Olive Oil Traceability

Analytical & Bioanalytical Chemistry
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Using Chemometric Resolution Methods For Fast Analysis Of

Some Phenolics In Olive Oil
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In Olive Oil to be considered for publication on the special issue of

Analytical and Bioanalytical Chemistry titled Analytical Sciences in
To the Editorial Office of
Italy (Editor: prof. Aldo Roda).
d Bioanalytical Chemistry
This manuscript presents an application of chemometric curve

resolution methods for the a posteriori deconvolution of coeluted
Dear
Editors,
chromatographic peaks resulting from the HPLC-DAD analysis of the

phenolic fraction in virgin olive oil samples. In particular, the
please
possibility of dealing with incomplete chromatographic resolution
find
through the use of mathematical processing of the signal allowed the
attached
use of fast gradients and the choice of a rapid extracting procedure. In
a copy of
terms of the achieved resolution, it was possible to separate up to 7
our
components in the chromatographic profile, even in the presence of
manuscri
spectral fingerprints which were rather similar among one another.
pt Using
The reduction of the analysis time and, correspondingly, in the amount
Chemom
of solvents used are outcomes that move towards the direction of
etric
greener procedures in analytical chemistry, thus representing a very
Resolutio
promising perspective. Moreover, these results are easily generalizable
to similar systems were a perfect chromatographic separation of the
Methods
peaks cant be achieved and were coelution stems from the complexity
For Fast
of the matrix or from the quest for reduced analytical times.
Analysis
In submitting the manuscript, I confirm on behalf of all the authors that
Of Some
the research proposed is original and that it hasnt been presented
Phenolics
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elsewhere.
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Best
regards,
Dept. of Chemistry
University of Rome La Sapienza
P.le Aldo Moro 5
00185 Rome
Italy
Tel +39 06 4991
3680 Fax +39 06
4457050 e-mail:
fmmonet@hotma
il.com
Federico
Marini
-Dr.
Federico
Marini
Page 2 of 21
USING CHEMOMETRIC RESOLUTION METHODS FOR FAST ANALYSIS

OF SOME PHENOLICS IN OLIVE OIL
Riccardo Nescatelli, Remo Bucci, Antonio L. Magr, Andrea D. Magr, Federico
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Marini*
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Dept. of Chemistry, University of Rome La Sapienza, P.le Aldo

Moro 5, I-00185
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Rome, Italy.
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*Corresponding author:
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Dr. Federico Marini
P.le Aldo Moro 5
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Dept. of
Chemistry
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Tel +39 06 4991 3680
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00185 Rome
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e-mail: fmmonet@hotmail.com
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USING CHEMOMETRIC RESOLUTION METHODS FOR FAST
ANALYSIS
OF SOME PHENOLICS IN OLIVE OIL
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Riccardo
Nescatelli,
Remo Bucci,
Antonio L.
Magr, Andrea
D. Magr,
Federico
properties. The phenolic compounds of virgin olive oils are currently
Marini*
component signals from coeluting chromatographic peaks in the
Dept.
of
a subject of great interest, due to their antioxidant action, their health

effects and how they affect the organoleptic characteristics of food. In
this study, the possibility of using a chemometrics, and in particular
multivariate curve resolution, for the a posteriori separation of the pure
analysis of the neutral polyphenolic fraction of olive oil was shown.
In particular, two groups of coeluting peaks were identified in the
Chemistry,
of
chromatogram and MCR-ALS in the multi-set configuration was used
La
to recover from the signals the elution and spectral profiles of the pure
Sapienza, P.le
components. Results were validated by comparison with a complete
Aldo Moro 5, I-
chromatographic separation of the substances giving rise to the two
00185
cluster of peaks: comparison of the spectral profiles showed a good
University
Rome
Rome,
Italy.
consistency, Additionally, when considering the relative amount of the
*e-mail:
fmmonet@hot
mail.com
different components in the analyzed samples, results were quite
Abstract
Keywords: Multivariate Curve Resolution (MCR-ALS); HPLC-DAD;
Phenolic
chemometrics; coelution; polyphenols; olive oil
promising as in almost all the cases errors lower than 5% were

