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CHROMB-19792; No. of Pages 11 ARTICLE IN PRESS

Journal of Chromatography B, xxx (2015) xxx–xxx

Contents lists available at ScienceDirect

Journal of Chromatography B
journal homepage: www.elsevier.com/locate/chromb

Chromatographic and mass spectrometric techniques in studies on

oxidative stress in autism
Joanna Kałużna-Czaplińska ∗ , Jagoda Jóźwik-Pruska
Department of Chemistry, Institute of General and Ecological Chemistry, Lodz University of Technology, Lodz, Poland

a r t i c l e i n f o a b s t r a c t

Article history: Healthy body is characterized by the presence of a dynamic and balanced equilibrium between the pro-
Received 30 July 2015 duction of reactive oxygen species (ROS) and the antioxidant capacity. In oxidative stress this balance
Received in revised form is switched to reactions of oxidation leading to increased production of ROS, exceeding the capacity of
17 December 2015
physiological antioxidant systems. Oxidative stress is known to be linked to many disturbances, disor-
Accepted 18 December 2015
ders and diseases. One of these is the autism spectrum disorder (ASD). ASD is a neurodevelopmental
Available online xxx
disorder manifested by abnormalities in social communication and interaction, as well as by occurrence
of repetitive, restricted patterns of behavior or activities. It is believed that adequate knowledge about
Keywords:
Autism
the oxidative stress biomarkers and the possibility of their reliable measuring could be useful in broad-
Biomarkers ening knowledge on various diseases including ASD. A high number of compounds have been proposed
Chromatography as biomarkers of oxidative stress. Some of these are connected with the severity of ASD. The present
Mass spectrometry review gives a summary of the chromatographic techniques used for the determination of biomarkers
Oxidative stress for oxidative stress in autism, and of other compounds important in this context. The first part of the
review focuses on the correlation between oxidative stress and autism. The second part describes appli-
cations of chromatographic and mass spectrometric methods to the analysis of different metabolites
connected with oxidative stress in biological fluids of autistic children. Advantages as well as disadvan-
tages of the application of these methods for the analysis of different types of oxidative stress biomarkers
are discussed.
© 2015 Elsevier B.V. All rights reserved.

1. Introduction ondary antioxidant enzymes include glutathione reductase and

Chemically, an atom is usually composed of a central nucleus
with pairs of electrons orbiting around it. Yet, some atoms in
molecules have unpaired electrons which tend to form pairs with
other electrons, thus making free radicals highly reactive and unsta-
ble [1].
In a normal and healthy body there is a dynamic balanced equi-
librium between the production of reactive oxygen species (ROS)
•
such as superoxide (O2 − ), peroxyl, hydroxyl, alkoxy, and nitric
oxide (NO) free radicals [2], and the antioxidant capacity of the cell
[3]. In states of elevated oxidative stress this balance is disturbed
and switched in favor of ROS [4].
ROS are inactivated within the cell by antioxidant defense
mechanisms. Primary antioxidant enzymes include catalase, super-
oxide dismutase (SOD) and glutathione peroxidase (GPx). These
enzymes are directly involved in the elimination of ROS. Sec-
∗ Corresponding author. Fax: +48 426313103.
E-mail address: jkaluzna@p.lodz.pl (J. Kałużna-Czaplińska).
glucose-6-phosphate dehydrogenase which help maintain a steady
concentration of NADPH and glutathione (GSH), which are essential
for the proper functioning of the whole antioxidant defense sys-
tem [5,6]. To ensure effective antioxidative mechanism and optimal
catalytic activity of these enzymes, the presence of micronutrients,
including selenium, copper, iron, zinc and manganese, is necessary.
Glutathione, ceruloplasmin, transferrin, vitamin E and vitamin C
also participate in the antioxidant defense system [7–9].
ROS have many harmful effects on numerous biomolecules
including lipids and proteins, which result in structural and func-
tional disorders at molecular and cellular levels [10,11]. The
mitochondrial respiratory chain is a natural endogenous source
of ROS in the human body. ROS may also have exogenous origin,
e.g., toxins present in the environment or cigarette smoke, drugs,
xenobiotics, or radiation [1].
Nevertheless, it should be highlighted that oxidative stress is
desirable in some cases. First of all oxidative stress induces apo-
ptosis to prepare the birth canal to delivery. Additionally, oxidative
stress affects strengthening the biological defense mechanisms
during ischemia [1].

http://dx.doi.org/10.1016/j.jchromb.2015.12.035
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Human brain consumes about 20% of metabolic oxygen and the

