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Toxicological hair analysis: Pre-analytical, ! The Author(s) 2018
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DOI: 10.1177/0025802418768305
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Himanshu Khajuria1 , Biswa Prakash Nayak1 and

Ashish Badiye2

Abstract
Background and aims: Hair analysis for drug detection is one of the widely accepted imperative techniques in the
field of forensic toxicology. The current study was designed to investigate the efficacy of chromatography for detection
of drugs of abuse in hair.
Method: A comprehensive review of articles from last two decades on hair analyses via PubMed and similar resources
was performed. Issues concerning collection, decontamination and analytical techniques are summarised. Physiochemical
nature of hair, mechanism of drug incorporation and its stability in hair are briefly discussed. Furthermore, various
factors affecting results and interpretation are elucidated.
Result: A hair sample is chosen over traditional biological samples such blood, urine, saliva or tissues due to its
inimitable ability to provide a longer time frame for drug detection. Its collection is almost non-invasive, less cumber-
some and does not involve any specialised training/expertise. Recent advances in analytical technology have resulted in
better sensitivity, reproducibility and accuracy, thus providing a new arena of scientific understanding and test
interpretation.
Conclusion: Though recent studies have yielded many insights into drug binding and drug incorporation in hair, the
major challenge in hair analysis lies in the interpretation of results, which may be affected by external contamination and
thus lead to false-positives. Therefore, there is a need for more sensitive and selective analysis methods to be developed
in order to minimise factors that induce the effect of melanin, age and so on, and this would certainly provide a new
dimension to hair analysis and its applications.

Keywords
Hair analysis, drug detection, dose–concentration relationship

Background
Drugs have been used and abused now for centuries. In
India, there have been three predominant reasons for
drug use: medicinal, religious and recreational. In
ancient times, drugs were primarily used either during
religious ceremonies or for treating ailments. There
were times when not only was using drugs one of the
status symbols amongst the wealthy, it was also a mode
of seeking pleasure. Drugs may modify one or more
bodily function in living organism. The number of
drug-facilitated crimes has increased in recent years.
As per the data provided by National Crime Record
Bureau (NCRB, India), the highest number of cases
pertaining to cannabis derivatives have been registered
under NDPS Act.1
Various methods have been developed to detect
prior drug abuse. Once incorporated into growing
hair, drugs can be detected long after they have been
eliminated from conventional samples such as blood
and urine.2–6 Hair samples are extensively being used
for drug detection for forensic analysis purposes, as
hair provides a longer detection window.7–13
Accordingly, hair analysis has found applications in
drug treatment programmes, workplace testing, crimi-
nal justice cases and child custody disputes.14–17
1
Amity Institute of Forensic Sciences, Amity University, India
2
Department of Forensic Science, Government Institute of Forensic
Science, India
Corresponding author:
Himanshu Khajuria, Amity Institute of Forensic Sciences, Amity
University, Sec 125, Noida-201313 U.P., India.
Email: hkhajuria@amity.edu
2 Medicine, Science and the Law 0(0)
Hair anatomy and physiology
Hair is a complex epidermal outgrowth, synthesised in
the hair follicle. It is composed of proteins (65–95%),
lipids (1–9%) and pigments (0.1–5%; melanin), as well
as small amounts of trace elements, polysaccharides
and water.18 Human hair contains two types of cells.
The cuticle is composed of overlapping scale cells, and
the cortex contains spindle-shaped cortical cells. In the
core of the cortex, there may be condensed cells form-
ing the medulla, which might be continuous or inter-
spersed with air spaces.19 The hair follicle is an
appendage of the skin that develops from the human
epidermal layer around the end of the third gestational
month in repeated cycles. Each of these cycles can be
divided into three different phases.
The anagen or growth phase
At any given time, approximately 85% of all hairs are
in the growing phase. The anagen phase can last from
two to six years. On average, a human hair grows
approximately 10 cm per annum, and it is unlikely
for an individual hair to grow >1 m in length.
