Journal of Pharmaceutical and Biomedical Analysis 147 (2018) 341–349

Contents lists available at ScienceDirect

Journal of Pharmaceutical and Biomedical Analysis

journal homepage: www.elsevier.com/locate/jpba

Review

The analytical process to search for metabolomics biomarkers

M.D. Luque de Castro a,b,c,∗ , F. Priego-Capote a,b,c
a
Department of Analytical Chemistry, Annex Marie Curie Building, Campus of Rabanales, University of Córdoba, E-14071, Córdoba, Spain
b
Institute of Biomedical Research Maimónides (IMIBIC), Reina Sofía University Hospital, University of Córdoba, E-14004, Córdoba, Spain
c
CIBER Fragilidad y Envejecimiento Saludable (CIBERfes), Instituto de Salud Carlos III, Spain

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

Article history: The scant number of available metabolomics biomarkers does not reflect the extent of the research in
Received 15 June 2017 this field. Looking for the reasons of failure, the authors, as analytical chemists, critically discuss each of
Received in revised form 19 June 2017 the steps in the analytical process that requires improvements. They find insufficient information about
Accepted 19 June 2017
how the experimental part is developed. After revising the steps from sampling to obtainment of the
Available online 6 July 2017
analytical sample (from typical samples such as blood and urine to others less common such as sweat or
saliva), the need for data and metadata for either reproduction of a given study or for taking the study as
Keywords:
starting point after biomarker discovery is criticized. The separation and analysis steps are also revised
Metabolomics
Biomarkers
as does data treatment. After the sources of errors from the analytical process are overcome, subsequent
Sample preparation steps in the implementation of biomarkers to reach the final aim of clinical adoption should be supported
Blood as required.
Urine © 2017 Elsevier B.V. All rights reserved.
Saliva
Sweat
Exhaled breath condensate

Contents

1. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 341
2. From sampling to the analytical sample . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 342
3. Sample preparation dependence on the subsequent analytical strategy . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 344
4. Analysis in the search for metabolomics biomarkers . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 345
5. Statistical analysis in the search for metabolomics biomarkers . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 346
6. Foreseeable and desirable trends in the search for metabolomics biomarkers . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 347
Acknowledgments . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 347
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 348

1. Introduction

It is each time more accepted that the majority of our chronic

diseases (e.g., diabetes, cardiovascular diseases, cancer) are poly-
Abbreviations: CA, clustering analysis; EBC, exhaled breath condensate; KNN, genic [1]. This seems to be the reason why approaches such as
k-nearest neighbors algorithm; LC, liquid chromatograph; MS, mass spectrometry;
MSTUS, Mass Spectrometry Total Useful Signal; NMR, nuclear magnetic resonance;
genome-wide association studies have so far explained only a
O-PLS, orthogonal partial least squares; PCA, principal component analysis; PLS- small number of these diseases, and contributed also in small
DA, partial least squares discriminant analysis; QC, quality control; ROC, receiver proportion to intervention strategies based on the involved mecha-
operating characteristic; SIMCA, soft independent modelling class analogy; SPE, nisms [2,3]. The necessity to test the relationship between genetics
solid-phase extraction; TOF, time-of-flight.
∗ Corresponding authors at: Department of Analytical Chemistry, Annex Marie
and xeno-factors (e.g., diet, the gut microbiome, drugs, physi-
Curie Building, Campus of Rabanales, 14071 Córdoba, Spain.
cal activity) makes almost mandatory to use metabolomics as a
E-mail addresses: qa1lucam@uco.es (M.D. Luque de Castro), q72prcaf@uco.es bridge, from which also proteomics and transcriptomics can ben-
(F. Priego-Capote). efit. The key role of the “omics of the small molecules” (as is

