Article - Hussain — USA v. Jittaphol (Dkt. 70-4, D. Mass.)

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U.S. District Court for the District of Massachusetts · No. 1:21-cr-10270-MLW · Doc. 70-4 · 2022-04-06 · Docket on CourtListener

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Sweat Collection for Metabolomics Analysis
Clin Biochem Rev 38 (1) 2017   13
Review Article
Working Up a Good Sweat – The Challenges of Standardising Sweat 
Collection for Metabolomics Analysis
*Joy N Hussain,1 Nitin Mantri,2 Marc M Cohen1
1School of Health and Biomedical Sciences, RMIT University, Bundoora, Vic. 3083; 2Health Innovations Research Institute, 
School of Applied Sciences, RMIT University, Bundoora, Vic. 3083, Australia.
*For correspondence: Dr Joy Hussain, joyhussain9@gmail.com
Abstract
Introduction 
Human sweat is a complex biofluid of interest to diverse scientific fields. Metabolomics analysis of sweat promises to improve 
screening, diagnosis and self-monitoring of numerous conditions through new applications and greater personalisation of medical 
interventions. Before these applications can be fully developed, existing methods for the collection, handling, processing and 
storage of human sweat need to be revised. This review presents a cross-disciplinary overview of the origins, composition, 
physical characteristics and functional roles of human sweat, and explores the factors involved in standardising sweat collection 
for metabolomics analysis. 
Methods 
A literature review of human sweat analysis over the past 10 years (2006–2016) was performed to identify studies with metabolomics 
or similarly applicable ‘omics’ analysis. These studies were reviewed with attention to sweat induction and sampling techniques, 
timing of sweat collection, sweat storage conditions, laboratory derivation, processing and analytical platforms.
Results
Comparative analysis of 20 studies revealed numerous factors that can significantly impact the validity, reliability and 
reproducibility of sweat analysis including: anatomical site of sweat sampling, skin integrity and preparation; temperature 
and humidity at the sweat collection sites; timing and nature of sweat collection; metabolic quenching; transport and storage; 
qualitative and quantitative measurements of the skin microbiota at sweat collection sites; and individual variables such as diet, 
emotional state, metabolic conditions, pharmaceutical, recreational drug and supplement use. 
Conclusion
Further development of standard operating protocols for human sweat collection can open the way for sweat metabolomics to 
significantly add to our understanding of human physiology in health and disease.
Introduction
Human sweat is a biological fluid (biofluid) that is generating 
increasing interest across a diverse set of fields including 
dermatology, paediatrics, toxicology, analytical chemistry, 
forensic pathology, psychiatry, illicit drug testing and infectious 
diseases. Currently sweat is primarily used in clinical medicine 
for chloride sweat testing which is used in the diagnosis 
of cystic fibrosis (CF). Additionally, some centres around 
the world use a sweat patch for monitoring drugs of abuse, 
while others have developed an indicator test (Neuropad) to 
detect peripheral neuropathy in the foot sweat of diabetics.1-3 
Aside from these applications, the use of sweat in medical 
practice is limited in part due to challenges involved with 
sweat collection and the range and reproducibility of testing. 
This is likely to change as advances in analytical technology 
methods within metabolomics and other related ‘omics fields 
allow more complex physiological information to be derived 
from smaller amounts of sweat with less arduous processing. 
This is leading to a greater understanding of the physiology of 
human sweating and the skin’s excretory pathways in relation 
to metabolites, pathogens, and xenobiotics.4 Incorporation 
of Bluetooth capabilities with some of the newer wearable 
sweat electrolyte and metabolite detecting systems reflects 
even wider trends in applications to enhance personalised 
analysis.5-7
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14   Clin Biochem Rev 38 (1) 2017
Each type of human biofluid or tissue sample has its own 
signature metabolome, but most of what is known about the 
human metabolome is based upon findings in the ‘serum/blood 
metabolome’ and the ‘urine metabolome’. Further study and 
standardised procedures are now required to characterise the 
‘sweat metabolome’ and how it fits into the bigger picture of 
the human metabolome, and whether the case exists for wider 
application of sweat metabolomic testing. 
When applying a metabolomics approach to analysing human 
sweat, a number of variables need to be examined within the 
context of the origins, composition, physical characteristics 
and functional roles of sweat. These variables include: 
sweat induction and sampling techniques, timing of sweat 
collections, sweat storage conditions, and laboratory aspects 
such as metabolic quenching, extraction, concentration, 
fractionation, separation and other processing methods 
applicable to sweat. Exploring these variables within the 
framework of newer laboratory analytical platforms that 
optimise qualitative and quantitative detection of sweat 
metabolites will pave the way forward to make more rigorous 
and meaningful comparisons of sweat metabolomics studies.8 
Standardising the collection, handling, processing and storage 
of sweat for further metabolomics analysis is vital to this 
endeavour and working out the further steps necessary to 
achieve this standardisation is the focus of this review.
Background – Metabolomics
Metabolomics is the multidisciplinary science involving 
the measurement and analysis of low molecular weight 
metabolites such as electrolytes, sugars, lipids and other 
compounds that exist in a selected biofluid, cell, tissue or 
organism under a given set of physiological conditions. It has 
its roots in the works of many biochemists who pioneered 
the discovery and detection of various vitamins in the 1940s 
and progressed the concepts of ‘metabolic variance’ and 
‘biochemical individuality’.9-12 
The exact number of unique metabolites in the human 
metabolome has yet to be firmly established, but it is generally 
thought that there is a lower number of metabolites in the 
human metabolome (>3,500) compared with the total number 
of genes (>30,000), RNAs/transcription factors (>30,000) and 
proteins (>100,000).13 Small changes in the transcriptome may 
translate into more amplified changes in metabolites.14 With 
presumed fewer total metabolites to analyse and a potentially 
more amplified signal to be detected, the power and potential 
of metabolomics to pick up minute but significant health-
related changes holds promise.
As with all newly emerging fields, within metabolomics 
there is multiplicity and various expansions of terminology. 
Although metabolomics and metabonomics are often used 
interchangeably in the literature, metabonomics technically 
refers to the study of the interactions of metabolites over 
a timeframe in a complex system.15 Fluxomics refers to an 
extension of metabolomics, in which metabolomics is applied 
at various experimental time points generating kinetic data 
which can then be used to study metabolic pathway fluxes.16 
Exposomics, another extension of metabolomics, refers to 
identifying metabolites linked to environmental risk factors 
for disease.17 Metabolites can be classified into two categories: 
endogenous metabolites (synthesised and utilised within a 
biological system) and exogenous metabolites (imported 
from outside the biological system into the cell, such as drugs, 
xenobiotics and nutrients).13,16 The Human Metabolome 
Project (HMP) led by Dr David Wishart of the University 
of Alberta in Canada published a first draft of the human 
metabolome in 2007 which consisted of 2180 metabolites, 
1200 drugs and 3500 food components.18 A growing list of 
findings additional to the HMP is being compiled and verified 
on the Human Metabolome Database – a freely accessible 
and continually updated web resource (http://www.hmdb.
ca/).11,13,19 Not all known human metabolites can be found 
in any given biofluid because different biofluids serve 
different functions and play different metabolic roles. As of 
November 2016, the HMP had identified and/or quantified 
over 3848 metabolites: 440 metabolites in cerebrospinal fluid, 
1233 metabolites in saliva, 2287 metabolites in blood, 1746 
metabolites in urine, 695 metabolites is faeces and over 172 
metabolites in other tissues and biofluids including sweat.19
The methodology of metabolomics can be divided into 
different conceptual approaches such as targeted analysis, 
global metabolite profiling, metabolomics and metabolic 
fingerprinting/metabolic 
footprinting.20 
A 
targeted 
metabolomics approach involves a targeted search and 
quantitative analysis of a set number of known metabolites or 
substances that play a particular role, much like a typical clinical 
laboratory test. Global metabolite profiling is untargeted 
and comprises an analysis of all measured metabolites or 
substances, including those known and unknown, which 
make up a metabolic profile of the total complement of 
metabolites in a particular sample.13,20,21 Metabolomics 
utilises complementary analytical methodologies such as 
liquid chromatography-dual mass spectrometry (LC-MS/
MS), gas chromatography-mass spectrometry (GC-MS), 
and nuclear magnetic resonance (NMR) spectroscopy 
in a coordinated attempt at global metabolite profiling.20 
Metabolic fingerprinting refers to the metabolic ‘signature’ or 
mass profile of the biofluid or tissue sample of interest which 
is then compared in a large sample population to screen for 
differences between samples. When signals or significant 
differences can be detected, the metabolites are then identified 
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Sweat Collection for Metabolomics Analysis
Clin Biochem Rev 38 (1) 2017   15
and the biological relevance of these particular metabolites 
can be more easily elucidated. Metabolic footprinting is 
analogous to metabolic fingerprinting except the differences 
detected involve focus on extracellular metabolites.15
Sweat Origins, Components and Functions
Sweat Definitions
Whole body sweat is a complex mixture of cumulative 
secretions from millions (1.6–5 million) of eccrine, apocrine, 
apoeccrine and sebaceous glands as well as bacteria, yeast, 
fungi, other microbiota and cellular debris that reside in 
and on the largest organ of the human body – the skin.22 
These microscopic glands dwell largely in the dermis and 
hypodermis layers with secretory canals through which sweat 
flows onto the skin surface and into hair follicles. Defining 
sweat precisely is complicated by confusing nomenclature 
across different disciplines in the scientific literature and 
a lack of a biological systems-based approach to studying 
sweat. Sweat collected from the skin surface in experimental 
studies, especially older studies, is often referred to as 
‘eccrine’ sweat because eccrine sweat glands are the most 
numerous and ubiquitous glands in the skin, however many 
sweat samples also contain potentially trace amounts of 
apocrine, apoeccrine and sebaceous gland secretions (called 
sebum), depending upon the body site of sampling. This 
imprecision of nomenclature is the case in some toxicology 
literature dealing with sweat patch testing of illicit drugs and 
physiology literature studying electrolyte changes in exercise. 
