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Cite this: Chem. Soc. Rev., 2011, 40, 3915–3940

www.rsc.org/csr CRITICAL REVIEW

Hair analysis as a biomonitor for toxicology, disease and health status
Ivan M. Kempson*a and Enzo Lombibc
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Received 19th January 2011

DOI: 10.1039/c1cs15021a

Hair analysis receives a large amount of academic and commercial interest for wide-ranging
applications. However, in many instances, especially for elemental or ‘mineral’ analysis, the degree
of success of analytical interpretation has been quite minimal with respect to the extent of such
endeavors. In this critical review we address the questions surrounding hair analysis with
specific intent of discovering what hair concentrations can actually relate to in a biogenic sense.
This is done from a chemistry perspective to explain why and how elements are incorporated
into hair and their meaning. This includes an overview of variables attributed to altering
hair concentrations, such as age, gender, melanin content, and other less reported factors.
Hair elemental concentrations are reviewed with regard to morbidity, with specific examples
of disease related effects summarized. The application of hair analysis for epidemiology and
etiology studies is enforced. A section is dedicated specifically to the area of population
studies with regards to mercury, which highlights how endogenous and exogenous incorporation
relies on species dependant metabolism and metabolic products. Many of the considerations are
relevant to other areas of interest in hair analysis, such as for drug and isotopic analysis.
Inclusion of a table of elemental concentrations in hair should act as a valuable reference
(298 references).

a
Institute of Physics, Academia Sinica, 128 Academia Road, Section 2, 1. Introduction
Nankang, Taipei 115, Taiwan. E-mail: ikempson@phys.sinica.edu.tw;
Fax: +886 (0)3 578 0281 (#3290) With increasing application and demand in medical diagnostics
b
Centre for Environmental Risk Assessment and Remediation, University and biomonitoring, interest in biological media/biopsies for
of South Australia, Building X, Mawson Lakes Campus, Mawson toxicology, epidemiology, and assessing individuals’ homeo-
Lakes, South Australia SA-5095, Australia.
E-mail: enzo.lombi@unisa.edu.au; Tel: +61 (08) 8302 6267 static state or morbidity is immense. With regard to toxicology
c
CRC CARE, P.O. Box 486, Salisbury, South Australia 5106, Australia and environmental related exposure to pollutants, biological

A. Prof. Kempson conducted Enzo Lombi is Associate

studies in Applied Physics at
the University of South
Australia before conducting
his PhD within a physical
chemistry institute (IWRI) in
the same organization. After
conducting research in Canada,
Japan and the USA, he is now
in the Institute of Physics,
Academia Sinica, Taiwan, and
proven diverse adaptability in
problem solving and analytical
approaches with emphasis on
Ivan M. Kempson organic and inorganic associa-
tions and interactions. His
current focus is on understanding such interactions for bio-
monitoring, biosensors and delivery of nanostructures in biologic
systems for a variety of applications. This has involved intense
characterization of materials with applied interpretation.
Professor and Australian
Research Council Future
Fellow at the University of
South Australia. He received
a PhD in agricultural chemis-
try from the Catholic Univer-
sity of Piacenza, Italy. Enzo
held positions at the Univer-
sity of Agricultural Science in
Vienna, at Rothamsted Research
(UK), at CSIRO Land and
Water in Adelaide and at the
University of Copenhagen.
Enzo Lombi His major research focus is
on the biogeochemistry of
trace elements with a special interest on synchrotron-based
techniques for the investigation of biological and environmental
processes. He is the current President of the International
Society of Trace Element Biogeochemistry.

This journal is c The Royal Society of Chemistry 2011 Chem. Soc. Rev., 2011, 40, 3915–3940 3915

samples are sought that provide accurate indications of con-
sumption or ingestion and metabolism of the particular xeno-
biotics. A sample providing after-the-fact analysis for
retrospective assessment is additionally attractive. For indicating
homeostasis, or deviation from, a convenient sample insensitive
to major short-term temporal fluctuations is desirable.
Successful analysis of a biopsy can assist in the diagnosis,
prognosis and treatment of disease and morbidity; indicate
diet and nutrition status which has relevance in modern-day
and archaeological contexts; and assess exposure to toxins and
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polluted environments. Currently, blood or urine sampling is
undertaken for monitoring such aspects of health. These
commonly encountered procedures are constantly being
improved and diversified with continuing research. However,
alternative media have been sought to extend beyond the
shortfalls of current sample choices to provide complementary
information or more convenient screening. In this regard, hair
analysis has the potential of offering a convenient and unique
medium for assessment of individuals and populations.
Hair has several distinct advantages and unique qualities
providing it with particular promise in a number of regards to
biomonitoring. It is well proven that hair composition is
influenced by blood composition and concentrations. This
can be taken as fact, however it is rather complicated to reveal
what an analyte concentration in hair actually means.
Extension of a measurement by way of hair analysis to actual
endogenous concentrations and biological relevance is far
from trivial. Nevertheless, hair analysis has become quite
prominent in recent years with numerous commercial services
providing promises of identifying nutritional deficiencies and
health status. The extent of commercial practices invokes
difficulty for those unfamiliar with the subject in knowing
what is factual and what could be unscrupulously driven.
Analysis of a biological specimen is complicated. A thorough
understanding of the material and the factors that influence
the specimens’ composition is critical. We attempt to highlight
the complexity and intertwined relationships associated with
hair elemental concentrations. We draw attention to a few of
the many physiological variables associated with attempts to
attribute elemental concentrations to homeostasis/morbidity,
etiology, or simply, as a bioindicator of health status. We
provide an overview of where hair analysis has been applied,
specifically for essential and non-essential elements. The non-
essential elements are encompassed by the term xenobiotics,
meaning here anything foreign to homeostatic function, with
particular emphasis on metals such as Pb, Hg, etc. Other elements
such as Ca, Cu, Zn are essential in acquiring homeostasis, but are
perturbed due to the effects of disease and other potentially
detrimental abnormalities in the biological system. Due to the
prevalence of publications regarding mercury in hair and the
success in regards to research assessing mercuric species, we have
dedicated a section specifically discussing this metal.
The review contained herein has been motivated by the
unique properties offered by hair for analysis of incorporated
elemental species and has been written with the chemistry of
hair and elements in mind. The sheer multitude of publications
regarding hair analysis means only a fraction of publications
can be referenced here. A brief description of hair structure
and growth is provided and an explanation of the advantages
3916 Chem. Soc. Rev., 2011, 40, 3915–3940
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of hair analysis, followed by a concise historical perspective
leading into the current status. What this review then attempts
is to summarise how and why metals’ and other elements’ fate
includes deposition into hair from systemic concentrations.
Hair is a complex medium and many of the variables impacting
these concentrations are summarised to provide greater awareness
of complicating considerations. These are presented with the
intention of providing factors to which a hair concentration
may need normalisation, to enable ascribing a meaningful
interpretation of the measurement. Basically, the fundamental
question with regard to measuring a concentration in hair is:
‘‘what does it mean?’’. This is really a multi-part question:
Firstly, why does a hair concentration relate to a blood
concentration, if it even does at all? Secondly, what then
would the blood concentration relate to? And subsequently,
how should this be interpreted with respect to a population, or
to an individual? The sections regarding the variables in hair
should be considered in any pursuit to deliver meaningful
interpretation of hair analysis with respect to elemental
analyses. These typically can also be extended to other areas
of hair analysis, such as for drug screening. Included is a
discussion of exogenous contamination. A major hurdle in
hair analysis is the decontamination, or washing, step to
remove the exogenous material.
After covering these aspects, different approaches to hair
analysis are covered in two different sections with regard to
more fundamental work utilising spatially resolved analysis
and highly applied bulk analysis (in particular for large
population studies). Included in the review of spatially
resolved analysis is the ability to perform longitudinal, or
temporal, analysis of individual hair strands, demonstrating a
distinct advantage of hair. The bulk analysis is primarily
discussed with regard to large population studies in discriminating
groups of people. A specific section is included here dedicated
to the analysis of mercury in hair. This topic has received a
large amount of attention and warrants a dedicated section.
The section on Hg is included as a mini-review within itself. The
review finishes with a final discussion of the status of hair analysis
in light of the reviewed material, the areas of most promising
application and possible future directions. Included within this
review are tables summarising concentrations of elements
determined in hair from the literature. These tables are provided
to serve as a reference for typical and atypical results from various
population groups, and should serve as a valuable resource.
1.1 Historical perspective
In 1966 a manuscript titled ‘‘Analysis of zinc levels in hair for
the diagnosis of zinc deficiency in man’’1 was published.
This was by no means the first proposed measurement of
nutritional elements in hair, but it did highlight the potential
for application as a biomonitor. In addition, the authors were
already mindful of variables impacting hair concentrations,
such as season. Since this time, many analytical methods have
been developed and optimised with regard to hair analysis and
applied to biomonitoring, disease detection, anthropological
studies, toxicology and so forth. Proficiency tests in 1997 by
the Society of Hair Testing demonstrated unsatisfactory
quality control between laboratories.2
This journal is c The Royal Society of Chemistry 2011

Numerous correlations with hair elemental concentrations
have been discovered since then. The application for isotopic
analysis has been quite successful, in particular for dietary
studies in archaeological contexts. Analysis for drug abuse in
the general community and dopants in the sporting community
has also begun to reach a general level of acceptance (however
some hurdles still remain). For organic xenobiotics, analysis
has benefited from the detection of metabolites and their ratios
to confirm consumption and relate to pharmacokinetics. For
elemental levels however, very little progress has been achieved
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in confirming ingestion and interpretation severely suffers
from exogenous contamination. The initial lure of hair analysis
has been to provide diagnostics for individuals; however
greater application in recent years has been in larger population
studies with regard to epidemiology and etiology, and even
animal populations at risk.3
1.2 Current status
It has been convincingly shown that any element and almost
any xenobiotic imaginable can be detected and quantitatively
measured in hair. Credibility for hair analysis is becoming
more apparent, in particular for drug and doping investigations.
Despite this, hair analysis undergoes considerable criticism
with regard to elemental analysis (see section on commercial
analysis below). This is due to the fact that such measurements
generally can’t be interpreted in a meaningful and consistent
manner. There remain many variables4,5 that appear to influence
concentrations in hair that have not been accurately
described.3 Sadly, many publications only provide incrementally
more information than was obtained in previous decades, i.e.
yes, differences in elemental concentrations in hair can be
observed between individuals or populations. Few publications
grapple with the fundamental incorporation mechanisms, or
to implement screening protocols, or provide epidemiological
and etiological information with regard to risk factors and
disease.
A quantitative measurement from a hair sample does not
represent a quantitative measurement of an ingested dose.
Many authors have found hair concentrations vary in a dose-
dependent manner, however too many variables impart greater
complexity to relate to blood concentrations. Incorporation
depends on many factors (discussed below). Other barriers
also need to be overcome by understanding incorporation
mechanisms, dose concentration relationships, hair treatment
effects, exogenous discrimination etc. A number of these
factors have previously been summarised4 and will also be
discussed further here. Many of these concerns can be argued
to originate from poor understanding of the incorporation
mechanisms and how substances are biogenically and/or
diagenetically bound into hair. Particularly in trace element
research, the role of exogenous contaminants is poorly
understood. Contamination, if present, must be discriminated
before analysis by conventional bulk methods. A multitude of
washing methods have been suggested, but it remains to be
seen whether any of these options remove all and only
exogenous contaminants.6,7 Again, this problem was identified
decades ago and little has progressed considering the
time span.
This journal is c The Royal Society of Chemistry 2011
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Techniques applied often involve the destruction of the hair
sample by way of digestion, and tend to require a relatively
large sample size severely limiting the identification of localised
concentrations. These bulk methodologies are rarely applied
to understanding specific details regarding localisation, binding
and incorporation or even loss from a hair sample. Microprobe
techniques have contributed further to a thorough under-
standing. Publications arising in the previous decade
have proven the value of microprobe analysis to assist in
understanding elemental localisations and longitudinal
variation.8–10
Many papers over recent years are reiterative of the areas
where hair analysis distinguishes population groups based on
elemental concentrations. What is required now is a collation
of the current knowledge and a fundamental enhancement in
understanding what these data mean and how they can be
practically applied. To apply hair analysis, the fundamental
incorporation must be understood, discrimination from
exogenous sources achieved and ideally a normalised process
relating hair concentrations to blood concentrations.
1.3 Commercial analysis
Despite the complexity involved in the interpretation of
elemental hair analysis, commercial providers are prevalent
and appear quite popular to the general public. Two particular
publications have assessed several commercial providers and
are summarised below.
Based on the study by Drasch and Roider,11 not only can
measurements and recommendations vary from one service
provider to the next, but even from the same provider from the
same samples at different times. Such poor quality control
with time appeared to be common amongst commercial
providers. They stated: ‘‘Hair mineral analysis from these
laboratories is unreliable. Therefore we must recommend to
refrain from using such analysis to assess individual
nutritional status or suspected environmental exposure’’. Very
little consensus was found between 7 laboratories. For one
volunteers’ samples, only 2 out of 23 elements were within
agreement between laboratories.
An earlier assessment of 6 laboratories by Seidel et al.12
found a similar conclusion: ‘‘Hair mineral analysis from these
laboratories was unreliable, and we recommend that health
care practitioners refrain from using such analyses to assess
individual nutritional status or suspected environmental
exposures’’. Normal reference ranges were highly variable,
nearly all elements were categorised differently and
recommendations were contradictory, as too the suggested
courses of action. Three laboratories offered proprietary
products for extra expense.
2. Why hair?
There is intense interest in conventional and unconventional
biosamples to assess homeostasis and abnormal exposure to
pollutants or toxins.13 Typically, urine and blood are the
samples of choice. They are well understood and routine
analysis generally offers reliable results. Urinalysis is a well-
known and practiced technique that can be used for the
diagnosis of a variety of diseases. For analysis of acute
Chem. Soc. Rev., 2011, 40, 3915–3940 3917

