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FOOD SCIENCE AND TECHNOLOGY

ADVANCES IN FOOD
ANALYSIS RESEARCH

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FOOD SCIENCE AND TECHNOLOGY


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FOOD SCIENCE AND TECHNOLOGY

ADVANCES IN FOOD
ANALYSIS RESEARCH

ANITA HAYNES
EDITOR

New York

Copyright 2015 by Nova Science Publishers, Inc.


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Library of Congress Cataloging-in-Publication Data


Advances in food analysis research / editor, Anita Haynes.
pages cm. -- (Food science and technology) Includes bibliographical references and index.

ISBN: (eBook)

1. Food--Analysis--Research. I. Haynes, Anita, editor.


TX541.A328 2015
664'.07--dc23
2015032806

Published by Nova Science Publishers, Inc. New York

CONTENTS
Preface
Chapter 1

Chapter 2

Chapter 3

Chapter 4

Chapter 5

Chapter 6

vii
Confirmation Strategies in Food Analysis by Liquid
Chromatography-Mass Spectrometry
lida Alechaga, Paolo Lucci and Oscar Nez
Novel Insights in the Methods for Measuring
the Antioxidant Capacity of Foods
Sergio Prez-Burillo, Silvia Molino,
Cristina Delgado-Andrade and Jos A. Rufin-Henares
Advantages and Pitfalls of the Methods for the Antioxidant
Activity Evaluation
Angela Fadda and Daniele Sanna
Recent Applications of Chiral Capillary Electrophoresis
in Food Analysis
Raquel Prez-Miguez, Mara Castro-Puyana
and Mara Luisa Marina

37

65

89

Chromatographic and Chemometric Methods


for the Authentication of Olive Oil Geographical Origin
Raffaella Preti, Simone Vieri and Giuliana Vinci

121

Application of Ultra-Performance Liquid


Chromatography-Photodiode Detector-Quadrupole/Time
of Flight-Mass Spectrometry (UPLC-PDA-Q/TOF-MS)
Method for the Characterization of Phenolic Compounds
of Products at the Manufacturing Stage of Olive Oil
Joanna Kolniak-Ostek, Agnieszka Kita,
Mamdouh Helmy El-Kalyoubi,
Mohamed Magdy Mostafa Khallaf
and Adel Gabr Abdel-Razek

135

vi
Chapter 7

Chapter 8

Chapter 9

Chapter 10

Index

Contents
Frozen Minced Fish from Lower-Value Species:
Evaluation of Fatty Acids Variability by Chemometrics
Maria Joo Ramalhosa, Simone Morais,
Susana Casal, Eullia Mendes, M. Rui Alves,
Cristina Delerue-Matos
and Maria Beatriz Prior Pinto Oliveira
Validation of a Matrix Solid Phase Dispersion Methodology
for the Determination of Triazines Herbicides in Fish
M. J. Gonzlez-Castro, V. Castro-Bustelo,
N. Rodrguez-Gonzlez and E. Beceiro-Gonzlez

151

167

The Use of DNA-based Methods for Detection of Plant-based


Adulterants in Plant Food Products
Nadia Haider

177

LC-MS/MS Identification of Unknown Species in Beverages


due to Interactions between Dyes and Other Additives
or due to Dye Degradation Reactions
Fabio Gosetti, Bianca Bolfi, Eleonora Mazzucco,
Marcello Manfredi and Emilio Marengo

211

241

PREFACE
Food products are produced and distributed worldwide, leading to very stringent
regulations to guarantee food quality and safety. The authors of the first chapter of this book
confirm strategies that work in food analysis by liquid chromatography-mass spectrometry.
Furthermore, antioxidant activity is well-known to have a great deal of beneficial impacts on
health due to the protection against oxidative damage. Novel insights are provided for the
methods for measuring the antioxidant capacity in foods, placing particular emphasis on the
advantages, pitfalls and possible developments in the various methodologies. The next
chapter highlights the interesting role that chirality plays when evaluating the quality and
safety of foods and points out the main practical aspects that should be considered to carry out
the chiral analysis of food components, additives and pesticides by capillary electrophoresis
(CE). Next, authors' review the literature of the application of chemometric methods and
chromatographic data for the characterization and authentication of the geographical origin of
olive oil. An identification and comparison of phenolic compounds in different products at the
manufacturing stage of olive oil is provided as well. Next, the long-chain omega-3 fatty acids
stability of three important fish species are characterized and evaluated by the authors. Fish
gender and catching season were taken into account as these factors influence the lipid
amounts and compositions. This is followed by a chapter on the validation of a matrix solid
phase dispersion methodology for the determination of triazines herbicides in fish.
Furthermore, food adulteration is an act of intentionally debasing the quality of food offered
by sale either by the admixture of substitution of inferior substances or by the removal of
some valuable ingredients. The next chapter examines the use of DNA-based methods for the
detection of plant-based adulterants in plant food products. Finally, the interactions among the
ingredients in food and beverages are still an open problem. The last chapter is devoted to
food safety, with particular attention devoted to the interactions between beverages and food
dyes.
Chapter 1 - Food products are produced and distributed worldwide, so leading to very
stringent regulations to guarantee food quality and safety. Food products are very complex
mixtures consisting of naturally occurring compounds and other substances generally
originating from technological processes, agrochemical treatments, or packaging materials.
Several of these compounds (e.g., pesticides, veterinary drug residues, environmental
contaminants, mycotoxins) are of particular concern because, although they are generally
present in very small amounts, they are nonetheless often dangerous to human health. So
today there is an increasing need for applications able to cope with a large number of analytes

viii

Anita Haynes

in very complex matrices. Laboratories are interested in cost-effective methodologies with


reduced analysis time.
Liquid chromatography coupled to mass spectrometry (LC-MS(/MS)) is one of the most
widely used techniques in food analysis, and several modern approaches such as ultrahigh
pressure liquid chromatography (UHPLC) methods enable the reduction of the analysis time
without compromising resolution and separation efficiency. However, the use of ultrafast
separations is not enough to develop a fast analytical method for food analysis. Moreover, the
possibility of analyzing multiple compounds for target or non-target screening, such as multiresidue methods in various matrices, minimizing the sample manipulation is demanded. So
sample extraction and treatment must also be optimized when considering reducing the total
analysis time. For example, QuEChERS is an attractive alternative method for food sample
treatment in multi-residue analysis. Nevertheless, by reducing the sample treatment more
matrix related compounds may be introduced into the chromatographic system and although
high resolution and separation efficiency is achieved, the possibility of interfering compounds
and matrix effects such as ion suppression or ion enhancement may increase. In this context,
confirmation of the identity and the presence of suspected target or non-target compounds in
food products are mandatory to prevent false-positive and, especially, false-negative results
because the presence of a possible harmful compound would be ignored.
Typically, conventional LC-MS/MS methods in food analysis (generally using triple
quadrupole mass analyzers) are proposing for multi-residue approaches two selective reaction
monitoring (SRM) transitions to comply with EU directive 2002/657/EC. However, the
application of this criterion does not completely eliminate the possibility to report a falsepositive or a false-negative result due to the low resolving power of conventional MS
analyzers (triple quadrupole, ion-trap) to base-line resolve isobaric ions. So today, alternative
confirmation strategies are frequently required in food analysis by LC-MS(/MS). In this
context, high-resolution mass spectrometry (HRMS) and accurate mass measurements are
emerging as the best options for multi-residue analysis in food to guarantee confirmation and
identification.
In this chapter, confirmation strategies in food analysis by LC-MS(/MS) and LC-HRMS
techniques will be reviewed. Traditional confirmation criteria as well as novel ones such as
the combination of several acquisition modes via dependent or independent data acquisition,
or the use of library searching engines would be discussed by means of relevant applications.
Coverage of all kind of applications is beyond the scope of the present contribution, so we
will focus on the most relevant applications published in the last years.
Chapter 2 - Antioxidant activity is well-known to have a great deal of beneficial impacts
on health due to the protection against oxidative damage. However, not only is important
because of the health benefits but also because of the suitability of antioxidant activity in the
food technology industry as a tool to prevent food oxidation and prolong shelf-life.
There are many different methods to measure antioxidant activity of foods with several
classifications. One of them distinguishes between the assays focused on lipid peroxidations
(for instance thiobarbituric acid assay (TBA) or beta-carotene bleaching assay) and the assays
which draw its attention to the scavenger activity against free radicals (i.e., ABTS assay or the
ferric reducing antioxidant power (FRAP) assay); whereas others take into account the
transfer of either hydrogen atoms or electrons. The formers are mostly based on the
competition of antioxidants for peroxyl radicals like the ORAC assay (oxygen radical

Preface

ix

absorbance capacity) or TRAP assay (total radical trapping antioxidant parameter). Among
the electron-transfer methods we have the Folin-Cioucalteu method or DPPH method.
Moreover, before measuring the antioxidant activity a correct extraction of the
antioxidant compounds from the food matrix is needed. This extraction can be done either
chemically or at physiological conditions.
The former is based on the extraction of antioxidant compounds with organic or acidic
solvents. The physiological extraction, however, is basically an in vitro gastrointestinal
digestion composed by an oral, gastric and intestinal phase. In some cases, extraction is not
performed and antioxidant activity is directly measured.
Chapter 3 - The beneficial influence on human health of many foodstuffs and beverages
originates from their antioxidant activity. The increasing interest on the role of natural
antioxidants in biological systems and their nutritional effects has determined continuous
efforts in the search of simple and reliable methods for the determination of antioxidant
activity.
In this paper an overview of the most used methodologies for the determination of
antioxidant capacity is presented, placing particular emphasis on the advantages, the pitfalls
and the possible developments of each method. In particular the methods based on the
Electron Spin Resonance (ESR) spectroscopy with direct measurement of relatively stable
radical species or unstable radicals, trapped to form stable adducts (spin trapping method), are
reviewed and compared with assays based on spectrophotometric and voltammetric methods.
All of these methods, widely used in the food industry, differ for the antioxidant
mechanism involved, the analytical conditions applied in terms of substrate, solvents and
range of concentrations, the instrumentation used and the outcome they provide. This review
thus focuses on the effect of each analytical parameter on the antioxidant activity evaluation.
When selecting an appropriate method for the estimation of the antioxidant activity, any
interference should be known in advance. In addition an overall evaluation of the antioxidant
activity should be based on a comparison of different methods in order to overcome the
limitations of the method itself. Hence this review provides a basis for the choice of the best
antioxidant activity method applicable to a particular nutraceutical or dietary supplement in a
particular matrix.
Chapter 4 - The determination of the enantiomers of chiral food components, additives or
contaminants presents a high interest in Food Science since it can provide information related
to food quality, authenticity and safety, also enabling to evaluate the effect of processing or
manufacturing procedures and possible contaminations. Capillary Electrophoresis (CE) has
demonstrated in the last years a very high potential to achieve enantiomeric separations due to
its high efficiency, versatility, feasibility and the low consumption of solvents, reagents and
samples which makes it an environmentally friendly analytical technique. In addition, the fact
that the chiral selector in CE can be added to the electrolytic solution makes very easy the
screening of different chiral selectors in order to select the most adequate for the enantiomeric
separation of a given compound which is the most difficult step in the development of a chiral
analytical methodology.
This chapter highlights the interesting role that chirality plays when evaluating the quality
and safety of foods and points out the main practical aspects that should be considered to
carry out the chiral analysis of food components, additives and pesticides by CE. The
characteristics of the CE separation modes mostly employed in chiral food analysis
(Electrokinetic Chromatography, EKC and Capillary Electrochromatography, CEC) are

Anita Haynes

summarized together with the main detectors employed to achieve the determination of
enantiomers. Moreover, a critical overview on the main applications of chiral CE
methodologies developed during the last ten years including the chiral analysis of protein and
non-protein amino acids, organic acids, phenolic compounds, sweeteners, and pesticides in
foodstuffs is presented. Experimental conditions enabling the chiral separations are presented
and some representative examples are illustrated. Finally, the main advantages and drawbacks
of these procedures are discussed with the aim of showing the potential of CE to solve
specific problems in the Food Science research field.
Chapter 5 - In recent years, the issues of food quality and safety have received a special
attention both from the producers and the consumers, in particular the geographical origin is
gaining importance as guarantee of quality.
On 2009 the European Union (EU) Member States agreed to require origin labeling for
virgin and extra virgin olive oils (EC Regulation 182/2009) to defend consumers need about
true characteristics and origin. This regulation, together with the well established laws
regarding denominations and protected indications of origin (POD PGI and STG), has led to a
new olive oil market where geographic origin labels are a big market competitive advantage.
To enforce these laws, a measure of the authenticity of samples must be made, most often
in the form of proving the presence/absence of adulterants, or verifying geographical or
cultivar origin by comparison with known and reliable samples. Many analytical methods
(NMR; DNA fingerprint, isotopic ratio, elemental analysis etc.) have been employed in the
last decades in order to verify the real compliance to the geographical origin declared in the
label.
Among them, chromatographic techniques are the most utilized for their widespread
availability, low cost and reliability, and their data have been successfully used for olive oil
traceability models building. These methods often include the use of multivariate statistical
techniques such as principal components analysis, linear discriminant analysis, canonical
variance analysis and partial least squares regression.
In view of the growing interest in this topic, this paper reviews the literature of the
application of unsupervised/supervised chemometric methods on chromatographic data for
the characterization and authentication of the geographical origin of olive oil.
Chapter 6 - The aim of the study was to identify and compare phenolic compounds in
different products at the manufacturing stage of olive oil. In addition the content of bioactive
compounds and antioxidant capacity of olive oil manufacturing products were investigated.
Identification of phenolic compounds was carried out using UPLC-PDA-Q/TOF-MS method.
Total phenolics was measured by the Folin-Ciocalteau method, and the antioxidant properties
have been determined by DPPH, ABTS and FRAP methods. Extraction was conducted using
two solvents: 70% methanol and 70% ethanol. Influence of the solvent type on the extraction
of phenolics and antioxidant properties of the olive extracts was also investigated. Forty two
compounds belong to the compound groups of simple phenols, hydroxycinnamic acids,
flavonoids, lignans and secoiridoids were identified. Total phenolics content of olive products
ranged from 24.05 to 235.60 mg of gallic acid/1 g DW. The highest antioxidant properties
were determined in olive leaves. Obtained results revealed that extracting solvent has a
significant influence on the polyphenolic profile, total phenolic concentration and antioxidant
properties of olive products, and that methanol is the most efficient extraction solvent.
Chapter 7 - Sardine (Sardine pilchardus), chub mackerel (Scomber japonicus) and horse
mackerel (Trachurus trachurus) are widely distributed in Atlantic Ocean, and represent some

Preface

xi

of the most important pelagic groups captured. They are classified according to their fat
content as medium (horse mackerel) and high (sardine and chub mackerel) fat fish. These fish
species are usually consumed fresh, due mostly to their high local availability, with little
industrial applications rather than caning. Effective preservations techniques are needed to
reduce fish losses by deterioration. Also, innovative strategies are necessary to enhance their
acceptability among the youngers. Studies determining the fatty acid profiles of minced
frozen fillets of these three species, in particular, are scarce. This aspect is very important
since minced fish is a valuable commercial product, which has a high number of possible
applications. However, it is less stable than whole fish requiring effective knowledge of the
variation during frozen storage to determine its shelf life and define complementary
preservation techniques, if necessary.
Thus, the main goal of this study was to characterize the long-chain omega-3 fatty acids
stability of these three fish species during frozen storage of minced fillets, for up to six
months. Fish gender and catching season were also taken into account, as these factors are
known to influence the lipid amounts and composition.
High variability on the lipid content was observed between seasons, particularly in
female specimens. All species were characterized by high omega-3 polyunsaturated fatty
acids (PUFA) contents (0.53 g/100 g - 2.37 g/100 g), particularly eicosapentaenoic acid
(EPA) and docosahexaenoic acid (DHA). Sardine fillets presented higher relative amounts of
EPA, while horse mackerel samples exhibited higher levels of DHA, and chub mackerel had
the highest nutritional scores regarding potential benefits on cardiovascular health. Reduced
differences were detected for up to six months of frozen storage, using principal components
and canonical variates analyses, indicating a good frozen stability.
These results highlight the potential use of these underutilized species for the preparation
of frozen fish alternatives, with high nutritional value. These preliminary findings will
support the evolution into industrial applications to reduce fish losses.
Chapter 8 - A method using dual process columns of Matrix Solid Phase Dispersion
(MSPD) and Solid Phase Extraction (SPE) has been validated for extracting and cleaning-up
of nine triazine herbicides (ametryn, atrazine, cyanazine, prometryn, propazine, simazine,
simetryn, terbuthylazine and terbutryn) in trout samples. For this purpose, freeze dried
samples (0.2 g) were blended with 2 g of octadecylsilyl-derivatized silica (EnviTM-18) and
transferred into a SPE cartridge containing ENVITM-Carb II/SAX/PSA (0.5/0.5/0.5 g) as clean
up co-sorbent. Then the dispersed sample was washed with 10 mL of n-hexane and triazines
were eluted with 20 mL ethyl acetate and 5 mL acetonitrile. Finally the extract was
concentrated to dryness, re-constituted with 1 mL methanol and injected into the HPLC-DAD
system. Recoveries varied between 88 and 105% with associate standard deviations below
8.6%, meeting the requirements stipulated by European Union legislation. The main
advantages of this methodology when compared with conventional methods of sample
preparation to screen herbicides in fish matrices are easy of work-up, fast, cheap, avoidance
of clean-up procedure, as well as the reduction of organic solvents and energy requirements in
agreement with the principles of Green Chemistry.
Chapter 9 - Food adulteration is an act of intentionally debasing the quality of food
offered for sale either by the admixture or substitution of inferior substances or by the
removal of some valuable ingredients. In addition to food adulteration, contamination of food
products as a result of inadequate company controls, or due to the nature of the product or the

xii

Anita Haynes

process has been reported. Food adulteration has been a major contributor to the poor
quality of industrial products, and the number of adulterants used to supplement expensive
ingredients has been continually increasing. A serious problem with the adulteration of food
products with declared or undeclared other foodstuff may cause severe health problems and
may even risk death to people who may have a severe allergenic response to some adulterants
(e.g., nuts and soya). This problem is particularly acute when the presence of adulterants (e.g.,
walnuts in bread) cannot be reasonably anticipated. In consequence, there is a constant and
continuing need for improved methods to detect the presence of adulterants in foodstuffs
(e.g., adulteration of single food commodities). Numerous methods based on DNA analysis
have contributed immensely in the food industry to monitor adulterations of food products of
plant or animal origin. In this chapter, reported examples of adulteration of plant food
products (mainly those which are economically important) with adulterants, of plant origin in
particular, and examples of DNA-based methods and markers used for detection of such
adulteration are presented without making detailed or critical analysis or evaluation of each of
those examples.
Chapter 10 - The interactions among the ingredients in food and beverages are still an
open problem. Unfortunately, much of these interactions give rise to unknown species that
sometimes are potentially dangerous.
In food safety, particular attention is devoted to food dyes. The risk is not ascribed to the
dye itself, whose use and permitted quantities are rigidly regulated by specific restrictions, but
to the degradation products that may form due to inadequate storage and management
conditions.
Different studies on the toxicity of food dyes have been performed, but very few
information has been collected about their possible degradation reactions in commercial
beverages undergone to sunlight irradiation and their chemical reaction with other ingredients
present in a specific drink. Since many food dyes are characterized by aromatic
aminosulfonate groups, the risk may regards, besides the possible presence in beverages of
impurities deriving from the industrial dye synthesis process, also the possible formation of
carcinogenic aromatic amines in the beverage itself exposed to sunlight irradiation, which
may take place along the different phases of the life cycle of the specific food product.
After a quick overview about the problem of additive interactions in food, the possible
causes of dye discoloration and their relevance in wastewater pollution, this chapter deals
with the potential modifications of permitted dyes taking place in beverages. These
modifications can be due to reactions occurring among different ingredients, to potential
transformations of the ingredients themselves and to environmental effects. Generally, these
interactions take place during the post-production phases and in particular during the preselling storage, conservation and commercialisation. They are often unpredictable, but they
can give rise to different kinds of contaminations with the formation of new species
potentially harmful to the consumer health.
It is useful to underline that the nature of these contaminations is not due to possible
frauds, adulterations or use of poor quality ingredients, but on a still scarce knowledge and
available documentation concerning these kinds of interactions. The sources of possible
contamination that have been considered can be classified into two types of reactions, taking
into account that some of their effects can also occur simultaneously: i) unpredictable
reactions that potentially take place among food ingredients, both naturally present or added,

Preface

xiii

ii) degradation reactions induced by not suitable preservation conditions, as the exposure of
the product to sources of intense sun light and/or high temperature.
This chapter describes the process of identification of these unknown species through
liquid chromatography coupled with tandem mass spectrometry, and in particular, through the
use of high-resolution mass spectrometry.
As a consequence of what described above, the attention to this type of problems is
extremely relevant for the consumers health safety and for the development of standardized
investigation protocols to be applied in the routine control for the permission of a certain
product or to establish management/transport/conservation procedures of the product in order
to avoid the appearance of the problems described.