However, in the dermatology and cosmetic literature, ‘sebum’ 
can figure in addition to ‘sweat’ with more emphasis on the 
underlying structures within the skin. The converse can also 
be true with studies focused on collection of sebum. The 
term ‘residual skin surface components’ (RSSC) is another 
synonym of ‘sweat’ as it comprises potential sweat glandular 
secretions and cellular debris (from stratum corneum – 
outermost epidermal skin layer).23
Mindful of the semantics surrounding sweat, it is useful 
to revisit the anatomy, histology and secretions of the four 
known gland types that can contribute to sweat. This sets the 
stage for better understanding and targeting of future studies 
to fully characterise the sweat metabolome.
Eccrine Sweat Glands and Secretions
Eccrine sweat glands exist at birth and can be located all over 
the body’s skin except on lips, on the nail bed and on some 
fields of the genitalia (e.g. glans penis). They can average 
100–200/cm2 body surface area, with higher densities (600–
700/cm2) on palms and soles, and at luminal diameters of 
20–60 μm at skin openings.24,25 Eccrine glands consist of 
single tubules ranging 4–8 mm in length that are generally 
divided into 3 parts: (i) deep, coiled secretory portion in deep 
dermis layers; (ii) upper dermal portion with straight and 
coiled parts; and (iii) intra-epidermal part often referred to as 
the acrosyringium. The dermal portion, or dermal duct, has 
epithelial cells connected at numerous sites by desmosomes 
and intercellular junctions that are believed to constitute a 
barrier between the luminal and extracellular compartments. 
The inner luminal cells contain various tonofilaments while 
the outer basal cells are surrounded by collagenous and 
fibrocyte-rich sheathes.22,26
Eccrine sweat glands are classed as merocrine glands (Figure 
1). Eccrine sweat gland secretions are released from cells as 
an aqueous fluid, without disintegration of cells, containing 
various electrolytes, elements, ions, amino acids, proteins 
and other known and unknown small molecules as outlined 
in Figure 2.27,4 Composition varies with many factors: rate 
of sweat production, transit time through the excretory 
duct, aldosterone activity, physical training, psychological 
states and acclimatisation to environmental temperatures.26 
These give hints to underlying functions that have not yet 
been fully determined. There also exists debate whether 
secretory eccrine sweat is perhaps an isotonic ultrafiltrate of 
plasma since sweat contains many of the same solutes found 
in plasma, but at much lower concentrations.28 However, 
based on a recent proteomics study of pooled sweat samples 
collected from schizophrenic and control subjects, only 6 
of 185 unique proteins identified in sweat were reported in 
serum. The authors therefore argue that sweat is not merely 
a plasma transudate and future metabolomics studies are 
required to shed more light on this topic.29
Apocrine and Apoeccrine Sweat Glands and Secretions
Apocrine sweat glands also exist at birth but do not become 
active until the androgenic stimulation of puberty.26 They are 
confined to hairy body areas (i.e. axilla, mammary areola, 
peri-umbilicus, perineal and genital areas) since they open and 
secrete into adjacent hair ducts (e.g. apopilosebaceous ducts) 
before secretions reach the skin surface. They are generally 
larger than eccrine sweat glands with apocrine coil diameters 
of ~800 µm compared to eccrine coil diameters of ~500 
µm, both located in the dermis and hypodermis.22 Apocrine 
ducts are relatively short and found in close proximity to hair 
follicles. The density of apocrine glands is highly variable 
with reports of 8–43/cm2 body surface area in one study of the 
axilla.30 Two different types of cells are visualised in apocrine 
glands: columnar secretory cells and myoepithelial cells. The 
secretory cells are generally noted to be full of mitochondria 
and different granules with convoluted cell membranes and 
microvilli presenting towards the lumen.22
Apoeccrine sweat glands are a mixed type gland as the name 
suggests and were first described in 1987 by Sato et al.30 They 
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are also presumed to develop during puberty and be restricted 
to hairy body areas. As many as 50% of all axillary sweat 
glands are thought to be apoeccrine. Component cells of 
apoeccrine glands include eccrine secretory cells, apocrine 
secretory cells and myoepithelial cells.30 Identification of 
these morphologically distinct glands can be made with 
specific protein markers (i.e. phalloidin,S-100, CD15).22
Apocrine and apoeccrine glands are both classed as apocrine 
glands. With apocrine glands, secretion occurs via pinching-
off of the cell’s plasma membrane producing membrane-
bound vesicles, which helps to account for comparatively 
Figure 1. Sweat gland secretion patterns.
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Sweat Collection for Metabolomics Analysis
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more viscous secretions.22,26 Sato et al. determined that Na+ 
and K+ concentrations obtained from the isolated ducts of the 
apoeccrine glands are curiously more similar to that of eccrine 
sweat compared to apocrine sweat.30 While the composition of 
apoeccrine sweat has not yet been fully elucidated, apocrine 
sweat has been demonstrated to contain carrier proteins for 
volatile odour molecules like volatile organic compounds 
(VOCs) and pheromones with amino acid conjugates 
produced by bacterial enzymes.22 Apocrine bromhidrosis 
(more commonly known as BO or body odour) is thought 
to be linked to large amounts (i.e. over 106 bacteria/cm2) of 
resident microflora such as aerobic cocci, diphtheroid species, 
Corynebacterium species and Staphylococcus epidermidis.26,31
Sebaceous Glands and Sebum
Sebaceous glands are located in the skin of all surface areas 
except for the palms of hands and soles of feet. They are 
particularly numerous on the forehead, scalp, midline back, 
chest, perineum and surrounding the orifices of the human 
body. Densities of up to 400–900 glands/ cm2 occur on the 
face, especially in the T-zone area of the face which starts from 
the midpoint and sides of the forehead, and extends downward 
toward the middle of the nose, including the sides of the 
nose and the midline part of chin.32 Sebaceous gland density 
decreases towards the extremities of the body. Sebaceous 
glands can be divided into two types: pilosebaceous glands, 
when associated with hair follicles, and free sebaceous glands 
seen mostly at transitional zones between skin and mucous 
membranes. A well-known example of a free sebaceous 
gland is the Meibomian gland of the eyelid. All sebaceous 
glands consist of single or multiple lobules, or acini, with 
ducts emptying into a main sebaceous duct. Secretory lobules 
contain sebaceous gland cells, or sebocytes, that are excreted 
in their entirety as part of the holocrine gland status. Maturing 
sebocytes have been visualised to accumulate high lipid 
content as they migrate from periphery to gland duct.33 
Like apocrine sweat, sebum is thought to play a role in the 
generation of pheromones and body odour in its interactions 
with skin-residing bacteria and yeast of the microbiome.34 
Sebum, however, is even more lipid-based, containing 
triglycerides and fatty acids (together 57% of contents) as 
well as cholesterol, wax esters, squalene, keratin, cellular 
debris, anti-microbial lipids, antioxidants, coenzyme Q10, 
vitamin E and other various metabolites of fat-producing 
cells.34,35 Interestingly, the lipids in sebum would seem to 
originate from both sebocytes and keratinocytes, with studies 
identifying differences based upon cholesterol and squalene 
conversion enzymes.34
Collected Sweat
Setting aside the above distinctions in gland origins, the 
vast majority of sweat studies in the literature have analysed 
a collective form of sweat with eccrine gland secretions 
predominating. Depending upon location of sweat sampling 
and various cleaning and collection strategies used during 
sampling or processing, trace amounts of sebum and/or 
apocrine and apoeccrine gland secretions, cellular matter from 
the epidermis and associated ~1012 skin microbes, as well 
as other metabolites like xenobiotics may feature in sweat 
samples.36 The systems-based approach of analysing sweat 
with metabolomics offers the prospect of uniting all these 
different subcomponents of sweat. With such a metabolomics 
approach, studies of ‘normal’ sweat obtained from ‘healthy’ 
people have detected highly variable metabolite compositions 
with large numbers of different small molecules, of both 
microbial and human origin, in a primarily water-based 
(~99%), relatively acidic (mean pH 6.3) solution (Figure 2).37-40
This rich complexity of sweat content hints at its functions 
both at the level of the skin and at the level of the organism 
as a whole.