exposure, urine generally only has a window of detection of up
to around 36 to 72 hours. Concentrations vary with hydration
of the individual and vary widely with dose and time. Its
complement with hair analysis, however, has interesting
potential.14
Blood analysis similarly can be routinely used for monitoring
of essential and non-essential elements, but again, concentrations
can decline rapidly in cases of acute exposure. It is invasive
with a risk of disease transmission and requires experienced
personnel for obtaining and handling samples (adding to
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expense). As with urinalysis it is most useful for very recent
and/or chronic exposure.
Toe and finger nails also have potential application. They
contain no melanin which simplifies interpretation but grow in
thickness and length at different stages of the growth cycle.
Therefore they reveal limited temporal information and also
suffer significantly from contamination. Nails have a poorly
defined ‘‘window’’ of analysis and may incorporate lower
concentrations than hair.15
Deciduous teeth can be informative of Pb exposure in
children and offer promise for general screening, but cannot
be collected as desired from any individual.16 A review of the
application of biomonitoring with various matrices has been
conducted for a particularly large population and over a long
time scale by Wilhelm et al.17 Other more specific reviews
include different media in relation to maternal/fetal exposure
highlighting various advantages and disadvantages,18 and for
Cd and Pb exposure.19
Human hair typically provides a good source of tissue
samples, being easy to obtain, is robust and sample storage
and preparation for analysis is simplified since no special
preservation techniques are required. The nutritional source
of growing hair is the blood supply, which contains traces of
anything ingested by the individual. Any xenobiotics and their
metabolites can become incorporated into the growing hair
matrix creating a time resolved profile. Hair analysis is commonly
utilised in the study of nutrition,20 dietary habits,21 exposure
to toxins,22,23 and drug abuse as evidenced by the numerous
publications in circulation. In many studies, hair has been a
medium of choice due to its representation of short- to mid-term
history prior to sampling.24,25 Hair analysis offers particular
advantages over urinalysis26 including a larger window of
detection (greater than 1–3 days), ease of collection and
stability of sample. A superior aspect of hair analysis is its
ability to provide a historical profile of the exposure to
xenobiotics which can provide particular interest for acute
exposures. No other medium provides information regarding a
period of time which hair represents. Biological fluids are only
suitable for short term representation, generally on the order
of hours to days. Bone sits at the other end of the spectrum
when used for the study of trace elements for it represents a
collective average of the prior ten or so years. Hair, depending
on length, may represent a period from a few days to months
or even several years. Hair analysis may miss consumption
within the previous few days before collection however, unless
the hair is plucked to include the region below the skin, but
this encounters difficulties in collection, especially if a large
number of hairs are needed. Otherwise, sufficient time is
required for the hair to emerge from the skin.
3918 Chem. Soc. Rev., 2011, 40, 3915–3940
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Biological samples, such as hair, that persist with time, while
others may degrade and decay, also offer superiority in the
anthropological study of ancient individuals. Hair analysis
potentially offers a number of analytical advantages over
conventional biological specimens. However, it suffers from
many complicating factors described below.
2.1 You are what you eat
The old adage of ‘‘you are what you eat’’ is especially
exemplified by the use of isotopic analysis of hair which can
provide an isotopic fingerprint of quite specific geographical
origin based on hair composition.27–29 Similarly seasonal
fluctuations are also apparent from longitudinal analysis,30
as well as geographical movements.31,32 These observations
stem from the isotopic variation in water sources and dietary
intake. This also applies for non-essential elements such as
inhaled particulate material (Be)33 and exposure to Pb (with
subtle correlations to blood concentrations).34 These elements
undergo incorporation via normal transport pathways at the
cellular level (for essential elements) and also via inhibitive or
mimicry properties of non-essential elements.
For essential elements, metals such as Ca, Cu and Zn are
vital at certain concentrations in the activity of proteins and
the like for cellular function, growth and division. Often these
metals play roles as catalysts in promoting energetically
favourable biological processes. Deficiencies in these elements
(representing poor nutrition, metabolic dysfunction or effects
of disease for example) are represented in blood concentrations,
indicating that insufficient quantities are being transported for
homeostasis. Following from this, cellular concentrations are
affected, and hence hair concentrations. Heavy metals have the
propensity to competitively complex with proteins such as
enzymes and become incorporated into cells through the
membrane. While positive correlations between hair and
blood concentrations exist, for Pb in one example,35 this is
not always observed.16 This can be attributed to the many
variables involved in the incorporation and different time
periods represented by each medium.
2.2 Why would hair reflect disease?
Here we provide a very brief introduction into some effects of
disease and effects of elements on homeostasis to give some
basic understanding of how hair concentrations can be linked
with biological processes. Blood concentrations of elements
can be correlated with morbidity, such as Mg with depression36
and Cd with hypertension.37 It is not always clear if these are
due to cause or effects however. Metals are involved in roles
associated with disease prevalence and progression, such as
competing roles of Se and Pb in mammary tumour development.38
Additionally, Cu and Zn play vital roles in carcinogenesis.39
Similarly altered Zn concentrations could have impact on
atherosclerosis by way of impacting on cellular regulation
and expression.40
The transport protein transthyretin and its transmembrane
receptor protein, Megalin, are multiligand endocytic receptors.
Megalin is expressed within the hair follicle. During the hair
cycle it is involved in mediating hair follicle signalling, the
degree of which varies over the cycle course and is also the case
This journal is c The Royal Society of Chemistry 2011

for transthyretin.41 Transthyretin has the capacity to bind with
Zn,42 thus could impact on cellular concentrations. While it
has not been shown that these proteins directly interact in the
accumulation of metals in hair, they help to demonstrate
the complexity of the follicle biology and reveal possible
mechanisms that could impart roles on the incorporation of
metals for purposes of hair analysis.
Subsequent to such biogenic function, various biomarkers
can be identified with analysis of hair.43 There are many
dependencies of homeostasis and morbidity upon essential
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and non-essential elements in human biology. The cellular
expression of proteins can be up- or down-regulated as a result
of disease. Of particular relevance can be changes in metallo-
protein expression with disease44 which can have severe
implications on metal transport. Within the follicle, changes
in cellular expression and demands on nutrition will also vary
due to the hair growth cycle,45 other hormonal changes and in
hair associated growth factors. All of these have dependencies
or could affect essential and non-essential element
concentrations.
Not only can elemental levels be perturbed due to a morbid
state, but their concentrations themselves can be linked with
the cause of morbidity such as in the case of various toxic
metals. For example, there is evidence of arsenic exposure
inducing impaired glucose tolerance.46 Obviously in this case,
As is most heavily linked to the etiology rather than vice versa
(i.e. the glucose intolerance would not cause a bioaccumulation
of As in the hair). Cellular dysfunction is induced by oxidative
stress or interference in signalling or gene expression due to the
As or its metabolites.47 Inter-personal differences could have a
significant impact.48 Needless to say, the impacts of the
glucose intolerance may have other compounding effects on
the transport and incorporation of other elements and
homeostatic cellular growth and expression. Correspondingly,
it is known that As metabolism, transport, excretion and
pathology are highly complex and far reaching within human
biology.49 Complexes in the body can create competitive
mechanisms of exclusion of other essential elements.
These too may then be reflected in the hair as decreased
concentrations.
3. Hair
3.1 Composition and structure
The composition and morphology of hair, particularly within
the follicle, is quite complex.50,51 While a genetic remnant of
history offering protective function and minor advantages for
homeostatic health,52 it is of major social and cultural
importance. Its composition is quite variable from one individual
to another. As a guide, the following figures have been
published:4 fibrous proteins which are mostly alpha keratins
(85–93%), melanins (complex eumelanin and pheomelanin
polymers derived from tyrosine), water (typically 3–5%, but
up to 15% by mass),53 lipids (1–9%) and mineral compounds
(0.25–0.95%). Typical levels of the constituents of hair are
C (45.7%), O (28.2%), N (13.6%), H (7.5%), S (4.1%) and Ca
(0.3%) resulting in a structure with a density of approximately
1.47 g cm 3.54 Hair colouration is derived from the melanin
This journal is c The Royal Society of Chemistry 2011
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pigmentation that can be eumelanin in black hair, or the
reddish/brown pheomelanin which can encompass different
pigment structures.55
The protein is composed of amino acids, 21 of which have
been reported in human hair. The amino acid content of hair
varies with the structural components of the hair and is
influenced by genetics, weathering, cosmetic treatment and
diet. Water content is largely influenced by the relative
humidity. Lipids in hair come principally from sebum and
consist of free fatty acids and neutral fat (esters, glyceryl, wax,
hydrocarbons and alcohols). The chemical structures within
hair create unique physical characteristics. Strong disulfide
bonds linking adjacent keratin chains produce a structure
significantly resistant to chemical and biological degradation.
The structure and chemical characteristics of the protein
(covalently linked amino acids) are determined by the
sequence, number and chemical nature of its constituent
amino acids. Condensation reactions between a-NH3+ and
a-COO terminal groups of amino acids lead to covalently
bound peptide chains. The resulting sequence of main chain
atoms is characterised by a –Ca–C–N–Ca– repetition. Each
Ca has a side chain functional group. In an a-keratin, the
favourable structure is that of a helical chain. The polypeptide
backbone follows a right-handed helical spring. Each carbonyl
group forms a hydrogen bond with the amide group of the
residue four amino acid units further along the chain. Each
3601 turn incorporates 3.6 amino acids and advances by
5.6 angstroms in the helical direction. Aligned dipoles form
hydrogen bonds and the helix radius dimension allows for Van
der Waals attraction across the helix.56 The amino acid side
chains are arranged in a spiral around the polypeptide, hence
when alpha helices bind together with two or three units they
form a strong, compact left handed twist. These fibrous
structures are composed of individual polypeptides laterally
cross-linked by several types of bonding. Because of their
structure, they are physically tough and water insoluble. The
basic structural unit of a-keratin usually consists of three
right-handed a-helical polypeptides in a left-handed coil that
is stabilised by cross-linking disulfide bonds. The formation of
a ‘cable’ with twisted alpha helices results from optimisation of
packing among the amino acid residue side chains. The thiol
(–SH) group of cysteine is highly reactive and will readily
oxidize to covalently bind with another cysteine residue to
form a disulfide bond (–S–S–), forming cystine. Such bonds
are a major contributor to the rigidity, mechanical properties
and tertiary structure of the protein. The individual alpha
helices covalently bind with a high level of cross-linking by
disulfide bonds between adjacent polypeptide chains. In hair,
these are organised into microfibrils of about 70 angstroms in
diameter. The microfibrils are embedded in a matrix of an
amorphous substance containing sulfur rich proteins to form a
macrofibrillar structure. Four type I acidic keratins and four
type II basic keratins are unique to living hair-forming cells
(trichocytes), mainly in the medulla and cortex.57–59
The following description of the hair structure is based on
the work of Swift60 and the references therein. The cuticle
layer (outer region of a mature hair) of the hair shaft is
comprised of overlapping cuticle cells with a total thickness
of roughly 5 mm but varies considerably. Initially there may be
Chem. Soc. Rev., 2011, 40, 3915–3940 3919

