State of The Art in Hair Analysis For Detection of Drug and Alcohol Abuse PDF

Clinica Chimica Acta 370 (2006) 17 49
www.elsevier.com/locate/clinchim
Invited critical review
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
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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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F. Pragst, M.A. Balikova / Clinica Chimica Acta 370 (2006) 1749
3.6.
Detection and quantification . . . . . . . . . . . . . . . . . . . . . . . . . .

3.6.1. Immunochemical techniques . . . . . . . . . . . . . . . . . . . . .
3.6.2. Gas chromatographymass spectrometry (GCMS) . . . . . . . . .
3.6.3. Liquid chromatographymass spectrometry (LCMS) . . . . . . . .
3.6.4. Other methods. . . . . . . . . . . . . . . . . . . . . . . . . . . . .
3.7. Quality control . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
4. Interpretation of analytical results . . . . . . . . . . . . . . . . . . . . . . . . . . .
4.1. Drug consumption or external contamination? . . . . . . . . . . . . . . . . .
4.2. Interpretation of hair concentrations . . . . . . . . . . . . . . . . . . . . . .
4.2.1. Conclusions about intensity of drug use . . . . . . . . . . . . . . .
4.2.2. Cut-off values . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
4.2.3. Pubic, axillary and body hair . . . . . . . . . . . . . . . . . . . . .
4.3. Interpretation concerning the time of drug intake . . . . . . . . . . . . . . .
4.3.1. Exclusion of drug use for a certain period of time . . . . . . . . . .
4.3.2. Time period presented by a certain hair lengths . . . . . . . . . . .
4.3.3. Testing for previous drug use around a certain date . . . . . . . . .
4.3.4. Additional analysis of pubic, axillary or body hair . . . . . . . . . .
4.3.5. Detection of a single dose in context of drug facilitated crimes . . .
5. Practical applications . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
5.1. Human performance toxicology . . . . . . . . . . . . . . . . . . . . . . . .
5.1.1. Workplace drug testing . . . . . . . . . . . . . . . . . . . . . . . .
5.1.2. Drug test in context of driving ability examination . . . . . . . . . .
5.1.3. Doping control . . . . . . . . . . . . . . . . . . . . . . . . . . . .
5.2. Criminal liability and drug addiction . . . . . . . . . . . . . . . . . . . . . .
5.3. Diagnosis of drug abuse and chronic intoxication . . . . . . . . . . . . . . .
5.3.1. Drug abuse history and withdrawal control . . . . . . . . . . . . . .
5.3.2. Smoking behavior . . . . . . . . . . . . . . . . . . . . . . . . . . .
5.3.3. Gestational drug exposure. . . . . . . . . . . . . . . . . . . . . . .
5.3.4. Chronic intoxication by environmental pollution or food adulteration
5.4. Postmortem toxicology . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
5.4.1. Health impairments caused by chronic drug abuse . . . . . . . . . .
5.4.2. Tolerance in opioid death cases . . . . . . . . . . . . . . . . . . . .
5.4.3. Chronic drug use and fatal accidents . . . . . . . . . . . . . . . . .
5.4.4. Repeated criminal poisoning . . . . . . . . . . . . . . . . . . . . .
5.4.5. Probability of self-administration . . . . . . . . . . . . . . . . . . .
5.4.6. Contribution to identification of a corpse . . . . . . . . . . . . . . .
5.5. Criminal assaults . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
5.5.1. Single drug application . . . . . . . . . . . . . . . . . . . . . . . .
5.5.2. Repeated or frequent administration. . . . . . . . . . . . . . . . . .
5.5.3. Control of regular intake by the offender . . . . . . . . . . . . . . .
5.6. Therapy compliance control . . . . . . . . . . . . . . . . . . . . . . . . . .
5.6.1. Single hair sampling, no segmentation . . . . . . . . . . . . . . . .
5.6.2. Single hair sampling, segmental analysis . . . . . . . . . . . . . . .
5.6.3. Repeated hair sampling, analysis of the proximal 1 cm segment . . .
5.7. Detection of excessive alcohol abuse . . . . . . . . . . . . . . . . . . . . .
5.7.1. Fatty acid ethyl esters (FAEE) . . . . . . . . . . . . . . . . . . . .
5.7.2. Ethyl glucuronide (EtG). . . . . . . . . . . . . . . . . . . . . . . .
6. Conclusions and future prospects in hair analysis . . . . . . . . . . . . . . . . . . .
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
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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development of analytical technologies, offering methods with

sufficient sensitivity, enabled hair analysis for organic substances [3]. In 1979, Baumgartner et al. [4] extracted hair of
heroin users and determined opiate content by radioimmunoassay (RIA). Following matrix disintegration with NaOH, the
first chromatographic detection of opiates in hair was performed
in 1980 by Klug [5]. This study found that the concentration
along the hair shaft differed and corresponded with the time
course of drug intake. Following these initial reports, two
decades of scientific research has led to the development of a
standardized hair testing approaches that were documented in

consensus papers and official guidelines [6,7]. Preliminary
immunochemical tests for selected drug groups may be
accepted as the first step [8,9]. Although gas chromatographymass spectrometry (GCMS) is generally the method of
choice [10], various tandem mass spectrometry methods (GC
MSMS or LCMSMS) are also used for targeted analyses of
low dose compounds such as fentanyl, buprenorphine, and
flunitrazepam [1115]. These methods are also useful for
detection of some important specific metabolites typically
present in hair at trace concentrations such as 11-nor-9-carboxydelta(9)-tetrahydrocannabinol (THCOOH) [16,17] or for retrospective detection of drugs administered as single doses [18,19].
As described above, hair analysis is a complex scientific
undertaking. Several books and comprehensive reviews have
been published on this topic (e.g., Refs. [2022]). Because of the
intense interest in this topic, a number of scientific conferences
have been held with their findings published as special journal
issues [2327]. Although the anatomy, physiology, physical and
chemical properties of hair have generally been described [28
33], hair remains an enigmatic structure to date [34,35]. A hair is
not a homogeneous fiber, but consists of keratinized cells glued
by the cell membrane complex that together form three
concentric structures: cuticle, cortex and medulla (Fig. 1a).
19
The pigmented cortex is responsible for the stretching stability

and color composition, whereas the 510 layers of shingle-like
cells of the non-pigmented cuticle is responsible for high
chemical and physical resistance and shine.
Hair originates from the hair follicle (Fig. 1b) located 3
5 mm below the skin surface. The hair follicle is surrounded by a
rich capillary system that provides the growing hair with
necessary metabolic material. The germination center around the
hair bulb papilla is formed by matrix cells (keratinocytes and
melanocytes) present on the basement membrane. This association gives rise to the different hair shaft layers including the
cuticle, cortex and medulla. The matrix cell cycle is one of the
most rapid of all human tissues. Rapid mitosis forces a migration
of the upper zones into the direction of the hair root mouth. In the
next higher zones the genes for formation of keratine are expressed. It should be noted that cell development occurs differently for the cortex and cuticle. Cortex cells change from
spherical shape at the germinative level to a spindle-like form.
Protein filaments are synthesized which fill the cell and fuse
together. In the zone of hardening, disulfide bonding, resorption
and dehydration, all cytoplasmic organelles disappear with cellular residue coupled by membrane structures. Cuticle cells
originate from matrix cells of the outer sphere of the papilla.
These cells change to a shingle-like structure and contain
Medulla
Cortex
Epidermis
Cuticle
Cell membrane
complex
Mature hair
1.2 1.5 mm ( 3 days)
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
amorphous protein. The cell membrane complex consists of

proteins and a proteinlipid complex originating from previous
cell membranes. This part of hair is most vulnerable to chemical
and mechanical attack and is the primary diffusion point for
incorporation and elimination of drugs. As can be expected
lipophilic drugs are preferentially deposited in the cell membrane complex.
Hair color is produced by melanocytes located in the basal
layer in contact with the basal membrane (Fig. 1c). They are
mostly found in the basal layer of the cortex in a ratio of about
1:5. Melanocytes produce melanin pigments in melanosoms and
have long dendrites that penetrate neighboring keratinocytes and
discharge vesicles with the melanosoms. Vesicular membranes
and melanosoms are digested by phagocytosis with the
remaining pigments imbedded in keratinocytes. Melanocytes
and pigmentation play an important role in the incorporation of
basic drugs into hair.
Each hair can be erected by muscular action. Each hair also
belongs to a sebaceous gland with the duct leading to the upper
part of the root to ensure that the mature hair is bathed in sebum
for two to three days prior to reaching the skin surface. The
eccrine sweat glands are nearby but separated from the hair root.
The sweat glands wet the hair shaft and can contribute to the
incorporation of hydrophilic drugs. Interestingly the hair shaft
can incorporate up to 30% water by radial swelling. In contrast,
apocrine sweat glands are only found in hair of the armpit and at
other intimate sites. These emerge into hair follicles superficial
to sebaceous gland ducts.
Hair grows in a cycle composed of the anagen (active
growing), catagen (transition) and telogen (resting) stages. The
individual length of hair depends on stage duration and growth
rate. The average values for these stages are 48 years, a few
weeks, and 46 months, respectively. Scalp hair growth ranges
0.61.4 cm per month in general [22,3638]. It should be
noted, however, that there are significant differences both in the
proportions anagen/telogen hair and in growth rate from various
anatomical sites. Both parameters are dependent on race, sex,
age and state of health (Table 1). At any one time,
approximately 85% of adult scalp hair is in the growing phase
Table 1
Literature data (ranges or mean values) about growth rate, duration of growth
cycle and portion in catagen + telogen stage of human hair (selection from Ref.
[22] and references given there)
Anatomical site
Scalp, non-bald
Scalp, alopecia
Beard
Axillary
Pubis
Eyebrow
Arm
Thigh
Trunk + extremities
Vellus, forehead
Duration of stages a
Cat + Tel Growths rate
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.
Fig. 2. Incorporation and elimination of drugs in hair.
(anagen) with the remaining 15% in the resting phase (telogen).

