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Journal of Chromatography B, xxx (2013) xxx–xxx

Contents lists available at ScienceDirect

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

Review

Chromatographic techniques coupled with mass spectrometry for the

determination of organic acids in the study of autism夽
Joanna Kałużna-Czaplińska ∗ , Ewa Żurawicz 1 , Jagoda Jóźwik 1
Institute of General and Ecological Chemistry, Department of Chemistry, Lodz University of Technology, Zeromskiego 116, 90-924 Lodz, Poland

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

Article history: Chromatographic methods find application in the diagnostics and prognosis of diseases. They are used in
Received 14 August 2013 finding new biomarkers, which may result in early medical intervention. Early diagnosis and intervention
Accepted 12 October 2013 are especially important in the case of diseases of unknown etiology. One of these is autism. Autism
Available online xxx
is a neurodevelopmental disorder characterized by severe impairment in reciprocal social interaction
and communication and a pattern of repetitive or stereotyped behavior. Organic acids are intermediate
Keywords:
metabolites of all major groups of organic cellular components and can play a role in the pathogenesis of
Chromatographic methods
autism. This review presents information about abnormal levels of some organic acids observed in the
Mass spectrometry
Organic acids
urine of children with autism and determination of acids with the use of chromatographic techniques. 342
Autism literature sources on frequency (2005–2012) of the use of chromatographic methods in the determination
of organic compounds in various body fluids were searched.
© 2013 Elsevier B.V. All rights reserved.

Contents

1. Scope . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 00
2. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 00
3. The health problems comorbid with selected organic acids . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 00
4. Chromatographic techniques coupled with mass spectrometry in the determination of selected organic acids . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 00
5. Biochemical anomalies connected with organic acids in autism . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 00
6. Positioning chromatographic techniques among other diagnostic methodologies in autism . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 00
7. Conclusions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 00
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 00

1. Scope (LC–MS/MS), liquid chromatography–nuclear magnetic

resonance-mass spectrometry (LC–NMR-MS) and capillary
This review focuses on the use of chromatographic techniques electrophoresis–mass spectrometry (CE–MS) find wide applica-
coupled with mass spectrometry for the determination of organic tions in clinical research. Chromatographic methods are applied
acids in the diagnosis and intervention in autism. in the diagnostics and prognosis of diseases. They are used to find
new biomarkers, which may help in early medical intervention.
2. Introduction Early diagnosis and quick intervention are especially important
in the case of diseases of unknown etiology such as autism.
Coupled techniques such as gas chromatography–mass Autism is a neurodevelopmental disorder characterized by severe
spectrometry (GC–MS), gas chromatography–tandem mass spec- impairment in reciprocal social interaction and communication
trometry (GC–MS/MS), liquid chromatography-mass spectrometry and in the pattern of repetitive or stereotyped behavior. Many
(LC–MS), liquid chromatography–tandem mass spectrometry investigations suggest that biomedical intervention may help in
the therapy and recovery of autistic children. Restrictive diets and
other nutritional or gastrointestinal therapies such as a gluten-
夽 This paper is part of the special issue on Acids edited by Alexander A. Zoerner free and casein-free diet, antifungal medications to treat fungal
and Dimitrios Tsikas. overgrowth in the gut and dietary supplementation with vitamins,
∗ Corresponding author. Tel.: +48 426313091; fax: +48 426313103.
minerals, omega-3 fatty acids and others have become very
E-mail address: jkaluzna@p.lodz.pl (J. Kałużna-Czaplińska).
1
Tel.: +48 426313117; fax: +48 426313103. popular in intervention in autism [1]. The analysis of metabolites

1570-0232/$ – see front matter © 2013 Elsevier B.V. All rights reserved.
http://dx.doi.org/10.1016/j.jchromb.2013.10.026