obtained.
For Peer Review
compounds are
related to the
stability of the
Introduction
oil, but also to
The phenolic fraction of olive oil is a complex mixture of compounds
its biological
with different chemical structures. The literature on these compounds

has increased exponentially
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over the last ten years for various reasons: the

phenolic compounds are related to the
stability of the oil, but also to its biological
properties. To date, there is greater interest in
this feature: indeed, many compounds have
been studied thoroughly with the aim to
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establish a relationship between their dietary intake and the risk of
various
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degenerative diseases. In this respect, the studies agree in

identifying a beneficial role
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to human health associated with the consumption of these

compounds. The
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phenolic compounds identified, which can be grouped according to

their structural
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characteristics in the following classes: alcohols, phenols,

secoiridoids, phenolic acids,
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differs in terms of
hydroxy-isocromans, flavonoids and lignans [1]. The quality of oil
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the qualitative and quantitative composition of the phenolic
fraction. The average
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concentration of phenolic compounds is 100-300 mg/kg [1]. There
are differences in
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cultivar and the
composition and concentration due to many factors. First, the olive
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region in which fruits grow, as it was shown that olive oils of
different quality but
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from the same geographical area have similar phenolic profiles [2].
Secondly,
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compounds which,
agricultural techniques also influence the concentration of some
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for instance, is affected by the level of irrigation [3]. The phenolic
compounds of
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virgin olive oils are currently a subject of great interest, due to their
antioxidant action,
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of food [4].
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Besides their antioxidant properties, polyphenols have other
features that make them
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important to our health. Indeed, they lower blood cholesterol
levels; slow down tumor
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growth; strengthen the immune system; inhibit certain cancer-causing
chemicals;
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inhibit cyclooxygenase and lipoxygenase enzymes; inhibit platelet

aggregation;
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inhibit the peroxidation of LDL; carry out anti-allergic and antiinflammatory
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activities [5-11].
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In this context, it is clear that the analytical determination of the

composition of the
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polyphenolic fraction of olive oil is becoming more and more

important, to the point
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that in recent years an increasing number of methods have been
proposed in the
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analysis time,
often using more than two solvents and
anyway do not allow a complete resolution of
all compounds under consideration. In this
respect, also taking the lead from an earlier
work by our group on the separation and
quantification of phenolic acids [16], the aim
of this study was to investigate the possibility
of using chemometric resolution methods to
improve the analysis of some compounds
present in the neutral
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polyphenolic fraction of olive oils. Indeed, the introduction of appropriate
chemometric techniques to resolve coeluting peaks has resulted in the possibility of
using faster gradients, simpler extractions and also, in the case of hyphenated
chromatographic techniques, the combination with a less selective (but also less
expensive and more widespread) spectroscopy as the UV-Visible. In these cases, the
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coupling
with
savings in time and cost of analysis. Therefore, this research has investigated the
chemometric
possibility of applying this concept, i.e. the mathematical resolution of coeluted
techniques allows
hyphenated chromatographic peaks to the analysis of the content of some
to resolve signals
polyphenols in samples of extra virgin olive oil. In particular, we assessed the
of
that
possibility of using a rather fast gradient for the HPLC-DAD analysis of some
coelute (even in
relevant polyphenolic fractions, making up for the co-elution of different groups of
the
peaks with the application of chemometric methods for resolution of the signal.
analytes
presence
of
significant
baseline
Materials and methods
contribution)
Samples
through
sample was collected directly from the oil mill just after pressing and then stored in
mathematical
analysis
posteriori
Thirteen extra virgin olive oils from Sabina (Italy) were analyzed in this study. Each
dark brown glass bottles at 4C until used. Each sample was analyzed in replicate.
that
does not require a
Extraction of phenolic compounds from virgin olive oils
perfect separation
A great number of procedures for the isolation of the phenolic fraction of VOO
of
peaks
utilizing two basic extraction techniques, LLE or SPE, have been published in the
already at the level
literature. In this study, LLE was chosen to isolate the phenolic fraction of oil, as
of
being less selective, allows to extract more analytes in less time. In particular, the
the
instrumental
analysis.
feature
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translates
protocol was optimized to use less solvents to extract most of the polar phenolic
compounds excluding phenolic acids.
considerable
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into
This
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Five grams of oil
hexane allowed to avoid the formation of emulsions and to eliminate the glycerides.
were dissolved in
Extraction of phenolic compounds was performed by adding 5 mL of CH 3OH:H2O
6 mL of n-hexane.
80:20 (v/v), and mixture was shaken for 3 min. After that, the two phases were
The presence of nPage 6 of 21
separated by centrifugation at 3000 rpm for 3min and the