The significant consequence of cyclic growth is hair age
heterogeneity with respect to distance from the skin.
Drug of abuse testing in hair has become routine practice in
forensic toxicological laboratories. This practice has now been
extended to therapeutic drugs and alcohol metabolites. In this
paper, we review practical concepts and experimental principles
of hair testing methods.
2. Incorporation and elimination of drugs in hair
The precise mechanisms involved in the incorporation of
drugs into hair remain unclear requiring further investigation.
Incorporation models typically assume that drugs or chemicals
enter hair by passive diffusion from blood capillaries into
growing cells over a length of 1.2 to 1.5 mm between the level
of matrix cells and end of the keratinization zone of the hair
follicle. This period would correspond to a timetable of drug
exposure of about three days. Experimental data, however,
indicates that drugs enter hair by various mechanisms in a
variety of locations, times and sources (Fig. 2). Besides
incorporation from blood, substances can be incorporated,
albeit with some time delay, from deep skin compartments
during hair shaft formation. The most important alternative
mechanism is, however, deposition by diffusion from sweat or
sebum secretions into the completed hair shaft. In addition,
substances can be deposited from the external environment.
This multi-compartment model has been specifically demonstrated by Henderson [39] and generally accepted in practice
[2022,40,41]. It is important to note, however that the nature
of the incorporated substance (structure, chemical properties) as
well as the physical/physiological characteristics of the

individual strongly influence which mechanism will dominate.
From structural point of view, three key factors influence the
drug incorporation. These include the melanin content of hair
and the lipophilicity and the basicity of the substance itself. For
example, grizzled individuals are particularly suitable to
demonstrate the effect of pigmentation. Gray hair is a mixture
of white and pigmented hair. Despite root exposure to the same
drug concentration in blood, the concentration of basic drugs in
pigmented hair was about 10-fold higher than non-pigmented
hair [4245]. In controlled studies, similar results were obtained
by comparison of black, brown, blond and red colored hair [46]
as well as in Caucasians and non-Caucasians [47]. Results
correlated with melanin concentration [48,49]. These findings
were also confirmed in several studies using animals [50,51] and
in in vitro experiments [52,53]. In contrast, no differences were
noted for drug incorporation for pigmented and non-pigmented
hair for neutral compounds such as carbamazepine [45].
Generally, the incorporation of drugs into hair from blood
is controlled by the pharmacological principles of drug
distribution. Lipophilic (uncharged) organic molecules can
easily penetrate membranes and diffuse according to the
concentration gradient in matrix cells. However, for hydrophilic molecules or organic ions of medium molecular mass,
membranes form an impermeable barrier. Basic or acidic drugs
ionized to a high degree at physiological pH can reach matrix
cells following deprotonation or protonation, respectively, to a
neutral state (Fig. 3). As such, the pKa of the compound and
pH of the matrix cells are both important. The intracellular pH
of keratinocytes has been found to be more acidic than plasma
[54] and the pH of melanocytes has been described to be
between 3 and 5 [50,51]. Furthermore, a significant melanin
affinity for basic drugs has been demonstrated in vitro [52,53].
Both effects, lower pH and binding to melanin, lead to the
accumulation of lipophilic and basic drugs in matrix cells with
clear preference for pigmented hair. For acidic drugs or
metabolites of salicylic acid, valproic acid or THC (11-nor-
9-tetrahydrocannabinol-11-carboxylic acid, THC-COOH), the

distribution equilibrium is clearly shifted in disadvantage of more
acidic matrix cells. As such, these compounds are found only in
very low concentrations in hair.
Nakahara and co-workers investigated systematically the
structural effects on the drug incorporation in hair in animal
experiments with DarkAgouti rats [5558]. These studies
introduced the incorporation rate (ICR, ratio of the drug
concentration in rat hair and the area under the plasma
concentration versus time curve AUC) as a measure for
quantitative description of the drug deposition efficiency in
hair. From twenty frequently used drugs or metabolites, cocaine
appeared to be the most efficiently incorporated drug, whereas
THC-COOH had the lowest ICR (3600-fold lower than cocaine)
[55]. In the group of thirty-two amphetamines, ICR ranged from
0.03 (p-hydroxyamphetamine) to 1.81 (clobenzorex) [56].
Lipophilicity and basicity were found to be the essential
characteristics that favored incorporation. The same was found
for benzodiazepines [58] in which highly basic flurazepam and
medazepam displayed the highest ICR and the weakly basic
flunitrazepam as well as the hydroxylated and dealkylated
metabolite oxazepam the lowest ICR.
In most cases, drug metabolism leads to increased hydrophilicity. Polar metabolites such as benzoylecgonine, morphine
and amphetamine enter hair to a lesser extent than their lipophilic
precursors (cocaine, 6-monoacetylmorphine or methamphetamine). In the same way, the tricyclic antidepressants amitriptyline, clomipramine, doxepine and imipramine accumulate
more in hair than their corresponding nor-metabolites [59].
Although the concentration of many illicit and therapeutic
drugs in hair has been described, only a small fraction of these
investigations were adequately controlled studies, i.e., in which
daily dose and duration of drug intake were actually known.
Several examples include hair testing for heroin [60], antiepileptics [6164], benzodiazepines [14] and neuroleptics
[43,65,66]. In other studies, self-reported data about previous
drug intake were evaluated in comparison to the measured hair
Plasma, extracellular
Melanocyte, intracellular
pHe
>
pHi
+H+
-H+
+
A-
BH+
B
Membrane
BH
21
AH
Binding
to melanin
AH
A-
Concentrations: BH+e << BH+i

A-e >> A-i
Fig. 3. Effect of basic or acidic properties on drug incorporation rate in hair. A = acidic drug; b = basic drug; e = extra-cellular; i = intra-cellular. The lower pH in
melanocytes and binding to melanin lead to an accumulation of basic drugs in pigmented hair.
22
Table 2
Selected literature data about concentrations of frequently abused drugs and their metabolites in hair
Drug or metabolite a
Concentration in hair, ng/mg

Range
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
Illicit cannabis use2

Illicit cannabis use
Heroin maintenance
Illicit heroin users
Chronic abuse
Illicit abuse
Methadone maintenance
Drug fatalities
Illicit drug users
Crack marker
Illicit drug use
Illicit drug use
MDMA + MDE use
One of 11 consumers
Illicit drug use
Chronic administration
Single administration
Psychiatric patients
Fatal drug cases

Fatal drug cases
Psychiatric patients
Fatal drug cases

Chronic pain treatment
Abusing anesthesists
Active smokers
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
A comprehensive collection of data is found in Ref. [21]. Metabolites are indented.

a
Abbreviations: AEME = anhydroecgonine methylester; CBD = cannabidiol; CBN = cannabinol; EDDP = 2-ethylidene-1,5-dimethyl-3,3-diphenylpyrrolidine;
EMDP = 2-ethyl-5-methyl-3,3-diphenylpyrrolidine; MDA = methylenedioxyamphetamine; MDE = methylenedioxyethamphetamine; MDMA = methylenedioxymethamphetamine; THC = 9-tetrahydrocannabinol; THC-COOH = 11-Nor-9-carboxy-9-tetrahydrocannabinol; LSD = lysergic acid diethylamide; GHB = gammahydroxybutyric acid; FAEE = fatty acid ethyl esters.
b
FAEE concentrations = sum of the concentrations of ethyl myristate, ethyl palmiate, ethyl oleate and ethyl stearate.
concentrations. Since illegal drugs were most frequently

analyzed, the concentrations measured after usual consumption
are typically described. Therapeutic drug concentrations in hair
have been surveyed [67] and a selection of important examples

of illegal and therapeutic drugs and their metabolites are shown
(Table 2).
Normalized concentration
The lack of inter-individual correlation between drug

concentration in hair and daily or cumulative dose is not,
however, surprising given the fact that correlation between
dose and blood concentration between individuals is typically
poor.
Retention and stability of drugs in hair is considered good.
This is clearly demonstrated in hair segments of patients
receiving constant dosage. Mean values of the normalized drug
concentrations in 82 hair samples are shown (Fig 4a).
Measurements were determined in 25 segments of 3 cm
length for 23 different drugs or metabolites [94]. As can be
seen, mean drug concentration slowly decreases with increasing distance from the hair root (i.e., increasing age of the hair).
Interestingly, after more than one year in the hair segment (i.e.,
1215 cm) about 4% of the drugs remain present. The duration
of substances in normally kept hair depends on the chemical
structure and increases with increasing polarity. For example,
the more polar nor-metabolites of tricyclic antidepressants are
more slowly eliminated than the parent drugs. Therefore, as a
rule, the concentration ratio metabolite/drug increases from
proximal to distal as shown for 29 samples (Fig. 4b) [94].
However, for metabolites formed by ester hydrolysis, this
longer detection of the metabolite can also be caused by a slow
hydrolytic degradation of the drug within hair. As a rule, in
segmental analysis of hair samples of cocaine or heroin
consumers, the ratio benzoylecgonine/cocaine or morphine/6monoacetylmorphine increases with increasing distance from
the hair root.
1,2
23
In regularly shampooed hair, i.e., not treated by aggressive

cosmetic agents such as oxidative dyeing, bleaching or
permanent wave, drugs are usually well detected at least one
year after intake. In fact, one long hair study found drug
presence in distal segments more than three years after ceased
consumption [95]. Although the cuticle becomes more
susceptible damaged by mechanical stress and increasingly
penetrable for drug elimination, it is affected to a much higher
degree by the cosmetic treatments described above [9698]. In
fact, decreases of 190% of the original drug concentration
can occur. In general, cocaine is less affected than morphine or
6-monoacetylmorphine. The extent of drug decline following
cosmetic treatment is dependent on its initial concentration
and the properties of the hair matrix. Long-term effects of
weather (sunshine, rain, wind) may cause the damage to the
hair shaft with subsequent impacts on drug concentration [99].
The findings indicate that not only the stability of a particular
drug is important but also the influence of UV light and water on
the hair pigment. For example, cannabinoids in hair are
particularly sensitive to sunlight. Of 11 positive THC hair
samples (0.111.72 ng/mg), only three remained positive (0.10
0.35 ng/mg) after 10 weeks in a quartz vessel exposed to daylight
[100].
3. Performance of hair analysis
Hair analysis for drugs is preferentially performed in
forensic cases. Because of its serious consequences, the analyst
(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
Distance from hair root, cm
(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

Fig. 4. (a) Mean values of the normalized drug or metabolite concentrations in 3 cm long hair segments of 82 hair samples of patients with long-term medical treatment
and constant dose [94]. The concentrations of each of the 23 drugs or metabolites were normalized to the mean of the corresponding sample. (b) Metabolite/drug ratio
in 29 hair samples investigated in 3 cm long segments from the same study [94]. The data were normalized to the proximal segment.
24
assumes a high responsibility for obtaining a correct result.

Therefore, the whole process from sampling to result
interpretation must be well organized and precisely performed
to avoid any potential error. For example hair sampling and
analysis are not allowed in locations where the drugs
themselves are handled. Practical steps of hair analysis are
outlined (Fig. 5). Analytical strategies were developed to
include screening and confirmatory testing and the use of
different methods to obtain as much information from typically
limited samples and take into account the nature and
extenuating circumstances of each case under investigation
[101,102]. In this chapter, the essentials of each step shall be
described.
3.1. Documentation on the case, collection and storage of hair

samples
The importance of pre-analytical steps in hair analysis is
often underrated. Because of the specific nature of testing, the
hair sample should be collected by laboratory staff. If this is not
possible, a clearly written collection protocol should be
provided. Because the selection of hair segment lengths and
subsequent analytical methods depend on case history and hair
sample characteristics, all relevant information should be
obtained and well documented. This information includes a
brief outline of the case history, aim of the investigation,
suspected drug(s), suspected time of drug intake, date of
Assignment of investigation
Information about case history and purpose of investigation
Choice of appropriate analysis strategy
Sampling (2 hair samples)

Documentation of sampling process, appropriate storage
Segmentation
Decontamination by washing
Storage and, if necessary, analysis of wash solutions
Cutting to small pieces or grinding
Extraction or digestion of hair matrix
Clean-up of hair extract
Pre-test by immunoassay
Qualitative and quantitative analysis

by GC-MS, GC-MS/MS or LC-MS/MS
Confirmation analysis
Interpretation of results, expertise