In: Advances in Food Analysis Research


Editor: Anita Haynes

ISBN: 978-1-63483-783-5
2015 Nova Science Publishers, Inc.

Chapter 1

CONFIRMATION STRATEGIES IN FOOD ANALYSIS BY


LIQUID CHROMATOGRAPHY-MASS SPECTROMETRY
lida Alechaga1, Paolo Lucci2 and Oscar Nez1,3, *
1

Department of Analytical Chemistry, University of Barcelona, Barcelona, Spain


2
Department of Food Science, University of Udine, Udine, Italy
3
Serra Hnter Fellow, Generalitat de Catalunya, Spain

ABSTRACT
Food products are produced and distributed worldwide, so leading to very stringent
regulations to guarantee food quality and safety. Food products are very complex
mixtures consisting of naturally occurring compounds and other substances generally
originating from technological processes, agrochemical treatments, or packaging
materials. Several of these compounds (e.g., pesticides, veterinary drug residues,
environmental contaminants, mycotoxins) are of particular concern because, although
they are generally present in very small amounts, they are nonetheless often dangerous to
human health. So today there is an increasing need for applications able to cope with a
large number of analytes in very complex matrices. Laboratories are interested in costeffective methodologies with reduced analysis time.
Liquid chromatography coupled to mass spectrometry (LC-MS(/MS)) is one of the
most widely used techniques in food analysis, and several modern approaches such as
ultrahigh pressure liquid chromatography (UHPLC) methods enable the reduction of the
analysis time without compromising resolution and separation efficiency. However, the
use of ultrafast separations is not enough to develop a fast analytical method for food
analysis. Moreover, the possibility of analyzing multiple compounds for target or nontarget screening, such as multi-residue methods in various matrices, minimizing the
sample manipulation is demanded. So sample extraction and treatment must also be
optimized when considering reducing the total analysis time. For example, QuEChERS is
an attractive alternative method for food sample treatment in multi-residue analysis.
Nevertheless, by reducing the sample treatment more matrix related compounds may be
introduced into the chromatographic system and although high resolution and separation
efficiency is achieved, the possibility of interfering compounds and matrix effects such as
*

E-mail address: oscar.nunez@ub.edu

lida Alechaga, Paolo Lucci and Oscar Nez


ion suppression or ion enhancement may increase. In this context, confirmation of the
identity and the presence of suspected target or non-target compounds in food products
are mandatory to prevent false-positive and, especially, false-negative results because the
presence of a possible harmful compound would be ignored.
Typically, conventional LC-MS/MS methods in food analysis (generally using triple
quadrupole mass analyzers) are proposing for multi-residue approaches two selective
reaction monitoring (SRM) transitions to comply with EU directive 2002/657/EC.
However, the application of this criterion does not completely eliminate the possibility to
report a false-positive or a false-negative result due to the low resolving power of
conventional MS analyzers (triple quadrupole, ion-trap) to base-line resolve isobaric ions.
So today, alternative confirmation strategies are frequently required in food analysis by
LC-MS(/MS). In this context, high-resolution mass spectrometry (HRMS) and accurate
mass measurements are emerging as the best options for multi-residue analysis in food to
guarantee confirmation and identification.
In this chapter, confirmation strategies in food analysis by LC-MS(/MS) and LCHRMS techniques will be reviewed. Traditional confirmation criteria as well as novel
ones such as the combination of several acquisition modes via dependent or independent
data acquisition, or the use of library searching engines would be discussed by means of
relevant applications. Coverage of all kind of applications is beyond the scope of the
present contribution, so we will focus on the most relevant applications published in the
last years.

1. INTRODUCTION
Food products are very complex mixtures consisting of a great variety of compounds
including natural ones such as lipids, carbohydrates, proteins, vitamins, organic acids, and
aromas, but also of other substances generally occurring from technological processes (such
as heating), agrochemical treatments, or packaging materials, that can be considered
contaminants. Several of these compounds (pesticides, veterinary drug residues, endocrine
disruptors, food additives, environmental contaminants), as well as contaminants of natural
origin such as mycotoxins and marine toxins are of particular concern because, although they
are generally present in very small amounts, they are nonetheless often dangerous to human
health [1].
The comparison of the various sources of food contamination with organic chemicals
suggests that among the public, but also among experts, the perception of risk is often
distorted. For instance, and as reported by Grob et al. [2], if you ask educated consumers
about the principal source of food contamination they will list pesticides, and then veterinary
drugs, as the first items.
This is understandable since in the case of pesticide residues in food there is a growing
concern because they are used world-wide on a broad variety of crops to control pests and
prevent diseases in order to increase agricultural production, improve the quality, and extend
the storage life of food crops [3]. Thus, nowadays the monitoring of pesticide residues in food
is a priority objective in order to get extensive evaluation of food quality and to avoid
possible risks to human health. For instance, in Europe, the European Union (EU) operates a
country-by-country monitoring program that oversees the quality of imported food products
[4].

Confirmation Strategies in Food Analysis

However, when dealing with possible sources of food contamination few people would
even consider food-packaging materials, although the amount of material migrating from food
packaging into food may well be 100 times greater than the contribution of pesticides or other
common environmental pollutants such as polychlorinated biphenyls (PCBs) [2]. Moreover, it
is difficult to compare the toxicity (primarily acute) of well-controlled pesticides with the
potential (primarily chronic) toxicity of frequently not even identified compounds entering
food from packaging materials.
Food packaging has become an indispensable element in the food manufacturing process
because it is an important way to store food at different temperatures, to extend the shelf-life
of the product, and to enable foods to travel safely for long distances from their point of
origin and still be wholesome at the time of consumption. Moreover, food packaging
safeguards foods from natural agents, such as air, and can retard product deterioration, retain
the beneficial effects of processing, and maintain or increase the quality, organoleptic
properties and safety of the food [5]. Nevertheless, the safety in the use of food packaging
materials, for instance polymeric materials, is a subject of concern due to the transfer of
materials constituents to the food by a diffusion process called migration [6]. Migration is a
term used to describe the transfer of components from a certain material to the foodstuff in
contact with this material.
In the last 20-25 years, modern science has also shown that the heating of food can
generate several potentially hazardous compounds, some of which are genotoxic and
carcionegenic. For instance, in April 2002 Swedish researchers shocked the food safety world
when they presented preliminary findings of acrylamide in some carbohydrate-rich foods,
especially in fried and baked products [7]. These findings attracted considerable interest and
wide attention all over the world due to the classification of acrylamide as a potential
carcinogen to humans [8].
Moreover, the Swedish findings on the high level of acrylamide in heat-treated foods
were quickly confirmed by a series of government agencies through official website
notifications. Regarding the formation of acrylamide in heated food, several theoretical
mechanisms have been proposed but the Maillard reaction between amino acids (mainly
asparagines) and reducing sugars such as glucose or fructose is accepted as the most probable
formation mechanism [9].
In order to protect the consumer from potential food hazards, risk analysis is mandatory,
and, for that purpose, hazard identification, hazard characterization, exposure assessment and
risk characterization is necessary. A very important prerequisite for performing risk
assessment adequately is the presence of data generated by reliable, fit-for-purpose analytical
methods to estimate the level of exposure and intake of the consumer to contaminants and
residues. Moreover, efforts on harmonization of legislation and regulations between countries
to achieve food safety are made [10, 11].
However, despite the efforts on food legislation and regulation, food-safety incidents
occasionally occur and can originate from different sources (e.g., microbial and chemical
contaminants). For instance, over the past 10 years, food safety incidents have occurred
frequently in China [12]. Melamine, a nitrogen-rich chemical, was purposely added to foods
by unethical manufactures in China in order to elevate the organic nitrogen content, thus
increasing the price and the profit made from such products [13]. About 294,000 children
became victims of melamine-tainted milk, and six deaths were reported in 2008 [14, 15].
Some food safety incidents have been directly related to packaging materials, e.g., the alert

lida Alechaga, Paolo Lucci and Oscar Nez

for food contamination by UV-ink photoinitiators on November 2005 in Europe [16]. The
Italian Food Control Authority detected that photoinitiator 2-isopropylthioxanthone migrated
into baby milk at concentrations of 120-300 g/L, resulting in the withdrawal from the market
of more than 30 million liters of milk.
Nowadays, reliable methodologies are therefore crucial for both industrial and
enforcement testing of compliance with the legislation. It is necessary to assess the
concentration levels of contaminants in food to evaluate the level of exposure according to the
diet. For these reasons, there is an increasing need for applications able to cope with the
determination of a large number of analytes in very complex matrices. Laboratories are
interested in cost-effective methodologies with reduced analysis times. In the analysis of
contaminants and chemical residues in food, gas chromatography (GC) and liquid
chromatography (LC) are the two main chromatographic methods employed in practice.
However, the complexity of food matrices often requires not only extensive sample
preparations, but also on-line coupling techniques, which are used for their superior
automation and high-throughput capabilities [17].
Liquid chromatography coupled to mass spectrometry (LC-MS) is one of the most widely
used techniques in food analysis, and several modern approaches such as ultrahigh
performance liquid chromatography (UHPLC) method enable the reduction of the analysis
time without compromising resolution and separation efficiency. But the use of ultrafast
separations is not enough to develop fast analytical methods in food analysis, and the
requirement of analyzing multiple compounds (multiresidue methods) minimizing sample
manipulation is demanded. This makes necessary to optimize simultaneously both sample
extraction and treatment procedures with the analytical methods in order to achieve an
important reduction on total analysis time. For example, QuEChERS, acronym of Quick,
Easy, Cheap, Effective, Rugged and Safe, is an attractive sample preparation technique used
in multiresidue analysis [18]. It entails solvent extraction with acetonitrile and partitioning
with magnesium sulfate alone or in combination with other salts followed by a clean-up step
using dispersive solid-phase extraction which have been widely employed in the multiresidue
analysis of pesticides and other compounds [19-24]. However, by reducing sample treatment
more matrix related compounds may be introduced into the chromatographic system and
although high resolution and separation efficiency is achieved by UHPLC methods, the
possibility of interfering compounds and matrix effects such as ion suppression or ion
enhancement may increase [25]. Is within this context that the confirmation of the identity
and the presence of suspected target or non-target contaminants in food products are
mandatory to prevent false-positive but, especially, false-negative results because the
presence of a possible harmful compound would be ignored.
Generally, conventional LC-MS/MS methods using two selective reaction monitoring
(SRM) transitions are being proposed in food analysis to comply with EU directive
2002/657/EC [26]. However, these criteria do not completely eliminate the possibility of
reporting false-positive or false-negative results, especially when isobaric species are
analyzed by low resolution MS techniques. In this context, high-resolution mass spectrometry
(HRMS) techniques and accurate mass measurements are emerging as the best options for
multiresidue analysis in food to guarantee identification and confirmation.

Confirmation Strategies in Food Analysis

2. TARGET ANALYSIS IN FOOD


2.1. Liquid Chromatography-Mass Spectrometry
Liquid chromatography coupled to mass spectrometry (LC-MS) and tandem mass
spectrometry (LC-MS/MS) are among the techniques of choice to cope with the increasing
need of analytical methodologies to analyze a large number of target compounds in very
complex food matrices [27, 28]. There are several modern approaches in LC methods which
enable the reduction of the analysis time without compromising resolution and separation
efficiency, being ultrahigh pressure liquid chromatography (UHPLC) methods either using
sub-2m particle packed columns [29, 30] or porous shell columns (with sub-3 m
superficially porous particles) [31, 32] among the most frequently proposed. Generally, LCMS/MS or UHPLC-MS/MS methods using triple quadrupole (QqQ) mass analyzers because
of their high sensitivity in multiple reactions monitoring (MRM) acquisition mode are
proposed for the multiresidue determination of food contaminants. Some examples are the
multiresidue analysis of pesticides [20, 33, 34], veterinary drugs [35, 36], and mycotoxins
[37-39] in food matrices by LC-MS/MS using triple quadrupole instruments. In general, when
working with triple quadrupole instruments, the selectivity of the analysis given by such
analyzers has prevailed over the possibility to give a general overview of the compounds in
the sample due to the limited sensitivity of these instruments when carrying out a full scan
acquisition. Thus, in these systems, collision energies are optimized for two selective reaction
monitoring (SRM) transitions for every compound and both SRM transitions are used for
confirmation analysis to meet the EU Decision 2002/657/EC [26]. The most sensitive SRM
transition is then used for quantitation purposes. In order to achieve confirmation of a given
targeted contaminant in food analysis, the EU Decision 2002/657/ED has established an
identification point system in which at least 3-4 (depending on the contaminant) identification
points are required to fulfill confirmation. In general, 2 identification points are obtained with
each SRM transition when working with LC-MS/MS, and for this reason triple quadrupole
instruments using two SRM transitions are the most frequently used low resolution MS
analyzers in food safety analysis, achieving 4 identification points [26]. However, this is not
enough to guarantee identification and confirmation of the presence of a given contaminant,
and other criteria must be fulfilled. For instance, the ratio of the chromatographic retention
time of a given analyte shall correspond to that of the calibration solution at a tolerance lower
than 2.5%. Regarding both LC-MS/MS SRM the ion-ratio tolerances are also established
depending on the relative ion intensities of both SRM signals. For a relative intensity >50%
of the base peak intensity, a tolerance of 2% is permitted, but as lower is the relative
intensity of a given SRM transition with respect to the base peak, higher is the tolerance
accepted. For instance, for relative intensities 10% of the base peak intensity a tolerance of
50% is accepted. However, the application of these criteria does not completely eliminate
the possibility to report a false-positive or a false-negative result [40]. There are contaminants
with low fragmentation or no fragmentation at all, and in some of these cases two SRM
transitions cannot be monitored. Moreover, because the resolving power of triple quadrupole
mass spectrometers is often insufficient to baseline-resolve isobaric ions [41-43], the presence
of an interfering compound coeluting with the target food contaminant may induce a falsepositive assignment. For instance, Schrmann et al. [41] reported a false-positive LC-MS/MS

lida Alechaga, Paolo Lucci and Oscar Nez

confirmation of the pesticide residue sebuthylazine using the identification point system of
EU directive 2002/657/EC due to the presence of a biogenic insecticide in a tarragon
(Artemisia dranunculus) sample. In this analysis, a coeluting interfering matrix compound
produced product ions in the MS/MS analysis perfectly corresponding to the two SRM
transitions used for monitoring sebuthylazine residues. In this case, using the EU directive
2002/657/EC which regulates the confirmation of suspected positive findings this sample
would have resulted in a false-positive finding. The use of a third LC-MS/MS transition with
a deviant ion ratio and a GC-MS/MS analysis revealed the false-positive results. The authors
were able to separate spiked sebuthylazine from the interfering matrix compound by using an
optimized high resolving ultra-high performance liquid chromatography (UHPLC) method.
Then, using its exact mass and isotope ratios from LC-time-of-flight (TOF) MS
measurements, the compound was identified as nepellitorine, an endogenous alkamide in
tarragon samples.
The possibility of reporting a false negative will be even more problematic because the
presence of a possible harmful compound will be ignored. In all those cases, further
confirmatory analysis by employing alternative strategies must be used to achieve
confirmation of a given food contaminant.
One of the simplest strategies to increase confirmatory information in LC-MS/MS
methods is the use of a third SRM transition, as reported by Schrmann et al. [41], but this is
only possible if the targeted food contaminants have MS/MS spectra with more than two
fragment ions. Additionally, LC-MS/MS methods using ion-trap mass analyzers are also used
as for the analysis of contaminants in food samples [44, 45]. For instance, Cheng et al. [44]
developed an on-line SPE method coupled to LC-ESI-ion trap-MS/MS for the analysis of
abamectin and ivermectin residues, two macrolide antibiotics, in milk. However, although
structural information is obtained because the full MS/MS spectra can be recorded, sensitivity
is lower than the one achieved with QqQ instruments, and in order to comply with EU
Decision 2002/657/ED, generally SRM transitions are also employed. In the above mentioned
method, the qualitative identification of both macrolid antibiotics with the low-resolution LCESI-ion trap-MS/MS method was performed by the LC retention time and the match between
the precursor ions and the relative abundance of two product ions in MRM mode. Therefore,
the identification of abamectin was confirmed by the precursor ion (m/z 895.4) at the
retention time of the extracted ion chromatogram and the presence of the product ion
fragment (m/z 751.3). The identification of ivermectin was confirmed by the precursor ion
(m/z 897.5) and its product ion fragment (m/z 753.4). Lately, the use of QTrap mass
analyzers, hybrid instruments combining a quadrupole and a linear ion-trap in a similar
configuration than a QqQ instrument, is gained popularity for the analysis of food
contaminants [46-49]. For instance, Banerjee et al. [49] proposed a multiresidue method for
the determination of 87 pesticides in mango by LC-ESI-MS/MS at concentration levels below
10 g/kg (see LC-MS/MS chromatogram in Figure 1).
The method involved extraction of 10 g of homogenized mango samples and a dispersive
SPE with primary secondary amine (PSA) and graphitized carbon black (GBC) sorbents.
Direct analysis (without clean-up) resulted in significant suppression in the ionization of
the majority of the pesticides over the electrospray ionization probe. However, the proposed
clean-up with the above combination of PSA+GBC reduced the matrix-induced signal
suppressions significantly, and the signals in the cleaned extracts were nearly equivalent to
the corresponding solvent standards.

Confirmation Strategies in Food Analysis

Figure 1. LC-MS/MS chromatogram of 87 pesticides in mango at 10 g/kg. Reprinted with permission


from reference [49]. Copyright (2009) American Chemical Society.