Nomenclature main portion of energy is used by neurons [12]. These cells are
characterized by a limited antioxidant capacity and require high
3CT 3-Chlorotyrosine amounts of energy, lipids and iron [13]. Therefore, it is prone to
3NT 3-Nitrotyrosine oxidative stress. The main role of antioxidants is to counter attacks
5cxP Pentacarboxyporphyrin of neurons by ROS, which is particularly important in the early criti-
8-OHdG 8-Hydroxy-2’deoxyguanosine cal period [14]. It can be assumed that children who have lower GSH
ASD Autism spectrum disorder levels are exposed more often to oxidative stress [9]. Additionally,
ATEC Autism treatment evaluation checklist other external factors such as environmental and metabolic factors
cP Coproporphyrin may intensify this process.
DLI Direct liquid introduction The role of oxidative stress is considered important in the
EC Electrochemical context of many diseases, including neuropsychiatric diseases
ECD Coulometric electrochemical detection [10,15,16], anxiety disorders [17], and major depressive disorder
ELISA Enzyme-linked immunosorbent assay [18]. According to the Diagnostic and Statistical Manual of Men-
EP European Pharmacopoeia tal Disorders (5th edition, DSM-5), the autism spectrum disorder
F2-IsoPs F2-Isoprostanes (ASD) is manifested by abnormalities in social communication and
GABA Gamma-aminobutyric acid interaction as well as by the occurrence of repetitive and restricted
GC/NICI-MS/MS Chromatography/negative ion chemical patterns of behavior or activities. ASD represents a single con-
ionization-tandem mass spectrometry tinuum of impairments with varying degrees of severity [19]. It
GC–MS Gas chromatography-mass spectrometry is suggested that this disorder may result from an interaction
GC–MS/MS Gas chromatography-tandem mass spectrome- between immunological, environmental and genetic factors, with
try oxidative stress being the linking mechanism [20].
GID Gastrointestinal dysfunction Oxidative stress can be detected by the examination of antioxi-
GLC–MS Gas liquid chromatography dant status, antioxidant enzymes, protein/DNA oxidation, and lipid
GPx Glutathione peroxidase peroxidation. All of them are considered significant in children
GR Glutathione reductase with ASD, including the glutathione system (reduced glutathione-
GSH Glutathione oxidized glutathione), methionine, cysteine, transferrin, urine
GSSG Glutathione disulfide 8-OHdG, ceruloplasmin, 3-chlorotyrosine (3CT), 3-nitrotyrosine
HCY Homocysteine (3NT) [21], and F2t-isoprostanes (F2-IsoPs) [22].
Hg Mercury This review focuses on the use of chromatographic techniques
HOCl Hypochlorous acid coupled with mass spectrometry for the determination of biomark-
HPLC High-performance liquid chromatography ers of oxidative stress in autism.
HPLC-TOF-MS High-performance liquid chromatography-
time of flight- mass spectrometry
LC–MS Liquid chromatography-mass spectrometry 2. Biomarkers of oxidative stress
LC–MS/MS Liquid chromatography-tandem mass spectrom-
etry 2.1. Biomarkers
LC-TOF-MS Liquid chromatography-time of flight- mass
spectrometry As long as the main cause of ASD is not clear, there is a need
LOD Limit of detection for searching for biomarkers being specific to this disorder. A lim-
LOQ Limit of quantification ited understanding of its etiology makes it difficult to diagnose in
MDA Malondialdehyde its earliest stage. However, studies on the determination and iden-
NADPH Nicotinamide adenine dinucleotide phosphate tification of ASD biomarkers are believed to be a powerful tool in
PDD-BI Pervasive development disorder behavior inventory predicting the development of this disorder. This assumption raises
prcP Precoproporphyrin the question of the definition of a biomarker. This term refers to a
ROS Reactive oxygen species wide range of medical sings. According to the definition presented
SAH S-Adenosylhomocysteine by The National Institute of Health Biomarkers Definition Working
SAM S-Adenosylmethionine Group [23], a biomarker is “a characteristic that is objectively mea-
SAS Severity of autism scale sured and evaluated as an indicator of normal biological processes,
SIM Selected-ion monitoring pathogenic processes or pharmacologic responses to a therapeu-
SOD Superoxide dismutase tic intervention”. Another definition assumes that a biomarker or
SST System-suitability Test a biological marker is a measureable substance, a structure or pro-
tGSH Total glutathione cess which occurs in the body, or its products having an influence
TOF Time of flight or making it possible to predict a disease [24]. WHO describes the
UPLC Ultra-performance liquid chromatography biomarker as “almost any measurement reflecting an interaction
UPLC–MS Ultra-performance liquid chromatography-mass between a biological system and a potential hazard, which may be
spectrometry chemical, physical, or biological. The measured response may be
WHO World Health Organization functional and physiological, biochemical at the cellular level, or a
molecular interaction” [25].
Searching for new biomarkers is currently a very popular direc-
Considering all the above, sound knowledge about oxidative tion in research. Hence, the question about abusing this term should
stress biomarkers and the availability of reliable analytical meth- be asked. Literature presents some requirements that should be
ods for the measurement in biological samples are indispensable met in order to name a potential factor “biomarker” [26].
for scientists from various disciplines to gain deep insights into the A wide range of compounds occurring in the human body are
pathological features of diverse diseases as well as to assess the characterized by reference ranges. Disturbances in their concen-
efficiency of medications. trations may lead to abnormal functioning of the body, various