http://dx.doi.org/10.1016/j.jpba.2017.06.073
0731-7085/© 2017 Elsevier B.V. All rights reserved.
342 M.D. Luque de Castro, F. Priego-Capote / Journal of Pharmaceutical and Biomedical Analysis 147 (2018) 341–349
also known metabolomics) is a consequence of both the loca-
tion of the metabolome in the flowchart of biological information
(metabolome is the “ome” closest to the phenotype) and the nature
of these small molecules (the metabolites, which are endowed with
well known characteristics as compared with the objects of older
omics).
In fact, metabolites are the most reliable indicators of phe-
notypes, located downstream of genomic, transcriptomic and
proteomic changes; therefore, metabolomic changes are frequently
more significant than those taking place at upstream levels, and
they are fast indicators of either non-homeostasis state and adap-
tation of the organism to a new homeostasis state [4]. In addition,
metabolomics data can easily be translated across species because
most metabolic pathways are hardly altered through evolution,
which is crucial to interrelate findings from experiments with lab-
oratory animals and human studies [5]; even more, most of the
metabolic processes in the body, such as energy metabolism and
amino acid catabolism, are common to all living cells.
Also the fact that metabolomics makes use of a variety of easily
accessible biological samples such as blood, urine, sweat, etc. is a
favorable aspect to reduce cost and analysis time, thus facilitating
application of a high number of samples and contributing to an
assessed study as a result.
Hence, metabolomics analysis offers a sensitive measure of bio-
logical status in health or disease as some metabolic pathways are
up- or down-regulated in diseased cells. The altered metabolic fin-
gerprints, which are unique to every individual, offer novel avenues
to better understand systems biology, detect or identify potential
risks for various diseases and ultimately help to achieve the goal of
“personalized medicine” [6].
Broadly, metabolomics investigations focused on human dis-
eases can be grouped into three categories:
(i) A major group of investigations is focused on understanding
the molecular basis of pathogenesis of diseases and identifying
altered metabolic pathways.
(ii) The second group is focused on identifying metabolite
biomarkers to classify diseases with sensitivity and specificity
as high as possible. The number of such studies is increasing
exponentially owing to the urgent need for discovering sensi-
tive biomarkers that can improve disease diagnostics, mainly
cancer biomarkers (e.g., breast cancer biomarkers based on
investigations of breast tissue metabolites) [7–10]; prostate
cancer biomarkers supported on low levels of citrate concen-
tration [11,12]; ovarian carcinoma biomarkers, including those
involved in purine, pyrimidine and glycerolipid metabolism
[13]; colon cancer biomarkers that include lactate, pyruvate,
malic acid and long-chain polyunsaturated fatty acids [14,15];
lung cancer biomarkers using samples as different as urine
[16], exhaled breath condensate [17,18] or sweat [19,20].
(iii) The third group of investigations describes translational
opportunities and applications in metabolomics. These inves-
tigations are far few, but they are also increasing exponentially
[21].
As being the subject matter of this review, the different types of
biomarkers deserve to be defined, as they can be:
(a) Features, that consists of measurable biological components
(e.g., a metabolite or more commonly a panel of metabolites) or
state of components (e.g., a primary, secondary defense metabo-
lite or that resulting from gene demethylation) that can be
analyzed as a candidate biomarker.
(b) Biomarkers as such or features indicative of disease states,
