Trends in Analytical Chemistry 135 (2021) 116157

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Trends in Analytical Chemistry

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

A guide to good practice in chemometric methods for vibrational

spectroscopy, electrochemistry, and hyphenated mass spectrometry
Manuel David Peris-Díaz*, Artur Kre˛ zel
_ **
Department of Chemical Biology, Faculty of Biotechnology, University of Wrocław, F. Joliot-Curie 14a, 50-383, Wrocław, Poland

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

Article history: Chemometric methods are powerful tools used in analytical sciences and beyond. Their application is
Available online 22 December 2020 continuously increasing due in part to the technological advances that allow exploration of new issues.
This review presents the most common chemometric tools utilized in the most recent research articles
Keywords: (2018 to now), which emerge at the interface between several disciplines and instrumental techniques
Chemometrics such as vibrational spectroscopy, electrochemistry, and hyphenated mass spectrometry techniques. The
Vibrational spectroscopy
review is divided into several sections: statistical design of experiments, signal pre-processing, explor-
Electrochemistry
atory data analysis, predictive modelling of the data, and statistical validation. In each section, we review
Mass spectrometry
Signal processing
the main mathematical models, then we examine the trends observed in the research articles, and finally
Design of experiments discuss the potential pitfalls to avoid during the application of the methods. We believe that bringing
Exploratory analysis together the main mathematical models with the possible experimental challenges will be of great use
Predictive modelling for non-experts chemometricians.
Validation © 2020 The Author(s). Published by Elsevier B.V. This is an open access article under the CC BY license
(http://creativecommons.org/licenses/by/4.0/).

1. Introduction
The technological developments in analytical chemistry are
accompanied by the generation of abundant and complex chemical
data. The introduction of new methods and instrumentation is
crucial for solving many fundamental questions. However, it also
presents a big challenge for data analysis. Understanding what
chemical information among all of the data obtained is in itself a
fundamental issue. The continuous development in transmission
technology, mass analyzers or activation methods in mass spec-
trometry has permitted their application in new areas [1,2]. Also,
the development of Raman spectroscopy methods enables their use
in biomedical sciences [3]. Such advances require more time and
effort to comprehensively analyze the data and extract the infor-
mation of interest. Here, bioinformatics, systems biology and che-
mometric disciplines overlap in order to develop mathematical and
statistical methods for the analysis of the data. The latter discipline
is especially useful for the design of experiments, the mathematical
treatment of the signal response, the exploration of the data, or for
* Corresponding author.
** Corresponding author.
E-mail addresses: manuel.perisdiaz@uwr.edu.pl (M.D. Peris-Díaz), artur.krezel@
_
uwr.edu.pl (A. Kre˛ zel).
predictive modelling [4], and will be the subject of this review
(Scheme 1). Therefore, chemometrics plays a key role in under-
standing the data and reaching the right conclusions. For example,
an early article that presented a meta-analysis of genomics studies
on schizophrenia concluded that “gene association studies are
typically wrong” [5]. Although this has been referred to as geno-
mics studies, statistical issues are still present in the post-genome
era. This was exemplified in a recent meta-analysis of metab-
olomics studies, which concluded that a third of them did not make
use of proper statistics [6]. Another study reported how often
methods cause bias in peptide identification [7].
This review summarizes the advances (2018 to present) in the
use of chemometrics in three main fields of analytical chemistry:
spectroscopy, electrochemistry and hyphenated mass spectrom-
etry. This period is rather narrow but clearly indicates trends in
chemometric tools and high interest of their application in multiple
physicochemical methods (Scheme 2). By clustering and creating a
bibliometric map, we quickly identified the main fields of appli-
cations, methods and/or instrumental techniques. For example, in
blue is clustered gas chromatography with volatile organic com-
pounds, biomarkers and metabolomics. This indicates the rela-
tionship between the technique and the field of application. On the
other hand, we see that food chemistry such as the analysis of
adulteration of olive oil and traditional Chinese medicines are fields
of application of great interest (Scheme 2).

https://doi.org/10.1016/j.trac.2020.116157
0165-9936/© 2020 The Author(s). Published by Elsevier B.V. This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/).
M.D. Peris-Díaz and A. Kre˛ z_ el Trends in Analytical Chemistry 135 (2021) 116157

Abbreviations AsLSSR asymmetric least squares splines regression

COW correlation optimized warping
DOE statistical design of the experiments icoshift interval correlation optimized shifting
OFAT one-factor-at-a-time GC gas chromatography
RSM response surface method LC liquid chromatography
PB Plackett-Burman MS mass spectrometry
CCD central composite design TIC total ion chromatogram
BBD Box-Behnken designs Q2 test set validation coefficient
ANOVA analysis of variance RMSECV root mean square error of cross validation
ML machine learning RMSEC root mean square error of calibration
MOO multi-objective optimization PLS-DA partial least squares-discriminant analysis
DPC N-dodecylpyridinium chloride PLS partial least squares
QU quercetin PCA principal component analysis
TA tannic acid PC principal components
NIR near-infrared HCA hierarchical clustering analysis
MIR middle infrared LDA linear discriminant analysis
SNV standard normal variate SVMs support vector machines
MSC multiplicative scatter correction RF Random Forest
EEM excitation-emission matrix NIPLS nonlinear iterative PLS
PARAFAC parallel factor analysis SIMPLS statistically inspired modification of PLS
MDR missing data recovery LV latent variable
SG Savitzky-Golay polynomial filters CART classification and regression tree
NW Norris-Williams derivation OOB out-of-bag
FFT fast Fourier transform SRM structural risk minimization
CWT continuous wavelet transform VIP variable importance in projection
DWT discrete wavelet transform CV cross-validation

In the first part of this review, we focus on the use of the sta-
tistical design of the experiments (DOE) and their optimization.
Here, we discuss and present the main DOE used throughout the
research analyzed. In the second part, we describe the main signal
pre-processing algorithms and strategies according to the afore-
mentioned three main experimental techniques. This section, and
the rest that are presented, are concluded by exposing the potential
pitfalls that one may encounter during application of these algo-
rithms and by presenting the current trends. In the third part, we
address the exploratory analysis of the data, giving a brief intro-
duction to the main techniques and concluding with the main
pitfalls and trends. The fourth section constitutes the predictive
modelling of the data and it is followed by the model validation.
These articles draw trends for each section directly from the
manuscripts examined, which were searched in the Scopus citation
database. The search was focused on articles’ titles and keywords
that contained chemometrics in combination with “electrochem-
istry”, or “spectroscopy” or “mass spectrometry”. The large number
of articles (almost 400) published in the last two and half years
confirms how widely chemometrics is used along with analytical
chemistry. Therefore, it is of utmost importance to reveal the
possible pitfalls at every step of the chemometrics pipeline, and to
extract conclusions derived from the trends.
2. Experimental design and mathematical optimization of
experimental responses
Optimization of the obtained signal response may be addressed
with a one-factor-at-a-time (OFAT) or with an experimental design
that accounts for multiple factors simultaneously. OFAT is a simple
experimental design in which one factor is changed while keeping
the rest of the factors fixed [8]. Commonly approached by non-
expert users, OFAT presents multiple disadvantages [9]. Mathe-
matically, OFAT usually requires more experimental runs than
factorial designs (either full or fractional factorial designs) to obtain
2
similar factor precision. Moreover, the experimental design de-
pends on the project lead, which can easily lead to false optimums.
The second main disadvantage is that OFAT cannot estimate
whether there are interactions between the factors studied.
Because one factor is changed while the rest are kept fixed, infor-
mation about how factors influence each other is lost. Therefore,
OFAT can only be used for screening of the main factors and informs
about the main effect of a factor on the output. This relates to the
third disadvantage, finding sub-optimal solutions for the optimi-
zation problem.
All of these issues can be solved by incorporating the DOE
methods [10,11]. DOE presents multiple advantages: (i) multiple
factors can be simultaneously studied and thus their interactions
estimated. This results in higher precision to estimate the effects of
a factor; (ii) the experiments are designed in factorial designs
(either full or fractional) and thus do not depend on the project
lead; (iii) DOE provides better optimization than OFAT, finding the
optimum solution; (iv) DOE offers the possibility to develop a
regression model that captures the relationship between the sig-
nificant factors and their interactions. Therefore, DOE is a much
more reliable and complete methodology for screening or optimi-
zation than OFAT experiments [11]. There are several types of DOE,
which according to the purposes are factorial designs for screening
and response surface method (RSM) designs for optimization pur-
poses [12,13]. Factorial designs generally attempt to identify the
factors that influence the response (output), and depending on the
experimental design also to identify the effect of the interactions
(Table 1) [14,15]. Mainly full factorial designs, two-level full facto-
rial designs, two-level fractional factorial designs, Plackett-Burman
(PB) and Taguchi's orthogonal array are screening designs. Full
factorial designs completely study all of the factors, evaluating the
main factors and all of the interactions between them. However,
the designs quickly become too large when incorporating multiple
factors or levels. For instance, selecting five factors (K) at three
levels (n) results in 35 ¼ 243 experimental runs (calculated
M.D. Peris-Díaz and A. Kre˛ z_ el Trends in Analytical Chemistry 135 (2021) 116157

Scheme 1. Overview of the chemometrics pipeline followed in this review. If the experimental data set (n) is large enough (a threshold of 40 indicated in the scheme), the data can
be split into a training set and test set. On the other hand, when having small datasets (n < 40), resampling algorithms for such cross-validation are preferred to split the data. If the
data set is divided into a training and test set, the preprocessing algorithms should be applied separately to these two data sets. The data preprocessing schemes have been
separated according to the experimental techniques considered herein, indeed electrochemistry, spectroscopies and hyphenated mass spectrometry. That is because each
experimental technique requires specific preprocessing steps in order to remove the noise from the true signal, and prepare it for the exploratory and/or predictive modelling
treatment. After the predictive model is built, it should be validated by means of the validation set, and the previous steps iteratively followed until achieving the optimal model. At
the end, the optimal model can be evaluated using a test set, that is independent of the collected experimental data. Abbreviations: NW: Norris-Williams derivation; FFT: fast
Fourier transform; CWT: continuous wavelet transform; DWT: discrete wavelet transform; AsLSSR: asymmetric least squares splines regression: COW: correlation optimized
warping; icoshift: interval correlation optimized shifting; SNV: standard normal variate; MSC: multiplicative scatter correction.