Functions of Sweat
Temperature and Fluid Homeostasis
Sweat is integral to the regulation of body core temperature 
by water evaporative heat dissipation. Blood flow regulation 
and vasodilation of superficial blood vessels largely 
contribute to this homeothermic control and the finding that 
eccrine sweat production is under the control of cholinergic 
and, to a lesser extent, adrenergic innervation is consistent 
with this hypothesis.41 Various stimuli of this system include 
temperature, emotions, intellectual stimulation and gustatory 
stimulation.42 Sweat volumes vary widely as a result. Global 
insensible fluid losses can be approximately 1000 ml daily 
for the whole human body, including more than half of fluid 
losses through the skin via perspiration with the remaining 
losses being through the lungs.4,40 However, there are reports 
of individuals perspiring up to several litres per hour, 12 L per 
day under certain extreme physiological conditions.4,35,43
Eccrine sweat activity appears intermittent over a large 
portion of the body: cycles of periodic discharges alternating 
with pauses occurring from <1 to 12 geyser-like emissions 
per hour with single sweat gland emissions recorded every 
3.3 min in one recent study.41 This activity differs among 
individuals, environmental circumstances and body sites 
with approximately 50% of total body volume of sweat being 
thought to be produced by the trunk, 25% by the legs and 
25% by the head and upper extremities.1,48 Even in cases of 
profuse sweating, it is thought that only approximately 50% 
of sweat pores release sweat at any given time, except for the 
palmoplantar regions where the sweat gland activity is largely 
synchronised.44 How these findings fit within sweat’s overall 
functions in the human body is still unclear.
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Figure 2. Metabolomic sweat content.
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In contrast to eccrine glands, apocrine gland activity is 
reported to be more continuous in its fluid secretions, while 
still receiving predominantly cholinergic and some adrenergic 
innervation.22,42 Apoeccrine gland stimulation by physiological 
and pharmacological stimuli appears to be distinct from those 
controlling eccrine or apocrine glands. Apoeccrine glands 
respond quickly to psychological stress and are thought to 
more significantly contribute to the abundant sweat produced 
in the axilla.25,30
Sweat Electrolyte Regulation
Another important component of sweat that impacts human 
fluid balances is sodium. The concentrations of Na+ in sweat 
can be highly variable, ~20–100 mmol/L, and some individuals 
can lose an estimated 4–6 g of Na+ per day, equivalent to 12–
15 g of NaCl daily through sweating, especially if working in 
moderately hot conditions.43 Eccrine gland duct cells reabsorb 
several ions, including Na+ and Cl-, via a number of known 
anion exchangers such as Na+/K+-ATPases (on basolateral 
membranes), cystic fibrosis transmembrane conductance 
regulators (CFTRs, mutations of which provide the basis for 
Cystic Fibrosis Sweat Chloride testing), carbonic anhydrases 
II, and vacuolar proton pumps (V-H+-ATPase).22 Sweat Na+ 
and Cl- concentrations have been documented to increase with 
age to 12–19 years then stabilise thereafter.45,46 Sweat Na+, 
Cl-, and K+ concentrations also have reported body regional 
variations.47
Acid-base homeostatic mechanisms are presumed to be 
involved in sweating since sweat is more acidic than plasma, 
with pH ranges of 4–6.8 reported in various studies.1,4 It is 
noted that with increased flow rates following exercise or at 
temperatures above 31 °C, sweat pH increases to upper limits 
of approximately 6.8, which is still more acidic than plasma.47 
Some non-ionised basic drugs diffuse into sweat and become 
ionised as a result of the lower pH of sweat, although the 
exact mechanisms have not been fully elucidated. This has 
led to projections of these non-ionised basic drugs displaying 
free-drug (or molecule) sweat-to-plasma (S/P) ratios of >1, as 
in the case of ammonia with reported S/P ratios of 20–50.48,49
Skin Protection
Sweat also provides lubricating, water-proofing, antimicrobial 
and skin barrier-promoting properties that support skin in the 
first line of defence against many environmental insults. In 
extreme hot conditions, the lipid-rich secretions of apocrine 
and sebaceous glands can emulsify sweat produced by 
eccrine glands to create a hydrolipid film that is not as readily 
evaporated. This is thought to be of importance in delaying 
dehydration. In colder conditions, the lipid nature of sweat 
becomes more solid and, in coating the hair and skin, sources 
of unwanted moisture like rain or snow can theoretically 
be more effectively repelled.50 Palmar hydration, which is 
directly linked to eccrine sweat production, increases the skin 
friction coefficient which therefore improves the adherence 
of hands to objects and contributes to a heightened sense of 
touch.41 Sweat also contains antimicrobial peptides (AMPs) 
like dermicidin, lactoferrin, and LL-37, an AMP of the 
cathelicidin family, which serve to control certain pathogenic 
bacterial counts on the skin surface.4,41 However, the precise 
qualitative and quantitative content of skin microbiota and 
associated microbe-microbe and microbe-host dynamics via 
sweat are areas of active research with early findings hinting 
at rich metabolic inter-relationships with impacts on skin 
integrity, especially in skin inflammatory states.36 
The free amino acid composition of sweat is curiously 
different from other biofluids. Data from a recent study 
suggest the amino acid content of sweat is remarkably similar 
to the amino acid content of an epidermal protein, profilaggrin. 
Since profilaggrin is thought to be the key contributor of free 
amino acids making up the natural moisturising factor within 
the stratum corneum, it is postulated that sweat plays a role 
via interactions with profilaggrin in maintaining the barrier 
integrity of human skin.51
Immune System 
Sweat has links to many immune-mediated mechanisms. 
Skin epithelial cells interact with various external stimuli to 
produce cytokines, and sweat directly activates epidermal 
keratinocytes to produce various cytokines using in vitro 
models with cultured human keratinocytes from surgically 
discarded neonatal skin samples.39 It is postulated that sweat 
may play both beneficial and pathological roles in immune-
mediated communications. For example, sweat is well-
recognised in exacerbating atopic dermatitis (AD) lesions 
and is associated with increased itching (pruritus) which 
has associations with enhanced expression of IL-31 (newer 
member of IL-6 family of cytokines) in tissue samples of 
exacerbated AD lesions.52 Sweat also contains cystatin A, a 
proteinase inhibitor of bacterial cysteine proteases. Given 
these exogenous proteases are known to break down the 
epidermal barrier, cystatin A in sweat may serve both immune 
and skin protective roles.53 Quantitative levels of IL-1α, IL-
1β, IL-6, TNF-α, IL-8 and TGF-β have been measured in 
human sweat although the precise cellular origin of these 
cytokines is still unknown and could be derived from sweat, 
blood or epidermal cells.54 
Excretion Functions and Drug Delivery Mechanisms
While the excretory function of sweat has previously been 
considered negligible compared to the kidney, recent studies 
challenge this notion. There is evidence that several toxic 
elements and xenobiotics may be preferentially excreted 
through human sweat.55-58 Some studies report arsenic, 
cadmium, lead and mercury being excreted in appreciable 
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quantities via sweat, with the rates of excretion matching 
or exceeding urinary excretion.58 Furthermore, while excess 
dietary nicotinamide cannot be eliminated through urine 
because of its reabsorption by the renal tubules, it can be 
effectively excreted by sweat glands.35 Many pharmaceutical 
drugs are also excreted via sweat and the role of sweat 
patch technology in monitoring illicit drug use is based on 
dozens of studies examining the pharmacodynamics and 
pharmacokinetics of amphetamines, cocaine, cannabis, 
opiates and associated metabolites excreted in sweat.1,48 Drug 
binding to various skin fractions and reabsorption of drugs 
from pooled sweat on skin has also been observed. The 
relative concentration of unmetabolised drugs is reported to be 
occasionally higher in sweat than in blood, urine or saliva.59,60
The above findings suggest that molecules of drugs/
metabolites/xenobiotics may reach the skin surface from 
blood by various proposed routes: via sweat or sebum by 
active or passive inter- and/or transcellular mechanisms; 
and transdermal migration through lipid bilayers of stratum 
corneum.40 The second mechanism could be linked to the 
concentration gradient in which only the free fraction of drug/
metabolite/xenobiotic, unbound to proteins, diffuses through 
lipid membranes from plasma to sweat. Thus, it seems that 
the physical nature (i.e. molecular mass, protein-binding, 
pKa and lipophilicity) of each particular drug/metabolite/
xenobiotic plays a role in how much ends up in sweat. In 
fact, dermatologists routinely take advantage of this scenario 
when treating cutaneous fungal infections with oral antifungal 
medications such as ketoconazole, terbinafine or fluconazole. 