Please cite this article in press as: J. Kałużna-Czaplińska, J. Jóźwik-Pruska, Chromatographic and mass spectrometric techniques in
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disorders, or diseases. Nowadays, a great number of databases
for those compounds are available. One of these is Human
Metabolomic Database [27].
2.2. Consequences of oxidative stress in ASD
It is well known that molecular oxygen is essential for aero-
bic life, yet excessive amounts of its metabolic by-products are
toxic. This “oxygen paradox” raises the theory of oxidative stress
[28]. Free radicals participate in cellular signaling, mitosis and
physiological immunological responses. However, because of their
instability and the presence of unpaired electrons, they can dam-
age lipids, proteins, nucleic acids and carbohydrates [29]. Due to the
fact that especially brain requires large amounts of oxygen, oxida-
tive stress is thought to be a significant factor in the pathogenesis
of psychiatric disorders [30]. The role of oxidative stress in autism
is also considered. Obtained results seems to be promising and may
become an important element both in pointing biomarkers of this
disorder as well as in establishing proper therapy aiming to the
mitigation of occurring symptoms [21,22,30,31].
One of the disorders that have been linked to elevated oxida-
tive stress and lower anti-oxidant capacity is autism [1,20,31].
Oxidative stress influences membrane phospholipids, the immune
system, metabolic pathways involved in energy, and excitotoxicity.
It is believed that this influence results in clinical symptoms and in
the pathology of ASD [30]. A high number of cell membrane pro-
teins are attached to membrane phospholipids or embedded in its
structure. Thus, membrane phospholipid abnormalities affect the
functioning of these proteins. Additionally, the production of lipid
peroxides and their by-products leads to the loss of integrity and
functions of the cell membrane [32].
Literature also reports on impaired immune system response
and increased inflammatory response in ASD patients connected
with oxidative stress [33,34]. Excitotoxicity is considered to con-
tribute to oxidative stress, simultaneously being the result of the
process. An increased concentration of extra-cellular glutamate
(confirmed in autism) and a reduced concentration of gamma-
aminobutyric acid (GABA) are reported to increase excitotoxicity
[35].
One of the most commonly found abnormalities in autism is
mitochondrial dysfunction which is marked by impaired energy
production. Mitochondrial dysfunction may result from many fac-
tors such as vitamin B6 or iron deficiency, elevated nitric acid,
exposure to environmental toxicants, or oxidative stress [21].
Patients with ASD are often supplemented with antioxidants in
order to minimize some symptoms of this disorder. Supplements
such as vitamin C, vitamin B6 (especially in combination with mag-
nesium), zinc, and fish oil are included in the diet [36].
Scheme 1 shows the relationship linking oxidative stress to ASD.
2.3. Oxidative stress biomarkers in ASD
Human body is not fully protected against oxidative damage.
Some of its constituents are at risk of injury by ROS. The reaction
of ROS with biomolecules results in the formation of oxidatively
modified products. Unlike the ROS, their reaction products with
biomolecules are chemically stable and are therefore often used as
biomarkers of oxidative stress [1].
Children with ASD are known to suffer from metabolic abnor-
malities which are related to the interconnected pathways of
glutathione, methionine and folate metabolism [37,38]. Among the
compounds which have been found to be important in ASD, GSH is
one of the most frequently studied. GSH is composed of cysteine,
glutamate and glycine and serves as a major intracellular redox
buffer. Under conditions of chronic ROS formation, GSH can become
depleted leading to disruption in redox homeostasis. Under nor-
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3
mal conditions, NADPH-dependent enzyme glutathione reductase
(GR) converts oxidized glutathione (GSSG) to GSH maintaining an
optimal GSH/GSSG redox ratio. Yet, under conditions of elevated
oxidative stress, GSSG is exported by the cell in order to regain
intracellular redox homeostasis. This may result in a decrease in the
total amount of GSSC available to recycle to GSH. Moreover, GSH
participates in the elimination of toxins, including xenobiotics and
heavy-metals, from cells [39,40]. According to the literature GSH
concentration and GSH/GSSG ratio are decreased, whereas GSSG
concentration is increased in ASD patients [31,38,41].
The regeneration of methionine to homocysteine occurs in the
methionine cycle. Methionine, activated by methionine adenosyl-
transferase, forms S-adenosylmethionine (SAM), which is the basic
methyl donor for most cellular methyltransferase reactions, includ-
ing the methylation of proteins, RNA, DNA, and neurotransmitters.
The methyl group from SAM can be transferred to various accep-
tors, leading to the formation of S-adenosylhomocysteine (SAH)
and adenosine. However, homocysteine may also be remethylated
to methionine or be removed from the methionine cycle. A decrease
in methionine results in a decrease in the synthesis of SAM, as well
as in a decreased synthesis of cysteine and GSH [31]. Methylation
abnormalities in children with ASD are reported in the literature
[38,42,43] (Scheme 2).
Homocysteine also serves as a biomarker for possible deficien-
cies of some vitamins, including vitamins B6 , B12 , and folic acid.
This amino acid is metabolized via remethylation to methionine or
transsulfuration to cysteine [44–46]. The former involves folic acid
and vitamin B12 , while the latter requires vitamin B6 . Accumulation
of homocysteine occurs when either of these pathways is defected
[46].
8-Hydroxy-2’deoxyguanosine (8-OHdG) can be measured in
urine or in DNA by various methods. This compound is biomarker
for oxidative damage to DNA [21,47]. It is commonly determined
despite there are confounding factors and intra individual varia-
tions. Additionally, the results obtained from independent studies
are inconsistent [21]. Literature reports that the elevated levels of
urinary 8–OHdG in ASD patients are not significant [20,21].
Isoprostanes are prostaglandin-like molecules produced in vivo
via nonenzymatic free radical-catalyzed peroxidation of arachi-
donic acid [48,49]. The first reports on the formation of these
compounds by auto-oxidation of polyunsaturated fatty acids date
back to the mid-1970s [50]. Yet, only about 20 years later it was
proved that they are formed in vivo in human body [48]. The F2 -
isoprostanes group may consist of up to 64 compounds that are
isomeric in the structure to the cyclooxygenase-derived PGF2 ␣
[51]. Measurement of F2 -isoprostanes is now considered one of the
most reliable tools to assess the status of oxidative stress in vivo.
These substances have been found to be elevated in ASD patients
[13]. However, it should be mentioned that some F2 -isoprostanes,
notably 8-iso-PGF2 ␣, may also be produced from arachidonic acid
by the cyclooxygenase, and that PGF2 ␣ may also be formed nonen-
zymatically from arachidonic acid.
Plasma 3-chlorotyrosine (3CT), a measure of myeloperoxi-
dase activity, is well known biomarker of chronic inflammatory
response. 3CT is formed by the reaction of p-tyrosine with
hypochlorous acid (HOCl) [52]. It is reported that the concentra-
tion of plasma 3CT is connected with mitochondrial dysfunction
and increases with age [53]. The literature [53] reports that plasma
levels of 3CT in ASD children are significantly higher as compared
with healthy children. Moreover, autistic patients who met crite-
ria for probable or define mitochondrial disease were characterized
by higher levels of 3CT than autistic patients without any biological
markers or symptoms of mitochondrial disease. However, elevated
concentration of 3CT in ASD children may suggest the presence of
chronic inflammation.
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Scheme 1. Pathway linking oxidative stress to ASD.