The advantage of this kind of instrumentation is that similar sensitivity is achieved than
with traditional QqQ instruments while obtaining more confirmatory information because full
MS/MS spectra can be recorded being able to follow more than two SRM transitions, if
necessary. For instance, Banerjee et al. [49] monitored three SRM transitions for 30 of the 87
analyzed pesticides, among them atrazine, buprofezin, diazinon or omethoate, and even four
SRM transitions were monitored for alachlor residues, which improves the confirmation for
these contaminants in the analyzed mango samples.
Obviously, if no fragmentation is obtained for a given food contaminant, the use of iontrap or QTrap analyzers will not be useful to achieve alternative confirmation. Highresolution mass spectrometry (HRMS) and accurate mass measurements are then emerging as
the best options for the analysis of food contaminants in order to guarantee confirmation and
identification (this will be addressed in the next section). However, several alternative
strategies with low resolution MS analyzers have been described in the literature to achieve
further confirmatory analysis. For instance, Esparza et al. [50] developed an UHPLC-ESIMS/MS method using a triple quadrupole instrument for the determination of naphthalenederived compounds, the isomers - and -naphthylacetic acid (NAA), naphthyloxy acetic acid
(NOA) and naphthylacetamide (NAD) which are used as growth regulators, in apples. Two

lida Alechaga, Paolo Lucci and Oscar Nez

SRM transitions were monitored for NAD and NOA, but only one product ion was observed
in the MS/MS spectra of both NAA isomers. However, the authors worked with a TSQ
Quantum Ultra AM (Thermo Fisher Scientific, San Jose, CA) triple quadrupole instrument
equipped with hyperbolic quadrupoles that can operate at relatively high resolution, up to
0.04 FWHM (full width half maximum) and accurate mass (AM) measurements can be
obtained with mass errors down to 5 mDa. For NAA, accurate mass of the product ion m/z
141.0701 was measured in order to get enough identification points to confirm -NAA and NAA isomers according to EU Decision 2002/657/EC [26]. This acquisition strategy is
described as highly-selective selected reaction monitoring (H-SRM) mode. To monitor the
AM and to correct the mass drift, two lock masses were used, m/z 59 for the acetate ion
present in the mobile phase and m/z 295 for the deprotonated ion of 3,3-dichlorobisphenol A
added post-column via the syringe pump integrated in the used QqQ instrument. The use of
this kind of strategy by taking advantage of new developed QqQ instruments able to work in
enhanced mass resolution in both high-selective single ion monitoring (H-SIM) and H-SRM
modes has also been proposed in the literature by other authors in the analysis of food
contaminants [20, 51-53].
The use of library search engines is also an alternative identification and confirmation
system that is becoming very popular in the multiresidue analysis of food contaminants.
Library search engines can also be used with conventional triple quadrupole instruments able
to perform data-acquisition experiments. For instance, Nez et al. [20] proposed an
alternative strategy for the multiresidue analysis of 100 pesticides in fruits and vegetables
using LC-triple quadrupole-MS by performing a data-dependent analysis.

Figure 2. Analysis of an orange sample by data-dependent analysis. Pesticides detected in H-SRM


mode (first scan event) and, as an example, the RER product ion scan spectrum of imazalil (second scan
event). Reprinted with permission from reference [20]. Copyright (2012) Elsevier.

For this purpose, H-SRM acquisition mode, which provides high sensitivity, was selected
as the first scan event. When the H-SRM ions were detected with a signal higher than a
threshold value of 103, a second scan, enabling the acquisition of a product ion MS/MS
spectrum, was activated.

Confirmation Strategies in Food Analysis

This product ion MS/MS spectrum was not obtained with a defined collision energy, but
by using reversed energy ramp (RER) mode and applying collision energies from 10 to
25 eV. The results were then a product ion scan spectrum average of spectra at the different
collision energies, which will better help in confirmation and identification of pesticide
residues. For example, Figure 2 shows the analysis of an orange sample by data-dependent
scan analysis where three pesticides, azoxystrobin, imazalil, and methidathion were detected,
and the RER product ion scan spectrum of imazalil (second scan event). The advantage of this
product ion scan spectrum with RER is that it provides higher structural information than a
simple ion scan spectrum obtained at specific energy. Since it is well known that low collision
energies are too weak to adequately fragment the precursor ions, while at high collision
energies few product ions are generated, the use of RER is more likely to generate fragmentrich MS/MS spectra that will be optimal for library entries and searching purposes.

Figure 3. Results generated by the library search engine with the RER product ion scan spectrum of
imazalil in an orange sample. Reprinted with permission from reference [20]. Copyright (2012)
Elsevier.

The authors then created a product ion scan spectra library and loaded the RER product
ion scan spectra for each pesticide into the Quantum Library of the Xcalibur software
(Thermo Fisher Scientific), and a user library for routine library searching was generated.
Figure 3 shows the results generated by the software library search engine for the
determination of pesticides in an orange sample.

10

lida Alechaga, Paolo Lucci and Oscar Nez

The library compares RER product ion scan spectra of the target sample (in the example
the pesticide imazalil) with those of the user library previously generated, and, as can be seen,
a match (with a probability of 98.61) was found, allowing the confirmation of the presence of
imazalil pesticide in the sample.
This data-dependent analysis combined with library search engines can then be proposed
as a further confirmatory strategy in the analysis of food contaminants.

2.2. Liquid Chromatography-High Resolution Mass Spectrometry


As previously commented, LC-MS/MS using mainly triple quadrupole instruments is
frequently proposed for the target multiresidue determination of food contaminants by
monitoring two SRM transitions. Such a performance helps the analyst comply with the EU
directive 2002/657/EC and confidently report a positive or negative finding [26]. The criteria
to report a positive or a negative result are based mainly on the monitoring of these two
transitions: the deviation of the relative intensity of the recorded product ions (which must not
exceed a certain percentage of the one achieved with a reference standard) and the retention
time of the precursor ion (variation from 1% to 5% with the one of a standard) [54]. However,
as previously addressed with some examples, the application of these criteria does not
completely eliminate the possibility to report a false-positive or a false-negative result [40].
Nowadays, high resolution mass spectrometry (HRMS) and accurate mass measurements
are emerging as the best options for the analysis of food samples in order to guarantee
confirmation and identification of the targeted food contaminant. Basically, there are four
types of HRMS instruments: magnetic sector, time-of-flight (TOF), Orbitrap, and Fourier
transform ion cyclotron resonance (FTICR) instruments. The most frequently used with LC or
UHPLC methods are TOF and Orbitrap analyzers. In general, TOF instruments present a
resolution (instruments ability to measure the mass of two closely related ions precisely) of
approximately 10,000 40,000 FWHM with accuracies in the mass determination of
1-5 ppm, while the resolution of Orbitrap instruments is in the range 10,000 140,000 with
1-2 ppm mass accuracy (for comparison, a conventional quadrupole MS instrument shows a
resolution of 1,000 FWHM and accuracies of 500 ppm) [55].
Recent advances in both LC-TOF-MS and LC-Orbitrap-MS methods have reduced
instrument costs, simplified analysis, and improved accuracy, and today these advances offer
bench-top instrumentation that is amenable to screening and identification of a great variety
of contaminants in food, not only of targeted ones, but also of non-target or unknown
contaminants [56].
Accurate mass measurements improve the probability of a correct analysis when
monitoring m/z ions in an MS spectrum, as the number of possible compounds with a specific
m/z value will decrease as the accuracy is improved (lower ppm errors). Moreover, HRMS
will allow removing interferences from other compounds and their fragment ions, adducts,
and stable isotopic peaks. So, as the mass resolution increases, the more selective the method
will be, at least to remove interferences.

Confirmation Strategies in Food Analysis

11

Table 1. Concentration of benzophenone (BP) found in 28 packaged food samples


analyzed by LC/(LR)-MS/MS. Experimental ion ratio obtained between signal intensity
of quantifier (m/z 105) and qualifier (m/z 77) ions. Reprinted with permission from
reference [57]. Copyright (2011) John Wiley and Sons, Ltd.
Sample
Pineapple juice 1
Pineapple juice 2
Pineapple juice 3
Orange juice 1
Orange juice 2
Orange juice 3
Peach juice 1
Peach juice 2
Sangria
Fruit-milk 1
Fruit-milk 2
Fruit-milk 3
Fruit-milk 4
Fruit-milk 5
Milk 1
Milk 2
Milk 3
Milk 4
Soy-milk 1
Soy-milk 2
Soy-milk 3
Soy-milk 4
Baby food 1
Baby food 2
Baby food 3
Baby food 4
Baby food 5
Baby food 6

BP (g/kg)
2.8
1.1
n.d.
2.3
n.d.
3.2
4.1
n.d.
3.4
3.6
3.1
3.9
3.8
4.7
3.1
4.8
3.2
3.0
n.d.
1.4
n.d.
4.0
8.8
0.7
4.2
0.7
2.9
3.3

Ion ratio (Standard*, Matrix**)


1.29 (C, C)
0.94 (NC, NC)
1.22 (C, NC)
1.03 (NC, NC)
1.33 (C, C)
1.19 (C, NC)
1.70 (NC, C)
1.54 (C, C)
1.58 (NC, C)
1.36 (C, C)
1.61 (NC, C)
1.08 (C, NC)
1.93 (NC, NC)
2.71 (NC, NC)
0.88 (NC, NC)
1.06 (C, NC)
1.67 (NC, C)
1.52 (C, C)
1.44 (C, C)
1.40 (C, C)
1.00 (NC, NC)
1.20 (C, NC)
0.92 (NC, NC)

Ion ratio confirmation range (C - Confirmed; NC - Not Confirmed):


Standard*: 1.04-1.56.
Spiked blank matrix**: Milk-based products 1.27-1.91; Baby food 1.24-1.86; Fruit juice/sangria 1.251.88.

As previously commented, the possibility of reporting false-negative results will be even


more problematic than reporting false-positive results because the presence of a possible
harmful compound would be ignored. In this aspect, HRMS instruments are playing an
important role. An interesting example is the benzophenone case reported by Gallart-Ayala et
al. [57]. Benzophenone (BP) is one of the many contaminants reported as present in
foodstuffs due to its migration from food packaging materials. LC-MS/MS using triple
quadrupole instruments and monitoring two SRM transitions is acknowledged in the literature
as the method of choice for its analysis together with other UV ink photoinitiators [21].
However, cases have been reported where the use of this methodology was insufficient to
unambiguously confirm the presence of a contaminant.

12

lida Alechaga, Paolo Lucci and Oscar Nez

In a previous work reported by Gallart-Ayala et al. [21], LC-MS/MS was applied for the
determination of photoinitiators, BP included, in different food matrices. Although there was
a strong indication of the presence of BP in the analyzed samples, in some of the cases, this
fact could not be completely confirmed due to ion ratio deviations. The authors analyzed 28
packaged food samples using a triple quadrupole instrument and following the EU Decision
2002/657/EC [26], two SRM transitions (m/z 183 105 and m/z 183 77) were monitored.
The ion recorded as m/z 105 was selected as the quantifier (Q) ion, while the ion m/z 77 was
selected as the qualifier (q). The ion ration Q/q was monitored as a confirmatory parameter of
the analysis and a deviation range of 20% was estimated in neat standard and matrix. The
results obtained (Table 1) show an ion ratio deviation higher than 20% for half of the
analyzed samples indicating the presence of BP. However, these samples could not be
confirmed due to this deviation, being a case of a false-negative result. As previously
addressed in section 2.1, to overcome this problem the monitoring of a third transition is
recommended [26]. However, this strategy could not be followed with BP because its
fragmentation pattern only reveals two product ions, m/z 105 and 77.
Since the occurrence of false negative results is normally attributed to the presence of
interfering compounds co-eluting with the analyte of interest, the authors investigated this
analytical problem using HRMS with an Orbitrap analyzer. Figure 4 shows the LC-HRMS
analysis of a baby food sample at three different resolutions. As can be seen, when a
resolution of 50,000 FWHM was used, BP could be detected with a mass error of 1.1 ppm
but, in addition, another compound with an assigned elemental composition of C12H11N2
(lately confirmed by the authors as Harman) was detected with a mass error of 0.5 ppm.
Harman is recognized as being present in foodstuffs at concentrations ranging from 1 to 200
g/kg [58].
Product ion scan experiments of Harman showed an ion at m/z 77 at low relative
abundance, confirming the authors suspicion that the concentration of Harman found in the
analyzed samples was sufficient to interfere with the confirmatory SRM transition of BP, m/z
183 77, preventing confirmation of BP in those samples and reporting false-negative
results. Thus, a direct comparison between LC-HRMS and LC-MS/MS data indicated better
selectivity when working with LC-HRMS at a resolution of 50,000 FWHM than when
monitoring two SRM transitions by LC-MS/MS.
So, today LC-HRMS either using TOF [59-62] or Orbitrap [63-69] mass analyzers are
frequently employed for the targeted determination of food contaminants such as pesticides,
veterinary drugs, mycotoxins among others in a great variety of food commodities. For
instance, Aguilera-Luiz et al. [65] proposed a fast and non-laborious method using turbulent
flow chromatography coupled to ultrahigh-performance liquid chromatography-Orbitrap mass
spectrometry (TFC-UHPLC-Orbitrap-MS) for the determination of veterinary drug residues
in honey samples. Figure 5 shows, as an example, some veterinary drug residues detected in a
spiked honey sample with the proposed method. Honey samples were only diluted with an
aqueous solution of Na2EDTA (0.1 M) and then they were injected into the TFG-UHPLCOrbitrap-MS system.
Mean recoveries ranging from 68 to 121% for most compounds and repeatability
(intraday precision) and interday precision (expressed as relative standard deviation RSD
values) lower than 25% were obtained. The proposed method showed limits of quantitation
(LOQ) values in the range 0.1 to 50 g/kg.

Figure 4. Baby food 1 (Table 1) sample analyzed by LC-HRMS at three different mass resolving powers: (A) 10,000 FWHM, (B) 25,000 FWHM, and (C)
50,000 FWHM. Reprinted with permission from reference [57]. Copyright (2011) John Wiley & Sons, Ltd.

14

lida Alechaga, Paolo Lucci and Oscar Nez

Figure 5. Chromatograms of some veterinary drugs detected in a honey sample spiked at 25 g/kg
analyzed by TFC-UHPLC-Orbitrap-MS. Reprinted with permission from reference [65]. Copyright
(2013) American Chemical Society.

Accurate mass measurements improve the probability of a correct analysis when


monitoring m/z ions in an MS spectrum, as the number of possible compounds with a specific
m/z value will decrease as the accuracy is improved (smaller ppm errors). Moreover, HRMS
will allow removing interferences from other compounds and their fragments ions, adducts,
and stable isotopic peaks. So, as the mass resolution increases, the more selective the method
will be, at least to remove interferences, as previously commented. However, it should be
pointed out that accurate mass measurements allow us to only determine the molecular

Confirmation Strategies in Food Analysis

15

formula for a target food contaminant, and a unique molecular formula separated from every
possible interference is not a unique compound [55].
Several compounds may have the same formula than the targeted food contaminant, even
those that are not structural isomers. Thus, alternative information such as fragmentation
spectra and chromatographic separation are needed to guarantee unambiguous identification.
Once the molecular formula for a targeted compound is reached, additional resolving
power and mass accuracy will be irrelevant. The studies performed by Thurmans research
group in the analysis of pesticides in foodstuffs [70, 71] have shown that for many vegetable
matrices a resolution between 6,000 and 10,000 FWHM is enough to remove interferences
based on the complexity of the food samples. This resolution is easily attainable by TOF and
Orbitrap MS instruments available today.
For example, Wang et al. [66] proposed a UHPLC-ESI-Q-Orbitrap MS method for the
targeted determination of 451 pesticide residues in fruits and vegetables. Pesticides were
extracted from samples using the QuEChERS procedure. UHPLC-ESI Q-Orbitrap MS in full
MS scan mode was employed to acquire full MS data for quantitation purposes, and Full
MS/dd-MS2 (i.e., data-dependent scan mode) obtaining product ion spectra were used for
identification purposes. For instance, Figure 6 shows the UHPLC-ESI-Q-Orbitrap Full
MS/dd-MS2 chromatograms and product ion spectra of secbumeton and prometon pesticide
residues. Thus two isomeric pesticide residues coeluted, secbumeton at 6.52 min and
prometon at 6.53 min (Figures 6A1 and 6A2), but possessed different fragment ions at m/z
170.10349 and 184.11919, respectively (Figure 6, panels C1 and D1 and C2 and D2,
respectively). Therefore, although accurate mass measurements were employed with the
Orbitrap MS analyzer, alternative information such as dd-MS2 product ion spectra was
required to differentiate between secbumeton and prometon residues. Among the 451 targeted
pesticides in this method there were 22 pairs or groups of isomers and among them only 5
pairs were not chromatographically resolved, and dd-MS2 product ion spectra were necessary
for unequivocal identification when these pesticide residues were detected in the full MS
mode.
Overall, the proposed UHPLC-ESI-Q-Orbitrap MS method demonstrated acceptable
performance for the quantification of pesticide residues in fruits and vegetables. This method
along with library matching showed great potential for the identification of pesticide residues
in complex food samples.
The necessity of performing fast sample screening of hundreds of food contaminants in a
great variety of food matrices by LC-HRMS with accurate mass measurements requires
databases of exact masses (and even of exact mass fragment ions) in order to rapidly look for
these substances via appropriate data software available with the HRMS instruments [72].
Moreover, one of the most important advantages of using HRMS in full-scan MS
acquisition mode (but also in combination with additional fragmentation information) is the
possibility of a post-processing analysis of previously analyzed samples if new target food
contaminants are intended to be determined. Thus, today the confirmation strategy of creating
accurate mass databases of food contaminants is fundamental in this type of analysis [55, 62,
65, 73-78]. For instance, Gmez-Prez et al. [76] created a database for the simultaneous
analysis of more than 350 pesticides and veterinary drugs (including antibiotics) in honey
samples using UHPLC-Orbitrap-MS. The customized database included exact masses of the
target ions and retention time data, and the probable adduct ions, and allowed the automatic
search of the targeted pesticides and veterinary drugs.

Figure 6. UHPLC-ESI Q-Orbitrap Full MS/dd-MS2 chromatograms and product ion spectra. Individual secbumeton or prometon (50 g/L in solvent buffer) was
injected and analyzed: (left) secbumeton; (right) prometon; (A1, A2) extracted ion chromatograms of m/z 226.16624 with a mass tolerance of 5 ppm; (B1, B2)
dd-MS2 chromatograms of m/z 226.16624; (C1, C2) dd-MS2 product ion spectra of m/z 226.16624 at 6.43 and 6.46 min, respectively; (D1, D2) dd-MS2 product
ion spectra of m/z 226.16624 at 6.61 and 6.63 min, respectively. Reprinted with permission from reference [66]. Copyright (2014) American Chemical Society.

Confirmation Strategies in Food Analysis

17

The authors used the database for screening analysis, but it was also evaluated for
quantitative purposes in routine analysis after a generic extraction procedure.
Turnipseed et al. [78] developed a LC-Q-TOF method to determine veterinary drug
residues in frog legs and other aquacultured species. Figure 7 shows the overlaid extracted ion
chromatograms for [M+H]+ ions of targeted veterinary drugs in frog legs fortified at the levels
of interest.

Figure 7. Overlaid extracted ion chromatograms for (A) [M + H]+ ions of target compounds in frog legs
fortified at the levels of interest (TMP, 10 ng/g; CIP, 5 ng/g; ENR, 5 ng/g; SMOZ, 10 ng/g; OXO, 10
ng/g; NAL, 10 ng/g; FLU, 10 ng/g), (B) [M + H]+ ions of target compounds in control frog legs, and
(C) [M H] ions of CAP in frog legs fortified at 1 ng/g. The mass window for extraction was set to 10
ppm and CIP, SMOZ, OXO, FLU, and CAP were not detected in the control frog legs. TMP,
trimethoprim; CIP, ciprofloxacin; ENR, enrofloxacin; OXO, oxolinic acid; NAL, nalidixic acid; FLU,
flumequine; and SMOZ, sulfamethoxazole. Reprinted with permission from reference [78]. Copyright
(2014) American Chemical Society.