(three or more factors) are not significant, so they are substituted

Scheme 2. Network topology generated from the documents retrieved in this review.
The yellow, blue, and green colors represent the three main clusters that correspond to
chemometric methods, instrumental techniques, and applications, respectively. The
sizes of the labels are scaled according to the occurrence of the word in the retrieved
documents.
according to nK), which is prohibitive to perform in terms of cost
and effort. Two-level full factorial designs (2K) are widely popular
for screening when the factors are lower than 4e5. Approaching
this number of factors increases the complexity of the matrix
(24 ¼ 16 or 25 ¼ 32 runs). One way to mitigate the large number of
experimental runs required is with fractional factorial designs.
They have an assumption that higher-order factor interactions
with new factors, thus reducing the number of runs required. The
standard notation for this design is 2Kp, where K is the number of
factors and p is the number of design generators. We may calculate
the fraction of experiments required concerning the full factorial
design as 1/2p. For example, considering an experiment with three
factors (K ¼ 3) and one design generator (p ¼ 1) leads to a 231
design with 1/21 or half of the runs required from a full factorial
design (23). So, the design was reduced from eight to four
experiments.
However, half fractional factorial designs can be practically used
only when the factors studied number at least three. This relates to
the resolution of the design, that is its ability to separate the main
effects from the interaction effects. Formally, the resolution is
defined as the length of the shortest word in the defining relation.
Our previous 231 design with three factors (e.g., factor “A”, factor
“B” and factor “C”), has resolution III because there is only one
possible design generator with the word “ABC”. A design with at
least resolution III is needed to estimate the main effects. However,
here one main factor is confounded with two-factor interaction. An
easy way to interpret it is by splitting the resolution number (III)
into the sum of two integer numbers. Resolution III would be
3 ¼ 1 þ 2. So, the main effects (1) and the two-factor interactions
(II) can be confounded. A 241 design with four factors has reso-
lution IV and may estimate the main effects, confounded or aliased
with third-factor interactions. This design can also estimate the
two-factor interactions but they are aliased with other two-factor
interactions. Similarly, splitting IV into two integers results in two