It is believed that both increased exposure to pro-oxidants, production of oxidative markers and decreased production of antioxidants may lead to production of free radicals
and the occurrence of oxidative stress in ASD patients.

Scheme 2. Pathway of formation of oxidative stress biomarkers.

Scheme 2 presents transmethylation and transsulfuration pathways. Transmethylation pathway results in formation of homocysteine. A methyl group is needed for methio-
nine regeneration and formation of homocysteine. Its donor are both methylcobalamin and SAM. Transmethylation and transsulfuration pathways are linked by homocysteine,
which can be used again in trasmethylation cycle or removed resulting in formation of cysteine (essential for synthesis of glutathione). Symbols ↓ and ↑ in the scheme refers
to noticed tendency in concentrations of specific compounds to decrease and increase, respectively.

3-Nitrotyrosine (3NT) is as a specific marker for the nitrating
species such as peroxynitrite from which it is formed. Additionally,
it can serve as a biomarker of neuron death and a measure of chronic
immune activation. Although it has been widely studied with the
use of various techniques, its levels are reported to differ 30-fold
among different studies [54]. It can be explained by the assumption
that 3-NT can be generated easily ex vivo in the process of sample
preparation and analysis [54,55]. Its measurement has been cor-
related with the measurement of the cognitive function, behavior
and the development for patients with ASD and mitochondrial dys-
function, but not for the patients with ASD without a mitochondrial
dysfunction [53].
The major antioxidant proteins synthesized in some tissues
including the brain are ceruloplasmin and transferrin. Cerulo-
plasmin is ␣2-serum glycoprotein responsible for the transport
of copper ions in blood. Its role in the metabolism of copper is
essential. Moreover, in red blood cell membranes ceruloplasmin
protects polyunsaturated fatty acids form ROS. Transferrin can be
found in body fluids, yet its higher concentrations occur in serum.
Its main role is the transport of iron to proliferating cells and it
serves as an antioxidant reducing the level of free ferrous ions [56].
Literature reports mainly about their quantification using a colori-
metric method for ceruloplasmin described by Karl et al. [57]. and
Buglanov et al. [58]. method for transferrin Plasma levels of ceru-

