in sebum, that there is no strong difference in their
concentrations between pubic, chest and scalp hair, and that they are detectable in hair segments after a 2 months period of
abstinence. From the results follows that the measurement of FAEE concentrations in hair is a useful way for a retrospective
detection of alcohol abuse. # 2001 Elsevier Science Ireland Ltd. All rights reserved.
Keywords: Alcohol markers in hair; Fatty acid ethyl esters; Gas chromatography-mass spectrometry; Hair analysis; Headspace solid-phase
microextraction
1. Introduction
Hair analysis has proved to be a suitable tool for the
retrospective detection of the consumption of illicit or
therapeutic drugs. The physiological basis, analytical methods and practical applications were described in books [1,2],
reviews [3,4] and special issues of journals on the occasion
of hair conferences [5]. Different from these advantages up
to now there is no practicable method for the detection of
chronically elevated alcohol consumption by hair analysis,
*
Corresponding author. Tel.: 49-30-2093-7320;
fax: 49-30-2093-7268.
E-mail address: fritz.pragst@charite.de (F. Pragst).
0379-0738/01/$ see front matter # 2001 Elsevier Science Ireland Ltd. All rights reserved.
PII: S 0 3 7 9 - 0 7 3 8 ( 0 1 ) 0 0 4 5 6 - X
77
Fig. 1. Formation routes and hydrolysis of fatty acid ethyl esters (FA: fatty acids; FAEE: fatty acid ethyl esters).
78
Retention time
(min)
Values of m/z in
SIM mode
D5-Ethyl myristate
Ethyl myristate
9.71
9.92
D5-Ethyl palmitate
Ethyl palmitate
10.73
10.75
D5-Ethyl oleate
Ethyl oleate
11.57
11.58
D5-Ethyl stearate
Ethyl stearate
11.67
11.68
79
80
Fig. 2. Mass spectra of ethyl myristate, ethyl palmitate, ethyl oleate and ethyl stearate in comparison to those of their D5-ethyl analogues.
81
Fig. 3. FAEE concentrations extracted from 50 mg of the alcoholic hair pool with n-hexane/DMSO at different extraction times (n 2).
82
Fig. 4. Effect of the sample amount on the HS-SPME extraction yields of the FAEE. A 1 ml phosphate buffer pH 7.6, 0.5 g NaCl and 40 ng of
each of the four FAEE were added to the extract from 0, 10, 20, 50 to 100 mg of the teetotaler hair pool.
Table 2
Calibration curves, LOD, LOQ and reproducibility in the analysis of FAEE in haira
FAEE
Ethyl
Ethyl
Ethyl
Ethyl
a
b
c a(Asample/Astandard) b
myristate
palmitate
oleate
stearate
LOD
(ng/mg)
R2
1.094
1.135
1.083
1.169
0.024
0.037
0.023
0.014
0.9993
0.9990
0.9987
0.9937
0.015
0.02
0.04
0.01
LOQ
(ng/mg)
0.05
0.07
0.12
0.04
Reproducibility (n 10)b
Medium concentration
(ng/mg)
0.067
0.796
0.387
0.133
S.D.
ng/mg
0.010
0.052
0.023
0.005
15.7
6.6
5.9
3.5
83
Fig. 5. GC-MSSIM chromatogram of the hair extract of case 169/99 obtained after HS-SPME. Measured concentrations: ethyl myristate
0.13 ng/mg, ethyl palmitate 1.31 ng/mg, ethyl oleate 2.76 ng/mg and ethyl stearate 0.37 ng/mg. Deuterated standards 0.8 ng/mg. For control of
the method 1.0 ng/mg ethyl pentadecanoate was added.
84
Table 3
Alcohol concentrations in blood and urine, cause of death and concentrations of FAEE in scalp hair of fatalities with known heavy alcohol
abusea
Case
No.