9 or 10 layers of cuticle cells but these are typically eroded
moving distally. Cuticle cells are laminated, roughly rounded
square shapes with sides of 50 mm and a thickness of 0.3 to
0.5 mm. Atomic force microscope studies also show cuticle step
heights to generally range between 0.3 and 0.5 mm with large
variations in an individual hair.61 The outer facing layer of the
cell is the A-layer which is approximately 110 nm of mechanically
tough and chemical resistant protein. Its strength originates
from isopeptide cross-linking and a high cystine content
(B11.8% sulfur). Inside from the A-layer is the exocuticle,
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accounting for about 50% of the cell volume. This region is
also of high cystine content but not to the extent of the
A-layer. Below this is the endocuticle which accounts for the
bulk of the remaining cell volume. This region is void of
cystine but possesses abundant free amino and carboxyl
groups. This may be because the proteins in this region are
of low molecular weight, or comprise high concentrations of
residues with acidic and basic functional groups. The inner-
facing surface of the cuticle cell is a layer of around 20 nm
thickness of similar composition to that of the exocuticle.
Inside the cuticle layer, the bulk of the hair volume is
constructed from cortical cells. Cortical cells are spindle-
shaped, 50–100 mm long and around 3–6 mm in diameter.
Centrally within the cortical cell exists the nuclear remnant,
which is typically around 40 mm long and 0.5 to 1 mm in
diameter. The nuclear remnant does not possess any cystine
and is rich in amino and carboxyl functional groups. The bulk
of the volume within a cortical cell is comprised of long
cystine-containing macrofibrils, each with a length of between
40 and 200 nm in diameter. Each of these macrofibrils are in
turn compiled from numerous microfibrils. The packing of the
macrofibrils within a cortex cell gives rise to one of two
different cortical cells, either paracortical or orthocortical.
Each of these contains similar levels of cysteine in human
hair. The microfibrils are a-helix proteins cemented together
by the intermicrofibrillar matrix comprising a random coiled
protein of high cysteine content.
At the centre of the hair shaft there may exist a medulla
where the structure becomes denatured and can consist of
vacant, air filled spaces. There is a coating on the hair of lipids
generated in the sebaceous glands.62 The majority are fatty
acids, in particular 18-methyl-eicosanoic acid comprising 40%
of the fatty acid content (4.3 mg g 1 hair). Other lipids include
cholesterol sulfate (3.3 mg g 1 hair), and to a lesser extent
cholesterol and fatty alcohol. All cell surfaces are covered in a
lipid layer covalently bound to the protein.
3.2 Growth
The shaft of a hair is formed within a hair follicle (simply
defined as a depression in the skin layers), within the pilose-
baceous unit. Hair growth occurs in a series of growing and
resting phases. During the growth (anagen) stage, the matrix
cells differentiate, keratinise and die. This occurs for 2–6 years
before going through a catagen stage of 2–3 weeks and resting
(telogen) stage for about 3 months after which it either falls
out or is forced out by the reinitiation of the growth cycle. At
any time, approximately 85% of scalp hairs are in the growth
stage. It is generally assumed that a delay of 2–4 weeks occurs
3920 Chem. Soc. Rev., 2011, 40, 3915–3940
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between the incorporation in hair and hair emergence from the
skin considering a growth rate of approximately 1 cm per month
(e.g. Myers et al.).63
Nutrition is supplied to the growing hair via the blood in the
dermal papilla. At the crest of the dermal papilla are melano-
cytes, which produce melanin granules that subsequently
become incorporated into the hair. As the keratinocyte cells
continue to grow and divide, the cylindrical arrangement is
forced towards the opening of the follicle. Within the follicle,
the hair shaft is constructed from a number of layers: from the
medulla moving radially outwards there exists the hair cortex,
hair cuticle, inner root sheath cuticle, inner root sheath Huxley
layer, inner root sheath Henle layer, outer root sheath,
basement membrane and the dermal sheath. Cells associated
with the development of hair are among the fastest dividing
cells in the body (dividing approximately every 24 hours).
Compounds are reportedly associated with the forming
fibrils in the dermal papilla within 5 minutes of systemic
administration.64 100–1000 cells an hour move from the
undifferentiated stage to the differentiated stage. A technical
review summarising hair growth factors has been presented by
Peus et al. and is recommended for a highly detailed account.65
Additionally, in vitro studies aid in understanding of growth
factors and the formation of the hair shaft.66
Sebaceous gland openings within the follicle excrete sebum
(a waxy lipid material) within the hair follicle and occasionally
to the surface of the skin, thus the hair is coated by this
material. Approximately one third of sebum comprises free
fatty acids, another of combined fatty acids and the last third,
unsaponifiable matter such as squalene, cholesterol and
waxes.67
4. Considerations and variables in hair analysis
Due to the nature of hair, in that it has highly inter-individual
chemical variability, it has significant exposure to the environment
and undergoes numerous treatments for purposes of hygiene
and cosmetic appeal, it is no surprise there exist many variables
that affect hair concentrations. In some instances, such as
dying, hair can offer little credibility. In the best case scenarios
there are still many variables that should be kept in mind when
interpreting hair analysis. This section highlights considerations
that may affect hair concentrations and highlights the
complexity in attempting to infer blood concentrations from
hair concentrations. References specifically regarding Hg with
respect to these factors are confined to the section dedicated to
Hg later in this review.
4.1 Colour and melanin
About the most dramatic difference in hair is the variability in
colour due to the melanin content which can affect elemental
composition of hair.68 Due to the massive variability in hair
based on colour, some extra consideration is included here.
Melanin is synthesized in melanocytes (dendritic cells) at the
crest of the papilla and is affected by hormones and local tissue
growth factors. Melanins (eumelanin and pheomelanin) are
polyanionic polymers which possess a high number of charged
carboxyl groups and o-semiquinones among the keratin fibrils.69,70
Levels of eumelanin and pheomelanin determine hair colour.
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Melanin concentrations may be quite different although two hairs
appear to be of the same colour.71 Their structure and synthesis
have been described in detail elsewhere.71 Eumelanin (a brown/
black polymer of dihydroxyindole subunits) is synthesized from
the precursor amino acid tyrosine, has a quinone like structure and
is highly anionic. Ito summarised that ‘eumelanins have the
properties of polyanions and may, in vivo and in vitro, bind with
various cations such as di- and tri-valent metal ions, polyamines,
and anionic drugs.’69
Pheomelanin (a reddish-yellow pigment) is also synthesized
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from the precursor tyrosine. Incorporation of cysteine results
in benzothiazine derivatives that polymerize to pheomelanin.
Pheomelanin possesses free carboxyl and amine groups
suggesting electrostatic forces or hydrogen bonding could
retain certain xenobiotics.
Melanins have been shown to influence elemental concentrations
in hair, however many fundamental studies regarding the
incorporation have revolved around analysis for drugs and
are used here to highlight considerations relevant to the
binding of elemental species. Organic molecules often show
that black or dark hair binds larger quantities than light or
white coloured hair even though blood plasma levels were the
same in the original subjects,72,73 eumelanin can be of greater
importance for binding xenobiotics than pheomelanin.71,74
Studies into the effect of melanin content as binding sites in
hair have long been investigated. Experiments from the 1970’s
studied the incorporation of D-amphetamine, L-dopa,
L-a-methyldopa and DL-isoproterenol in pigmented and non-
pigmented guinea pig hair.75 Drugs access melanocytes via
arteriovenous and lymphatic circulations.76 The drug molecules
enter the melanosomes interacting with melanin and melano-
somal protein and become incorporated into the hair pigment
granules. A number of other studies have investigated the
affinity of drugs for melanin, including studies into the
competition between drugs for the same binding sites. For
example, quantification of the capacity and affinity of a variety
of drugs for synthetic levodopa melanin have been
presented,77 and fastest association has been by the most
lipophilic compound.78
Xenobiotics with cationic properties appear to be bound to
melanin by electrostatic forces between the positively charged
form and anionic sites on the melanin polymer.79 For organic
molecules, ionic bonding may be strengthened by non-electrostatic
contributions, such as van der Waal’s forces and interactions
with the aromatic indole nuclei of the melanin. The binding is
typically complex with Scatchard analyses showing that more
than one type of binding is involved. Carboxyl, quinol and
amine groups are all possible binding sites.70 Codeine and
morphine can deposit in both pigmented and non-pigmented
hair, however fail to bind in non-pigmented hair and diffuse
out.80 Two binding sites were proposed in black rat hair, but
only one in brown. Differences in melanin content based on
gender may also impart variability.81,82 Bleaching samples of
Africoid and Caucasoid hair dramatically reduced the binding
capacity of cocaine but had little effect on blonde hair.82 Other
drugs however appear to have no dependence on melanin
content.83,84
In cross-sectional elemental imaging of hair, there is strong
association of various elements with melanin granules.
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This has been shown with high resolution images by SIMS85
and X-ray fluorescence spectroscopy.86 P, Fe, Al, Mn and Cr have
been quantitatively shown to vary in hair based on colour.68
4.2 Contamination and natural deterioration
Hair is subject to numerous external sources of contamination,
intentionally and unintentionally. Atmospheric dust and
pollution as well as water borne content are particularly
important considerations. In many case studies examining
hair concentrations, the analyte being tested with regard to
exposure is the same that can contribute most to contamination.
For example, investigating hair for biomonitoring of smelter
workers to test for workplace exposure, the same atmospheric
route of exposure can deposit as contamination on hair. In the
case of smelter workers, SEM coupled with EDX revealed
deposition of particulate gold, lead and steel onto hair.86
Mass spectrometric imaging also revealed non-particulate
deposition of Pb. Similarly, in cases of using hair to test for
exposure to arsenic polluted drinking water where the same
water is used for washing, contamination should be considered
as problematic.87 Tap water can contain many other metals
and other elements, particularly copper and calcium. Lead can
also be problematic in the case of lead plumbing.
The external surface of hair has distinct regions of different
chemical composition and can accumulate exogenous
products differently.88 Topically applied material can readily
penetrate hair.89 Longitudinal analysis of hair in fundamental
studies with regard to the impact of contamination reveals
significant penetration of contaminants into hair.90 In one
study, hair was sectioned longitudinally and the internal
longitudinal distribution was compared to the external
distribution. Contaminants increased on the outer surface of the
hair and within the internal structure moving longitudinally.91
The follicle also has a degree of permeability which has
been described with regards to pharmaceuticals92 highlighting
an additional route for exogenous material to enter the
pilosebaceous unit.
Hair undergoes natural deterioration with loss of its
mechanical and chemical protective integrity, such as commonly
seen with split ends and increasing fragility of long hair. A
particularly interesting publication in this regard is that by
Thibaut et al. in the rather unique study of hair 2.4 m long that
had not been exposed to chemical treatments.93 Deterioration
obviously will increase as a function of distance away from the
scalp, i.e. time exposed to the external environment. This
begins with mechanical deterioration by abrading of the outer
cuticle and following loss of the protective lipids, in particular
18-methyl eicosanoic acid. This is followed by progressive loss
of hair keratin proteins. As such mechanical strength decreases
longitudinally as with hydrophobicity and shine. It can be
expected that elemental concentrations will be affected by
these chemical and physical changes.
Particularly in archaeological contexts, a variety of processes
can occur that act to detract from the informative value of hair
analysis as a result of fungus, bacteria, chemical degradation
and even mineralisation.94–96 Burial environments can have a
massive impact of hair composition and should be assessed in
hand with hair analysis to aid interpretation.97
Chem. Soc. Rev., 2011, 40, 3915–3940 3921