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such as carboxylic acids, amino acids, carbohydrates, opioid
peptides in body fluids of autistic children plays an important
role in the screening, diagnosis, and monitoring of a variety of
anomalies in autism. Opioid peptides are a potential marker of
autism and thus they are very useful in predicting and monitoring
responses to a casein and gluten-free diet. The determination of
opioid peptides in the urine of autistic children is possible due
to chromatographic techniques such as liquid chromatography
coupled mass spectrometry (LC–MS/MS), liquid chromatography
and matrix assisted laser desorption ionization-time of flight mass
spectrometry (MALDI-TOF-MS) [2]. Moreover, a chromatographic
method was studied for the quantification of ␤-carbolines in hair
as potential biomarkers in autistic children [3]. Elevated levels
of trans-indolyl-3-acrylolglycine (IAcrGly) have been reported in
the urine of people with autism. The results suggest that urinary
IAcrGly can be a diagnostic indicator of autism. Chromatographic
method LC–MS/MS makes it possible to detect and quantify
IAcrGly [4]. Biochemical anomalies connected with organic acids
have been identified in many people with autism [5]. A profile of
the biochemical acid may play an important role in understanding
the etiology of autism and different developmental disorders in
children. In the case of autistic children, the quantitative organic
acid profiling can assess: mitochondrial energy production, fatty
acid metabolism, carbohydrate metabolism, B-complex sufficiency,
methylation of co-factors, neurotransmitter metabolism, oxidative
damage, detoxification status and bacterial and yeast overgrowth.
3. The health problems comorbid with selected organic
acids
Since the first use of organic acid profile in the diagnosis of lactic
iso-valeric in 1966 [6], the list of disorders resulting from abnormal
excretion of organic acids has widely grown [7]. The determina-
tion of organic acids is the key for the diagnosis of inborn errors of
metabolism (IEM). Organic acidurias are a biochemically heteroge-
neous group of inborn errors of metabolism. They are characterized
biochemically by the accumulation of metabolites which are not
present under physiological conditions, produced from the activa-
tion of alternative pathways in response to the loss of function of a
specific gene product (enzyme), or by the accumulation of patho-
logical amounts of normal metabolites. More than 65 inherited
metabolic abnormalities are known to yield a characteristic urinary
organic acid pattern, which is essential for diagnosis [8].
In the case of neurological diseases and disorders, homovanillic
acid (HVA) and vanillylmandelic acid (VMA) play a crucial role. The
analysis of the levels of VMA and HVA in body fluids is used in the
diagnosis of various health problems (Table 1), as well as to monitor
the progress of therapy.
Studies of metabolic fingerprints present the correlation
between the occurrence of bacterial infection and the levels of
succinic acid in clinical samples. Although the succinic acid gives
information about the bacterial infection of the organism, it can-
not serve as an indicator of differentiation of aerobic and anaerobic
bacteria [17].
Since the publication of the information (25 years ago) about
the deficiency of carnitine and the deficiency of muscle carnitine
palmitoyltransferase, the fatty acids oxidation disorders (FAOD)
has become one of the most important problems in the group
of congenital disorders with a broad spectrum trial (from major
malformations or sudden infant death syndrome, to the defects
in adulthood nearly asymptomatic) [18]. Among mitochondrial
dysfunctions of fatty acids oxidation we can distinguish, for exam-
ple: very long-chain acyl-CoA dehydrogenase (VLCAD) deficiency,
mitochondrial trifunctional protein (MTP) deficiency, carnitine
palmitoyl transferase (CPT 1) deficiency, carnitine palmitoyltrans-
ferase 2 (CPT 2) deficiency, carnitine-acylcarnitine translocase
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Table 1
Health problems associated with abnormal levels of homovanillic acid (HVA) and
vanillylmandelic acid (VMA) in body fluids [9–16].
Health problem connected with high Health problem connected
level of metabolites in body fluids with low level of metabolites
in body fluids
HVA, VMA in urine – neuroblastoma HVA, VMA in urine and
cerebrospinal fluid –
depression, sleep disturbances,
anxiety and fatigue
HVA in plasma – delusional symptoms HVA in cerebrospinal fluid –
bipolar disorder type 1
HVA/VMA in urine – Menkes syndrome HVA in cerebrospinal fluid –
Parkinson’s disease
HVA, VMA, HVA/VMA in
urine–anorexia nervosa
VMA in urine – familial
neurodegenerative disorder with
hypertension and paroxysms of
irritability and sweating
HVA in urine – autism
(CACT) deficiency. FAOD disorders affect the functioning of organs
with high energy demand and insufficient supply of energy can
result in severe clinical symptoms, including neurological dysfunc-
tion and risk to life. Despite the relative abundance of new research
on FAOD, there is evidence that many of these disorders remain
undiagnosed due to the diversity of their symptoms. Thus, there
is a need to study the metabolic profiles in blood and urine sam-
ples of patients [18]. The diagnosis of FAOD relies mainly on the
determination of the levels of organic acids in urine with the appli-
cation of GC–MS, and on the analysis of the blood acylocarnitine
profile using tandem mass spectrometry (MS/MS) [19]. Due to the
fact that carnitine is essential for the transportation of long-chain
fatty acids into the mitochondrial matrix, which is the place of reac-
tion of the ␤-oxidation, its lack leads to the degradation of fatty
acids through alternative less energy efficient ␻-oxidation path-
way and, consequently, to increased urinary excretion of adipic
acid and suberic acid. These abnormalities are common in children
with attention disorders. Among the group affected with dicar-
boxylic acidosis symptoms of Reye’s syndrome are observed, which
also is associated with the features that affect mitochondrial toxic
metabolic products of viruses [14]. In addition, deficits of vitamin
B2 (Riboflavin) contribute to the weakening of the ␤-oxidation and
increased urinary excretion of adipic acid and suberic acid [20].
Studies of levels of dicarboxylic acid in patients suffering from Zell-
weger syndrome indicate that in the absence of ketoacidosis the
complete excretion of adipic, suberic and sebacic acid was signifi-
cantly higher by approximately 100%, 200%, 350% as compared with
the reference group. In the treatment of disorders associated with
high levels of adipic acid and suberic acid, the literature reports
mainly about application of l-carnitine, acetyl-l-carnitine, vitamin
B2 and B5, magnesium, coenzyme Co10. The diet low in fat and
protein and high in carbohydrates is also recommended [20,21].
4. Chromatographic techniques coupled with mass
spectrometry in the determination of selected organic acids
Coupled chromatographic techniques are reliable and suit-
able to determine organic acids present in biological fluids,
especially in urine. Organic acids are mostly analyzed by gas
chromatography–mass spectrometry and tandem mass spectrom-
etry (GC–MS and GC–MS/MS, respectively) [22,23]. These methods
are characterized by high sensitivity, peak resolution and repro-
ducibility. Compounds analyzed by GC/MS should be volatile and
thermally stable. Therefore, polar and non-volatile compounds
require chemical derivatization at the polar functional groups to
reduce the polarity, increase the thermal stability and volatility of
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Fig. 1. The diagram of reports on frequency (2005–2012) of the use of chromatographic methods: GC–MS and LC–MS and CE-MS in the determination of organic compounds
in various body fluids. Literature review was based on PubMed sources. 342 literature sources were searched. Used phrases: ‘body fluids’ or ‘urine’ or ‘blood’ or ‘saliva’ or
‘cerebrospinal fluid’ or ‘CSF’ or ‘semen’ or ‘sperm’ or ‘plasma’ or ‘serum’ or ‘sweat’ or ‘tears’ and ‘gas chromatography’ and ‘mass spectrometry’ or ‘GC-MS’ or ‘GC/MS’ or ‘gas
chromatography tandem mass spectrometry’ or ‘GC-MS/MS’ and ‘liquid chromatography’ and ‘mass spectrometry’ or ‘LC-MS’ or ‘LC/MS’ or ‘liquid chromatography tandem
mass spectrometry’ or ‘LC-MS/MS’ and ‘capillary electrophoresis’ and ‘mass spectrometry’ or ‘CE-MS’ or ‘CE/MS’ or ‘capillary electrophoresis tandem mass spectrometry’ or
‘CE-MS/MS’ and ‘organic acids’.