It is often recommended to exercise to induce sweating while 
taking these oral antifungals since the drugs are transported 
to the skin surface by eccrine sweat and/or sebum and then 
often reabsorbed, thus optimising drug delivery to site of 
infection.61-63
Metabolic and Infectious Diseases
The alteration of sweat with different pathological conditions 
makes sweating a useful clinical indicator for various 
conditions. Over-sweating (hyperhidrosis) and under-
sweating (hypohidrosis), whether regional or systemic, may 
represent warning signs for systemic conditions or diseases. 
Decreased sweat production involving the feet is the basis 
for the Neuropad indicator test for diabetic peripheral 
neuropathy.3 Impaired overall sweating is associated with pre-
eclampsia and thought to be related to decreased clearance of 
plasma vasoactive amines.64 Female menopause is commonly 
associated with ‘hot flushes’ linked to increased sweat 
production.65 
Night sweats can indicate serious systemic infections (e.g. 
tuberculosis) and malignancy, while local hyperhidrosis 
around a bite site can indicate toxic envenomations such as 
occurs with Australian redback spider bites.66 Hypoglycaemia, 
hyperthyroidism, hypercapnia and vagus nerve stimulation 
can all lead to stimulation of eccrine sweat production and 
alterations in local sweating may arise directly from certain 
skin conditions.26 For example, some hyperkeratotic disorders 
such as pityriasis versicolor and psoriasis interfere with 
the excretion of sweat and are associated with decreased 
sweat output as visualised with skin capacitance imaging of 
lesions.44 Abnormalities in the transport of sweat onto the 
skin’s surface may also cause a severe prickly sensation and 
skin inflammation resulting in the intra-epidermal retention 
of sweat, such as occurs with miliaria rubra which has been 
linked to elevated levels of IL-1 and IL-31detected in sweat.39
Therapeutic and Wellness Functions of Sweat
It is hypothesised that sweat produced by different activities 
may differ in composition. For example, IL-1 concentrations 
are increased in sweat induced by both exercise and sauna 
bathing,39 yet exercise is linked to increases in the generation 
of several end-metabolites like reactive oxygenated species 
that are in turn linked to oxidative stresses. This is thought not 
to be the case with sauna-induced sweat although this remains 
to be validated by further studies.35
Lipid Homeostasis
Sebum production changes have been linked to diet. Caloric 
deprivation in the setting of obesity decreases sebum 
production while a high fat diet in the setting of psoriasis 
increases it.67,68 Increases in energy intake have been associated 
with increased excretion of triglycerides, cholesterol and 
associated esters in sebum.35 As newer studies in sweat and 
skin surface lipidomics are being done, more definitive 
information regarding these links and potential mechanisms 
of action are likely to emerge.69
Methods
Pubmed, Medline, Google Scholar, Embase, Science Direct, 
Scopus, Ovid, Web of Science, Proquest, Toxline and UpToDate 
databases were initially searched with keywords ‘sweat’ and 
‘metabolomics’ with restrictions of English language and 
of dates 2006–2016. These records were then supplemented 
with searches for other research by key authors, searches of 
citations and reference lists of key papers, and additional 
searches with expanded keywords relating to sweat including 
perspiration, sauna, exercise, secretion and/or excretion from 
human skin and residual skin surface components as well 
as expanded keywords relating to metabolomics including 
exposomics, xenometabolomics, toxicometabolomics and 
fluxomics. Older studies of sweat (before 2006) have been 
used in compiling background information, but not for the 
detailed analysis of sweat collection methods.
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Sweat Collection for Metabolomics Analysis
Clin Biochem Rev 38 (1) 2017   21
Of the 1320 records identified for review as of 1 June 2016, 
all 17 studies presenting quantitative human data utilising a 
sweat metabolomics methodology of analysis between the 
dates 2006–2016 were included, regardless of quality of 
experimental design. An additional three sweat proteomics-
based studies were identified that utilised similar laboratory 
platforms relevant to metabolomics and were also included in 
the comparison analysis. 
Results
The Table presents a summary of the pertinent information 
regarding sweat induction and collection methods extracted 
from the 20 identified studies for comparison. 
Discussion
Sweat Induction Protocols
Induction of perspiration represents a phenomenon involving 
a complex chain of metabolic reactions, with many possible 
triggers, as already discussed. Exercise, temperature, 
stress, psychological state, relative humidity, hormonal and 
sympathetic/parasympathetic nervous system parameters, 
diet, skin colonisation factors, xenobiotics exposure – both 
purposeful and non-purposeful – can influence sweat volumes 
and content.40 Refer to the fourth column in the Table 
describing sweat induction modes utilised in the reviewed 
studies. A number of important factors are apparent when 
obtaining sweat for metabolomics analysis: (i) ensuring 
adequate amounts of sweat are available to complete the 
analysis, including enough volume for controls and potential 
further analysis; (ii) ensuring the mode of sweat induction 
does not interfere with the utility of the results; and (iii) 
ensuring that sweat induction and sweat collection happen 
in a timely manner that optimises metabolic quenching and 
metabolite stability.8,70
Pilocarpine Iontophoresis
Several active research groups rely on a chemical pilocarpine 
iontophoresis method of inducing sweat.29,37,38,71,72 This 
method takes advantage of the bioelectric properties of skin 
which allow the application of low intensity electrical current 
(i.e. 1.5 mA) for 5 min. The resulting opposition offered by 
skin to this electrical current, called bio-impedance, is present 
in intra-and extra-cellular fluids and the capacitive reactance 
of cell membranes. For a topically applied chemical such as 
pilocarpine (0.5% pilocarpine nitrate solution), a drug with 
cholinergic parasympathomimetic activity which aims to 
stimulate primarily the muscarinic receptors of eccrine sweat 
glands, to be absorbed through human skin, the electrical 
current must overcome the bio-impedance imposed on its 
flow to reach the target tissue of sweat glands with sufficient 
intensity. This bio-impedance can be influenced by a range of 
factors, some of which are electricity source-dependent such 
as the distance between electrodes positioning, pulsed direct 
current vs constant direct current source, and size and content 
of iontophoresis electrodes (typically containing 70% copper, 
30% zinc with diameter of 30 mm).27,73
Some of the important host-dependent factors involved 
with this mode of sweat induction include the amounts of 
keratin and the variable thickness of stratum corneum (SC) 
at different body sites, fluctuating amounts of fluid in skin 
layers with overall hydration status, ambient temperature 
increasing or decreasing hydration of keratin, adipose tissue 
thickness (especially with some sweat glands residing in deep 
dermis/ subcutaneous fat) and individual pain/tolerance to 
the electric current. All of these factors can alter biological 
responses, thereby potentially confounding metabolic results. 
Therefore, the argument can be made that using pilocarpine 
with iontophoresis induces production of a particular type of 
primarily eccrine sweat but whether the detailed metabolomic 
contents of this type of sweat are the same as physiologic 
sweat and/or thermally-induced sweat and/or exercise-
induced sweat remains unknown. 
After all, the original method of cholinergic stimulation 
with pilocarpine iontophoresis on the skin to facilitate sweat 
production dates back to the 1959 Gibson and Cooke publication 
describing implementation and standardisation of the ‘classic 
sweat test’ targeting sweat chloride levels for the purposes of 
diagnosing cystic fibrosis (CF).74 The Webster Sweat Inducer 
system coupled with a patented Macroduct Sweat Collector 
used in more recent sweat metabolomics studies originates 
from a further enhancement of the pilocarpine method, again 
designed to specifically improve the classic sweat test for 
CF.75 The quantitative pilocarpine iontophoresis test (QPIT) 
remains the gold standard for sweat induction in terms of 
CF-related testing and now has over 50 years of progressive 
standardisation.76 Despite better uniformity in collecting 
sweat samples and improved reference intervals based on 
age, defined rates of sweating and the volume of sweat to be 
collected at standardised sites as well as newer confirmatory 
CFTR-based testing, there are still complicating factors.77-79 
Documented reports of false positive and false negative sweat 
chloride tests are in the literature, hypothesised to be due 
to such wide ranging factors as contaminating topical gels, 
interfering dermatological lesions (i.e. atopic dermatitis), 
autonomic nervous system dysfunction, prostaglandin 
use and other medication uses (e.g. topiramate), arsenic 
toxicity, malnutrition states, immunoglobulin deficiencies, 
autoimmune disorders such as systemic lupus erythematosus, 
and various abnormal endocrine states such as untreated 
hypothyroidism and Addison’s Disease.27, 80, 81
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Hussain JN et al.