hydroalcoholic phase was transferred to a balloon. This step
was repeated three times and the hydroalcoholic extracts were
collected and evaporated to dryness by a rotary evaporator;
the
4 temperature was always controlled (<40 C) to avoid the deterioration of phenols. The
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residue was dissolved with 3 mL of CH3OH. The extracts were then filtered
through a
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13-mm PTFE 0.45 m membrane filter from Waters (Waters, Milford, MA) and 20
L were injected into the liquid chromatograph for HPLC-DAD analysis.
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HPLC-DAD analysis
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Methanol extracts prepared according to what described in Section 2.2 were then
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analyzed by HPLC-DAD on a Thermo Quest Spectrasystem LC (Thermo Fisher
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array detector, and a SN4000 interface to be operated via a personal computer.

Instrument software ChromQuest 5.0 (Thermo Fisher Scientific, Waltham, MA)
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flow
used for data acquisition. Water/methanol mixture was used as the mobile phase,
16 rate was 1 mL/min, and the column was kept at 25C. The column was an Eclipse xdb
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CA). As the scope of the study was to take advantage of the potential of
chemometric
C18 (5 m particle, 0.46 mm i.d., 15 cm length; Agilent Technologies, Santa Clara,
19 a posteriori resolution methods, the gradient chosen was faster than those proposed in
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the literature, allowing the analysis of the polyphenolic fraction in less than 45
mins.
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Moreover, MeOH was preferred to acetonitrile, being cheaper and less toxic. In
detail,
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the initial composition of the mobile phase was 80% water/20% methanol and this
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ratio was maintained for 5 min; after that, the amount of methanol was linearly
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increased to 100% in 30 min, and this percentage was maintained for additional 10
25
min. The chromatograms were registered from 258 to 360 nm at 2 nm intervals using
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was
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Scientific, Waltham, MA) equipped with a P4000 pump, a UV6000 UVVis diode
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diode array detector.
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Use of this gradient resulted in coeluting peaks that were resolved by chemometric
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curve resolution methods. In a further stage of the study, to validate the results of
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chemometric analysis, the same groups of peaks that appeared as coeluted using the
gradient reported above, were separated chromatographically.
In order to do so, the corresponding eluting fractions were
collected when coming out from the detector and stored. After
preconcentration, they were then injected in the HPLC
apparatus
and
separated
using
different
gradient:
water:acetonitrile 90:10 was chosen as the initial mobile

phase and this ratio was kept constant for 5 minutes;
successively, the amount
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of acetonitrile was linearly increased to 100% in 25 mins and kept at 100% for
further 10 mins.
Chemometric analysis
Whenever an hyphenated technique is used for the analysis, the experimental
outcome of each measurement is a data matrix, having one elution and one spectral
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dimensions; accordingly, when more than one sample is analyzed, the resulting data
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could be someway reduced to lower dimensional data arrays and analyzed by
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advantages in the use of the full landscape when performing the calibration stage:
structure is a three dimensional (three-way) array. In principle, this kind of data

standard chemometric techniques; however, there are some relevant theoretical
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this is the socalled second-order advantage [17], that stems from the fact that
second-order tensors (data matrices) are used to describe experimentally each
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sample. Simply stated, second-order advantage means that calibration can be done in
the presence of unknown interferences, calibration samples may be pure, and
25
identification/confirmation of compound identity through the pure response
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detection profile is possible.