the analytes. Availability of GC/MS electron impact (EI) spectral
library further facilitates the identification of diagnostic organic
acids and aids the subsequent mechanistic elucidation of biolog-
ical and pathological variations [24]. Gas chromatography without
the positive identification of components of the complex profile is
prone to errors and should be discouraged. Before the identifica-
tion of organic acids, three steps are required: isolation/separation
of the organic acids from the physiological specimen, derivatiza-
tion of the organic acids to more stable and volatile compounds,
GC/MS separation and identification of the derivatized compounds.
Although high-performance liquid chromatography (HPLC) and
capillary electrophoresis (CE) show some advantages such as
shorter time of analysis compared to gas chromatography, a sig-
nificant disadvantage is the lower resolving power of HPLC and
limitations of sensitivity in the case of CE [25,26].
Fig. 1 shows the frequency of using chromatographic methods:
GC/MS, LC/MS and CE/MS in the determination of organic acids in
biological samples in the period from 2005 to 2012.
The measurement of organic acids in urine evaluates four critical
areas of metabolism: gastrointestinal function, cellular energy and
mitochondrial metabolism, neurotransmitter metabolism, amino
acid/organic acid balance influenced by vitamin/mineral co-factors
[27]. The accumulation of organic acids can also provide useful
information for an early diagnosis of neurological diseases. For this
purpose the concentrations of two acids: homovanillic and vanil-
lylmandelic in body fluids are most often determined. Due to the
coexistence of metabolites of a similar structure and characteristics
of the VMA and HVA, it is necessary to use the steps of separating
the analytes from the matrix prior to their identification. The stages
are based on a solid phase extraction (SPE) [28,29], or liquid–liquid
extraction (LLE) [30] (Table 2). SPE and LLE are time-consuming,
and are prone to analytical errors. Quantification is often per-
formed using gas chromatography (GC) and liquid chromatography
(LC). Gas chromatography methods require the time consuming
step of transforming HVA and VMA acids into volatile derivatives
(derivatization) [32]. The methods using liquid chromatography
are based on the electrochemical detection [28,43]. Liquid chro-
matography with electrochemical, amperometric or coulometric
detection, makes it possible to avoid the time-consuming step of
extraction. The advantages of the methods described in literature,
which are employed to analyze HVA and VMA using liquid chro-
matography, are the short time of analysis and a simple procedure
of sample preparation. However, the disadvantage is their high cost.
Another acid which is often helpful in medical diagnostics
is succinic acid, which is a dicarboxylic acid, being an interme-
diate product of the citric acid cycle (tricarboxylic acid cycle,
known as TCA or Krebs cycle), which is the final common path-
way of oxidation of carbohydrates, lipids and proteins occurring
in mitochondria. Succinic acid is determined in urine, as well as
in plasma, serum and saliva. In order to isolate this acid from the
complex matrix, like body fluids, LLE [40,42] and SPE [37] are used.
Liquid chromatography permits an analysis of a larger number of
samples per unit of time in comparison with gas chromatography
[42]. In the case of GC analysis [38,42] the step of transforming
analytes into their volatile derivatives can be avoided using liquid
chromatography or capillary electrophoresis [36,40].
Fatty acids and related disorders are nowadays a very impor-
tant issue in clinical chemistry. Despite the relative abundance of
new research on the fatty acids oxidation disorders (FAOD), there is
evidence that many of these disorders remain undiagnosed due to
the diversity of their symptoms. Thus, there is a need to study the
metabolic profiles in the blood and urine samples of patients [18].
FAOD diagnosis is based mainly on the determination of the levels
of organic acids in urine using GC–MS, but also on the analysis of
the profile of blood acetylcarnitine by tandem mass spectrometry
(MS/MS) [19].
Dicarboxylic acids: adipic acid and suberic are the products of
␻-oxidation of fatty acids by cytochrome P450 and they accumu-
late when the ability of mitochondria to ␤-oxidation of fatty acids
is exceeded [44]. Azelaic acid is an endogenous C9-dicarboxylic
acid synthesized in the ␻-oxidation of fatty acids excreted from
the body in urine [45]. Adipic acid, suberic acid and azelaic acid
are determined in urine, as well as in cerebrospinal fluid, blood
plasma, serum and saliva. To isolate these acids from the matrix,
LLE [35,38–40] is usually used. SPE [35,37], mainly after prior
acidification and salting, is less often used. Despite the need for