3
M.D. Peris-Díaz and A. Kre˛ z_ el Trends in Analytical Chemistry 135 (2021) 116157

Table 1 interactions are considered. Thus, prior knowledge of the system

Overview of the most commonly used statistical design of experiments (DOE) ac- is required to identify which interactions might be significant for
cording to the main objective, either screening or optimization.
the design.
Objective DOE Features Once the important factors have been screened, the optimiza-
Screening Full factorial design - Allow all factors and levels (nK) tion and finding the optimum value of the response can be per-
- Prohibitive in terms of cost and effort formed with the RSM designs [16]. There are two main types of
Screening Two-level full - Factors < 4-5 RSM designs, central composite design (CCD) and Box-Behnken
factorial design - Two-level: 2K
design (BBD). CCD is often used in optimization designs because
Screening Two-level fractional - 3 factors
factorial design 231 - Resolution III: One main factor is it can account for up to five levels for each factor and also it can
confounded with two-factor include runs from fractional factorial designs. BBD is a three-level
interaction design that results in a less expensive design and thus is
Screening Two-level fractional - 4 factors
preferred to CCD when three factors are involved in the optimiza-
factorial design 241 - Resolution IV: Estimate the main
effects with no confounding, but the
tion. However, BBD cannot include runs from fractional factorial
two-factor interactions are aliased designs and thus does not allow sequential experiments. The
with each other screening designs presented, since they have only two levels for
Screening Two-level fractional - 5 factors each factor, only allow modelling a linear response surface. The
factorial design 251 - Resolution V: main effects and two-
responses obtained are fitted to a linear function (Equation (1)):
factor interactions are estimated
with no aliasing to any other main
effect or two-factor interaction X
k
Screening Plackett-Burman - N-1 factors with N experiments, y ¼ b0 þ bi xi þ ε (1)
where N is equal to a multiple of i¼1
four with eight as starting point.
- Two levels per factor where k is the number of variables, b0 is a constant term or inter-
- Resolution III: One main factor is
confounded with two-factor interac-
cept that represents the overall mean effect, bi are the linear co-
tion. Thus, it is desired for cases efficients, xi are the variables and the residual error is ε. That is, bi is
where we do not expect interactions the effect of the factor xi. On the other hand, both designs CCD and
between effects and we aim to BBD can estimate the interaction between the experimental vari-
investigate main effects.
ables through interaction terms (Equation (2)):
Screening Taguchi's orthogonal - Estimate main effects and two-factor
array interactions
- Prior knowledge of what interactions X
k X
k
might be significant y ¼ b0 þ bi xi þ bij xi xj þ ε (2)
Optimization CCD - Up to five levels each factor i¼1 1ij
- It can include runs from fractional
factorial designs
where bij represents coefficients for the interaction between the
- Estimation main effects, interaction
and quadratic terms factor xi and the factor xj. The terms k, b0 , ε and bi are the same as
Optimization BBD - Three-level each factor for Equation (1).
- Estimation main effects, interaction These models may incorporate quadratic terms in a regression
and quadratic terms model to account for the curvature in the response. This implies
CCD: Central composite design; BBD: Box-Behnken design. that they are able to determine critical points in the surface (i.e.,
maximum, minimum, and saddle points). In order to do so, the
response is fitted to a polynomial function that contains quadratic
possibilities, 4 ¼ 1 þ 3 or 2 þ 2. Therefore, the main factors are terms (Equation (3)):
aliased with three-factor interactions, or the two-factor in-
teractions are aliased with other two-factor interactions. Increasing X
k X
k X
k
to resolution IV leads to estimating the main effects with no con- y ¼ b0 þ bi xi þ bij xi xj þ bii x2i þ ε (3)
founding, but the two-factor interactions are aliased with each i¼1 1ij i¼1
other. A 251 design with 5 factors would have resolution V. Here,
the main effects and two-factor interactions are estimated with no where k; b0 ; ε; bi ; bij , xi and xj are the same than for Equation (2).
aliasing to any other main effect or two-factor interaction. How- Thus, modelling the results obtained can be accomplished by
ever, the two-factor interactions are aliased with three-factor using the simplest linear function (Equation (1)) to a full model that
interaction. Explained as above, V can be split as 1 þ 4 or 3 þ 2, accounts for linear, interaction, and quadratic terms (Equation (3)).
meaning that main factors are aliased with four-factor interaction Generally, the mathematical model should be selected based on the
or two-factor interactions are aliased with three-factor interaction. application of analysis of variance (ANOVA). Based on the p-values
The higher the resolution is, the lower is the confounding, but obtained for each coefficient regression obtained from ANOVA, the
requiring more experimental runs. linear (A, B, C), interaction (AB, BC, AC), or quadratic terms (A2, B2,
Plackett-Burman designs are a two-level fractional factorial C2) are included or excluded in the final model. Once the terms to
design with resolution III, and thus aimed at investigating the main include in the equation have been selected, the regression problem
effects (Table 1). This design allows one to include up to N-1 factors can be solved with a least-squares estimator with machine learning
with N experiments, where N is equal to a multiple of four. PB (ML) algorithms [17e20]. The multiple regression model is assessed
designs offer a lower number of runs than fractional factorial de- based on the determination of the coefficient (R2). The R2 increases
signs for the screening of the main effects. Thus, for cases where we when adding new terms to the model. Therefore, one should
do not expect interactions between factors, PB is desired. On the examine instead the adjusted R2, which takes into account the
other hand, if we assume that there will be an interaction between number of terms in the regression model. The adjusted R2 will in-
the factors, we might want to choose fractional factorial designs. crease only if the new term added to the regression model im-
The last design for screening is Taguchi orthogonal array, a frac- proves its explanatory power. Another parameter that should be
tional factorial design where only the main effects and two-factor checked is the lack-of-fit of regression, which assess the variability
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M.D. Peris-Díaz and A. Kre˛ z_ el
Table 2
Overview of the DOE employed for the different analytical techniques, that is vibrational spectroscopy, electrochemical and hyphenated mass spectrometric techniques.
Analytical method DOE
Spectroscopies Fractional factorial design resolution IV
Full factorial design
Electrochemistry 33 full factorial design
Mixture design
L36 fractional factorial design
23 full factorial design
CCD
PB
BBD
Four-level orthogonal array
LC-GC/MS CCD
Full factorial design
BBD
Statistical mixture design
CCD: Central composite design; BBD: Box-Behnken design; PB: Plackett-Burman; LC: liquid chromatography; GC: gas chromatography; MS: mass spectrometry.
due to random error. In order to be able to calculate it, the design
must incorporate replicates or central points. If the error provided
by the lack-of-fit of regression is lower than the random pure error
(p-value > 0.05), the regression model is well fitted. ML algorithms
have proved higher accuracy, handling complex nonlinear re-
lations, and automatically pick up the interaction effects between
the variables [17e20].
When there are multiple responses to simultaneously optimize,
finding the optimum solution is not a trivial task. Multi-objective
optimization (MOO) strategies represent an approach for consid-
ering all of the responses simultaneously in order to provide a so-
lution. Several approaches such as Bayesian models, genetic
algorithms, Pareto optimality, and desirability functions have been
used for solving MOO [11,17,18,21e24]. The most often used crite-
rion is the desirability function or Derringer function is in which
each response has attributed a desirability function that ranges
from 0 (undesirable response) to 1 (fully desired response). Overall
desirability is obtained as the weighted geometric average of the
individual desirability functions. The individual desirability func-
tion can be constructed based on minimization/maximization
criteria establishing target values for the response. An interesting
approach that we have recently employed is the Pareto front
approach [17]. The Pareto approach identifies a set of optimal so-
lutions according to a trade-off selected (i.e., maximization of a
particular response without degeneration of the rest of responses).
2.1. Applications, trends and potential pitfalls in DOE
DOE has been used for the screening and optimization of
chromatographic conditions [25,26], optimization of extraction
methods [28,29], in screening stages during drug development
[29e31] optimization of chemical synthesis [32e34], and for
determination of other compounds of interest [35e40]. DOE has
become a preferred choice for rational pharmaceutical develop-
ment. For example, a full factorial design and a four-level orthog-
onal array have recently been used in the screening of ramipril and
prediction of drugs used to treat hypertension [29,33]. Another
common application is in the screening of instrumental parameters
affecting signal acquisition. In this sense, PB has been used for
screening the parameters influencing the electrochemical detec-
tion of Sm(III) in its complex with diethylenetriaminepentaacetic
acid. Furthermore, the authors identified significant factors that
were then optimized with a BBD design [40]. Other application of
BBD designs is the optimization of mass spectrometry conditions
[17,18]. In both reports, the authors employed BBD for the optimi-
zation of preselected instrumental factors related to mass spec-
trometry instruments. Multiple responses were simultaneously
Trends in Analytical Chemistry 135 (2021) 116157
Objective Experimental aim Reference
Screening Synthesis of silica nanoparticles [32]
Screening Drug development [29]
Screening Drug quantification, phenol quantification [30,35,36]
Optimization Synthesis biosensor [33,34]
Screening Determination metals in soils and plants [161]
Screening Determination flavonol in drink, determination of folic acid in urine [37]
Optimization Detection of Sm(III) [38,39]
Screening Determination of Sm(III) [40]
Optimization Drug quantification [40]
Screening [31]
Optimization Optimization chromatographic and MS conditions [25,162,163]
Screening Optimization chromatographic conditions [26]
Optimization Optimization MS parameters, compound extraction [17,18,27,28]
Optimization Compound extraction [164]
optimized, solving the MOO problem through Pareto optimality.
Generally, we may observe how BBD is more common employed
than CCD for optimization purposes in hyphenated MS techniques
(Table 2). Herein, the focus will be set on using CCD and BBD for
optimization purposes.
A recent application in the development of analytical methods,
based on supercritical fluid chromatography coupled to time-of-
flight mass spectrometry in order to identify lipid markers from
traditional Chinese medicine, used a CCD to optimize the chro-
matographic separation [25]. In chromatography separations usu-
ally one is interested in the evaluation of several responses, such as
the separation between peaks, the analysis time of characteristics
of the peaks such us peak width, and symmetry or resolution. To do
so, multiple response optimization through desirability function or
Pareto efficiency is of great interest. The authors studied the effect
of the solvent type, flow rate, column temperature and back pres-
sure at five levels, which required 30 experiments. The experi-
mental design included six repetitions of the central point to
estimate the variance of the model, and the experiments were
randomized [25]. Several responses were simultaneously studied:
resolution between peaks, analysis time, total number of peaks, and
number of peaks between triglycerides and diglycerides. The four
responses obtained from the CCD were evaluated using a Derringer
function, which showed for each response the best set of condi-
tions. The individual desirability values were combined into the
global desirability function that ranged from 0 to 0.22. Then, a
quadratic model with a R2 ¼ 0.78 was determined and subse-
quently evaluated with ANOVA. Only the flow rate and the inter-
action between black pressure and column temperature were
found as significant factors (p < 0.10). The investigated procedure
allowed the best settings of the experimental variables to be
determined for investigation of the lipid profile of coix seeds [25].
Several comments and conclusions can be made based on this
report. If using desirability functions, the authors should report the
criteria for the individual desirability (i.e., maximization of the
resolution while minimizing the analysis time). The report showed
a low global desirability function around 0.2, which may be
explained by very restrictive criteria to for the individual desir-
ability, although it was not explained. An interesting question is
whether the authors should use a screening design instead of an
optimization design in order to determine the experimental factors
and interactions that have a significant influence. The answer is in
fact yes. During the analytical method development, thus investi-
gating the effect of particular factors and later their optimization,
screening designs are executed in order to determine whether a
factor should be later included in an optimization design. There-
fore, a recommendation would be to remove those factors that are
5
M.D. Peris-Díaz and A. Kre˛ z_ el
not significant from the experimental design that are reducing the
accuracy of the predictors, and obtain a simplified model.
Another application in extraction procedures includes the use of
ultrasound-assisted extraction of RAs (RA-V, RA-VII and RA-XII) in
Rubia plants [28]. RSM combined with BBD design with four vari-
ables was applied in this study to evaluate the optimal methanol
concentration, liquid to solid ratio, extraction time and frequency.
Then, ANOVA was performed to test whether the models were
significant. The regression models were significant (p < 0.05) for all
three analytes, and the lack of fit was not significant (p > 0.05) for
RA-V and RA-VII. Herein, the authors provided adjusted R2 of 0.96
and a low coefficient of variance (<10%), which shows high preci-
sion and repeatability. Moreover, the regression coefficients were
also found to be significant (p < 0.05). Finally, a set of optimized
extraction conditions obtained were used to predict the maximum
content of the RAs (2.46, 0.35 and 3.68 mg/g). Five additional ex-
periments obtained 2.47, 0.35 and 3.57 mg/g, which is in excellent
agreement with the predicted values, and validates the model [28].
Mollaei et al. recently developed an electrochemical method for
the simultaneous determination of folic and folinic acid based on
the application of N-dodecylpyridinium chloride (DPC) as a modi-
fier [38]. Here, four-factor CCD and two-factor CCD at five levels
were applied to optimize several variables (the first model included
scan rate, DPC, step potential and pre-concentration time, and the
second design included the pulse height and pH). Quadratic models
were fitted and the responses surfaces were obtained and evalu-
ated. No significant lack of fit was obtained for all models with a
good adjusted R2 > 0.91. Finally, the set of optimal values was
elucidated and evaluated using real samples [38].
Mosleh et al. proposed a method for the determination of
quercetin (QU) in the presence of tannic acid (TA) in drinks
based on a nano-structured sensor for achieving better detection
limits of carbon nanotubes [39]. CCD and RSM were used to
evaluate the effect of pH, scan rate, step potential and amount of
multi-walled carbon nanotubes on the determination of QU in
presence of TA with 27 experiments. The model was evaluated
with visualization of the normality probability plot, which shows
the normal distribution of the residuals. The straight line ob-
tained indicates that the residuals follow a normal distribution,
which is a requirement for the validity of ANOVA. Then, the
authors evaluated the homogeneity of the variance with the
visualization of the residuals of the response, randomly distrib-
uted. Once it was verified that ANOVA could be applied, it was
used to evaluate the quadratic model, although the results were
not presented in the manuscript. A final adjusted R2 of 0.98
indicated a good agreement between predicted and observed
values. Finally, the method was evaluated using real samples and
showed good performance [39].
3. Signal pre-processing
3.1. Pre-processing spectroscopic data
The application of pre-processing methods is of vital impor-
tance to prepare the data for the subsequent analysis (explor-
atory, regression, or classification analysis). Generally, one is
seeking to transform the data in a way that will better follow
Beer's law. Depending on the spectroscopic method, different
physical and chemical phenomena may deviate the signal from
the linear relationship between the concentration and the
absorbance. For instance, scatter is more pronounced in near-
infrared (NIR) than in middle infrared (MIR) spectra, although
baseline and noise are common issues for spectroscopic tech-
niques. The pre-processing method aims to improve this linear
relationship. In spectroscopy the most commonly used pre-
6
Trends in Analytical Chemistry 135 (2021) 116157
processing methods are reference-independent and they do
not require reference values. Reference-independent pre-pro-
cessing methods can basically be divided into two groups:
scattering correction methods and spectral derivatives (Scheme
1). Light scatter is a common effect for all analytical techniques
using light, such as NIR or IR. When the size of the particles in
the sample matches with the magnitude of the spectroscopic
wavelengths, the baseline shifts, and non-linearities affect the
signal. Thus, applying proper scatter correction techniques, the
physical variability in the samples and the baseline shifts can be
removed. Normalization methods such as mean-centering or
Euclidean distances, baseline correction methods, standard
normal variate (SNV) [41,42] and multiplicative scatter correc-
tion (MSC) [43,44] are commonly used for this purpose. SNV
does auto-scaling on the rows of the matrix (samples in rows
and wavenumber in the column) whereas MSC performs back-
ground correction and normalization simultaneously using a
reference spectrum or using the entire data set if the reference
spectrum is not available. Then, the spectrum is corrected by
regressing each spectrum against a reference spectrum or the
average spectrum from the data set. Although these two tech-
niques often provide similar results, if SNV is followed by
baseline correction likely it will give higher baseline correction
due to the fact that MSC performs a simultaneous correction. A
dedicated attention has been focused on reducing Rayleigh and
Raman scattering from fluorescence excitation-emission matrix
(EEM) [45e47]. In fluorescence experiments, part of the incident
energy is absorbed and converted to vibrational and rotational
energy, producing scattering bands. Therefore, it is important to
remove such artifacts prior to any chemometric analysis,
commonly parallel factor analysis (PARAFAC) [47]. One simple
approach is to remove these scattering bands and add missing
values [48]. Another approach consist in the use of interpolation
methods to reduce the scattering effects [46]. Notwithstanding, a
weighted PARAFAC may model the EEM fluorescence spectra in
the presence of Raman scattering [49]. Among the current
strategies reported, one made use of a missing data recovery
(MDR) coupled to principal component analysis or PARAFAC to
correct Rayleigh scattering [48]. Extended information is pre-
sented in a recent review article [50].
On the other hand, baseline correction techniques subtract the
baseline offset or slope. Detrending is one of the most common
techniques in spectroscopy for baseline correction, especially in NIR
or IR, although other algorithms such as wavelet transformations or
polynomial baseline fittings are sometimes used.
Spectral derivatives have been commonly used to reduce the
additive and multiplicative effects on spectral data, including finite
differences, Savitzky-Golay polynomial filters [51] (SG), and Norris-
Williams (NW) derivation. The first one, finite differences, calcu-
lates a difference spectrum between adjacent points for the first
derivate, but in practice it is not used due to the inflation of the
noise. SG and NW, on the other hand, are popular choices that
include a previous smoothing step before derivation avoiding
reduction of the signal-to-noise ratio. The use of a derivate provides
a means to remove the baseline but also the scattering effects. NW
performs a moving average over the data and calculates a finite
difference on the smoothed spectrum. Although NW might provide
similar estimates as SG, the latter one is more widely used, giving
better results.
Generally, to find the optimal pre-processing method, one may
apply a trial-and-error approach where one combines different
pre-processing techniques and selects those that give the best
model performance [52,53]. However, other two approaches are
commonly employed to select the proper pre-processing method.
These are the simple visual inspection of the data before and after
M.D. Peris-Díaz and A. Kre˛ z_ el
applying a pre-processing method, and the use of quality param-
eters that assess the quality of the pre-processing. All of these ap-
proaches will be explained in more detail in section 3.4. Due to the
fact that the diverse spectroscopic techniques are based on
different phenomena, their background signals are different and
require particular pre-processing (Scheme 1). Although it is true
that there are predetermined combinations that work sufficiently
well for a particular spectroscopic technique, the investigation of
other pre-processing methods might be worthwhile. For example,
NIR spectroscopy exhibits pronounced scattering effects that might
be reduced enough with a derivate [52,53]; IR exhibits less scat-
tering effects than NIR and thus pre-processing might not be
necessary in some cases; Raman spectroscopy is strongly influ-
enced by the intrinsic fluorescence background, which might be
removed by baseline correction [54]. These effects need to exceed
the detector noise and the intensity fluctuation of the radiation
source. Moreover, the scattering algorithms are designed to be
applied over the raw spectrum and thus should be applied before
differentiation or baseline correction. Whilst these only represent
basic guidelines, in practice, one iteratively applies a set of pre-
processing methods and checks the performance achieved
(Scheme 1). As we may observe, the proper pre-processing tech-
nique is intrinsic to the particular spectra, and many combinations
are found in the literature (Table S1). Approximately 15% of the
manuscripts published in the time period analyzed used a combi-
nation of pre-processing methods (Fig. 1A). Savitzky-Golay differ-
entiation filters represent the most often used method, providing a
means to remove the baseline but also the scattering effects. Briefly,
SG infers derivatives from local polynomials used for each data
point.
3.2. Pre-processing electrochemical data
In contrast to spectroscopic methods, the linear relationship
between signal and concentration is not featured for electro-
chemical methods in which the signal is understood under elec-
trochemical processes rather than chemical species. Another main
difference with respect to spectrophotometric methods is the
dynamic character of many electroanalytical methods that adds an
extra complex dimension. The aforementioned reasons have for a
Fig. 1. Trends in the number of publications observed for the pre-processing techniques used in spectroscopic (A) and electrochemical methods (B). Frequency is calculated as the
percentage of publications where a particular method has been used, considering the total sum of all of them. SG: Savitzky-Golay; SNV: standard normal variate; MSC: multi-
plicative scatter correction; ALS: asymmetric least squares; Combination stands for any combination of pre-processing methods. BC Poly: polynomial baseline correction. MC: mean
centering; COW: correlation optimized warping; FFT: fast Fourier transform; DWT: discrete wavelet transform; AsLssr: asymmetric least squares splines regression.
7
Trends in Analytical Chemistry 135 (2021) 116157
long time delayed the use of chemometrics methods, and still,
nowadays the number of publications reporting their use is small
in comparison with spectroscopy. Typical signal processing steps
are noise removal, baseline correction, potential shift correction
(alignment of the voltammograms), and separation of overlapping
signals (Scheme 1). Each one of these steps aims at focusing on a
particular artefact, and is isolated or consecutively applied in the
data. As for other techniques, there exists no specific order that
may be generally applied, but the order in which they are used is
particular to the experimental data and should be carefully
inspected. The noise affecting electrochemical data is commonly
classified according to three types: 1) quasi-random high-fre-
quency noise; 2) spikes that are short pulses with large amplitude
and; 3) baseline drifts or low-frequency signals. In order to remove
the noise and enhance the signal, algorithms based on smoothing
are commonly used. These include Savitzky-Golay [55], splines,
fast Fourier transform (FFT), continuous wavelet transform (CWT),
and discrete wavelet transform (DWT). The baseline correction is
rather a difficult issue in electrochemistry, due to the complexity of
developing a theoretical mathematical model that describes the
baseline. The background current is constituted by the capacity
and faradaic components from electrolytes, solvents, or side redox
reactions. Moreover, the background current is affected by the pH,
oxygen, and electro-sensitive components. Experimental methods
such as subtractive anodic stripping voltammetric have been
designed to reduce or eliminate the background current. On the
other hand, common mathematical algorithms are widely
accepted and employed. Among them are splines [56e59], asym-
metric least squares splines regression (AsLSSR) [60e63] and CWT
[64e66] or wavelet-based [67e71] (Scheme 1). Another issue with
electrochemical data is the horizontal or potential shift. The sig-
nals obtained from electrochemical techniques usually suffer from
shifts in potential values. The nonlinearity effects result in de-
viations from the linearity that hinders the application of linear
data processing algorithms. Therefore, the alignment of voltam-
mograms is often required. In order to correct the potential shift,
correlation optimized warping (COW) and interval correlation
optimized shifting (icoshift) are popular algorithms. Table S2 and
Fig. 1B shows the algorithms used for the different electrochemical
techniques in the latest research (2018e2020).
M.D. Peris-Díaz and A. Kre˛ z_ el Trends in Analytical Chemistry 135 (2021) 116157