responses to therapeutic treatments, or other relevant biological
states.
(c) Biosignatures or collection of features, which together define
given biomarkers.
(d) Risk biomarkers that identifies patients who are likely to develop
given diseases.
(e) Diagnostic biomarkers, for detection of early disease state, clas-
sification into disease subtypes or characterization of response
to treatment.
(f) Prognostic biomarkers, for prediction of disease progression, pre-
diction of disease recurrence or identification of patients who
are likely to respond to a treatment.
After establishment of solid definitions and a wide development
of research in this field for more than a decade, it must be admit-
ted we cannot name a single resulting metabolomics biomarker
routinely used in the clinical field apart from those used for many
years in typical clinical tests (creatinine or glucose in blood, and uric
acid, chloride, ammonia, creatine or glucose in urine). This situation
can be explained by the lack of either analytical validation or any
other step towards clinical implementation. Looking for the reasons
for failure authors working in this field have revised the different
steps involved in the obtainment of clinically accessible biomark-
ers with clinical utility, and informing clinical decision-making to
improve patient outcomes [22,23]. Thus, the steps from biomarker
discovery, biomarker assay development and analytical validation,
validation of clinical utility, to clinical implementation have been
reviewed [24] and they appeared in Fig. 1. Recently, more steps have
been included by considering not only the metabolites by them-
selves, but also the pathways in which they are involved and the
mechanisms from which they result. The publication by Jonhson
et al. [25] provides a detailed workflow of what the authors con-
sider – and it should be – a holistic approach of the involved steps,
as shows Fig. 2. In this figure pathways to relate the metabolites to
each other and within interconnected biological pathways, as well
as the combination of metabolomic, orthogonal biological analy-
sis and isotope-assisted deciphering of pathways have been taken
into account. Nevertheless, a step, considered as insignificant for
most of the researchers working in this field – the analytical pro-
cess that starts with sampling and finishes with data treatment to
provide the information for “untargeted metabolomics” or “high-
throughput profiling” in Fig. 2 – is crucial as its development has a
cascade repercussion on the final results. No person pays attention
to a very complete reproduction of all the steps and sub-steps of
the analytical process, such as sampling, deproteination, centrifu-
gation, sample preparation, and so on. A critical discussion of these
steps is below.
2. From sampling to the analytical sample
Establishment of an appropriate experimental design and use
of patient questionnaires with subsequent population stratification
are mandatory prior to sampling. This is one of the critical steps to
guarantee the success in biomarkers discovery. Control of external
variability factors such as drug administration or the identification
of exclusion criteria is crucial to associate metabolite patterns to
pathological states.
Concerning sampling, the physical state of the sample condi-
tions this and subsequent steps leading to obtain the analytical
sample. This last is the fraction of the original or raw sam-
ple that remains after the different necessary steps – e.g.,
solid–liquid extraction, protein precipitation, centrifugation, solid-
phase extraction (SPE) or liquid–liquid extraction –, and is ready for
being introduced into either the detector or high-resolution equip-
 M.D. Luque de Castro, F. Priego-Capote / Journal of Pharmaceutical and Biomedical Analysis 147 (2018) 341–349 343