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Table 2
Examples of application of chromatographic techniques coupled with mass spectrometry in determination of homovanillic acid, vanillylmandelic acid, succinic acid, adipic,
suberic and azelaic acids in body fluids.

Organic acid Body fluid Analysis Stages of sample preparation References

technique

HVA Blood GC–MS Not given [31]

Urine LC–MS/MS SPE extraction [29]

HVA, VMA Urine GC–MS Dilution, acidification, LLE extraction, transformation into volatile [32]
derivatives
Extraction, enzymatic hydrolysis, transformation into volatile [33]
derivatives
GC–QqQ MS Adjusting pH, salting-out, shaking, transformation into volatile [34]
derivatives, extraction SPME
CSF, urine GC–MS (1) Acidification, salting-out, LLE extraction, transformation into [35]
volatile derivatives
(2) SPE extraction, acidification, salting-out, shaking,
transformation into volatile derivatives

Succinic acid Plasma LC–MS/MS Centrifugation, incubation, ultrafiltration [36]

Urine GC–MS Derivatization of oxo-acids, acidification, SPE extraction, [37]
transformation into volatile derivatives
Oximation of 2-keto acids, acidification, salting-out, LLE extraction, [38]
evaporation, transformation into volatile derivatives
Acidification, LLE extraction, evaporation, transformation into [39]
volatile derivatives
HPLC–TOF/MS Centrifugation, ultrafiltration, dilution [40]
HPLC–FLD/MS Filtration, fluorescence derivatization [41]
Serum, plasma, GC–MS, Evaporation, transformation into volatile derivatives [42]
urine LC–MS/MS
Saliva HPLC–TOF/MS LLE extraction, centrifugation, ultrafiltration, evaporation, [40]
dissolving in mobile phase