Fig. 5. Steps of hair analysis.
sampling, questions to be answered by the analysis, and

anticipated use of the expertise.
With respect to sample identity, avoidance of sample mix-up
and data security, the same requirements established for other
forensic samples have to be fulfilled. The individual must be
unambiguously identified (i.e., passport) and confirm the sample
identity by written signature. In addition to personal data, hair
sample characteristics should be recorded including date and site
of collection (on the head), residual length on scalp, hair length
and color. History of cosmetic hair treatment can generally be
detected at the hairline on the scalp prior to collection and should
additionally be requested from the individual.
Although more sensitive methods were introduced in the last
years, the practical performance of hair sampling has not
essentially changed. A tuft of hair with a diameter of 3 to 4 mm is
fixed by a string and carefully cut directly at the skin surface. For
forensic purposes, it is advisable to collect at least two separate
hair samples. One sample must be sufficient for the comprehensive analysis including confirmation and any necessary
repetitions. The second hair sample is stored separately and is
left untouched for future use, i.e., in cases of objection. The
preferred site on the scalp is vertex posterior because the
proportion of telogen hair is lowest and the growth rate relatively
uniform. Hair sampling kits are commercially available
(Forensic Technologies, Inc., Woodbury, Minnesota, USA).
These kits generally consist of a piece of aluminum foil for
holding and stabilizing the hair sample, a suitable envelope
with imprinted instructions and tamper-evident mailer seal.
The proximal end of the sample must be clearly marked. If
scalp hair is not available, alternative sources including pubic,
axillary and body hair should be collected. In important cases
or in subjects with very short scalp hair, these alternative sources
should also be collected to provide additional information. It
should be noted that these alternative sources are traditionally
subject to less exposure to cosmetic and environmental influence
and represent another growth time period. These samples do not
need to be fixed by a string because segmental analysis is not
useful due to a high proportion of telogen hair.
Hair samples should be stored under dry and dark conditions
at room temperature. The simplest way is in an appropriately
labeled paper envelope. Storage in plastic bags should be
avoided because of contamination by softeners (i.e., plasticizers)
and since the plastic can potentially extract lipophilic substances
from the hair. A practical procedure is to wrap the dried hair
sample in aluminum foil prior to placement in the paper
envelope. Under these conditions, most drugs or metabolites in
hair are very stable and can be detected after years of storage.
3.2. Division into segments
Despite the limitations described in Section 2, segmental hair
analysis can provide important information with respect to time
course of drug use by the individual in many cases. A
comprehensive review on this topic has been published [22].
Prerequisite is very careful sampling. In order to avoid single
hair shifts within the collected tuft, decontamination after
segmentation is strongly recommended. In order to choose the
25
appropriate segmental lengths, expected time resolution,

available sample amount and analysis costs must be considered.
Analysis of segments of equal length (root to tip) has been
described in most published cases and literature sources.
Because the time resolution of a hair segment in a hair tuft
decreases with increasing distance from the root, it is generally
recommended to investigate progressively increasing segmental
lengths. For example, 45 cm length sample (proximal to distal)
would be composed of segments: 4 0.5, 3 1.0, 2 2.0, 2 3.0,
2 5.0, 2 10.0 cm. A convenient method that has proven
especially suitable for very short segments is to arrange the
exactly aligned hair tuft into folded graph paper. Using the
gradations listed on the graph paper, the enclosed hair can be
systematically cut to the appropriate lengths with a suitable razor
blade [22].
3.3. Decontamination
Cleaning the hair sample of external contamination is
necessary for two reasons. First, residues of hair care products
(wax, shampoo, hair sprays) as well as sweat, sebum and dust
typically present on hair lead to increased analytical noise/
background. Second, drugs could adhere from the environment
of the individual thus potentially contributing to incorrect test
results. This type of environmental contamination is not
unlikely for individuals involved with illegal drugs. To exclude
that a positive analytical result originated from this type of
environmental contamination, the first wash solution should be
tested or stored for subsequent analysis.
Solvents used for hair decontamination should remove
external impurities as completely as possible, but not extract
drugs from the hair matrix. There is no general consensus with
respect to the hair washing procedure. For example, one
washing sequence for post-mortem hair samples is composed of
0.1% sodium dodecylsulfate in water, distilled water and
acetone. Other procedures include one or two washes with
dichloromethane [103], sequences of different organic solvents
[104] or a brief wash with methanol [105]. Non-protic solvents
such as dichloromethane or acetone are advantageous because
they do not swell the hair thereby extracting materials from the
hair. In contrast, protic solvents such as phosphate buffer or
methanol promote extraction in the washing step by swelling
the hair. Sophisticated multiple step wash procedures coupled
with wash analysis have been described to distinguish between
external contamination from systemic incorporation [106,107].
This validity of these approaches, however, has been questioned
[108,109]. It has been shown in a systematic study that the
analytical outcome of hair analysis can be strongly affected by
the wash procedure used [110].
3.4. Separation of drugs from the hair matrix
There are currently no direct methods for the detection of
organic drugs in the hair matrix. Therefore, drugs must be
extracted by solubilization or digestion of the hair matrix itself.
In order to make the appropriate choice, the chemical structure of
the drug and its sensitivity to agents used for sample preparation
26
must be considered. In many cases, hair is screened for wide

panels of substances including virtually any illegal drug. As
such, sample preparation should be applicable to different drug
groups. Prior to extraction, the hair is typically cut to 13 mm
lengths. Alternatively, hair may be processed by grinding.
However, this latter approach generally results in loss of sample
material and does not improve the extraction process.
3.4.1. Extraction with methanol
A procedure compatible with almost all drug substances is methanol extraction (518 h) in an ultrasonic bath [101,111,112].
Hydrophilic methanol penetrates the hair matrix leading to
swelling and drug liberation via diffusion. As an organic solvent,
methanol dissolves neutral and lipophilic compounds. Ultrasonication causes a strong degradation of the hair structure and
supports this process. Drugs sensitive to hydrolysis such as heroin
[60,113] and lipophilic drugs such as THC [112] can be
determined from the same extract. For drugs at high concentrations, the methanol extract can be directly injected for GCMS. A
disadvantage of this approach, however, is relatively high
impurity level in the extract. Therefore, a secondary clean-up
procedure involving liquid/liquid extraction or solid-phase
extraction is generally recommended.
Despite extended methanol extraction ultrasonic bath, drug
recovery is incomplete and frequently lower in comparison with
other procedures. Formation of methyl esters was observed as a
consequence of this procedure during determination of fatty
acid ethyl esters, i.e., alcohol markers [114]. Re-esterification is
believed influenced by high local cavitation energy produced
during ultrasonication within the hair matrix, because this
phenomenon was found at a substantially lower level in the
absence of ultrasonic treatment.
3.4.2. Extraction by aqueous acids or buffer solutions
Basic drugs (opiates, cocaine and its metabolites, amphetamines, methadone) are well extracted by aqueous 0.01 to 0.5 M
HCl [115] or phosphate buffer (pH 6.4 or 7.6) [75] due to
protonation. An enzymic mixture of glucuronidase/arylsulfatase
is sometimes used in order to elaborate any deposited metabolites. These aqueous extracts are much cleaner than methanol
extracts. Despite this improvement, partial hydrolysis of cocaine
to benzoylecgonine and 6-monoacetylmorphine to morphine
typically result and as a consequence require acknowledgement.
For example, heroin (diacetylmorphine) is never detected in
aqueous hair extracts although it is well deposited.
3.4.3. Treatment with urea and thioglycolate
Hair extraction of drugs in aqueous medium is facilitated in
the presence of denaturants such as urea (8 mol/L) and
thioglycolate (0.2 mol/L) under acidic conditions (pH 3.2).
Exposure to these agents results in breakdown of hydrogen and
disulfide bonds within hair. This unique approach has proven
particularly successful for extraction of benzodiazepines [116].
3.4.4. Supercritical fluid extraction
The use of supercritical fluids has been applied to hair
extraction due to their unique properties of low viscosity and
rapid extraction with high mobility [117121]. The powdered

hair sample was treated at 300 bar and 60 C with CO2.
Modifiers such as ethyl acetate, chloroform or isopropanol can
be used to improve CO2 as a solvent. Despite the advantages of
a rapid extraction, high yield, miniaturization and automation,
this method is seldom used due to high expense.
3.4.5. Enzymatic digestion of the hair matrix
The enzymes pronase and Proteinase K can be used to
hydrolyze hair protein. Because of its ability to reduce disulfide
bonds, enzymatic digestion can also be substantially improved
in the presence of dithiothreitol during or prior to enzyme
treatment [14,122,123]. For example, the digestion of 1030 mg
hair with Proteinase K (100 mg) and dithiothreitol (200 mg) in
60 mL Tris buffer pH 8.0 was complete after 46 h at 37 C
[123]. Other enzymatic methods have been described [124].
3.4.6. Digestion of the hair with aqueous NaOH
For drugs that are stable under alkaline conditions, a
convenient method of hair extraction is digestion with aqueous
NaOH (1 mol/L) for one hour at 80 C. Basic extraction is
particularly advantageous for nicotine [125,126], amphetamines
[82,127131], THC [16,6870,132,133], antidepressants and
neuroleptics [134137], as well as several other drugs.
Generally, quantitative recovery of drugs from the hair matrix
can be assumed. In addition, this technique can be automated
with headspace solid phase microextraction (HS-SPME) for
semi-volatile substances [136].
3.5. Clean-up of hair extracts
Except for methanol or supercritical fluid extractions,
analysis via direct GCMS injection is not possible due to
impurities present in the aqueous extract or digestion solution.
Clean-up methods used for this purpose are similar to those
used in drug isolation from plasma or urine. Although
procedures for liquidliquid as well as solid phase extraction
(SPE) have been described, the latter method is typically used
due to availability of automated systems and improved
extraction materials. A detailed survey of clean-up techniques
used in hair analysis has been published [21].
Solid-phase microextraction (SPME) in combination with
gas chromatographymass spectrometry (GCMS) has provided some new possibilities for hair analysis with respect to
miniaturization and automation [131,136,138,139]. The principle of headspace version (HS-SPME) is shown (Fig. 6).
Lipophilic substances, even those with relatively low
volatility (i.e., tricyclic antidepressants, phenothiazines, lidocaine or methadone and its metabolites) can be extracted by
the SPME fiber directly from the headspace above the NaOH
digestion solution [77,136,140]. Absolute recoveries have
been reported from 0.417.4% with this technique. The
presence of amphetamines has been determined from less than
1 mg hair [128]. Depending on drug lipophilicity and
volatility, fibers with different coatings can be used. At
higher temperature, drugs are transported to the fiber via tiny
water droplets as well as gaseous phase. An advantage of this
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
Hair digestion and

preheating (30 min)
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.
method is that it can be performed without organic solvents.