Screening of samples was accomplished by collecting TOF (MS1) data and comparing the
accurate mass and retention times of compounds to a database containing information of
veterinary drugs. An evaluation of the MS data in fortified frog legs indicated that the target
compounds could be consistently detected at the levels of concern. MS/MS data were also
generated from precursor ions, and the mass accuracy of the product ions for each compound
was compared with theoretical values. The authors also evaluated the data from these samples
for non-target analytes such as residues of metabolites and other chemotherapeutics.
However, the non-target analysis of food contaminants will be addressed in the next sections.
Recently, Ser et al. [73] developed a desorption electrospray ionization-high resolution
mass spectrometry (DESI-HRMS) method for the screening of veterinary drugs in crosscontaminated feedstuffs. The reliable detection was performed working at high resolution

18

lida Alechaga, Paolo Lucci and Oscar Nez

(70,000 FWHM) using an Orbitrap mass analyzer. To evaluate the applicability of the
developed DESI-HRMS method, 50 feed samples were analyzed in order to detect those
samples suspected of being cross-contaminated by veterinary drugs.
Feed samples were screened and the acquired mass spectral raw data were interrogated
by a custom-made database that included more than 60 veterinary drugs (anthelmintics,
antibiotics, coccidiostats, hormones, etc.) commonly used to produce medicated feedstuffs.
For each substance, the compound name, the CAS number, the elemental composition,
and the chemical structure were included. The ionization mode and the expected ions
(protonated and deprotonated molecules, adduct ions, in-source fragments, etc.) that can be
generated in the DESI source were also included by the authors to the custom-made database.
The sample raw data files were processed using the Exact Finder software (Thermo
Fisher Scientific) and interrogated by the custom-made database to automatically identify the
veterinary drugs in the feedstuffs. The criteria applied by the authors to confirm the presence
of suspected compounds were the following: a mass accuracy of less than 5 ppm on the exact
mass, a minimum signal-to-noise ratio of 3:1, and an isotope cluster fit higher than 80% (both
mass relative deviation and relative intensity differences, for each isotope peak within the
cluster ion, were taken into account). Veterinary drugs at dose levels between 37 and 107
mg/kg in medicated feed were easily detected and confirmed by the proposed DESI-HRMS
screening method, and in one sample an unexpected cross-contamination of monensin (3.5
mg/kg) was detected. Additionally, the authors reported that the results obtained for nonmedicated feed indicated that cross-contamination occurs quite frequently and values above
the legislated levels were detected in 28% of the samples analyzed by DESI-HRMS.
It should be mention that the match probabilities of these databases is based on the exact
mass being measured correctly and compared against the one in the database, and this will
depend on the stability and accuracy of the mass calibration of the HRMS instrument. For
more accurate mass measurements, TOF and Q-TOF instruments frequently used a lock mass
correction, which consists of the infusion of a reference compound, while Orbitrap usually
has higher accurate mass stability and lock masses can be omitted. Moreover, some analyst
skill in MS is required when using databases in order to use the M+1 and M+2 isotopic
information to achieve a correct formula, and to lower the probability of error as much as
possible to low ppm values [55].
Summarizing, these accurate mass databases together with the use of library search
engines are very useful alternative confirmatory strategies for target food contaminants
analyses, as it has been described in this section. However, these strategies are also employed,
and are almost a requirement, when non-target analysis of food contaminants is intended,
topic that will be addressed in the next sections.

3. NON-TARGET ANALYSIS IN FOOD


3.1. Liquid Chromatography-Mass Spectrometry
In recent years, there has been growing movement to analyze food beyond target
compounds, and a shift towards non-targeted or unknown screening approach. This is mainly
due to the fact that non-targeted analysis provides greater scope than a targeted approach. For

Confirmation Strategies in Food Analysis

19

example, it is possible to extend monitoring to certain metabolites or other transformation


products, for which reference standards might not be available, or to cover old or
unauthorized substances no longer in use as well as very recent compounds not yet included
into current monitoring plans [79]. Unfortunately, whilst all LC-MS instruments can perform
full spectral acquisition, not all analyzers can provide sufficient sensitivity in this mode for
non-target analysis. As a result, non target analysis is commonly based on the use of HRMS,
such as TOF or Orbitrap, that can give the advantages of using the full-scan acquisition mode
with high sensitivity, high resolving power (>30,000 FWHM) and accurate mass
measurements (1-5 ppm) [80].
So far only a few examples have been described in the literature dealing with the use of
low resolution MS in non-target analysis. For instance, the identification of lipid molecular
species with low resolution instrumentation can be achieved by MSn analysis (using both
linear ion trap and triple quadrupole technology), because this kind of analysis may provide a
high degree of structural information. On the other hand, a good compromise between
targeted and non-targeted analysis on a triple quadrupole instrument operating in low
resolution mode are precursor ion and constant neutral loss scans. Although sensitivity for
such scans might not be as high as in MRM mode, it opens the possibility to find unexpected
species within the lipid classes surveyed by the respective precursor or constant neutral loss
scans [81]. Such methodologies are quite often used with ESI and reversed phase HPLC
coupling. An interesting example of this approach has been reported by Retra et al. [82]. In
this study, neutral loss of 141 amu and 87 amu have been used for identifying
glycerophosphoethanolamine and glycerophosphoserine molecular species. The use of
constant neutral loss scans have also been later used by Clark et al. [83] for
phosphatidylinositol phosphate identification and quantitation. In a previous study, the
neutral loss of 35 amu i.e., the loss of ammonia plus water was used for the selective
detection of tetracycline antibiotics (TCAs). In the same way, for sulfonamides analysis, a
characteristic m/z 156 ion representing the sulfanilyl fragment has been employed for the nontargeted detection of compounds within this class [84].
In another example, Tao et al. [85] proposed the use of constant neutral loss with
reversed-phase LC-MS/MS for the identification and quantification of gingerols and related
compounds in Ginger dietary supplements. Figure 8 shows the negative ion ESI LC-MS/MS
chromatograms of a chloroform extract of ginger roots/rhizomes using constant neutral loss
analysis. The cleavage of the C4-C5 bond with a neutral loss of 194 uma and benzylic
cleavage leading to to the neutral loss of 136 uma were found to be class-characteristic
fragmentation patterns of the pharmacologically active gingerols or shogaols, respectively.
On the basis of these results, an assay using LC-MS/MS with neutral loss scanning (loss of
194 and 136 uma) was developed that was suitable for the fingerprinting of ginger dietary
supplements bases on the selective detection of gingerols, shogaols, paradols, and
gingerdiones.
In addition, a quantitative method based on LC-MS/MS with SRM was developed by the
authors for the quantitative analysis of 6-gingerol, 8-gingerol, 10-gingerol, 6-shogaol, 8shogaol, and 10-shogaol in ginger dietary supplements, showing excellent sensitivity,
accuracy and precision.

20

lida Alechaga, Paolo Lucci and Oscar Nez

3.2. Liquid Chromatography-High Resolution Mass Spectrometry


HRMS has been proven a powerful tool for the identification of analytes. Thus, many
applications can be found in the literature for the screening of "suspects" (without having
reference standards, but a priori information about the compounds to be detected is provided)
and also for the identification of "unknowns" (signals for which no information is available)
[86]. Another interesting feature is the possibility to perform retrospective (also called posttarget) analysis, that is, the screening for additional compounds that had not been considered
when the analysis of the sample took place [87]. A general workflow of a LC-HRMS
screening method for target, suspect and unknown (non-target) analysis can be found in
Figure 9. As it can be seen, the complexity of the analysis workflow increases as the
information about the analyte is reduced. In all cases, the first step is the extraction of a list of
signals (peak picking) by means of an ion list (suspect) or by supplementary criteria (nontarget), followed by an elemental composition match. Additional search for structure
candidates is needed in the case of unknown analysis. The proposed structure for the suspect
and non-target screening is then checked using tandem mass spectral information, to obtain
good confidence in the identification. And the last step, which is not shown in Figure 9, is the
confirmation of the identification by the analysis of a standard and matching both the
retention time and the fragmentation spectra with that of the sample.
Generally, non-target analytical methods rely on the use of acquisition modes combining
a non-directed full scan step, and a scan performing a fragmentation step in order to obtain
additional spectral information. The objective of the full scan step is to detect all ionizable
substances and to provide sufficient information for the elucidation of the elemental
composition of the ionized substance, often based on the accurate mass of the ion and the
observed isotope pattern. To this end, it is important to obtain high quality data, with good
precision and accuracy [88], and to perform the acquisition at a resolution that reduces the
possibility of interferences due to isobaric ions. When two adjacent masses differ by less than
twice de FWHM in mass, merging of both signals occurs, and as a result the accurate mass of
the signal is distorted. For this reason, the resolution at which the full scan is performed
should be adjusted accordingly depending on the complexity of the matrix, recommending
working at resolutions above 50,000 FWHM in worse-case scenarios [56, 89]. The product
ion scan is generally used to provide additional information that can help with the
identification of the compounds or for confirmation purposes. This fragmentation scan can be
performed at lower resolution than that of the full scan to obtain a faster duty cycle.
In regard to the fragmentation step, two main approaches have been described, datadependent (DDA) and data-independent (DIA) acquisitions. In the first one, also addressed as
information-dependent acquisition (IDA), the most abundant ions in the full scan at a specific
time are successively selected as precursors for product ion scan. Only precursor ions above a
specific intensity threshold are selected to avoid experiments in background noise ions.
Moreover, "dynamic exclusion, which is a temporary exclusion of an already selected
precursor ion from further DDA experiments, is usually set, to avoid repeated experiments
and to perform product ion scans on less intense precursor ions instead [90]. The main
drawback of the DDA approach is that only some of the accessible ions from the full scan are
selected, and as a consequence, false-negative results can occur, either because the signal
intensity of the precursor ion is below the established threshold, or due to the increased duty
cycle time as a result of the multiple scan events. Another flaw of DDA methods is the

Confirmation Strategies in Food Analysis

21

increased spectral variability of the product ion scan, due to the fact that only one or two
scans are performed, and that said scans are not acquired at the apex of the chromatographic
peak, which corresponds to the highest intensity of the precursor ion. In the DIA approach,
data acquisition is independent from prior information from the full scan, and the
fragmentation is performed either in the full m/z range (all-ion fragmentation -AIF-, MSE or
MSALL) or by dividing the full m/z range into smaller isolation windows that are
consecutively selected for fragmentation. This second option, named "Sequential Windowed
Acquisition of All Theoretical Fragment Ion Mass Spectra" (SWATH), has been mainly used
in proteomics, although some works applying SWATH for small molecules in biological
samples can also be found in the literature [91, 92]. On the one hand, DIA methods allow the
obtention of fragmentation information of all precursor ions available, dwindling the
possibility for false-negative results. Additionally, fragmentation spectra are acquired
throughout the whole chromatographic peak, resulting in lower variability and increased
signal-to-noise ratios for the product ion scan spectra. On the other hand, as many precursor
ions are selected, the tandem MS spectra obtained might not be selective enough to ensure the
identification of the analyzed species [93], especially if there are a high number of coeluting
compounds due to the analysis of complex matrices and/or the use of fast chromatographic
methods. SWATH acquisition strategy improves purity of the fragmentation spectra by using
relatively narrow isolation windows, but still data deconvolution softwares are needed to
ensure correct interpretation of the fragmentation spectra [94, 95].
As a result of the combined acquisition mode, a massive amount of data is obtained, and
some type of reduction process is needed to make the number of signals to be identified
manageable. Peak picking is the most common strategy, which can be based on the
comparison of the sample with control or blank samples [96], or even selecting the most
discriminant signals by principal component analysis [97]. Also, more specific criteria can be
used to select the most relevant signals to be analyzed. For instance, chlorine-containing
organic compounds can be easily distinguished by their particular isotopic signature, and their
presence in honey samples is assumed to be, or to come from, human produced xenobiotics
[98]. Another option, usually performed in suspect analysis, is to interrogate full scan data
with commercial or in-house databases containing structural information of the analytes of
interest [37, 67, 99, 100]. Mass defect filtering is commonly performed when screening for
substances related to a specific compound, such as metabolites or transformation products
[101]. It is based on the fact that the difference between the exact mass and the nominal
(integer) mass of a compound usually falls within a narrow window from a related parent
compound.
Next step is to assign the elemental composition to each of the detected signals.
Theoretically, by means of the accurate mass it is possible to identify the exact elemental
composition of an ion, provided that its mass is determined with sufficient accuracy [102].
However, if non priori information of the compound is available, several considerations must
be taken into account. For instance, for an ion of nominal m/z of 118, only one candidate can
be found within a mass accuracy tolerance of 34 ppm, whereas for an ion of m/z 750 the
tolerance window for only one possible candidate is reduced to 0.018 ppm [103]. However,
this statement is true if only the elements C, H, N and O are considered, and when additional
elements commonly present in organic molecules such as S, F or P are included the number
of possible elemental compositions within a fixed tolerance level increases exponentially
[104]. Kind and Fiehn proposed some years ago the "seven golden rule, which allows an

22

lida Alechaga, Paolo Lucci and Oscar Nez

automatic exclusion of molecular formulas that are either wrong or that contain unlikely high
or low number of elements [105]. Briefly, these rules are as follows:
1. restriction of the maximum number of atoms of each element, according to the total
molecular weight
2. compliance with LEWIS and SENIOR valence rules
3. match of the isotope pattern corresponding to the elemental composition
4. restriction of the H/C ratio between 0.2 and 3.1
5. restriction of the heteroatom/C ratios
6. for heteroatom combinations with H, N, O, P and S, multiple element count
restriction
7. compliance with trimethylsilylation (only in GC-MS if derivatization of the analytes
is performed)
Several of these rules are already implemented in most commercial softwares for
elemental composition elucidation. Thus, generally the most common practice is to check that
the identification of the elemental composition complies with a mass error tolerance of 5 ppm
and provides a good match for the isotope pattern. Some authors recommend to set mass error
tolerance values according to the experimental mass accuracy of the method (i.e., 1.96m/z
or 2.58m/z, corresponding to a 95% and 99% confidence value, respectively) in order to
minimize both false-positive and false-negative results [106], but the 5 ppm criteria is the
most widespread.
As for the isotope pattern comparison, it can be done visually although most softwares
provided by the supplier of the MS instrument used include algorithms for isotope pattern
comparison. However, it is important to perform the comparison with a theoretical isotope
pattern generated using the same resolution than for data acquisition, as merging of low
intensity isotopic peaks can occur.
In order to assign a structure to the detected elemental compositions, a list of candidates
needs to be constructed. To this end, interrogation of chemical databases, such as ChemSpider
(www.chemspider.com/), KEGG (http://www.genome.jp/kegg/) or PubChem (https://pub
chem.ncbi.nlm.nih.gov/), with the elemental compositions can be performed. In this case,
fragmentation data can be used to confirm the structure identification [98].
Fragmentation information can also be used to generate a candidate list or to rank the
obtained candidates. In metabolomics there are five basic approaches to use tandem MS data
for the identification of a compound (Figure 10) [107]: (a) search in spectral libraries; (b) insilico fragmentation spectrum prediction; (c) mapping of the fragmentation spectrum to the
compound structure (combinatorial fragmentation); (d) prediction of structural features and
compound classes; and, (e) use of fragmentation trees.
Some of these strategies can be applied to the identification of any compound in food
matrices as well. For instance, tandem mass spectra can be searched in free spectral libraries
such as NIST (http://chemdata.nist.gov/), HMDB (www.hmdb.ca/), mzcloud (http://www.mz
cloud.org/), MetLin (http://metlin.scripps.edu/index.php) or Massbank (http://massbank.ufz.
de/MassBank/) [37, 99, 108, 109], and the information from the parent compound (e.g.,
molecular formula, substructures for metabolite or transformation products) can be used to
restrict the search.

Figure 8. Negative ion electrospray LC-MS/MS chromatograms of a chloroform extract of ginger roots/rhizomes using constant neutral loss analysis of (A) 194
uma showing the detection of gingerols and (B) 136 uma showing the detection of shogaols, gingerdiones, hydroxyshogaols, dehydrogingerols, and paradols.
Reprinted with permission from reference [85]. Copyright (2009) American Chemical Society.

Figure 9. Comparison of systematic workflows for (i) quantitative target analysis with reference standards, (ii) suspects screening without reference standards,
and (iii) non-target screening of unknowns in environmental samples by using LChigh resolution (tandem) mass spectrometry. Reprinted with permission from
reference [87]. Copyright (2010) Springer.

Figure 10. The five basic approaches of dealing with metabolite fragmentation data: (a) searching spectral libraries; (b) fragmentation spectrum prediction; (c)
combinatorial fragmentation; (d) predicting structural features; and, (e) fragmentation trees. Reprinted with permission from reference [107]. Copyright (2014)
Elsevier.

26

lida Alechaga, Paolo Lucci and Oscar Nez

Mzcloud also offers the possibility to compare spectral trees. However, it must be taken
into account that recorded fragmentation spectra in LC-HRMS are instrument-dependent, and
although mass spectral libraries are constructed using different ionization sources and
analyzers, and working at different collision energies, high probability matches should not be
always expected. Additionally, tandem MS information of the candidate structures from the
literature can be compared to that of the sample in order to identify the compounds [96], but
this strategy is only achievable if the candidate list is short enough. Fragmenter softwares
have been used to obtain simulated tandem mass spectra of a candidate structure to be
compared with the experimental mass spectra [97]. These softwares use general
fragmentation rules, literature information and/or combinatorial approaches based on bond
energies to perform in-silico fragmentation of given structures. Some examples of fragmenter
softwares are MOLGEN-MS (http://www.molgen.de/), ACD/MS Fragmenter (www.acdlabs.
com/products/adh/ms/ms_frag), MassFrontier (www.highchem.com/index.php/massfrontier)
and MetFrag (http://msbi.ipb-halle.de/MetFrag/). MetFusion (http://msbi.ipb-halle.de/Met
Fusion/) is an improvement from MetFrag software that combines in-silico fragmentation
with spectral database searching, and MassFrontier also offers the possibility to construct
custom made spectral trees for comparison with the samples, which has been used to identify
illegal adulterants in health foods [110].

CONCLUSION
Reliable methodologies are crucial for both industrial and enforcement testing in
compliance with the legislation. It is necessary to assess the concentration levels of
contaminants in food to evaluate the level of exposure according to the diet. For these
reasons, there is an increasing need for applications able to cope with the determination of a
large number of analytes in very complex matrices. Laboratories are interested in costeffective methodologies with reduced analysis times. The complexity of food matrices often
requires not only extensive sample preparations, but also on-line coupling techniques, which
are used for their superior automation and high-throughput capabilities. However, by
reducing the sample treatment more matrix related compounds may be introduced into the
chromatographic system and although high resolution and separation efficiency is achieved,
the possibility of interfering compounds and matrix effects such as ion suppression or ion
enhancement may increase. In this context, confirmation of the identity and the presence of
suspected target or non-target compounds in food products are mandatory to prevent falsepositive and, especially, false-negative results because the presence of a possible harmful
compound would be ignored.
The present chapter has reviewed confirmation strategies in liquid chromatography-mass
spectrometry and liquid chromatography-high resolution mass spectrometry for target and
non-target analysis. Typically, conventional LC-MS/MS methods in food target analysis
(generally using triple quadrupole mass analyzers) are proposed for multiresidue approaches
by using two selective reaction monitoring (SRM) transitions to comply with EU directive
2002/657/EC. However, the application of this criterion does not completely eliminate the
possibility to report a false-positive or a false-negative result due to the low resolving power
of conventional MS analyzers (triple quadrupole, ion-trap) to base-line resolve isobaric ions.

Confirmation Strategies in Food Analysis

27

So today, alternative confirmation strategies such as the use of additional SRM transitions,
additional fragmentation information, or the use of product ion scan libraries are being
employed in food analysis for the LC-MS(/MS) target analysis. In this context, highresolution mass spectrometry (HRMS) and accurate mass measurements (for instance by
using accurate mass databases) are emerging as the best options for multiresidue analysis in
food to guarantee confirmation and identification. Relevant examples addressing these points
have been described.
Regarding non-target analysis, low resolution LC-MS/MS can also be employed by
working in full scan acquisition modes. Although sensitivity for such scans might not be as
high as in MRM mode, it opens the possibility to find unexpected non-targeted species by
performing precursor or constant neutral loss scans. Some examples have been described in
the present work. Obviously, HRMS is the ideal tool for non-target analysis of food
contaminants. Generally, non-target analytical methods rely on the use of acquisition modes
combining a non-directed full scan step, and a scan performing a fragmentation step in order
to obtain additional spectral information. The objective of the full scan step is to detect all
ionizable substances and to provide sufficient information for the elucidation of the elemental
composition of the ionized substance, often based on the accurate mass of the ion and the
observed isotope pattern. To this end, it is important to obtain high quality data, with good
precision and accuracy and to perform the acquisition at a resolution that reduces the
possibility of interferences due to isobaric ions. Regarding the fragmentation step, examples
of the two main approaches described in the literature, data-dependent (DDA) and dataindependent (DIA) acquisitions have been described in the present chapter. Finally, strategies
to assign a structure to the non-targeted detected elemental compositions have been
addressed.