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loplasmin and transferrin were shown to be decreased in autistic
patients [56,57,59].
SOD is found in the cytosol of most cells. Ceruloplasmin and SOD
are reported to play a significant role in some neurodegenerative
diseases such as Parkinson’s disease, Alzheimer’s disease, Down’s
syndrome [60]. In the older literature [61], an increased activity
of superoxide dismutase was found. Yet, in more recent reports
[62] the activity of SOD in erythrocytes has been noticed to be sig-
nificantly lower in ASD patients. Other studies present normal SOD
activity in the plasma of ASD subjects compared to controls [60,63].
The most studied product of polyunsaturated fatty acids perox-
idation is malondialdehyde (MDA) [64,65]. The literature reports
that the mean plasma level of MDA is significantly higher among
patients with ASD compared to controls, but only for patients being
younger than 6 years of age. In older subjects there is no significant
difference between these two groups regarding MDA [65].
It should be highlighted that levels of some metabolites may
be related to autism severity. Scientists [66] associated several
of the biomarker groups, including vitamins (vitamin B6, vita-
min C, n-methyl-nicotinamide), minerals (calcium, iron, zinc),
primary amino acids, secondary amino acids and the group of Sul-
fation/Methylation/Glutathione/Oxidative Stress, with severity of
this disorder (assessed using the Pervasive Development Disorder
Behavior Inventory (PDD-BI), Autism Treatment Evaluation Check-
list (ATEC), and Severity of Autism Scale (SAS)).
2.4. Positioning chromatographic and mass spectrometric
methods in the studies of oxidative stress in autism
Many potential biomarkers have been considered in oxidative
stress assessment and many analytical techniques have been pro-
posed and used for its measurement. The Yagi [67] method is
widely applied in both clinical and experimental studies, and is one
of the most famous methods used to evaluate lipid peroxidation.
The approach is based on the measurement of substances which
react with thiobarbituric acid (TBA), the so called thiobarbituric
acid reactive substances (TBARS). Recently, chromatography-based
techniques have become a powerful tool in the determination of
oxidative stress biomarkers.
In order to ensure reliability of obtained results, some
requirements must be met. For this purpose scientists use a system-
suitability test (SST) which is an essential part of chromatography.
Requirements for chromatographic analyses are specified by Euro-
pean Pharmacopoeia (EP) and demonstrate that a chromatograph
is fit for the analysis. System suitability tests are run each time an
analysis is undertaken and each SST is specific for an individual
method [68]. SST is applied to verify whether a planned analy-
sis will be performed by a chromatographic system with correct
detection sensitivity, reproducibility and resolution. The quality of
the results is demonstrated by using quality control (QC) systems
involving procedures and techniques performed to ensure that
quality requirements (see for example Good Laboratory Practice,
quality-assurance system (ISO 9001) and testing and calibration
(ISO 17025) containing general rules [69]).
A wide range of techniques, including for example spectropho-
tometric analyses [59,61,70], ELISA [71–73], radioimmunoassay
[73], have been used to measure oxidative stress biomarkers. Not
only do they allow identification of these markers but they also
enable their reliable quantification in different biological matrixes
including whole blood, plasma, and erythrocyte.
Mass spectrometry has been used for more than 60 years. By
the 1950s, this technique was a well-established technology. It
enabled analyses of volatile compounds in the petroleum, chem-
ical and pharmaceutical industries. Yet, the desire for availability
of rapid, online separation method was growing. Chromatography,
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5
then just coming to the market, appeared to be a key to success
[74,75].
The first coupling of chromatography was explored in 1955
by Roland Gohlke and Fred McLafferty of Dow Chemical Com-
pany, who combined homemade gas chromatograph with a
time-of-flight (TOF) instrument. In 1968 Victor Tal’rose published
information (in Russian Journal of Physical Chemistry) about the
first attempt to connect LC–MS. In 1973 Baldwin and McLafferty
developed direct liquid introduction (DLI) LC–MS interface, which
was capable of generating a stable ion beam in the mass spectrom-
eter [74,75].
Chromatographic methods are among the most frequently used
coupled techniques in the field of study of oxidative stress in autism.
Chromatographic techniques are characterized by many advan-
tages especially in describing the composition of many biological
matrix including body fluids.
GC–MS has been employed in clinical biochemistry and urinary
analysis for more than 30 years. Generally, this technique has slight
sample requirements and enables rapid analyses and reduced use of
expensive labeled substrates. Additionally, it allows obtaining mea-
sureable results. Yet there are some disadvantages, such as lengthy
sample preparation (hydrolysis/derivatization), and incapability of
detection of non-volatile, polar and thermally labile compounds
[76–78]. LC–MS and LC–MS/MS are among the most powerful tech-
niques used in the study of organic compounds. LC–MS has been
applied in the study of urine for about 30 years. The improvement of
liquid chromatography by the development of ultra-high-pressure
or ultra-performance techniques resulted in high chromatographic
peak resolution and reduced time of analysis while maintaining
high-quality mass spectrometry [76,80].
GC–MS/MS, UPLC–MS, HPLC–TOF–MS, LC–TOF–MS have also
been used for the measurement of oxidative stress biomarkers. Yet,
GC–MS, HPLC–MS, LC–MS, and LC–MS/MS are of particular interest.
The most common coupling technique for chromatography is
mass spectrometry (MS). In clinical studies almost all mass spec-
trometers are single MS or tandem mass (MS/MS) coupled to LC or
GC. Tandem mass spectrometry is characterized by high selectiv-
ity, sensitivity and minimum of interferences. Yet, when used in
the same study population, the obtained results were reported to
be similar [79].
Homocysteine (HCY) was measured with the use of GC–MS in
urine of autistic (n = 34) and healthy (n = 21) children and found to
be higher in the ASD group (2.36 ± 1.24 vs. 0.76 ± 0.31 mmol/mol
creatinine). The method was validated for recovery, precision,
limit of quantification (LOQ), limit of detection (LOD), and linear-
ity. Sample preparation was based on a simultaneous extraction
(chloroform) and derivatization (pyridine, ethanol and ethylchlo-
roformate). Elevated HCY concentration may correlate with
deficiencies of folic acid and vitamins B6 and B12 in nutrition [80].
Further studies showed the impact of supplementation with either
folic acid or vitamins B6 and B12 . The concentration of HCY in urine
samples was measured in autistic patients before (n = 30) and after
intake (n = 24) of these supplements or after supplementation with
vitamins B6 and B12 alone (n = 6). The obtained results revealed that
both supplementations led to the decrease in the HCY level, yet the
intake of the mentioned vitamins and folic acid was more effective
in lowering the urinary levels of HCY [81]. In order to determine
HCY in blood samples, James et al. [31]. employed HPLC. Plasma
concentration of HCY was lower in ASD individuals (5.8 ± 1.0 M;
n = 20) compared to healthy subjects (6.4 ± 1.3 M; n = 33).
Oxidized and reduced glutathione (total glutathione tGSH) in
plasma samples of ASD and healthy patients were measured by
HPLC with electrochemical detection. Authors used metaphospho-
ric acid to precipitate protein. The tGSH level was significantly
lower in ASD individuals (4.1 ± 0.5 vs. 7.6 ± 1.4 M), while higher
levels of oxidized glutathione in autistic subjects were measured
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Table 1
Determination of the selected biomarkers of oxidative stress in human body fluids with the use of chromatography-based techniques, applied parameters and exemplary results.

