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Chapter

From Targeted Quantification to

Untargeted Metabolomics
Veronica Lelli, Antonio Belardo and Anna Maria Timperio

Abstract

Metabolomics is an emerging and rapidly evolving technology tool, which

involves quantitative and qualitative metabolite assessments science. It offers
tremendous promise for different applications in various fields such as medical,
environmental, nutrition, and agricultural sciences. Metabolomic approach is based
on global identification of a high number of metabolites present in a biological
fluid. This allows to characterize the metabolic profile of a given condition and to
identify which metabolites or metabolite patterns may be useful in the discrimina-
tion between different groups. The use of one mass spectrometry (MS) platform
from targeted quantification to untargeted metabolomics will make more efficient
workflows in many fields and should allow projects to be more easily undertaken
and realized. Metabolomics can be divided into non-targeted and targeted. The first
one can analyze metabolites derived from the organisms comprehensively and
systematically, so it is an unbiased metabolomics analysis that can discover new
biomarkers. Targeted metabolomics, on the other hand, is the study and analysis
of specific metabolites. Both have their own advantages and disadvantages, and are
often used in combination for discovery and accurate weight determination of dif-
ferential metabolites, and allow in-depth research and analysis of subsequent meta-
bolic molecular markers. Targeted and non-targeted metabolomics are involved in
food identification, disease research, animal model verification, biomarker discov-
ery, disease diagnosis, drug development, drug screening, drug evaluation, clinical
plant metabolism and microbial metabolism research. The aim of this chapter is to
highlight the versatility of metabolomic analysis due to both the enormous variety
of samples and the no strict barriers between quantitative and qualitative analysis.
For this purpose, two examples from our group will be considered. Using non-
targeted metabolomics in opposite Antarctic cryptoendolytic communities exposed
to the sun, we revealed specific adaptations. Instead, through the targeted metabo-
lomics applied to the urine during childbirth, we identified a different distribution
of specific metabolites and the metabolic differences allowed us to discriminate
between the two phases of labor, highlighting the metabolites most involved in the
discrimination. The choice of these two approaches is to highlight that metabolomic
analysis can be applied to any sample, even physiologically and metabolomically
very distant, as can be microorganisms living on Antarctic rocks and biological
fluids such as urine.

Keywords: urine, metabolite, HPLC–MS, fungus, biomarkers, system biology

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Metabolomics - Methodology and Applications in Medical Sciences and Life Sciences

1. Introduction

1.1 Metabolomics: the medicine of the third millennium

Metabolomics is a discipline thanks to whose analysis it is possible to ascertain the

presence of biochemical imbalances caused by the lack of nutrients that are the basis
of the functions of our body. With this research it is therefore possible to identify the
true causes of any chronic disease and restore the biochemical balance of our body.
The use of “omics” sciences, especially metabolomics, has been having posi-
tive implications in recent years in the main actions of daily life, as monitoring the
metabolism helps to keep energy levels, sleep and body weight under control. The
operation is very simple: by measuring the metabolites present in the body, prob-
lems are identified and action is taken in a targeted and relevant way. An action of
extraordinary effectiveness if you think about the current situation, for example in
the food sector; nutrition today is very rich in calories but poor in nutrients, with
the real risk of being overfed, but undernourished (Figure 1).
It is essential to remember that this is not an alternative medicine, but is
complementary to other disciplines: in the face of even important pathologies, such
as neoplasms, it will support the oncologist, improving the responses to cancer
treatment and helping to defeat the disease without interfering with the pathways
of oncological treatment. A normal blood draw or a simple urine sample is suf-
ficient to check the metabolites. An extraordinarily precise picture of the situation
will emerge, a sort of fingerprint of our body and of how many external factors,

Figure 1.
Metabolomic workflow.