The catagen or transitional phase
This phase succeeds the anagen phase in the hair-
growth cycle and lasts approximately one to two
weeks. During this phase, the hair follicle shrinks to
about one-sixth of its length, subsequently resulting
in the destruction of the lower part that leads the
dermal papilla to break away and rest below.
The telogen or resting phase
The telogen phase is the last phase of the hair-growth
cycle and lasts about five to six weeks. At any given
time, approximately 10–15% of all hairs are in this
phase. During this phase, whilst the dermal papilla
stays in a resting phase below, the hair does not grow
staying attached to the follicle. As the resting phase
approaches its end, the hair-growth cycle begins with
the anagen or growth phase once again joining the
dermal papilla and the base of the follicle, in turn lead-
ing to the formation of new hair. If the old hair has not
already been shed, the new hair pushes it out, starting
the growth cycle all over again.20,21
Drug incorporation into the hair
Over the years, several studies have been conducted to
understand the mechanism of the incorporation of
drugs into the hair from the bloodstream. However,
the mechanism of incorporation of drugs into the
hair remains unclear and requires further investiga-
tion.22–27
The following three models have been proposed to
explain the mechanism of incorporation of drugs into
the hair:
• Active or passive diffusion from the capillaries that
feed the dermal papilla,
• Diffusion from biological secretions such as sweat
that bathe the developing or developed hair or
• External drug deposition caused by vapours or pow-
ders that diffuse into the fully grown hair.
Although a combination of the above-mentioned
models may be the most realistic model for explaining
the mechanism of drug incorporation into the hair, the
relative importance of other routes or models is not yet
clear and hence cannot be dismissed.28–31
Various components of the hair have been suggested
as the potential molecular locations for the binding and
interaction of drugs. Out of these components, the ones
that have been analysed in detail to assess the binding
mechanisms are keratin and melanin. A report about
the binding of drugs to proteins was first published
more than five decades ago.32 Since then, a consider-
able amount of research has been conducted on various
drugs and their physicochemical properties to evaluate
the binding mechanism. These studies have highlighted
the efficacy of melanin as an absorber of drugs, as it
can bind to both charged and neutral species. Several in
vitro and in vivo studies have been carried out to
understand thoroughly the process of binding of a
selection of drugs and inorganic cations to
melanin.24,33,34
The process of drug–melanin surface binding was
also demonstrated by Bridelli et al.35 They investigated
the binding of three different drugs: gentamicin
(MW ¼ 462, water soluble, basic), methotrexate
(MW ¼ 454, almost insoluble in water, acidic) and
chlorpromazine (MW ¼ 319, water solubility ¼ 0.4 g/
mL, pKa 9.3).
In 1991, the scalp hair of eight Chilean mummies
with ages ranging from 2000 BC to 1500 BC were pos-
itively tested for presence of benzoylecognine in a very
stable form. Because of the exceptional stability dem-
onstrated by hair over such a longer period, its analysis
may be regarded as extremely advantageous for the
detection of various drugs.36
Collection, preparation and analysis
of hair
The most common site for collection of hair remains
the vertex of the head due to uniformity regarding
Khajuria et al.
growth, age and sex, although non-scalp hairs lack sub-
stantial growth in comparison to scalp hair and pose
challenges regarding invasiveness.37–41 Hair grows
approximately 1 cm per month. Therefore, its segmen-
tal analysis can help in detection of past exposure to
drugs, which can be used as a ‘calendar’.42–44 Hair
samples should be collected approximately one month
after exposure, especially in Drug facilitated assault
(DFA) cases.44 Sample size may vary from anything
between 50 and 200 mg of hair, which is sufficient for
screening and drug confirmation.45–47 The Society of
Hair Testing (SoHT) issued the following guidelines
and recommendations for the collection of
hair samples:48
The legal, ethical and human rights of the subject
should be respected;
• The sample should be collected by a trained individ-
ual, not necessarily a physician;
• The environment should be free from drug
contamination;
• Collection should be done from the vertex region
and close to the scalp; and
• The sample should be wrapped in aluminium foil
and stored in a dry place to avoid contamination.