Adipic acid, suberic acid Urine GC–MS Derivatization of oxo-acids, acidification, SPE extraction, [37]
transformation into volatile derivatives
Acidification, LLE extraction, evaporation, transformation into [39]
volatile derivatives

Adipic acid, suberic acid, azelaic acid Urine, CSF GC–MS Acidification, salting-out, LLE extraction, transformation into [35]
volatile derivatives
SPE extraction, acidification, salting-out, shaking, transformation
into volatile derivatives
Urine GC–MS Oximation of 2-keto acids, acidification, salting-out, LLE extraction, [38]
evaporation, transformation into volatile derivatives

Azelaic acid Urine, plasma HPLC–MS Acidification, salting-out, LLE extraction, evaporation [46]
Plasma GC–MS, LC–MS LLE extraction, transformation into volatile derivatives [47]
Serum CE-TOF/MS LLE extraction, centrifugation, filtration [48]

Adipic acid Urine HPLC–TOF/MS Centrifugation, ultrafiltration, dilution [40]

Saliva HPLC–TOF/MS LLE extraction, centrifugation, ultrafiltration, evaporation, [40]
dissolving in mobile phase

CE, capillary electrophoresis; FLD, fluorescence detector; HPLC, high performance liquid chromatography; LC, liquid chromatography; LLE, liquid–liquid extraction; MS, mass
spectrometry; MS/MS, tandem mass spectrometry; SPE, solid phase extraction; TOF, time of flight detector; UV/VIS, ultraviolet-visible spectrophotometry detection; CSF,
cerebrospinal fluid.

transforming analytes into their volatile derivatives, gas chro-
matography is a separating technique widely used for the studies
of the levels of dicarboxylic acids in body fluids. Both in the case
of gas chromatography and liquid chromatography or capillary
electrophoresis, mass spectrometer is the most commonly used
detector in the determination of the levels of adipic, azelaic, suberic
acid (Table 2).
5. Biochemical anomalies connected with organic acids in
autism
The determination of organic acids is necessary in the diag-
nosis of inborn errors of metabolism, but sometimes it is also
used for identification of dietary abnormalities. Organic acids are
intermediate metabolites of all major groups of organic cellular
components and play a role in the pathogenesis of autism spectrum
disorders. In the urine of autistic children abnormal levels of some
metabolites were identified, which suggests a strong connection
between the abnormal level of organic acids in the urine of chil-
dren with autism and the growth of some autistic symptoms. The
application of the urinary organic acid test helps to determine
where the imbalance in the metabolic cycle occurs. Coupled chro-
matographic techniques are more reliable and suitable to deter-
mine concentrations of organic acids in biological fluids, especially
in urine. The samples of the most important organic acids in autism
were presented in Table 3. The measurements of urine organic acids
are used in modern nutritional medicine as a simple and sensi-
tive test, which can demonstrate functional inadequacy of specific
nutrients [27,64]. The presence of the urinary organic acid test helps
to understand how nutrient metabolism is executed and determine
where the imbalance in the metabolic cycle may occur. Differenti-
ated levels of urinary organic acid excretion for autistic and healthy
children are reported [5,65]. Such results do not provide strong evi-
dence to support the theory that abnormal metabolites play a role
in the pathogenesis of autism spectrum disorders; however, they
suggest some metabolic or dietary abnormalities in autism.
In the case of autistic children quantitative organic acid pro-
filing can assess: mitochondrial energy production, fatty acid
metabolism, carbohydrate metabolism, B complex sufficiency,
methylation of co-factors, neurotransmitter metabolism, oxidative

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Table 3
Examples of significant organic acids in study of autism.