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which we can define as part of the so-called epigenetics, can directly influence the
chemical aspect. The ultimate goal is to bring the body back to perfect efficiency,
taking into account that the body tends by itself, by its nature, to the best possible
state of health. However, when the body has difficulty repairing the damage or is
facing a progressive problem, which does not have time to fix or lacks adequate
resources, metabolomics comes to its rescue. Our body must be considered like a
car: to function at its best, the best fuel is needed together with winning strategies
of suitable equipment. This discipline, which is progressively affirming itself, has
set itself the task of identifying the optimal conditions to support the architrave of
human existence.

2. Targeted and untargeted metabolomics

The metabolome is the final downstream product of the genome and consists of all
low molecular weight molecules (metabolites) in a cell, tissue or organism [1, 2]. The
metabolic profile can provide a complete picture of that cell’s physiology. As emerg-
ing data suggest an important role for the microbiome and its metabolic products,
the potential size of the metabolome is often highly controversial. Given the variety
of chemical classes and physical properties that characterize metabolites and the
dynamic range of metabolite concentrations over large orders of magnitude, a wide
range of analytical techniques are required for metabolomics research. Metabolomics
aims to identify and quantify multiple molecules in the context of physiological
stimuli or in disease states. The “omics” revolution of the 1980s and 1990s provided
new methodologies for the study of interactions on a global level and offered an
alternative means of investigation to the more reductionist one in molecular biology.
Omics is a field that aims to study the abundance and/or structural characterization of
a wide range of molecules in organisms in distinct scenarios. In the clinical field, high-
throughput omics techniques are used for disease characterization to better predict
the clinical course of organisms and to evaluate the efficacy of existing or developing
therapies [3]. In food science, for example, omics plays a significant role in trying to
improve human nutrition [4]. On the other hand, concerning the environment, omics
studies aim to evaluate the alterations that organisms could undergo after exposure to
environmental stressors [5, 6]. In recent years, a variety of omics subdisciplines have
emerged (eg Fluxomics, lipidomics, glycomics, foodomics, interactomics and metal-
lomics), demonstrating that omics is a continuously evolving discipline and among all
these platforms, metabolomics is becoming increasingly popular [7].
The first definition of metabolomics dates back to the 1990s, describing tech-
niques aimed at identifying existing metabolites within a cell, tissue or organism
during a genetic alteration or physiological stimulus [8, 9]. Metabolomics has been
shown to be complementary to other omics techniques, thus identifying - called
silent phenotypes - genes that when perturbed have no apparent influence on physi-
cal characteristics or behavior [10]. The metabolomic approach can be conducted in
two distinct ways; non-targeted approach and targeted approach [11].
The reason for this differentiation is due to the different types of data generated
in these two approaches, which must be handled accordingly (Figure 2). Targeted
studies focus research on a number of known metabolites, while non-targeted studies
allow for a more comprehensive evaluation of metabolomic profiles. Most of the
methodologies used in the first targeted studies only allowed for the identification
of a limited number of metabolites. However, recent targeted methodologies allow
for the creation of large-scale metabolic profiles, including hundreds of compounds.
However, the number of compounds analyzed in non-targeted studies is even greater.
This is because entire datasets, including thousands of metabolic signals, need to be
processed, and of these, few are finally identified as candidate biomarkers.
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Metabolomics - Methodology and Applications in Medical Sciences and Life Sciences

Figure 2.
Targeted and untargeted approach.

3. Untargeted approaches

Untargeted approaches provide the most correct path to detect unexpected

changes in metabolite concentrations. The goal is to maximize the number of
metabolites detected and thus provide the opportunity to observe unexpected
changes. However, a single analytical method cannot detect all metabolites in a bio-
logical system. It is therefore necessary to combine multiple analytical approaches
(such as complementary HPLC methods) to maximize the number of metabolites
detected and improve metabolome coverage.
Sample preparation in non-targeted studies consists of extracting the metabo-
lites from the biological sample in a suitable solvent for analytical analysis. The
extracted sample is analyzed with an appropriate analytical method (for example,
LC–MS). The result of the mass spectrometry analysis is a chromatogram and the
peak area of each metabolite is used as a parameter in the statistical analysis to
define the concentration differences between the different biological samples mea-
sured. This is called relative quantification as there is no comparison with calibra-
tion curves constructed from chemical standards. The use of calibration curves is
indispensable for full quantification. The biological significance of each metabolite
is determined during data analysis and metabolite identification, and biological
interpretation is performed at the end of the experimental pipeline. Currently, one
of the main limitations in non-targeted approaches is the identification of metabo-
lites. It may not be possible to identify the metabolites highlighted in the statistical
analysis as significant changes between biological classes in the study. The identifi-
cation of metabolites is currently one of the hot topics of metabolomics.