ACKNOWLEDGMENTS
This work has been funded by the Spanish Ministry of Economy and Competitiveness
under the project CTQ2012-30836, and from the Agency for Administration of University
and Research Grants (Generalitat de Catalunya, Spain) under the project 2014 SGR-539.

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In: Advances in Food Analysis Research


Editor: Anita Haynes

ISBN: 978-1-63483-783-5
2015 Nova Science Publishers, Inc.

Chapter 2

NOVEL INSIGHTS IN THE METHODS FOR MEASURING


THE ANTIOXIDANT CAPACITY OF FOODS
Sergio Prez-Burillo1, Silvia Molino2, Cristina Delgado-Andrade3
and Jos A. Rufin-Henares1,*
1

Departamento de Nutricin y Bromatologa, Facultad de Farmacia,


Universidad de Granada, Spain
2
Dipartimento di Biologia e Biotecnologie L. Spallanzani, Pavia, Italy
3
Departamento de Fisiologa y Biqumica de la Nutricin Animal (INAM),
Estacin Experimental del Zaidn (CSIC), Armilla, Granada, Spain

ABSTRACT
Antioxidant activity is well-known to have a great deal of beneficial impacts on
health due to the protection against oxidative damage. However, not only is important
because of the health benefits but also because of the suitability of antioxidant activity in
the food technology industry as a tool to prevent food oxidation and prolong shelf-life.
There are many different methods to measure antioxidant activity of foods with
several classifications. One of them distinguishes between the assays focused on lipid
peroxidations (for instance thiobarbituric acid assay (TBA) or beta-carotene bleaching
assay) and the assays which draw its attention to the scavenger activity against free
radicals (i.e., ABTS assay or the ferric reducing antioxidant power (FRAP) assay);
whereas others take into account the transfer of either hydrogen atoms or electrons. The
formers are mostly based on the competition of antioxidants for peroxyl radicals like the
ORAC assay (oxygen radical absorbance capacity) or TRAP assay (total radical trapping
antioxidant parameter). Among the electron-transfer methods we have the FolinCioucalteu method or DPPH method.
Moreover, before measuring the antioxidant activity a correct extraction of the
antioxidant compounds from the food matrix is needed. This extraction can be done
either chemically or at physiological conditions.
*

Corresponding author: Prof. Jos A. Rufin-Henares; Departamento de Nutricin y Bromatologa, Facultad de


Farmacia, Campus Cartuja S/N; Universidad de Granada; 18071, Granada; Spain; Tel: +34-958-241000 Ext:
20463; Fax: +34.958-249577; e-mail: jarufian@ugr.es.

38

Sergio Prez-Burillo, Silvia Molino, Cristina Delgado-Andrade et al.


The former is based on the extraction of antioxidant compounds with organic or
acidic solvents. The physiological extraction, however, is basically an in vitro
gastrointestinal digestion composed by an oral, gastric and intestinal phase. In some
cases, extraction is not performed and antioxidant activity is directly measured.

INTRODUCTION
An antioxidant is a molecule capable of slowing or reducing the oxidation of other
compounds. This is because antioxidants are oxidized instead of the compound they are
protecting. The damage caused by that oxidation is called oxidative damage and is due to the
action of free radicals which are chemical species that have unpaired valence electrons. For
this reason, they are highly reactive against other molecules or even against themselves. Free
radical production can happen exogenously through smoking, ionizing radiation, certain
pollutants, organic solvents and pesticides, although free radicals are also physiologically
produced (Berker et al., 2010). Hence, mitochondria which consume more than 90% of the
oxygen are the main source of free radicals (Lee J. et al., 2004). Due to their ability to oxidize
biological molecules such as proteins, fatty acids or nucleic acids, free radicals have an
important role on health. Accordingly, they have been related with diseases whose progress or
development occur in oxidative stress conditions, most of them chronic disorders such as
cancer, liver disease, aging, diabetes, AIDS, Alzheimer's disease, Parkinson's disease,
arthritis, inflammation and atherosclerosis among others (Moon, Joon-kwan and Shibamoto,
Takayuki, 2009). Because of this, great attention has been paid to antioxidant activity since
90s when such capacity was firstly related to health (Vitaly Roginsky and Eduardo A. Lissi,
2004).
Consequently, many different methods have been developed through the years in order to
measure antioxidant power of food stuff though at the beginning just vegetables focused most
the attention due to their higher content in compounds such as polyphenols or carotenoids.
However, currently it is known that any kind of food could exert antioxidant power due to the
fact that any food has proteins, carbohydrates or lipids which are digested releasing smaller
molecules, some of them exerting antioxidant activity (Pastoriza et al., 2011).
A key factor to properly measure the antioxidant activity is to perform the correct
extraction procedure in order to make the antioxidant compounds accessible from the food
matrix. At this point many different procedures are described in the scientific literature, being
most of them chemical extractions which consist on the contact between the sample and
different solvents to drag antioxidant molecules. Less employed, due to the tedious process,
but very valuable is the physiological extraction which consists on an in vitro digestion with
an oral, gastric and intestinal fluids and enzymes. Obviously, physiological extraction has
more resemblance to what really happens when food are ingested and molecules are released
inside our body. Finally, it must be also mentioned that some authors, depending on the food
matrix, choose not to perform the extraction step and work directly with the solid matrix
(Pastoriza et al., 2011). Once the extraction is conducted the antioxidant activity can be
measured by many different methods depending on the particular interest (measuring lipid
peroxidations or scavenger activity, reduction capacity, etc.).

Table 1. Advantages and disadvantages of the different extraction methods


Method

Conventional extraction

Advantages

-very easy to carry out


-good extraction
-widely used
-cheap

Disadvantages

-long extraction time


-artificial far from reality
-long time under heat may
denature antioxidant
compounds (soxhlet)
-molecules extracted depend
on the kind of solvent used
-toxic solvents
-risk for the environment

Ultrasound-assisted
extraction (UAE)
-cheap reactants
-cheap apparatus and easy to
handle
-high yield of extraction
-strong correlation between
samples extracted by UAE
-green process to obtain
extracts rich in antioxidant
compounds
-effective for the extraction
of phenolic compounds
-faster than classical
methods such as soxhlet
-can be useful for extracting
thermolabile compounds
-artificial far from reality
-long time under heat may
denature antioxidant
compounds
-long sonication time may
alter the extract
-has a high dependence on
the sample matrix needing a
carefully design for each
matrix

Soxhlet extraction
-good for lipidic antioxidants
-cheap and easy to carry out
-widely used for fatty foods, though
not much to measure antioxidant
activity
-possibility to recover the solvent
-there is no need to filtrate the
extract

-molecules extracted depend on the


kind of solvent used
-artificial far from reality
-long time under heat may denature
antioxidant compounds
-long extraction time and high
volume of solvents

Microwave assisted
extraction (MAE)
-low cost
-simplicity
-low volume of solvent
needed
-high yield
-short time

Boiling and
organic solvents
-cheap

-need for centrifugation


-not suitable if either the
solvent or the target
compounds are non-polar

-molecules
extracted depend
on the kind of
solvent used
-artificial far from
reality
-long extraction
time
-tedious

Table 2. Continuation of Table 1


Method

Advantages

Disadvantages

Microwave/ultrasonic
assisted enzymatic
extraction (SMU-AEE)
-antioxidant values obtained
are higher than that of
solvent extraction methods.
-better extraction of
phenols, flavonoids and
anthocyanins.
-a way to make the best of
by-products

-artificial far from reality


-more expensive than most
methods
-more complicated than
others

Supercritical fluid extraction (SFE)

Pressurized liquid extraction

In vitro gastrointestinal
digestion

-the solubility of a certain chemical on those


fluids can be changed just by modifying either
the pressure or the temperature of the
supercritical fluid
-target molecules can be recovered from the
solvent without losing volatile compounds
thanks to the high volatility of the supercritical
fluid
-the higher diffusion coefficient of
supercritical fluids allows a greater extraction
rate than the one achieved with other solvents.
-low temperature making it ideal for
thermolabile compounds
-environmentally friendly due to the low use
of organic solvents
-artificial far from reality
-more expensive than most methods
-more complicated than others

-easy to carry out


-short time
-green solvents
-cheaper than SFE

-it is close to what really


happens inside our body
-it takes into account the
insoluble fraction which can
exert antioxidant activity in
the large intestine thanks to
microbiota fermentation

-artificial far from reality


-extraction yield is not higher
than that of other methods
-high temperature might
destroy some molecules

-it is more expensive than


conventional extraction
through organic solvents.

Novel Insights in the Methods for Measuring

41

But going back to the extraction step, first of all we must focus on features of the target
molecules whose antioxidant activity is tested, especially in their solubility, thermolability
and yielding of our extraction procedure. That will allow us to choose the proper solvent and
the proper method. Accordingly, a variety of extraction methods will be discussed pointing
out their advantages and disadvantages (Table 1 and 2).

CHEMICAL EXTRACTION
1. Conventional Extraction
Chemical extraction with different solvents is the most widely used extraction method.
This extraction can be performed with several solvents depending on the sample matrix and
the aim of the study. Also, it is essential to take into account what kind of molecule is
expected to be extracted, since this will determine the election of the solvent. Even though
several solvents could be used to extract the same molecules, each one of them will achieve a
different extraction yield. Most common solvents are purified water, methanol, ethanol,
acetone, ethyl acetate, petroleum ether, hexane or some acids. The samples are crushed or
powdered in order to improve the accessibility of antioxidant compounds and put in contact
with the solvent. Simply by leaving the sample in touch with the solvent, the solvent will pass
through the sample dragging the aimed compounds separating them from the sample matrix;
however, sometimes the process is accompanied by constant stirring to favor the solventantioxidant compounds contact and improve the yielding. Afterwards the mixture is filtered
or centrifuged to separate to the clean liquid fraction from the solids. Possibly, this step will
be needed more than once. In that case the solid fraction is submitted again to the solvent
addition, stirring, etc., and the liquid fraction unified with the previous one as many times as
considered necessary. Since antioxidant compounds are often thermolabile, almost the
extractions are carried out at room temperature for 24 h, but occasionally low temperatures
can be applied. Some authors, as a previous phase to the extraction, perform a defatting step
through soxhlet. Also, a purification step through glass microfiber filter is performed
sometimes (0.7 m) (Contini, Marina et al., 2008). After extraction, it is usual the removing
of the solvent to concentrate the antioxidant extract, typically with a rotary evaporator for
chemical solvents or a lyophilizer if water was used for the extraction. The residue is
dissolved then in some solvent such methanol and stored for further analysis. It has been
reported that polar protic solvent such as methanol and water, are the most effective solvents
to extract phenolic compounds from aqueous, alcoholic or lipidic medias (Rezaie, Mitra et al.,
2014). This is due to the fact that phenolic compounds are dissolved in polar solvents such as
water and methanol whereas they are not in nonpolar solvents such as hexane. Thus,
antioxidant activity, which is strongly related to phenolic content, is usually much higher in
water or methanolic extracts than in nonpolar ones (Basiri, 2013; Mihaylova et al., 2013).
However, if the sample contains an important lipidic fraction, the overall extract yield would
be higher when using nonpolar solvents though phenolic compounds will not be extracted
with them (Figures 1, 2 and 3).

42

Sergio Prez-Burillo, Silvia Molino, Cristina Delgado-Andrade et al.

Figure 1. Total phenolic content in pomegranate seeds and extract yield achieved with different
solvents. Source: Basiri, 2013.

Figure 2. The value of half maximal effective concentration of antioxidant (EC50) of pomegranate seeds
(PS). Source: Basiri, 2013.

Figure 3. FRAP assay of pomegranate seeds (PS). Source: Basiri, 2013.

Novel Insights in the Methods for Measuring

43

2. Ultrasound-Assisted Extraction (UAE)


This extraction is carried out in an ultrasonic cleaner. UAE appears as a method for
making extraction time shorter using low temperatures and that aim to improve compound
recovery with a lower cost. Physics mechanisms of ultrasound waves make possible a deeper
penetration of the solvent inside the sample allowing it to reach cells inner space and also
promote mass transfer (Luque-Garcia and Luque de Castro, 2004). Moreover, ultrasounds
break cell walls releasing many more compounds being this effect and the mass transfer two
of its major advantages (Wang, Lijun and Weller, Curtis L., 2006). Thus, the solvent is able to
penetrate into the cells making the extraction easier and yield more compounds. However,
UAE also needs to heat up the sample though at a lower temperature than other methods.
Moreover, although this method can achieve a higher extraction yield than the classic
chemical extraction, some authors have found the same compounds in comparable quantities
with both UAE with ethanol and chemical extraction with ethanol.
As previously described, it is essential to consider the kind of sample, the particle size
and the solvent, being the tips mentioned above about the solvent also apply here. Also, this
method will need to set some parameters such as pressure, temperature, frequency and time
(Wang, Lijun and Weller, Curtis L., 2006). Ultrasound frequency can have a great impact on
yield extraction and according to Wang, Lijun and Weller, Curtis L. (2006) this effect will
depend on the nature of the sample. In some samples minimum frequency changes will
greatly vary the yield while in some other the yield could keep practically unaffected.
Something interesting is that since the use of ultrasounds allows lower temperature and
pressure values, this method could be suitable for the extraction of thermolabile compounds
(Wang, Lijun and Weller, Curtis L., 2006).
The procedure consists on putting the sample, previously grinded, and the solvent inside
the ultrasonic cleaner with a ratio raw material:solvent which will depend on the authors
criteria, the target compounds and the sample. For example a ratio of 1:60 has been used to
extract phenolic acids and flavonoids from plants (Mihaylova et al., 2013). Next it is
necessary to set parameters related to the ultrasound treatment, a step which usually requires
several tests aimed to optimise the conditions. Thus, Chen, Mingshun et al., (2015) has
documented that acidic methanol at the concentration of HCl 1.55-1.72 M and ethanol 5763% (v/v) is the best solvent to reach a proper extraction of phenolic compounds, anthocyans
and other antioxidant compounds from sugar beet molasses, setting the temperature at 4148C during 66-73 min. Other authors apply this method with other solvents such as purified
water, methanol, acetone, ethyl acetate, petroleum ether or hexane depending on the
composition of the sample and the target molecules.

3. Soxhlet Extraction
This method is mostly used for fatty foods though it is also used for bioactive compounds
extraction from plants (Norsyamimi Hassim et al., 2015). Soxhlet apparatus consists on a
heater, solvent flask, sample chamber and condenser. First, the sample has to be grinded to a
fine powder, since particle size is a key factor for the success of the method (Wang, Lijun and
Weller, Curtis L., 2006). Thus, an extraction with a particle size of 2 mm would last 12 h
whereas if the particle size is 0.4 mm the extraction time is reduced to 2 h (Luque-Garcia and

44

Sergio Prez-Burillo, Silvia Molino, Cristina Delgado-Andrade et al.

Luque de Castro, 2004). Then the sample is placed in a cartridge inside the soxhlet on the
sample chamber and the solvent is placed in a flask contacting hot water so the solvent can
evaporate, go up through the soxhlet and finally condensates an falls on the sample. When the
cartridge with the sample is filled with solvent, it falls to the flask. Extraction time depends
on the sample and can oscillate from 4 to 12 hours or even more. Once the extraction is
complete the solvent can be removed on a rotatory evaporator or through lyophilisation. The
solvents used could be either organic or non-organic such water and the one used depends
once again on the sample features. Accordingly, it is also possible to use solvents such as
water or methanol to extract polar molecules like phenols. As mentioned for the previous
extraction procedures, the right election of the solvent is crucial, because the extraction yield
will depend on that. The extraction capability of the solvent depends on its chemical structure
and its polarity (Nadeem Asghar, Muhammad et al., 2013). As said above, this method is
widely used to extract fat from different foodstuffs using mostly hexane as the solvent which
presents an excellent yielding (Basiri, 2013), although according to Wang and Weller (2006)
a mix of hexane and d-limonene reached a higher yield than hexane. Unfortunately, hexane is
one of the most dangerous air pollutants and for that reason the application of other solvents
such as isopropanol, ethanol or water has increased. Nevertheless, those alternatives do not
reach the yield that hexane does and so, the cost would also be higher. On the other hand,
when the release of phenolic compounds is the aim, a polar solvent is always advised.
Equation 1. Soxhlet yield.

100

4. Microwave Assisted Extraction (MAE)


Microwaves are electromagnetic radiations which work at frequency values between 0.3
and 300 GHz. Microwaves promote an efficient and homogenous heating of the sample and
the solvent leading to a cell break which allows a good release of compounds and
consequently a good extraction (Wang and Weller, 2006). There are two different MAE
systems, one consisting on a close structure with controlled pressure and temperature whereas
the other is used at atmospheric pressure. The first one is chosen to carry out extractions at
extreme conditions, basically at high temperature because the pressure will depend on the
volume and boiling point of the solvent. The second one is applied at boiling point
temperature of the solvent and atmospheric pressure (Wang and Weller, 2006). Although high
temperature usually improves the extraction yield, it is absolutely unadvised when
antioxidants are thermolabile compounds.
A positive aspect of MAE is the significant reduction of the extraction time compared
with other solid-liquid extraction methods, from hours to just some minutes. It is important to
take into account that MAE depends on the dielectric susceptibility of the sample and the
solvent what means that wet samples will achieve a much higher extraction yield than dry
ones. Thus, hydration of dry samples will be needed. For the same reason, a non-polar solvent
such as hexane is not suitable. Hence, again it is extremely important to choose the proper
solvent, which in this case is not only based on the molecules of interest, but also in its

Novel Insights in the Methods for Measuring

45

dielectric constant. Polar solvents such as water, methanol or ethanol are appropriate whereas
non-polar as toluene or hexane are not. However by mixing non-polar with polar solvents is
possible to make them more suitable for the method. Particle size is another important aspect
since the more contact surface the easier the molecules can be extracted. Often the particle
size is 0.1-2 mm (Wang and Weller, 2006).

5. Boiling and Organic Solvents


It is usually chosen for meat and similar samples. Firstly the samples are boiled in water
for 10 minutes and then the meat samples are removed from the water and 70% ethanol is
added to the resulting broth in order to precipitate it. The supernatant is centrifuged at 10000
rpm during 10 minutes and an aliquot is concentrated in vacuum. After this concentration, the
sample is dissolved in purified water and extracted twice with hexane. Water fraction can
undergo further extractions with other organic solvents such as chloroform or ethyl acetate
(Watanabe et al., 2011).

6. Microwave/Ultrasonic Assisted Enzymatic Extraction (SMU-AEE)


This method has been developed to optimize antioxidants extraction from juice byproducts. It mixes three different procedures for extraction: ultrasounds, microwaves and
enzymes. The enzymatic attack helps to degrade cell structures and subsequently to release
target compounds.
Briefly, 1 g of dried sample is mixed with phosphate buffer (pH 3.0-6.0) and 1-10 mg of
Celluclast 1.5 L. This mixture is incubated at 45oC during 20 minutes in a rotatory shaker at
160 rpm. After the incubation, the necessary volume of absolute ethanol to reach a
concentration of 70% is added. The mixture is then simultaneously treated with microwaves
at 100-600W and with ultrasounds at 200-1200W at a temperature between 50-90oC for 10-60
minutes. The mixture is then centrifuged at 10000 rpm during 10 minutes and the supernatant
is vacuum drying. This extract can undergo then several extraction steps before measuring
antioxidant activity. According to the authors, it is initially dissolved in water and then
purified through a macroporous resin column with several elutions with water and ethanol.
The ethanolic phase is collected and vacuum dried, dissolved in water and finally measured
(Wu et al., 2015).