Furthermore, the fiber is conveniently regenerated during
sample desorption in the GCMS injection port. With
appropriate derivatization, both amphetamines and THC
could be determined with this method [129,138,139,141].
An efficient variation of this technique is headspace solid
phase dynamic extraction (HS-SPDE). In this method, the
gaseous phase is drawn through a steel capillary coated with
an adsorbent [138,142,143]. This alternative approach appears
to be more robust in comparison to the fragile SPME fiber
and in some cases has demonstrated higher extraction yield.
Beside the headspace version, SPME can also be used for
extraction from the aqueous solution obtained by hair
digestion or extraction (direct immersion of the fiber, DISPME). This method has been described for methadone (and
its metabolite EDDP) after pronase digestion [123] as well
as for cannabinoids (THC, CBD and CBN) and other drugs
after NaOH digestion [144]. An important disadvantage of
the DI-SPME method is the short lifetime of the relatively
expensive fibers.
3.6. Detection and quantification
Instrumental methods used in hair analysis must be suitable
for unambiguous drug identification and quantitation. Although
procedures for a systematic toxicologic analysis in hair have
been described [145], these methods were typically developed
for application to one drug or limited number of drugs. General
drug screening methods for hair (i.e., similar to urine or blood)

are generally difficult to develop due to low drug concentration
and small sample size.
3.6.1. Immunochemical techniques
Although the first hair analyses for drugs were performed
by radioimmunoassay (RIA) [4], immunoassays have not
gained general acceptance because they are available for a
limited number of drugs (or metabolites) and not generally
specific. Immunoassay drug testing kits originally developed
for urine are of insufficient sensitivity for use in hair extracts.
However, over the last few years some ELISA tests for opiates,
cocaine, cannabinoids, benzodiazepines and methadone have
been developed and proven to be sufficiently sensitive for use
in hair analysis [9,102]. The Cozart microplate opiate ELISA
test demonstrated a sensitivity and specificity of 98% and 93%,
respectively, for 106 hair samples with methanol extraction
(cut-off of 0.2 ng/mg) [146]. Another example is the use of the
One-step ELISA kit for buprenorphine with a cut-off of
10 pg/mg [147]. In laboratories with a large sample numbers,
these kits are used as screening devices. For example, Harrison
et al [148] described an automated ELISA procedure for
cocaine, opiates, methamphetamine, phencyclidine and cannabinoids in hair. This system had a capacity of 500 samples per
day and was staffed by 8 persons for sample preparation and 4
persons as ELISA technicians. As can be appreciated, the
exclusive application of ELISA in forensic cases is inadmissible. Sensitivity must be adequate to ensure that no false
28
negative results are reported. Positive results have to be

confirmed by GCMS. In addition, negative drug testing
results should also be controlled if they are detrimental to the
accused.
hydroxyl (OH) or carboxyl (COOH) groups. Therefore, the

hair extract must usually be derivatized prior to GCMS [149].
For simultaneous analysis of several drugs with different
groups to be protected, mixed derivatization reactions can be
applied. These include derivatization with pentafluoropropionic anhydride/pentafluoropropanol [75] or N-methylbistrifluoroacetamide/N-methyl-tert-butylsilyltrifluoroacetamide [150].
Differences in sample preparation, particularly derivatization, appear to limit application to a generalized approach for a larger
number of drugs.
Increased sensitivity with LOD between 0.2 and 15 pg/mg
has been achieved with positive or negative chemical ionization
(GCPCI/MS and GCNCI/MS) for many compounds such as
benzodiazepines [18,151153] or polyfluoroacyl derivatized
substances such as ethylglucuronide [93]. A consequence of this
approach, however, is loss in specificity due to missing
fragmentation. Sensitivity and specificity have been increased
by GCMS/MS techniques in which the molecular ion or a
fragment ion after the first MS separation is exposed to a second
fragmentation with a collision gas. These secondary fragmentations can then be separated and detected (daughter ion
spectrum). As a result of these advances, GCMS/MS is
increasingly used in hair analysis [11,12,79,154156].
3.6.2. Gas chromatographymass spectrometry (GCMS)

Capillary gas chromatographymass spectrometry is the
method most frequently used in hair analysis. The advantages of
GCMS include high resolution of the capillary GC and high
specificity of electron impact ionization (EI) mass spectra.
These features are enhanced by measurement in the selected ion
monitoring mode (SIM) and use of deuterated internal
standards. Together these features enable the development of
specific and sensitive procedures for a large variety of drugs or
metabolites with sufficient accuracy at very low concentration.
Furthermore, a large number of substances can be measured in
the same run by dividing the chromatogram into several time
windows with different SIM masses (Fig. 7). The limit of
detection (LOD) of GCEI/MS is about 0.03 ng/mg for most
drugs.
A prerequisite for performing GCMS is that the substance
is sufficiently volatile and stable at the high temperature. These
characteristics not found for drugs with free amino (NH2)
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
Time, min -->

Fig. 7. Simultaneous GCMSSIM analysis for 15 illicit drugs or drug metabolites of the hair sample of a 26 year old polytoxycomanic drug user. Hair extraction:
30 mg hair + 200 ng of each deuterated standard, 5 h by phosphate buffer pH 6.0 in ultrasonic bath. Clean-up: SPE on mixed-mode columns (Chromabond Drug
200 mg, MacherayNagel). Combined derivatisation by trifluoroacetanhydride + hexafluoroisopropanol. Measurement in 12 time windows. Three characteristic m/z
were measured for each drug, metabolite or deuterated standard. For clarity reasons, only the ion chromatograms for the quantifier m/z are shown in this figure. m/z of
substance and corresponding deuterated standard (marked by -d3, -d5, -d6 or -d9): A = amphetamine 144/149, MA = methamphetamine 154/158, ME = methylecgonine
182/185, MDA = methylenedioxyamphetamine 135/136, MDMA = methylenedioxymethamphetamine 154/158, MDE = methylenedioxyethamphetamine 168/174,
BE = benzoylecgonine 318/321, EDDP = primary methadone metabolite 276/279, Met = methadone 72/78, Coc = cocaine 303/306, CE = cocaethylene 317/320,
Mo = morphine 364/367, Cod = codeine 282/285, DHC = dihydrocodeine 284, 6-AM = 6-acetylmorphine 364/367. Cannabinoids are not included in this method and
are tested in a separate analysis. The following substances were identified in this case: amphetamine 24 ng/mg, MDMA 39 ng/mg, MDA 2.1 ng/mg, cocaine 0.4 ng/
mg, benzoylecgonine 0.10 ng/mg, 6-acetylmorphine 1.0 ng/mg and morphine 0.13 ng/mg.
3.6.3. Liquid chromatographymass spectrometry (LCMS)

The combination of liquid chromatography and mass
spectrometry is useful for hair analysis because the GCdependent complications of volatility, stability and issues
with derivatization are avoided. Because of its lower
chromatographic resolution (versus GC), an efficient use of
LC in hair analysis is only possible with coupled tandem
MS/MS. A number of papers using LCMS/MS in hair
analysis have been published including detection of ethylglucuronide [157], methadone and metabolites [158], benzodiazepines [14,159], neuroleptics [15,160] and sildenafil
[161]. In one report, the hair was extracted with the mobile
phase solvent that was then analyzed for 14 drugs without
additional treatment [13]. In another screening for opioids,
the methanol extract was only evaporated and dissolved in
the mobile phase before injection [101]. Advances in data
processing have enabled the measurement of more than 100
drugs in a single chromatographic run. Because of low LOD
(about 1 pg/mg), LCMS/MS was used for detection of a
single dose of bromazepam and clonazepam [162] as well as
zopiclone [163]. Despite its advantages, LCMS/MS remains
limited to a small number of forensic toxicological
laboratories because of high expense.
3.6.4. Other methods
Although mass spectrometric identification of the drugs
is regarded as the gold standard in forensic hair analysis,
other detection systems have been successfully used in GCand HPLC-based techniques. Examples include GC-NPD for
amphetamines [128] and chloroquine [164] and GC-ECD
for organochlorine pollutants [165]. HPLC with UV
detection has been described for carbamazepine [166],
phenytoin and lamotrigin [61,62,167,168] as well as
indinavir [169]. HPLC with diode array detection has
been used in a screening procedure [145] and for detection
of tricyclic antidepressants [170], benzodiazepines [171] and
dextropropoxyphene [172]. HPLC has been used with ECD
for haloperidol [173]. HPLC with fluorescence detection has
been reported for flecainide [174], 5-fluorouracil after
derivatization [175] and ciprofloxacine and analogs [176].
In most cases drug concentrations were usually above 1 ng/
mg. The analysis of LSD by HPLC with fluorescence
detection after separation by immunoabsorption [83] was,
however, a specific example in which an alternative method
was superior to MS-based methods.
3.7. Quality control
The general guidelines and recommendations for quality
management in analytical laboratories [177180] can be
applied to hair analysis. General requirements include
laboratory staff, instruments and equipment, sample handling,
chain of custody or documentation. Accuracy assessment of
qualitative and quantitative drug test results is of particular
importance.
Unambiguous substance identification has the highest
priority in hair analysis. Because MS confirmation is
29
compulsory in forensic cases, the guidelines refer mainly to

GCMS or LCMS. Guidelines vary by organization. These
include the European Commission EC [177], Food and Drug
Administration FDA [178], Gesellschaft fr Forensische und
Toxikologische Chemie GTFCh [179], World Anti Doping
Agency WADA [180]. In general, a maximum allowable
retention time deviation of 25% (LC) and 0.52% (GC) has
been established between the analyte and reference standard.
The mass spectra in EI-MS must agree for a minimum of three
ions specific to the analyte and that originate from different
parts of the molecule. The intensity ratio of these mass
fragments must agree from 10% to 20% between analyte
and standard. Similar criteria have been established for PCI/
MS or NCI/MS and MS/MS. Although a points system in the
EC document enables the combination of different methods
for identity assessment, a final decision with respect to
substance identity cannot exclusively depend on formal
criteria but must include judgment by a specifically educated
and experienced analyst.
Quality control for quantitative analysis in hair is generally
more difficult in comparison to blood and urine. This is
largely due to the solid nature and heterogeneous composition
of the sample material and typically insufficient amount of
authentic reference material. As described above, differences
in sample preparation procedures can lead to differences in
concentration yields due to variable extraction yield and
partial substance degradation. To date, there has been a
general lack of consensus with respect to hair decontamination
and extraction procedures. It is, however, recommended to
perform calibration and validation including reproducibility,
precision, accuracy and determination of LOD and LOQ with
spiked hair samples. Despite this recommendation, this
approach does not involve decontamination and is thus unable
to simulate actual extraction conditions. If available, deuterated internal standards should be used. For internal quality
control, a drug-free and drug-positive hair sample should be
included in every analytic series. The best drug-positive
quality control material is a thoroughly mixed pool of hair
segments from a variety of drug consumers [181]. In lieu of
this approach, a reference hair quality control material may be
prepared by extending soaking in an appropriate solution of
drugs with subsequent, but thorough washing [182,183].
For external quality control, proficiency tests are available
in Europe from the Society of Hair Testing (SoHT) and from
the Society of Toxicological and Forensic Chemistry (GTFCh)
[184188]. Hair, from a variety of authentic drug cases, is cut
to 13 mm lengths and mixed. Samples are analyzed by
reference laboratories to determine target values. Results of 23
participants for the 2004 SoHT proficiency testing for cocaine
and benzoylecgonine are shown (Fig. 8). Although drugs or
metabolites were correctly identified in all cases, concentrations varied considerably. For example, reported cocaine
concentrations ranged from 0.52.8 ng/mg (approximately 6fold range). Procedures used by the participants were also
reported in the proficiency testing reports. To evaluate
reference laboratory performance, mean values of the participants were used. This participant comparison enabled
30
Cocaine
Reported concentration in hair, ng/mg
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.
examination or validation of in-house procedures for sources of

error in order to improve inter-laboratory performance.
4. Interpretation of analytical results
After hair analysis, answers to the following questions are
usually expected:
Did the individual use drugs?
Which drugs were used?
Was it single, occasional, regular or excessive use?
When were the drugs used?
These answers, however, are not typically derived on the sole
basis of analytical results. In general, these issues require expert
and critical examination of the case history, variability of hair
growth (cf. Section 2) and the hair sample itself, drug
pharmacology and thorough review of findings in other cases
previously investigated or described in literature.
4.1. Drug consumption or external contamination?
Frequent objections often arise to hair results that test
positive for a drug. A common claim is that the individual did
not consume the drug but that their hair was passively exposed
to smoke or dust in commercial establishments or at home due
to the presence of a drug-consuming companion. In fact,
deposition of cocaine in hair has been experimentally shown for
both the solid hydrochloride form as well as the evaporated base
[108,182,189,190]. In one study, cocaine hydrochloride powder
(10 mg) was uniformly applied to the scalp of four volunteers