4. Targeted approaches

Targeted studies investigate a relatively limited and specific number of metabo-

lites. At the start of the study, before data acquisition is performed, the metabolites
are chemically characterized and biochemically annotated. Targeted methods have
greater selectivity and sensitivity than non-targeted methods. A targeted study

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can only be performed if a genuine chemical standard of the metabolite is avail-

able. The quantification of metabolites is performed using internal and chemical
standards to construct calibration curves for each of the metabolites under study.
Sample preparation in targeted studies applies methods that can be optimized to
retain metabolites of interest and to remove other biological species and analytical
artifacts that are not performed via downstream analysis.
Therefore, data analysis strategies for non-targeted studies require very exten-
sive chromatogram processing. A large number of data analysis strategies are found
in the literature, but none of them can be considered the optimal choice in all cases,
which makes data analysis an open task in bioinformatics research. In fact, the field
of MS-based metabolomics is quite young and new methods, software and plat-
forms are regularly published or updated.
The data are produced by chemical–physical investigation techniques such as
magnetic resonance spectroscopy, chromatography and mass spectrometry applied
to biofluid samples or suitably selected solid tissues. These methodologies find
today numerous possibilities of use in the field of medical sciences where there are
numerous variables detectable on human and animal subjects that present a specific
pathology. This approach is valid both for the description of existing pathologies
and for the identification of pre-pathological stages. The use of metabolomic meth-
ods can help provide an overall - holistic - view of the problem, highlighting the
relationships between variables and their relative importance, and can also high-
light differences and similarities between samples. Considering individual biologi-
cal processes as isolated processes expresses a reductionist view of vital functions,
an abstraction that at times makes it possible to considerably simplify the problem
under consideration but which inevitably leads to models of limited value.

5. Metabolomics techniques

There is a range of analytical chemistry tools applied in metabolomics research.

Each instrument has advantages and limitations, and no single instrument or
instrumental method can detect all of the metabolites present in a metabolome, but
multiple instrumental methods or multiple different instruments are required to
provide the largest number of metabolites detected.
Several analytical techniques have been developed for each of the omics plat-
forms, including techniques based on DNA microarray and RNA sequencing [12],
nuclear magnetic resonance (NMR) spectroscopy [13, 14] and mass spectrometry
(MS) [15, 16]. NMR and MS are the most used in the field of metabolomics. High
resolution proton NMR spectroscopy (1H-NMR) has proven to be one of the best
technologies for examining biofluids and studying intact tissues, as the result is a
complete signal profile of metabolites without separation, derivatization and pre-
selected measurement parameters [17, 18]. On the other hand, MS methods, both by
direct injection [19] and coupled with chromatographic techniques [20], have also
evolved into an excellent technology for metabolomics due to their ability to analyze
low molecular weight compounds in biological systems. These two approaches
(NMR and MS) are complementary and the integration of both technologies can
provide more comprehensive information in the field of metabolomics.
Applications of metabolomics have expanded in line with genomics, proteomics
and transcriptomics with the aim of determining gene function in microbes [21],
plants [22] and animals [23]. Many different applications are also used today,
such as the determination of metabolic biomarkers that change as an indicator of
the presence of a disease or in response to a pharmacological intervention or the
determination of the effect of biochemical or environmental stress on plants or

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Metabolomics - Methodology and Applications in Medical Sciences and Life Sciences

microbes, genetically modified plants [24], bacterial characterization [25], health

status assessments [26] and metabolic engineering [27].

