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www.elsevier.com/locate/clinchim
State of the art in hair analysis for detection of drug and alcohol abuse
Fritz Pragst a,, Marie A. Balikova b
a
Institute of Legal Medicine, University Hospital Charit, Hittorfstr. 18, D-14195 Berlin, Germany
Institute of Forensic Medicine and Toxicology, 1st Medical Faculty and Hospital, Charles University in Prague, Kateinsk 32, CZ 12108, Czech Republic
Received 9 November 2005; received in revised form 11 January 2006; accepted 8 February 2006
Available online 6 March 2006
Abstract
Hair differs from other materials used for toxicological analysis because of its unique ability to serve as a long-term storage of foreign
substances with respect to the temporal appearance in blood. Over the last 20 years, hair testing has gained increasing attention and recognition for
the retrospective investigation of chronic drug abuse as well as intentional or unintentional poisoning. In this paper, we review the physiological
basics of hair growth, mechanisms of substance incorporation, analytical methods, result interpretation and practical applications of hair analysis
for drugs and other organic substances. Improved chromatographicmass spectrometric techniques with increased selectivity and sensitivity and
new methods of sample preparation have improved detection limits from the ng/mg range to below pg/mg. These technical advances have
substantially enhanced the ability to detect numerous drugs and other poisons in hair. For example, it was possible to detect previous
administration of a single very low dose in drug-facilitated crimes. In addition to its potential application in large scale workplace drug testing and
driving ability examination, hair analysis is also used for detection of gestational drug exposure, cases of criminal liability of drug addicts,
diagnosis of chronic intoxication and in postmortem toxicology. Hair has only limited relevance in therapy compliance control. Fatty acid ethyl
esters and ethyl glucuronide in hair have proven to be suitable markers for alcohol abuse. Hair analysis for drugs is, however, not a simple routine
procedure and needs substantial guidelines throughout the testing process, i.e., from sample collection to results interpretation.
2006 Elsevier B.V. All rights reserved.
Keywords: Criminal assaults; Driving ability examination; Drug abuse; Hair analysis; Postmortem toxicology; Workplace drug testing
Contents
1.
2.
3.
Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Incorporation and elimination of drugs in hair . . . . . . . . . .
Performance of hair analysis . . . . . . . . . . . . . . . . . . .
3.1. Documentation on the case, collection and storage of hair
3.2. Division into segments . . . . . . . . . . . . . . . . . .
3.3. Decontamination. . . . . . . . . . . . . . . . . . . . . .
3.4. Separation of drugs from the hair matrix . . . . . . . . .
3.4.1. Extraction with methanol. . . . . . . . . . . . .
3.4.2. Extraction by aqueous acids or buffer solutions .
3.4.3. Treatment with urea and thioglycolate . . . . . .
3.4.4. Supercritical fluid extraction . . . . . . . . . . .
3.4.5. Enzymatic digestion of the hair matrix. . . . . .
3.4.6. Digestion of the hair with aqueous NaOH . . . .
3.5. Clean-up of hair extracts . . . . . . . . . . . . . . . . .
Corresponding author. Tel.: +49 30 450 525031; fax: +49 30 450 525904.
E-mail address: fritz.pragst@charite.de (F. Pragst).
0009-8981/$ - see front matter 2006 Elsevier B.V. All rights reserved.
doi:10.1016/j.cca.2006.02.019
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18
20
23
24
25
25
25
26
26
26
26
26
26
26
18
3.6.