Organic acid Body fluid/used Result References

method

4-Hydroxyphenylacetic acid, 4-hydroxyhippuric acid Urine/high- ↑ 4-Hydroxyphenylacetic acid [49]

resolution liquid ↑ 4-Hydroxyhippuric acid
chromatography
Homovanillic acid Urine/GLC, GC ↑ Homovanillic acid [50]
Indoleacetic acid Cerebrospinal n. [51]
Fluid/HPLC
Homovanillic acid Plasma, ↑ Homovanillic acid in autistic patients [52]
Urine/HPLC treated with neuroleptics
n. Homovanillic acid in untreated
autistic patients
Homovanillic acid, 5-hydroxyindoleacetic acid Cerebrospinal n. [53]
Fluid/HPLC
Citramalic, tartaric (3-OH-Malic), and 3-oxoglutaric acids, Urine/GC–MS ↑ Analogs of Krebs cycle metabolites [54]
Compounds identified as a citric acid analog and
partially identified as a phenylcarboxylic acid
5-Hydroxyindoleacetic acid Blood, Urine/HPLC n. [55]
Arachidonic acid, docosahexaenoic acid, linoleic acid, Plasma/TLC, GC ↓ Docosahexaenoic acid, ␣-Linolenic [56]
␣-linolenic acid acid
5-Hydroxyindoleacetic acid, homovanillic acid, lactic acid, Cerebrospinal ↑ Orotic acid [57]
orotic acid Fluid, Urine/HPLC
5-Hydroxyindoleacetic acid Urine/HPLC n. [58]
Polyunsaturated fatty acids, lactic acid Plasma/GC ↑ Lactic acid [59]
↓ Docosahexaeinoic acid
↓ Linolenic acid
↓ Arachidonic acid
Homovanillic acid, vanillylmandelic acid Urine/GC–MS ↑ Homovanillic acid [60]
↑ Vanillylmandelic acid
Acetic, valeric, hexanoic, stearidonic, propionic, butyric, Plasma/GC ↑ Most of the saturated fatty acids [61]
caprylic, decanoic, lauric, palmitic, stearic, arachidic, except for propionic acid
␣-linolenic, eicosapentaenoic, docosahexaenoic, linoleic, ↓ Most of polyunsaturated fatty acids
␥-linolenic, arachidonic, oleic, elaidic acids
Succinic, adipic, suberic acids Urine/GC–MS ↑ Succinic acid [62]
↑ Adipic acid
↑ Suberic acid
Homovanillic acid, 5-hydroxyindolacetic acid Cerebrospinal ↓ 5-HIAA [63]
Fluid/HPLC

↓, decreased level of acid; ↑, increased level of acid; n., level of acid in the normal range.

damage, detoxification status, and bacterial and yeast over- of autistic children is shown in Fig. 2. The levels of succinic,
growth. Biochemical anomalies connected with organic acids adipic, and suberic acids in the urine of autistic children were
were identified in children with autism [5,66–68] a typical total 41.47 ± 50.40 ␮mol/mmol creatinine, 15.61 ± 15.31 ␮mol/mmol
ion chromatogram of derivatized organic acids in the urine creatinine, and 8.02 ± 6.08 ␮mol/mmol creatinine [62].

Fig. 2. The typical total ion chromatogram derivatized organic acids in the urine of autistic children IS (internal standard).