22   Clin Biochem Rev 38 (1) 2017
Table. Sweat induction and collection methods for metabolomics.* 
Study
Aims 
n
Sweat Induction Mode
Methods
Adewole et al., 
201672
Identify diagnostic 
biomarkers of active 
tuberculosis in eccrine sweat 
83
Webster Sweat Inducer – 
pilocarpine iontophoresis  x 
5 min
Macroduct® Sweat Collector – part of 
Macroduct® Sweat Analysis System – covers 
volar forearm x 15–35 min; sweat transferred
to micro-centrifuge tube
Jia et al., 201694 Assess feasibility of using 
HPLC-MS/MS for accurate 
quantifying of cortisol in 
human eccrine sweat
4
Hot room set at 41°C and 
~55% humidity
Leg skin cleansed with alcohol pads, followed 
by dH2O and drying; sweat collected off skin 
into Eppendorf LoBind micro-centrifuge tubes
Sheng et al., 
201693
Monitor elimination of bio-
accumulated heavy metals in 
humans with exercise
17
Exercise; no specific 
instruction as to type or 
location
Direct collection of sweat from any part of 
the body into glass bottle with cover; then 
transferred into 50 mL glass vials with lid. 
Referenced methods from Genuis et al. 2011 
utilised
Tang  et al., 
201695
Compare levels of 5 heavy 
metals (Cr, Cu, Zn, Cd, Pb) in 
human sweat and urine after 
physical exercise
9
Exercise; playing badminton 
x 2 h 
Upper bodies cleansed with ultrapure 
H2O before exercise; sweat scraped into 
polyethylene sample bottles. Samples allowed 
to stand for 30 min, then filtered using 9-mm 
filter paper into test tube
Delgado-
Povedano, 
Calderon-
Santiago et al., 
201671
Develop and validate a 
method for metabolomic 
analysis of human sweat 
using GC-TOF/MS
6
Webster Sweat Inducer –  
Pilogel® Iontophoretic discs; 
1.5 mA electric current x 5 
min
Macroduct® Sweat Collector – part of 
Macroduct® Sweat-Analysis System – covers 
forearm skin x 15 min; sample transferred into 
micro-Eppendorf tube
Calderon-
Santiago  et al., 
201538
Identify metabolic markers 
of lung cancer in sweat to 
develop screening tool for 
diagnosis of lung cancer
96
Webster Sweat Inducer – 
Pilogel® Iontophoretic discs; 
1.5 mA electric current x 5 
min
Macroduct® Sweat Collector – part of 
Macroduct® Sweat Analysis System – covers 
forearm skin x 15 min; sample transferred into 
micro-Eppendorf tube
Porucznik  et 
al., 201589
Targeted detection of BPA in 
sweat in comparison to urine 
for biomonitoring
50
Passive sampling – no 
artificial modes of sweat 
induction
Sweat patches (PharmChek®) applied after
skin cleansed with alcohol wipes, to either 
upper-outer arm or front/back midriff 
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Sweat Collection for Metabolomics Analysis
Clin Biochem Rev 38 (1) 2017   23
Timing
Amount
Storage
Sweat 
Preparation 
Protocols
Analytical Chemistry 
Platforms
5 min induction 
+ 15–35 min 
collection
~10–30 µL
Samples placed 
on dry ice 
immediately, stored 
at  -70°C until 
analysed
Solubilised, reduced, alkylated, and digested 
with protease; then dried, desalted, dried again, 
then resuspended in ACN, formic acid
LC – MS/MS, untargeted 
proteomics; FT mode for MS 
detection, ion trap mode for 
MS/MS detection
25–30 min 
collection times
>200 µL
Samples placed 
on dry ice 
immediately, stored 
at -80°C until 
analysed
Sample mixed with ACN/ammonium acetate; 
addition of internal standard (in ACN); ethyl 
acetate extraction repeated twice, evaporated to 
dryness, re-constituted in ACN
HPLC-MS/MS, SRM mode, 
targeted
Same day as 
urine sample 
collection
>20 mL
Stored at -20°C 
until analysis
Samples dried in oven for standardised weight, 
ashed in furnace, cooled in dryer; residue 
reconstituted in HNO3 with heat
Flame atomic absorption 
spectrophotometry, targeted
~2 h
>20 mL
Stored at 4°C until 
testing
3 methods:
(i) direct dilution with HNO3
(ii) wet digestion with HNO3 + HClO4, heated 
to 200°C; cooled, with HNO3 re-added, final 
dilution with ultrapure H2O
(iii) microwave digestion with HNO3 added, 
microwaved, cooled, diluted with ultrapure H2O
ICP-MS, targeted
5 min induction 
+ 15 min 
collection
>70 µL each 
participant – 
pooled into one 
sample
Frozen at -80°C
Pooled sweat into each of 3 protocols:
(i) deproteinisation with methanol-ACN; 
(ii) extraction with dichloro-methane; 
(iii) extraction with ethyl acetate. 
Each followed with methoxymation + silylation
GC-TOF/MS, full scan 
mode, untargeted
5 min induction 
+ 15 min 
collection
>10 µL 
Frozen at -80°C 
until analysed
Diluted with formic acid and vortexed
LC-QTOF MS/MS, 
untargeted
7 days
Not specified
Sweat patches 
stored and 
transported in 
sterile, BPA-
free 4-oz poly-
propylene 
sample cups; 
no temperature 
specified
Sweat patches extracted with methanol; 
evaporated in Turbovap®; reconstituted with 
ammonium bicarbonate:
ACN (mobile phase)
UHPLC-MS-MS, targeted; 
using methods initially 
designed for urine samples
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Hussain JN et al.
24   Clin Biochem Rev 38 (1) 2017
Study
Aims 
n
Sweat Induction Mode
Methods
Dutkiewicz et 
al., 201459
Untargeted metabolomics 
profiling of human sweat to 
evaluate hydrogel micropatch 
collection linked with direct 
mass spectrometry
9
Passive sampling – in room 
temperature, ~25°C, 45% 
relative humidity
Skin pre-wiped with cellulose tissue soaked 
with isopropanol: H2O; fabricated agarose 
hydrogel micropatch embedded with PTFE 
probe attached to forearm area with adhesive 
bandage tape
Calderon-
Santiago  et al., 
201437
Untargeted global 
metabolomics profiling of 
human sweat to optimise 
laboratory methods and 
chemometrics
96
Webster Sweat Inducer – 
Pilogel® Iontophoretic discs; 
1.5mA electric current x 5 
min
Macroduct® Sweat Collector – part of 
Macroduct® Sweat Analysis System – covers 
forearm skin x 15 min; sample transferred into 
micro-Eppendorf tube
Shetage  et al., 
201423
Identify collection methods 
for RSSC and evaluate effects 
of ethnicity, gender and age 
on amount and composition
315
Passive sampling at room 
temperature: 18–25°C, 50–
60% relative humidity 
Forehead pre-wiped with cotton soaked in 
diethyl ether, allowed to dry. Cigarette paper 
applied, held in place with elastic headband, in 
duplicate x 1 h, fresh cigarette paper replaced 
every hour for total 3 h
Mark  et al., 
201351
Detailed amino acid analysis 
of sweat to better understand 
key biological mechanisms 
governing its composition
12
Hot room; 40°C, 60% relative 
humidity x 15–40 min
Sweat droplets removed from axilla 
with positive displacement pipette using 
polypropylene tips; sample transferred directly 
into ‘low binding’ Eppendorf tube kept at 4°C
Raiszadeh  et 
al., 201229
Untargeted and targeted 
analysis of healthy control 
and schizophrenic patient 
sweat, to identify candidate 
biomarkers of disease
78
Webster Sweat Inducer – 
pilocarpine iontophoresis 
applied to volar forearm 
Macroduct™ Sweat Collector – Macroduct 
™ Sweat Stimulation and Sweat Collection 
System (Elitech/WESCOR, Inc., Logan, UT, 
USA); sample transferred into
micro-centrifuge tubes
Genuis  et al., 
201257 
Targeted profiling of phthalate 
compounds in blood, sweat 
and urine
20
Self-determined by 
participants – infrared sauna, 
steam sauna, exercise
Direct collection from any body site into 500 
mL glass jar using stainless steel spatula; 
participant-delivered to commercial laboratory; 
transferred to 4 mL glass jars at laboratory
Genuis  et al., 
201255
Targeted profiling of BPA in 
blood, sweat and urine
Genuis  et al., 
201156
Targeted profiling of 120 
compounds  (toxicants) in 
blood, sweat and urine
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Clin Biochem Rev 38 (1) 2017   25
Timing
Amount
Storage
Sweat 
Preparation 
Protocols
Analytical Chemistry 
Platforms
1 min–1 h
‘single droplet’ 
– unable 
to estimate 
volume of 
sweat sample 
accurately
Hydrogel 
micropatch probe 
covered with glass 
slide, stored at 4°C
Direct coupling of hydrogel micropatch probe 