In these framework, multi-set Multivariate Curve Resoultion (MCR) was used in this
study.
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and the spectral fingerprint of individual components. This means that, ideally, if
Multivariate Curve
Resolution [18-20]
The hypothesis of
MCR is that the
overall
sample
corresponding chromatographic landscape resulting in overlapping signals can be

separated into the individual contribution for the two chemical species. In
mathematical terms, if X is the 2D signal measured on a sample, it is decomposed in
the invidual component elution and spectral matrices, C and S, respectively,
chromatographic
landscape
two analytes are coeluting and their spectral profile is sufficiently different, the
for
can
decomposed
according to:
a
be
into
the contribution of
the elution profile
X=CST
(1)
What is relevant is that the search for the pure contributions is normally done on a
data-driven basis, meaning that it is not necessary (even if when possible it can help)
to know in advance the number of species giving rise to overlapping bands and their
individual spectra. As a consequence, in non-ideal cases more components than the
Page 8 of 21
expected chemical rank can be added to the model to account for

baseline effect or the presence of unknown interferents.
When more than one sample is measured, since the method works on matrix
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decomposition, an unfolding step is necessary: operationally this means that the

individual landscapes corresponding to the different samples are aligned one after
the
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other, providing that they share the same spectral dimension: this corresponds to
the
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hypothesis that the same component are present in all the samples but allows for the
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elution profiles to be different not only in terms of analyte concentration but also of
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peak positions (therefore allowing to deal with the presence of shifts) [20]. With
the
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same nomenclature as before, if Xi is the chromatographic landscape

measured on the
19 th
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i sample, then the decomposition is performed according to:
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Xi=CiST
(2)
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S
being
the
matrix
composed of the spectral loadings
that are common to all samples
24 th
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and Ci being the elution

profiles estimated for the i sample.
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Algebraically,
chemical
meaningfulness of the solution is
achieved by imposing
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mathematical constraints on the algorithm used to compute the components: non-
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negativity
chromatographic
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of
concentration
and
spectral
profiles
peaks are just the most common that can be implemented.
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and
unimodality
of
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Software
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All chemometric computations were run under Matlab R2011a (The Mathworks,
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Natick, MA) environment. In particular, MCR-ALS computations were performed
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using the MCR toolbox developed by the chemometric group of the University of
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Barcelona (freely downloadable at http://www.mcrals.info) [21].
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3. Results and discussion
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As anticipated, the aim of this study was to show the potential of chemometric
28
resolution methods to improve the quality of the HPLC-DAD analysis of some
29
phenolics in extra virgin olive oil samples. To this purpose, HPLC analysis using the
conditions reported in the methodological section was carried
out on each sample and the corresponding chromatograms
were recorded in the wavelength range 258-360 nm. In Figure
1, the profiles recorded at three wavelengths chosen as
representative of the different absorption of phenolic
substances (258, 280 and 320 nm) are reported. It is evident
from the Figure that the choice of a fast gradient using
methanol as modifier
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result in some peaks being coeluted and not fully resolved. Chemometric curve
resolution methods were then used to a posteriori separate the coeluted signals into
their single component profiles. In particular, as an example of the potential of curve
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resolution methods
at the lower and less significant at the higher wavelengths. Therefore, prior to
for
the
operate multivariate curve resolution on the two selected retention time windows,
identification and
the 2D HPLD-DAD landscapes from each sample were baseline corrected using the
separation of the
penalized asymmetric least squares approach proposed by Eilers [22]. Figure 2
contributions from
shows the effect of baseline correction on the chromatographic signals reported in
individual
Figure 1. It is apparent from the Figure that the contribution of baseline was
coeluting
completely removed at all wavelengths.
components,
two
the
clusters
peaks
of
Starting from these corrected data, MCR was operated separately on the two
chromatographic windows 19.99-22.65 min and 23.00-25.21 min.
between
around 20 and 25
MCR on the first cluster of peaks
minutes
The first chromatographic window considered in this study comprised the retention
were
considered.
time interval between 19.99 and 22.65 min. In this region, samples present highly
It can be seen in
overlapping peaks as shown in Figure 3a, where the signals recorded at 280 nm for
Figure 1 that there
the analyzed oils are reported. On the other hand, as multiple wavelengths were
is a not negligible
recorded during the chromatographic runs, for each sample the signal takes the form
contribution of the
of a 2D landscape as the one reported in Figure 3b. Accordingly, the experimental
baseline
data corresponding to the chromatographic landscapes in this retention time window
to
the
chromatographic
recorded for all samples were organized into an array of dimension 26 (number of
signals and that
runs) x 161 (number of retention time points) x 52 (number of wavelengths). This
this contribution is
array was the basis of the following chemometric analysis.
wavelength
In a first stage, Multivariate Curve Resolution analysis was carried out on the single
dependent,
being
data matrices corresponding to the chromatographic landscapes measured on
more pronounced
individual samples. For each sub-matrix, initialization of the ALS algorithm was
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performed
using
the purest spectra
extracted by the SIMPLISMA algorithm [23], while Principal Component Analysis