Compound Function in human body Disturbance in ASD Reported levels in ASD Matrix Chromatography- Chromatographic conditions Ref.
patients patients based
technique

Glutathione Critical endogenous Decreased 4.1 ± 0.5 ␮mol/L Plasma HPLC Reverse-phase [31]
antioxidant & detoxifer 5-␮m C18 column
Decreased Data presented in figure Cerebellum and HPLC Reverse-phase [41]
∼5 nmol/mg protein temporal cortex 5-␮m C18 column; isocratic
elution sodium phosphate
monobasic, monohydrate,
ion-pairing reagent OSA,
acetonitrile, pH 2.7 (85%
phosphoric acid)

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Oxidized glutathione Increased Increased 0.55 ± 0.2 ␮mol/L Plasma HPLC Reverse-phase [31]

ARTICLE IN PRESS
concentration of GSSH 5-␮m C18 column
is connected with Increased Data presented in figure Cerebellum and HPLC Reverse-phase [41]
decreased levels of ∼1 nmol/mg protein temporal cortex 5-␮m C18 column; isocratic
glutathione- a major elution sodium phosphate
antioxidant monobasic, monohydrate,
ion-pairing reagent OSA,
acetonitrile, pH 2.7 (85%
phosphoric acid)
Increased 0.447 ± 0.13 nmol/ml Plasma HPLC-MS/MS Not given [65]

Homocysteine Essential for proper Decreased 5.8 ± 1.0 ␮mol/L Plasma HPLC Reverse-phase [31]
development of 5-␮m C18 column
nervous system. source Increased 2.36 ± 1.24 mmol/mol Urine GC–MS Full scan (m/z 50–500) SIM (m/z [78]
of methyl groups creatinine 56,128,234)

Isoprostanes Formed in vivo by Increased F2␣2-VI: Urine TLC with According to method described by [85]
nonenzymatic free 5.2 ± 0.5 ng/mg creatinine GC–NCI–MS Lawson et al. [89]
radical-catalyzed Increased F2-IsoPs: Plasma GC–NCI–MS Ion m/z 569 to ion m/z 573 ratio [22]
peroxidation of ASD-GID 53.6 ± 24.4 pg/mg method described by Milne [90]
arachidonic acid total protein and
ASD-only Morrow [91]
36.5 ± 13.3 pg/mg total
protein

3NT Biomarker of neutron Increased Data presented in figure Cerebellum and HPLC 5-␮m C18 column; isocratic [41]
death and a measure of ∼80 pmol/mg protein temporal cortex elution sodium phosphate
chronic immune monobasic, monohydrate,
activation ion-pairing reagent OSA,
acetonitrile, pH 2.7 (85%
phosphoric acid)
Increased 16.6 ± 7.8 ␮g/l Plasma HPLC–MS/MS Not given [65]

3CT Serves as a marker for oxidative Increased Data presented in figure Cerebellum and HPLC Reverse-phase [41]
damage; has been shown to cause ∼70 pmol/mg protein temporal cortex 5-␮m C18 column; isocratic
endothelial dysfunction and elution sodium phosphate
production of free-radical monobasic, monohydrate,
ion-pairing reagent OSA,
acetonitrile, pH 2.7 (85%
phosphoric acid)
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Table 1 (Continued)

J. Kałużna-Czaplińska, J. Jóźwik-Pruska / J. Chromatogr. B xxx (2015) xxx–xxx

Compound Function in human body Disturbance in ASD Reported levels in ASD Matrix Chromatography- Chromatographic conditions Ref.

ARTICLE IN PRESS
patients patients based
technique

Methionine Essential amino acid; Decreased 19.3 ± 9.1 ␮mol/L Plasma HPLC Reverse-phase [31]
builds various protein 5-␮m C18 column
molecules, participates 22 ± 9 ␮mol/L Plasma HPLC with photodiode  = 254 nm [81]
in the synthesis of array detection
cysteine

SAM Common cosubstrate Decreased 75.8 ± 16.2 nmol/L Plasma HPLC Reverse-phase [31]
involved in methyl 5-␮m C18 column
group transfers, 214.5 ± 15 ␮mol/dl RBC HPLC–MS/MS not given [65]
transsulfuration, and
aminopropylation
SAH Formed by the Increased 28.9 ± 7.2 nmol/L Plasma HPLC Reverse-phase [31]
demethylation of SAM; 5-␮m C18 column
intermediate in the 44.6 ± 8.0 ␮mol/dl RBC HPLC–MS/MS Not given [65]
synthesis of cysteine
and adenosine.
Adenosine Plays important role in Increased 0.39 ± 0.2 ␮mol/L plasma HPLC Reverse-phase [31]
energy transfer and 5-␮m C18 column
signal transduction; 23.2 ± 5.9 10–8 mol/l plasma HPLC–MS/MS Not given [65]
neuromodulator;
regulates a blood flow

7
 8

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studies on oxidative stress in autism, J. Chromatogr. B (2015), http://dx.doi.org/10.1016/j.jchromb.2015.12.035
Please cite this article in press as: J. Kałużna-Czaplińska, J. Jóźwik-Pruska, Chromatographic and mass spectrometric techniques in

Table 2
Pitfalls in the determination of the selected biomarkers of oxidative stress with the use of various methods.