7. Supercritical Fluid Extraction (SFE)


Supercritical state means that a substance has undergone a temperature and a pressure
over its critical, sharing at that point characteristics of both fluids and gases. Thus,
supercritical fluids have some advantages over classic solvents: their dissolving power
depends on their density making it easier to modify it just by changing the pressure or the
temperature and secondly, a supercritical fluid has lower viscosity and surface tension and
higher diffusion coefficient meaning better mass transference (Wang and Weller, 2006).

46

Sergio Prez-Burillo, Silvia Molino, Cristina Delgado-Andrade et al.

Supercritical carbon dioxide is the most common solvent used for SFE. Frequently,
supercritical CO2 is used along with polar co-solvents such as water, ethanol, methanol or
water-ethanol mixtures. That is because polar molecules will not be extracted just with CO2
due to their low solubility in this solvent. Before the extraction with co-solvents, a previous
extraction with pure supercritical CO2 is carried out for 15 minutes in order to extract the low
polarity CO2-soluble compounds. This pre-treatment is important to increase polyphenols
extraction yield since lipophilic and non-polar substances will be removed (Solana et al.,
2015). The SFE extraction can be carried out in two phases: an static period where the
supercritical CO2 and the sample are closed in the extraction vessel and a dynamic stage
where the system is opened. To carry out this method it is necessary to control CO2 flow,
pressure and temperature and depending on the combination made with such parameters the
extraction yield will change. According to several investigators, increasing the pressure at a
steady temperature will increase the extraction yield but over certain pressure value the yield
will decrease (Norsyamimi et al., 2015, Botelho et al., 2015). The extraction yield improves
because as pressure increases, supercritical CO2 solvent density also increases which means
that the extraction yield is better. However, on the other hand increasing the pressure too
much will decrease CO2 diffusivity meaning a decrease of the extraction yield. Thus,
according with Norsyamimi et al., (2015), a pressure over 200 bars at 40oC will decrease the
yield. Moreover, they stated that factors such as solvent flow rate and porosity of the sample
can affect the extraction yield as well. Based on Rodriguez-Solana et al., (2014), SFE
capacity for extracting volatile compounds is similar to that of soxlhet and accelerated solvent
extraction (ASE). Moreover, antioxidant activity measure with DPPH method showed that for
three of four samples SFE was the best extraction method since antioxidant values were
higher. However, quantification of total phenolic content on SFE extracts showed
significantly lower results (200 to 500 times lower) than that found using soxleht or ASE
methods and consequently the antioxidant power of the SFE extracts was lower.

8. Pressurized Liquid Extraction


This is a solid-liquid extraction that is carried out under temperatures between 50 and
200oC and pressures around 10-15MPa. Thus, is similar to SFE except that in this method the
solvents stay in their liquid state. High temperatures and pressure help the solvent to penetrate
into the matrix sample and cells and thus, allowing a good extraction yield in a shorter time
than that needed for example in soxhlet (Wang and Weller, 2006). As most methods, this one
needs the sample to be fine powder. Usually the sample is previously lyophilized and then
finely crushed. Thus, the sample is placed in the extraction compartment and the solvent in a
reservoir. The solvent flow starts reaching the proper temperature for the extraction (50200oC) and is usually controlled by a HPLC pump. The fluid pressure is around 10MPa and
the flow rate is usually 2 ml/min during 30 minutes. Finally the solvent is removed from the
sample in a rotatory evaporator (Solana et al., 2015).

Novel Insights in the Methods for Measuring

47

PHYSIOLOGICAL EXTRACTION
9. In Vitro Gastrointestinal Digestion
Extracts obtained through the methods described above are often very good in terms of
yield extraction. However, the important question is what yield is our body able to reach. In
this sense, in order to know the antioxidant activity that certain foodstuffs could exert in our
body a model simulating in vivo conditions is needed. Pastoriza et al. (2011) developed the
GAR (Global antioxidant response) method consisting on an in vitro gastrointestinal digestion
followed by antioxidant activity measurement. The difference between this method and other
previous digestion based extraction methods is that in the later, the solid (insoluble) fraction
is systematically discarded whereas in the GAR approach that part is also considered. So that,
the GAR method takes into account the antioxidant activity of both fractions, the soluble
which would be absorbed through the small intestine and the insoluble one which would
reach the large intestine. Thus, the procedure consists on an oral, a gastric and an intestinal
phase carried out in saliva, gastric and intestinal simulated fluids mimicking salts
composition. The oral phase lasts two minutes at 37oC with salivary alpha-amylase. The next
step is the gastric phase which lasts two hours at 37oC using pepsin solution at pH 3. Then,
after raising the pH up to 7, the intestinal fluid with bile salts and either pancreatin or a
mixture of lipase, colipase, amylase and trypsin is added. This phase will last also two hours
at 37oC. Finally, the reaction is stopped applying a thermal treatment, freezing or by adding
enzymatic inhibitors depending of the sample susceptibility. After centrifugation two
fractions are obtained: the bioaccesible one, the supernatant, able to be absorbed through the
small intestine and the insoluble one, the solid and initially non-absorbed fraction. The
singularity of the GAR method is the consideration of the global antioxidant activity present
in the food summarising the antioxidant power of both fractions. For that task different
antioxidant methods can be applied.

ANTIOXIDANT METHODS
Methods of assessing antioxidant behaviour of food systems are focused on appraising
the efficacy of an antioxidant(s) in providing protection for the food against oxidative
spoilage. Furthermore, in the last years, a great interest for identifying alternative natural and
safe sources of food antioxidants to prevent the radical chain reactions of oxidation in humans
has developed. For this reason, a branch of these evaluations involves measurements of
activity in foods aimed at predicting dietary burden and in vivo activity (Antolovich et al.,
2002). For determination of antioxidant ability of food components, the terms of antioxidant
activity and antioxidant capacity are often used interchangeably, but it should be recognized
that they have different meanings. Activity refers to the rate constant of a reaction between a
specific antioxidant and a specific oxidant. Capacity is a measure of the amount (as a mole) of
a given free radical scavenged by a sample (Glin, 2012). The capacity of the natural
antioxidants contained in foods, fruits, beverages, and supplements has been evaluated in
numerous reviews and monographs (Huang, Ou and Prior, 2005; Moon and Shibamoto, 2009;
Miguel, 2010; Glin, 2012) by different methods under different conditions. Nonetheless,

Sergio Prez-Burillo, Silvia Molino, Cristina Delgado-Andrade et al.

48

because multiple reaction features and mechanisms are involved nowadays theres not a
single assay that reflects an appropriate and standardized analytical method to measure the
antioxidant capacity (AOC) (Prior, Wu and Schaich, 2005). Performance characteristics of a
standardized method for AOC should:
(1) measure chemistry actually occurring in potential application(s);
(2) utilize a biologically relevant radical source;
(3) be simple;
(4) use a method with a defined endpoint and chemical mechanism;
(5) have instrumentation readily available;
(6) have good within-run and between-day reproducibility;
(7) be adaptable for assay of both hydrophilic and lipophilic antioxidants and use of
different radical sources; (8) be adaptable to high-throughput analysis for routine quality
control analyses (Prior et al., 2005).
According to a IUPAC technical report antioxidant assays can classified on the base of
the type of reaction: (i) hydrogen atom transfer (HAT)-based assays (ii) electron transfer
(ET)-based assays. The end result is the same, regardless of mechanism, but kinetics and
potential for side reactions are different. ET and HAT mechanisms almost always occur
together in all samples, with the balance determined by antioxidant structure and pH. Table 3
lists the major antioxidant capacity assays (Gorinstein et al., 2013). Oxygen radical
antioxidant capacity (ORAC) and Trolox equivalent antioxidant capacity (TEAC) assays are
the most popularly used HAT and ET methods, respectively (Huang et al., 2005; Pinchuk et
al., 2012). Nowadays, the most widely used methods for measuring antioxidant activity are
those that involve the generation of radical species and the presence of antioxidants
determining the scavenging of these radicals (Huang et al., 2005; Barba et al., 2013). Many
assays have been developed to test and rank antioxidants until now. Some of these methods
are modifications of same basic assay and only a small number of methods were considered
for standardization in The First International Congress on Antioxidants Methods in 2004
(Prior et al., 2005).
Table 3. Antioxidant methods according to IUAPC
HAT Assays

X + AH

XH + A

ET Assays
Mn + AH

AH+ + M(n -1)

Oxygen radical absorbance capacity (ORAC)


Total radical-trapping antioxidant parameter (TRAP)
Inhibition of induced LDL oxidation
Total oxyradical scavenging capacity (TOSC) assay
Croton-bleaching assays
Photochemiluminescent (PLC) assay
Total phenolics assay by Folin-Ciocalteu (F C) reagent assay
Trolox equivalence antioxidant capacity (TEAC) or others ABTS
assays
Ferric ion reducing antioxidant power (FRAP) assay
2,2-Diphenyl-1-picrylhydrazyl radical (DPPH) scavenging
Cupric ions (Cu2+) reducing antioxidant power (CUPRAC) assay

Novel Insights in the Methods for Measuring

49

Following, the most commonly used analytical method for determine AOC and with
certain degrees of influence and applications are described. It will be pointed out as a
summary the main characteristics of the methods that will be discussed in terms of simplicity,
instrumentation required, mechanisms, endpoint, quantitation method, and biological
relevance (Table 4). Moreover, tables 5 and 6 show the main advantages and disadvantages of
these methods.

HAT-Based Assays
HAT-based methods measure the classical ability of an antioxidant to quench free
radicals (generally, peroxyl radicals considered to be biologically more relevant) by hydrogen
donation (Prior et al., 2005):
X + AH XH + A
In particular, the HAT mechanisms of antioxidant action in which the H-atom of a phenol
(ArOH) is transferred to a ROO radical can be summarized by the reaction:
ROO + ArOH ROOH + ArO
where the aryloxy radical (ArO) formed from the reaction of antioxidant phenol with peroxyl
radical is stabilized by resonance. The AH and ArOH species denote the protected
biomolecules and phenolic antioxidants, respectively (Huang et al., 2005).
HAT-based methods generally are composed of a synthetic free radical generator, an
oxidizable molecular probe, and an antioxidant. HAT reactions are pH and solvent
independent and are very fast and usually completed in seconds to minutes. The presence of
reducing agents, including metals, is a complication in HAT assays and can lead to
erroneously high apparent reactivity. Most HAT-based assays monitor competitive reaction
kinetics, and the quantitation is derived from the kinetic curves. Since in HAT-based
antioxidant assays, both the fluorescent probe and antioxidants react with ROO, the
antioxidant activity can be determined from competition kinetics by measuring the
fluorescence decay curve of the probe in the absence and presence of antioxidants, integrating
the area under these curves, and finding the difference between them (Prior et al., 2005;
Huang et al., 2005; Miguel, 2010).
Methods based on the HAT reaction include the following methods (Huang et al. 2005):
1. Oxygen radical absorbance capacity (ORAC) assay
2. Total radical-trapping antioxidant parameter (TRAP) assay
3. Inhibition of induced LDL oxidation assay
4. Total oxyradical scavenging capacity (TOSC) assay
5. Croton-bleaching assays
6. Photochemiluminescent (PLC) assay

Table 4. Summary of the main characteristics of the different antioxidant assays in terms of simplicity, instrumentation
required, mechanisms, endpoint, quantitation method, and biological relevance
Assay

Mechanism

Measurement
principle
Fluorescence
Fluorescence
Absorbance (234 nm)
Luminescence
Chromatography
Absorbance (470 nm)

Measurement
instrument
Microplate reader
Microplate reader
Spectrophotometer
Luminometer
Gas chromatography
Spectrophotometer

Time
required
++
++

Instrumentation
required
+
+

++
+++

Biological
relevance
++
+++

++

+++

+++

HAT
HAT

Radical or
oxidant
AAPH
AAPH
Cu2+ or
AAPH
ABAP
-carotene

ORAC
TRAP
LDL
oxidation
TOSC
Croton
bleaching

HAT
HAT
HAT

Simplicity

+++

Absorbance (443 nm)


Chemiluminescence

F-C
TEAC
FRAP

ET
ET
ET

Absorbance (765 nm)


Absorbance (234 nm)
Absorbance (595 nm)

DPPH
CUPRAC

ET
ET

MoO4+
ABTS+
Chelated
Fe3+
DPPH
Cu2+

Spectrophotometer
Photochem
(luminometer)
Spectrophotometer
Spectrophotometer
Spectrophotometer

Photoquemical

crocin
O2-

++

PCL

Absorbance (515 nm)


Absorbance (450 nm)

Spectrophotometer
Spectrophotometer

+++

++

+
+++

+++
+++

++
+++

++
+

+++

+++

++

+++
+++

+++
+++

++
+++

+
++

+: poor; ++:medium; +++:very good.

Table 5. Advantages and disadvantages of antioxidant methods


Method
Advantages

ORAC
- As pyrogallol red reacts
faster than fluorescein with
ROO radicals, its
consumption does not
present induction times,
even in the presence of
very reactive antioxidants,
with the exception of
ascorbic acid.
- The nutritional relevance
of these data has recently
been questioned.

TRAP
-This assay is useful for
measurements of in-vivo
antioxidant capacity in
serum or plasma because it
measures nonenzymatic
antioxidants such as
glutathione, ascorbic acid,
R-tocopherol and Bcarotene (Huang et al.,
2005).

LDL oxidation
-Biological relevant target.

TOSC
-Is able to evaluate
different antioxidants
with different
biologically relevant
radical sources.

Croton bleaching
- Carotenoid bleaching
is readily adaptable to
high-throughput
methodology such as
microplates.

Method
Disadvantages

ORAC
- Fluorometers, may not be
routinely available in
analytical laboratories.
- Temperature control
decreases reproducibility
(Badarinath et al., 2010).
- It has recently been
reported a re-examination
of the ORAC assay that
indicates that antioxidantmetal .

reactions could result in a


lower concentration of
antioxidants and therefore
to an under- estimation of
the ORAC value (Nkhili &
Brat, 2011).

TRAP
-Many different end points
have been used, there is a
great variability so
comparisons interlaboratories are difficult.
-It is relatively complex and
time consuming.
- It also requires a high
degree of expertise and
experience (Badarinath et
al., 2010).

LDL oxidation
-Variability between lot to lot
of the LDL sample due to
their complex composition.
- When cupric sulfate is
employed as initiator of
oxidation, it should be
considered that the
antioxidant effect can also
derive from the copperchelating capacity of the
sample (Camilo LpezAlarcn & Denicola, 2013).

TOSC
- Long reaction time
and the necessity of
multiple
chromatographic
analyses for each
experiment.
-There is not a linear
relationship between
the percentage
inhibition of TOSC by
the antioxidant source
and antioxidant
concentration or
dilution..
- Comparison between
foods becomes difficult
because of these
multiple endpoint
parameters (Prior et al.,
2005)

Croton bleaching
-The thermicallyinduced oxidation is not
controlled.
-Crocin is not available
commercially and so
must be extracted.
-There are no standard
formats for expressing
results.

-It is not easy to


interpret the results
because -carotene itself
is an oxygen-sensitive
antioxidant (Laguerre,
Lecomte, & Villeneuve,
2007; Prior et al., 2005).
-Interferences with other
molecules that also
absorb at the same
wavelength.

Table 6. Continuation of Table 5


Method
Advantages

PCL
- This system is
marketed as a timeand cost-effective
system for the
determination of the
integral antioxidative
capacity toward
superoxide.

F-C
-Operationally
simple, reproducible.
-Procedure is rather
standardized.
- The relationship
between the FC
method and ORAC, a
HAT-based assay, is
usually good
(Magalhes et al.,
2008).

Disadvantages

-Only one sample can


be measured at a time
-Expensive
instrumentation.

- The presence of
interfering substances
requires different
methodological
approaches to avoid
the problem.
-Needs high time.

TEAC (ABTS)
-Operationally simple.
-Reactions are rapid
(most methods use 30
min or less).
-Run over a wide range
of pH.
-It has been used in
multiple media to
determine both
hydrophilic and
lipophilic antioxidant
capacity.
-The results provided by
this assay are dependent
of time of analysis.
- ABTS+ radical is not
representative of
biomolecules.
-Thermodynamically,
any compound that has a
redox potential lower
than that of ABTS+ may
react with the radical.
-The high extinction
coefficient of ABTS+
limits the useful
antioxidant
concentration range that
can be analysed
accurately to about 1.5
70 M final
concentrations.
Antioxidant
concentrations outside
this range require too
much or too little
ABTS+ for accurate
optical measurements.

FRAP
-Simple
-Speedy
-Inexpensive
-Robust
-It does not required
specialized equipment.

DPPH
-Simple
-Speedy
-Inexpensive
-DPPH radical is stable
and commercially
available.

CUPRAC
-Cost-effective
-Rapid
-Stable
-Selective
-Suitable for a
variety of
antioxidants

- FRAP cannot detect


species that act by
radical quenching (H
transfer), particularly
SH group containing
antioxidants like
thiols, such as
glutathione and
proteins.
- The absorption ( =
593) slowly increase
for polyphenols such
as caffeic acid, tannic
acid, ferulic acid,
ascorbic acid, and
quercetin, even after
several hours of
reaction time. Thus, a
single-point
absorption endpoint
may not represent a
completed reaction.

- Interpretation is
complicated when the
test compounds have
spectra that overlap
DPPH at 515 nm.
Carotenoids, in
particular, interfere.
- The assay is not a
competitive reaction
because DPPH is both
radical probe and
oxidant.
-Small molecules have
better access to the
radical site than larger
ones meaning that the
latter show their activity
more slowly and may
even not show any
activity at all.
- DPPH also is
decolorized by reducing
agents as well as H
transfer, which also
contributes to inaccurate
interpretations of AOC

-Requires high
time for
complex
molecules.

Novel Insights in the Methods for Measuring

53

1. Oxygen Radical Absorbance Capacity (ORAC) assay


Among the methodologies developed to estimate antioxidant capacity, this assay is one of
the most employed. In fact, this method has been used for estimating the antioxidant capacity
of the mainly consumed foods and beverages in USA (Lpez-Alarcn and Denicola, 2013).
The Oxygen Radical Absorbance Capacity (ORAC) method measures the oxidative
degradation of a fluorescent molecule such as -phycoerythrin (B-PE) after being mixed with
free-radical generators such as azoinitiator compounds (Moon and Shibamoto, 2009). The
original assay was developed by using the fluorescent protein B-PE as the radical target
(Glazer, 1988; Cao, Alessio and Cutler, 1993; Ghiselli et al., 1995). Nowadays fluorescein or
dichlorofluorescein are also used as substrates and the reaction progress is followed by
fluorescence (Ou et al., 2013) or pyirogallol and pyranine for an improved colorimetric assay
(Lpez-Alarcn and Lissi, 2006; Atala et al., 2013). The peroxyl radicals will react with a
fluorescent substrate to form a non-fluorescent compound. In the ORAC assay the antioxidant
capacity is evaluated from the area under the curve (AUC) of the kinetic profiles of the target
molecule consumption. AUC values are commonly compared with acid gallic or Trolox (a
hydrosoluble vitamin E analog) and data are generally reported as Trolox equivalents [M
Trolox equivalents (TE)/g]. The assay can be used to evaluate peroxidation in body fluids
including plasma, serum as well as tissue samples and food products (Prior et al., 2005;
Huang et al., 2005; Lpez-Alarcn and Denicola, 2013).