and Evolving Factor Analysis [24] were used to
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estimate the overall number of components and the local

rank , i.e. how many species are present in the different
portions of the retention time window. Then, MCR-ALS
optimization was carried out using the non-negativity
constraint on both spectra and
4
concentration profiles and the unimodality constraint on concentration profiles only.
10 5
Comparison of the optimal resolved profiles for the different sub-matrices showed
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a
11 6
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7 Therefore, based on this considerations in a second stage the analysis was repeated on
15 8
the complete data set.
16 9
In order to be analyzed by MCR in a multi-set arrangement, the 3-way
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10 chromatographic data were unfolded in a column-wise augmented fashion, by putting

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good consistency and suggested that 7 could be the optimal number of components.
11
sub-matrices on one another keeping the spectral dimension constant. Accordingly,
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the
estimate of the spectral profiles to be used in the ALS optimization, the average of
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the
spectral profiles obtained on the individual sub-matrices in the previous stage of
16
analysis was used. Then multi-set MCR was run on the augmented matrix using non-
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negativity constraint for spectra and concentration and uni-modality constraint for
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concentration only, and incorporating the information on local rank obtained by
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Evolving Factor Analysis.
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Eventually, the cluster of coeluted peaks was resolved into the contribution of 7
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that
components, whose spectra are reported in Figure 4b. It can be seen in the Figure
22
there is a very close similarity among the spectra, as expected, considering the
40 23
structural similarity of the phenolic compounds and the low selectivity of UV
41 24
spectroscopy. Anyway, even with a so close similarity, it was possible to obtain a
25
very good resolution of the chromatographic peaks in this retention time window, as
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x number of runs) x 52 (number of wavelengths). In this case, to have an initial
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the resulting augmented matrix had dimensions 4186 (number of retention time
points
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evidenced in Figure 4a, where the concentration

profiles of the 7 resolved components
46 27
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for one of the analyzed samples is reported. 47

As all the components were unknown to us (we are at present trying to identify at
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least some using HPLC-MS), chemical validation of the results was done by
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comparing the outcomes of curve resolution with those