[108]. Cocaine was detectable (1.8 ng/mg) ten weeks post
exposure. Interestingly, benzoylecgonine (cocaine metabolite
and hydrolysis product) increased during this time period from
0.0 to 0.8 ng/mg. In another study, the hair of toddlers who lived
in a household where crack was smoked was also examined
[191]. This study found toddler hair cocaine concentrations
nearly as high as the smokers themselves [191]. Controlled
experiments with environmental marijuana smoke exposure
demonstrated that false-positive or falsely increased drug test
results could be obtained in hair [192].
Based on the above studies, environmental hair contamination is likely complication given by the appropriate circumstances. In lieu of passive exposure, hair can be intentionally
treated with powdered or dissolved drugs to portray addiction as
a mitigating circumstance following arrest. In order to
distinguish passive contamination from active consumption, a
thorough examination of the wash solutions obtained during hair
decontamination is required. Baumgartner and Hill proposed the
presence of inaccessible domains in hair into which the drug is
permanently incorporated during keratinization and from which
it cannot be extracted without matrix decomposition [193]. A
specific washing sequence and ratio of drug concentrations in
the wash versus hair may be used to differentiate passive
contamination and systemic incorporation [107]. Although this
model is not generally accepted [194,195], recent drug
contamination is likely if the first wash drug concentration is
higher than that in hair. Previous contamination with subsequent
shampoo activity cannot, however, be clearly differentiated by
this approach.
Unequivocal proof for systemic drug origin is metabolite

detection. This is unambiguously true only for metabolites
that cannot be formed by hydrolysis. For example, cocaine
metabolites benzoylecgonine, ecgoninemethylester and norcocaine can typically be found in the hair of a cocaine user
(Fig. 9). In cases of simultaneous alcohol consumption,
cocaethylene can additionally be found. With respect to
cocaine, only norcocaine and cocaethylene are exclusively
endogenous metabolites whereas benzoylecgonine and ecgoninemethylester can also be formed by exogenous hydrolysis. The
concentration of norcocaine found in hair is about 13% of the
cocaine concentration (0.03 to 5 ng/mg) [110,189,196,197].
The criteria for endogeneous cocaine incorporation is a
benzoylecgonine/cocaine ratio greater than 0.05. With respect
to heroin abuse, the metabolites 6-monoacetylmorphine and
morphine can also arise from exogenous hydrolysis (after
contamination with heroin) whereas only normorphine is an
exclusively endogenous metabolite that is typically found at low
concentrations in hair [198].
For cannabinoids, the detection of THC, cannabidiol (CBD)
and cannabinol (CBN) in hair only indicates that the individual
had contact with cannabis or cannabis products [16,69,133,199].
In this case, drug consumption is only confirmed by the
detection of THC-COOH, a metabolite formed from THC in
several steps in the liver [16,69].
H3C
31
Methamphetamine and the ecstasy drugs methylendioxymethamphetamine (MDMA) and methylenedioxyethamphetamine

(MDE) are only endogenously N-dealkylated to amphetamine
and methylendioxamphetamine (MDA) [82,200,201]. The
presence of these metabolites can be used as evidence of active
intake. However, it should be noted that amphetamine
and MDA are used as illicit drugs themselves. To date, hair
amphetamine metabolites have not been detected. Identification
of unique and low abundance drug metabolites in hair remains
an active field of research. The development of highly sensitive
and specific analytical methods will continue to improve
detection. Metabolite / drug ratios expected in hair for frequently
abused drugs are shown (Table 2).
4.2. Interpretation of hair concentrations
4.2.1. Conclusions about intensity of drug use
What does the concentration of a certain drug or metabolite
in hair mean? As described earlier, there is no inter-individual
correlation between frequency of drug use or drug dose with
hair concentration. As can be expected, prospective studies for
illicit drugs of abuse are rather rare [60].
Retrospective studies using self-reported data about drug
consumption habits have provided an estimation of drug
concentrations in hair for users of heroin [204], cocaine [204],
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]

6-Acetylmorphine
Cut-off
Mean
Minimum
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
Because of methodical differences, every laboratory should evaluate its own

data as a basis for interpretation.
Second, cut-off values are used to decide whether a correct

analytical result is caused by a certain reason (i.e., by relevant
drug use) or not [211]. These cut-off values can be far above the
LOD and depend on the aim and the conditions of the decision
to be made. This must also be taken into account in hair analysis
in view of steadily improving method sensitivity.
The values given in Table 5 were chosen mainly in context of
the driving ability examination and should exclude a single or
very rare drug use as well as levels caused by environmental
contamination. Occasional drug use should be detected with a
high probability. Because a positive result is disadvantageous to
the individual, false positives must be excluded. Until present,
no relevant statistical analyses have been performed for
determination of appropriate drug cut-off concentrations in
hair. Because of these limitations, a revision of these drug cut
off values is expected when better substantiated data are
available.
4.2.3. Pubic, axillary and body hair
A comparison of drug concentrations between scalp hair
and hair from other sites has been described in several papers.
Higher drug concentrations for have been found for a number
of drugs in pubic, axillary, arm, chest or thigh hair versus scalp
hair. These drugs include opiates [103,212,202], methadone
[213], cocaine [202,212], methamphetamine [200], cannabinoids [214] and fatty acid ethyl esters [215]. These differences
are explained by increased incorporation from sweat or sebum
during the longer telogen stage, representation of another time
period because of the different growth cycle, differences in
pigmentation, and less exposition to light, weather and
cosmetic treatments. In general, these differences are in the
order of the inter-individual variations of scalp hair concentrations. Therefore, apart from a different time period they
represent, drug concentrations in hair from other body sites can
be interpreted in a similar fashion as scalp hair.
4.3. Interpretation concerning the time of drug intake
Under ideal conditions, the position of drug molecules in a
hair sample can be used to calculate the date of intake by Eq. (1)
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
SoHT [7] cut-off or LOQ, ng/mg
IC: 0.2 CH: 0.2 for 6-AM, MOR

and others
IC: 0.5 CH: 0.5 for COC 0.05
for Met.
Amphetamines: A, MA, MDMA, IC: 0.2 CH: 0.2
MDE, MDA
Cannabinoids
IC: 0.1 THC CH: 0.1 THC 0.2 pg/mg
for THC-COOH
Opiates: 6-acetylmorphine
morphine
Cocaine metabolites (Met.)
GTFCh [179,210] cut-off or LOI, ng/mg
SFTA [207] lower limit, ng/mg
IC: 0.2 CH: 0.2 for 6-AM, MOR and

others
IC: 0.2 CH: 0.2 COC 0.1 Met
0.5 for MOR, COD, 6-AM and others

0.5 for COC, BE and CE
IC: 0.2 CH: 0.2
0.5 for A, MA, MDMA, MDE, MDA
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.
from growth rate, date of sampling and length of the residual

stubble [22].
ti ts 1i =vh 1r =vh t0
ti
ts
t0
li
lr
vh
time of the drug intake

time of the hair sampling
time between incorporation of the drug into the hair
root and appearance at the skin surface
distance of the drug position in hair from the proximal
end of the hair sample
length of the residual hair shaft from the skin surface
after sampling
hair growth rate.
Prerequisites are a uniform, constant and known hair

growth rate, and that the incorporation of the drug occurs
only in the hair root. The resulting theoretical drug distribution
along the length of a hair tuft is compared versus time scale
(Fig. 10a and b) for a one month-long drug intake. Time
resolution would be about three days corresponding to a
distance of 1.21.5 mm between matrix cells and end of the
keratinization zone (Fig. 1a) and can be increased for drugs
with longer half-life in blood.
Many examples that demonstrate this agreement between the
drug history and drug concentrations in hair segments have been
published [176,216219]. Conversely, there are also many
experimental results that emphasize the limits caused by
heterogeneous physiology of hair growth and alternative drug
incorporation mechanisms as described earlier [82,92,164,220].
Alterations of the drug distribution along the hair sample
may result from a variety of causes (Fig. 10cg). Slowly
growing strands (Fig. 10c), catagen and telogen hair (Fig.
10d), and delayed incorporation from tissues (Fig. 10e) cause
an extension of the drug zone in proximal direction, whereas
fast growing strands (Fig. 10c) and incorporation from sweat
(Fig. 10f) or sebum (Fig. 10g) lead to an extension of the drug
zone in the distal direction. The degree to which these effects
influence hair drug distribution depends on the individual
(large portion of telogen hair), the pharmacokinetics of the
drug (high excretion rate in sweat or sebum) and on the
occasion of drug use (extreme sweating during a rave party)
and varies from nearly the ideal curve (clearly defined region)
to a distribution over the whole hair length. However, there is

no axial migration of the incorporated drug within the hair
strands and there are no indications for drug transport (i.e.,
distal to proximal) [221]. Therefore, proximal hair must be
drug-free after several months of abstinence.
As a rule, a complete drug history of a person is
generally not required with more attention focused on the
details of the circumstances surrounding the case history.
This focus must already be taken into account at the
beginning of the analysis for a proper choice of the
segmentation. Some examples shall be discussed below. In
generally, it is common to assume a hair growth rate of 1
cm/month, although a growth rate of 1.1 0.2 cm/month
would be more accurate.
4.3.1. Exclusion of drug use for a certain period of time
For regranting the driver's license after drug abuse
suspension, one year of abstinence has been imposed by
German authorities. This practice is monitored by hair analysis
[222]. To the benefit of the applicant, only the proximal 6 cm of
hair are subject to investigation. As shown earlier (Fig. 10), a
positive drug result may be obtained at 7 cm in extreme cases
despite complete abstinence for 12 months. In case of a positive
result of the proximal 6 cm segment, drug exposure unambiguously occurred within the last 12 months.
4.3.2. Time period presented by a certain hair lengths
In general, males have shorter scalp hair than females. Not
surprisingly, individuals with hair shorter than 6 cm frequently
apply for drug hair analysis. As such it is important to note the
time period actually represented by sample length. Under these
circumstances, the type of hair used and dates of hair
samplings are extremely important. Because of these limitation
and the effects shown earlier (Fig. 10), only a time period range
can be estimated. The minimum time period in months
corresponds to sample length (cm) if the scalp was totally
shaved and then allowed to grow until sampling. In contrast,
the maximum time period is obtained when the longer hair
sample was cut to the investigated length on the sampling day.
Under these circumstances, up to six months (maximum
duration of telogen stage) can be added to the age calculated
from the hair length. Of course, the concentrations measured in
the sample must also be taken into account. It is improbable
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
Relative drug concentration in hair
12
13
10
11
12
13
e) Effect of incorporation from tissues
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
Distance from proximal end, cm