4.3 UV-degradation
UV light has the ability to severely damage hair structure and
integrity. It is involved in the damage of both the keratin
structure and the melanin pigment molecules.98 Certain
amino-acid residues demonstrate photosensitivity and have
been shown to lead to cross-linking of protein fragments with
amorphous domains of the hair.99 White, brown, red and
blonde hair have all been shown to undergo colour change
after radiation exposure.100 UV degrades melanin, while infra-
red wavelengths can act to degrade tryptophan which also
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alters hair colour, thus white hair undergoes yellowing. Such
alteration has been shown to significantly alter elemental
composition in wool fibers of sheep,101 and could contribute
to the longitudinal variation observed for many elemental
concentrations.91
4.4 Hair treatments
Cosmetic treatments offer a significant source of contamination.
Henna dyes can all but rule out meaningful analysis, similarly
for many other dyes,102 especially of a mineral base. Typically
dyed hair should be omitted from serious consideration.
Shampoos can have several inorganic ingredients, in particular
selenium and zinc in anti-dandruff formulations. Hair colour-
ing can have a number of chemical and compositional effects,
such as for Cu and Ca.103 Bleaching can have quite dramatic
effects on elemental compositions, as demonstrated in a
longitudinal analysis of individual hairs traversing bleached
to untreated regions of hair.91 The disulfide bonds are
destroyed in the evolution of cysteic acid and cortical proteins
also convert to a random coil structure.104 Interestingly, the
disulfide bond cleavage is accelerated for melanin-rich hair
which may act to compound any influence on elemental
incorporation or loss, as it becomes increasingly vulnerable
to external aqueous solutions.105
4.5 Curly or straight
While there do not appear to be published data regarding the
impact of hair curliness on elemental concentrations, it is
worth consideration and may contribute to some perceived
racial differences. Hair that is curly or straight may bind
metals differently. Genetic differences lead to differences in
hair structure.106 Additionally, altered expression of a growth
factor binding protein (IGFBP-5) impacts expression of several
extracellular matrix proteins and disruption of adhesional
junctions, leading to differences in the hair morphology leading
to curliness.107 In this case, if there are changes in cellular
expressions related to hair shape, then there is potential that
curly hair has different capacity to bind metals than
straight hair.
4.6 Age
With age various changes such as hormonal and steroidal
levels change, as too with numerous other proteins and
physiological processes. These changes can influence blood
concentrations and thus hair concentrations. In addition,
changes at the dermal papilla can also induce variation in hair
composition. Age-related oxidative stress affects melanin
production, ultimately seen as greying. Many melanin associations
3922 Chem. Soc. Rev., 2011, 40, 3915–3940
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in hair have been reported for metals, metalloids, drugs and
organo-pollutants (see above). Animal studies have been used
to monitor changes in biomarkers with age.43
For children under 4 years old, Zn concentrations were
significantly lower and the rate of low Zn concentrations was
increased in one study.108 Other authors have reported Ca
decreasing with age,109 Ca, Mg and Fe decreasing with age,110
and for a Korean population of children, Zn, Ca, Na, P, Mn,
and Li were positively correlated with age, while Cr, V and
U were negatively correlated.111
4.7 Gender
Several publications have identified differences in elemental
concentrations in hair based on gender.112 For example, Ca
and Mg were higher in females.109 However, Cu and Mg were
lower in girls suffering from Autism as opposed to boys.113 In
other studies, Zn, Na and Pb were higher in boys, while Fe and
Bi were higher for girls.111 Au, Ca, Mg and Sr were higher
while Cl, Fe, I, Sc, Se, and Sm were lower in females’ hair
compared to males’.114 Other correlations with age for Ce, La
and Th increasing with age were only significant for females
and not males. These results highlight discrimination based on
gender and should be considered to impact hair
concentrations.
4.8 Ethnicity
On the basis of structural differences hair is usually categorised
into three ethnic groups (African, Asian and Caucasian).
There are obvious differences between hair of different ethnic
origins. Colour and hence melanin content is highly variable,
but there are also differences specific to structure, such as the
thickness and composition. Some differences may exist due to
hair shape (see section above on straight and curly hair), but
other differences also exist. Rhodamine B has been shown to
rapidly incorporate into extensively bleached hair, followed
(less dramatically) by Chinese hair, African hair, and Caucasian
blonde hair demonstrating differences in permeability.115
However it is also important to discriminate between what is
actually an ethnic difference rather than a related cultural
difference.116 Quite simply, different cultures can have
different life-style habits with regards to washing, cosmetic
treatments and diet. African hair may have a much larger
permeability to environmental exposure due to a greater
proportion of amorphous regions.105 Another study compared
Korean hair to Caucasian hair, showing a greater number
density of cuticle scales and a greater number of cuticle cell
layers in the Asian hair.117 These factors add variation in
relative and total amounts of particular proteins and the
accessibility of the internal structure to contamination.
4.9 Personal habits and seasonal fluctuations
Personal use and frequency of use of shampoos and
conditioners are an obvious inter-individual difference. However
other factors can also have impact. Smoking has been identified
to actually restrict the ingestion of essential minerals and lead
to a decrease in Ca, Mg, Fe, Zn, and Cu.110 Some personal
habits such as having ‘‘hair painted’’ obviously dismisses an
individual from being accurately assessed by hair analysis.118
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Season can also impact hair concentrations as previously
mentioned to be observed as early as the 1960’s.1 Particularly
in non-tropical regions this may have a reliance on changes in
personal activity with season, sunlight exposure and especially
dietary change.
4.10 Growth/cycle stage
Hair growth is a cyclic phenomenon based on changes in
hormone levels, local regulation of growth factors and ultimately
cellular activity. During these different stages, it is foreseeable
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that individual hairs will undergo changes in their ability and
capacity to incorporate essential and non-essential elements.
For example, by mapping micro-compositional elements within
the hair follicle, the activation of vanilloid receptor-1 (TRPV1)
(which is a Ca permeable channel) at the instigation of the
catagen stage was found to correspond with an increase in
Ca.119 Thus hairs, particularly at the beginning and end of
their growth cycle may incorporate metals and minerals
differently. While this would be averaged over the analysis
of a large number of hairs it will add to the intra- and
inter-individual variability in concentrations.
4.11 Speciation
The simple presence of an element being ingested by an
organism is not sufficient to predict its fate within that organism.
It is commonly known that a metals’ transport and interaction
in biology is strongly dependent upon its speciation.120 An
elements’ species is highly influential on biological toxicity.
Similarly, its biological transport and fate is highly dependent
upon the chemical form.
Thus, an element may or may not be preferentially absorbed
by an individual depending upon its chemical species. The
presence of other metals can also inhibit absorption depending
on species. Even upon entering the blood stream, metals will
exist in different forms with different capacities to interact with
other parts of the human body and its constituents such as a
myriad of proteins.121 What’s more is that the relative fractions
of species can vary as a function of concentration, particularly
in cases of high chronic or acute exposure. Furthermore, the
incorporation into hair occurs with additional biochemical
and redox interactions with the forming proteins and other
components. It could therefore be expected that the chemical
form, availability and affinity will have a dependence on the
elemental species involved. This in turn will depend upon the
source, extent and route of exposure. Also see the section
dedicated to mercury below.
4.12 Inter-element correlations and competition
As is typical in chemistry, elemental competition, substitution,
and correlations exist. For example Pb and Zn in children’s
hair have shown a negative correlation,16 Ca and Mg can be
correlated,109,122,123 and also Al and V, Al and Mn and Mg
and V.123 Mg and Ca, Mn and Ca, Sr and Ca, Sr and Mg,
U and Na, Ni and Zn, Mn and Sr, Cd and Ni, Sb and Pt have
also shown strong correlations.124 Gender effects again may
impart influence, for instance Ca and Fe were correlated for
men, and Mg and Na in women.125 For a more detailed study
into correlations based on gender, see Chojnacka et al.126
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In rats fed a Zn deficient diet, hair concentrations were
correspondingly lower. An interesting observation was that
Cu, Fe and Mn concentrations simultaneously increased.127
For many of these relationships it is not clear at what level
they occur, i.e. at the point of ingestion, in the transport
mechanisms, cellular incorporation or after the hair has
formed by way of contamination or substitution. Various
competitive and substitution mechanisms can be expected.
4.13 Genetic polymorphism
Very simply, it is observed within biology that there is large
variation of responses to identical conditions. With regard to
endogenous metal concentrations, genetic variability has been
shown to vary As, Cd, Cu, Pb, Hg, Se and Zn concentrations
in erythrocytes.128 Thus blood and hair concentrations could
vary due to inter-individual as well as inter-cellular genetic
differences.
4.14 Structural change
Hair structural changes can occur with morbidity and
nutritional deficiencies.129 Si may also play a role in maintaining
healthy growth of hair.130 In cases of severe poisoning, metals
can impact hair structure, for example in the case of thallium.131
Structure and nutrition within the hair follicle are likely to be
interconnected. Structural analysis of hair from breast cancer
patients shows distinct differences, possibly offering greater
convenience and earlier diagnosis of breast cancer than
conventional methods.132–134 Such structural changes may
be related to the cause or effect of altered elemental
concentrations.135
5. Exogenous vs. endogenous elements: washing
Ineffective washing procedures will leave remnants of
contaminants and results may be artificially high. Alternatively,
a procedure that is too rigorous may remove elements from the
hair reducing the true biogenic signal. Diffusion into human
hair has been modelled previously,136 and also shown for dye
(rhodamine B) to uniformly penetrate hair in as little as
30 minutes in aqueous solution.115 Hair is not as impervious
to external contamination as would be convenient to assume.
Large variance may be induced by non-standard washing
procedures employed.137–139
The real impact of exogenous contamination on extracting a
meaningful result from hair analysis based on the endogenous
incorporation is far from trivial. The decontamination or
washing step in the analysis of hair is vital, yet no approach
is universally followed.
The importance of an effective technique to discriminate the
endogenous contribution from contamination is obvious. The
simplest approach would be a cleansing procedure that would
remove the contamination and leave the endogenously
incorporated elements unaltered, rather than chemically distinguish
endogenous and exogenous material. Many authors have
published statements such as: ‘‘. . .hair samples were cleaned
of surface contamination. . .’’; or ‘‘To exclude the influence of
external contamination. . .’’; etc., often without any mention
about how effective the treatment actually was. It is a bold
assumption that all influence of exogenous sources will be
Chem. Soc. Rev., 2011, 40, 3915–3940 3923

removed by a simple rinsing procedure when the binding of
elemental and molecular species in hair has been shown to be
quite complex and far from fully understood.
Authors of papers critically reviewing decontamination
have demonstrated that these procedures are an important
and vital step in producing credible, indisputable results.
Results have shown to depend significantly on the washing
procedure utilised6 and external contamination is indeed a
‘‘stumbling block’’ for hair analysis.140 Such authors have
identified issues involved and have developed what they have
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deemed to be the most suitable and effective procedures for
their analysis. Many following these instructions bear no
mention of the warnings and questions that still remain and
should be addressed. The possibility that any cleansing process
has not performed ideally, i.e. removed all and only that which
has contaminated the sample, should always be considered.
Washing procedures are most stringently discussed in
papers dealing with the detection of drugs. In these cases,
contamination is perhaps less likely than in elemental
investigations, but however, is of greater importance that false
results (false positives in particular) do not occur. Studies
investigating the most or least suitable washing method remain
very difficult to effectively examine. It remains that in these
studies, the component of trace elements being removed
cannot be distinguished as being of either endogenous or
exogenous origin. Therefore it is difficult to distinguish
whether a method that removes the greatest proportion is
the best since it has removed the most contamination, or the
worst since perhaps there was no contamination and the
method succeeded in only removing elements biogenically
bound in the hair. This question is extremely difficult to
answer due to the many uncertainties in the security of the
hair matrix and the indistinguishability of many biogenic and
diagenetic species.
Washing procedures remain as one of the major ‘unknowns’
for hair analysis. The large number of washing approaches
and the different measurements obtained, depending on the
procedure used, demonstrate this.6,141,142 However where
contamination is restricted such as the case for fetal hair, the
procedure does not appear as influential.143 Many publications
bear little mention of the washing procedure used or specific
details, and many demonstrate little consideration with regard
to the solvents, duration and use of any agitation. Most
commonly, the procedures presented by Rodushkin and
Axelsson,144 and the IAEA145 are used. However, the specific
elements of interest should also be regarded in the approach
used according to Hawkins and Ragnarsdóttir for sheep
wool.146
The method proposed by Rodushkin and Axelsson involves
washing in a sequential order with acetone, deionised water
and 0.5% Triton X-100 solution in an ultra-sonic bath and
laboratory shaker, allowing a few minutes for each stage. The
hairs are then repeatedly rinsed with ultrapure water and air
dried in a clean room.
The IAEA recommend washing in a non-polar solvent
(double distilled acetone), followed by two washings in a polar
solvent (deionized or redistilled water) and finally a wash with
acetone, with each stage lasting for approximately ten minutes.
This approach was also advocated by Bencko.22
3924 Chem. Soc. Rev., 2011, 40, 3915–3940
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6. Hair analysis
Chronic and acute exposures represent themselves distinctively
in hair analysis. Acute exposure is effectively indistinguishable
by bulk analysis and longitudinal ‘‘time’’ resolved analysis is
critical for the interpretation. A distinct band due to an acute
exposure of a metal, if diluted along the entire hair length by
bulk analysis, could simply appear to be the same as a lower-
level chronic exposure. Typically hair will be cut at skin level
from the posterior vertex or occipital region, however, for
fundamental studies or particular case studies, the information
provided below the skin level can be highly informative. It
assists in identifying what contributes to hair concentrations
as far as incorporation via the dermal papilla, sebaceous
deposition or exogenous accumulation.90,91 Additionally, in
specific case studies this region can divulge information in the
immediate hours to days prior to sampling, or death.147
Packaging can be an important consideration depending on
elements of interest. Many plastics are covered in siloxanes
and will add Si-based contamination to the hair. This however
is most likely to be removed easily but is preferable to avoid.
An alternative is aluminium foil providing it also is not
covered in contaminants originating from lubricating oils used
in the foil manufacture. If Al itself is to be analysed then this
would be better avoided. Some foresight and consideration is
obviously needed regarding packing material with respect to
the elements that will be analysed. If the samples are only to be
stored for a short term, there is little threat of the hair
deteriorating. However for long periods of time, fungi,
keratinolytic microbes and even insects can consume hair.
To avoid this, it may be desirable to store hair in freezing
conditions.
Instances of acute exposure can be readily analysed in
longitudinal hair analysis,147 but such analysis requires more
specialised equipment and would not be suitable for screening
large numbers of samples. These approaches are also important
for studying fundamental aspects of hair elemental incorporation,
distribution and associations. Bulk analysis is suitable for
running large numbers of samples routinely which is important
for population studies. The following sections provide examples
of the usefulness and applications of high-resolution and bulk
techniques. A small section is included to mention historical
and archaeological studies, before a comprehensive section on
the analysis of Hg in hair is provided. The following section on
Hg exemplifies the use of hair for epidemiological studies and
with the benefit of analysing different species.
6.1 Spatial and longitudinal analysis
Spatial analysis is important for hair analyses in two major
respects and regimes. A resolution of tens to hundreds of
microns will provide sufficient resolution to monitor acute vs.
chronic exposures. Such resolutions are appropriate to identify
increase and decay curves along the hair that could relate to
pharmacokinetics. High resolution imaging on the order
of B100 nm to a few microns provide more for fundamental
studies into hair analysis, composition, elemental associations
and understanding incorporation mechanisms. In many
biological contexts there is particular desire to measure
elemental distributions for fundamental understanding.148
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Ion beam probes for example can offer good spatial resolution.
Micro-PIXE has been used for studying nutritional elements
in the hair follicle.119
A significant amount of work has been performed with
Secondary Ion Mass Spectrometry (SIMS) and synchrotron
based X-ray Fluorescence (XRF). Synchrotron sources can
provide a variety of analytical techniques for very sensitive
analysis for material characterisation.149 Synchrotron XRF
offers excellent sensitivity for elemental imaging,150 and coarse
XRF probes are ideal for longitudinal analysis of individual
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hairs.147 This approach is non-destructive and due to the depth
penetration of X-rays will probe the entire hair thickness.
Transverse line scans can also be interpreted with basic
algorithms to infer transverse distributions.90 The longitudinal
analysis of the hair plucked from a Pb smelter employee
clearly showed longitudinal increase of Pb in the hair above
skin level, while Zn remained relatively constant. Subsequent
analysis of hair cross-sections with a higher resolution
(B130 nm) revealed associations of metals within the hair,
such as the cuticle layers, nuclear remnants or melanin
granules and corticle proteins (Fig. 1).86
In attempts to address the fundamental questions regarding
contamination of hair fibers, hairs were sectioned longitudinally
and had the outer surface and inner surface analysed with Time-
of-Flight Secondary Ion Mass Spectrometry (ToF-SIMS).151
ToF-SIMS has monolayer surface sensitivity, thus is a truly
surface analysis technique that does not suffer from having to
interpret what contribution underlying material may have to
spectra. In this regard, it is thus possible to section an individual
hair and analyse the external surface of the hair and then also
the corresponding internal section of the same hair; the primary
motivation being to address fundamental questions regarding
the influence of longitudinal accumulation of exogenous
products on internal concentrations and hence, to infer the
integrity of the internal structure, or assess factors regarding
decontamination of hair.91 ToF-SIMS can acquire elemental as
well as molecular information and provides interesting informa-
tion of inorganics in organic matrices and vice versa,152–154 as
well as chemical and structural information.155,156 With this
approach, elemental distributions can be analysed with regard
to organic and molecular fragments88,91,97 and have been used
to identify localised phosphorus based depositions in ancient
hairs.157 Analysis with nano-SIMS can achieve spatial
resolution around 50 nm with excellent sensitivity and provide
interesting analysis of hair cross-sections.85,103
Fig. 1 Elemental maps constructed with an X-ray fluorescence microprobe from a hair thin-section. Bar = 10 mm. Reproduced with permission
from the American Chemical Society from Kempson et al.86
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One of the most novel aspects of hair is the nature of its
growth and potential in recording a historical timeline of
blood xenobiotic concentrations along its length.158–160 This
aspect is one of the most impressive approaches of hair
analysis with respect to the information withheld by individual
strands. The time span of the longitudinal analysis depends on
the length of hair, which grows at a rate of roughly 1 cm per
month, thus a strand of hair can reflect a period of the prior
hours (at the bulb of the hair) up to several months or even
years. Scanning along a hair can be performed by cutting the
hair into segments and running sensitive, bulk analysis of each
segment. However, with greater spatial resolution comes
greater ‘time’ resolution and microprobes can resolve
concentration fluctuations on the order of hours. Such analysis
can yield quite dramatic and visually interesting exposure
profiles, as demonstrated in several examples.
Laser Ablation coupled with ICP-MS can also be successfully
applied to longitudinal analysis with interesting affect,161,162
and has the powerful added advantage of isotopic sensitivity.31,163
Its depth sensitivity is in between that of XRF and SIMS
techniques, thus scans can be performed on the outer region of
a hair and then an internal region by ablating away more outer
material.164 The surface sensitivity of ToF-SIMS is far superior,
but this approach does not require sectioning of the hair. The
laser ablation is partially destructive to the sample, but this is
rarely a concern considering the ease of collecting multiple
hairs from the same individual. In cases where limited sample
is available, such as in historical contexts, this requires
consideration. Such analysis has distinctly shown workplace
uranium exposure over the course of a three day period.165
Those authors present interesting quantified results and
detailed analytical considerations for such analysis with
LA-ICP-MS as well as fundamental considerations for hair
analysis. Similarly, exposure to U in drinking water has been
monitored in longitudinal scans.163
Another dramatic example of longitudinal analysis was in
the profiles of arsenic and its specific chemical species to
indicate poisoning of Phar Lap (Fig. 2). Strong debate over
the cause of death of the famous race horse Phar Lap led to
hair analysis in an attempt to quell speculation. At the time of
his death, Phar Lap exhibited symptoms that could be attributed
to three different proposed causes of death, including arsenic
poisoning. Longitudinal analysis of his hair was performed
with a synchrotron based X-ray fluorescence microprobe with
a resolution on the order of 10 microns. Mapping of his hairs
Chem. Soc. Rev., 2011, 40, 3915–3940 3925