obtained by chromatographic resolution of the coeluted
cluster, achieved by changing the experimental conditions (see
Materials and Methods). In Figure 5, the results of
chromatographic resolution of the overlapping peaks is shown
for one of the samples chosen as example. It can be seen that,
as estimated by MCR-ALS, the cluster was made of the
contribution of 7
Page 11 of 21
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species. Moreover, comparison of the spectral profiles obtained by chemometric resolution and those after
chromatographic separation confirmed the consistency of the results. As far as the quantitative analysis is
concerned, as no standard was available only relative concentrations could be estimated, i.e. only the
relative amount of each analyte from sample to sample and not its absolute quantity. Also under this
respect, very good results were obtained as the integrated area of the peaks obtained by MCR were in very
good agreement with the areas of the corresponding peaks after chromatographic resolution (relative errors
being in almost all cases less than 5%).
MCR for the second cluster of peaks
The data coming from the HPLC-DAD analysis of the second cluster of peaks were treated analogously. In
this case, a data cube of dimension 26 (runs)x133(retention times)x52(wavelengths) was processed. Also
in this case, analysis was at first performed on the individual sub-matrices to have hints about the number
of components in each chromatographic run and the local rank, using EFA. Successively, the 3-way data
cube was unfolded in a column-wise fashion and the resulting augmented data matrix was processed using
multi-set MCR-ALS. Investigation of the results obtained on the individual sub-matrices suggested that 8
components could be optimal for the chemometric resolution and, as in the previous case, the algorithm
was initialized using the averages of the optimal spectral profiles obtained in the analysis of the single 2D
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landscapes. Non-negativity (spectra and concentrations), unimodality (concentration) and local rank were
used as constraints for the ALS algorithm. The final results are reported in Figure 6.
As it was for the other cluster of coeluting peaks, it can be observed that even if the spectral profiles were
very similar (Figure 6b), it was possible to achieve a good resolution of the peaks (an example is reported
in Figure 6a, for one of the samples). Also in this case, in the absence of standards and lacking the
knowledge about the identity of the coeluted compounds, validation of the obtained results was achieved
by comparison with the outcomes of complete chromatographic separation under different experimental
conditions. A very good consistency between the chemometrically resolved spectral profiles and those
recorded on the chromatographically separated analytes was found. Moreover, as observed for the other
group of peaks, the error in the quantification of the relative amount of the
Page 12 of 21
analytes in the different samples was almost always less than 5 %, thus
confirming the goodness of the multivariate resolution approach.
4
8
9
Conclusions
10 5
In this study, the possibility of using a chemometrics, and in particular multivariate
11 6
curve resolution, for the a posteriori separation of the pure component signals from
coeluting chromatographic peaks in the analysis of the neutral polyphenolic fraction
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of olive oil was shown.
16 9
In particular, two groups of coeluting peaks were identified in the chromatogram
and
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MCR-ALS in the multi-set configuration was used to recover from the signals the
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elution and spectral profiles of the pure components. A good resolution was
obtained
21
12
in both cases, even if the spectroscopic fingerprints of the analytes were very
similar
13
with one another.
1
2
25
3
14
of
Results were validated by comparison with a complete chromatographic separation
26
15
the substances giving rise to the two cluster of peaks: comparison of the spectral
16
profiles showed a good consistency, Additionally, when considering the relative
27
28
29
30
17
amount of the different components in the analyzed samples, results were quite
31
18
promising as in almost all the cases errors lower than 5% were obtained.
19
Based on this considerations, it is possible to conclude that the use of chemometric
35
20
curve resolution methods can help and improve chromatographic analysis, by
36
21
allowing the a posteriori separation of the pure signals from coeluting compounds.
22
This can result in the possibility of using fast gradients and cheaper and/or more less
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harmful solvents (towards a greener analytical chemistry) without losing in
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accuracy, and with a corresponding saving of time and money.
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References
46 27
[1] Servili M, Montedoro GF (2002) Contribution of phenolic compounds to virgin
oil
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28 quality. Eur J Lipid Sci Technol 104: 602-613.
49
50 29 [2] Esti M, Cinquanta L, La Notte E (1998) Phenolic Compounds in Different
Olive 30 Varieties. J Agric Food Chem 46: 32-35.

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31 [3] Servili M, Selvaggini R, Esposito S, Taticchi A, Montedoro GF,

Morozzi G 32 (2004) Health and sensory properties of virgin olive oil
hydrophylic phenols:
33 agronomic and technological aspects of production that affect the occurrence in the
oil.
34 J Chromatogr A1054: 113-127.
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[4] Skevin D, Rade D, Strucelj D, Mokrovcak Z, Nederal S, Bencic D (2003) The
influence of variety and harvest time on the bitterness and phenolic compounds
of olive oil. Eur J Lipid Sci Technol 105: 536-541.
[5] Visioli F, Bellomo G, Galli C (1998) Free radical-scavenging properties of olive
oil polyphenols. Biochem Biophys Res Commun 247: 60-64.
[6] Visioli F, Galli C (1995) Natural antioxidants and prevention of coronary heart
disease: the potential role of olive oil and its minor constituents. Nutr Metab
Cardiovasc Disease 5: 306-314.
[7] L. Fremont (2000) Biological effects of resveratrol. Life Sci 66: 663-673.
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[8] Stuart
Scandlyn
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genistein: Evidence from epidemiological and laboratory studies. Toxicol Lett
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Rosengren RJ
[10]
Yokoyama M, Noguchi M, Nakao J, Pater A, Iwasaka T (2004) The tea
(2006) Role of
polyphenol, (-)-epigallocatechine gallate effects on growth, apoptosis, and
epigallocatechi
telomerase activity in cervical cell lines. Gynecol Oncol 92:197-204.
gallate
(EGCG) in the
treatment
breast
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and
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Bell DR, Gochenaur K (2006) Direct vasoactive and vasoprotective
properties of antocyanin-rich extracts. J Appl Physiol 100: 1164-1170.