Compound Pitfalls/interferences in Description Studied body fluid Type of studies Used method Ref.
determination

15(S)-8-iso- Artefactual formation Formation of 15(S)-8-iso-PGF2 ␣ from Plasma In vitro GC–MS/MS [94]
prostaglandin prostaglandin (PG) h synthases

J. Kałużna-Czaplińska, J. Jóźwik-Pruska / J. Chromatogr. B xxx (2015) xxx–xxx

F2 ␣ (PGHS)-catalysed peroxidation of
arachidonic acid (AA) is dependent

Artefactual formation
Method validation
challenges
ARTICLE IN PRESS
upon GSH
Plasma concentration of Plasma In vivo ELISA commercial kit [95]
15(S)-8-iso-prostaglandin F2 ␣
increased by N-acetyl-cysteine (NAC)
(precursor of GSH) and vitamin C.
probable reason may be GSH- and/or
vitamin C-promoted and
pghs-mediated formation of
15(S)-8-iso-prostaglandin F2 ␣
Low concentrations of compounds Plasma, urine – LC–MS, LC–MS/MS, GC–MS and [93]
cannot be determined because of lack GC–MS/MS allow for accurate,
of sensitivity of applied method sensitive quantification

GSSG Artefactual oxidation
Artefactual oxidation
Formation of GSSG in GSH-containing Plasma In vitro GC–MS/MS an HPLC–UV [94]
PGHS incubation mixtures. process
dependent upon PGHS concentration
and incubation time.
Sample acidification may result in Body fluids In vitro Chromatography-based techniques [93]
overestimating concentration.

GSH Artefactual oxidation Oxidation during blood withdraw, Blood In vitro Chromatography-based techniques [93]
centrifugation and handling.

8OHdG Artefactual formation Oxidative modification of guanine to HeLa cells vs human In vitro HPLC–ECD, HPLC–MS/MS [93,96]
8OHdG. lymphocytes

3- Artefactual formation Oxidative modification of tyrosine and In vitro – [92,93]