1. Introduction
Hair differs from other human materials used for toxicological analysis such as blood or urine because of its substantially
longer detection window (months to years) enabling retrospective investigation of chronic and past consumption. Because of
its solid and durable nature, hair analysis can be performed even
centuries after growth. Toxic metal ions such as Tl, As, Pb or Hg
were the first poisons that could be analyzed in hair to document
historic exposure (for reviews see Refs. [1,2]). Later on, gradual
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27
27
28
29
29
29
30
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31
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32
32
33
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35
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19
Medulla
Cortex
Epidermis
Cuticle
Cell membrane
complex
Mature hair
Degradation of the
inner root sheath
Sebum gland
Basement
membrane
Zone of hardening
disulfide bonding,
resorption and
dehydration
(a)
Dendrites
Keratine gene
expression,
incorporation of
pigments
Melanin
pigments
Exocytosis
Vesicles with
pigments
Cell proliferation
and differentiation
Papilla
(b)
Basement
membrane
Melanocyte
Keratinocytes
(c)
Fig. 1. a) Structure and constituents of the human hair shaft. b) Formation of hair in a follicle from matrix cells on the basement membrane to the mature hair shaft.
Drug incorporation from blood should occur in a 1.21.5 mm zone before completion of keratinisation. c) Melanocytes on the basement membrane of the cortex
synthesize melanine in melanosomes that are discharged in vesicles into the keratinocytes by an exocytotic mechanism. There, the membranes of the vesicles and
melanosoms are digested and remain the melanin pigments.
20
Scalp, non-bald
Scalp, alopecia
Beard
Axillary
Pubis
Eyebrow
Arm
Thigh
Trunk + extremities
Vellus, forehead
Duration of stages a
Ana
Cat + Tel
Total
(%)
26 y
1422
1118 m
4777 w
6w
615 w
26 m
63 d
26 m
912
1217 m
5173 w
15 w
824 w
24 m
65 d
26 y
2334
21 w
1534 w
77240 d
128 d
618
5384
40
50
50
51
mm/day
0.320.46
0.080.15
0.250.29
0.290.33
0.3
0.150.16
0.130.25
0.27
0.03
a
Abbreviations: d = days, w = weeks, m = months, y = years, Ana = anagen
stage, Cat = catagen stage, Tel = telogen stage.
Plasma, extracellular
Melanocyte, intracellular
pHe
>
pHi
+H+
-H+
+
A-
BH+
B
Membrane
BH
21
AH
Binding
to melanin
AH
A-
22
Table 2
Selected literature data about concentrations of frequently abused drugs and their metabolites in hair
Drug or metabolite a
THC
THC-COOH
CBD
CBN
Heroin
6-Acetylmorphine
Morphine
Codeine
Acetylcodeine
Codeine
Dihydrocodeine
Buprenorphine
Norbuprenorphine
Methadone
EDDP
EMDP
Cocaine
Benzoylecgonine
Methylecgonine
Cocaethylene
Norcocaine
AEME
Amphetamine
Methamphetamine
Amphetamine
MDMA
MDE
MDA
LSD
Phencyclidine
GHB
GHB
Clonazepam
7-Aminoclonazepam
Diazepam
Flunitrazepam
7-Aminoflunitrazepam
Nitrazepam
7-Aminonitrazepam
Nordazepam
Tramadol
Fentanyl
Nicotine
Cotinine
Nicotine
Cotinine
Ethanol
FAEE b
Ethyl glucuronide
0.067.6
0.00050.013
0.5318.4
0.554.54
0.004.53
0.0064.8
0.0053.7
0.0015.1
0.0010.5
9.012.3
1.231.2
0.0030.124
0.0051.518
0.042
0.05.0
0.180.84
0.5216.5
0.133.7
0.112.8
0.110.3
0.000.70
0.22.4
0.026.52
0.8756.4
0.123.5
0.18.3
0.1215
0.020.89
0.001
0.3314
3.15.1
0.212
0.020.04
0.070.24
0.012.21
0.0310.12
0.0030.15
0.050.13