Fig. 2 Longitudinal analysis of arsenic in a single hair from
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Phar Lap.
showed a dramatic profile of arsenic increasing rapidly and
then decaying in a typical pharmacokinetic-like curve. The
half-life of arsenic is quite short and complete clearance occurs
in approximately 24 hours. This leads to a very distinct band
of high arsenic-concentration at a location just above the bulb
and below the skin level. It is worthwhile to note that for this
analysis it was critical that the hair was plucked, rather than
cut at the skin level. This assisted in assessing external
contamination and in providing information from the very
hours before death. The authors concluded that the only
reasonable way to obtain such a profile was via ingestion
(rather than the use of arsenic-based preservatives in the
taxidermy). Another particularly interesting result to come
from that work was to apply a longitudinal analysis of arsenic
speciation by way of X-ray absorption spectroscopy (XAS).
XAS can provide information on speciation of elements even
at a trace level in hair.166 The analysis was performed with the
same X-ray probe used for the XRF mapping and by acquiring
data across the absorption edge at varying longitudinal points.
This technique revealed two dominating species of arsenic in
the hair, but the astounding result was that the longitudinal
variation in the ratio of the species demonstrates ability to
observe metabolic alteration of the arsenic. Thus, it was not
only possible to see the longitudinal arsenic profile, but to also
probe the time-resolved changes in the incorporation of
arsenic based on metabolism.
Such analysis can also be applied to following therapy
regimes utilising metals or metalloids such as in the application
of As167,168 and Pt based therapeutics.169 However, longitudinal
analysis requires somewhat more technically exclusive access
to analytical techniques. The approaches would typically be
applied to either case studies of acute exposures and/or
fundamental studies elucidating basic mechanisms of
incorporation, loss and various effects of specific considerations.
For example geographical translocation, changes in diet, onset
of disease, effect of a hair treatment, etc.
6.2 Bulk analysis
Bulk analysis is useful for the study of chronic exposure and
morbid effects on elemental concentrations due to disease.
Population studies can provide insights into epidemiology,170
etiology,171 endemic diseases172 and the influence of risk
factors by way of monitoring effects on elemental concentrations
in hair.110,173 A summary of elemental concentrations
measured in hair is provided in Table 1. These concentrations
can then assist to infer physiological conditions contributing
to the perturbed concentrations. While many studies identify
3926 Chem. Soc. Rev., 2011, 40, 3915–3940
View Article Online
hair elemental concentrations affected by a variety of conditions
(Table 2), this has rarely been extended to identify
fundamental mechanisms that result in the changes. Rather,
such studies have added supporting data to proposed underlying
mechanisms. It is important to note that the concentrations
discriminating groups in Table 2 are only relevant within each
specific study in comparison to the specific control group,
hence we have not provided quantitative data in this table. In
some instances, the morbid population from one study
completely overlaps the control population from a different
study. Population studies are also useful for assessing
geographically distinct groups which may have exposure to
pollutants, such as being proximal to industrial activities.174
Quantitative bulk analysis has diminished relevance in the
study of acute exposure. As seen with longitudinal analysis, an
element of interest can be highly concentrated within a band in
the hair.147 This concentration will be diluted relative to the
size of the hair segment analysed.165 Table 2 briefly
summarises a selection of publications identifying statistically
significant differences in elemental concentrations in population
groups based on morbidity compared to a homeostatic state.
An emphasis has been put upon essential elements, however
the ubiquitous Pb is often involved.
Bulk analysis techniques typically require a variety of
instrumental consideration critical for obtaining accurate
results.144,165,175 Instrumentation must be rigorously set up
for analysis of hair for optimal analysis and accurate
interpretation. Cheaper, faster and reliable digestion176 and
analysis are desirable in developing high throughput analysis
of samples to ensure confidence, and to minimise cost and
time. Non-destructive, quantified bulk analysis enables other
analysis to be performed which may be of interest to probe
different levels of information from a hair, or to preserve the
sample in legal or cultural heritage applications.89
6.3 A retrospective look
Due to the nature of hair growth and the ability for its
structure to persist for a long period of time, there exists
opportunity in retrospectively assessing individuals based on
hair analysis.147 In archaeological contexts, hair can potentially
provide an indication of dietary and cultural habits205,206 and
exposure in ancient individuals. For example, As exposure of
Chilean mummies.207 Hair can be preserved extremely well
even for hundreds or thousands of years in good environments.
However, the hair will have prolonged exposure to contamination
and degrading effects over the decades to centuries or even
millennia. It can be a remarkable feat simply for the hair to
even exist after such periods and not to have succumbed to
degradation. These can include breaking down of the proteins
by hydrolysis and consumption by fungi or insects and other
keratinolytic microbes.95 Contamination is highly dependent
upon the local environment and can be highly influential on
altering elemental concentrations.95,97 Accumulations may
even occur in the medullary canal.157
Many analyses have been performed on the hair of Napoleon
in attempts to determine a cause of death being attributed to
arsenic poisoning.208–211 These interpretations are complicated
by many factors. In some instances the credibility of the hairs’
This journal is c The Royal Society of Chemistry 2011
 View Article Online

Table 1 Elemental concentrations in hair

1 a
Mean(n)  s.d.[range]/mg g Population Reference
Li 0.017(114)  0.013[0.005–0.046] Swedish pop. 21
0.01(655)  0.01[0.00–0.0] Korean children 111
0.016(45)  [0.003–0.042] 177
Be 0.0060–0.0076(5) 178
0.0013(114)  0.0009[0.0004–0.0042] Swedish pop. 21
0.007(45)  [0.003–0.012] 177
B 0.67(114)  0.620[0.13–3.30] Swedish pop. 21
0.54(45)  [0.26–1.87] 177
Na 497.0(1236) Moscow women (40–60 yo) 179
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118(100)  113[7–643] Iran pop. 180

147(114)  149[17–670] Swedish pop. 21
27.14(655)  19.07[1.18–153.00] Korean children 111
Mg 118.7(1236) Moscow women (40–60 yo) 179
96(100)  45[19.9–208] Iran pop. 180
46(114)  38[8.5–141] Swedish pop. 21
12.29(655)  13.39[3.30–29.80] Korean children 111
Al 26(100)  18[4.0–77.5] Iran pop. 180
8.2(114)  4.8[2.7–25.6] Swedish pop. 21
8.78(655)  3.94[1.05–25.55] Korean children 111
1.63(45)  [0.26–5.30] 177
Si 33(114)  31(o4.6–132] Swedish pop. 21
P 133(114)  17[102–169] Swedish pop. 21
121.21(655)  19.33[65.84–193.90] Korean children 111
S 4.7%(100)  0.60[3.34–6.83] Iran pop. 180
4.77%(114)  4.1%[4.07–5.50] Swedish pop. 21
Cl 1231(100)  1081 Iran pop. 180
8600(114)  5900[1350–27000] Swedish pop. 21
K 282.2(1236) Moscow women (40–60 yo) 179
185(100)  181[15–754] Iran pop. 180
81(114)  83[7.4–420] Swedish pop. 21
34.10(655)  13.48[4.49–117.10] Korean children 111
Ca 1162.6(1236) Moscow women (40–60 yo) 179
955(100)  503[210–2353] Iran pop. 180
750(114)  660[113–2890] Swedish pop. 21
212.47(655)  92.33[70.23–1001.00] Korean children 111
Sc 0.0014(114)  0.0010[0.0004–0.0045] Swedish pop. 21
Ti 0.830(114)  0.680[0.12–2.71] Swedish pop. 21
V 0.13(100)  0.09 [0.02–0.54] Iran pop. 180
0.027(114)  0.024[0.005–0.134] Swedish pop. 21
0.08(655)  0.03[0.02–0.25] Korean children 111
0.016(45)  [0.001–0.051] 177
Cr 2.2(113)  0.5 Indian group with diverse medical status 181
0.167(114)  0.118[0.046–0.527] Swedish pop. 21
0.47(655)  0.21[0.14–1.09] Korean children 111
0.20(45)  [0.11–0.52] 177
Mn 3.76(20)  2.12 Indian pop. 182
0.74(100)  0.49[0.13–2.46] Iran pop. 180
0.560(114)  0.550[0.080–2.41] Swedish pop. 21
0.29(655)  0.18[0.05–2.61] Korean children 111
0.067(45)  [0.016–0.57] 177
Fe 43.42(20)  13.33 Indian pop. 182
9.6(114)  4.4[4.9–23] Swedish pop. 21
12.62(655)  4.33[4.16–32.48] Korean children 111
Co 0.043(1236) Moscow women (40–60 yo) 179
0.30(20)  0.18 Indian pop. 182
0.013(114)  0.011[0.002–0.063] Swedish pop. 21
0.01(655)  0.001[0.00–0.19] Korean children 111
0.023(45)  [0.004–0.14] 177
Ni 4.9(113)  0.8 Indian group with diverse medical status 181
1.13(20)  0.28 Indian pop. 182
0.430(114)  0.400[0.11–1.60] Swedish pop. 21
0.23(45)  [0.08–0.90] 177
Cu 17.1(20)  12.5 183
18.0(2) 184
44.4(113)  6.0 Indian group with diverse medical status 181
16.6(150)  3.4 5–15 yo males 185
11.07(20)  4.16 Indian pop. 182
12(100)  9[4.6–66.7] Iran pop. 180
25(114)  21[8.5–96] Swedish pop. 21
15.51(655)  10.08[5.82–104.70] Korean children 111
20.3(45)  [9.0–61.3] 177

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 View Article Online

Table 1 (continued )
1 a
Mean(n)  s.d.[range]/mg g Population Reference
Zn 159(20)  80.1 183
137.8(2) 184
186.1(1236) Moscow women (40–60 yo) 179
183.7(113)  24.0 Indian group with diverse medical status 181
245.0(150)  17.8 5–15 yo males 185
91.29(20)  20.34 Indian pop. 182
166(100)  48 [36–329] Iran pop. 180
137(67)  [34–543] 6 yo children in Poland 16
142(114)  29[68–198] Swedish pop. 21
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69.99(655)  28.89[13.82–170.60] Korean children 111