3.3. Pre-processing hyphenated mass spectrometry data
Common hyphenated mass spectrometry experiments, partic-
ularly gas chromatography (GC)- and liquid chromatography (LC)-
mass spectrometry (MS), have become indispensable tools in all
branches of science [72]. The chromatography step that separates
components of a mixture based on physical or chemical properties
coupled with the mass measurement at specific time points gen-
erates a large amount of data, which usually requires several pre-
processing steps. Moreover, the use of GC-MS and LC-MS in omics
studies where multiple samples are analyzed increases the
complexity of the pre-processing setup. For example, due to the
several hours or days that the analysis of a batch of samples might
take, the signal instrument may fluctuate, and thus one needs to
consider this and corrected it in the post-processing [72]. System-
atic variation in the signal appearing in time can be modelled and
corrected through the use of appropriate experimental design [72].
Typical signal processing steps are noise removal, baseline correc-
tion, deisotoping or charge-state deconvolution, peak alignment,
and peak detection [73e75] (Scheme 1). The filtering of the noise
highly depends on the analytical platform and instrument used.
Generally, the noise can be classified as chemical noise from the
buffers and solvents or random noise generated from the detector
electronics. As for spectroscopies and electrochemical methods,
Savitzky-Golay or wavelet-based algorithms are commonly
employed as a smoothening filter [55]. Correction of the baseline is
required since it may affect further quantification or chemometrics
analysis [76]. Usually, a baseline offset is calculated and then sub-
tracted from the original spectrum. This can be achieved through
derivatives with a SG filter or by applying asymmetric least squares
[77] to approximate the baseline. Another aspect is peak detection
that aims to identify the true signals from the background. A large
number of peak detections algorithms have been released (e.g.,
XCMS Matched filter [78], centWave within XCMS [79], cen-
troidPicker in MZmine [80], enviPick standalone [81] or R-Metab-
oList [82] and MAVEN [83]). Deisotoping or deconvolution identify
isotopic peaks that correspond to the same compound, removing
redundant information and obtaining a simplified data matrix [84].
Here, the members of an isotopic envelope should be first identified
in order to reduce it to the monoisotopic peak with an intensity that
results from the intensities of all of the envelope. Certainly, a great
number of algorithms have been developed running either for-
wards using a theoretical model or backward from the experi-
mental mass spectra. They are mainly classified as peak assignment
or simulation algorithms (e.g., CHAMP [85], Massign [86], UniDec
[87], PeakSeeker [88] or MetaOdysseus [89]). Peak assignment al-
gorithms are based on performing a prior peak detection and
assigning a specific charge state to each peak based on multiple
charge states (e.g., MaxEnt [90,91], AutoMass [92], Z-Score [93]) or
isotope peak spacing (e.g., TRASH [94], Z-Score [93]). Although
these algorithms are relatively fast, difficulties to perform a peak
picking of complex spectra might hamper its use. On the other
hand, the simulation algorithms simulate a multiple hypothetical
mass and charge state distributions and select the combination that
fits the data best. Simulation algorithms produce quantitative re-
sults, but they are computationally intensive. Peak alignment is
often required in omics studies in order to correct the retention
time differences between runs. The alignment methods may or may
not use the retention time to correct these differences. There are
several main alignment strategies: i) the direct alignment of the
total ion chromatogram (TIC) with another used as a reference. The
correlation optimized warping method has been widely used for
this purpose [95]; ii) aligning detected peaks by clustering the
chromatographic peaks [96]. Although this method does not
require retention time correction, multivariate analysis is per-
formed in order to cluster the peaks; iii) two-step alignment in

Fig. 2. Trends observed for the exploratory (A), the predictive modelling (B) and the validation techniques (C) used in spectroscopy, electrochemistry and hyphenated mass
spectrometry methods. PCA: principal component analysis; HCA: hierarchical cluster analysis; PLS-DA: partial least squares-discriminant analysis; PLS; partial least squares; LDA:
linear discriminant analysis; MCR-ALS: multivariate curve resolution-alternating least squares; SIMCA: soft independent modelling of class analogies; SVM: support vector ma-
chines; RF: random forest; PCA-LDA: principal component analysis-linear discriminant analysis; PCA-SVM: principal component analysis-support vector machines.