5.1 Gas chromatography–mass spectrometry (GC–MS)

GC–MS is a combined system in which volatile and thermally stable compounds

are first separated from the GC and then the eluted compounds are detected
by electron impact mass spectrometers. During the run, aliquots of derivatized
samples (injection volumes of 1 μl or less) are analyzed using split and splitless
techniques on different polarity GC columns (DB-5 or DB-50 or similar capillary
columns in stationary phase are most commonly used). These provide both high
chromatographic resolution of compounds and high sensitivity (typical limits of
detection are concentrations of pmol or nmol). Quantification is provided by exter-
nal calibration or response ratio (metabolite peak area/internal standard peak area).
The coverage of the metabolome is largely characterized by the volatility of the
non-derivatized or derivatized sample components. The identification of metabo-
lites is provided by matching the retention time and mass spectrum of the sample
peak with those of a pure compound previously tested on the same or a different
instrument under identical instrumental conditions [28]. Since the electron impact
mass spectrometer provides the standard fragmentation of molecular ions during
ionization, structural identification can be performed through the interpretation of
fragment ions and fragmentation patterns.

5.2 Liquid chromatography-mass spectrometry (LC–MS)

LC–MS provides separation of metabolites by liquid chromatography followed

by electrospray ionization (ESI) or, less typically, atmospheric pressure chemical
ionization (APCI) [29]. This technique differs from GC–MS for several reasons
(lower assay temperatures and unsolicited sample volatility) and this simpli-
fies sample preparation. In most non-pharmaceutical applications, samples are
prepared after intracellular extraction and/or protein precipitation by dilution
in an appropriate solvent. The chemistry and HPLC column size used will affect
chromatographic resolution and sensitivity. Analytical columns do not provide the
chromatographic resolving power to separate these complex samples and run times
of 10 minutes followed by chemometric instruments are used to extract the chro-
matographically unsolved data and classify the differences between the samples
[30]. The application of very high-pressure chromatography systems can improve
the chromatographic resolution. The most common column chemicals used today
are reverse phase C 18 or C 8 columns. However, for polar metabolites injected on
these columns, the retention of these metabolites is minimal, thus reducing the
volume of interpretive data. To overcome this problem, other chemical columns are
needed, such as HILIC [31] and other weak ion exchange chemicals. The sample
then, once the chromatographic separation has been carried out, reaches the source
of the mass spectrometer. Electrospray instrumentation operates in positive and
negative ion modes (as separate experiments or by polarity switching during analy-
ses) and detects only those metabolites that can be ionized by adding or removing
a proton or adding another species of ions. Metabolites are generally detected in
one but not both ion modalities, so broader metabolic coverage can be achieved by
analysis in both modalities. Quantification is performed by external calibration. ESI
does not cause molecular ion fragmentation as observed in electron impact mass
spectrometers, thus it does not allow direct identification of metabolites by com-
paring ESI mass spectra, as ESI mass spectral libraries are not commonly available
(as in the case by GC–MS). However, with the use of accurate mass measurements

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and/or tandem MS (MS/MS) to provide collision induced dissociation (CID) and

correlated mass spectra (MS/MS), correct identification of metabolites can be
achieved [30].

5.3 Direct injection mass spectrometry (DIMS)

DIMS is shown as a high-throughput screening tool (hundreds of samples per day

with a run time generally of 1 minute). The extracts of the crude sample are injected
or infused into an electrospray mass spectrometer and results in a mass spectrum
per sample, which is representative of the composition of the sample. The ioniza-
tion capacity of the metabolite determines metabolic coverage, as for LC–MS. The
applications of DIMS are mainly concentrated in the microbial [32] and plant areas.