2. Total Radical-Trapping Antioxidant Parameter (TRAP) Assay


TRAP method was developed by Wayner et al. (1985). The basic reactions of the assay
are similar to those of ORAC. This method is based on the protection provided by
antioxidants on the fluorescence decay of R-phycoerythrin (R-PE) during a controlled
peroxidation reaction. The fluorescence of R-phycoerythrin is quenched by APPH or ABAP
[2,2-azobis(2-amidinopropane) dihydrochloride] as a radical generator. This quenching
reaction is measured in the presence of antioxidants. The antioxidative potential is evaluated
by measuring the decay in decoloration (Alam, Bristi and Rafiquzzaman, 2013).
Requirements for the assay are that the probe must be reactive with ROO at low
concentrations, there must be a dramatic spectroscopic change between the native and
oxidized probe (to maximize sensitivity), and no radical chain reaction beyond probe
oxidation should occur. TRAP values are usually expressed as a lag time or reaction time of
the sample compared to corresponding times for Trolox (Prior et al., 2005) .

3. Inhibition of Induced LDL Oxidation Assay


Ex vivo oxidation of LDL was developed primarily as a measure of antioxidant status,
but applications of LDL oxidation have also been adapted to assess antioxidant capacity in a
more physiologically relevant system. LDL is isolated fresh from blood samples. In vitro
assays usually employ Cu2+ or AAPH as initiators of LDL oxidation and the lipid
peroxidation processes are easily followed by UV spectroscopy and/or chemiluminiscence
techniques. In the first case, the formation of diene conjugates at 234 nm is followed, while in

54

Sergio Prez-Burillo, Silvia Molino, Cristina Delgado-Andrade et al.

the second one, the emission of photons related to the formation of final oxidative products is
measured (Prior et al., 2005; Lpez-Alarcn and Denicola, 2013).

4. Total Oxyradical Scavenging Capacity (TOSC) Assay


Developed by Winston et al. (1998), this method permits quantification of the absorbance
capacity of antioxidants specifically toward three potent oxidants: hydroxyl radicals, peroxyl
radicals, and peroxynitrite. TOSC assay is based on the oxidation of alpha-keto-gamma(methylthio) butyric acid (KMBA) to ethylene by peroxyl radicals produced from AAPH. The
antioxidant capacity is quantified from the ability of the antioxidant to inhibit ethylene
formation by measuring the area under the curve (AUC) of ethylene concentration versus
time. The time course of ethylene formation is followed by headspace analysis of the reaction
cell by gas chromatography (Prior et al., 2005).

5. Croton-Bleaching Assays
Carotenoids bleach via autoxidation, oxidation induced by light or heat (50C), or
oxidation induced by peroxyl radicals (e.g., AAPH or oxidizing lipids), and this
decolorization can be diminished or prevented by classical antioxidants that donate hydrogen
atoms to quench radicals. The -carotene bleaching test consists of measuring the decay of
the absorption at 470 nm due to -carotene under a flux of free radicals, in the presence or
absence of antioxidants. The antioxidant activity is expressed as percentage of inhibition with
reference to the control, after 60 min of incubation. Although -carotene is often used as the
target, its decolorization at 470 nm can occur by multiple pathways, so interpretation of
results can be complicated. In contrast, crocin, a natural water-soluble carotenoid contained in
flowers of the Crocus genus, first championed by Bors, Michel and Saran (1984), has
straightforward reactions and bleaches only by the radical oxidation pathway, so it has
become the reagent of choice over -carotene. The crocin bleaching test is based on the
reaction between crocin and peroxyl radicals generated by thermal decomposition of AAPH.
Antioxidants are able to retard the decrease of the 443 nm absorbance of crocin caused by the
attack of peroxyl radicals. The antioxidant activity is expressed as the ratio between the rates
of crocin bleaching in the absence and in the presence of antioxidants (Prior et al., 2005;
Amorati and Valgimigli, 2015). Recently, Prieto, Vzquez, and Murado (2015) developed a
consistent routine for microplate assay, avoiding over-standardization. Furthermore, they
proposed a kinetic model for quantifying anti- and pro-oxidant activity.

6. Photochemiluminescent (PCL) Assay


This assay was described by Popov and Lewin (1999), commercialized by Analytik Jena
AG (Jena, Germany), and sold as a complete system under the name PHOTOCHEM. In the
PCL assay the process of photochemical generation of free radicals is coupled to the detection
step, which is by means of chemiluminescence. The assay involves the photochemical

Novel Insights in the Methods for Measuring

55

generation of superoxide O2- free radicals combined with chemiluminescent detection. The
assay is initiated by optical excitation of a photosensitizer (S), such as luminal, resulting in
the generation of the superoxide radical anion. The process is described by the equation:
S + light + O2 (S* O2) O2+ + O2The complete reaction mechanism is not known. Ascorbic acid and Trolox are typically
used as calibration reagents for hydrophilic and lipophilic AOC, respectively, at measuring
ranges of 0-2 nmol. In contrast to other commonly used AOC assays, the PHOTOCHEM
method is not restricted to a specific pH value or temperature range(Prior et al., 2005).

ET-BASED ASSAYS
Spectrophotometric ET (SET)-based methods detect the ability of an antioxidant to
transfer one electron to reduce any compound, including metals, carbonyls, and radicals
(Wright, Johnson and DiLabio, 2001):
X + AH X- + AH+
AH+ A + H3O+
H- + H3O+ XH + H2O
Mn + AH AH+ + M(n -1)
Relative reactivity of the SET method is based on deprotonation and in ionization
potential of the reactive functional group (Lemanska et al., 2001), so SET methods are pH
dependent (Wright et al., 2001; Prior et al., 2005). Generally, ionization potential decreases
with increasing pH values, which reflects a higher electron-donating capacity with
deprotonation. ET reactions are usually slow and can require long times to reach completion,
so antioxidant capacity calculations are based on percentage of decrease in product rather
than kinetics. When AH+ has a sufficient lifetime, secondary reactions become a significant
interference in assays (Sartor, Henderson and Schuster, 1999). ET methods are very sensitive
to ascorbic acid and uric acid, which are important in maintaining plasma redox tone, and
reducing polyphenols are also detected. Importantly, trace components and contaminants such
as metals interfere with ET methods and can account for high variability and poor
reproducibility and consistency of results (Huang et al., 2005).
In the reaction mixture of the method two basic components are involved antioxidants
and oxidant (also the probe). Hence, the electron-transfer reaction will be:
probe (oxidant) + e- (from antioxidant) reduced probe + oxidized antioxidant
The probe itself is an oxidant that abstracts an electron from the antioxidant, causing
color changes of the probe. The degree of the color change is proportional to the antioxidant
concentrations. The reaction end point is reached when color change stops (Huang et al.,
2005). In most ET-based assays, the antioxidant action is simulated with a suitable redoxpotential probe, namely, the antioxidants react with a fluorescent or colored probe (oxidizing

Sergio Prez-Burillo, Silvia Molino, Cristina Delgado-Andrade et al.

56

agent) instead of peroxyl radicals. In summary, SET-based assays measure the capacity of an
antioxidant in the reduction of an oxidant, which changes its color when reduced. The degree
of color change (either an increase or decrease of absorbance of the probe at a given
wavelength) is correlated to the concentration of antioxidants in the sample (Gorinstein et al.,
2013).
The ET-based methods include the following assays:
1)
2)
3)
4)
5)

Total phenolic content by Folin-Ciocalteu reagent assay


Trolox equivalence antioxidant capacity (TEAC) or others ABTS assays
Ferric ion reducing antioxidant power (FRAP) assay
2,2-Diphenyl-1-picrylhydrazyl radical (DPPH) scavenging assay
Cupric ions (Cu2+) reducing antioxidant power (CUPRAC) assay

1. Total Phenolic Content by Folin-Ciocalteu (F-C) Reagent Assay


The test was originally designed to titrate polyphenols in wine, but it has since then been
arbitrarily extended as a method to measure total phenolic content (TPC) in food or vegetable
extracts. Folin-Ciocalteu (F-C) method is based on the number of phenolic groups or other
potential oxidizable groups present in compounds in the sample. The exact chemical nature of
the F-C reagent is not known, but it is accepted that it contains phosphomolybdic/
phosphotungstic acid complexes. The electron-transfer reaction occurs between antioxidants
and molybdenum (Mo), which is reduced in the complex, leading to the formation of blue
colored molybdenum ions, MoO4+, that can be detected spectrophotometrically at 750
765nm. The results are reported relative to gallic acid, although other phenols have also been
used (Amorati and Valgimigli, 2015). This reaction only occurs in an alkaline environment
(pH 10), therefore the addition of sodium carbonate is crucial (Magalhes et al., 2008;
Miguel, 2010). Normally this assay is performed in aqueous phase, but new methods for
lipophilic compounds/matrices are developing (Berker et al., 2013). It is well-known that
apart from polyphenols other types of substances that may be present in high abundance in
plant and food extracts (particularly aromatic amines, sulfur dioxide, ascorbic acid and other
enediols and reductones, organic acids, and Fe2+) can also reduce the F-C reagent, skewing
the results of TPC. Therefore, different methodological approaches to improve the specificity
of the F-C assay have been proposed. Those methodological approaches include:
I.
II.
III.

the partial purification of phenolic extracts using solid phase extraction columns
before the F-C assay is performed;
the calculation of a corrected TPC (total phenolic content) value by subtracting the
reducing activity from the TPC quantified in the plant food extract and
the treatment of phenolic extracts with oxidative agents such as hydrogen peroxide
(H2O2) at levels that oxidize the interfering compounds and do not affect phenolic
compounds (Snchez-Rangel et al., 2013).

Reaction equation:
Mo4+ (yellow) + e (from AH) Mo5+ (blue)

Novel Insights in the Methods for Measuring

57

where the oxidising reagent is a phosphomolybdic/phosphotungstic acid complex in which


the hypothesized active center is Mo4+ with max = 765 nm.

2. Trolox Equivalence Antioxidant Capacity (TEAC) or Other ABTS Assays


TEAC assays use intensely colored cation radicals of ABTS to test the ability of
antioxidants to quench radicals. The original assay, TEAC I, was first reported by Miller et al.
(1993) and based on the activation of metmyoglobin-H2O2 in the presence of ABTS [2,2azinobis(3-ethylbenzothiazoline-6- sulfonic)acid], to produce in the presence of antioxidants
the cation radical, ABTS+. However, the quantification of the antioxidant effects were
equivocal because antioxidants could react with the original radical oxidant as well as the
ABTS+, causing an overestimation of antioxidant activity. Re et al. (1999) simplified this
assay to cleanly generate the greenish blue ABTS+ by first oxidizing ABTS with potassium
persulfate, then adding antioxidants and measuring direct reaction with the radical. This
format, called TEAC II, constitute the basis for the current TEAC assays.
TEAC II assays are quite simple to perform. Some modified methods have not used the
name TEAC, but they actually share the same reaction mechanism and use the same radical
cation, ABTS+. According to Cano et al. (2002), ABTS+ can be generated by either chemical
reaction (e.g., manganese dioxide, ABAP, potassium persulfate) or enzyme reactions (e.g.,
metmyoglobin, hemoglobin, or horseradish peroxidase). Generally, the chemical production
requires a long time or high temperatures, whereas enzyme generation is faster and the
reaction conditions are milder. The antioxidant activity is determined with the initial
absorbance at 734 nm and the drop in absorbance is measured after reaction periods varying
from four minutes to several hours (Gorinstein et al., 2013). The extent of decolorization,
expressed as percentage of inhibition of ABTS+ (Equation 2), is determined from the ratio of
test compound reaction to that of Trolox reaction, the reference standard (Miguel, 2010).
Equation 2. Percentage of inhibition of ABTS+

Inibitionbytestcoumpounds
InibitionbyTrolox

100

The strong point of this assay is that it can be applied for both water-soluble and lipid
soluble antioxidants. There is a method similar to that of TEAC, where ABTS+ is replaced by
the colored and stable DMPD+(N,N-dimethyl-p-phenylenediamine) (Prior et al., 2005;
Magalhes et al., 2008; Pinchuk et al., 2012).
Reaction equations:
ABTS + K2S2O8 ABTS+
ABTS+ + ArOH ABTS + ArO + H+

58

Sergio Prez-Burillo, Silvia Molino, Cristina Delgado-Andrade et al.

3. Ferric Ion Reducing Antioxidant Power (FRAP) Assay


The FRAP assay measures the ability of antioxidants to reduce the ferric 2,4,6-tripyridyls-triazine complex [Fe3-(TPTZ)2]3+ to the intensely blue colored ferrous complex [Fe2TPTZ)2]2+ in acid medium (pH 3.6). This method measured the reducing power in plasma, but
the assay has been subsequently adapted and used for the measurement of antioxidants in
different food or beverages, spices, vegetables juice and fruits (Glin, 2012). FRAP values
are calculated by measuring spectrophotometrically the absorbance increase at 593nm after a
fixed time (4 min) and relating it to a ferrous ions standard solution or to an antioxidant
standard solution (ascorbic acid, for instance). The absorption slowly increases for
polyphenols such as caffeic acid, tannic acid, ferulic acid, ascorbic acid and quercetin, even
after several hours of reaction time. Thus, a single-point absorption endpoint may not
represent a completed reaction (Prior et al., 2005). This method has also been adapted to 96well microplate reader, giving better reproducibility and higher sample throughput (Benzie
and Strain, 1996). The mechanism of FRAP assay is totally electron transfer rather than
mixed SET and HAT, so in combination with other methods can be very useful in
distinguishing the dominant mechanisms with a pool of different antioxidants in the food
matrix. In addition, because reduced metals are active propagators of radical chains via
hydroperoxide reduction to RO, sometimes it is interesting to evaluate whether high FRAP
values correlate with the tendency of polyphenols to become pro-oxidants under some
conditions. This has been shown for some flavones and flavanones, which also have high
FRAP values (Prior et al., 2005; Magalhes et al., 2008; Glin, 2012).
Reaction equation:
Fe(TPTZ)2 3+ + ArOH Fe(TPTZ)2 2+ + ArO + H+

4. 2,2-Diphenyl-1-Picrylhydrazyl Radical (DPPH) Scavenging


Color changing of DPPH (2,2-diphenyl-1-picrylhydrazyl) from purple to yellow is the
consequence of the reducing ability of antioxidants toward DPPH stable radical. The bluepurple chromogen radical DPPH absorbs at 515 nm and if free radicals have been scavenged
by an antiradical compound DPPH will change color to yellow, which also lead to the
disappearance of its absorption. The scavenging capacity is generally evaluated in organic
media by monitoring the absorbance decrease until the absorbance remains constant or by
electron spin resonance. As occurs in other ET-based assays, the scavenging capacity against
DPPH radical is strongly influenced by the solvent and the pH of reaction. Stasko et al.
(2007) suggested that the 50% (v/v) aqueous/ethanol solutions are a suitable choice for
lipophilic and hydrophilic antioxidants and the reaction rate between DPPH and the
antioxidant may increase considerably with increasing water ratios. Methanol and ethanol
solvents are strongly hydrogen bond-accepting; therefore the hydrogen-abstracting reaction
occurs very slowly. Generally, the results are reported as the efficient concentration (EC50),
which is the amount of antioxidant necessary to decrease by 50% the initial DPPH
concentration. The time needed to reach the steady state with EC50 concentration is calculated
from the kinetic curve and defined as TEC50. In recognition of the effect of both parameters

Novel Insights in the Methods for Measuring

59

the antiradical efficiency may be determined by calculating the reciprocal of EC50TEC50.


Therefore, the lower EC50 and TEC50, the higher is the antiradical efficiency. Finally, the
DPPH scavenging reaction is time-consuming and it may take 20 min up to 6h. Recently,
Magalhes et al. (2006) applied a mathematical model to the data collected within the first
3 min of DPPH scavenging reaction to estimate the total DPPH consumed (Prior et al., 2005;
Magalhes et al., 2008; Miguel, 2010). Milardovi, Ivekovi, and Grabari (2006) also
proposed the determination of antioxidant capacity based on the amperometric reduction of
DPPH at the glassy carbon electrode.
Reaction equation:
DPPH + ArOH DPPH + ArO + H+

5. Cupric Ions (Cu2+) Reducing Antioxidant Power (CUPRAC) Assay


CUPRAC is a variant of the FRAP assay using Cu instead of Fe. This method is a novel
hydroxyl radical scavenging antioxidant activity assay for water-soluble antioxidants. The
putative CUPRAC method was developed by Apak et al. (2006). It is based on the reduction
of Cu2+ to Cu+ by the combined action of all antioxidants or reducing in aqueous-ethanolic
medium (pH 7.0) in the presence of neocuproine (2,9-dimethyl-1,10-phenanthro-line) by
polyphenols, yielding a Cu+ complexes with a maximum absorption peak at 450 nm. The
reaction with the chromatic solution can be monitored by measuring absorbance, and the
antioxidant capacity can be easily calculated. The method can be used for the determination
of the antioxidant capacity of food constituent by the Cu2+-neocuproine (Cu2+-Nc) reagent as
the chromogenic oxidizing agent. CUPRAC chromogenic redox reaction is capable of
measuring thiol-type antioxidants such as glutathione and non-protein thiols unlike the widely
applied FRAP test, which is non-responsive to -SH group antioxidants. This method is
capable of measuring both hydrophilic and lipophilic antioxidants (Prior et al., 2005; Glin,
2012).
Reaction equation:
nCu(Nc)2 2+ + Ar(OH)n nCu(Nc)2+ + Ar(=O)n + nH+
where the polyphenol with suitably situated ArOH groups is oxidized to the corresponding
quinone, and the reduction product, i.e., bis(neocuproine)Cu(I) [Cu(I)-Nc] chelate. It should
be noted that not all phenolic OH are reduced to the corresponding quinones, and the
efficiency of this reduction depends on the number and position of the phenolic OH groups
as well as on the overall conjugation level of the polyphenolic molecule (Gorinstein et al.,
2013).

60

Sergio Prez-Burillo, Silvia Molino, Cristina Delgado-Andrade et al.

ACKNOWLEDGMENTS
This work was supported by the project AGL2014-53895-R from the Spanish Ministry of
Economy and Competitiveness.

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In: Advances in Food Analysis Research


Editor: Anita Haynes

ISBN: 978-1-63483-783-5
2015 Nova Science Publishers, Inc.

Chapter 3

ADVANTAGES AND PITFALLS OF THE METHODS


FOR THE ANTIOXIDANT ACTIVITY EVALUATION
Angela Fadda1 and Daniele Sanna2,
1

Institute of Sciences of Food Production,


2
Institute of Biomolecular Chemistry
National Research Council (CNR), Regione Baldinca,
Li Punti, Sassari, Italy

ABSTRACT
The beneficial influence on human health of many foodstuffs and beverages
originates from their antioxidant activity. The increasing interest on the role of natural
antioxidants in biological systems and their nutritional effects has determined continuous
efforts in the search of simple and reliable methods for the determination of antioxidant
activity.
In this paper an overview of the most used methodologies for the determination of
antioxidant capacity is presented, placing particular emphasis on the advantages, the
pitfalls and the possible developments of each method. In particular the methods based on
the Electron Spin Resonance (ESR) spectroscopy with direct measurement of relatively
stable radical species or unstable radicals, trapped to form stable adducts (spin trapping
method), are reviewed and compared with assays based on spectrophotometric and
voltammetric methods.
All of these methods, widely used in the food industry, differ for the antioxidant
mechanism involved, the analytical conditions applied in terms of substrate, solvents and
range of concentrations, the instrumentation used and the outcome they provide. This
review thus focuses on the effect of each analytical parameter on the antioxidant activity
evaluation.
When selecting an appropriate method for the estimation of the antioxidant activity,
any interference should be known in advance. In addition an overall evaluation of the
antioxidant activity should be based on a comparison of different methods in order to
overcome the limitations of the method itself. Hence this review provides a basis for the

Corresponding author: Daniele Sanna, e-mail: Daniele.Sanna@icb.cnr.it

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Angela Fadda and Daniele Sanna


choice of the best antioxidant activity method applicable to a particular nutraceutical or
dietary supplement in a particular matrix.