[12]
Buiarelli F, Di Berardino S, Coccioli F, Jasionowska R, Russo MV (2004)
Determination of phenolic acids in olive oil by capillary electophoresis. Ann

Chim 94: 699-705.
[13]
Bendini A, Bonoli M, Cerretani L, Biguzzi B, Lercker G, Toschi TG (2003)
2329-2336.
Liquid-liquid and solid phase extractions of phenols from virgin olive oil and
[9] Park OJ, Surh
their separation by chromatographic and electrophoretic methods. J Chromatogr
YJ
(2004)
Chemopreventi
A 985: 425-433.
[14]
Bianco A, Buiarelli F, Cartoni G, Coccioli F, Jasionowska R, Margherita P
ve potential of
(2003) Analysis by liquid chromatography-tandem mass spectrometry of
epigallocatechi
biophenolic compounds in virgin olive oil, Part II. J Sep Sci 26: 417-424.
ne gallate and
[15]
Servili M, Baldioli M, Selvaggini R, Miniati E, Macchioni A, Montedoro GF
(1999) High performance liquid chromatography evaluation of phenols in olive

fruit, virgin olive oil, vegetation waters and pomace and 1D- and 2D-nuclear
magnetic resonance characterization. J Am Oil Chem Soc 76: 120-126.
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Maeder
M (1987)
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analysis for the resolution of overlapping
chromatographic
peaks.
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59: factor
527-530.
[24]
Chem 65: 1425-1432.
[16]
Windig W, Guilment J (1991) Interactive self-modeling mixture analysis. Anal

Marini F, DAloise A, Bucci R, Buiarelli F, Magr AL, Magr AD (2011) Fast analysis of 4 phenolic
acids in olive oil by HPLC-DAD and chemometrics.

Chemometr Intell Lab Syst 106: 142-149.
[23]
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8
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[17] Booksh KS, Kowalski BR (1994) Theory of analytical chemistry. Anal Chem 66:
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782-791A.
Eilers PHC (2004) Parametric time warping. Anal Chem 76: 404-411.
11 6
[18] Tauler R (1995) Multivariate curve resolution applied to second order data.
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[22]
Chemometr Intell Lab Syst 30: 133-146.
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Chemometr
Intell Lab Syst 76: 101:110.
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interface for MCR-ALS: a new tool for multivariate curve resolution in MATLAB.
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[21]
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Jaumot J, Gargallo R, De Juan A, Tauler R (2005) A graphical user friendly
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[20]
Tauler R, Smilde A, Kowalski BR (1995) Selectivity, local rank three way data
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concepts and applications. Crit Rev Anal Chem 36: 163-176.
Figure Captions
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Figure 1 - Raw chromatographic profiles of the analyzed oil samples at three selected
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wavelengths: 258 nm, 280 nm and 320 nm.
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Tauler R, De Juan A (2006) Multivariate curve resolution (MCR) from 2000:
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[19]
Figure 2 Effect
in the retention time window corresponding to the first cluster of coeluted peaks
of
(signal recorded at 280 nm); (b) 2D elution-spectral landscape of one sample, chosen
baseline
correction
using
penalized
Figure 4 Results of MCR-ALS analysis on the first cluster of coeluted peaks. (a)
asymmetric
squares
as example, in the same chromatographic window.
on
least
Elution profiles of the 7 components identified as significant for one of the analyzed
the
samples chosen as example; (b) spectral profiles of the 7 resolved components.
chromatographic
Figure 5 Chromatographic separation of the components coeluting in the first
signals reported in
cluster of peaks using an additional chromatographic step (Materials & Methods
Figure 1.
Section) evidenced the same number of constituents as estimated by MCR.
Figure
(a)
Figure 6 Results of MCR-ALS analysis on the second cluster of coeluted peaks. (a)
Chromatographic
Elution profiles of the 8 components identified as significant for one of the analyzed
profiles
samples chosen as example; (b) spectral profiles of the 8 resolved components.
of
the
analyzed samples
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Raw chromatographic profiles of the analyzed oil samples at three selected wavelengths: 258 nm, 280 nm