Fig. 1. Conventional scheme for biomarkers discovery with special emphasis on statistical analysis and validation.

Fig. 2. Metabolomics workflow for biomarkers discovery. The workflow involves metabolomics analysis using two different approaches: untargeted analysis or high-
throughput profiling, pathways analysis, validation, orthogonal biological analysis and mechanistic interpretation by connection to the phenotype. Reproduced with
permission of Macmillan Publishers Limited, Ref. [25].

ment such as a gas or liquid chromatograph on-line coupled to the
detector (usually a mass spectrometer).
The types of samples usual in metabolomics studies encompass
mainly biofluids such as blood (either plasma or serum) and urine,
and in a lesser proportion saliva, tears, sweat or even exhaled breath
(either condensate or constituted only by volatile components); all
them characterized for a less invasive and simple sample retrieval
than tissue and providing a representative analysis or a ‘snapshot’ of
metabolism [26]. However, the concentrations to what metabolites
are present in biofluids as compared to tissue extracts may be disad-
vantageous. Other samples no frequent in metabolomics studies are
344 M.D. Luque de Castro, F. Priego-Capote / Journal of Pharmaceutical and Biomedical Analysis 147 (2018) 341–349
cerebrospinal fluid [27], seminal fluid [28] or even bronchoalveolar
lavage fluid [29].
The main problems to which the biomarkers researcher faces
from sampling to obtainment of the analytical sample are related to
the absence in the publications of crucial information (sometimes
consisting of metadata) that hinders no only to take the results
in the given publication as the starting point to support subse-
quent research, but even to reproduce the experiments in it. By
discussing the steps involved from sampling of typical samples to
obtainment of the analytical sample, examples of what must be
thoroughly explained and what results from previous publications
must be taken into account are given below.
Blood is the most common biofluid in metabolomics [30], jus-
tified by its minimally invasive sampling [31], homogeneity as
compared to urine, sweat or saliva; all them strongly influenced
by the collected volume [32]. The type of sample obtained from
blood – plasma or serum – depends on allowing or blocking
coagulation, respectively. The impact of the different commer-
cial tubes for collection of serum or plasma on variations in the
concentration of the metabolites (both primary and secondary
metabolites) in plasma or serum has been widely investigated
[33,34]; nevertheless, the type of collection tube used for sample
collection is rarely included under “experimental”. This infor-
mation can be crucial even in dealing with metabolites from
the same precursor (vitamin D and its mono- and dihydrox-
ymetabolites) for which very different behavior has been reported
depending on the given metabolite when serum or plasma were
used as sample. Thus, statistical analysis revealed similar phys-
iological levels for vitamin D3 , 24,25-dihydroxyvitamin D3 and
25-dyhydroxyvitamin D3 in serum and plasma, but significant
different levels for 1,25-dihydroxyvitamin D3 [35]. Also sample
preparation has demonstrated to be crucial for some vitamin
D3 metabolites as is the case with 1,25-dihydroxyvitamin D3 :
this metabolite was not detected after deproteination, but it was
quantified after SPE [35]. Thus, closely adoption of the optimum
procedure in all steps from sampling to obtainment of the analytical
sample is mandatory.
Urine is also a common biofluid in biomarker discovery by
metabolomic approaches [36]. Starting with sampling, urine can
be collected as random samples, timed samples, 24-h samples,
and even longer sampling times when searching for biomarkers
of pharmacokinetics behavior [36]. Unlike serum or plasma, urine
does not require containers with special characteristics as usu-
ally bare polypropylene containers of the required volume suffice.
Critical discussions involve the doubtful necessity of a quenching
step, preservative addition, volume correction by normalization
using the sometimes controversial approaches, pH adjustment,
deproteination, stability and storage conditions, among the most
influential. All these steps are commonly detailed in the exper-
imental section, but never the material from which the sample
container is made has been specified in this section, despite non-
specific adsorption to surfaces causes metabolites losses; therefore,
to ensure the use of containers of the same type is not a trivial
aspect. The presence of urinary sediment in the urine sample has
led to spurious results in the sarcosine–creatinine ratio related to
urine supernatant [37]; a fact that makes of paramount importance
to report the g value in centrifugation steps.
Saliva is a low frequent sample in the search for metabolomics
biomarkers, despite salivary metabolites may be transferred into
saliva by different cells in the oral cavity and salivary glands, includ-
ing cancer cells if present [38]. Saliva is at present successfully
emerging as a tool to diagnose or screen oral cancer [38], after
previous application for leukoplakia and Sjogren’s syndrome stud-
ies [39,40]. Subsequent research to validate promising biomarkers
requires a solid establishment of the conditions under which each
step is developed, including all data and metadata that can con-
tribute to create a work protocol as reproducible as possible.
Semen is also an infrequent sample in metabolomics, mainly
devoted to infertility studies [41], with a recent and interesting
contribution to comprehensive metabolites identification by both
nuclear magnetic resonance (NMR) and mass spectrometry (MS)
[28]. While the latter contribution provides wide information that
allows almost complete reproduction of the experiments, the infor-
mation on the sample containers (sterile containers) does not