0.190.54
0.131.83
0.17616.3
0.0080.644
0.933.9
0.094.99
0.541.82
0.010.13
0.060.37
0.200.85
0.9213.5
< 0.25
0.0723.38
Remarks
References
Methadone maintenance
Drug fatalities
Illicit drug users
Crack marker
Illicit drug use
Illicit drug use
One of 11 consumers
Illicit drug use
Chronic administration
Single administration
Psychiatric patients
Psychiatric patients
Passive Smokers
[68]
[69]
[70]
[70]
[60]
[71]
[71]
[71]
[71]
[72]
[73]
[74]
[74]
[75,76]
[75,76]
[77]
[78]
[78]
[78]
[78]
[79]
[78]
[80]
[81]
[81]
[82]
[82]
[82]
[83]
[84]
[85]
[86]
[14]
[14]
[87]
[88]
[88]
[14]
[14]
[87]
[89]
[90]
[91]
[91]
[91]
[91]
Teetotallers
Social drinkers
Heavy alcohol abuse
Social drinkers
Heavy alcohol abuse
[92]
[92]
[92]
[93]
[93]
Mean
0.97
0.002
1.3
1.2
7.2
3.7
1.0
0.3
10.7
10.9
1.2
12.9
3.7
1.8
1.6
0.26
0.6
0.84
18.3
0.86
3.25
2.38
0.31
0.060
0.064
0.49
4.41
0.16
0.41
4.0
Normalized concentration
1,2
23
(a)
n = 82
n = 82
n = 39
n = 30
0,8
n = 21
0,4
0
0 -3
3 -6
6 - 19
9 - 12
12 - 15
(b)
n=6
ratio metabolite/drug
Normalized concentration
n=4
2
n=9
n = 29
n = 29
0 -3
3 -6
0
6 - 19
9 - 12
12 - 15
24
Assignment of investigation
Information about case history and purpose of investigation
Choice of appropriate analysis strategy
Segmentation
Decontamination by washing
Storage and, if necessary, analysis of wash solutions
Pre-test by immunoassay
25
26
27
SPME-Device
Syringe Needle
Stainless Steel Rod
Coated Fused Silica Fiber
SPME-Device
Headspace vial (4 ml)
10 mg Hair
Internal Std.
1 ml 4% NaOH
0.5 g Na2SO4
80 C
80 C
Headspace SPME
(15 min)
250 C
Desorption in
GC-injection port
(5 min)
Fig. 6. Use of headspace solid phase microextraction (HS-SPME) in hair analysis. Semi-volatile drugs which are stable under alkaline conditions such as
amphetamines, methadone or tricyclic antidepressants can be extracted directly from the headspace above the solution obtained by NaOH digestion of the hair sample.
28
Abundance x 10-4
28
26
BE x 25
Mo x 20
Coc x 4 0
M DMA
24
22
20
MET-d9 Mo-d3
18
16
MA-d5
ME-d3
14
12
10
8
C o d - d 3 6 - A M - d3
C E - d3
MDMA-d5
4
2
BE-d3
Mat rix
MDA-d5
A-d5
0
Sens.: x 4 x 10 x 20
7 .0 0
8 .0 0
MDE-d6
BE
MDA
x 1.0
x1.3
x 1.3
DHC
Coc-d3
EDDP-d3 Coc
x 1.3
Mo
x 1.3
x 4 x 5 x 10
6- A M
x 10
9 .0 0 1 0 .0 0 1 1 .0 0 1 2 .0 0 1 3 .0 0 1 4 .0 0 1 5 .0 0 1 6 .0 0 1 7 .0 0 1 8 .0 0 1 9 .0 0
29
30
Cocaine
Median of
Ref. Labs
3
2
1
Median of
Part. Labs
Participant No.
5
4
Benzoylecgonine
Median of
Part. Labs
3
2
1
Median of
Ref. Labs
Participant No.
Fig. 8. Concentrations of cocaine and benzoylecgonine determined for a real hair sample in 23 laboratories in the proficiency test of the Society of Hair Testing 2004.
31
H3C
C OH
C O
CH3
O
O C
H
Benzoylecgonine
H3C
OH
H
Ecgonine methylester
H
C O
CH3
C O
H3C
C O
C2H5
O
H
Cocaethylene
CH3
O C
H
Norcocaine
Cocaine
+ C2H5OH
Crack smoking
H3C
O
C O
CH3
C
Anhydroecgonine methylester
Fig. 9. Biotransformation of cocaine. The hydrolytic metabolites benzoylecgonine and ecgonine methylester are formed in blood and can be formed also under
hydrolytic conditions in hair or during sample preparation. Norcocaine is formed only by action of cytochrom oxidases in the liver. Cocaethylene arises from
reesterification at simultaneous cocaine and alcohol consumption. Anhydroecgonine methylester is formed by thermal elimination of benzoic acid and, therefore, is a
marker for crack smoking.