5.4 Fourier transform infrared spectroscopy (FT-IR)

Vibrational spectroscopy techniques such as Fourier transform infrared spec-

troscopy (FT-IR) and Raman spectroscopy have been used to analyze metabolic
changes in biological samples. The methodologies consist of passing ultraviolet or
infrared light through a sample before it is detected. The vibrations and rotations of
the bonds relating to different chemical groups resulting from the interaction of the
sample with ultraviolet or infrared light are mainly measured.
The problem with these techniques is the inability to detect each metabolite sep-
arately; instead, it is a specific technique for single molecules that will absorb ultra-
violet or infrared light at specific wavelengths. In FT-IR, a metabolic fingerprint is
taken with a single absorption spectrum collected for each sample and consisting of
information for many metabolites. The result is similar to data produced by direct
infusion mass spectrometry where a single mass spectrum is collected rather than
an absorption spectrum for each sample. The metabolic fingerprints produced in
these approaches lack the sensitivity of mass spectrometry but are a useful tool for
high throughput screening since the FT-IR analysis time is approximately one min-
ute per sample. To date, the vast majority of metabolomic studies undertaken using
vibrational spectroscopy have been performed with FT-IR spectroscopy. However,
the work was done using Raman and, in terms of metabolomics, this is an emerging
technology with significant potential for metabolite monitoring [33].

5.5 Nuclear magnetic resonance (NMR)

Nuclear magnetic resonance (NMR) spectroscopy applies the magnetic proper-

ties of atomic nuclei in a metabolite. Only some atoms are active NMRs and include
1H, 13C and 31P. Proton (1H) NMR spectroscopy is the most frequently applied
in metabolomics. The technique works by inserting a liquid sample into a small
internal diameter tube (about 5 mm), where it is pulsed with a range of radio
frequencies that cover all possible energies required to excite the selected type of
nuclei. Nuclei absorb energy at different radio frequencies depending on their
chemical environment and then the release of this energy is measured, forming
what is called a free induction decay (FID). This FID is converted from a time
domain data set to a frequency domain - using a Fourier transformation - and an
NMR spectrum is constructed as the absorption energy plotted against the peak
intensity. The NMR spectrum (in particular the chemical shift) depends on the
effect of the shielding by the electrons orbiting the nucleus. The chemical shift for 1
H NMR is determined as the difference (in ppm) between the resonance frequency
of the observed proton and that of a reference proton present in a reference com-
pound (for 1 H NMR experiments, tetramethyl-silane in solution, fixed at 0 ppm).

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Metabolomics - Methodology and Applications in Medical Sciences and Life Sciences

The intensity of the signal depends on the number of identical nuclei and the
presence of complex samples does not interfere with the measured intensity, as does
the suppression of ionization with electrospray ionization. NMR spectroscopy is a
high-speed fingerprinting technique. Raw samples are mixed with a solution of the
reference compound added to an NMR probe (usually less than 2 mL), inserted into
the instrument and analyzed. NMR probes are generally based on a large volume of
μl and this adds constraints to the required sample volume. However, the introduc-
tion of 1 mm μl probes allowed to analyze volumes of 2 μl, thus enabling invasive
sampling of smaller volumes of study subjects, which is important for small animal
studies [10]. Spectra are complex and contain thousands of metabolic signals. For
data processing, the spectrum is generally divided into chemical shift ranges with
widths of 0.02–0.04 ppm. All signals in this bucket are added together. Chemical
changes can be assigned to specific metabolites and the pure metabolite can be
added for further clarification. However, the spectrum model is generally used in
sample classification, similar to that used for FT-IR and DIMS.