8
M.D. Peris-Díaz and A. Kre˛ z_ el
which first retention times are corrected and then the detected
peaks are clustered [97]. To correct the retention time deviation, a
reference sample is used, and then the rest of the samples are non-
linearly fitted; iv) two-step alignment in which first the detected
peaks are clustered and then the retention times corrected [78].
Regarding hyphenated MS methods (Table S3) there are a dispersed
number of combinations and also we observed the lack of appro-
priate description in many research articles, which complicates the
evaluation of the existence of any trend (Scheme 1).
3.4. Applications, trends and potential pitfalls in signal pre-
processing
Data pre-processing is a substantial challenge in a chemo-
metrics workflow. Sequentially, several steps are performed and
thus the number of possible combinations grows exponentially.
Every individual step possibly influences each other, which
complicates the development of a general protocol that will al-
ways work. One of the possible pitfalls is bad data pre-processing.
Once this has happened, the subsequent analysis of the data will
be influenced and thus wrong conclusions may be obtained.
Mainly, there are three approaches that have been used most to
select the best pre-processing strategy: (1) trial and error; (2)
visual inspection of the data and; (3) the use of an objective
function that measures the quality of the pre-processed data. In
the first one, trial and error, several pre-processing methods are
applied to the data and subsequently the pre-processed data are
used as an input for a classification or regression modelling [98].
The pre-processing method that gives the best performing is
selected. For example, Bhavana et al. performed a comparative
study by Raman spectroscopy, NIR and MIR of pre-processing
methods aimed at improving the signal-to-noise ratio [99]. That
is, they followed a trial-and-error approach in order to select the
best pre-processing approach. In their study, the spectra were
divided into spectral regions and submitted to a set of pre-
processing techniques and combinations. These were MSC, first
derivative and MSC followed by first derivative. After, a quanti-
tative model was built by using PLS regression and the quality of
the model was evaluated using R2, test set validation coefficient
(Q2), root mean square error of cross validation (RMSECV) and
root mean square error of calibration (RMSEC). All of the pre-
processing methods assayed produced high values, e.g., R2 in the
range 0.963e0.995, while MSC yielded to slightly lower RMSEC
and RMSECV. Another report employed NIR and MIR spectroscopy
to characterize the varietal origin of olive oil, where SNV provided
better performance of the partial least squares-discriminant
analysis (PLS-DA) model [100]. Another approach used to select
the pre-processing method is through the use of visual inspection
of the data before and after applying any pre-processing tech-
nique. The pre-processed data besides decreasing the number of
artifacts, something that it might be difficult to assess, should
show more spectral overlap than the raw data. The possibility to
assess the pre-processing by visual inspection definitely depends
on the type of the data and the pre-processing scheme. Looking at
an SNV-processed NIR spectra is not as complicated as evaluating
pre-processed mass spectrometry data. In any case, it is rather
complicated and inaccurate to rely only on the use of visual in-
spection to evaluate the signal pre-processing. One approach to
ameliorate the visual inspection is the use of a dimensional
reduction technique. In other words, instead of looking at the raw
spectra, the data is reduced to a lower dimensional space with for
example applying a principal component analysis. Then, visual-
izing a PCA score plot and/or loading plots can determine the
optimal pre-processing approach [101,102]. However, it has been
demonstrated how the use of PCA does not always determine
9
Trends in Analytical Chemistry 135 (2021) 116157
whether or not the pre-processing method is optimum [103]. In
any case, the visual inspection of the data before and after
applying a pre-processing strategy is a good idea. The relevant
information should be kept while the pre-processed data should
remove unwanted variations in the signal. The use of dimen-
sionality reduction tools such PCA can also help to assess the pre-
processed data. The third and last way to assess the quality of the
pre-processed data is thorough the use of an objective function
that measures the quality of the pre-processed data. Although it
does not yet seem common for spectroscopies or electrochemical
data, chromatography and mass spectrometry have experienced a
different situation. Pearson's correlation coefficient or a scoring
system has been used to assess the deconvolution quality of mass
spectra. A second major pitfall that may occur relates to the
application of pre-processing methods to a whole data set and
splitting the data set into a training and validation set. As will be
discussed further in this review, the validation of the predictive
model is an essential step. If the data set is divided into two
training and test set, the pre-processing algorithms should be
applied separately into these two data sets.
Certainly, the most used algorithm is a stand-alone SG filter,
covering almost 30% of the research (Fig. 1A, Tables S1e2). The use
of SG filters as a signal smoothing step reduces the enhancement of
the high-frequency noise coming from the derivation. Simply, a
polynomial interpolation is carried out through the signal. Here, the
polynomial degree (usually set to 2 or 3) and the data-point size are
the crucial parameters to select for the moving window. Decreasing
the polynomial degree increases the extent of the smoothing. A
simple rule is to keep fixed a 3rd degree polynomial, and vary the
data-point windows size, inspecting the final results that will
depend on the total number of signal points, the S/N and the
resolution.
In comparison with spectroscopy, the use of chemometrics in
electrochemistry still remains rare, as we can observe by the
number of research articles that have used it (Table S2). The low
number of reports using chemometrics makes its proper statistical
analysis difficult. However, the analysis of these data suggests that
normalization is frequently employed along with electrochemical
methods (Fig. 1B). For example, Baldo et al. developed a method to
determine the acidity of the extra-virgin olive oil by voltammetry
combined with partial least squares (PLS) regression [104]. Because
of the complexity of the electrochemical signal, no raw pre-
processing was applied and the data were only mean-centered
prior to PLS regression.
No trends can be extracted from hyphenated MS methods due to
both the dispersed number of combinations but also the lack of
appropriate description of the methods employed. Notwith-
standing, we may easily conclude, observing the data, that trans-
formation of the data through normalization prior to multivariate
regression analysis is common (Table S3). Here, it is interesting to
present the use of wavelet transforms to filter the noise and reduce
the data size [105e107]. This is mainly achieved by the use of CWT
or DWT, being the latter more efficient than CWT. Removal of un-
informative data reduces the computational time required for
posterior chemometric analysis.
4. Exploratory analysis
The exploratory data analysis represents the first step for che-
mometric processing after the data have been pre-processed if
needed (Scheme 1) [108]. Without the need of any formulated
hypothesis, the exploratory analysis gives an overview of the data,
and observes its variation. Through this, one can observe well
defined substructures of the data or diagnosed outliers, and also
identify which variables describe the system.
M.D. Peris-Díaz and A. Kre˛ z_ el Trends in Analytical Chemistry 135 (2021) 116157

4.1. Principal component analysis

Principal component analysis (PCA) is probably the most

exploratory method used in the chemical and physical sciences. It
defines new variables named principal components (PC) from
linear combinations of the original ones, allowing them to find
patterns in two-dimensional space [109,110]{Wold, 1987 #126}.
PCA can be applied where the number of variables exceeds the
number of samples without misleading results. The original data
matrix (X) is decomposed into a matrix of scores t and loadings p.
The scores (t) allow one to find patterns in the samples while the
loadings (p) give the weights of the original variables in the PCs,
thus giving information about which variables are part (or not) of
the trend. PCA also has the aim of dimensionality reduction,
removing redundant information while retaining all nonzero PCs
[111]. A dimension reduction tool tries to provide a discriminant
function by the total variability. This approach reduces and com-
pacts the original data, expressed in terms of the scores (t) vector
that can be used for further modelling. This approach might for
instance help to improve the classification performance. However,
the number of PCs retained should be considered carefully since
there is a risk that useful information will be neglected [109].

4.2. Cluster analysis

Although PCA represents the most used exploratory method, it

does not explicitly define clusters in the data but it is left for the
criteria of the user. More formal methods that define specific data
aggrupation are clustering methods [112]. Commonly, hierarchical
clustering analysis (HCA) methods are employed because very
often the data follow a hierarchical structure [113]. Importantly, the
choice of the distance function to calculate the distance between
the clusters and observations can dramatically alter the results. If
there is a correlated group of variables in the data, these variables
may get a too large weight while uncorrelated variables may fail to
be recognized. HCA can be approached from bottom up or
agglomeratively where each observation forms a cluster and then
the clusters are joined or not. The second HCA type is a divisive or
top-down strategy, where all observation starts in one cluster,
followed by division into smaller clusters as the hierarchy moves
down [114]. Currently, one runs several clustering routines and
selects the one that seems to give the most logical results.

4.3. Applications, trends and potential pitfalls in exploratory

analysis

The selection of PCs in PCA for exploratory purposes is not

critical; however, when PCA is used to identify outliers or as a
reduction step prior to predictive modelling the situation is
different. Here, the choice of the number of PCs and thus how much
variance is captured should be carefully chosen [115]. One approach
could use the number of components that provide the best classi-
fication model or select PCs by cross-validation. Another relevant
point to consider is the visualization of the Score plot. If one is
interested in observing the natural aggrupation of the data, the PCs
should capture a large proportion of the variance. PCs with low
percentage do not explain the raw data. Another issue is the need
for pre-processing the data before applying PCA. The selection of
the pre-processing method depends on the nature of the data and
should be investigated. Three recent pitfalls that have been greatly
covered in a recent review are [116]: (1) use of wrong measurement
units for the exploratory analysis. In order to avoid mis-
interpretations, the units used should directly be related to the
concentration; for example, convert from transmittance to absor-
bance; (2) misinterpretation of the PCA loadings from a first- or
10
Fig. 3. Chemometric analysis for case study 1 that dealt with the regression task of a near-
infrared spectra from 618 soil samples. The parameter total nitrogen (Nt) has been measured
for all of the samples and constituted the y vector for the regression. A) NIR spectra for the
first ten samples colored according to their intensity; B) Multiplicate scatter correction
(MSC)-transformed spectra; C) The spectra pre-processed by Savitzky-Golay polynomial
filters (SG) and second derivative; D) PCA score plot for the first and second PC applied to the
MSC-transformed spectra. The scores are colored according to the Nt content in the samples.
The two sample outliers are observed in the score plot; E) PCA score plot for the first and
second PC applied to the pre-processed spectra by SG (2nd der). The spectra are colored by
the Nt content in the samples; F) Loading plots for the first component obtained from the PLS
regression applied on the MSC-preprocessed spectra. We see how the information con-
tained in the loadings about the original variables in A is not lost; G) Loading plots for the first
component obtained from the PLS model applied on the spectra pre-processed by SG (2nd
der). The information in the loadings about the original variables in the raw spectra is
retained. We maysee how the positive derivate gives a positive contribution to the loadings.
H) The predictive quality obtained for the validation data pre-processed by MSC (30% of the
training data set) with an R2 of 0.60. I) The predictive quality obtained for the validation data
pre-processed by SG (2nd der) (30% of the training data set) with an R2 of 0.60.
M.D. Peris-Díaz and A. Kre˛ z_ el Trends in Analytical Chemistry 135 (2021) 116157

second-derivative pre-processed signal. The classical PCA rule 5. Predictive modelling

dictates that a sample with a high score value at a particular PC is
characterized with variables that are positively correlated with The main goal of predictive modelling is to build a model with
high loading values for the same PC. In the case of derivative predictive capabilities for new data. Usually, predictive modelling is
spectra, this is not always true. For correct interpretation of the PCA performed after performing an exploratory analysis of the data
loadings from the first or second derivative is not straightforward, (Scheme 1). Depending on the type of variable that we want to
and requires inspection of the derivative signal and the loadings. A predict, continuous or discrete, the problem can be classified as
solution proposed the use of antiderivative function on the PCA regression or classification, respectively. In regression, we are
loadings [116]; (3) normalization methods for pre-processing the interested in constructing a model that predicts about continuous
signal such as SNV or MSC can produce artifacts that leads to variables, while classification aims to assign a determined class
misinterpretation of the PCA loadings. The normalization can
remove not only unwanted variations in the signal but also valuable
information from the original spectra.
Potential pitfalls regarding clustering methods are mostly
based on the selection of the distance metric and the clustering
method selected. Because of that, clustering sometimes can lead
to misleading results without knowledge about the data.
Notwithstanding, the effects of these parameters on the clustering
results should be inspected and the clusters validated using
appropriate metrics [117]. For example, the use of ensemble
clustering can remove uncorrelated noise from the clustering re-
sults [117,118]. Table S4 lists the exploratory methods used in the
research articles for the three techniques explored in this review
(spectroscopies, electrochemistry, and hyphenated MS). These are
grouped and analyzed in a bar plot in Fig. 2A. As expected, PCA is
by far the most frequently used exploratory method among all of
the techniques presented. Approximately, 80% of the research
articles used PCA. One of the issues is illustrated by Tfaili et al.
who investigated the lipid modifications in J774 macrophages
after the addition of eicosapentaenoic acid into cells, loaded or not
with cholesterol [119]. The changes in the cell membrane upon
addition of lipids was studied thorough Raman spectroscopy and
IR. The visualization of the PCA loadings showed a negatively
correlated band at 2824-2865 cm1 assigned to the vsym(CH2) of
lipids, which is more present in the cytoplasm than in the nucleus.
This clear separation in component 1 that captures most of the
variance (75.8%) in the PCA score plot was attributed to this band.
Another popular use of PCA is in dimensional reduction
[120e127]. Zukovskaja et al. employed UV-Raman spectroscopy
for the identification of fungal spores that are implicated in res-
piratory diseases worldwide [124]. The UV-Raman spectra were
background corrected and vector normalized prior to dimensional
reduction with PCA. The optimal number of principal components
was selected according to the maximum accuracy, and the score
matrix (t) was used as an input for further modelling [124].
Another application recently reported is the application of Raman
and FT-IR spectroscopy for follow-up immunoadsorption therapy
treatment of dilated cardiomyopathy [125]. The authors reported
that the first principal component (PC1) separated the data ac-
cording to the time points, capturing about 19.9% of the spectra
variance. However, the results clearly show that TP2 is different
from the rest (TP1, TP3-5). Moreover, the PCA model obtained is
rather informative, since it captures a low amount of variance
(19.9% for the PC1 and 10% for the PC2) [125].
Clustering methods, in particular HCA, are used more frequently
in hyphenated MS studies, mainly because heat maps are especially
useful in omics studies to observe the correlation between samples
and variables simultaneously (Fig. 2A). For example, Negra ~o et al.
used a mass spectrometry-based proteomics approach in order to Fig. 4. Support vector machines (SVM) regression for case study 1. The NIR spectra
identify proteins related to infection with Leishmania spp [128]. were MSC preprocessed. A) Optimization of the penalty error (C) and the spread of the
Here HCA was used for clustering the samples according to the 50 kernel (s) for the non-linear kernel radial basis function (RBF). The blue dots are for
most significant proteins combined with a heatmap for visualiza- s ¼ 0.0001 the red triangles are for the s ¼ 0.001. The RBF achieved the lowest root
mean square error (RMSE) for C ¼ 6 and s ¼ 0.001; B) The prediction quality obtained
tion of the proteins and samples. To conclude, we observed that by the SVM-RBF for the calibration data (70% of the training data set) with an R2 of
other clustering analysis tools (e.g., k-means, k-NN) are employed 0.80. C) The prediction quality obtained by the SVM-RBF for the validation data (30% of
in spectroscopic studies (Fig. 2A). the training data set) with an R2 of 0.74.