provide enough data to check possible interaction between the con-
tainer’s wall and some potential interesting metabolites. Harder
to reproduce is the preliminary study by Chen et al. on seminal
plasma from infertile males [42] as the authors do not provide
information about sample containers, storage, etc. In addition, the
authors express the conditions for centrifugation as rpm, which
is not enough to reproduce the experiment and poorly express the
effectiveness of the separation achieved in this step that the authors
consider a “purification step”.
Tissue samples are subjected to multiple steps to obtain the tar-
get analytical sample. Information about each of the steps should
be better established after discovery of metabolomics biomarkers
for appropriate biomarkers validation, clinical validation and clin-
ical assay. Most of the sampling and sample preparation protocols
lack of the required data for reproduction of a given experiment
[38,43].
Two samples that deserve separate discussion as sources of lung
cancer biomarkers are exhaled breath condensate (EBC) [44] and
sweat [45]. Despite the excellent values of sensitivity and selec-
tivity they have provided [18,20,46,47], implementation of the
subsequent steps of validation, etc., makes mandatory better and
reproducible sampling devices and protocols, checking of contain-
ers, and exhaustive stability studies, among others.
Duplicate efforts in reproducibility are required when a
biomarker panel is based on two different samples, as is the case
with saliva and tissue samples that provide an integrated panel for
oral cancer [38]. After entering in the validation step, an exhaus-
tive study of sample stability, and target metabolites features (such
as thermolability and/or volatility that require modification of the
working conditions to ensure stability) is mandatory.
Especial working conditions and tools are required in dealing
with small sample volumes, as is the case with EBC, sweat or saliva
samples. Solid-phase extraction (SPE) using centrifugal micro-spin
cartridges with different sorbents as a function of the metabolites
characteristics or sample lyophilization have shown to be useful
as sample preparation steps for small sample volumes [48]. The
protocols required in these cases are even more restrictive as the
errors increase by decreasing the sample volume involved.
3. Sample preparation dependence on the subsequent
analytical strategy
The three metabolomics strategies – fingerprinting, untargeted
and targeted analyses – can be used in the way from biomarkers
discovery to clinical use according to the available instrumenta-
tion and methodologies. Each strategy requires different sample
preparation. As they must be used in the following order: finger-
printing and/or untargeted analysis and, then, targeted analysis,
also discussion of the specific sample treatment follows the order.
Metabolomics fingerprinting uses a global screening approach to
classify samples in terms of metabolite patterns (fingerprints) that
change in response to a specific factor. This approach is frequently
used prior to a more comprehensive strategy to find discrimina-
tion patterns among groups of samples. This preliminary approach
is performed by NMR, MS or even infrared or Raman spectroscopy,
which leads to a low identification rate in dealing with complex
 M.D. Luque de Castro, F. Priego-Capote / Journal of Pharmaceutical and Biomedical Analysis 147 (2018) 341–349
samples as compared to untargeted metabolomics analysis. How-
ever, metabolomics fingerprinting with an NMR detector based
on direct analysis of samples also contributes to biomarkers dis-
covery with key benefits such as direct analysis in most cases,
high-throughput in 1 H NMR, and non-destructive analysis, of espe-
cial interest in dealing with valuable samples or microsamples [49].
Some examples of the use of this approach are the study carried
out by Wu et al. to find candidate biomarkers of functional dys-
pepsia [50] and that developed by Paixao de Santana-Filho et al. to
find melanoma biomarkers by analysis of murine melanocyte and
melanoma cell lines [51]. It is worth mentioning that despite NMR
fingerprinting metabolomics is useful for biomarkers discovery, it
needs generally a confirmatory analysis before validation to obtain
a more comprehensive view of the biological effect–biomarker rela-
tionship.
Untargeted metabolomics analysis, global metabolomics analysis
or metabolomics profiling aims at detecting/identifying the broad-
est number of metabolites present in an analytical sample without a
priori knowledge of the metabolome (which is particularly achieved
when several analytical platforms are combined). This approach
assumes that the significant metabolites are by definition unknown
prior to analysis, as are their physico-chemical characteristics,
which usually encompass a wide variability range. The main aims of
the sample preparation step in this case are therefore to keep intact
the sample as much as possible and to avoid fractionation and/or
losses. Nevertheless, potentially important diagnostic differences
might remain undetected due to confounding factors (e.g., instabil-
ity of the analytical system, chemical and biochemical differences
that are unrelated to the condition under study).
An ideal sample preparation procedure to be subsequently used
to search for metabolomics biomarkers should be: (i) unselec-
tive, by avoiding from the very beginning preferential metabolites
that can lead to ignoring some others which should be key or
complementary biomarkers of the final panel; (ii) simple and fast
with the minimal number of steps as the increase of them also
increases losses of potential biomarkers, with danger of lowering
the concentration to undetected values, or even total removal; (iii)
reproducible that requiring the thoroughly application of the proto-