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The organic acid profile may play an important role in a better
understanding of the cases of autism and different developmen-
tal disorders observed in children. The analysis of the metabolic
organic acid profile determines: 39 organic acids, including 8
gastrointestinal metabolites, 13 cellular energy metabolites, 4
neurotransmitter metabolites, and 14 amino acid metabolites.
Collecting cerebrospinal fluid and blood from children (espe-
cially healthy ones) is an ethical and methodological problem.
This problem can be partly overcome through the use of urinary
assays. Numerous studies have demonstrated the utility of urinary
analysis in the case of autistic children [66,69,70]. The urinary
organic acids test measures selected metabolites, which serve as
important diagnostic indicators of abnormal metabolism in the
case of autistic children. Shaw [54] tested urinary organic acid
profiles in two autistic brothers. These tests revealed a consistent
excretion of a number of compounds of possible microbial ori-
gin identified as the carbohydrate arabinose, analogs of normal
Krebs cycle intermediates, including 3-oxoglutaric acid, tartaric
(3-hydroxymalic) acid, citramalic (methylmalic) acid, and a new
tentatively identified analog of citric acid (carboxycitric acid), not
previously reported in medical literature. The hydroxymethylfu-
rancarboxylic acid, furandicarboxylic acid, furancarbonylglycine,
and 3-(3-hydroxyphenyl)-3-hydroxypropionic acid were found in
urine as chemicals of possible microbial origin. Oxalates and oxalic
acid come from the diet, fungus (such as Aspergillus, Penicillium,
and Candida), and also from human metabolism. They seem to be
especially important in autism therapy because higher values of
oxalates are observed in the urine of autistic children than in that of
non-autistic children [71]. An increased level of citric acid can sug-
gest an amino acid deficiency or problems with protein metabolism
in autistic children. Succinic acid cannot play its role in the produc-
tion of cellular energy via the citric acid cycle when coenzyme Q10
(CoQ10) is inadequate. Elevated succinic acid excretion is a marker
for deficiency of CoQ10 and riboflavin in children with autism. Adi-
pate and suberate are the products of incomplete oxidation in the
omega-oxidation pathway.
The analysis of urinary dicarboxylic acids is a very important
tool used in the diagnosis of several metabolic disorders. The rea-
son for the disturbances in the levels of dicarboxylic acids in autism
is not clear. Several metabolism defects, which cause the excre-
tion of succinic, adipic, and suberic acid were reported [8]. One of
the reasons for elevated concentrations of adipic and suberic acid
in urine can be riboflavin responsive C6-C10 dicarboxylic aciduria.
Higher levels of these acids probably result from the deficiency of
octanoyl- and butyryl-CoA dehydrogenases, which affects ␻- and
␤-oxidation [72]. Adipic and suberic acids are the products of the ␻-
oxidation pathway. Without riboflavin, coenzymes in mitochondria
fatty acids cannot undergo oxidative metabolism, which results in
an incomplete ␻-oxidation. Thus, in the case of increased urinary
excretion of adipic and suberic acid, the ministration of riboflavin
can be considered a potential intervention. Increased levels of
adipic and suberic acids in the urine of children with autism were
reported [5,73]. Succinic acid is an intermediate product of the
Krebs cycle. To convert succinic acid into fumaric acid, flavin ade-
nine dinucleotide derived from riboflavin is needed. In general, to
improve the functioning of the Krebs cycle, vitamin B2, vitamin B6,
and magnesium are required [74].
Organic acids may also result from exogenous sources such
as diet, drugs, or bacterial contamination. Several organic acids
are known to be specific products of bacterial metabolic action
on dietary polyphenols or unassimilated amino acids or carbo-
hydrates. The detection of abnormally elevated levels of these
products is a useful diagnostic tool for children with gastrointesti-
nal or toxicological symptoms. Urinary organic acids and other
urinary compounds associated with microbial overgrowth include:
benzoate, hippurate, phenylacetate, phenylpropionate, cresol,
Please cite this article in press as: J. Kałużna-Czaplińska, et al., J. Chromatogr. B (2013), http://dx.doi.org/10.1016/j.jchromb.2013.10.026
hydroxybenzoate, hydroxyphenylacetate, hydroxyphenylpropi-
onate and 3,4-dihydroxyphenylpropionate, indican, tricarballylate,
d-lactate. Effective treatments for the associated microbial over-
growths may be directed at reducing microbial populations,
introducing favorable microbes, and restoring intestinal mucosal
integrity.
Among others significantly higher 4-hydroxyphenylacetic lev-
els in the urine of autistic children are observed This may reflect
increased gut metabolism of tyrosine secondary to bacterial over-
growth. Urinary 2-oxoglutaric acid may be influenced by many
factors, including urinary tract bacterial activity [75].
The urinary neurotransmitter analysis is a useful tool in the
therapy of some autistic children. Neurotransmitter metabolism
imbalances in serotonergic or adrenergic functioning are frequently
associated with neuroendocrine disorders such as insomnia,
depression, adrenal fatigue, eating disorders, and irritable bowel
syndrome (IBS). Neurotransmitter imbalances may indicate nutri-
ent deficiencies or methylation impairments, which may have an
impact on all the body systems [27,64]. The metabolome analysis
can be effective for detecting abnormal metabolic and/or nutri-
tional conditions in autistic children, including acquired vitamin
deficiencies and the potential biological response to drugs or excess
nutrient loading.
6. Positioning chromatographic techniques among other
diagnostic methodologies in autism
The diagnosis of autism involves many stages of medical
research that aimed to identify comorbid disorders in autism.