The fundamental part of hair analysis, which will
significantly affect the data quality, is sample prepara-
tion. Hair analysis requires long procedures due to its
complex matrix. The preparation and analysis of hair
include the following steps.
Sample decontamination
To achieve precision and accuracy in hair testing and to
improve analytical performance, it is important to
remove the residue of hair care products, dust, oils
and lipids, sweat and so on from the hair sample by
washing.49–51 Drugs may bind to the hair or hair matrix
due to passive environment exposure, though this
depends upon the porosity of the hair.52–59
Decontamination procedures help to remove these
loosely bound drugs and avoid false-positive results.
Decontamination procedures include washing the hair
with methanol, acetone, sodium dodecyl sulphate,
dichloromethane, other organic solvents, detergents
and phosphate buffers.60
Digestion or extraction from the hair sample
Bound drugs can be extracted from the hair matrix by
three different methods (described in Table 1):
• Alkaline digestion, which involve long incubation of
samples in alkaline solutions such as sodium
3
hydroxide followed by extraction procedures such
as solid phase extraction (SPE) and liquid–liquid
extraction (LLE);
• Acid extraction, which involves long incubation of
samples in acidic solutions such as sulphuric acid
followed by extraction procedures such as SPE and
LLE; and
• Enzymatic digestion, which involves the use of
enzymes such as b-glucoronidase/arylsulfatase to
release the drug from the hair.
Some of the recently developed techniques such as
ultrasonic-assisted extraction and microwave assisted
extraction has accelerated and improved the efficiency
of hair analysis.64,75 Miniaturised techniques such as
headspace solid-phase micro extraction, hollow-fibre
liquid-phase micro-extraction and micro-extraction by
packed sorbent dramatically reduce the quantity of
organic solvents used and toxic residue generated
during the clean-up processes.3,76–78
Quantification of the various analytes
Chromatographic methods are widely accepted and fre-
quently used for hair analysis (Table 1). Various ana-
lytical methods have been developed for quantification
of opiates, amphetamines, cocaine, diazepam and
nordiazepam from hair using gas chromatography–
electron impact/mass spectrometry (GC-EI/MS).69
GC-EI/MS-based methods for simultaneous quantifi-
cation of several phenylalkylamine derivatives in hair
specimens have been developed and validated.73
Segmental hair analysis has been carried out for the
evaluation of drug abuse, including opioids, cocainics
and amphetamines using liquid chromatography atmo-
spheric pressure chemical ionisation tandem mass spec-
trometry. The investigators were able to distinguish
between the concentration and origin of heroin.62
A solid-phase micro extraction coupled with gas chro-
matography–mass spectrometry was developed to
detect long-term exposure of 17 drugs in the hair.70
Montesano et al. validated a method for screening
and quantification of 96 drugs, including opiates,
amphetamines, hallucinogens, benzodiazepines, anti-
histamines, antidepressants, antipsychotics, barbitu-
rates and other sedatives, muscle relaxants and so on
in the hair using ultra-performance liquid chromatog-
raphy–tandem mass spectrometry.61
Factors affecting hair analysis
Hair analysis has found applications in proving chronic
intoxication in an individual, helping to solve drug-
facilitated crimes and child custody cases, conducting
post-mortem drug screening, workplace drug testing
 4

Table 1. Chromatography-based methods for drug determination form hair samples.

LOD/LOQ
Washing Digestion/extraction Detection (ng/mg) Substance Ref.