32
ecstasy [82] and THC [112]. For example, 6-monoacetylmorphine hair concentrations (0.3131.1 ng/mg, n = 61 cases) were
divided into four consumption classes: negative (< 0.5 ng/mg,
cut-off), low (0.52.0 ng/mg), medium (210 ng/mg) and
high (> 10 ng/mg) [204].
It is general practice to compare the drug concentration in
hair from actual cases with usual consumers (Table 3). It should
be noted, however, that these data were obtained from cases
without knowledge of consumption habits and, as such, cover
variation from occasional (e.g., once per week) to regular (daily
or almost daily) and excessive (several times per day) drug use.
Because the data is likely to contain extreme hair drug
concentrations, the use of a statistical distribution rather than
the whole range is recommended.
In fact, Jurado and Staub [205] proposed a statistic
evaluation of hair drug concentration data obtained from
heroin addicts [205] (Table 4). The ranges proposed were:
lower (minimum to the 25th percentile); middle (25th to 75th
percentile); and upper (above the 75th percentile). For
example, in an actual case in which 4.2 ng/mg 6-monoacetylmorphine and 1.1 ng/mg morphine were detected in hair,
the interpretation would read as follows: The concentration of
drugs in the hair of this individual is in the middle range of
those drug concentrations usually found in cases of heroin
abuse. To conclude that these findings are consistent with
regular heroin consumption and that the individual is addicted
to the drug are not justified. Although these are reasonable
assumptions, these findings require confirmation by other
symptoms. Possible reasons for exceptionally low drug
concentrations such as very fair or cosmetically treated hair
should be taken into account.
4.2.2. Cut-off values
Similar to the analysis of drugs in urine, cut-off values have
been recommended for some drugs in hair (Table 5) [204,207
210]. Cut-off values are generally used for two reasons. First, to
avoid false positive analytical results for methods such as
immunoassays where matrix effects and cross reactivity to other
compounds may set a lower limit for an accurate use [146].
These analytical cut-offs are statistically determined and
provide an optimal compromise of sensitivity and specificity.
In chromatographicmass spectrometric methods analytical
cut-offs are not used. Under these circumstances, cut-off limits
are described by the LOD and LOQ.
Table 3
Typical metabolite / drug concentration ratios of frequently abused drugs in hair
Metabolite/drug
THC-COOH/THC
Morphine/6-acetylmorphine
EDDP/methadone
Norbuprenorphine/buprenorphine
Benzoylecgonine/cocaine
Amphetamine/methamphetamine
MDA/MDMA + MDE
Cotinine/nicotine
Concentration ratio
Typical range
Mean
0.0010.01
0.210.74
0.060.50
3.312.3
0.050.62
0.0150.14
0.030.20
0.49
0.26
0.16
0.050
0.1
References
[68,69]
[154]
[76]
[74]
[202]
[81]
[82]
[203]
Table 4
Statistical evaluation of illegal heroin consumers according to Jurado and
Staub [205]
0.1
7.2
0.0
0.1
3.7
0.0
Percentile 25
1.3
0.9
Median
3.3
1.9
Percentile 75
6.3
4.1
Maximum
65
Interpretation
Morphine
54
Low range
Medium range
High range
33
Table 5
Cut-off values (immunochemical pre-test, IC) and required LOQ or LOI (chromatographic determination, CH) recommended for interpretation of hair results
Drug or metabolite
IC: 0.1 THC CH: 0.1 THC 0.05 pg/mg 0.1 for THC, CBD
for THC-COOH
Abbreviations: A = amphetamine; 6-AM = 6-acetylmorphine; BE = benzoylecgonine; COC = cocaine; CE = cocaethylene; COD = codeine; LOI = limit of inclusion;
LOQ = limit of quantification; MA = methamphetamine; MDA = methylenedioxyamphetamine; MDE = methylenedioxyethamphetamine; MDMA = methylenedioxymethamphetamine; Met. = metabolites; MOR = morphine; THC = 9-tetrahydrocannabinol; THC-COOH = 11-Nor-9-carboxy-9-tetrahydrocannabinol.