6. Examples of metabolomics studies

6.1 Urine metabolomics

The metabolic profile of biofluids has emerged as an important tool in the

diagnosis of numerous diseases that remain silent until late progression of the
disease [34]. Because urine is such a rich source of biomarkers, the metabolic
profile is a promising tool for assessing therapeutic efficacy. Urine collection is
also non-invasive, does not require patient preparation and substantially improves
compliance. Recent results clearly demonstrate the potential of urine metabolomics
in diagnosis by providing new insights into the biochemistry of its pathophysiology
[35]. The qualitative/quantitative analysis on the urine of pregnant women between
two different stages of labor called OL (out of labor) and IL-DP (in labor in the
dilation phase), using as a technique the ultra-performance liquid chromatography
of hydrophilic interaction coupled with mass spectrometry (HILIC-UPLC–MS),
a highly sensitive, accurate and unbiased approach, are an example [36]. The list
of metabolites is shown in Table 1. The urinary metabolites showing the great-
est differences belong to the steroid hormone, in particular conjugated estrogens
and amino acids, much of this difference being determined by fetal contribution.
The increased excretion of conjugated estrogens in the DP stage may confirm the
coordinated role played between fetus, mother and placenta during labor. It is
reasonable to consider this terminal phase of pregnancy not only as a mechanical
event linked to the increase in uterine contractions, but as a more complex process.
Figure 3 shows the major compounds excreted in the urine (such as Estradiol
Glucoronide, Estrone 3-Sulphate and Estriol Glucuronate) which are downstream
of a more intricate process. These compounds originate from an interconnected
metabolism between mother-fetus and placenta involving steroid hormones. As
seen in Figure 3, the metabolites excreted can come directly from the mother,
or with the contribution of both the placenta and the fetus. In the latter case,
the excretion of metabolites occurs through the degradation of intermediates, in
particular the hormone pregnenolone.

6.2 Antarctic cryptoendolithic communities

Antarctic cryptoendolite communities are microbial ecosystems that dominate

the biology of most ice-free areas in mainland Antarctica. These are complex and

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Compounds Molecular weight Percentage

3 hydroxy2-methyl-1H-quinolin-one 175.05 3% - up
19 chloro19-Chloro-3beta-hydroxyandrost-5-en-17- 365.1695 95% - up
one = dehydroepiandrosterone

Androst-5-ene-3beta,17beta-diol = androsterone 290.1736 21% - down

Androsterone 290.1736 21% - down

Dehydroepiandrosterone Sulfate 369.0992 92% - down

Dehydroepiandrosterone 288.1873 3% - down

Pregnanediol 321.2145 25% - down

3-Hydroxy-1-methylestra-1,3,5(10),6-tetraen-17-one 283.1551 63% - up

Tetrahydrocortisone 365.1592 88% - down

Estrone 3 sulfate 351.1085 >100% - up

Estrone gluconoride 447.1790 87% - down

Estradiol 17 beta 3 gluconoride 449.1945 42% - up

Ser 105.09 59% - down

Val 117.15 35% - up

His 155.15 23% - up

Arg 174.20 11% - up

Cys 121.16 47% - up

Ala 89.09 47% - down

Glu 147.13 85% - up

Gln 146.14 25% - up

Leu 131.17 38% - up

Lys 146.19 60% - up

Ile 131.17 38% - up

Thr 119.12 18% - up

Phe 165.19 11% - down

Tyr 181.19 12% - down

Table 1.
Estrogen and amino acid amount extracted from the urine in the two stages of pregnancy (OL and IL-DP). The
table refers to the relative concentration of the metabolites calculating as a percentage of the total compounds by
comparing the intensity of deconvolution of each compound.

self-supporting assemblies formed by the association of autotrophic and hetero-

trophic microorganisms such as Bacteria Chlorophyta and Fungi, which live at the
limit of their physiological adaptability and this represent the only possibility of
survival before extinction. They live inside the pores of the rocks creating an envi-
ronment that protects them from environmental stress, they are extremely tolerant
and remarkably resistant to stress and finally they adapt perfectly to the lithic life
(Figure 4A).
The study was conducted on the basis of the different solar exposure, verifying
how this factor influences the production of key metabolites and therefore on the
adaptation strategies implemented by these microorganisms to survive in extreme
conditions (Figure 4B). The result is the presence of 331 altered and differentially
expressed metabolites [37]. All intermediates of melanogenesis are found in the
highest concentration in south facing rocks (Table 2). Organisms have developed

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Metabolomics - Methodology and Applications in Medical Sciences and Life Sciences

Figure 3.
A coordinated cycle between mother fetus and placenta through the biosynthesis of steroid hormones.

Figure 4.
A - Communities stratified on rocks. B - Antarctic cryptoendolite communities. The yellow arrow indicates the
north-facing surface, while the white arrow indicates the south-facing surface. (Figure 4B comes from Coleine
et al. [37].