11
M.D. Peris-Díaz and A. Kre˛ z_ el Trends in Analytical Chemistry 135 (2021) 116157

label to a particular sample. Herein, our focus will be set on para-
metric classification algorithms (linear discriminant analysis (LDA)
and PLS-DA) and two machine learning algorithms that are
nonparametric (support vector machines (SVMs) and Random
Forest (RF)).
5.1. Linear discriminant analysis
Linear discriminant analysis [129] is a method that aims to find a
linear separator between classes of objects and use this decision
function for assigning the class membership to unknown samples
[130]. Other more complex decision surfaces (e.g., non-linear in
quadratic discriminant analysis) also exist but require more pa-
rameters to tune. The classification may be expressed either in the
Bayesian form or Mahalanobis distance. First, the Mahalanobis
distance is calculated for all samples to the class centers using the
pooled covariance matrix, and the samples are assigned to the
lowest class distance [131]. LDA is a well-established technique that
works well even when the data distribution moves from normality.
However, LDA should not be applied for matrices where the num-
ber of variables exceeds the number of samples, especially in the
presence of correlated variables. To overcome this limitation, LDA

Fig. 5. Chemometric analysis for case study 2 that dealt with the classification task of mid-infrared spectra from the 60 authenticated extra virgin olive oils from three different
countries (Spain, Italy and Greece). A) MIR spectra for the first ten samples; B) PCA score plot for the first and second principal component; C) PLS-DA score plot for the first and
second latent variable; D) Histogram of the Q2Y values obtained by 1000 permutation tests of the response variable vector (y) from the PLS-DA model. The red dot is the original Q2Y
(0.73) obtained in the PLS-DA model along with its confidence interval. All of the permutated Q2Y are lower than the original Q2Y that indicates the validity of our PLS-DA model
with an empirical p-value of 0.001. E) PLS-DA loadings plot for the first and second latent variable. The loadings are colored according to their variable in the importance (VIP)
values.