NT tyrosine residues to 3-NT
Method validation Low concentrations of compounds
challenges cannot be determined because of lack
of sensitivity of applied method
Plasma and tissue – HPLC–UV, HPLC–ECD and HPLC–FL do [92,93]
not provide adequate sensitivity;
LC–MS/MS and GC–MS/MS allow for
accurate, sensitive quantification
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(0.55 ± 0.2 vs. 0.32 ± 0.1 nmol/L) were observed [31]. A validated
(LOD, accuracy, precision, recovery values were given) HPLC
method with coulometric electrochemical (EC) detection showed
decreased levels of GSH and of the GSH/GSSG ratio in both cere-
bellum and temporal cortex of autistic children (n = 15) compared
to the control group (n = 12). Proposed method eliminates time-
consuming precolumn derivatization step [41]. Lower levels of
plasma glutamine and higher levels of plasma glutamate in autis-
tic children (n = 23) compared to healthy controls (n = 22) were
reported by Shimmura et al. with the use of the same analytical tool
[82]. The concentration of plasma glutamic acid measured by HPLC
was found to be elevated in ASD children (120 ± 89 vs. 83 ± 35 M)
[83]. Other studies based on the analysis of plasma samples by HPLC
confirmed these results and demonstrated that the increase of glu-
tamic acid is also maintained in the elderly [84]. Supplementation
with methylcobalamin and folinic acid has been reported to result
in the increase in GSH concentrations and the GSH/GSSG redox ratio
[42].
Plasma methionine was determined by Naushad et al. [83].
with the use of HPLC with photodiode array detection. Children
with ASD were reported to have lower concentrations of methi-
onine (22 ± 9 M, n = 138) in comparison with heathy controls
(28 ± 9 M, n = 138). Similar results were obtained by other sci-
entists (19.3 ± 9.1 ␮M in the ASD group vs. 31.5 ± 5.7 M in the
healthy group) [31].
Quantification of tryptophan in plasma samples was performed
with the use of many chromatographic tools. The level of tryp-
tophan was reported to be decreased in plasma samples of ASD
patients compared to controls (24 ± 11 vs. 41 ± 16 M) as measured
by HPLC with photodiode array detection [83]. Others confirmed
these results with the application of LC–MS/MS [66]. Similar results
were obtained for tryptophan in urine samples using GC–MS. For
this purpose authors applied simultaneous extraction and deriva-
tization. Scan range of the total ion chromatogram was from m/z
50 to 550. The mean level of tryptophan in ASD children with a
restricted diet low in casein and gluten (1.98 ± 1.17 ␮g/mL, n = 10),
as well as without a diet (7.44 ± 1.33 ␮g/mL, n = 23) was lower than
in healthy controls (14.24 ± 2.01 ␮g/mL, n = 21) [85].
The identification and quantification of 3-nitrotyrosine were
performed using a wide range of chromatographic methods, includ-
ing HPLC, GLC–MS [55]. Studies conducted by Rose et al. [41]. with
the use of HPLC with coulometric electrochemical (EC) detection
presented a significant increase in 3NT and 3CT in cerebellum and
temporal cortex of autistic patients (n = 15) compared to healthy
controls (n = 12). Researchers eliminated precolumn derivatization
step.
Application of gas chromatography/negative ion chemical
ionization-tandem mass spectrometry (GC/NICI–MS/MS) allowed
the determination of F2 -isoprostanes in the erythrocyte mem-
brane of ASD patients (n = 15) and healthy subjects (n = 15), and
revealed significantly elevated levels of these compounds [86].
Consistent results were obtained for urinary analysis of the F2-
isoprostane F2 ␣-VI (ASD group 5.2 ± 0.5; controls 3.1 ± 0.3 ng/mg
creatinine) [87]. Validated GC–NICI–MS and GC–MS methods were
used by Gorrindo et al. [22]. to quantify F2 -isoprostanes in plasma
samples of four groups ASD-GID (gastrointestinal dysfunction)
(n = 27), ASD-only (n = 29), GID-only (n = 21) and controls (n = 10).
F2 -Isoprostane plasma levels were elevated in the ASD-GID group
(53.6 ± 24.4 pg/mg total protein) compared to healthy controls
(17.3 ± 4.7 pg/mg protein). The ASD-only and GID-only groups
were not significantly different with respect to F2 -isoprostanes
(36.5 ± 13.3 and 46.4 ± 22.3 pg/mg).
Employment of techniques based on chromatography enabled
the determination of other compounds. Levels of plasma pheny-
lalanine (45 ± 20 vs. 59 ± 18 M) and histidine (45 ± 21 vs.
58 ± 15 M) measured with the use of HPLC with photodiode
Please cite this article in press as: J. Kałużna-Czaplińska, J. Jóźwik-Pruska, Chromatographic and mass spectrometric techniques in
studies on oxidative stress in autism, J. Chromatogr. B (2015), http://dx.doi.org/10.1016/j.jchromb.2015.12.035
9
array detection were lower in ASD patients [83]. Literature also
reports on decreased levels of plasma total cysteine (163 ± 15
vs. 201 ± 17 M), and SAM (75.8 ± 16.2 vs. 96.9 ± 12 nM). In the
same study, significantly higher levels of SAH (28.9 ± 7.2 vs.
19.4 ± 3.4 nM) and adenosine (0.39 ± 0.2 vs. 0.27 ± 0.1 ␮M) in
plasma samples were observed [31]. Similar results were obtained
by other scientists by LC–MS/MS. SAM turned out to be signifi-
cantly lower in ASD patients, while SAH did not significantly differ
between ASD vs. control groups. The SAM/SAH ratio was lower in
autistic individuals. Additionally, adenosine was slightly higher in
children with ASD [66]. Other compounds determined in ASD indi-
viduals are porphyrins. Specific porphyrin profiles are associated
with high exposure to mercury and other toxic compounds. Sci-
entists studied pentacarboxyporphyrin (5cxP), precoproporphyrin
(prcP), and coproporphyrin (cP) in urine samples and the analy-
sis of these compounds in autistic patients (n = 20) and healthy
controls (n = 20) showed their elevated levels in the first group
(5cxP 5.1 ± 1.3 vs. 4.45 ± 2.09; prcP 21.1 ± 6.8 vs. 17.9 ± 9.3; cP
253 ± 74.4 vs. 195 ± 88 nmol/g creatinine). Yet, no significant differ-
ences were found in non-Hg associated urinary phorphyrins (e.g.,
heptacarboxyporphyrin) [88]. 8-Oxo-deoxyguanosine, a biomarker
of oxidative DNA damage, was determined in brain tissue of ASD
individuals and unaffected controls with the use of HPLC–MS. The
level of 8-oxo-deoxyguanosine was elevated in ASD patients and
correlated inversely with GSH/GSSG in the cerebellum [41].
The studies cited and discussed above clearly indicate the role
of oxidative stress in ASD. Reported levels of the biomarkers of
oxidative stress and the compounds differ among the investigator
groups due to differences in the populations studied, the severity of
ASD symptoms and due to other conditions including the analytical
methods used. Nevertheless, the levels of biomarkers show a clear
tendency in differences between ASD and healthy controls.
Table 1 summarizes data for the determination of compounds
which are significant in oxidative stress with the application of
chromatographic techniques.
It should be highlighted that analysis and quantification of
biomarkers of oxidative stress (both well-established and poten-
tial) involves the risk of occurrence of pitfalls and mistakes. The
origin of these difficulties may relate to analytical process, includ-
ing artefactual generation of biomarkers during sample preparation
and the lack of sensitivity, selectivity and specificity of the proposed
method. MS-based analytical methods, especially LC–MS/MS and
GC–MS/MS, are known to be more selective and specific. In order to
avoid interferences additional chromatographic steps can be used
(prior to detection). Artefactual formation of biomarkers may also
arise from enzyme-catalysed reactions, which are not necessarily
associated with oxidative stress. In Table 2 we summarized data
about pitfalls, which may occur in the determination of oxidative
stress biomarkers [92–94].
3. Conclusions
Chromatographic and mass spectrometric techniques are use-
ful for the quantification of potential oxidative stress biomarkers
in health and disease. The use of these techniques helped identify
biological disturbances, new biomarkers and potential factors that
may contribute to the development of ASD. Moreover, these meth-
ods may help create novel diagnostic methods and improve the
therapy of patients suffering from ASD.
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