Drug dose
34
a) Drug
administration
Time before sampling, months
1
10
11
12
13
b) Constant
growth rate
Distance from proximal end, cm
0
10
11
12
13
c) Variation of
hair growth rate
12
13
10
11
12
13
x 10
11
d) Effect of catagen
+ telogen hair
x 10
10
10
11
12
13
f) Effect of incorporation
from sweat
3
2
10
11
12
13
11
12
13
g) Effect of incorporation
from sebum
0
2
3
5
10
February 12
1st Hair Sampling
Length 2.5 cm
Shaving of head
November 15
Letter from the authorities
Stopped drug use
Shaving of head
Habitual
marihuana
&
occasional
cocaine
use
Nov. 00
Dec. 00
Jan. 01
35
April 02
2nd hair sampling
length 1.5 cm
Feb. 01
Mar. 01
Apr. 01
Fig. 11. Delayed incorporation of THC in hair of a 26 year old man in context of driving ability examination after habitual marihuana and occasional cocaine use [223].
After three months abstinence and twice shaving the head, THC is still detected in newly grown hair.
Benzoylecgonine
Cocaine
Offence
Arrest
2
0
0-2
2-5
5-9
36
Ecgonine methylester
10
Benzoylecgonine
8
Cocaine
Scalp hair
(0-2 cm)
Pubic hair
(0-2.3 cm)
Fig. 13. Concentrations of cocaine and benzoylecgonine in scalp and pubic hair
of a 42 year old man [223]. Both samples were collected directly above the skin
4-1 / 2 months after arrest (termination of drug use). The small concentration in
the 2 cm long scalp hair sample can be explained by telogen hair. The 2.3 cm
long pubic hair sample had natural tips and, therefore, represented the whole
growth cycle (up to more than one year) including the period of drug use before
arrest.
often accepted. Cut-off values for testing are listed (Table 5). In
contrast to other illicit drugs, recreational cannabis use is not
generally incompatible with possessing a driving license in
Germany. Therefore as a prerequisite, every case with a positive
cannabis hair result must be checked in a medical psychological
examination whether the applicant is able to strictly separate
cannabis use and driving.
In Italy, hair analysis is included in a panel of clinical and
laboratory tests to investigate the toxicological behavior of the
drivers license applicant. Tagliaro et al. reported on an
integrated diagnostic strategy to check the physical and mental
fitness of former users in order to reissue a driving license after a
period of abstinence [234,235]. According to Italian law, these
individuals must provide evidence of drug cessation and
demonstrate no risk of relapse. These subjects undergo medical
examination involving hair and urine analysis on a series of
eight specimens collected over 40 days. Hair samples (45 cm
length from vertex posterior) are first screened for opiates,
cocaine and ecstasy by RIA (cut-off level of 0.1 ng/mg).
Positives are then confirmed by HPLC, CE or GCMS. In
1998, the percentage of positive drug results for morphine,
cocaine and ecstasy in these individuals was 4.8%, 11.3% and
2.6%, respectively.
The sampling protocol described by Montagna et al.
consisted of collection of one hair (5 cm length) and one
urine sample analyzed for opiates and cocaine [105]. When
samples were both positive or negative the result was definitive.
However, in cases of disagreement, a second hair sample was
collected 6 weeks later and the 1 cm proximal segment
analyzed. In the Italian province of Brescia, a program including
analysis of opiates and cocaine in two hair segments (03 and
36 cm) and in urine was adopted in order to reissue the driving
license to former drug addicts or occasional abusers [236].
Testing for cannabinoids was not part of these programs due to
its slow clearance.