Fig. 10. Schematic pattern of the drug concentration along a scalp hair tuft after one month of drug use. General conditions used for calculation: hair growth rate
vh = 1.1 cm/month (except for curve c); time between drug incorporation and appearance at the cutting point (proximal sample end) t0 = 0.5 months; width of the
incorporation zone in hair root: 1.2 mm. a) Drug administration between 7 and 8 months before sampling. b) Incorporation only from blood, constant hair growth rate.
c) Incorporation only from blood, variation of hair growth rate of the strands within the tuft between 0.7 and 1.5 cm/month, Gaussian distribution. d) Incorporation only
from blood, false positive result in proximal segments caused by hair in catagen and telogen stage (18%, duration 36 months). e) False positive results in proximal
segments caused by additional delayed incorporation from tissues (20% at end of administration, half-life in tissue 1 month). f) False positive results in distal segments
caused by additional incorporation from sweat. 1 = weak sweating, evaporation within 1 cm from the root. 2 = strong sweating, evaporation within 10 cm from the root.
3 = accumulation in distal segments because of evaporation from surface of hair style. g) False positive results in distal segments caused by additional incorporation
from sebum. 1 = accumulation during the drug administration period. 2 and 3 = preferred incorporation in distal segments because of increasingly damaged hair
structure and more intense removal from proximal hair during shampooing.
that a cocaine concentration in the medium range (15 ng/mg)

originated from the small portion of hair that was in telogene
stage for 6 months, whereas a low concentration (1 ng/mg)
could be explained in this way.
Delayed incorporation from depots in surrounding tissues

may lead to an increased time period following shaving. This
phenomenon appears particularly true for lipophilic THC stored
in fat tissues (Fig. 11) [223]. A 26 year-old male with habitual
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
THC 1.7 ng/mg

Cocaine 0.3 ng/mg
Nov. 00
Dec. 00
Jan. 01
35
April 02
2nd hair sampling
length 1.5 cm
THC 0.25 ng/mg

Cocaine negative
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.
marijuana use was summoned to hair analysis because of doubts

regarding his driving ability. He shaved his head and stopped
drug use. Analysis of a 2.5 cm long hair sample (about 3 months
later) revealed the presence of THC (1.7 ng/mg). Despite
shaving his hair again, analysis of a 1.5 cm long hair sample
(about 1.5 months later) contained THC (0.25 ng/mg). Despite 4
months abstinence, THC can be incorporated into newly grown
hair at concentrations consistent with occasional drug
consumption.
4.3.3. Testing for previous drug use around a certain date
This inquiry frequently occurs in cases of manslaughter,
murder or armed robbery. At some later point, the suspect is
arrested and claims to have been under the effect of drugs
during the crime and as such applies for mitigating circumstances. Although hair analysis cannot conclusively prove
retrospective drug use to a specific date, it can provide
approximate dates of alleged drug use. Under these circumstances, the approximate position on the sample length
corresponding to the date of the crime should be calculated
by Eq. (1). Analysis of the hair sample should be performed in

at least 3 segments with one covering the approximate crime
date as well as segments proximal and distal to it.
A 53 year old male accused for kidnapping was observed by
the hostage to have used drugs during the crime (Fig. 12). He
was arrested one month later. A 9 cm long hair sample (gray
with a large proportion of white hair) was then obtained for hair
analysis (approximately 3 months after the crime). Hair analysis
was performed in three segments as described above to estimate
the approximate time of drug use. Cocaine and benzoylecgonine
were detected in all three segments with similar concentrations
in the distal and the middle segment corresponding to the time
before and at the time of the crime. The lower drug
concentration measured in the proximal segment corresponded
to time in jail and should originate from telogen hair. No other
drugs were found. These results indicate that cocaine use during
the time period of the crime is probable. Although the drug
concentrations are relatively low (Table 2), frequent drug use is
not excluded because of the large proportion of non-pigmented
hair in this individual.
Benzoylecgonine
Cocaine
Offence
Arrest
2
0
0-2
2-5
5-9

Fig. 12. Detection of cocaine and benzoylecgonine in the grey hair sample of a 53 year old kidnapper arrested one month after the offence [223]. The sample was
collected three months after the offence.
36
4.3.4. Additional analysis of pubic, axillary or body hair

Although these alternative hair samples are generally
unsuitable for time-resolved hair analysis, their additional
analysis can be helpful because of the extended time period that
they represent. Under these circumstances, it is important to
confirm that the hair has natural tips to indicate that it has not
been shaved and thus represents a complete growth cycle time
(Fig. 13) [223]. For example, a 42 year-old male was arrested
while buying a large amount of cocaine. He claimed that the
purchase was for personal use. His last reported cocaine use was
the day prior to his arrest. Scalp and pubic hair samples (2 cm
length) were collected 4.5 months after arrest. Scalp hair
demonstrated very low drug concentration. In contrast, the drug
concentration was 10-fold higher in the pubic hair sample (with
natural tips). Because of the high proportion of telogen hair, this
sample represented a substantially longer time period (i.e., more
than a year). This finding indicated that the majority of the hair
was grown prior to arrest and supported previous and frequent
drug use history.
4.3.5. Detection of a single dose in context of drug facilitated
crimes
This problem will be dealt with in Section 5.5 in greater
detail. A suitable procedure, however, in these cases is to
collect the hair sample approximately one month after the
offense. The hair is then subjected to short segment analysis
(35 mm length). Positive drug results are expected to be
found approximately 1 cm from the hair root whereas the
more distal segments should be negative and exclude previous
voluntary drug use.
An alternative method for detection of single drug doses is
available, but unfortunately limited to male individuals since it
12
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.
involves analysis of daily shavings (approximately 1 mm

segments). Using this technique, several studies have demonstrated that drug concentrations in the ng/mg range were
detectable for a variety of drugs 114 days following intake
[224228].
5. Practical applications
5.1. Human performance toxicology
Drugs seriously interfere with human physical and/or
psychological performance. These effects have serious negative
consequences for many common activities ranging from
industrial safety to driving ability. Recently, drug use has
been highlighted as a means to enhance sports performance.
Because of its potential as a long-term index of drug use history,
hair analysis provides a mechanism to monitor and control
abuse in all these cases.
5.1.1. Workplace drug testing
Many employers now require staff to undergo drug testing to
ascertain illicit drug use or excess alcohol consumption. The
need to achieve a drug-free workplace in order to ensure
public safety, corporate security, and minimize immense
productivity losses overrides individual civil liberties. To date,
there is no specific legislation in Europe or other parts of world
and no generally accepted guidelines for the use of hair testing
for this purpose, although some recommendations have been
issued [229]. The European Workplace Drug Testing Society
(EWDTS) was founded in 1998 to ensure that workplace drug
testing is performed to a defined quality standard in a legally
secured way to provide an independent forum on this issue
[230].
For the last 40 years, urine drug testing has been the most
common technique for detection of drug use in the workplace.
Hair has been used to an increasing extent from the beginning
of the 1980s, and in the last years, the focus is on alternative
specimens such as oral fluid or sweat [231]. Hair should
preferentially be chosen for pre-employment or random tests.
Large numbers of samples are routinely analyzed by firms such
as Psychemedics that received FDA clearance for hair testing
in all the major drugs-of-abuse categories [193,196,232]. In
fact, a major United Sates metropolitan police department
reported that the mean rate of drug positive specimens was
1.36 times higher for hair than urine on a large number of
subjects (several thousand per year) tested between 1985 and
1999 [233].
5.1.2. Drug test in context of driving ability examination
Generally, drug addiction and regular drug abuse are
inconsistent with driving ability. Hair analysis is a part of the
procedure for granting or re-granting the driver's license in
Germany and Italy [105,222,234236]. In Germany, the
proximal 6 cm segment of the hair sample from vertex posterior
is analyzed in order to cover a 12 months time period (cf.
Section 4.3). Because hair analysis is only one part of the
complex examination system, results from shorter samples are
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
5.2. Criminal liability and drug addiction

Although hair analysis is frequently used in criminal cases in
order to investigate whether the accused regularly used drugs in
the time period of the crime, this approach has not received
much attention in literature. These cases typically involve drug
trafficking, crimes committed to support personal drug
consumption and crimes committed by addicts under the
influence of drugs. The diagnosis of drug addiction is an
essential part in the examination of the accused by a forensic
psychiatrist and may be supported or made improbable by the
hair drug testing result. Two examples have been described in
Section 4.3. Drug addiction or being under the influence during
the crime are extenuating circumstances commonly espoused
by the defendant in order to lower the sentence. Therefore, it is
advisable to collect a hair sample immediately after arrest if any
connection to drugs is observed or assumed.
5.3. Diagnosis of drug abuse and chronic intoxication
Hair samples are particularly useful to prove chronic
exposure to drugs or other poisons. Therefore, it can be used
as a diagnostic tool for clinical detection of drug abuse, for
gestational drug exposure in neonates and for elucidation of
other chronic poisonings.
5.3.1. Drug abuse history and withdrawal control
Hair samples of patients in withdrawal treatment were
analyzed in several studies in order to determine the identity of
abused drugs before treatment and to control change in
consumption behavior after treatment [73,75,104,245247].
Segmental hair analysis can provide a retrospective calendar of
an individual's drug use or period of abstinence. This historical
perspective can provide substantial evidence of behavior
including switching from one drug to another or mixing of
various drug (illicit and pharmaceutical) combinations, e.g.,
heroin, dihydrocodeine, and hydrocodone [73,75,104].
Furthermore, the knowledge of previous drug abuse is
important to obtain an accurate and correct diagnosis of
psychotic patients. Despite the advantages of hair testing, the
use of this technique has not received widespread application
due to increased cost and longer analytical time in comparison
to urine.
5.3.2. Smoking behavior
Nicotine and its main metabolite cotinine are well deposited
in hair [44,91,126,203,248]. Active smokers have substantially
higher concentrations of nicotine (0.933.9 ng/mg) and cotinine
(0.094.99 ng/mg) in hair versus passive smokers 0.541.82
and 0.010.13 ng/mg, respectively [91]. Because these are
systemically incorporated in passive smokers, the metabolite
cotinine cannot be used to distinguish tobacco use. High
concentrations of these markers were also found in the hair of
children who lived in a smokers household [249]. In vitro
experiments have demonstrated that nicotine is incorporated
from side-stream smoke and leads to a concentration gradient
(proximal to distal) in hair [250].
38
5.3.3. Gestational drug exposure

Alcohol, smoking and illegal drug use during pregnancy are
serious hazards to the fetus and may lead to miscarriage,
premature birth, increased peri- and neonatal mortality rate,
retarded physical and mental development, learning difficulty or
hyperactivity. A neonatal withdrawal syndrome may appear in
cases of maternal opiate or methadone abuse. Meconium
analysis is well-established approach to investigate in utero
drug exposure [251]. However, if drug abuse is suspected only
at some later time analysis of hair obtained from the baby and
mother can be performed. Under these circumstances, however,
informed consent may be required from the mother. Of
particular note is that fetal hair growth and hair cycle [38] and
the steady substance exchange with amniotic fluid before birth
must be taken into account when performing hair analysis on
the neonate.
Nicotine and cotinine have been determined in neonatal hair
in several studies [125,252254]. Investigation of 40 mother/
infants pairs found nicotine concentrations of 0.1511.8 and
0.3763.5 ng/mg in neonatal and maternal hair, respectively
[252]. This study found significant correlation between neonatal
and maternal nicotine hair concentration (r = 0.83). Although
nicotine and cotinine have been found in newborn hair of babies
from both active and passive smoking mothers, the concentrations of both substances in newborn hair were about equally
high [253]. Maternal hair concentration of nicotine decreased
during pregnancy without any reported reduction in smoking,
while the cotinine concentration remained constant [125]. This
finding may indicate increased nicotine metabolism during
pregnancy.
Hair analysis has been described as a sensitive means to
detect intrauterine exposure to cocaine [255,256]. In a study of
newborn twins, researchers reported that genetics appears to
influence drug incorporation rate into prenatal hair [257].
Although equal drug concentrations were found in monozygotic
twins, drug concentrations differed by approximately 3-fold for
dizygotic twins. Similar findings have been reported for
methamphetamine [258].
Opiates in hair of 17 maternal/neonatal couples were
assessed [259]. Newborns exposed to opiates in utero can
suffer withdrawal syndrome to varying degrees a few days after
the birth. Neonatal withdrawal syndrome, however, is not easy
to recognize when maternal addiction has not been identified. In
these patients, the gestational opiate exposure profile was
substantially correlated to the appearance of neonatal withdrawal syndrome.
5.3.4. Chronic intoxication by environmental pollution or food
adulteration
In the presence of clinically pathologic symptoms (hepatotoxicity, neurotoxicity) that cannot be explained, poisoning
with an unknown substance must be considered in the
differential diagnosis. Poisoning may result from a variety of
sources including long-term environmental pollution, food
adulteration with some ingredients or unknown criminal
activity. As alluded to above, general toxicological screening
of hair is not yet possible. Therefore, a specific suspicion
regarding the substance in question is of vital importance in