Genetic tests which are designed to identify genetic abnormali-
ties that can cause abnormal development of a child are the most
frequently recommended tests. Also, multistage metabolic tests
and neurologic tests are often applied. Metabolic screening tests
are recommended in the case of children who, apart from autism
symptoms, suffer from metabolic disorders (e.g., lethargy, cyclic
vomiting). Metabolic studies are also used for the evaluation of rare
diseases affecting fewer than 5% of children with ASD [76]. In the
study of clinical metabolic disorders in autistic children an analy-
sis of blood, urine and feces is recommended. In order to describe
abnormalities responsible for the difficulties in speech and social
relationships, hearing tests and the examination of the construction
and functioning of the brain (electroencephalography (EEG), mag-
netic resonance imaging (MRI), computed tomography) are per-
formed. Screening EEG tests are suggested because of the extremely
high prevalence of epileptiform abnormalities in children with ASD.
Studies using magnetoencephalography (MEG) provide an excel-
lent opportunity to locate bioelectric discharges and are used to
illustrate the relationship between the behavior and the location
of epileptiform discharges. Professionals, including mainly a child
psychiatrist and a psychologist and a pediatrician, are involved in
the diagnostic process of autism. The interview with the parents
is an important step in the diagnostic process. It makes it possible
to gather the necessary information about any alarming symptoms
in the behavior and functioning of a child, the course of develop-
ment in different areas, possible developmental problems related
to family predispositions, health condition, children’s preferences
(e.g., favorite forms of activity, food) and many other issues [77].
Using the correctly prepared samples of body fluids and tissues,
diagnostic techniques offer the possibility of measuring metabolic
endpoints (metabolic profiles).
In metabolic studies coupled techniques are widely used,
especially gas chromatography–mass spectrometry (GC–MS),
gas chromatography coupled to tandem mass spectrometry
(GC–MS/MS), liquid chromatography coupled with mass spectrom-
etry (LC–MS), liquid chromatography coupled with tandem mass
spectrometry (LC–MS/MS) mass, liquid chromatography coupled
 ARTICLE IN PRESS
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CHROMB-18593; No. of Pages 8
J. Kałużna-Czaplińska et al. / J. Chromatogr. B xxx (2013) xxx–xxx
Fig. 3. The diagram of frequency of scientific reports on the use of chromatography in the study of autism in 2005–2012. Literature review was based on PubMed sources.
108 literature sources were searched. Used phrases: ‘chromatography’ or ‘chromatographic’ or ‘autism’ or ‘autistic’.
with nuclear magnetic resonance and mass spectrometry, LC–MS
and NMR-capillary electrophoresis–mass spectrometry (CE–MS)
[5,23,78]. The studies of abnormal accumulation or deficiency of
specific metabolites in a particular route can provide information
on the genes responsible for the abnormal condition and/or the
environmental exposure. Such knowledge can also be helpful in
planning medical intervention strategies in order to restore the
metabolic balance and potentially to improve the health of an
autistic patient [79]. A better understanding of the condition of
metabolism is possible due to the research of biomarkers.
The use of chromatographic techniques in the study of autism
results from a broad spectrum of this disease, which goes far beyond
simple developmental disorders [80]. According to the trend shown
in Fig. 3, it can be expected that the importance of chromatography
in the studies of autism will grow steadily. The technique suitable
for testing metabolites in body fluids should meet the three basic
requirements: selectivity of separation, which allows the deter-
mined forms of the analyte to reach a detector (separated from
the matrix interferents), the sensitivity of detection and identifi-
cation of the chemical form of the determined analyte. So far, in
the studies of autism chromatographic techniques, especially those
coupled with mass spectrometry, have occupied an important posi-
tion, meeting all the above criteria, for example the identification
and quantitative determination of markers of neurological disor-
ders and metabolic diseases.
7. Conclusions
Organic acids are metabolic intermediates, which are pro-
duced in pathways of central energy production, detoxification,
neurotransmitter breakdown, or intestinal microbial activity. Accu-
mulation of specific organic acids detected in urine is the result
of health disorders. The metabolic block takes place due to nutri-
ent deficiency, an inherited enzyme deficit, toxic build up, or drug
effect. Organic acid testing provides important information in the
areas which are very important in the case of autistic children such
as: neurotransmitter metabolites to assess the functioning of the
central nervous system (CNS), vitamin and mineral insufficiencies,
the functional markers of B-complex deficiency, oxidative damage
and antioxidant sufficiency markers, indicators to assess detoxifi-
cation sufficiency, mitochondrial energy production assessment via
citric acid cycle components, specific dysbiosis markers for bacte-
rial and yeast overgrowth.
Please cite this article in press as: J. Kałużna-Czaplińska, et al., J. Chromatogr. B (2013), http://dx.doi.org/10.1016/j.jchromb.2013.10.026
7
Currently, the measurements of the urine organic acids pro-
file with use of chromatographic techniques coupled with mass
spectrometry are a sensitive test, which can reveal evidence of
functional inadequacy of specific nutrients, especially important in
autistic children. The chromatographic techniques are particularly
important in finding potential biomarkers of autism and establish-
ing inadequacy in the diet of autistic children as a new method of
intervention in autism.
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