to nanospray desorption electrospray ionisation 
mass spectrometer
ESI + IT + FT-ICR-MS
5 min induction 
+ 15 min 
collection
>5μL
Frozen at -80°C
Pooled samples diluted with formic acid:H2O 
with additional protocols: 
(i) hydrolysis with 0.1M NaOH or HCL in H2O, 
vortexed, evaporated to dryness, reconstituted 
in chromatographic mobile phase A;
(ii) solid phase extraction using C18 and 
hydrophilic centrifugal Micro SpinColumn™
LC-QTOF MS/MS, 
untargeted
3 h
Totals not 
specified: 
peak amounts 
0.11–0.12 +/- 
0.06–0.07 mg/
cm2 of RSSC 
collected in first 
hour
None specified
Cigarette papers dehydrated x 2 h; extracted 
with hexane; extract filtered through 0.2 micron 
PTFE membrane, concentrated by purging 
nitrogen
GC/MS, untargeted
~20 min 
>500 µL
Frozen at -70°C
Two methods:
i) ninhydrin derivatisation for amino acid 
automated analyser
(ii )oximation and trimethyl-silylation for 
GC-TOF/MS
Targeted amino acid analysis; 
automated amino acid 
analyser + GC-TOF/MS, 
targeted
30 min
50–60 µL 
Stored on dry ice
Pooled samples:
reduction (dithio-threitol/urea), alkylation 
(iodo-acetamide), overnight enzymatic 
digestion (trypsin/ammonium bi-carbonate), 
quenching (glacial acetic acid, then angiotensin 
II), desalting (C-18 Zip Tips), drying in vacuum 
concentrator, reconstitution in 0.1% formic acid
LC-MS/MS;
LC-MS/MS + spectral 
counting;
MRM-MS verification
No time 
parameters 
around sweat 
collection 
except 
conditional 
within 1 week 
of blood 
collection 
(before/after)
100 mL
Stored at -20°C; 
shipped frozen 
on dry ice from 
Canada to Sweden 
for analysis
Not specified
HPLC/MS, targeted; 
GC/MS, targeted
LC-MS-MS, targeted
ICP-MS, targeted
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Hussain JN et al.
26   Clin Biochem Rev 38 (1) 2017
Exercise/Sauna/Hot Rooms
Other forms of sweat induction used in the studies presented 
in the Table include exercise and sauna activity or exposure 
to elevated temperatures with varying humidity levels. Older 
non-metabolomics studies have suggested distinct differences 
in metabolic content when sweat is obtained from exercise or 
sauna activity, especially in Ca2+ and Mg2+ concentrations.82 
As this is an active area of ongoing research, it cannot be 
assumed that metabolomics studies using exercise and/or 
sauna-produced sweat have interchangeable results. A further 
potential confounder for sweat studies is the humidity level 
of sauna or hot rooms as this may contaminate sweat samples 
with condensation of airborne water droplets potentially 
containing bacteria, viruses, fungi, and/or xenobiotics. This is 
Study
Aims 
n
Sweat Induction Mode
Methods
Lee  et al., 
201191
Untargeted metabolomics 
analysis to determine 
biochemical composition of 
exercise sweat 
48
Exercise on ergometer for 60 
min
Collection with skin patch placed on the lower 
back 
Kutyshenko  et 
al., 201110
Untargeted metabolomics 
analysis to determine 
biochemical composition of 
human sweat 
10
Natural environmental heat
Direct collection from forehead, upper chest, 
upper/lower back, arms using glass pipette or 
glass roller, rolled in tray with dH2O and/or 
sterile spray gun filled with D2O sprayed 
Michael-Jubeli  
et al., 201169
Develop simple analytical 
protocol for qualitative 
characterisation of individual 
SSLs and quantitative 
evaluation of lipid classes
1
Passive sampling
Lipid-free absorbent papers placed on 6 areas 
– forehead, back, thorax, forearm, thigh, calf 
– maintained for 30 min with medical tape; 
removed with tweezers and placed into closed 
vials. Collections repeated 4 times
Penn  et al., 
200792
Test the validity of individual 
odour hypothesis by analysing 
VOCs in sweat, urine and 
saliva
197
Passive sampling
Axillary sweat sampled with devised twister 
PDMS-coated stir bars, held by special rollers, 
placed directly on skin; samples transferred to 
glass vials
Harker et al., 
200628
Untargeted metabolomics 
analysis of human eccrine 
sweat
60
Hot room at 43.3°C, 65% 
relative humidity x 15–40 min
Underarm area wiped, then sweat collected 
with plastic-tipped pipette, sample transferred 
into sealed glass vials
BPA – Bisphenol A; PTFE – polytetra-fluoro-ethylene; PDMS – polydimethyl-siloxane; RSCC – residual skin surface components; SSLs 
Chromatography-Tandem Mass Spectrometry; HPLC-MS/MS – High Performance Liquid Chromatography-Tandem Mass Spectrometry;
Time of Flight/ Mass Spectrometry; LC-QTOF MS/MS – Liquid Chromatography-Quadripole Time Of Flight-Tandem Mass Spectromet
MS – Multiple Reaction Monitoring-Mass Spectrometry; UHPLC-MS-MS – Ultra High Performance Liquid Chromatography-Tandem M
Spectrometry; 1H NMR – Proton (Hydrogen -1 nuclei) Nuclear Magnetic Resonance Spectroscopy; ICP-MS – Inductively Coupled Plasma
*See Appendix (online supplement) for an expanded version of this table including more detailed information of sweat preparation protoco
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Clin Biochem Rev 38 (1) 2017   27
especially the case if direct analysis of sweat without detailed 
cleaning and extraction methods is employed. There is also a 
documented progressive decline in sweat rates when the skin 
is thoroughly wetted and/or with higher humidity conditions, 
referred to as hidromeiosis. This can be followed by an after-
effect of increased sweating when the skin is outside the 
exposed high humidity environment. The timing and rate of 
sweating therefore depends upon ambient temperature and 
humidity.83
Passive Sampling/Physiological Sweating
Some of the studies in the Table used only passive sampling 
of sweat without any imposed modes of sweat induction. 
Most of the time, longer periods of sweat collection were 
Timing
Amount
Storage
Sweat 
Preparation 
Protocols
Analytical Chemistry 
Platforms
Sweat patches 
removed at 
3 time-points: 
10–20min, 
30–40min, 
50–60min of 
exercise; placed 
on dry ice
Not specified
Frozen at -80°C 
until analysis.
Not specified
GC/MS and LC/MS/MS, 
untargeted
 3–5 min 
collection 
+ 7–10 min 
sample 
preparation
>0.56 mL
Sample storage not 
specified; analysis 
performed 10–15 
min after sweat 
collection
Diluted with D2O, centrifuged, transferred to 
standard NMR tube
1H NMR Spectroscopy 
– high resolution, both 
one dimensional and two 
dimensional, untargeted
30 min 
collection
Not specified
Storage of 
unprocessed 
samples not 
specified
Extracted with diethyl ether twice, concentrated 
with rotary evaporation, transferred into 
2 mL vials, dried under nitrogen stream; 
dried extract stored at -20°C until analysis; 
extracts derivatised/trimethyl-silylated; rotary 
evaporated, residue dissolved in isooctane
HTGC-MS, with electron 
impact and chemical 
ionisation
Once each 
fortnight 
sampling over 
10-week period; 
unspecified 
sweat collection 
timing
Not specified
Stored at ~4°C; 
shipped in cooler 
each week from 
Austria to USA for 
analysis
Samples directly analysed with SBSE in 
connection with thermal desorption GC-MS
SBSE with thermal 
desorption GC-MS
15 min 
collection
>50µL
Frozen at -20°C 
until analysis
Samples diluted and deuterated phosphate 
buffer (pH 7.4, 0.1M); transferred into 5 mm 
OK NMR tubes
1NMR Spectroscopy – high 
resolution, one dimensional, 
untargeted
– surface skin lipids; VOCs – volatile organic compounds; ACN – acetonitrile; SBSE – stir bar sorptive extraction; LC-MS/MS – Liquid 
; SRM – selected reaction monitoring; ICP-MS – Inductively Coupled Plasma mass Spectrometry; GC-TOF/MS – Gas Chromatography-
try; ESI – Electrospray Ionisation; IT – Ion Trap; FT-ICR-MS – Fourier Transform Ion Cyclotron Resonance mass spectrometry; MRM-
Mass Spectrometry; GC / MS – Gas Chromatography / Mass Spectrometry; HTGC-MS – High Temperature Gas Chromatography-Mass 
a Mass Spectrometry; PTFE – polytetrafluoroethylene; dH20 – distilled water; -D2O – deuterated, heavy water
ols, chemometrics, databases and key findings pertaining to studies.