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chosen as example, in the same chromatographic window.
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a) Chromatographic profiles of the analyzed samples in the retention time window corresponding to the first
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identified as significant for one of the analyzed samples chosen as example; (b) spectral profiles of the 7
Results of MCR-ALS analysis on the first cluster of coeluted peaks. (a) Elution profiles of the 7 components

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estimated by MCR.
chromatographic step (Materials & Methods Section) evidenced the same number of constituents as
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Olive Oil Traceability

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Analytical & Bioanalytical Chemistry

Using Chemometric Resolution Methods For Fast Analysis Of

Analytical & Bioanalytical Chemistry

Analytical & Bioanalytical Chemistry

In Olive Oil to be considered for publication on the special issue of

To the Editorial Office of

Italy (Editor: prof. Aldo Roda).

This manuscript presents an application of chemometric curve

chromatographic peaks resulting from the HPLC-DAD analysis of the

possibility of dealing with incomplete chromatographic resolution

through the use of mathematical processing of the signal allowed the

use of fast gradients and the choice of a rapid extracting procedure. In

terms of the achieved resolution, it was possible to separate up to 7

components in the chromatographic profile, even in the presence of

spectral fingerprints which were rather similar among one another.

The reduction of the analysis time and, correspondingly, in the amount

of solvents used are outcomes that move towards the direction of

greener procedures in analytical chemistry, thus representing a very

promising perspective. Moreover, these results are easily generalizable

to similar systems were a perfect chromatographic separation of the

of the matrix or from the quest for reduced analytical times.

In submitting the manuscript, I confirm on behalf of all the authors that

the research proposed is original and that it hasnt been presented

For Peer Review

Analytical & Bioanalytical Chemistry

USING CHEMOMETRIC RESOLUTION METHODS FOR FAST ANALYSIS

Dept. of Chemistry, University of Rome La Sapienza, P.le Aldo

Dr. Federico Marini

P.le Aldo Moro 5

Analytical & Bioanalytical Chemistry

For Peer Review

Analytical & Bioanalytical Chemistry

Tel +39 06 4991 3680

Fax +39 06 4457050

Analytical & Bioanalytical Chemistry

Analytical & Bioanalytical Chemistry

properties. The phenolic compounds of virgin olive oils are currently

component signals from coeluting chromatographic peaks in the

a subject of great interest, due to their antioxidant action, their health

chromatogram and MCR-ALS in the multi-set configuration was used

components. Results were validated by comparison with a complete

chromatographic separation of the substances giving rise to the two

cluster of peaks: comparison of the spectral profiles showed a good

consistency, Additionally, when considering the relative amount of the

different components in the analyzed samples, results were quite

Keywords: Multivariate Curve Resolution (MCR-ALS); HPLC-DAD;

chemometrics; coelution; polyphenols; olive oil

promising as in almost all the cases errors lower than 5% were

For Peer Review

oil, but also to

The phenolic fraction of olive oil is a complex mixture of compounds

with different chemical structures. The literature on these compounds

Analytical & Bioanalytical Chemistry

over the last ten years for various reasons: the

establish a relationship between their dietary intake and the risk of

degenerative diseases. In this respect, the studies agree in

to human health associated with the consumption of these

7 composition of the polyphenolic fraction is very heterogeneous, with at

phenolic compounds identified, which can be grouped according to

characteristics in the following classes: alcohols, phenols,

hydroxy-isocromans, flavonoids and lignans [1]. The quality of oil

Analytical & Bioanalytical Chemistry

composition and concentration due to many factors. First, the olive