162(45)  [129–209] 177
Ga 0.0025(114)  0.0015[0.0008–0.0067] Swedish pop. 21
0.011(45)  [0.002–0.068] 177
Ge 0.0046(114)  0.0031[o0.0022–0.015] Swedish pop. 21
0.004(45)  [0.001–0.039] 177
As 0.3 Cambodia, Mekong delta pop. 186
0.83(150)  0.1 5–15 yo males 185
0.085(114)  0.054[0.034–0.319] Swedish pop. 21
0.11(655)  0.05[0.01–0.91] Korean children 111
0.05(45)  [0.03–0.08] 177
Se 0.280(20)  0.058 183
0.830(114)  0.280[0.48–1.84] Swedish pop. 21
0.75(655)  0.14[0.33–1.82] Korean children 111
0.54(45)  [0.37–1.37] 177
Br 3.2(100)  1.8[0.23–7.7] Iran pop. 180
Br 37(114)  33[5.6–221] Swedish pop. 21
Rb 0.006(45)  [0.003–0.03] 177
Sr 1.20(114)  1.00[0.14–5.54] Swedish pop. 21
0.89(45)  [0.17–4.63] 177
Y 0.023(114)  0.029[0.003–0.104] Swedish pop. 21
Zr 0.155(114)  0.237[0.011–1.21] Swedish pop. 21
Nb 0.0019(114)  0.0012[0.0005–0.0058] Swedish pop. 21
Mo 0.042(114)  0.020[0.021–0.046] Swedish pop. 21
0.07(655)  0.05[0.02–0.71] Korean children 111
0.021(45)  [0.01–0.028] 177
Ru 0.000028(114)  0.000040[o0.00002–0.000039] Swedish pop. 21
Rh 0.000021(114)  0.000047[o0.00001–0.00004] Swedish pop. 21
Pd 0.00032(114)  0.00078[o0.00006–0.0021] Swedish pop. 21
0.01(45)  [0.004–0.049] 177
Ag 0.231(114)  0.298[0.025–1.96] Swedish pop. 21
0.08(45)  [0.02–1.31] 177
Cd 1.4(57)  0.2 187
0.5(113)  0.08 Indian group with diverse medical status 181
3.2(150)  0.69 5–15 yo males 185
0.32(20)  0.12 Indian pop. 182
0.05(63)  [0.0–2.0] 6 yo children in Poland 16
0.08(655)  0.12[0.00–2.21] Korean children 111
0.011(45)  [0.004–0.17] 177
Sn 0.320(114)  0.390[0.06–1.41] Swedish pop. 21
0.046(45)  [0.007–0.34] 177
Sb 0.008(45)  [0.003–0.13] 177
Te 0.00034(114)  0.00033[o0.00007–0.001] Swedish pop. 21
0.0003(45)  [0.0003–0.001] 177
I 7.20(1236) Moscow women(40–60 yo) 179
0.57(100)  0.44[0.13–2.45] Iran pop. 180
0.680(114)  0.580[0.13–3.31] Swedish pop. 21
Cs 0.00067(114)  0.00046[0.00017–0.0019] Swedish pop. 21
Ba 0.640(114)  0.490[0.16–1.92] Swedish pop. 21
0.32(655)  0.29[0.05–3.75] Korean children 111
0.28(45)  [0.05–1.58] 177
La 0.035(114)  0.046[0.0046–0.106] Swedish pop. 21
Ce 0.039(114)  0.050[0.007–0.164] Swedish pop. 21
Hf 0.0054(114)  0.0081[0.0004–0.037] Swedish pop. 21
Ta 0.0044(114)  0.0034[o0.002–0.020] Swedish pop. 21
W 0.0053(114)  0.0049[0.001–0.021] Swedish pop. 21
0.0013(45)  [0.0001–0.007] 177
Re 0.000037(114)  0.000034[0.000005–0.00016] Swedish pop. 21
Ir 0.00003(114)  0.00011[o0.00001–0.000045] Swedish pop. 21
Pt 0.00015(114)  0.00017[0.0002–0.00061] Swedish pop. 21
0.00035(45)  [0.0004–0.0008] 177
Au 0.030(114)  0.0028[0.003–0.200] Swedish pop. 21

3928 Chem. Soc. Rev., 2011, 40, 3915–3940 This journal is c The Royal Society of Chemistry 2011
 View Article Online

Table 1 (continued )
1 a
Mean(n)  s.d.[range]/mg g Population Reference
Hg 1.73(263)  2.12 Taiwan pop. 188
3.4(19)  2.8 Exposed Cambodia p. 189
1.51(59)  0.91 19–20 yo Japanese Fem 190
1.03(100)  0.82[0.2–4.8] 123
0.261(114)  0.145[0.053–0.927] Swedish pop. 21
0.49(655)  0.29[0.05–1.83] Korean children 111
0.66(45) [0.31–1.66] 177
Tl 0.00061(114)  0.00032[0.0002–0.0016] Swedish pop. 21
0.0002(45) [0.0001–0.0004] 177
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Pb 5.7(113)  2.9 Indian group with diverse medical status 181

12.7(150)  3.5 5–15 yo males 185
1.43(42)  [0.31–12.07] From maternal/fetal pairs 191
1.25(245)  1.20[0.2(DL)–9.9] 8–10 yo in Germany 34
1.96(20)  1.25 Indian pop. 182
1.00(112)  [0.2–14.6] 6 yo children in Poland 16
0.960(114)  0.850[0.22–7.26] Swedish pop. 21
1.68(655)  1.12[0.10–7.09] Korean children 111
0.41(45)  [0.13–4.57] 177
Bi 0.019(114)  0.025[0.002–0.255] Swedish pop. 21
0.04(655)  0.06[0.00–0.51] Korean children 111
0.009(45)  [0.0004–0.14] 177
Th 0.0013(114)  0.0010[0.0003–0.0044] Swedish pop. 21
U 0.057(114)  0.065[0.006–0.436] Swedish pop. 21
0.04(655)  0.07[0.00–0.67] Korean children 111
0.009(45)  [0.002–0.03] 177
a
In some instances the median has been quoted where the mean value was not provided.

Table 2 Elemental correlations with morbidity

Condition Observation with reference to hair concentrations relative to control groups Ref.
Retardation Pb was higher in children with retardation. Cu correlated with severity 192
IQ Pb inversely correlated with IQ 35
Neurological disorders Pb was elevated in children with neurological disorders 193
ADHD Pb correlated with instances of ADHD 35
Autism Fe decreased and Se elevated with autism. Girls also had decreased Cu and Mg 113
Parkinson’s disease Fe was lower 109
Hyperactive disorders Mn was associated with oppositional and hyperactivity indices 194
Body mass index Pb and Cd correlated with BMI in children 16
Hypertensive diabetes K, Mg and Ca were lower while Na was higher 171
Osteomuscular disorders Pb and Zn were elevated and Cu lower for children with rheumatic diseases 195
Amblyopia Ca and Mg were elevated and Mn decreased for child patients 196
Bone mineral density Mg was elevated while serum Mg was lower for higher bone mineral density 197
Cardiovascular disease Ca and Mg correlated with a marker for arterial stiffness associated with vascular calcification 122
Myocardial infarction Fe and Zn were lower while Pb, Cd and Ni were elevated 198
Hepatitis Cu and Fe were elevated and Zn was lower in blood, serum and hair of viral hepatitis patients 199
Kidney failure Al, Ni, Cd and Pb were elevated 200
Anaemia Fe, Cu and Zn were lower while Cd and Pb were elevated for anaemic children 201
Premenstrual syndrome Zn/Cu and Hg were elevated while Fe, K and Mg/Ca were lower 202
Psoriasis Zn was lower while Cd, Cr, Ni and Pb were elevated 203
Metabolic syndrome Ca, Mg and Hg corresponded with prevalence of metabolic syndrome 173
Breast cancer Ca, Mg, Fe, Cu, Mn and Zn were lower while Na, As and K were elevated 204

origins are vague and was common practice at the time to treat
hair with arsenic for preservation. Thus order-in-magnitude
differences in arsenic concentrations have been reported.212
However, little information exists on these samples regarding
the longitudinal or transverse position which may also account
for some variation in measurements in the case of endogenous
uptake.
Another application for analysis in retrospect of exposure,
where other media are not suitable, is in the study of fetal hair.
Assessment of blood lead for example can be difficult, as 95%
of Pb in a person can be incorporated in the bone structure
and released when an individual is under Ca stress. In this case
fetal blood may not be representative of fetal exposure and
hair could be a better option for assessment,213 with specific
short term exposures identifiable.214 However, Black et al.
only found minor evidence of Pb crossing the placenta based
on hair analysis.191
7. Hair analysis for mercury
7.1 Occurrence in the environment
Mercury, as with every naturally occurring element, is wide-
spread throughout the environment. As such, a global cycle of

This journal is c The Royal Society of Chemistry 2011 Chem. Soc. Rev., 2011, 40, 3915–3940 3929

Hg has always been active at local as well as at a global scale
due to various processes such as volatilisation/condensation,
adsorption/desorption, methylation/demethylation and different
forms of transport. For instance, Hg in its monoatomic
gaseous form has a residence time in the atmosphere of about
one year and therefore it is hardly surprising that Hg is
ubiquitous in the environment. However, human activity has
dramatically increased the biogeochemical cycle of this
element. Dietz et al.215 have recently reviewed the literature
concerning the anthropogenic contribution to Hg levels in
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Arctic animals. Their findings clearly indicate that an order of
magnitude increase in Hg in Arctic marine foodweb-based
animals has been evident from the mid to late 19th century.
This increase has continued in the 20th century due to
human activities that today account for over 90% of the Hg
concentrations in Arctic animals.
Currently Hg is employed in a number of industrial
operations including small-scale mining of Au and Ag and
chlor-alkali production. While both these activities are still of
concern in developing countries, they are being phased out in
advanced economies. However, Hg is still present in a number
of products including manometers, thermometers, electrical
switches, fluorescent lamp bulbs, some batteries and dental
amalgam fillings. Furthermore Hg is released during the
combustion of fossil fuels.216
In terms of human health, two pathways of exposure are
widespread across the general population in many countries.
These are the exposure to methyl Hg through fish consump-
tion (and possibly rice)217 and to Hg vapour from amalgam
tooth fillings. An additional form of widespread exposure is
due to the use of an ethyl Hg derivative (thimerosal) as an
antiseptic in some vaccines.218 However, the use of Hg in most
vaccines is being phased out.219 Other pathways of exposure
are pertinent to specific occupations. Among these the above-
mentioned small scale Au mining activities and chlor-alkali
productions are responsible for significant occupational
exposure to inorganic forms of Hg.
7.2 Speciation and pharmacokinetics
As it will be discussed below, the mobility, toxicity and
accumulation of Hg are strictly dependent on its speciation.
This element is in fact characterised by a considerably
complex chemistry due to the existence of different oxidation
states [mercury (Hg0), inorganic mercurous (Hg+) and
mercuric (Hg2+)] and organic species (e.g. methyl-, ethyl-
and phenyl-mercury). Methyl-Hg is produced by saprophyte
aquatic organisms from inorganic Hg. As it is readily
absorbed by animals, methyl-Hg bioaccumulates in the
aquatic food chain reaching the largest concentrations in
predatory fish. While exposure to methyl-Hg through seafood
consumption is by far the most widespread pathway,
contamination of other foodstuff such as cereal seeds and
meat by methyl-Hg (or other alkyl-Hg) based fungicides has
also been reported.220,221
The absorption and disposition in the body of Hg species
has been thoroughly reviewed elsewhere,218 however a brief
summary is provided below as this information is relevant in
terms of the interpretation of hair analysis.
3930 Chem. Soc. Rev., 2011, 40, 3915–3940
View Article Online
The absorption of Hg0 is strictly dependent on its physical
state. While liquid Hg0 is very poorly sorbed by the gastro-
intestinal tract, Hg0 in the form of vapour is readily sorbed
through the lungs.222 Similarly, there is some evidence that
aerosols of HgCl2 can be sorbed to some extent.223 The uptake
of inorganic ionic species of Hg is dependent on the diet and
the solubility of the compound involved.222 Methyl-Hg is
almost completely sorbed in the gastrointestinal tract.
In a similar fashion as observed in the case of uptake, the
pharmacokinetics of Hg is highly dependent on the species
involved. Alkyl-Hg, such as methyl-Hg, and Hg0 can cross
both placental and blood-brain barriers.222 In contrast,
inorganic ionic forms of Hg do not efficiently pass these
barriers.
Once in the bloodstream Hg0 is readily transported to
several organs including the cranial region and the kidneys
which represent the main accumulation organ.224 Within the
cells elemental Hg is oxidised to its divalent ion which has a
half-time of several weeks.222 The half-time of methyl-Hg in
the human body is approximately 1.5–2 months.225,226
The half-times of different Hg species, and in particular,
methyl-Hg, have to be considered when hair and other
biomarkers are compared.
7.3 Toxicity
The acute toxicity of Hg has been recognised for a long time.
For instance, the Romans used slaves to extract cinnabar
(Hg sulfide) at the Almaden mine in Spain, where acute
mercurialism caused miners’ life expectancy to be as short as
three working years.227
Methyl-Hg compounds were first synthesized in the 1860s.
Their acute toxicity was immediately evident as two of the
laboratory technicians died in the process.228 More recently,
acute poisoning episodes were related to the misuse of
Hg-containing compounds. Noteworthy is the mass poisoning
that occurred in Iraq at the beginning of the 1970s. In this
case, methyl-Hg was used as a fungicide on wheat and barley
seed stocks and poisoning occurred when these grains were
directly used to produce flour.221
Besides acute toxicity, chronic effects linked to the
consumption of fish and other seafood are by far the occurrences
of most concern. This scenario reached dramatic consequences
when a factory manufacturing acetaldehyde discharged
methyl-Hg in the Minamata Bay of Japan from the 1950s to
1968 (e.g. Ninomiya et al.).229 The most well known effect of
methyl-Hg relates to its ability to disrupt neuronal cell division
and migration during brain development.230 For this reason,
and for the ability of this compound to cross the placental
barrier, prenatal exposure is considered as the most susceptible
stage of life. Prenatal exposure to Hg has in fact been linked to
learning delays and alterations of the autonomic nervous
system.231 However, it should be noticed that chronic
exposure to Hg, and methyl-Hg in particular, could have
deleterious effects on the neurological activities of adults
as well.
In addition to the effects on the central nervous system,
methyl-Hg has also been implicated in risk of cardiovascular
disease. For instance, methyl-Hg derived from fish consumption
This journal is c The Royal Society of Chemistry 2011