12
M.D. Peris-Díaz and A. Kre˛ z_ el
can be applied in the score (t) matrix obtained after applying PCA
and retaining all nonzero principal components. Another signifi-
cant disadvantage is that LDA considers that all classes have the
same pooled covariance matrix. Thus, dealing with groups with
different variance structures is not appropriate [132]. In this case, it
is more appropriate to use non-linear methods.
5.2. Partial least squares
Partial least squares discriminant analysis, in the PLS1 version, is
a linear two-class method that aims to find a linear separation to
classify the group of samples in the spacing algorithm where the y
vector takes values þ1 and 1/0 for each class, as opposed to PLS
regression, where the y vector takes continuous values [133]. Other
PLS versions such as PLS1 modified or PLS2 algorithms allow
modelling of more than two classes [134]. In this sense, a range of
algorithms such as the nonlinear iterative PLS (NIPLS) [134], kernel
algorithms [135] and statistically inspired modification of PLS
(SIMPLS) have been developed [136]. Of note, kernel algorithms
work with the variance-covariance matrices and are more advan-
tageous when there is a much lower number of samples than var-
iables [135]. Given a set of independent variables (matrix X), we are
interested in building a mathematical model that allows the pre-
diction of dependent variables (vector y). Briefly, the PLS1 model
works using a matrix Xij and a vector yik, where i is a sample, j is a
variable and k is the class coded. The PLS components are obtained
sequentially, where the first one is obtained from the cross-product
matrix w ¼ XTy. The X-scores t can be computed as t ¼ Xw, which is
a linear combination of the weights (w) and the matrix X. In the
next step, the normalized loadings (p) for X are calculated as
p ¼ XTt/(tTt). Similarly, the normalized loadings (q) for y are
calculated as q ¼ yTt/(tTt). To estimate the second component or
latent variable (LV), first, the variation related to the previous
component is removed from X and y. To do so, the outer products
tpT or tqT for X or y are subtracted from the current data matrices,
respectively. Then, the next component is computed from these
deflated matrices [137]. Sometimes the Y-scores (u) are used for the
model interpretation, calculated as u ¼ yq/(qTq). The optimal
number of components or LVs is usually determined by using cross-
validation techniques in a way that maximizes the covariance be-
tween X and y. After every interaction, the vectors (w, t, p, q, u) are
gathered in matrices. The prediction that a sample belongs to a
particular class is calculated as y ¼ tA þ F, where A is the regression
coefficient and F is the abscissa vector. To calculate A, we use the
scores t, A ¼ (tTt)1tTy. Values above 0 are assigned to one class (i.e.,
“A”), and below to another class (i.e., “B”), using the þ1/-1 criteria.
The deviation between experimental and modelled may be
assessed by calculating the y-residuals as TqT þ F. The normality
plot of the y-residuals gives insights into the quality of the model
and helps in the identification of outliers [138]. The common way to
show the PLS-DA results is with the score plot, although it requires
a proper validation to guarantee that the samples did not group by
chance [132]. As for LDA, PLS-DA is a linear method and therefore
does not consider that each group has a different structure.
5.3. Random forest
Random Forest, introduced by Breiman and Cutler (2001) [139],
is a machine learning technique based on the combination of
bagging and tree-based methods. There are several algorithms of
RF according to how the tree is constructed or how the data is
preliminarily divided. The most widely algorithm used is a classi-
fication and regression tree (CART) [140] that uses Gini impurity as
a splitting criterion [141]. First, each tree is built from a bootstrap
sample of the data, and for each bootstrap sample, a tree is grown
13
Trends in Analytical Chemistry 135 (2021) 116157
until it is fulfilled, by choosing at each node the best split among
random variables. The process is repeated until a collection of trees
is obtained. It can be applied when the number of variables is much
larger than the number of samples not leading to overfitting, in
non-linear situations, and can be used both for two-class and multi-
class problems as well [139]. Another great feature of Random
Forest is that it only has several parameters to tune, the most
important being the number of trees in the forest and the number
of variables in the random subset at each node. Random Forest
provides a metric of the error rate considered as a good estimator of
the error, based on prediction of the data that were not in the
bootstrap sample, called out-of-bag (OOB), using the tree grown
with the bootstrap sample. Finally, the OBB error is the average
error frequency obtained [142]. The algorithm also includes several
measures of the variable's importance, such as the Gini index and
classification accuracy [143].
5.4. Support vector machines
Support vector machines are a classification and regression
method derived from statistical learning theory [144,145]. It at-
tempts to form either a linear or non-linear boundary between the
groups by minimizing the structural risk minimization (SRM,
Equation (4)):
vffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffi
    ffi
u  
u h
udVC log 2N þ 1  log 4
t dVC
Re  Remp þ (4)
N
where dVC is the Vapnik-Chervonenkis dimension related to the
complexity of the classifier, Re is the complete error and Remp is just
the error using the N samples of the training set, in fact the
misclassification obtained during the model building. This principle
reduces the risk of overfitting since it is not just focused on the
empirical error Remp as other classification methods do, but it fo-
cuses on the true error Re. The difference between both errors (Re
and Remp ) is based on the number of the samples used in the
training set (N) and the complexity of the boundary of the model
(dVC ). Higher model complexity and number of samples lead to a
larger and smaller difference between the errors, respectively.
Thus, it is essential to control the complexity of the model ac-
cording to the number of samples [146]. SVMs aim to find a sepa-
rating hyperplane maximizing the distance or margin between the
samples and thus dVC comes down, hence reducing the general
error Re. A generic hyperplane can be written as Equation (5):
wT x  b ¼ 0 (5)
where x represents a set of points or sample, w the related weight
or the normal vector to the hyperplane, and b a bias parameter.
Since many hyperplanes can be obtained, the optimal that sepa-
rates the data is chosen in such a way that the margin is maximal
between the samples and minimizes the empirical error. The
optimization results in the following function (Equation (6)):
X X
LD ¼ 0:5 ai aj yi yj xTi xj  ai (6)
i;j i
where yi is the class label, either þ1 or 1, and a is a vector of
Lagrange multipliers that are used to calculate the weight vector w
and thus to determine the optimal hyperplane. The optimization of
LD is a quadratic programming problem, minimizing it with respect
to the coefficient a (Equation (6)). It should be noted that it is a
reproducible solution since the minimum is unique. The scalar
M.D. Peris-Díaz and A. Kre˛ z_ el
Table 3
Comparison between several preprocessing methods that were applied to the data from the case study 1. Subsequently, the preprocessed data were used as an input to PLS
regression modelling.
MSC SG (1st der) SG (2nd der)
Variance explained (%) 81 81 78
RMSEP 0.70 0.63 0.73
Adjusted R2 0.71 0.70 0.65
N. comp 12 6 7
RMSEP validation set 0.48 0.44 0.36
Adjusted R2 validation set 0.62 0.46 0.60
SNV: standard normal variate; MSC: multiplicative scatter correction; SG: Savitzky-Golay; RMSEP: root mean square error prediction; N. comp: number of components.
product xTi xj makes the method suitable when the number of var-
iables exceeds the number of samples. The boundary between the
classes is determined by only a limited number of those samples
that lie close to the margin, called support vectors. Only for these
points will the coefficient a not be zero and therefore will be
involved in the optimal boundary. The samples that are far away
from the margin will have the value 0 in the coefficient a, not
influencing the separating hyperplane but used in the model
building. The model includes a penalty error C term that de-
termines the importance of the margin errors or deviations that can
become training errors when having a value higher than 1. The
smaller the C value, the higher are the deviations obtained and
margin maximization has more importance in the computation.
The opposite is having a high C value, with lower deviations but
minimizing the training error Remp. This leads to no error with a
narrow margin, indeed a poor model. Thus the C value can be used
to obtain the optimal trade-off between the complexity of the
model and the training error of the SRM equation (Equation (4)).
The Lagrange multiplier a is also subject to the penalty error
(Equation (7)):
0  ai  C
The classification function to separate two classes includes the
support vector si by means of the following Equation (8):
X
Ns  selection method is the variable importance in projection (VIP).
y ¼ sgn ai yi sTi x þ b
i¼1
The prediction of the class a sample belongs to is determined by
the sign of the y value obtained. Leading with non-linear situations
SVMs return more satisfactory separations than the linear
discriminant functions as PLS-DA [146]. Substituting the scalar
product xTi xj with the kernel functions Kðxi ; xj Þ ¼ 4ðxi Þ4ðxj Þ it is
possible to determine more complex class boundaries [29]. For such
an aim, the data are mapped into a higher-dimensional space
where the samples are projected by means of a feature function
4ðxÞ and a suitable hyperplane is found. Then the complex class
boundary is translated into the original space.
5.5. Applications, trends and potential pitfalls in predictive
modelling
One of the main misuses that we may observe in the literature is
the use of PLS-DA without considering what the structure of the
data looks like. Often, PLS-DA is applied over matrices with small
sample sizes and a large number of variables or with groups with
different variance. One needs to have in mind that the linear sepa-
rator is the same for LDA as for PLS-DA with all non-zero compo-
nents retained. Therefore, the same results are achieved with LDA or
PLS-DA when the class sizes are equal, but LDA requires fewer de-
cisions to be made about the parameters and thus less risk of the
equivoque results [132]. We should emphasize that PLS-DA has the
Trends in Analytical Chemistry 135 (2021) 116157
SNV Autoscaling MSC þ Autoscaling SG (2nd der) þ Autoscaling
73 81 83 83
0.84 0.63 0.66 0.65
0.60 0.80 0.70 0.65
12 7 13 5
0.52 0.77 0.68 0.82
0.48 0.66 0.53 0.59
same main disadvantage as LDA: it considers that each class has a
similar variance structure [132]. In this case, other non-linear
methods such as Random Forest or SVM are more appropriate. For
example, Li et al. attempted to identify potential biomarkers of ce-
rebral infarction using GC-MS and chemometrics [147]. Random
Forest was used to discriminate among healthy, cerebral infarction
patients and quality control groups. In another example, we recently
optimized the mass spectrometry instrumental settings by using a
BBD, and modelling the signal response by a set of regression
models [17]. From all of the algorithms assayed, SVM achieved the
best metrics with R2 of 0.95 and RMSEP of 0.05. Therefore, superior
findings were achieved with nonlinear alternatives.
One disadvantage of using LDA versus PLS-DA is that if the
number of variables exceeds the number of samples, LDA requires a
prior step for reduction dimension with for example PCA. In the
aforementioned study performed by Zukovskaja et al. the authors
applied LDA over the scores (t) matrix obtained from PCA while
retaining the optimum number of principal components [124]. Out
of 1079 spectra, only 27 were misclassified, which resulted in 97.5%
of accuracy. The largest number of misclassifications comes from
the lack of separation between Aspergillus species and Penicillium.
(7) Another advantage of PLS-DA over PCA-DA is that it may provide
insights into the variables via the loading weights. Direct infor-
mation obtained about the variables is often desired in the omics
technologies, where PLS-DA is ruled out. Here, a common variable
(8) Briefly, it accumulates the importance of each variable reflected by
the weight from each component. Usually, the variables with a VIP
value greater than one are selected as important. For example, Luo
et al. performed an untargeted metabolomics study by LC-MS in
order to find biomarkers for early diagnosis of lung cancer [148].
The authors applied orthogonal PLS-DA in order to identify po-
tential metabolic biomarkers. Selection of the ions with a VIP value
greater than one revealed 31 key metabolites involved in seven
metabolic pathways [148]. The last and common pitfall is pre-
senting the PLS-DA score plot of the training sets for data visuali-
zation. It can give misleading results, especially when the number
of variables exceeds the number of samples. Moreover, because
there are many variables presented in the data, the existence of
correlation between them may occurs just by chance. This can lead
to obtaining a false separation between groups in a PLS-DA score
plot. Therefore, it is recommended to assess the natural aggrupa-
tion of the data through a PCA score plot, since it can be used when
there are more many variables than samples. Yan et al. differenti-
ated traditional Chinese medicines by using gas chromatography-
mass spectrometry. Wisely, the authors used PCA in order to
visualize differences between different samples, obtaining two
clear clusters that represent two plant species. To get insights into
the variables, in their case volatile compounds that are responsible
for the difference between both species, they employed a PLS-DA
variant named orthogonal PLS-DA [149]. Another way to assess
the reliability of the classification model is the use of permutation
tests over the classifier. The group label is randomly permuted, and
14
M.D. Peris-Díaz and A. Kre˛ z_ el
Table 4
Comparison between several preprocessing methods that were applied to the data from the study 2. Subsequently, the preprocessed data were used as an input to PLS-DA
classification modelling. 3-group modelling.
R2X R2Y Q2Y
MSC 0.774 0.723 0.702
SNV 0.774 0.723 0.702
SG (1st der) 0.702 0.759 0.699
SG (2nd der) 0.651 0.729 0.649
Autoscaling 0.802 0.712 0.667
Mean centering 0.888 0.780 0.730
SNV: standard normal variate; MSC: multiplicative scatter correction; SG: Savitzky-Golay; RMSEP: root mean square error prediction; N. comp: number of components; MC:
number of misclassifications.
statistics calculated. After a large number of permutations, an
ensemble of statistics obtained from permutations is compared
again to the unpermuted data set.
Table S5 lists the predictive modelling methods used in spec-
troscopic, electrochemical, and hyphenated MS methods for
2018e2020. A bar plot containing this information clearly indicates
that PLS-DA is by far the most frequently used method in hy-
phenated MS studies (Fig. 2B). It is not surprising that the devel-
opment of mass spectrometry-based omics sciences has been
accompanied by the abusive use of PLS-DA. When properly used,
PLS-DA can give great insights into the discovery of potential bio-
markers in metabolomics studies [150]. On the other hand, PLS has
been widely used in electrochemical methods for regression
(Fig. 2B). This finding suggests that electrochemical methods have
been poorly used for classification purposes but extensively for
building regression models for quantification aims. In spectroscopic
studies, the situation is intermediate. PLS-DA but also PLS in its
regression mode are both equally employed.
6. Model validation
The performance of the mathematical model used for modelling
the data may be influenced by several factors (i.e., the number of
samples in contrast to the number of variables, the representa-
tiveness, extreme outliers) [4]. The overfitting of the model can give
the wrong idea that the model can predict the properties of interest
better than it does. It is common to apply a set of modelling models
to a specific problem and select the one that gives better perfor-
mance (Scheme 1). However, a proper validation of the model is
needed to assess how well it can predict. For example, selecting too
many components or LVs in a PCA-DA, PLS-DA or PLS can lead to
overfitting [4]. The chemometrics strategies that address the
assessment of the quality of a predictive model are referred to as
validation [151]. To evaluate the quality of the model, a diagnostic
metric should be defined. It could be based on either a model