these cases.
Analysis of hair for pesticides and other pollutants is mainly
limited to organochlorine compounds such as DDT, lindane and
polychlorinated biphenyls [260263]. The hair concentration of
these pollutants in the normal population is in the range of 0.5
5 pg/mg [260,263]. Because these substances are typically
inhaled, external hair incorporation is regarded as more
important than systemic incorporation. The use of hair testing
to indicate chronic environmental intoxication is still, however,
widely unexplored and difficult to interpret in general.
There are few published reports that involve hair analysis
after ingestion of adulterated products. One case reported the
adulteration of Chinese herbal medicine with N-nitrosofenfluramine, an appetite suppressant [264]. In this study, the
metabolites fenfluramine (431389 pg/mg) and norfenfluramine (18680 pg/mg) were identified in the hair of exposed
patients hospitalized with hepatic dysfunction. Based on
these findings the authors concluded that these patients
ingested N-nitrosofenfluramine for a period of approximately
5 months.
5.4. Postmortem toxicology
Over the last years hair samples are regularly collected
during autopsy. Although hair is not generally useful for
proving a lethal intoxication, it has been demonstrated that
acute poisoning can be established with hair root examination
[265]. In this study, the authors found extremely high
methamphetamine concentrations (30.5134.6 ng/mg) in the
hair roots of four deceased men suspected of acute exposure.
However, the predominant value of hair analysis in postmortem cases is to support death diagnosis by proving or excluding
chronic substance abuse. Other applications include information regarding pathological alterations of internal organs, to
provide evidence for drug tolerance in cases of opiate or
methadone overdose, to explain withdrawal symptoms or as an
indication that, in case of a longer survived accident, the
deceased was probably under the influence of drugs. Finally,
hair analysis can contribute to the identification of an unknown
corpse.
5.4.1. Health impairments caused by chronic drug abuse
Long-term drug abuse or chronic poisoning can gradually
induce certain harmful effects on the human organism and
can exacerbate preexisting disease. For example, chronic
abuse of methamphetamine is known to be associated with
cardiovascular diseases [266]. During autopsy morphologic
alterations are commonly found in the hearts of stimulant
addicts. The rapid increase in blood pressure after intravenous
methamphetamine dose can place addicts with arteriosclerosis
at risk. However, lifestyle information regarding the deceased
is not always available to help explain pathological
cardiovascular alterations observed during the autopsy and
to classify cause of death correctly. As such, drug findings in
hair segments may be useful. For example, the drug analysis
of four 2 cm segments and fifth distal 7 cm segment has been
successfully used to explain pathological alterations found

during autopsy of a 31 year-old deceased male with bleeding
into cerebellum [267]. These results provided clear evidence
of methamphetamine abuse for more than 8 months. Similarly, hair analysis for anabolic steroids could be useful to
explain unexpected death of young athletes as a consequence
of chronic abuse [244,268].
5.4.2. Tolerance in opioid death cases
A significant factor in interpretation postmortem blood
morphine or methadone concentrations is whether or not this
finding is indicative of chronic abuse or single consumption
[41]. Tolerance to poisoning by these drugs can increase by
orders of magnitude after chronic use. As such, a measured
drug blood concentration may be quite lethal for a novice
user but be quite harmless for a long-term addict. In fact,
many drug death cases occur after first use or following long
abstinence. Because hair analysis can accurately resolve the
question of whether or not drug use was chronic, this testing
should be mandatory in the toxicological investigation of any
drug fatality.
5.4.3. Chronic drug use and fatal accidents
In fatal traffic or industrial accidents, drug use of the
deceased should be examined. Hair analysis may provide
important information if the victim survived several days and/
or if blood was not immediately collected. In case of a
negative blood drug result, a positive hair result may be an
indication for impairment by withdrawal symptoms. Also a
negative result of hair analysis can be very important [41].
For example, a man crashed his car against a tree resulting in
immediate death. Toxicological examination revealed the
presence of 7-aminoflunitrazepam at a blood concentration
of 59 mg/mL. Subsequent hair testing, however, was negative
for flunitrazepam and its metabolites by GC/MS/MS with a limit
of detection of 1 pg/mg. These findings substantiated medical
records the man had not been previously treated with
flunitrazepam and demonstrated that the deceased was not a
drug addict. These facts contributed to the confession of a
jealous partner who admitted to sedating the victim to cause fatal
impairment.
5.4.4. Repeated criminal poisoning
Hair analysis may be useful in cases of repeated poisoning.
For example, the repeated application of an increasing dose of
clozapine to a nearly two year-old girl by her mother with fatal
outcome was confirmed by segmental hair analysis despite a
one year burial in soil grave [269]. Confronted with the result
the mother admitted the crime.
5.4.5. Probability of self-administration
Detection of chronic drug abuse is very important in some
cases. For example, a female nurse was found dead at home
with high blood concentrations of anesthetics [270]. Segmental
analysis of a 6 cm long hair strand revealed the presence of
midazolam and propofol in all three 2 cm segments proving
long-term and most likely self-adminstered drug abuse.
39
5.4.6. Contribution to identification of a corpse

Hair analysis can be used also as a tool to identify population
sub-type postmortem (psychiatric patient, smoker or nonsmoker, drug abuser, male or female) prior to final identification by
DNA [41]. In one case, a totally skeletonized corpse was
identified by comparison of the drugs detected in hair with the
known medication history of a psychotic patient missing for
more than one year [145].
5.5. Criminal assaults
It is not a new phenomenon to use drugs to govern other
person's perception and behavior. In this context, drugs are
often used to achieve a number of goals including personal
criminal profit (i.e., robbery, sexual assault and child abuse).
Drugs involved in such crimes can be pharmaceutical
(sedatives, hypnotics and anesthetics), ethanol or drugs-ofabuse (cannabis, LSD, GHB, ecstasy, etc). In fact, the number of
drug-facilitated sexual assaults has dramatically increased over
the last few years [271].
5.5.1. Single drug application
As a rule, the drug is applied in single dose format in drugfacilitated crimes. Therefore, the preferred means of toxicological investigation is blood or urine analysis. The specimen
should be collected as early as possible after the crime and before
the drug is completely eliminated. However, in reality, hours or
days can pass until the victim becomes conscious or has some
indication that an assault occurred. Although the majority of
these cases are reported to the police after a few days, blood and
urine may not be immediately collected for subsequent
toxicological examination. Due to improved analytical sensitivity, hair analysis can be useful also for detection of single drug
doses exposure [18,19,85,86,152,162,163,272275]. In systematic prospective studies, 7-aminoflunitrazepam was determined by GCMS/NCI in the proximal 1.5 cm hair segments of
10 volunteers after application of 2 mg flunitrazepam [18]. In
this study, hair drug concentrations ranged from 0.48.0 pg/mg
following previous (128 day) exposure. In contrast, 7aminoclonazepam was only detected in 6 of 10 hair samples
(1.223 pg/mg) after exposure to 3 mg clonazepam [152].
Following administration of 10 mg zolpidem, hair drug
concentrations in three volunteers ranged from 1.89.8 pg/mg
in the proximal 2 cm segments one-month post exposure [273].
In a similar study following administration of 7.5 mg zopiclone
to 2 volunteers, the determination of these hair drug concentrations ranged from 5.49.9 pg/mg by LCMS/MS. Bromazepam
(0.828 pg/mg) was measured in three volunteers following
6 mg ingestion [162,272]. Interestingly, oral administration of
2.5 mg lorazepam was not, however, detectable in hair by these
methods [276].
-Hydroxybutyric acid (GHB) is one of the most frequently
used substances in drug-facilitated crimes [271]. Because of its
very short half-life (0.31 h), GBH is particularly difficult to
detect in blood or urine. Furthermore, it is also endogenously
formed leading to the presence of low but varying natural GHB
levels in human hair [85]. Nevertheless, it is possible to detect a
40
was considered unlikely if positive drug results were obtained in

more distal hair segments (Fig. 14c).
Thiopental and its metabolite pentobarbital were found in
scalp and pubic hair samples of a woman sexually assaulted
during hospitalization [274]. In this case, the hair samples were
collected four weeks following the assault. In the 1.5 cm
proximal segment of three scalp hair samples, thiopental (0.15
0.30 ng/mg) and pentobarbital (0.200.40 ng/mg) were
identified by GCMS/MS. In the distal hair segments no
barbiturates were detected.
Toxicological investigation of 128 drug-facilitated crimes
(12 month period) in Paris, France demonstrated that less than
20% (n = 23) of these cases could be confirmed by hair analysis.
Although hair analysis can verify exposure, the identity of the
drug and substantiate the victim's statement, it cannot
differentiate administration by a perpetrator and self-administration. Furthermore, it cannot indicate exact date of administration provide and can only provide an approximate time
period of exposure.
single dose by investigation of 3 mm segments of the hair sample

collected 1 month after the incident (Fig. 14a) [19,86]. Analysis
is performed by digestion of the hair sample with NaOH,
extraction with ethyl acetate after acidification, silylation and
GCMS/MS. GBH administration is clearly indicated by
increased concentrations in the corresponding segments above
baseline levels.
It should be pointed out that the drugs typically used in
these crimes are not known. In order to circumvent this
obvious limitation, a method for simultaneous measurement
of 23 benzodiazepines or benzodiazepine-like hypnotics by
LCMS/MS (LOD < 2 pg/mg) in hair was developed
[19,275]. Three hair segments are collected approximately
one month after the incident. These correspond to the crime
(02), prior to (24 cm), and drug-free (46 cm) time
periods. An example corresponding to a sample collection
time of 9 weeks after the assault is shown (Fig. 14b).
Additionally, the hair must be tested for GHB by GCMS/
MS as described above.
The use of this technique has been instrumental in a number
of cases involving detection of 7-aminoclonazepam (3.2 pg/
mg), 7-aminoflunitrazepam (5.2 pg/mg) [19], bromazepam in
four cases (4.110.3 pg/mg) [162,272], zolpidem (4.4 pg/mg)
[19,273] and zopiclone (4.2 pg/mg) [163]. Single drug exposure
5.5.2. Repeated or frequent administration

Criminal cases with repeated administration can be treated in
the same way as cases of drug abuse. More efforts are necessary
in cases with low dosed drugs such as buprenorphine [277]. A
GHB in hair, ng/mg
(a)
2
0
0
20
(b)
3
2
1
< 0.3
0
0-3
3-5
5-7
Bromazepam in hair, ng/mg
Zopiclone in hair, ng/mg
(c)
15
10
5
0
0-2
2-4
4-6
6-9
9-12