cols without forgetting metadata (e.g., the g value if centrifugation
is used, ultrasound frequency, power and duty cycle if this type
of energy is applied); and, (iv) including a metabolism-quenching
step, if required. In short, the sample preparation procedure prior
to untargeted metabolomics should transform the sample repro-
ducibly into a format compatible with the given analytical platform
while maintaining the original metabolite composition of the sam-
ple as much as possible. The main risk of failure comes from: (a)
the removal or losses of key metabolites by inappropriate prepa-
ration of the analytical sample; (b) the wide diversity of chemical
families of metabolites, which makes impossible to obtain a com-
plete metabolomics profile with only one analytical method; (c) the
detection of significant metabolites present at different concentra-
tion levels in the same sample, which makes difficult to determine
them in a unique analysis (d) the integration of data provided by
combination of methods including different techniques; (d) the lack
of databases offering information on many metabolites.
Targeted metabolomics analysis aims at qualitative and quantita-
tive studying one metabolite or, more frequently, a small group
of chemically similar metabolites. This analysis provides higher
sensitivity and selectivity than its untargeted counterpart, but the
target metabolites are analyzed on the basis of a priori information.
Therefore, the physico-chemical characteristics of the metabo-
lites are known and an exhaustive separation of them from the
matrix is usually required for a quantitation as free of interferents
as possible. Thus, in addition to requirements common to untar-
geted metabolomics analysis (viz., reproducibility and quenching),
the targeted approach requires a sample preparation procedure
345
as selective as possible to the metabolite(s) that constitute the
selected panel. Preconcentration and cleanup steps are usually
involved in sample preparation for targeted metabolomics analysis.
The main causes of failure can be attributed to insufficient
cleanup of the sample that results in ionization suppression. While
this effect can be overcome by the use of isotopically labeled stan-
dards when the ionization suppression phenomenon is partial [52],
the absence of signals by total ionization suppression makes useless
the practice of labeled isotopic standards.
An optimal way for development of sample preparation
when the sample volume is small – particularly for targeted
metabolomics analysis – is by connection with the subsequent
step (either detection or high-resolution separation). In this way,
the whole analytical sample resulting from sample preparation is
inserted into the detector or chromatograph, respectively [53,54].
4. Analysis in the search for metabolomics biomarkers
While analysis has traditionally been the name applied to the
whole analytical process, presently it is applied to the process
occurring after insertion of the analytical sample either into the
detector or the high-resolution equipment online connected to the
detector.
Direct insertion of the analytical sample into the detector to
search for metabolomics biomarkers has mainly involved NMR
instruments [27,38], with very few cases dealing with connec-
tion of the instrument to a liquid chromatograph (LC) [55,56]. On
the contrary, mass detectors have traditionally been connected to
chromatographs and in a lesser extent to capillary electrophore-
sis devices. The type of analyzer differs as a function of the
metabolomics strategy, being time-of-flight (TOF) and Orbitrap
analyzers (usually provided with a quadrupole unit) involved in
profiling analysis, while the triple quad is the preferred choice for
quantitation and confirmatory analysis. In this way, metabolites
can be tentatively identified with a resolution that is a function
of the equipment used. After checking the usefulness of the pan-
els (viz., their appropriate values of both sensitivity and specificity)
the metabolites involved in them are absolutely identified by the
corresponding standards, providing they are commercial or easy
to synthesize. After selection of a given panel with the consequent
reduction of the number of analytes under study, application of
a targeted approach is desirable after optimization of the sam-
ple preparation procedure and the analytical platform. In this
way, the accuracy, repeatability, and intra-days and inter-day vari-
ance can be evaluated, followed by clinical validation. This last
step should involve a number of samples as high as possible and
from patients with different disease development. The ideal case
for a metabolomics biomarker (either a panel or an individual
biomarker) is that in which the alteration of the involved metabo-
lite(s) is detected at the very incipient state of the disease, thus
allowing an early diagnostic.
Aspects that can jeopardize the desired development of
metabolomics biomarkers attributable to the analysis step are
related to: (i) the sensitivity of the detector that can be not enough
to reach the detection limit of the target metabolites (e.g., the
amount of sperm cells necessary for gas chromatography (GC)–MS
analysis is 15 millions, but 150 millions are needed for a 1 H NMR
experiment [28]). (ii) The absence of quality control (QC) samples
that should be periodically analyzed throughout the analytical run
to check the reproducibility of the data produce by the instrument
used; thus providing quality assessment for each metabolite peak.
(iii) the lack of selectivity to discriminate isomers when only one of
them possesses biomarker capability. (iv) the unavailability of com-
mercial standards of the biomarkers for preparation of calibration
models, applicable also to internal standards, isotopically labeled
346 M.D. Luque de Castro, F. Priego-Capote / Journal of Pharmaceutical and Biomedical Analysis 147 (2018) 341–349