Compound Molecular weight Regulation

Gentisyl alcohol 140.14 Down

Hypoxanthine 136.11 Down

6- Methoxymellein 208.21 Down

Allantoin 158.12 Down

DOPA 197.18 Up

5,6-Hydroxy indole 133.15 Up

Anthocyanin 3-O-β-D glucoside 449.38 Up

Plastoquinone 749.2 Up

Xanthine 152.11 Down

Uric acid 168.11 Down

Tyrosine 181.19 Up

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Compound Molecular weight Regulation

Dopaquinone 195.17 Up
Cysteinyldopa 316.33 Down

Dopachrome 193.16 Up

5,6-Dihydroxyindole 149.15 Up
Indole 5,6- quinone 147.13 Up

Table 2.
Regulation of metabolites in Antarctic cryptoendolithic communities exposed to the south in the absence of sun
compared to the north in the presence of minimal solar radiation.

metabolic profiles responding to the condition of deprivation of sunlight, in fact

in fungi, melanin performs functions such as photoprotection, energy conver-
sion, protection from thermal stress, metal chelation, resistance to drying and cell
strengthening. Therefore, every organism that lives in non-optimal conditions has
the ability to modify its biological functions and its structures according to external
dynamics. The adaptation analysis allowed to demonstrate how the environment,
and in particular the solar exposure, can strongly influence the olic phenotype of
these microbial communities, demonstrating how the direct incidence of sunlight in
one case, or their absence in the other, it has determined the acquisition of various
adaptations by the colonizing species aimed at favoring their growth and guaran-
teeing their survival in such a hostile environment.

7. Conclusion

Starting from a holistic approach, with a high-throughput non-targeted

metabolomics without prior knowledge of the metabolome, it is possible to find
biologically relevant metabolites (potential biomarkers), characteristic of targeted
metabolomics as shown by the two examples described in this chapter. In the case
of urinary metabolites hundreds of metabolites are represented but the major
differences between OL and IL-DP belong to the steroid hormone, in particular to
conjugated estrogens and amino acids. These compounds could predict the time to
delivery in pregnant women.
As for the Antarctic cryptoendolytic communities, among the candidates to be
considered biomarkers, the precursor metabolites of the melanin and allantoin
pathways emerged as these were the most affected by exposure to the sun. These
paths can be considered directly involved in the response to environmental
pressure. In conclusion, both targeted and non-targeted metabolomics are then
used in combination for the detection and accurate weighting of differential
metabolites. Furthermore, the types of metabolites that are recovered are
influenced by the extraction and the analytical method chosen. In this chapter
the main devices used have been summarized, subsequently the results will
require computational tools to identify and correlate the metabolites among the
samples to examine their connection in the metabolic pathways in relation to the
phenotype.

Conflict of interest

The authors declare no conflict of interest.

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Metabolomics - Methodology and Applications in Medical Sciences and Life Sciences

Abbreviations

CID Collision induced dissociation

DIMS Direct Injection Mass Spectrometry
FID Free induction decay
FT-IR Fourier Transform Infrared Spectroscopy
GC–MS Gas Chromatography–Mass Spectrometry
HILIC Hydrophilic interaction chromatography
HPLC High performance liquid chromatography
IL-DP In labor in the dilation phase
LC–MS Liquid Chromatography-Mass Spectrometry
MS Mass spectrometry
NMR Nuclear magnetic resonance
OL Out of labor

Author details

Veronica Lelli†, Antonio Belardo† and Anna Maria Timperio*

Department of Ecological and Biological Sciences, University of Tuscia,
Viterbo, Italy

*Address all correspondence to: [email protected]

† These authors contributed equally to this work.

© 2021 The Author(s). Licensee IntechOpen. This chapter is distributed under the terms
of the Creative Commons Attribution License (http://creativecommons.org/licenses/
by/3.0), which permits unrestricted use, distribution, and reproduction in any medium,
provided the original work is properly cited.

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