61
Isopropanol, water MeOH/acetonitrile (ACN)/ammonium UPLC–ESI–MS/MS 2.0–5.0 APs, BZDs, opiates, hallucinogens,
formate pH 5.3 (overnight, 37 C) antihistamines, antidepressants,
filtration through a PTFE filter antipsychotics, barbiturates
62
n-hexane, acetone MeOH (1 h, 50 C) LC–APCI–MS/MS 0.05–2.0 MOR, COD, 6-AM, AP, MP, BZE, COE,
COC, MDA, MDMA, 3,4-methylen-
dioxyethylamphetamine (MDEA)
63
DCM MeOH, 0.1 M HCl (overnight, 45 C) Column-switching LC–ESI–MS/MS 0.012–0.05 COC, BZE, COE
64
0.1% Tween 80, water MAE with MeOH (9 min, 60 C) HPLC–DAD 0.200 COC, BZE, COE, MOR, 6-AM, COD
65
DCM (2 mL, 3 MeOH (15 h, 55 C) UHPLC–ESI–MS/MS 0.06–0.027 MOR, 6-AM, COD, AP, MP, MDA,
min, twice) MDMA, MDEA, BZE, COC, THC,
MTD, buprenorphine
66
DCM (1 mL, 3 times) MeOH (4 h, 40 C) LC–ESI–MS/MS 0.001–0.010 MOR, COD, 6-AM, COC, BZE
67
10% SDS, Micropulverized extraction with water/ HPLC–ESI–HRMS 0.030–0.150 Opiates, APs, COC, BZDs, antide-
water, acetone ACN/1 M TFA (80:10;10, v/v/v), (10 pressants, hallucinogens
min) Filtration using an Acrodisc with
GHP membrane, 13 mm disposable
syringe filter
68
0.1% SDS, water, DCM MeOH/25% NaOH (20:1), (sonicated 1 LC–ESI–MS/MS 0.1 KET, NKET
h and left at room temperature
overnight) MISPE
69
DCM, isopropa- 0.1 M HCl (overnight, 50 C) SPE GC–EI/MS 0.1–0.2 MOR, 6-AM, COD, hydrocodeine
nol, acetone (mixed-mode cation exchange, (HCOD), COC, BZE, EME, COE,
Bakerbond Narc-2) two-step deriva- DZP, nordiazepam (NDZP), MP,
tization with MBTFA and MDMA, MDA
MSTFA þ 1% TCMS (80 C, 30 min)
70
DCM MeOH (overnight, 56 C) SPE (mixed- GC–EI/MS 0.13–0.20 AP, MP, MDA, MDMA, COD, 6-AM,
mode cation exchange, Oasis MCX) COC, BZE, COE, NCOC, MTD,
Derivatization with BSTFA– oxycodone, oxymorphone, hydroco-
MSTFA þ 1% TMCS HS-SPME done, meperidine, hydromorphone
(PDMS, 100 mm)
71
Diluted soap solution MSPD-enzymatic hydrolysis, SPE GC–EI/MS 0.04–0.18 COC, BZE, COD, MOR, 6-AM
at physiological (reversed-phase cartridge, Oasis
pH, water HLB), Derivatization with
BSTFA þ 1% TMCS (20 min, 100 C)
(continued)
Medicine, Science and the Law 0(0)
Table 1. Continued
LOD/LOQ
Washing Digestion/extraction Detection (ng/mg) Substance Ref.
 7
Khajuria et al.

Water, acetone, 1 N NaOH (10 min, 95 C) LLE with n- GC–EI/MS 0.02–0.05 THCA-A, THC
petroleum ether hexane/ethyl acetate (9:1, v/v) deri-
vatisation with MSTFA
72
Water Incubation in MeOH (16 h, 38 C), SPE GC–EI/MS 0.16–0.33 Diazepam, lorazepam, midazo-
(mixed-mode, Clean Screen1), lam, zolpidem
Derivatisation with MSTFA (20
min, 90 C)
73
Water, acetone Ultrasonication in 0.25 M HCl (1 h, GC–EI/MS 0.002–0.240 AP, phentermine, MP, cathinones,