was linked with risk of acute myocardial infarction in adult males
in Finland.232 Hg in fish may diminish the cardioprotective
effects generally associated with a diet rich in fish.233
7.4 Incorporation of different Hg species in hair
During keratinisation of hair, Hg is incorporated in the protein
structure of the hair through bonding with S-containing amino
acids.234 Methyl-Hg is readily incorporated into the hair
during its formation in the follicle and the concentration of
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methyl-Hg in the hair is proportional to the blood concentration.
This proportional relationship results in a concentration in the
hair which was generally considered to be B250 times higher
than in the blood (when the concentration in the hair is
expressed in mg per g hair and mg per L blood).235 However,
the blood-to-hair ratio has been demonstrated more recently
to vary with age.236 Once methyl-Hg is incorporated in the
hair structure, it remains stable for long periods of time. This
allows a retrospective analysis of the exposure to Hg on the
basis of the rate of hair growth.
In contrast to methyl-Hg, the incorporation of inorganic
forms of Hg during hair growth is much more limited. This
situation has been convincingly supported by Cernichiari
et al.237 who produced a brief but powerful article showing
that inorganic Hg concentrations in blood and urine do not
correlate to Hg concentrations in hair. This work was
produced as a response to the claim by another group who
suggested that the 7th President of the USA, Andrew Jackson,
may not have suffered from Hg poisoning even though he was
therapeutically administered calomel (mercurous chloride).238
This conclusion was drawn by Deppisch et al.238 based on the
analysis of 2 samples of Jackson’s hair which showed low
levels of Hg. However, Cernichiari et al.237 reported the case of
5 individuals exposed to inorganic Hg (in the form of vapour
or salts) whose blood and urine Hg content was highly
elevated despite the Hg hair concentrations remaining at
nearly background levels. This convincingly supports the case
made by Suzuki that Hg in hair is not a robust marker for
dietary exposure except in the case of methyl-Hg.239
The situation is more complex when volatile species of
inorganic Hg are present in the environment such as in the
case of chlor-alkali plants or small scale artisanal mining
operations. When this is the situation, elemental Hg could
accumulate in hair through simple sorption from the atmosphere
as well as through blood circulation since approximately 80%
of the inhaled Hg0 is absorbed by the lungs and remains in the
body.240 Little is known about the lung absorption of other
inorganic Hg species such as HgCl2 or Hg oxide aerosols. In
dogs, Hg oxides aerosols were shown to be cleared by the body
in less than 24 hours while the remaining Hg had a half-time of
approximately a month.241 Again it is expected that these
inorganic species of Hg could accumulate in hair through
sorption (i.e. without Hg entering the blood circulation). Hair
of workers and non-workers near artisanal mercury mines in
China showed enhanced elevated concentrations of Hg in
hair.217 Interestingly, while in populations exposed to Hg
through a fish diet methyl-Hg in hair is strongly correlated
to total Hg (representing 80% of it, e.g. Pellizzari et al.),242 in
these Chinese studies a large proportion of Hg in the hair
This journal is c The Royal Society of Chemistry 2011
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(490%) was present as inorganic species. In this case,
inorganic Hg could have accumulated in the hair through
the blood circulation, upon lung absorption, or through
deposition. Spatially resolved analysis of hair strands including
the follicle could possibly provide a way to differentiate
between these two accumulation pathways as the portion of
hair not yet emerged through the scalp should be largely
protected from atmospheric Hg.
7.5 Other factors affecting the Hg content in hair
Various factors, such as those described earlier, may play roles
in the accumulation of Hg in hair. So far no distinctive ethnic
differences have been reported in regard to the degree of
accumulation of Hg in hair or in relation to different
blood-to-hair Hg ratios.236 Hair colour has also been investigated
in a few studies but the results appear to be inconclusive.
Ando et al.243 reported that total and methyl-Hg in elderly
men and women were statistically higher in grey hair than in
dark hair. In contrast, a study conducted in Spain on a cohort
of 233 school children failed to reveal a difference in hair Hg
concentrations due to hair colour.244
The effect of age has been investigated in a number of
epidemiological studies again with contrasting results. This
issue is discussed in detail by Budtz-Jorgensen et al.236 who
reported a change in the blood-to-hair Hg ratio between
children at the age of 7 and 14 years. While the blood
Hg concentrations doubled with increase in age, the hair
concentrations only increased by 50%. The results were
explained on the basis of changes in elemental composition
of hair during childhood. In particular, finer hairs at the age of
7 seem to accumulate more Hg, on a weight base, than adult
hair. In addition to these changes due to age-dependent hair
structures, changes related to the age of individuals were found
to be relevant in some cases245–247 but not in others.248,249
Another important issue is regarding the effect of gender on
Hg concentration. Again this is a factor that has been
considered in a number of studies and conflicting evidence
can be found in the literature. For instance, Yasutake et al.250
and Grandjean et al.251 found that females had lower Hg
concentration than males. However, other studies244,245,252,253
reported an opposite trend with females having higher
concentrations of Hg in hair than males. These differences
have been tentatively explained on the basis of differences in
the diet, and in particular in the amount of consumed fish, or
hormonal control in Hg uptake.245 In this regard Hirayama
et al.254 reported differences in methyl-Hg uptake and
elimination between male and female mice. In other studies
however, no differences related to gender were found.217,246
All the above mentioned factors are however likely to play a
role that is less marked than other determinant factors such as
dietary or other habits (e.g. smoking)255 and conclusive
evidence of their importance would be difficult to achieve.256
One factor that should be considered, as it has the potential
to invalidate epidemiological studies if not taken into account,
is the effect of permanent hair treatment on the Hg hair
concentration. This effect may also be correlated to gender
differences as women are more likely than men to have hair
treatments. In a comprehensive study of 327 women, ranging
Chem. Soc. Rev., 2011, 40, 3915–3940 3931

in age from 24 to 49 years, artificial hair-waving reduced total
Hg concentration in hair by approximately 30% (after correcting
for other factors including daily Hg intake).257 A similar result
was obtained by Yasutake et al.250 who showed a decrease of
the same order after a single hair treatment with further
decrease in successive treatments. These results are explained
by the fact that waiving lotions contain thioglycolate which is
an effective agent in Hg removal from hair.258 On the basis of
these findings Dakeishi et al.257 correctly point out that
‘the myth that Hg concentration in hair reflects the methyl-
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Hg concentration circulating in the blood, may collapse if a
study population includes subjects with such hair treatment’.
7.6 Analysis of Hg in hair
Generally, total Hg is measured in hair. This is considered
sufficient in most cases as most of the studies focussing on
methyl-Hg, which are the most common, indicate that methyl-Hg
represents the preponderant majority of Hg in hair. For
instance, 490% of the total Hg in hair was in the form of
methyl-Hg in the hair of Faroese women exposed to Hg
through whale meat consumption.251 Issues arise when total
Hg is also analysed in the blood as the percentage of inorganic
Hg in the blood could be significant and would decrease the
correlation with Hg in hair (as inorganic species are incorporated
in the hair to a lesser degree than methyl-Hg).236 Furthermore,
the analysis of total Hg may not be a good predictor of
exposure when vapour/atmospheric Hg is a determinant factor
as direct adsorption to the hair could occur. In this case,
speciation of Hg in the hair would provide additional and
important information.
The most commonly used method to assess total Hg in hair
is based on the digestion of a sample of at least 5–10 mg
followed by cold vapour atomic fluorescence spectrometry
(CV-AFS) or inductively couple plasma mass spectrometry
(ICP-MS) (e.g. Gill et al.).259 However, a method for single
human hair analysis has also been proposed.260 This method is
based on a combination of combustion, Hg collection with
Au amalgamation and detection by atomic absorption
spectrometry (C-GA-AAS). Legrand et al.158 reported a
satisfactory 1 : 1 relationship between these two methods.
Interestingly, the standard deviation in Hg concentration
between individual hair strands was reported to be 6.5%
which is not much larger than what can be expected when
CV-AFS is employed. It should be pointed out again here that
large variability can occur in the results obtained between
commercial laboratories. For instance, variation in results
obtained from 6 commercial laboratories in the USA ranged
from 0.1 to 2 mg kg 1 for a split sample of hair.12 It is
therefore essential that accurate quality controls are in place
when analysing hair samples for Hg.
A detailed review of the analytical techniques that can be used
for the speciation of Hg species and the determination of methyl-Hg
is beyond the scope of this review. Gas chromatography
techniques hyphenated to a variety of detectors have proven
popular due to the convenient conversion of Hg species in
volatile compounds.261 However, an increasing number of
techniques have been developed that achieve separation through
liquid chromatography and detection through ICP-MS.262
3932 Chem. Soc. Rev., 2011, 40, 3915–3940
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Besides these ‘bulk’ analysis techniques, a few applications
of spatially resolved techniques to assess the distribution of Hg
along hair strands have also been employed. For instance,
X-ray fluorescence (XRF) was used to analyse a single hair
strand with a longitudinal resolution of 2 mm.263 This study
was conducted on a hair sample of a chemistry professor
who died of dimethyl-Hg exposure several months after the
poisoning accident happened. This analysis confirmed the date
of the accident and also detected the subsequent increase in Hg
concentration caused by the chelator used to treat the patient.
High-resolution synchrotron-based XRF has been used to
analyse the spatial distribution of Hg in the hair of rats
exposed to methyl-Hg through an endogenous and exogenous
pathway.264 Cross sections of the hair clearly show the
different distribution caused by the two pathways of exposure
while a longitudinal analysis could describe the timeline of
exposure. Zareba et al.265 visualised the distribution of Hg in a
human scalp/nude mouse model through autoradiography
with radiolabelled CH3203 HgCl. These studies demonstrate
the potential of in situ analytical techniques to understand
the mechanism of Hg accumulation in hair and to provide
valuable information regarding the pathways and timelines of
exposure.
7.7 Case studies, potentials and limitations of Hg hair analysis
The validity of hair analysis to assess methyl-Hg exposure has
been recently investigated by a human scalp/nude mouse
model.265 This model system consists of grafting human scalp
hair on to nude mice and subsequent exposure of the animal to
methyl-Hg. The results showed that methyl-Hg is rapidly
incorporated in the human hair and Hg concentrations in
the hair were 2 orders of magnitude higher than in blood.
The observed hair-to-blood ratio (217 : 1) is similar to that
observed in human studies. As mentioned above, the useful-
ness of hair testing in the case of other Hg species is not as
established and it should be treated carefully as various path-
ways of exposure may be present at the same time.
The analysis of Hg hair concentrations offers several
advantages: (a) the prolonged life-time of Hg in blood produces
easily interpretable timelines of exposure and Hg concentrations
are largely stable once incorporated in the hair structure;
(b) hair samples are easy to collect, store and transport as
described earlier; and (c) methyl-Hg is concentrated in the hair
in comparison to the blood, facilitating analysis.277 These
advantages have made hair analysis an attractive option to
assess Hg exposure in epidemiological studies.278,279 However,
the situation is different when Hg exposure of single patients is
of concern. Nuttall (2006) provides a comprehensive review of
the issues related to the interpretation of hair Hg levels in
individual patients.280 When individual patients are concerned, Hg
concentrations in the hair need to be interpreted taking into
account the patient’s history, signs and symptoms and possible
alternative explanations. In this case, blood or urine Hg
concentrations are possibly a more robust measurement of
exposure as exogenous contamination can be excluded.
However, hair analysis can be valuable in order to reconstruct
a pattern of early exposure. As Nuttall points out, the risk is
that of attributing subtle symptoms to modest concentrations
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Table 3 Mercury hair concentrations and selected variable in a number of epidemiological studies
Location Source No. of subjects Age
Minamata Fish 3038 10–470
Iraq Hg-dressed 2147 1–450
seeds
Seychelles Fish 779 6.5 months to
(mother–child pairs) 10.7 years
Faroe Whale meat 1022 0–14 years
Islands (mother–child pairs)
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Germany Amalgam 245 8–10 years
USA Amalgam 534 6–10 years
China Mining 66 23–67 years
of Hg in hair. In fact, while acute toxicity symptoms can be
more safely related to large Hg concentrations in hair, chronic
and subtle symptoms are more difficult to diagnose and
differentiate from other causes.
As mentioned above, hair analysis for Hg is much more
established and accepted when epidemiological studies are
concerned. A large number of these studies, especially in the
case of methyl-Hg exposure, have been conducted worldwide;
a selection of some of the most relevant studies is reported in
the following sections. The main parameters investigated in
the studies reported are summarised in Table 3.
7.7.1 Methyl-Hg release in Minamata Bay, Japan. The first
patient with a neurological syndrome, referred to as Minamata
disease, was identified in 1942.281 This disease was associated
with a number of neurological effects ranging from numbness
in the extremities to paralysis. The Minamata disease was
caused by the continuous discharge of methyl-Hg in the
Minamata Bay by a local chemical company producing
acetaldehyde over a period of several decades (ending in 1968).
Methyl-Hg accumulation in fish, which represented a significant
input in the local diet, was the main pathway of exposure. As
early as 1960 a survey showed high levels of Hg, ranging from
97 to 705 mg g 1, in hair of patients affected by Minamata
disease.282 Other patients showed elevated levels of Hg in hair
(up to 191 mg g 1) without showing apparent symptoms.
However, it should be noted that in 1960, residents in the area
were already aware of the cause of Minamata disease and their
fish consumption was already reduced, it is therefore likely
that Hg concentrations in hair would have been larger in
previous years.283 Since then a large number of studies have
focussed on the effects of this pollution and investigations are
continuing to assess the long-term effects of it. Recently,
Yorifuji et al.266 reanalysed data collected during two surveys
in 1960 and 1971. They identified 120 residents in the exposed
area that were included in both surveys. Based on these data
they reported that neurological signs of Hg poisoning were
found among residents having Hg content in hairo50 mg g 1.
The same set of data was also used to investigate the effect of
Hg exposure in relation to cardiovascular disease (by using
hypertension as a proxy). A dose–response relationship was
found between the hair Hg content and hypertension
outcomes.284 A more recent survey showed that the Hg
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Other Adverse
Median/mean (mg g 1) measurements effect(s) Reference
2.1 (control) 30.0 (exposed) — Y 266
(and ref. therein)
5 (control) 136 (exposed) Blood Hg Y 267 and 268
6.9 (mothers) — N 63 and 269–272
4.2 (mothers) Cord blood Y 236 and
273–275
0.18 Urine, saliva — 238
0.3–0.4 (over 5 years) Urine — 276
Total: 0.8 (control), 69 (exposed) Urine Y 249
methyl-Hg: 0.6 (control), 2.3
(exposed)
content in hair of 191 fishermen and their families living in
the Hg polluted area was, with a few exceptions, in the
normal range (o10 mg g 1).283 However, various neurological
symptoms of mild Minamata disease were still prevalent
suggesting that, due to the time elapsed, the current hair Hg
content is not a useful criterion for the diagnosis of chronic
Minamata disease.
7.7.2 Fungicide-treated wheat and barley in Iraq. In
1971–1972 a large amount of barley (22 000 t) and wheat
(73 000 t) were distributed in Iraq. These grains were to be
used as seeds and, as such, were treated with methyl-Hg
fungicides.285 Organomercury compounds were introduced
in 1915 for seed-dressing to prevent seed and soil-born fungi.
Unfortunately, an unknown quantity of these seeds was used
to produce bread and as feed for domestic animals. Several
studies were conducted following this accident. In one study,
the Hg concentration in hair of 184 individuals was investigated
against clinical symptoms of Hg poisoning.286 Generally, no
effects were detected when Hg concentrations in hair were
below 300 mg g 1. However, individual data were not always
consistent as some elevated concentrations of Hg in hair were
not accompanied by symptoms. These false-positive data
points were possibly related to external contamination from
the fungicide.267 A similar study in which a total of 2147
people, from both highly exposed and control areas, were
examined and Hg was measured in hair and blood.268 Using
these data Kazantzis et al.267 concluded that hair Hg
concentrations allowed a better discrimination between different
categories of exposure than blood Hg. The main reason of this
finding was attributed to the retrospective capability of hair
Hg analysis as several months had elapsed between the onset
of the poisoning episode and the survey. Another important
outcome of studies related to this accident is the finding that
concentrations as low as 10 mg g 1 in maternal hair were
associated with adverse effects to the foetus.287
7.7.3 The Seychelles child development study. This long-
term longitudinal cohort study was designed to investigate the
developmental outcomes of prenatal methyl-Hg exposure in a
fish-eating population. This study started in 1989–1990 when
779 infant–mother pairs were enrolled. The Seychelles diet is
heavily reliant on fish and the mothers taking part in this study
Chem. Soc. Rev., 2011, 40, 3915–3940 3933