Age
(sex)
169/98
252/98
325/98
4/99
88/99
156/99
169/99
223/99
451/99
453/99
102/00
132/00
140/00
173/00
185/00
188/00
232/00
241/00
260/00
276/00
287/00
a
45
57
35
40
59
43
57
65
51
40
34
65
50
40
64
38
45
46
49
56
53
(m)
(m)
(f)
(m)
(m)
(m)
(m)
(m)
(m)
(m)
(m)
(m)
(m)
(m)
(f)
(m)
(m)
(m)
(m)
(m)
(f)
Postmortem alcohol
concentration (mg/g)
Blood
Urine
0.1
0.0
1.4
0.0
0.0
2.3
1.5
3.2
4.8
0.0
0.1
0.1
0.0
5.1
0.1
0.0
0.0
0.0
0.0
0.0
0.0
0.2
0.0
0.0
0.0
2.2
3.1
6.1
0.1
0.0
0.0
5.6
0.1
0.0
0.1
0.0
0.1
Cause of death
Hepatic cirrhosis
Pneumonia and lung cancer
Hypothermia during alcoholic intoxication
Withdrawal syndrome, ketoacidosis
Hypothermia during alcoholic intoxication
Alcoholic intoxication and loss of blood
Suicide by jumping from 11th floor
Suicide by electricity in bathtub
Acute alcohol intoxication
Withdrawal syndrome
Suicide by electricity in shower cubicle
Hepatic cirrhosis
Hyperglycemic coma at known diabetes
Acute alcohol intoxication
Heavy coronary arteriosclerosis
Withdrawal syndrome and liver failure
Pontine myelolysis
Bronchopneumonia
Hepatic cirrhosis, pancreatitis
Gastric hemorrhage, hyperglycemia
Streptococcus infection
Ethyl
myristate
Ethyl
palmitate
Ethyl
oleate
Ethyl
stearate
0.47
0.22
0.08
0.03
0.09
0.27
0.13
2.44
0.88
0.72
0.045
0.42
0.19
0.20
0.11
0.17
0.38
0.11
0.11
0.06
0.14
1.73
1.37
0.39
0.57
0.40
1.25
1.31
13.5
3.76
2.28
0.46
1.17
0.49
1.15
0.35
0.86
3.33
0.60
0.45
0.73
0.99
1.09
1.23
0.65
0.68
0.40
2.14
2.76
7.07
6.88
2.75
0.25
1.47
1.25
2.89
0.62
1.08
8.94
0.79
1.02
1.23
1.01
0.22
0.35
0.10
0.25
0.08
0.19
0.37
3.85
0.68
0.36
0.05
0.28
0.11
0.27
0.10
0.26
1.39
0.26
0.15
0.47
0.47
m: male; f: female.
Table 4
Concentrations of FAEE in hair of teetotalers (T) and social drinkers (SD)
No.
Age (sex)
T01
T02
T03
T04
T05
T06
T07
T08
T09
T10
SD01
SD02
SD03
SD04
SD05
SD06
SD07
SD08
SD09
SD10
a
b
9
11
13
15
30
32
54
56
65
68
19
22
25
25
26
26
29
30
35
35
(m)
(m)
(f)
(m)
(f)
(f)
(f)
(f)
(f)
(f)
(m)
(f)
(f)
(m)
(f)
(m)
(m)
(f)
(f)
(f)
Drinking behaviora
Ethyl myristate
Ethyl palmitate
Ethyl oleate
Ethyl stearate
0
0
0
0
0
0
0
0
0
0
6
6
5
6
6
5
3
3
6
6
n.d.
n.d.
n.d.
n.d.
n.d.
n.d.
n.d.
n.d.
n.d.
n.d.
pos.
n.d.
n.d.
n.d.
pos.
n.d.
pos.
0.05
pos.
n.d.
n.d.
n.d.
n.d.
n.d.
n.d.
pos.
pos.
pos.
n.d.
n.d.
0.40
pos.
0.07
0.12
0.09
0.08
0.07
0.22
pos.
pos.
n.d.
n.d.
n.d.
n.d.
n.d.
n.d.
n.d.
n.d.
n.d.
n.d.
0.32
n.d.
pos.
pos.
pos.
n.d.
0.12
0.23
0.13
pos.
n.d.
n.d.
n.d.
n.d.
n.d.
n.d.
n.d.
n.d.
n.d.
n.d.
0.12
n.d.
n.d.
n.d.
n.d.
n.d.
pos.
0.04
n.d.
n.d.
Self-reported data, one beverage unit is 250 ml beer, 100 ml wine or 20 ml high percent spirits.
Here, n.d.: not detected; pos.: between LOD and LOQ (cf. Table 2); m: male; f: female.
85
Fig. 6. GC-MSSIM chromatogram of the hair extract of a child (11 years, male, No. T04 in Table 4) obtained after HS-SPME. Deuterated
standards 0.8 ng/mg. For control of the method 1.0 ng/mg ethyl pentadecanoate was added. FAEE from the sample were not detected and
should be seen at the retention times marked by arrows.
86
Fig. 7. Concentrations of FAEE in hair samples of alcoholics: (a) comparison of sebum and hair matrix for the fatalities 102/00 and 173/00;
(b) comparison of scalp hair, pubic hair and chest hair for the fatality 241/00; (c) segmental analysis of the hair sample of an alcoholic (63-oldfemale), which stopped drinking 2 months before sampling.
the routine analysis of the FAEE in hair. With the multipurpose sampler MPS 2 the manual work was minimized
and the reproducibility was improved. Further investigations
are in progress to examine the applicability of the method in
forensic and clinical practice of the detection and treatment
of alcohol abuse.
Table 5
Drinking behavior and total FAAE concentrations in hair
Drinking behavior
Teetotalers
Social drinkers (three to six drinking units per week)
Fatalities with known heavy alcohol consumption
Sum of the concentrations of ethyl myristate, ethyl palmitate, ethyl oleate and ethyl stearate.
Acknowledgements
The authors thank the Deutsche Forschungsgemeinschaft
(DFG) for supporting these investigations.
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