50 C), derivatization with TFAA (30 methcathinone, fenfluramine, des-
min, 70 C) methylselegiline, MDA, MDMA,
MDEA, NKET, mescaline, 4-bromo-
2,5-dimethoxyphenethylamine
74
DCM (1 mL, 3 times) 0.1 M HCl (overnight, 37 C), SPE 2D GC– TOF–MS – COD, MOR, 6-AM, AP, MP, MDA,
(mixed-mode cation exchange, Bond MDMA, MTD, BZP
Elut Certify) derivatization with
MTBSTFA þ 1% TBMCS
2D GC– TOF–MS: Two-Dimensional Gas Chromatography with Time-of-Flight Mass Spectrometric analyses; 6-AM: 6-Acetylmorphine; AP: Amphetamines; BSTFA: N,O-bis[trimethylsilyl] trifluor-
oacetamide; BZD: Benzodiazepines; BZE: Benzoylecgonine; BZP: Benzylpiperazine; COC: Cocaine; COD: Codeine; COE: Cocaethylene; DCM: Dichloromethane; DZP: Diazepam; EME: Ecgonin
Methylester; GC–EI/MS: Gas Chromatograph–Electron Impact-Mass Spectrometer; GHP: GH Polypro (GHP) Membrane; HCl: Hydrochloric Acid; HLB: Hydrophilic-Lipophilic-Balanced; HPLC–DAD:
High-Performance Liquid Chromatography with Diode-Array Detection; HPLC–ESI–HRMS: High Performance Liquid Chromatography-Electrospray Ionization-High Resolution Tandem Mass
Spectrometry; HS-SPME: Headspace Solid Phase Micro-extraction; KET: Ketamine; LC–APCI–MS/MS: Liquid Chromatography–Atmospheric Pressure Chemical Ionization-Tandem Mass Spectrometry;
LC–ESI–MS/MS: Liquid Chromatography– Electrospray Ionization -Tandem Mass Spectrometry; LLE: Liquid Liquid Extraction; LOD: Limit of Detection; LOQ: Limit of Quantification; MAE: Microwave-
Assisted Extraction; MBTFA: N-methyl-bis-triflouroacetamide; MCX: Mixed-mode Cation-eXchange; MDA: 3,4-Methylenedioxyamphetamine; MDMA: 3,4-Methylenedioxymethamphetamine; MeOH:
Methanol; MISPE: Molecularly Imprinted Solid-Phase Extraction; MOR: Morphine; MP: Mercaptopurine; MSPD: Matrix Solid Phase Dispersion; MSTFA: N-methyl-N (trimethylsilyl) trifluoroacetamide;
MTBSTFA: N-Methyl-N-tert-butyldimethylsilyltrifluoroacetamide; MTD: Methadone; NaOH: Sodium Hydroxide; NCOC: Norcocaine; NKET: Norketamine; PDMS: Polydimethylsiloxane; PTFE:
Polytetrafluoroethylene; SDS: Sodium Dodecyl Sulfate; SPE: Solid Phase Extraction; TBMCS: Tert-Butyldimethylchlorosilane; TMCS: Trimethylchlorosilane; TFA: Trifluoroacetic Acid; TFAA:
Trifluoroacetic Anhydride; THC: Cannabis; THCA-A: Tetrahydrocannabinolic Acid; TMCS: Trimethylchlorosilane; UHPLC–ESI–MS/MS: Ultrahigh Performance Liquid Chromatography Coupled with
Electrospray Ionization Tandem Mass Spectrometry; UPLC–ESI–MS/MS: Ultraperformance Liquid Chromatography Coupled with Electrospray Ionization Tandem Mass Spectrometry.