parameter (e.g., variance explained) or the calculation of residuals.
One needs to bear in mind that the predictive model will only be
relevant when the data are used as representative for the system
studied. In this context, validation of the model is usually based on
splitting the sample into a training set and test set or by using
resampling methods (bootstrapping, jack-knifing and cross-
validation) (Scheme 1). When dealing with large data sets,
dividing the data into a training set and a test or validation set is the
most conservative approach. Here, the application of Kennard-
Stone algorithm [152] provides a way to divide the sample. On
the other hand, when having small datasets (smaller than 40
samples) [151], resampling algorithms is preferred to split the data.
However, it is assumed that the data used for resampling algo-
rithms are representative of the data that will be used in the future.
The most popular resampling algorithm is cross-validation (CV)
[153]. CV divides the data into k-folds; one fold is left out of the data
as a validation set, and the model is built using the remaining k-1
Trends in Analytical Chemistry 135 (2021) 116157
RMSEP No LV MC calibration set MC validation set
0.250 2 4 0
0.250 2 4 0
0.233 3 6 0
0.248 3 6 0
0.255 3 4 0
0.224 4 4 0
folds. The predictive performance of the model is evaluated using
the validation set [154]. All of this is repeated k times so each k-fold
is used once as a validation set, and the predictive performance is
calculated as the average [151].
6.1. Applications, trends and potential pitfalls in model validation
We can observe in the literature a disagreement over the
definition of external validation [96,97]. Some authors defined
external validation as simply the case when the validation of the
model was carried out by using objects that were not used for
building the model. These samples were randomly pulled out
from the data set [155]. However, the strict definition of external
validation implies the use of a new and external set of data, that is
independent but representative [4,156]. For instance, the new
external data set has been measured in a different period than the
training set, so it was not available at the time [4]. On the other
hand, splitting the data that have been collected at once into
training and validation set falls into the category of internal vali-
dation. External validation does not always guarantee better
model validation than using resampling methods [156,157]. That
is because it requires a large number of samples and they must be
representative and independent samples. We collected and
analyzed the validation protocol implemented in the last years
(2018e2020) for the three experimental techniques (Table S6).
Hyphenated MS approaches mostly used resampling algorithms
for validation of the predictive model (Fig. 2C). We may argue that
this is due to the low number of samples available for the exper-
iments in omics studies, because of the difficulty to get biological
samples. In those cases, the use of resampling methods is more
recommended than a split-sample protocol. In the case of elec-
trochemical methods, validation using external data sets appears
the most frequent. Traditionally, electrochemical methods have
been used for regression modelling of pharmaceutical or chemical
compounds with PLS regression, as we commented above.
Because of the facility to collect such samples and the nature of
the studies, validation of the models is performed with a test set.
Finally, spectroscopic studies matched in an intermediate situa-
tion (Fig. 2C). Split-sample, resampling and test set are almost
equally employed along with these studies. This may be due to the
fact that spectroscopies have been used for different aims, from
building classificatory models in metabolomics studies to cali-
bration of pharmaceutical compounds. The trends observed in the
validation protocols according to the experimental techniques
correspond to their field of application. It is easy to understand
and find a correlation between the predictive modelling and the
validation protocols (Fig. 2BeC). Hyphenated mass spectrometry
has been mostly used for applications aimed at building classifi-
catory models, and because of this PLS-DA is the most used. The
main drawback of these studies is the low number of samples
available, and this is reflected in the validation technique.
Resampling is the most employed approach. The opposite
15
M.D. Peris-Díaz and A. Kre˛ z_ el
scenario occurs with electrochemical methods. Traditionally used
for calibration purposes, PLS regression dominates over the rest of
the methods (Fig. 2B) and the ease of collecting samples allows for
a more robust and independent validation thorough the test set
(Fig. 2C).
7. Case study 1: A regression model
We have incorporated a case study that consisted of an NIR
dataset of 618 soil samples provided in the “Chimiome trie 2006”
meeting [158]. Here we aim to build a predictive model for the Nt
parameter (total nitrogen in g/kg of dry soil) that has been deter-
mined for each sample. Thus, the Nt parameter is our vector y, and
the NIR dataset is the matrix X. To follow the case study, the R code
can be found in the Supplementary information.
In the first step, the 618 training samples were divided into 70%
for the calibration (433 samples) and 30% for the validation set (185
samples) by means of the Kennard-Stone algorithm. In brief, KS
starts by selecting the pair of samples that are the farthest apart.
These points are assigned to the calibration set and removed from
the original training set. Then, the algorithm assigns the remaining
samples to the calibration set by computing the Euclidean or
Mahalanobis distance between each unassigned samples. The
points that are the farthest from their closest neighbors are selected
from the training set and assigned to the calibration set. The
Mahalanobis distance can be used by performing a PCA and
computing the Euclidean distance on the score matrix. The number
of PC retained can be specified when the increase in the cumulative
explained variance in the next components is lower than some
percentage. Sample selection based on the Kennard-Stone algo-
rithm in the principal component space is presented in Fig. S1. The
red solid points are the selected samples for the calibration set
(70%) and the empty black points correspond to the validation set
(30%). Following the pipeline in Fig. 1, the calibration set was then
subjected to several preprocessing algorithms. These included first
and second derivatives with SG filters, MSC and SNV and a com-
bination with column scaling (Fig. S2 and Fig. 3 and 4A-C). In the
first step, we attempt to select the best pre-processing method by
trial and error. This is based on the performance for building a
predictive model, in this case by PLS for the Nt parameter (total
nitrogen in g/Kg of dry soil). To examine the natural aggrupation of
the data, and identify possible outliers, PCA was used. PCA scores of
the MSC-transformed spectra revealed the presence of two outliers
(samples no 313 and 402), while applying SG (2nd der) resulted in
the appearance of two clusters along the PC1 (Fig. 3DeE, Fig. S3).
However, the aggrupation was not related to the Nt content or any
of the parameters available.
Next, we consider building a PLS regression model for the Nt
parameter as the real-valued dependent variable. The number of
PLS components was selected by 7-fold CV, and the validation re-
sults are the root mean square error of prediction (RMSEP)
(Figs. S4AeB). The decision of how many components to retain
remains subjective and therefore several strategies have been
developed to deal with that. Here, we used a permutation
approach, which tests whether the addition of a new component
improves the model [159]. The algorithm starts backwards and
continues by removing components until the model deteriorates.
Table 3 lists the number of components selected according to this
strategy along with the RMSEP, the adjusted R2 for the prediction
based on CV, and the RMSEP and adjusted R2 for the validation set
(30% of the training data). The use of either MSC or SG (2nd der)
provided the models with the highest prediction capabilities, that is
the lowest RMSEP and highest adjusted R2 (Table 3). Twelve and 7
components were selected with this strategy, yielding ca 80% of the
variance explained, which gives an RMSEP of 0.48 and 0.36, and an
16
Trends in Analytical Chemistry 135 (2021) 116157
adjusted R2 of 0.62 and 0.60 for MSC SG (2nd der), respectively
(Table 3, Fig. S4).
The PLS score plot did not show any clear grouping or outliers
(Figs. S5AeB.) Plotting the loadings on component 1 showed
agreement with the profile of both the MSC-transformed spectra
and those preprocessed by SG (2nd der) (Fig. 3FeG). We can see
how the information contained in the loadings about the orig-
inal variables is not lost. For example, the positive derivatives
gave a positive contribution of the peaks on component 1
(Fig. 3A, F-G).
The validation set (30% of the training data) was preprocessed as
for the calibration set, and the constructed PLS model used to
predict the Nt parameter. The predictions of the validation set
achieved an RMSEP of 0.48 and 0.36 with an adjusted R2 0.6 for
MSC, and SG (2nd der), respectively (Table 3, Fig. 3HeI). These are
similar values to those obtained at the “Chimiome trie 2006”
meeting [158]. Another source of information is the relationship
between the X-scores and Y-scores, which did not follow a linear
relation as observed in Figs. S5CeD (see section 5.2). Therefore, we
decided to use the SVM regression in order to check whether we
would obtain lower RMSEP and higher R2 for the predictions. The
linear kernel and the non-linear kernel radial basis function (RBF)
were tested with appropriate optimization of the main parameters
by means of a grid of tuning values and thorough 7-fold CV, that is,
the penalty error (C) and the spread of the kernel (s) for the RBF
and C for the linear kernel. The RBF achieved the lowest RMSE
cross-validation (0.56) and highest R2 (0.80) with optimal values of
C ¼ 6 and s ¼ 0.001 (Fig. 4AeB). When using the model to predict
the unseen data (validation set), an adjusted R2 and RMSEP of 0.74
and 0.28 were obtained, respectively (Fig. 4C).
In conclusion, we have shown how to follow the pipeline pre-
sented in Scheme 1, and to interpret the results obtained for each
step. We have discussed several main possible pitfalls that may
occur in such outlier detection, the interpretation of derivative
signals and the use of linear or non-linear regression methods.
Moreover, the example can be easily reproduced using the R code
included in the Supplementary information.
8. Case study 2: A classification task
In the second case study, we used 120 mid-infrared spectra from
60 authenticated extra virgin olive oils (EVOO) from three different
countries (Spain, Italy and Greece) (Fig. 5) [160]. The main objective
is to build a classificatory model that will be able to distinguish
between the EVOO provenance. Also, the R code is available in the
Supplementary information.
Similar to case study 1, sample selection was based on the
Kennard-Stone algorithm and the best pre-processing technique
was selected via a trial-and-error approach by means of PLS-DA
modelling. It is evident that no single pre-processing method
stood out over the rest (Table 4). Among them, mean centering
provided 88% accuracy for the calibration set with four mis-
classified samples. We can observe that the first and second PCA
component captured ca 70% of the variance and could show some
trend for clustering according to their provenance (Fig. 5B). For
example, samples from Spain and Italy or Greece are “separated”
along the PC1, while Greece was separated from them along the
PC2. To determine whether the three groups of samples can be
distinguished we employed a PLS-DA classification model, the
second most often used algorithm (Fig. 2B). As previously dis-
cussed, presenting the PLS-DA score plot can give misleading
results. Visualizing a separation of the samples does not mean
that there is a chemical reason behind it. Simply the correlation
between many variables is a simple reason why a PLS-DA score
plot can show separation between groups [132]. To visualize the
M.D. Peris-Díaz and A. Kre˛ z_ el
natural aggrupation of the data, one should use instead a PCA
score plot. We can see how in our example the score plots ob-
tained by PLS-DA modelling (Fig. 5C) closely agree with the PCA
score plot (Fig. 5B). Therefore, there exists a pattern visualized by
both score plots. The model achieved a cumulative predictable Y
ability (Q2Y) of 0.73. The metric evaluates the error between the
predicted and measured response variable vector (y) from the
regression model. However, the metric is not a robust diagnostic
criterion since the model can be overfitted when the variables
highly exceed the number of samples [132]. A solution can be the
use of permutation testing for the response variable vector (y)
and the subsequent calculation Q2Y. We performed 1000 per-
mutations, obtaining a set of permuted Q2Y that could be
compared with the original Q2Y (Fig. 5D). The maximum Q2Y for
the permutated sets is 0.04, which is considerably lower than the
original Q2Y of 0.73, which validates the metric. As a simple
definition of the p-value, 1000 permutations (n) corresponds to
the level 0.001 (1/n). In order to characterize those wavenumbers
that contribute most to the separation observed in the PLS-DA
score plot, we extracted the loadings for the first and second
LV and colored them according to the VIP score (Fig. 5E). VIP
reflects the importance of each variable or wavenumber in the
model. The loading plot follows a similar triangle shape as the
PLS-DA score plot, where LV1 discriminates Spain from the rest
and LV2 discriminates between Greece and Italy. Examination of
the VIP values demonstrates that the interval 1650-1800 cm1
highly contributes to the model and these positively contribute
to both LV1 and LV2 to the LV1 (Fig. 5C and E). Therefore, this
interval is important for the discrimination between Greece and
Italy. The second interval that corresponds to the 1110-1200 cm1
match with the position of Spain clusters in the LV space. This
interval contributes less to the model according to the VIP values
but still contributes to the Spain cluster (Fig. 5E). To conclude,
this second study dealt with a classification task that was solved
by using the popular PLS-DA algorithm. We have presented the
main steps that should be followed to construct, interpret and
validate the model, discussing at each step the possible pitfalls
and their solution.
9. Conclusions
Chemometrics provides plenty of techniques for the experi-
mental design, exploratory analysis, building calibration and clas-
sification models and also for their validation. The quantitative and
qualitative prediction of the responses based on the experimental
signals acquired from the samples is of utmost importance. For
example, chemometrics coupled to various spectroscopies repre-
sents a highly versatile tool for pharmaceutical analysis. In the
current omics era, chemometrics has exploited and captured the
attention of a broad scientific community. Now more than ever,
there is an awareness of what chemometrics or data analysis can
offer. As the development of chemical instruments is advancing and
they are being exported to all of the fields of science, the need to
incorporate chemometric methods also increases.
We have reviewed the latest research where chemometrics has
been employed in electrochemistry, spectroscopy and hyphenated
mass spectrometry for the 2018e2020 three-year period. Partic-
ularly, we focused on a pipeline that started with the experi-
mental design, followed by the exploratory analysis, then
predictive modelling and finishing with the validation of the
optimal model. Our search resulted in more than 300 research
articles. This vast number represents an ongoing revolution where
chemometrics takes part and cannot be disregarded. We envisage
that chemometrics will remain close to the current technological
revolution.
17
Trends in Analytical Chemistry 135 (2021) 116157
Declaration of competing interest
The authors declare that they have no known competing
financial interests or personal relationships that could have
appeared to influence the work reported in this paper.
Acknowledgements
This work and AK were supported by the Polish National Science
Centre (NCN) under Opus grant no. 2019/33/B/ST4/02428 (to AK)
and Preludium grant no. 2018/31/N/ST4/01909 (to MDPD). Publi-
cation of this article in open access was financially supported by the
Excellence Initiative - Research University (IDUB) program for the
University of Wroclaw.
Appendix A. Supplementary data
Supplementary data to this article can be found online at
https://doi.org/10.1016/j.trac.2020.116157.
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