Fig. 3. Random forest analysis provided by analysis of EBC from current smokers (CS) and never smokers (NS). Reproduced with permission of Nature, Ref. [18], http://
creativecommons.org/licenses/by/4.0/.

or not. (v) the presence of matrix effects with direct influence on accurate enough to identify the appropriate normalization method
accuracy and, therefore, on the final results. for some datasets. However, normalization methods influenced
relevantly the kind and number of potential biomarkers. These
results led the authors to propose a two-part strategy to deter-
5. Statistical analysis in the search for metabolomics
mine the suitability of the normalization method. The first part
biomarkers
was targeted at understanding comprehensively the compatibil-
ity between data and normalization approaches, while the second
Apart from the experimental plan and the analytical method,
part was used to handle biomarkers by comparison of normaliza-
there is one other step that needs for scientific rigor to ensure
tion approaches [59]. The same conclusions could be extracted from
the quality of the final results: statistical analysis, which means
the applicability of other operations for data transformation such
the application of univariate and multivariate tools to reveal the
as centering or scaling, which transform the original data sets to
biomarker capability of molecular entities or metabolites, provid-
ensure comparisons. These pretreatment operations depend on the
ing they have been previously identified.
data distribution, thus hindering establishment of guided work-
The conventional scheme for statistical analysis in the search for
flows.
biomarkers typically involves four main steps, as Fig. 1 shows: data
The data treatment step is generally initiated by testing unsu-
preconditioning, pretreatment, treatment and validation. Although
pervised and supervised analysis. PCA and clustering analysis
this scheme seems to be very well defined, its main limitation is the
(CA) are the two main alternatives to reveal discrimination pat-
huge variability of implementable software, algorithms and statis-
terns occurring between cases and controls. Partial least squares
tical operations. Thus, it is difficult to find common protocols in
discriminant analysis (PLS-DA) is the most frequent supervised
the literature for biomarkers discovery. This lack of standardiza-
approach to prove that the studied factor explains a representa-
tion particularly affects to data preconditioning and pretreatment.
tive part of the total variability. Other frequent alternatives are
One example is normalization, which can be carried out at a pre- or
orthogonal PLS (O-PLS), k-nearest neighbors algorithm (KNN), soft
post- data acquisition step to ensure comparability among groups
independent modelling class analogy (SIMCA), neural networks-
of samples [57]. It is well-known that normalization should be
based algorithms, logistic regressions, supported vector machines
compatible with the experimental design, research purpose and
and random forest analysis [60–62]. Since all these supervised
data mining methods [58]. However, the selection of an appropri-
approaches generate classification models, they can be subjected
ate method is still confusing, the prerequisites for a normalization
to internal and external validation. The former, typically cross-
method are difficult to confirm in some cases, and evaluation
validation, is essentially selected when the number of samples used
of normalization results is unclear [59]. Extended practices are
to create the models is limited. Therefore, it is preferably tested in
the MSTUS (Mass Spectrometry Total Useful Signal) approach,
a screening phase. External validation is a more robust approach
QCs correction or logarithmic transformations. Nevertheless, few
since the samples of the validation set are not used to develop the
researchers have evaluated the influence of normalization proto-
classification models. The highest variability level is to use sam-
cols on the final biomarker candidates. Recently, Chen et al. tested
ples from different locations and combine them in training and
five representative normalization methods in three real datasets
validation sets.
obtained by GC–MS affected by different variability sources. The
Once discrimination patterns are revealed, parametric or non-
tested approaches were MSTUS, Median, Probabilistic Quotient
parametric tests (ANOVA, t-test versus Kruskal-Wallis test) allow
Normalization, Remove Unwanted Variation-random and System-
highlighting the metabolites more statistically affected by the fac-
atic Ratio Normalization. Typical evaluation parameters such as
tor under study. The selection of these tests is influenced by the
the values of relative standard variation of the metabolites among
success in the normalization process. An extended alternative is the
QC samples, principal component analysis (PCA), within-group and
analysis of fold change ratios to check the concentration variations
across-group relative log abundance plots were applied to compare
that contribute to differentiate groups of samples. However, these
normalization effects. Surprisingly, these evaluation tests were not
 M.D. Luque de Castro, F. Priego-Capote / Journal of Pharmaceutical and Biomedical Analysis 147 (2018) 341–349
Fig. 4. ROC curves of the panel to discriminate control and risk factor groups
(black) and the best individual marker —indole— (grey). The panel included five
metabolites: monopalmitin, monostearin, benzyl alcohol, 2,4-diphenyl-4-methyl-
2-E-pentene and p-cresol. Reproduced with permission of IOP Publishing, Ref. [64].