5
6 Medicine, Science and the Law 0(0)
and so on, but its scope depends upon the detection of
drugs and their metabolites followed by their quantifi-
cation in the hair matrix. Several factors influence or
affect the accumulation of drugs in the hair.74
Contamination
The availability of extensive literature on hair analysis
makes hair a non-controversial biological sample, but
the most important issue – false-positive results –
always detracts it from its benefits..41,79 There is
always a question mark on the efficiency of the de-
contamination procedure. There is no agreement on
the fact that after washing of hair, externally deposited
drugs are completely removed.47,80–84 Hair samples
have been treated with methanol and isopropanol/
phosphate buffer with three to five washes to remove
drugs externally deposited on the hair.49,85–88 Several
studies have reported that washing procedures can
remove >90% of externally deposited drugs from
hair. Therefore, the criteria for the concentration
ratio between the last wash and hair sample were
established.87,89,90
Detection of endogenous metabolites of drugs may
also minimise false-positive results.91–94 Organisations
such as SoHT and the Substance Abuse and Mental
Health Services Administration established metabolite
cut-off levels such as for a cocaine-positive sample:
COC 500 pg/mg and at least one metabolites of
COC 50 pg/mg.48,95 Drug markers were also used
to determine active and passive exposure.96
Hair colour
The mechanism of a drug binding to melanin and pheo-
melanin pigments has also been elucidated by several
studies.33,97–99 Darker hair has more melanin which
leads to a greater accumulation of drugs compared to
light hair. However, studies are still being carried out to
determine the binding mechanism between different
types of drugs (acid and base) and melanin.22,100
Researchers have not ruled out the possibility of inher-
itance playing an important role in melanin concentra-
tion which influences drug incorporation into
the hair.101
Cosmetic treatments
Cosmetic treatment history of hair should always be
taken into consideration while interpreting the results,
as studies have indicated that low concentration of
drugs may not be detected in hair that has undergone
chemical treatment, whereas studies have also reported
that chemical treatment damages the hair, making it
external drug contamination more likely.91,98,102,103
Dose–concentration relationship
Though some studies have established a weak relation-
ship between the dose and hair concentration, it is very
important to find out whether the detected quantity can
indicate the ingested amount.104,105 Hair samples col-
lected from subjects in a case-control study did not
yield significant information on dose and concentration
of opiates.106
In view of the various findings and modern techni-
ques mentioned above, hair analysis interpretation is a
very complex subject with various biases and pitfalls. A
single, unique and accurate method of interpretation is
still not available to date. There are several aspects
which may influence the detection of drugs such as
the purity of the drug, the mechanism of drug incorpo-
ration into the hair, the stability of the drug in the hair,
the frequency of abuse, cosmetic treatment, hair
colour, the section of hair tested and so on.107,108
Conclusion
The crucial part of any toxicological analysis is selec-
tion of an appropriate sample or specimen. The collec-
tion and preservation of hair samples is cost-effective,
as less training/expertise is needed in comparison to the
collection of body fluid for analysis. In conjunction
with the biological specimens, hair analysis provides
tangible information on previous drug exposure of a
subject due to a wider detection time frame and greater
stability versus body fluids or other tissues. Hair anal-
ysis has found applications in human performance tox-
icology, chronic intoxications, post-mortem toxicology,
criminal assaults to modify human behaviour, drug-
facilitated crimes, workplace testing, criminal justice
cases and child custody disputes, drug abuse in sports
and so on. Though recent studies have yielded many
insights into drug binding and drug incorporation in
the hair, the major challenge in case of hair analysis is
interpreting the results, which may be affected by exter-
nal contamination leading to false-positives. Recent
advances in analytical technology have resulted in
better sensitivity, reproducibility and accuracy, thus
providing a new arena for scientific understanding
and test interpretation. Current generations of chro-
matographic techniques such as gas chromatography–
tandem mass spectrometry and liquid
chromatography–tandem mass spectrometry technolo-
gies may be standardised for toxicological laboratories.
These improved methods will further promote the use
of hair analysis as a useful and objective tool of evi-
dence. Further research is required to develop sensitive
and selective methods of analysis that minimise factors
such as melanin, age and so on that affect the determi-
nation of drugs in hair. Moreover, studies should focus
Khajuria et al.
more on the mechanism of drug incorporation with
factors affecting it such as age, sex and so on which
will ultimately help in identifying the dose–concentra-
tion relationship. Modernisation and advancement of
analytical techniques will provide accurate detection of
drugs at lower limits, thus avoiding misinterpretations.
Furthermore, hair analysis may play a crucial role in
the examination of harmful substances not measurable
by present techniques, thus enabling its application in
large-scale testing and more general use.
Declaration of conflicting interests
The authors declared no potential conflicts of interest with
respect to the research, authorship and/or publication of
this article.
Funding
The authors received no financial support for the research,
authorship and/or publication of this article.
ORCID iD
Himanshu Khajuria http://orcid.org/0000-0002-2438-0007
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