5.1.3. Doping control
The official rules in various sport disciplines state that a
positive case is established by unequivocal chemical
detection of a banned substance in urine. In a 1999
consensus, the Society of Hair Testing stated that hair drug
analysis can supplement but not substitute for routine urine
drug testing [206]. For example, a positive urine drug result
cannot be overruled by a negative hair drug result. However,
the positive hair drug result can demonstrate exposure during
the period prior to sample collection even in case of a
negative urine test. Hair analysis in sports drug testing is
important to test for substances permanently prohibited such
as anabolic steroids. In contrast, substances prohibited only
temporarily, i.e., during the period of competition, in a certain
application form (oral intake of salbutamol), or above a
certain limiting dose (caffeine) can generally not be
controlled in this fashion. In addition to several reviews
[237239], special methods for detection of anabolic steroids
in hair [240244] have been described. Steroid concentrations between 0.6 and 84 pg/mg have been found in hair
samples from body builders.
37
38
39
40
(a)
2
0
0
20
(b)
3
2
1
< 0.3
0
0-3
3-5
5-7
(c)
15
10
5
0
0-2
2-4
4-6
6-9
9-12
14-year old boy was found dead at the home of well-known sex
offender. At autopsy only pulmonary and visceral congestion
were noted. LCMS analysis revealed concentrations of
buprenorphine of 1 ng/mL in blood and 23 pg/mg in hair. The
hair concentration of buprenorphine was consistent with
chronic administration. For example, in a controlled long-term
study (maximum 180 days), administration of buprenorphine
8 mg sublingual to 12 subjects resulted in hair concentrations of
3.1123.8 pg/mg [74]. In this case, actual death was attributed
to accidental asphyxia in a facilitated repetitive sexual abuse
situation.
5.5.3. Control of regular intake by the offender
Claims are often made that the drug is for personal
consumption in drug-related crimes. This explanation, however,
can be investigated by hair analysis of the defendant. In one
case, passengers on a long-range bus ride were intoxicated by a
drink containing a mixture of diazepam, midazolam, levomepromazine and carbamazepine [278]. The victims were then
robbed while unconscious. The woman suspected of drugging
the passengers claimed that the drugs found were for high-dose
personal use. However, no drugs were detected in her hair.
5.6. Therapy compliance control
The potential use of hair analysis in therapeutic drug control
has been explored in several publications [258,279,280]. The
opinions of the authors range from a worthless tool [279] to a
proposal to establish standardized methods of hair analysis for
all newly introduced pharmaceuticals [280]. Hair testing is
unsuitable for individual adjustment of drug dosing (therapeutic
drug monitoring, TDM). Detailed monitoring to ascertain
therapeutic compliance is difficult due to enormous intra- and
inter-individual variation and relatively poor relationship
between dose, plasma concentration and hair concentration.
Several investigations have, in fact, been performed with
antiepileptics and different groups of psychopharmaceuticals
[14,43,5966,74,166168,174,175,281]. From a practical point
of view, several experimental approaches may warrant continued pursuit.
5.6.1. Single hair sampling, no segmentation
In this scenario, it is only possible to state that the patient has
repeatedly taken the drug or that drug intake was improbable
during the time period corresponding to the hair length segment.
Due to poor correlation no quantitative conclusions can be
drawn between dose and hair concentration and that there is no
therapeutic hair concentration range.
5.6.2. Single hair sampling, segmental analysis
In this scenario, the sample is investigated in 1 cm long
segments nearly constant or equal from proximal to distal.
Decreasing hair drug concentrations can be interpreted as a
good compliance whereas fluctuations in drug hair concentrations are an indication for non-regular intake. Examples of this
approach have been published in a number of studies
[43,94,217,281,282].
41
42
43
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45
46
[182]
[183]
[184]
[185]
[186]
[187]
[188]
[189]
[190]
[191]
[192]
[193]
[194]
[195]
[196]
[197]
[198]
[199]
[200]
[201]
[202]
[203]
[204]
47
48
49