variations are not statistically supported. In any case, statistically
significant metabolites or compounds experiencing a relevant con-
centration change must not be labeled as candidate markers prior
to assessment of their predictive capability.
The predictive capability is one of the most critical tasks in
biomarker discovery. This information can be deduced from any
of the supervised approaches previously indicated. Nevertheless,
the most used approach with this purpose is random forest anal-
ysis, which allows obtaining a list of top-n metabolites classified
by this parameter. One example is found in Fig. 3 that shows
the top-12 metabolite candidates found in EBC for discrimination
of current smokers versus never smokers, in which the classifi-
cation is carried out by independent predictive capability [18].
Complementarily, receiver operating characteristic (ROC) analy-
sis quantifies the marker capability of each compound in terms
of sensitivity and specificity, which means identification of true
positive and true negative cases. ROC analysis is considered an
essential tool in the identification of biomarkers for all proto-
cols. In this aspect, it is quite difficult to find a unique biomarker
with high predictive behavior in terms of sensitivity and selectiv-
ity and the strategy is generally guided to multivariate analysis
by combination of biomarkers in panels. Combination of metabo-
lites to enhance biomarker performance can be carried out with a
targeted approach, by selecting metabolites with the highest pre-
diction capability, or in an iterative manner by configuring panels
of n compounds from the list of significant metabolites. Within
this second strategy it is worth mention the role of software such
® reproduced are known.
as Panelomix [63], which accelerates considerably the searching
process. Additionally, panels can be configured according to the
final aim to maximize accuracy, sensitivity and/or specificity. This
approach has been recently applied to find candidate biomarkers
in EBC to discriminate lung cancer patients, risk factor individuals
and control individuals. Fig. 4 illustrates the ROC curves obtained
by combination of five metabolites – monopalmitin, benzyl alcohol,
monostearin, 2,4-diphenyl-4-methyl-2-E-pentene and p-cresol –
347
and by indole (this one the best individual marker, which curiously
was not in the panel) to discriminate risk factor and control individ-
uals. As can be seen, the performance was considerably enhanced
as compared to indole when several metabolites were combined
[64].
The final step in the workflow is the validation of biomarkers
in the analytical context. This step is basically focused on analyti-
cal characterization of the biomarkers to be quantified in absolute
terms in order to find differences between groups of samples versus
controls. Clinical validation is out of the scope of this review as it
typically involves a large scale study dealing with cohorts subjected
to different variability sources.
6. Foreseeable and desirable trends in the search for
metabolomics biomarkers
The numerous successful developments on metabolomics have
shown the power of this discipline that encompasses from
biomarker discovery to understanding both the mechanisms
underlying phenotypes and the key contribution of microbiota to
the individual state.
Once development of the analytical process for metabolomics
biomarkers discovery is achieved with the required information
referred to both data and metadata, its perfect reproduction in any
laboratory will facilitate participation of as many research groups
as possible. It would be followed by the accurate development of
the steps following biomarkers discovery (see Fig. 1). Therefore:
(i) Biomarker validation by different laboratories will give
robustness to this step.
(ii) Clinical validation by multiple hospitals from which a huge
number of samples can be obtained will facilitate knowledge
of the lowest concentration of metabolites involved in the
panel for disease detection.
(iii) Further improvement can even be achieved by polishing the
analytical process after checking the presence of interferents
or masking agents when the panel components are at very
low concentrations.
(iv) Integration of orthogonal omics [25] can provide insight into
the mechanisms of the given disease by providing up- and
down-stream information [48].
(v) Development of workflows to assign biological meaning to
metabolites and to move towards finding mechanisms of dis-
ease to determine the metabolic pathways perturbed by the
aberrant conditions.
(vi) Targeting of metabolomics pathways to influence/interfere
metabolite levels can even be directed to genes and silencing
gene expression.
(vii) Also, metabolic pathways can be influenced at the protein
level by using antimetabolites.
(viii) Manipulation of sources of exposure to different stimuli can
also influence the metabolome, providing further mechanis-
tic insights.
Without forgetting that all subsequent research should be sup-
ported on a solid analytical process of which all data for being
Acknowledgments
The Spanish Ministerio de Economía y Competitividad
(MINECO), Junta de Andalucía and FEDER program are grate-
fully acknowledged for financial support (Projects “Development
of methods for early cancer detection”, FQM-1602, and CTQ2015-
348 M.D. Luque de Castro, F. Priego-Capote / Journal of Pharmaceutical and Biomedical Analysis 147 (2018) 341–349
68813-R). CIBER de Fragilidad y Envejecimiento Saludable
(CIBERfes), an initiative of ISCIII, Spain, is also acklowledged.
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