Fig. 14. Detection of a single drug administration in drug facilitated crimes by hair analysis. The dates of the crimes according to a growth rate of 1.1 cm/month are
marked by an arrow. a) -Hydroxybutyric acid (GHB) in the hair sample from a 19 year old woman who became unconscious and was sexually assaulted after a soft
drink [19]. Sampling 1 month after the rape, analysis in 3 mm segments. The concentrations in the segments 4 and 5 (2.4 and 2.7 ng/mg) was clearly above the
individual endogenous GHB level (0.60.8 ng/mg) in the other segments. b) Zopiclone in the hair sample of a 16 year old girl who claimed to be raped while sedated
by a drug [163]. Sampling 9 weeks after the crime. The positive result in both proximal segments and the negative third segment (LOD 0.3 ng/mg) were consistent with
the administration of a single dose at the date of the crime. c) Bromazepam in the hair sample of a 25 year old woman collected 6 weeks after an alleged assault [272].
The positive result in all five segments is inconsistent with a single exposure to the drug.
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
5.6.3. Repeated hair sampling, analysis of the proximal 1 cm

segment
In this scenario, the integrated storage of the drug can be
exploited by repeated sampling. The main advantage of this
approach is for monitoring compliance. It has been shown in
several papers that hair drug concentration remains almost
constant if the same dose is taken for a long period of time
[14,61,62,166]. Although each patient has individual doserelated hair concentrations, this issue can be addressed during
controlled drug application in the clinic. Deviations in hair
drug concentrations during outpatient treatment can be
interpreted as evidence of non-compliance. The high costs
and labor intensive nature of this approach, however, prevents
its utilization in large-scale daily treatment programs. Despite
its advantages versus blood, this technique is limited to
special case studies.
5.7. Detection of excessive alcohol abuse
Although alcohol is the most frequently abused substance, it
has played only a minor role in hair analysis because it is a
volatile substance and not durably incorporated. Alcohol
consumption is legal and as such generally tolerated.
Therefore, a hair alcohol test must be able to differentiate
social drinking from abuse. In addition, other routine and
relatively low cost laboratory biomarkers for chronic alcohol
abuse have been well established including gamma-glutamyltransferase (GGT), mean corpuscular volume of erythrocytes
(MCV), carbohydrate deficient transferrin (CDT). Because of
these limitations, alcohol abuse via hair analysis is limited and
generally associated with determination of its minor metabolites including fatty acid ethyl esters and ethyl glucuronide
[283].
5.7.1. Fatty acid ethyl esters (FAEE)
Fatty acid ethyl esters (FAEE) are formed in presence of
ethanol from free fatty acids, triglycerides, lipoproteins or
phospholipids. These compounds are formed by action of
specific as well as non-specific enzymes present in the liver,
pancreas, heart, adipose tissue, brain and white blood cells. More
than 15 ethyl esters of linear and branched saturated and
unsaturated fatty acids have been identified in hair. Of these, ethyl
myristate, ethyl palmitate, ethyl oleate and ethyl stearate have
been chosen as markers of ethanol intake [92,93,114,215,284
286]. The sum of the concentration of these four esters CFAEE
has, in fact, been used for quantitation. For quantitative determination, the decontaminated hair sample was extracted with a
two-phase mixture of dimethylsulfoxide and n-heptane. The nheptane phase is then evaporated and the residue analyzed by
headspace solid phase microextraction and GCMS using the
deuterated four esters as internal standards [114].
Investigation of the lipids from the hair surface and sebum
collected from the forehead as well as from the comparison
between segmental analysis and drinking history has demonstrated that FAEEs are mainly incorporated into hair from
sebum [92,287]. Therefore, time-resolved interpretations with
respect to previous drinking and abstinence are not possible.
42
The analysis of hair samples from a variety of individuals

including non-drinkers, social drinkers, patients in withdrawal
treatment and death cases with excessive alcohol consumption
resulted in CFAEE between 0.05 and 30 ng/mg. Interestingly, low
FAEE concentrations (0.060.37 ng/mg) were also found for
non-drinkers. A CFAEE cut-off value of 0.5 ng/mg has been
proposed for chronic excessive alcohol consumption with 90%
sensitivity and specificity by ROC analysis of a large number of
cases [211]. Although hair shampooing, permanent wave,
dyeing, bleaching or shading did not affect results, frequent use
of hair lotions containing ethanol was found to increase the false
positivity rate.
5.7.2. Ethyl glucuronide (EtG)
Ethyl glucuronide (EtG) is a phase II-metabolite of ethanol
mainly formed in the liver. About 0.020.06% of ingested
alcohol is eliminated as EtG. Following extraction with water or
aqueous methanol, this metabolite can be determined in hair by
GCEI/MS [288290], GCNCI/MS [93] and LCMS/MS
[157]. Sensitivity of this analytical approach could, however, be
substantially increased using clean-up by solid-phase extraction
with aminopropyl columns [93]. The cleaned extract was
directly injected into LCMS/MS. Alternatively, the extract
was derivatized with BSTFA for GCEI/MS or with a mixture
of pentafluoropropionic anhydride/pentafluoropropanol for
GCNCI/MS. Using this technique, detection limits of 2 and
50 pg/mg have been determined for GCMS/NCI and LCMS/
MS, respectively.
In these studies, EtG concentration for individuals with
known heavy alcohol abuse ranged from 0.0313.3 ng/mg. In
contrast, all hair samples from non-drinkers and social drinkers
were negative. Therefore, the detection of EtG in hair above
0.03 ng/mg is a strong evidence for alcohol abuse. It should be
noted, however, that a negative hair EtG result does not
completely exclude abuse because no EtG was detected in some
hair samples obtained from alcoholics. Although increased
analytical sensitivity has generally decreased the number of
false negative results, positive EtG results have also been found
in hair from social drinkers (private communication from M.
Yegles, Luxembourg). A temporal relationship has not been
demonstrated for segmental hair EtG concentration and time
course of self-reported alcohol consumption [93]. Being acidic
and extremely hydrophilic, EtG is likely incorporated into hair
to some degree from sweat.
The advantage of both FAEE and EtG is that they are direct
alcohol markers that possess the unchanged ethyl group of
alcohol in contrast to the indirect biochemical markers currently
in use. The measurement and combined interpretation FAEE
and EtG in hair is very helpful in cases of driving ability
examination.
6. Conclusions and future prospects in hair analysis
Hair as a medium for diagnosis of previous and chronic drug
exposure has received increased attention because of a wider
detection time frame, less embarrassing circumstances of
collection, and greater stability versus body fluids or other
tissues. Improved analytical technology has resulted in

improved sensitivity and accuracy thus providing better
scientific understanding and test interpretation. These advances
will further promote the use of hair analysis as a useful and
objective tool of evidence. Due to their high sensitivity and
specificity, newer generations of GCMS/MS and LCMS/MS
technologies will become standardized tools in toxicological
laboratories. In addition, the use of hair analysis will be
extended to investigation of harmful substances not measurable
by present techniques. Trends to miniaturization in analytical
chemistry [291] will continue to find application in hair
analysis. The investigation of a single anagen hair in short
segments [292] will be possible for organic substances and will
provide additional information and improved interpretation.
Introduction of automation techniques and improved methods
for sample preparation will enable application of hair analysis
for large-scale testing and more general use.
Further progress is also expected in applications of hair
analysis already established. Weak points in the present hair
tests for illicit drugs must be overcome by 1) preparation of
reliable reference standards for calibration and inter-laboratory
quality control, 2) standardization of procedures for decontamination and extraction procedures, 3) establishment of performance standards for drug and metabolite identification, 4) use
of scientifically founded drug cut-off values. In order to
facilitate consistent interpretation and consensus approach, the
Society of Hair Testing (founded in 1995,www.soht.org) has
made some recommendations about specimen collection,
decontamination and specimen handling procedures, criteria
for obtaining positive results, metabolites to be assayed and
metabolite-to parent drug ratios [7].
Despite these advances, assay performance will continue to
be challenged by biologic variables including differences in hair
growth and mechanisms of drug incorporation. As can be
expected, the complex nature of the issues addressed in this
review will require continued research in this field and
appropriately trained and experienced scientists now as well
as in the future.
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Clinica Chimica Acta 370 (2006) 17 49

Invited critical review

F. Pragst, M.A. Balikova / Clinica Chimica Acta 370 (2006) 1749

Detection and quantification . . . . . . . . . . . . . . . . . . . . . . . . . .

development of analytical technologies, offering methods with

F. Pragst, M.A. Balikova / Clinica Chimica Acta 370 (2006) 1749

standardized hair testing approaches that were documented in

The pigmented cortex is responsible for the stretching stability

1.2 1.5 mm ( 3 days)

F. Pragst, M.A. Balikova / Clinica Chimica Acta 370 (2006) 1749

amorphous protein. The cell membrane complex consists of

Cat + Tel Growths rate

Fig. 2. Incorporation and elimination of drugs in hair.

(anagen) with the remaining 15% in the resting phase (telogen).

F. Pragst, M.A. Balikova / Clinica Chimica Acta 370 (2006) 1749

well as the physical/physiological characteristics of the

9-tetrahydrocannabinol-11-carboxylic acid, THC-COOH), the

Concentrations: BH+e << BH+i

F. Pragst, M.A. Balikova / Clinica Chimica Acta 370 (2006) 1749

Concentration in hair, ng/mg

Illicit cannabis use2

MDMA + MDE use

Fatal drug cases

Fatal drug cases

A comprehensive collection of data is found in Ref. [21]. Metabolites are indented.

concentrations. Since illegal drugs were most frequently

have been surveyed [67] and a selection of important examples

F. Pragst, M.A. Balikova / Clinica Chimica Acta 370 (2006) 1749

The lack of inter-individual correlation between drug

In regularly shampooed hair, i.e., not treated by aggressive

Distance from hair root, cm

Distance from hair root, cm

F. Pragst, M.A. Balikova / Clinica Chimica Acta 370 (2006) 1749

assumes a high responsibility for obtaining a correct result.

3.1. Documentation on the case, collection and storage of hair

Sampling (2 hair samples)

Cutting to small pieces or grinding

Extraction or digestion of hair matrix

Clean-up of hair extract

Qualitative and quantitative analysis

Interpretation of results, expertise

F. Pragst, M.A. Balikova / Clinica Chimica Acta 370 (2006) 1749

sampling, questions to be answered by the analysis, and

appropriate segmental lengths, expected time resolution,

F. Pragst, M.A. Balikova / Clinica Chimica Acta 370 (2006) 1749

must be considered. In many cases, hair is screened for wide

rapid extraction with high mobility [117121]. The powdered

F. Pragst, M.A. Balikova / Clinica Chimica Acta 370 (2006) 1749

Hair digestion and

method is that it can be performed without organic solvents.

drug screening methods for hair (i.e., similar to urine or blood)

F. Pragst, M.A. Balikova / Clinica Chimica Acta 370 (2006) 1749

negative results are reported. Positive results have to be

hydroxyl (OH) or carboxyl (COOH) groups. Therefore, the

3.6.2. Gas chromatographymass spectrometry (GCMS)

Time, min -->

F. Pragst, M.A. Balikova / Clinica Chimica Acta 370 (2006) 1749

3.6.3. Liquid chromatographymass spectrometry (LCMS)

compulsory in forensic cases, the guidelines refer mainly to

F. Pragst, M.A. Balikova / Clinica Chimica Acta 370 (2006) 1749

Reported concentration in hair, ng/mg

examination or validation of in-house procedures for sources of