consumed a mean of 12 fish meals per week.269 The concentrations
of methyl-Hg in local fish are similar to those commonly found
in commercially available fish in the USA.288 However, due to
the diet, the concentration of Hg in maternal hair (6.8 mg g 1)
is much higher than what is commonly found in the hair of
women of child-bearing age in the USA (1 mg g 1).289
The cohort was investigated several times from 6.5 months
to 17 years of age (for references of the individual studies see
Davidson et al.270 and Schoeman et al.).290 Results of this
longitudinal study did not reveal a relationship between
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prenatal Hg exposure and neurodevelopmental deficits.63 Only
1 out of 21 endpoints that were measured showed a slight
adverse association with prenatal methyl-Hg, and this was
only observed in boys.63 Similar conclusions were reached
when scholastic achievements were investigated at age 9 and
17 years.270 In contrast, language and functional attention
were beneficially influenced by methyl-Hg levels.271 Recent
studies conducted in the Seychelles seem to point to the
counteracting role of micronutrients (Fe, Zn, Se and I)
and polyunsaturated fatty acids present in fish which could
mitigate the detrimental effects of methyl-Hg.269,272
7.7.4 Faroe Islands study. Another long-term study similar
to the Seychelles Child Development Study was conducted at
the Faroe Islands. In this case a cohort of 1022 consecutive
singleton births was generated in 1986–1987. The source of
prenatal methyl-Hg exposure in this case was due to the
traditional consumption of pilot whale meat. Mercury
concentrations in cord blood and in hair of the mothers were
measured. Hair concentrations in the mothers ranged from
o0.2 to almost 40 mg g 1 (geometric average of 4.2 mg g 1).
The children were tested for a number of indicators of
cognitive performance at 7 and 14 years. In contrast to the
results reported for the Seychellois children, adverse effects
related to pre-natal exposure were reported at both ages
investigated.273,274 Post-natal methyl-Hg exposure did not
have any significant effects. The deficits due to pre-natal
exposure were similar at 7 and 14 years and affected the
motor, attention and verbal parameters tested. These results
indicated that the methyl-Hg caused permanent effects. The
differences between the results of this study and the study
conducted at the Seychelles may be due to various reasons. In
the Faroe Islands, the consumption of whale blubber could
provide an exposure pathway to other co-contaminants such
as organochlorine compounds. However, Grandjean et al.273
showed a lack of association between PCB exposure and
neuropsychological test results even though an additional
study subsequently indicated that PCB could aggravate the
symptoms of methyl-Hg exposure in children already exposed
to high Hg levels.275 Another possibility is that the nutritional
value of a rich fish diet, as that of Seychellois, could counteract
the negative effects of methyl-Hg in comparison to the
Faroese diet.
7.7.5 Mercury vapour exposure: studies of dental amalgam.
Mercury represents approximately 50% of the metal content
of dental amalgam. As the Hg in the amalgam can be released
in the form of vapour, concerns have been raised in terms of
Hg inhalation through this exposure route. This inorganic
3934 Chem. Soc. Rev., 2011, 40, 3915–3940
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form of Hg is largely retained by the human body and is
carried by the blood cells and crosses both the placenta and
blood-brain barriers. However, inorganic Hg that circulates
through the blood system is not significantly incorporated in
hair. For this reason some studies have used Hg in hair as a
measure of exposure to methyl-Hg through fish consumption
while inorganic Hg in urine was used as a measure of exposure
to Hg vapour.276 In general, while it has been observed that
Hg concentration in urine increases with the number of
amalgam and defective amalgam fillings (e.g. Pesch et al.),247
the concentrations of Hg in tissues due to this exposure
pathway are below those considered of concern (for review
see Clarkson and references therein).218
7.7.6 Occupational exposure in artisanal small scale mining.
Comprehensive reports of occupational exposure in artisanal
small scale mining are available (e.g. Veiga and Baker)291 and
a thorough review of the matter is beyond the scope of this
review. Briefly, while most modern gold mining operations
rely on a cyanide leaching process of the ores, artisanal small
scale mining in developing countries is often still reliant on the
use of liquid Hg to bind Au (or Ag). The amalgam is then
heated and the Hg released as vapour. Another case relates to
the exposure of workers at artisanal Hg mines where cinnabar
is smelted.249 Inhalation as in the case of dental amalgam
is therefore the principal exposure pathway even though
contamination of the surrounding areas by Hg can generate
secondary exposure pathways. As in the case discussed above,
incorporation of inorganic Hg through the blood stream is not
an efficient process. However, extremely large concentrations
of Hg in hair have been reported for artisanal workers. For
instance, hair Hg concentrations above 300 mg g 1 were
reported in workers at Hg mines in Wuchuan, China.217 As
expected in these cases, direct exposure of hair to Hg vapours
makes the use of this biomarker hardly suitable to assess Hg
body burden.
8. Delivering a meaningful result. The use and
future of hair analysis
The optimal goal of bulk hair analysis will be to provide a
quantitative measurement which can be related to a blood
concentration. This can only occur with discrimination
between contamination and endogenous content. One possibility
is the assessment of metabolites, which is a distinct advantage
of drug analysis in hair,292–295 i.e. that a metabolite will only
exist with consumption and hence proves ingestion.
In the interest of a standard biomonitor such as in
commercial provision, this requires reduction of the contribution
of exogenous sources, consideration of the many variables
influencing endogenous incorporation to the extent that inter-
individual variability is removed allowing assessment of the
concentrations pertaining to an individual. As discussed
earlier there are many variables influencing the incorporation
into hair which vary greatly from one individual to another.
These include age, gender, hair colour, lipid content etc., as
well as any other variables based on the genetics of the
individual and the actual source of exposure. For this to be
realised, a great deal of normalisation needs to take place and
This journal is c The Royal Society of Chemistry 2011

it is not sufficient to simply measure the hair elemental
concentrations.
Hair analysis has had considerable debate over its
application and has remained controversial, particularly in
the commercial sector. There is definitive evidence that
essential elements and xenobiotics that find their way into an
individual are subsequently incorporated into hair, the
concentrations of which have a dependence upon the extent
of consumption/exposure and can provide information
regarding chronic and acute exposure.
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The value for hair analysis has been demonstrated, for
mineral analysis, with particular success in the cases of acute
exposure, exemplified in several cases including the death of Phar
Lap. For the investigation of chronic exposure levels, particular
ability has been shown for epidemiological studies where data
from a population group can be pooled in comparison to other
groups and deconvolve important factors relating to the sources,
exposure routes and extent of exposure to xenobiotics.
With respect to morbidity, hair analysis has shown distinct
ability to reflect changes in endogenous metal concentration
and/or transport. In this sense there is promise for etiological
studies, discriminating cause and effects, and identifying
environmental or habitual risk factors. However, to classify
an individual is much more problematic. For instance, if an
individual returned a result of a perturbed Zn concentration,
certain aspects may be normalised for, such as gender, age and
a crude estimation of hair colour. The measured concentration
will also be altered based on lipid content and genetics
(as examples), and other local variables such as water
concentrations when washing, swimming and other hair care
products. Assuming all these aspects can be normalised and
accounted for, then what does the concentration of this metal
actually indicate? Based on the references indicating perturbed
Zn concentrations in hair with morbidity, it could be due to a
multitude of reasons (Table 2). A general ‘screening’ of an
individual may give an indication of homeostasis, or lack
thereof, but offering a diagnosis as to the cause of an abnormal
concentration is currently not feasible and is difficult to see as
realistic. In addition, hair analysis could only show homeo-
static deviation, not the problem. For example, if cancer
reduces Zn in hair and hair analysis indicates a deficiency, it
is not a cure for cancer to provide Zn supplements.
The methods employed for hair analysis are greatly varied
and there’s unlikely a need for an individual approach to be
the one and only standard. This is particularly evident in
comparing analysis for acute versus chronic exposure. The
analysis for acute exposure requires high resolution and
typically more exclusive approaches (such as XRF micro-
probes or LA-ICP-MS). These approaches are time consuming
and viable only for a very limited number of samples. Analysis
for chronic exposure will typically involve large populations
and require a high throughput of samples. These utilise bulk
methodologies which yield no, or very limited, temporal
resolution. The approach (including washing procedure)
should be proven and robust. Ideally the analyses will undergo
interlaboratory comparisons for confirmation of protocols.
The future would also ideally see normalisation with a multitude
of factors to provide a comparison for blood concentrations.
However this would dramatically increase expense if other hair
This journal is c The Royal Society of Chemistry 2011
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components were required to be quantitatively measured and
could make the approach prohibitive.
The following paragraphs highlight areas of hair analysis
where pursuit holds specific value.
8.1 Fundamental studies
Greater understanding of the fundamental incorporation of
elements into hair is required. This is needed with respect to
localisation and association of the elements in the hair structure
with sub-cellular information. The chemical characterisation
as a function of these associations will assist in the discrimination
of exogenous content, enabling proper assessment of washing
protocols and furthering ability to normalise hair concentrations
to blood concentrations. Normalisation to blood concentrations
would be a major and significant accomplishment. This may
come with further development of quantitative methods with
ability to assess elemental speciation.
In vitro growth methods66,296 could also add control over
variables in the study of hair growth and the incorporation
and effects of elemental concentrations297 and speciation.
Hairs from the same individual would give unprecedented
information with the removal of inter-individual variability.
8.2 Longitudinal/temporal analysis
Longitudinal analysis of hair offers unique possibilities for
toxicology and monitoring acute exposures and/or changes in
lifestyle, metabolism and impact of other variables. Information
from the hair analysis can be correlated with the pharmaco-
kinetics known for the blood concentrations and also assist in
hair–blood concentration normalisation. Such studies also
reveal information regarding incorporation mechanisms and
with speciation can probe the effects of metabolic processes.147
8.3 Metabolic discrimination
Analysis for metabolites, organoproteins for example or
complexes that can be identified as originating from biogenic
incorporation can discriminate contamination. This is particularly
vital for toxicology in a forensic context. An understanding of
the influence of metabolites on incorporation will also assist in
achieving normalised concentrations to relate to blood
concentrations. Value exists in developing methodologies
capable of assessing elemental species and identifying meta-
bolic products.298
8.4 Epidemiological and etiological studies
As demonstrated in this review, hair elemental concentrations
from large population studies can identify correlations with
lifestyles, morbidity and risk factors. This information can
then be extended for the pursuit of underlying mechanistic
phenomena for disease state and development. For instance,
identifying deficiencies in trace elemental levels may reveal
metabolic dysfunction pertaining to the impact of the disease
on other organs, which may not initially be apparent. The
advantages of hair analysis could hold value in such studies
rather than attempting to identify a disease itself.
Chem. Soc. Rev., 2011, 40, 3915–3940 3935

8.5 Economical remote and proof of concept studies
For epidemiological and other studies, hair analysis offers
benefits for remote sampling and screening of large population
groups for a broad range of elemental concentrations with
beneficial economics. This enables cheaper investigative and
proof-of-concept studies before conducting more rigorous and
expensive programs such as including blood sampling. The
benefit of hair here is for the ease in collection and storage with
respect to remote communities and little influence from hourly
or daily fluctuations. However, many considerations still
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remain for interpretation and may not be as sensitive as blood
or urine analysis in discriminating population groups.
Acknowledgements
The authors would like to thank all colleagues and collaborators
and all the authors of the cited publications as well as those
not referenced here that are too numerous to include.
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