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Hussain JN et al.
28   Clin Biochem Rev 38 (1) 2017
deemed necessary to obtain the same or smaller amounts of 
sweat or residual skin surface components (RSCCs). This 
may compromise the results, especially in terms of metabolic 
quenching of enzymatic reactions and metabolite stability. 
With untargeted metabolomics analysis, this is highly relevant 
due to unknown metabolites and thus unknown metabolite 
stability profiles. With more targeted metabolomics, as in 
the case of sweat testing for various drugs of abuse, this 
would depend on desired levels of quantification and the 
relative stability of the target molecule in certain specified 
conditions, necessitating controlled stability studies to be 
done, as discussed more extensively in a recent review by 
de Giovanni and Fucci.1 With newer technological trends 
of sweat analysis requiring smaller amounts of sweat, the 
need for complicated sweat induction methods will likely be 
reduced or redundant.6,59,84,85
Sweat Collection Protocols
A brief overview of the sweat collection methods in the Table 
reveals a diverse range of techniques employed to collect sweat 
for metabolomics studies. These techniques vary from simple, 
direct collection of sweat off skin into microcentrifuge tubes 
or glass jars, to elaborate specifically-designed implements 
(i.e. glass pipettes and rollers, hydrogel micropatches) to 
more commercially-available products like the Macroduct® 
Sweat Collector or PharmChek® sweat patches. Large 
variations between individuals in the amounts and location of 
sweat produced create major difficulties for those attempting 
to design a universal sweat collection device. Skin irritation, 
alterations of skin pH, disruptions to skin barrier properties 
and interactions with differing individuals’ skin microbiota 
are just some of the difficulties to be encountered in designing 
an ideal sweat collecting apparatus.1
Commercial Sweat Collection Devices
The Macroduct (ELITECH Wescor® Inc., Logan, UT, USA) 
is a popular commercially-available sweat collector that 
employs a plastic capillary-coil device of 29 mm diameter to 
wick the sweat off the skin surface, usually of the forearms.86 
Since its introduction in 1986, it has been used in several 
sweat studies, including several addressing sweat metabolome 
optimisation.37,71 The Macroduct® is a component of the 
Macroduct® Sweat Analysis system, involving a commercial 
apparatus covering the skin after iontophoresis stimulation 
using pilocarpine29,37,38,71,72 that was developed to reduce 
the problems encountered with older filter pad- and tissue 
paper-based sweat collections of sweat chloride testing for 
CF.86 Macroduct® helps to overcome issues of background 
contamination, encapsulation (which increases local skin 
temperature and sweat gland secretion) and hidromeiosis 
(the progressive decline in sweat rates that occurs when 
skin is thoroughly wetted and/or with higher humidity) 
encountered with the older methods.86 The Macroduct® has 
a capacity of ~0.1 mL of sweat collection per device session. 
Some researchers have considered placing more than one 
Macroduct® simultaneously to collect larger amounts of 
sweat that are then pooled for analysis, but differing sweat 
rates at different collection sites, amplification of inconsistent 
dilutional effects and difficulty in attaching the Macroduct® 
to other body sites produces confounding results.29
The same corporation (ELITECH Wescor®, Inc., Logan, 
UT, USA) developed a larger version of the Macroduct® 
called the Megaduct®. This is a round, plastic concave-based 
device with a larger collection area of 22.1 cm2 and a central 
aperture through which sweat collects into coiled capillary 
tubing. While the Megaduct® has an increased sweat volume 
capacity of ~0.5 mL,40,87 its utility is limited by the duration of 
heat and/or exercise necessary to sweat long enough to fill the 
Megaduct® reservoir. For example, in one study, it required 
65–75 min to collect the full 0.5 mL reservoir of sweat in 10 
healthy men, with varying exercise intensities (VO2 = 0.5–2.0 
L/min), temperatures (20–40 °C) in a controlled 50% humidity 
environment.87 Increasing sweat collection times to this range 
(>60 min) can potentially impact the power of metabolic 
findings, especially with the issues of metabolic quenching 
and time course of metabolic changes.87 For example, it is 
known that concentrations of sweat electrolytes and minerals 
such as zinc and iron change in relatively short periods of 
time (<30 min).87,88 Furthermore, both the Macroduct® and 
Megaduct® are designed primarily for forearm placement. As 
discussed already, the human body does not have a uniform 
sweat rate or composition over all skin locations. In fact, 
results of one study suggest forearm sweat rate is 30–60% 
less than that of the chest or back.87 
Another popular commercial sweat collection device is the 
PharmCheck® or PharmChek® (PharmChem Inc., Fort 
Worth, TX, USA) sweat patch which has been available 
since 1990 (refer to the sweat collection method used by 
Porucznik et al., 2015 in the Table).89 This device is a non-
occlusive patch consisting of a medical-grade cellulose paper 
absorption pad covered by a thin layer of polyurethane and 
acrylate adhesive. To use it, the skin site must first be cleaned 
with a tolerable solvent (e.g. isopropyl alcohol swabs) and 
thoroughly dried before application. The adhesive film of the 
patch is a semipermeable barrier that allows oxygen, carbon 
dioxide and water (vaporised by body heat) to diffuse freely. 
Larger non-volatile molecules (such as drugs, metabolites, 
metals and other xenobiotics) are retained on the inert 
cellulose absorption pad of the patch. Contaminants from 
the environment cannot penetrate the adhesive barrier from 
the outside once it is in place, enabling the patch to be worn 
during normal activities, including bathing, swimming and 
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Clin Biochem Rev 38 (1) 2017   29
other exercise. It has a release liner that allows removal of 
the collection pad only once from the adhesive layer after 
use thereby preventing removal, reapplication or tampering 
with the patch. Underneath the polyurethane layer is a unique 
9-digit number printed on the patch that is visible through a 
purpose-made window for legal (or research) applications. 
These features make this device useful in sweat testing for 
illicit drugs.1 
Some of the disadvantages of PharmChek® include high 
inter-subject variability (potentially due to variable body 
site placement), high cost, possibility of environmental 
contamination either before patch application or after patch 
removal, risk of accidental removal before desired monitoring 
period and differing rates of drug/metabolite/xenobiotic 
penetration through the membrane, depending upon charged 
or uncharged state. Molecules in an uncharged state have 
been recorded to migrate more rapidly than charged species 
in studies of PharmChek®.1,90
Non-Commercial Sweat Collection Techniques
A newer form of sweat patch with commercial potential 
described by Dutkiewicz et al. is a specifically-designed 
agarose hydrogel micropatch with polytetrafluoroethylene 
(PTFE) support that has been developed for simplified 
collection of very small amounts of sweat that can be analysed 
directly within minutes using various MS platforms.59 
This new method of sweat collection shows promise, but 
still requires further validation and optimisation of signal 
sensitivity and performance at higher temperatures and at 
increased sweat rates.59
Other noncommercial techniques of sweat collection for 
metabolomics studies are also documented in the Table. Lee 
et al. describe a ‘sweat collection patch’ placed on the lower 
back with sweat collected at three time points (10–20 min, 
30–40 min, 50–60 min) while participants exercised on a 
cycling ergometer.91 Sweat was frozen on dry ice, and then 
stored at –80 °C until prepared and analysed. Unfortunately, 
there is limited mention of skin preparation, the type of sweat 
collection patch used, how the sweat is frozen, either intact 
in patch or transferred to another collection tube, or how 
sweat is prepared for untargeted metabolomics analysis.91 
Occlusive skin patches consisting of 2–3 layers of filter paper 
or gauze have been used in other sweat collection studies 
but limitations of excessive pH variations and skin irritation 
with some degree of presumed skin disruption have been 
significant detractors.40
Shetage et al. and Michael-Jubeli et al. both use passive 
sampling with ‘cigarette paper’ and ‘lipid-free absorbent paper’ 
to collect the desired RSCCs or surface skin lipids (SSLs), 
respectively. These collection methods have advantages of 
economics and simplicity but still have the disadvantages of 
encapsulation and hidromeiosis already discussed as well as 
long collection times of 3 h and 30 min respectively.23,69
Kutyshenko et al. describe specially-designed glass rollers 
and glass pipettes for sweat collection. The rollers were used 
on lower sweat-producing regions (e.g. arms) moisturised 
with a sterile distilled water spray gun beforehand whilst the 
glass pipettes were used on heavier sweat-producing areas, 
namely forehead, chest and back.10 Although the use of glass 
is compelling with its relative inertness and is certainly of 
benefit when metabolomically targeting plastics-related 
xenobiotics, the confounders of varied locations of sweat 
harvesting, dilutional effects of adding sprayed distilled water 
and a lack of standardisation of temperature and humidity are 
likely to complicate the untargeted findings of this study.
Penn et al. describe another unique, specially-designed 
method of collecting sweat with a polydimethylsiloxane-
coated stir bar that is rolled directly onto skin. The fact that 
sweat samples can then directly go through the necessary 
extraction step with a thermal desorption GC-MS setup is 
attractive. However, the fact that samples had to be shipped 
at 4 °C overseas to a special laboratory is a limitation and 
raises the issues of sample contamination and metabolite 
degradation during transportation.92
Unsupervised Sweat Collection Techniques
In studies by Genuis et al. and Sheng et al., participants were 
instructed to collect perspiration from any site on their body 
directly into a laboratory-provided, pre-cleaned, acid- and 
water-rinsed 500 mL glass jar or by using a stainless steel 
spatula against their skin to transfer perspiration directly 
into the same laboratory glass jar.55-57,93 Sweat was collected 
within one week before or after specified blood collection and 
participants delivered the collected sweat sample themselves 
to a laboratory without any specified storage or transport 
timeframes. The choice of glass for storage container concurs 
with the previous study discussed above. Including the option 
of stainless steel spatulas for sweat collection is intriguing. 
A grade of stainless steel was reportedly chosen to match 
the composition of laboratory needles used in standard 
blood collections since sweat was being directly compared 
to similarly-targeted detections of compounds in blood and 
urine in these studies. Recognising that stainless steel contains 
varying amounts of primarily iron, nickel and chromium that 
can also be found in trace amounts of physiological sweat, this 
is indeed important to factor in with future sweat metabolomics 
studies.56 However, the relative absence of controlled timing, 
temperature, humidity and storage conditions of sweat 
samples and the presumed delay of a metabolic quenching 
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Hussain JN et al.
30   Clin Biochem Rev 38 (1) 2017
step make the sweat collection protocol of these series of 
studies less than ideal for metabolomics studies.
Direct Sweat Sampling Techniques
In a yet different approach, Harker et al. detailed subjects 
rinsing and drying their axillae with water just before entering 
a hot room (set at 43.3 °C and 65% relative humidity) for 
15–40 min.28 Subsequently, the underarms were ‘wiped’ in an 
unspecified way and sweat was collected with a plastic-tipped 
pipette and transferred directly into glass vials with a collection 
period of approximately 15 min. The glass vials were then 
immediately sealed and stored frozen at –20 °C until analysis. 
This method included several commendable presampling 
controls by limiting use of pharmaceutical medications and 
topical applications of antiperspirant and soap products, 
detailing dietary limitations and specifying shaving of the 
axillary hair. However, the wiping of underarms immediately 
before sweat collection introduces potential issues of altered 
skin integrity on a molecular level which may impact the 
content of sweat analysis with 1H NMR spectroscopy. As 
mentioned in the section discussing sweat functions, the skin 
integrity is thought to influence pathways of water and other 
molecules/metabolites either via transmembrane proteins or 
lipid membranes or sweat glands during fluid transport from 
plasma to skin surface. Not specifying the exact material the 
underarms were wiped with and mixing plastic with glass 
in the sweat collection and storage before analysis create 
additional uncertainty.28
Some of the researchers associated with the Harker et al. 
study went on to publish another study of axillary sweat with 
similar direct sweat collection techniques, but optimised and 
targeted for amino acid analysis with a different metabolomics 
platform of GC-TOF (Time of Flight)/MS instead of 1H NMR 
spectroscopy.51 While direct sampling with minimal handling 
time and prompt metabolic quenching are advantages of this 
method, wiping the armpit before sampling and the use of 
a positive displacement pipette that might disrupt the skin 
surface along with the requirement for trained personnel to 
perform the sampling task remain as drawbacks.51 The choice 
of harvesting sweat from the axillae, rich in apocrine and 
apoeccrine sweat as well as eccrine sweat, in both of these 
studies complicates the comparisons to be made with other 
metabolomics studies harvesting sweat from other specific 
areas of the body with minimal apocrine or apoeccrine 
contributions. 
Some similar advantages and disadvantages are appreciated 
with the sweat collection methods of Jia et al.94 Leg skin 
cleansing with alcohol pads followed by distilled water 
rinsing and drying precede the direct collection of sweat into 
microcentrifuge tubes which were placed immediately on dry 
ice to effect metabolic quenching.94 The cleansing and rinsing 
beforehand, as well as the physical, direct contact with the 
microcentrifuge tube may however disrupt the skin surface 
and again potentially alter skin integrity with its possible 
effects on fluid migrations from plasma to skin surface. 
Summary
A diverse range of sweat induction modes and sweat 
collection methods are presented in the Table, all with 
their own advantages and disadvantages. Issues of variable 
location, timing and amounts of sweat induction and sampling 
as well as inconsistent sample processing steps and storage 
conditions confound most comparisons between methods. 
Optimising these parameters and exploring newer identified 
concepts surrounding sweat collection based upon updated 
information about sweat glands and the collective contents 
of their secretions will generate more meaningful results to 
build and improve our knowledge of the sweat metabolome. 
Standard operating protocols (SOPs) for collecting human 
biofluids like urine, blood and sweat for metabolomics studies 
are crucial to help control for the wide variety of factors that 
can influence metabolite concentrations. The SOPs for human 
sweat collection require updating beyond cystic fibrosis and 
illicit drug testing models to optimise metabolomics results. 
The following considerations need attention in future studies:
•	
Specifying body sites of human sweat collection is 
of utmost importance in future comparisons of both 
targeted and untargeted metabolomics studies. Not all 
sweat collected anywhere on the body can be assumed 
homogenous in metabolic content. 
•	
Until further comparative studies are done, consideration 
should be given to subclassifying sweat based upon 
induction approaches – i.e. pilocarpine-induced sweat 
vs physiological sweat vs thermally-induced sweat vs 
exercise-induced sweat. 
•	
Measures to ensure adequate skin integrity other than 
mere visual inspection at sweat collection sites need 
further development and study.
•	
Examining the molecular content of sweat induction, 
collection and storage devices for potential adsorption 
and metabolic reactivity requires further attention and 
attempts at standardisation.
•	
Minimising the timing of sweat collection, transport 
and storage as well as ensuring a timely and adequate 
metabolic quenching step is important for comparing 
future sweat metabolome studies.
•	
Environmental factors of temperature and humidity 
significantly impact the metabolic parameters of sweat and 
need to be specified and ideally standardised. Furthermore, 
the potential inter-relationship between overall core body 
temperature and local skin temperature at sweat collection 
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Sweat Collection for Metabolomics Analysis
Clin Biochem Rev 38 (1) 2017   31
sites may impact sweat metabolomics results.
•	
Attention to controlling individual variables such as 
diet content, fasting vs postprandial state, exercise state, 
emotional state, pharmaceutical and/or recreational drug/
supplement use and underlying medical conditions that 
could impact the pH or overall metabolic state is important 
when interpreting any metabolomics results. This applies 
to controls instituted both preceding and during collection 
of sweat.
•	
Defining the minimum amounts of sweat necessary to 
overcome intra-individual and inter-individual global 
metabolomic differences, stretching beyond guidelines 
based upon CF-specific testing of pilocarpine-induced 
sweat, is still a work in progress. Clarifications between 
physiological sweating and exercise- or thermally-induced 
sweating within this context are also necessary.
•	
Age-specific influences on sweat metabolomics results 
will require further investigation.
•	
Attempts at simultaneously characterising the individual 
skin microbiota (colonising bacteria, viruses, fungi, etc.) 
both quantitatively and qualitatively at sites of sweat 
collection might further elucidate suspected important 
metabolic relationships.
Conclusion
Better standardising of human sweat induction and collection 
methods to address the important challenges identified in this 
review is a key step to furthering sweat metabolomics. If this 
can be achieved, it is anticipated that sweat may become a 
more utilised biofluid capable of delivering easily accessible, 
individualised 
and 
instantaneously 
useful 
metabolic 
information that significantly enhances our knowledge of 
human health and disease. 
Acknowledgements: This manuscript was developed as 
part of study conducted by Dr Joy Hussain during her PhD 
candidature. We wish to thank the Judy Jacka Foundation of 
Natural Therapies for providing an academic scholarship to 
support her candidature and therefore this study.
Competing Interests: None declared.
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Case 1:21-cr-10270-MLW     Document 70-4     Filed 04/06/22     Page 22 of 22

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