INTRODUCTION
The free radicals and the dietary antioxidants have received much attention in the last
years since it has been claimed that they are involved in the regulation of several biological
processes. Before considering the available methods for the determination of the antioxidant
activity it is better to clarify what an antioxidant is and what antioxidant activity means.
An antioxidant can be defined as any substance that when present at relatively low
concentrations, compared with those of the oxidizable substrate, significantly delays or
inhibits oxidation of that substrate [1]. This is a broad definition that encloses the chemical,
biochemical and biological meaning of the word antioxidant. In its chemical significance an
antioxidant is a compound that stops or delays oxidation by breaking down radical chain
reactions, therefore it is mainly associated to a radical scavenger. In food science antioxidants
are claimed to have a double role: they protect food from lipid oxidation, which is the major
cause of deterioration, and as dietary antioxidants they protect cells and tissues from the
adverse effects of reactive species. In the first meaning they act essentially as radical
scavengers but as dietary antioxidants the phytochemicals present in fruits and vegetables
may, not only act as radical scavenger but have different targets inside the cells. Very recently
Forman et al. [2] shifted the action of nutritional antioxidants inside the cell system from free
radical scavengers to stimuli in the cellular signaling pathways that results in the induction of
the expression protective enzymes and in the increase of substrates like glutathione,
thioredoxin and NADPH.
In many papers dealing with food antioxidants the terms radical scavenging activity
and antioxidant activity are often, improperly used as synonyms. The first is related only
with the capacity to react with free radicals preventing the propagation of chain reactions
while the second is more general and includes also the reactivity towards oxidant species.
Moreover other terms, such as antioxidant activity and antioxidant capacity are normally
used as synonyms but a different meaning for the two terms has been proposed: antioxidant
activity can be related to the kinetics of the reaction with an antioxidant, whereas
antioxidant capacity deals with the thermodynamic conversion efficiency of the same
reaction [3].
In biological systems, it has been suggested that the antioxidants may exert their action
by: (i) quenching free radicals; (ii) chelating redox metals; (iii) regenerating other
antioxidants within the antioxidant network; (iv) displaying a positive effect on gene
expression; (v) being readily absorbed; (vi) having a concentration in tissues and biofluids at
a physiologically relevant level; (vii) working in both the aqueous and/or membrane domains
[4]. The capacity of antioxidants to chelate redox metals is frequently claimed as one of the
requirements of a good antioxidant. However this is an oversimplification because the
chelation of a metal ion not necessarily modifies its redox activity. Chelated Fe(II) ions can
certainly be oxidized to the corresponding Fe(III) complex or vice versa, what has changed
after the complex formation is the standard reduction potential of the couple Fe(III)/Fe(II).
Therefore a redox active metal ion could become inactive only if the standard reduction
potential of the couple changes considerably, otherwise it would remain active. Furthermore

Advantages and Pitfalls of the Methods for the Antioxidant Activity Evaluation

67

in solution the aqua-ions exist only in very acidic solutions, whereas at physiological pH, the
metal do not exist as free or aqua-complex (i. e. Fe3+ or [Fe(H2O)6]3+, respectively) but as a
complex coordinated by ligands eventually present in solution.
Six major reactive oxygen species (ROS) have been identified in biological systems
causing oxidative damage. These species are: superoxide (O2), hydrogen peroxide (H2O2),
peroxyl radicals (R-OO), hydroxyl radical (OH), singlet oxygen (1O2) and peroxynitrite
(ONOO) [5].
Cellular systems protect themselves by the effects of ROS with enzymatic and nonenzymatic antioxidants. Enzymatic antioxidant systems consist of Superoxide Dismutase,
Catalase and Glutathione Peroxidase, while between non-enzymatic antioxidants can be
included vitamin C, vitamin E, carotenoids, glutathione, thioredoxin, -lipoic acid, flavonoids
and others.
Typically, the efficiency of an antioxidant is influenced by its chemical structure and by
other factors like concentration, type of substrate and pH.
All the analytical methods, developed so far, that measure the efficacy of an antioxidant
vary for the type of oxidant involved and for the antioxidant reaction mechanism. In this
contest, the proper method for the estimation of the antioxidant activity should be chosen on
the basis of the aim pursued. For example if we examine the biological significance of an
antioxidant the study should focus on very dangerous radical species such as hydroxyl and
superoxide and not on relatively stable radical like DPPH or ABTS which give meaningless
results from a biological point of view. By contrast the spin trapping method coupled with
Electron Spin Resonance (ESR)1 spectrometry allows the direct determination of extremely
reactive radical species once trapped to form relatively stable adducts.
Usually antioxidant assays are classified considering the type of reaction: (i) hydrogen
atom transfer (HAT) and (ii) electron transfer (ET) based assays. This classification is
sometimes misleading because the difference between the two reactions is a proton and, at
least in protic solvents, the reduction reaction, that is the addition of one electron, can be
easily followed by the abstraction of a proton from the solvent and because in some assays
both mechanisms can be operating.
This review will provide an overview of the most used methodologies for the
determination of antioxidant capacity, placing particular emphasis on the advantages, the
pitfalls and the possible developments of each method. In particular the methods based on the
Electron Spin Resonance (ESR) spectroscopy with direct measurement of relatively stable
radical species or unstable radicals, trapped to form stable adducts (spin trapping method), are
reviewed and compared with assays based on spectrophotometric and voltammetric methods.

SPECTROPHOTOMETRIC METHODS
Spectrophotometric methods are based on the increase or decrease of absorbance at a
specific wavelength, usually in the visible range. The absorbance decrease is the result of the
reduction of the free radical by the antioxidants leading to a species which absorb at a
different wavelength as in the DPPH and ABTS assays. However the reactions can be more
complicated and involve a series of reductions and oxidations which generate, at the end, a
1

Also known as Electron Paramagnetic Resonance (EPR) spectroscopy.

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Angela Fadda and Daniele Sanna

color increase or decrease. The main disadvantage of these methods is that one is not sure
whether or not the color change is really due to the reaction which should take place or if
there are interfering substances which lead to the same experimental result. On the other side
these methods have the advantage, once all the reactants have been prepared, of being very
simple by the experimental point of view and the treatment of the data is very easy. Moreover
the instrumentation required to perform these assays is a spectrophotometer, which is
relatively common in analysis or research laboratories.

DPPH Assay
The DPPH (1,1-diphenyl-2-picrylhydrazyl) method was first proposed by Blois [6].
DPPH is an intensely colored radical species and the assay is based on the measurement of
the decrease of absorbance at 517 nm, corresponding to the amount of radical reduced to
hydrazine (see Figure 1).
The reduction takes place after the formal addition of a hydrogen atom.

Figure 1. Reduction of the stable radical 1,1-diphenyl-2-picrylhydrazyl (DPPH).

Usually the parameters reported for the quantification of the antioxidant activity
measured with the DPPH assay are trolox or L-ascorbic acid equivalents, % of inhibition and
the EC50 value. This latter value expresses the concentration of antioxidant necessary to
decrease of 50% the concentration of DPPH initially present in solution and can be obtained
by drawing a plot percentage of inhibitionconcentration of the antioxidant. The percentage
of inhibition, in its turn, is defined by the following expression: % inhibition = 100 % (A0
AS)/A0, where A0 is the absorbance at 517 nm of the DPPH solution without the antioxidant
and AS is the absorbance of the sample.
The main pitfalls of the DPPH assay are reported below and some of them are better
understood if the results of the spectrophotometric method are compared with those obtained
with ESR [7]. Specific interferences in the spectrophotometric assay are due to the absorption
at 517 nm by the antioxidants or their oxidation products. The reduction product of DPPH has
low absorption at 517 nm, but this should be quantified and taken into account for data
correction (the following expression for calculating the % of inhibition = 100 % (A0 AS)/(A0
Ai) where Ai is the absorbance of the solution when 100% of DPPH is reduced, should be
used).
UV-Vis spectra in a large enough wavelength range instead of single reading at 517 nm
should be measured; in this way it is easier to identify and recognize precipitation phenomena
(see beyond).

Advantages and Pitfalls of the Methods for the Antioxidant Activity Evaluation

69

To attribute the real importance to the antioxidant activity measured with this assay we
should examine what we are really measuring. In this assay we are measuring the ability of an
antioxidant to reduce the DPPH radical to the corresponding hydrazine (see Figure 1). To do
this the antioxidant should be able to formally donate a hydrogen atom under the
experimental conditions in which the assay is carried out, using the solvents where DPPH is
soluble. For hydrophilic antioxidants the assay is not applicable and in different solvents the
reactivity of the antioxidants could be distinct. Moreover steric effects should be considered
in the DPPH assay, since only unhindered antioxidants can easily reduce this free radical. In
any case this assay has nothing to do with reactivity towards biologically important radical
species such as hydroxyl or superoxide. Therefore the biological importance of this assay is
limited and its frequent use is only due to its simplicity.
Another parameter poorly considered when measuring the antioxidant activity with this
assay is the time needed to reach the end of the reaction. It was shown in the literature [8] that
the kinetics of the reaction between DPPH and the antioxidants can be extremely variable and
the speed of the reaction can be classified as slow, intermediate or rapid. In order to quantify
this different reactivity a new parameter was introduced, TEC50, the time needed to reach the
steady state. Therefore it is necessary to know in advance the kinetics of the reaction between
DPPH and the antioxidant or plant/food extract in order to measure reliable values of the
antioxidant activity [9]. The EC50 values measured after the same time span for the same class
of compounds, i.e., for antioxidants which show the same kinetic behavior, can be profitably
compared, while the comparison of EC50 values obtained at very different time spans has low
practical utility. In order to have directly comparable EC50 values, exactly the same method,
that is DPPH concentration and solvent or mixture of solvents, should be applied to different
antioxidants, since it was recently shown that these variables influence the experimental
results [9]. Therefore the need for standardization of the method is perceived [3].
To try to overcome the limitations of the DPPH assay and to take into account both the
EC50 values and the kinetic of the reaction between DPPH and the antioxidants, a new
parameter was introduced: the antiradical efficiency (AE), defined as AE = 1/(EC50 x
TEC50) [8]. The lower the EC50 value (higher antioxidant activity) and the lower the TEC50
value (faster kinetic reactivity), the higher the AE value. This new parameter allows for
discriminating between two antioxidants which have similar EC50 values but react with DPPH
with different kinetics (different TEC50 values). Between these two antioxidants the one
having the higher AE value is considered better than the other; in fact free radicals are very
reactive species and an antioxidant which reacts quickly is certainly more efficient than one
which reacts slowly. For antioxidants having a complex mechanism with a fast kinetic step
followed by a slow one it was supposed that the formal abstraction of an hydrogen atom (or
the electron transfer if ET mechanism is supposed) proceeds quickly, while the subsequent
decay was attributed to secondary slow reactions between the products of reaction derived
from the antioxidants, which in some cases are relatively stable radicals [10].

ABTS Assay
This assay is based on the reduction of the radical cation of 2,2-azinobis-(3ethylbenzothiazoline-6-sulfonic acid), ABTS+, obtained by the oxidation of ABTS with

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Angela Fadda and Daniele Sanna

potassium peroxydisulfate [11] (see Figure 2).2 This assay can be seen and considered as a
measure of the capacity of antioxidants to reduce the radical cation ABTS+ (or in aqueous
media, where the sulfonic groups are deprotonated, of the radical anion ABTS) to ABTS (or
ABTS2). This capacity is directly related to the reduction potential of the antioxidants, and
only those with E0 values lower than that of the couple ABTS+/ABTS (E0 = 0.68 V) are able
to perform this reduction.
This assay can be used to determine the TEAC, trolox equivalent antioxidant activity, of
a specific antioxidant or plant extract. TEAC is an dimensionless value, particularly useful
because it allows to compare the antioxidant activity of different antioxidants, all referred to
trolox (6-hydroxy-2,5,7,8-tetramethychroman-2-carboxylic acid) used as a standard. However
in many cases, when plant extracts or fruit juices are analyzed, the reported TEAC values are
not dimensionless but expressed as mg of trolox/100 g of fresh weight or mg of trolox/100
mL of extract or juice.

Figure 2. ABTS and its oxidation product ABTS+.

For the determination of the TEAC value a plot percentage of inhibitionconcentration


for the standard, trolox, should be drawn in advance. The percentage of inhibition is defined
as previously shown for DPPH, that is % inhibition = 100 % (A0 AS)/A0, where A0 is the
absorbance at 734 nm of the ABTS+ solution without the antioxidant or plant extract and AS
is the absorbance of the sample.
If an analogous plot is obtained for the antioxidant or antioxidant mixtures under
evaluation, the TEAC value is simply obtained by dividing the slope of the line of the
antioxidant by that of trolox.
If only one measurement of percentage of inhibition is available for the antioxidants, the
TEAC value can be obtained by dividing the concentration of the antioxidant by the trolox
concentration which gives the same percentage of inhibition. Therefore the assay is very
2

Since sulfonic acid groups are strongly acidic can easily deprotonate in the presence of proton acceptors; in this
case ABTS exists in solution as the dianion, ABTS2, and therefore its oxidized form as radical anion, ABTS.

Advantages and Pitfalls of the Methods for the Antioxidant Activity Evaluation

71

simple and only one measurement (with at least three replications) is sufficient for the
determination of the TEAC value of a particular antioxidant.
The ABTS radical cation has an intense blue-green color with different absorption
maxima (645, 734 and 815 nm) in the visible region, but usually the reading at 734 nm is
used in the assay. However if the antioxidants or their oxidation products absorb at this
wavelength one can decide to use another absorption maximum or correct the results for the
absorbance of the interfering substances. Another problem which could be encountered when
applying this assay is due to the solubility of antioxidants.
According to Re et al. [11] the assay is applicable to lipophilic and hydrophilic
antioxidants, by mixing stock solutions of these latter in ethanol, dichloromethane or water,
depending on the solubility, with the water/ethanol solution containing the radical cation.
Another potential pitfall is connected with the comparison of TEAC values of lipophilic and
hydrophilic antioxidants. Since for lipophilic antioxidants probably dichloromethane is used,
while for hydrophilic ones water or ethanol is employed, the influence of the solvent
composition of the final mixture on the measured TEAC values was investigated [12, 13],
showing that this is an essential parameter on chemical behavior of antioxidant compounds.
The main disadvantages of the method are connected with the preparation of the radical
cation solution which isnt immediate since the oxidation reaction by peroxydisulfate takes at
least 1216 hours; another limitation is that this solution is stable for a relatively short period
of time.
One of the advantages of the assay is that, once the ABTS+ solution has been prepared,
the measurements are very rapid and the readings at 734 nm are made after 1, 4 or 6 minutes.
However, for antioxidants which react slowly, the reaction could be not completed after these
short time spans [10].

FRAP Assay
The ferric ion reducing antioxidant power assay (FRAP) is a typical ET based method
that measures the reducing power of the antioxidants. It was originally employed to measure
the reducing power in plasma, but its use has been extended to other biological fluids, foods
and plant extracts.
The method is based on the ability of antioxidants to reduce the yellow ferric
tripyridyltriazine complex (Fe(III)-TPTZ, TPTZ = 2,4,6-tripyridyl-1,3,5-triazine) to the blue
ferrous complex Fe(II)-TPTZ (Figure 3).
The reaction is carried out under acidic conditions (pH= 3.6) in order to maintain iron
solubility. The ferric reducing capacity of the antioxidant compounds is measured
spectrophotometrically reading the absorbance at 593 nm after 4 minutes of reaction. The
change of absorbance (A = A4min A0min) is calculated and results are expressed as
micromolar Fe(II) equivalents or relative to an antioxidant standard. Typically a FRAP unit is
defined as the reduction of 1 mol of Fe(III) to Fe(II). Likewise TEAC assay the use of such a
short endpoint (4 minutes) may underestimate FRAP results of those polyphenols, like caffeic
acid, ferulic acid or quercetin that show slow reaction kinetics. In these cases the absorption
at 593 nm does not stop growing after 4 minutes but even increases after several hours of
reaction [14].

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Angela Fadda and Daniele Sanna

Figure 3. The iron complex of TPTZ (2,4,6-tripyridyl-1,3,5-triazine). Iron changes its oxidation state
from +III to +II during the reaction with antioxidants changing its color from yellow to blue.

The advantage of the FRAP assay is that is simple, rapid and does not require specialized
equipment. However the information obtained by this assay is comparable to that of ABTS as
the redox potential of the Fe(III)/Fe(II) pair is 0.70 V, quite similar to that of ABTS radical.
Moreover if in the extract to analyze is present any electron donating substance, with a redox
potential lower than the Fe(III)/Fe(II) couple it can overestimate the FRAP results. The same
could occur if the food mixture contains other Fe(III) species that can react with antioxidants.
Although tripyridyltriazine (TPTZ) is the most used iron-binding ligand, alternative
ligands such as ferrozine (3-(2-pyridyl)-5,6-diphenyl-1,2,4-triazine-p,p-disulfonic acid) and
more recently potassium ferricyanide ([Fe(CN)6]3) have also been employed. In the
ferricyanide FRAP assay the Prussian Blue is the end product of the reaction and is measured
spectrophotometrically.
The disadvantage of this ligand is that Prussian blue precipitates and stains the cuvette
leading to erroneous results. To avoid precipitation a surfactant, sodium dodecyl sulphate,
was added to the reaction mixture and the pH was reduced to 1.7 to maintain the redox
activity of the ferric ion. According to Berker et al. [15], this modifications allow to extend
the antioxidant evaluation even to thiol type nonenzymatic antioxidant like glutathione which
is able to reduce Fe(III) to Fe(II) but forms a stable complex with Fe(II) [16] that does not
absorb at the same wavelength as the complex Fe(II)-TPTZ and therefore is not quantified in
the assay.

CUPRAC Assay
The CUPRAC (Cupric Reducing Antioxidant Capacity) method of antioxidant capacity
measurement [17] uses the bis-chelated copper(II) complex of neocuproine (2,9-dimethyl1,10-phenanthroline). [Cu(neocuproine)2]2+ is reduced by the antioxidants eventually present
in the system under examination to the corresponding copper(I) complex
(see Figure 4).

Advantages and Pitfalls of the Methods for the Antioxidant Activity Evaluation

73

Figure 4. Reduction of [Cu(neocuproine)2]2+ to [Cu(neocuproine)2]+ in the CUPRAC reaction.

The Cu(II) complex has a light blue color, while its reduction product,
[Cu(neocuproine)2]+, is yellow-orange with an absorption maximum at 450 nm. The
absorption increase at this wavelength is a measure of the capacity of the antioxidants to
reduce Cu(II) to Cu(I). The antioxidants are able to reduce Cu(II) to Cu(I) only if their
standard reduction potential is lower than 0.6 V, the E0 value of the couple
[Cu(neocuproine)2]2+/[Cu(neocuproine)2]+.
If the standard reduction potential of an antioxidant is larger than 0.6 V this wont be able
to reduce Cu(II) to Cu(I) and therefore its antioxidant capacity measured with this assay will
be negligible. However the same antioxidant could be an efficient scavenger of free radicals if
different assays are considered instead of CUPRAC. The reduction potential of the couple
Cu(II)/Cu(I) can be slightly changed if neocuproine is replaced by similar ligands. ABTS,
FRAP and CUPRAC methods have the common feature of measuring the antioxidant
capacity against a redox couple whose reduction potential is fixed and known in advance.
Therefore they measure the antioxidant activity only by an electrochemical point of view and
have nothing to do with the ability of antioxidants to scavenge very reactive species (hydroxyl
radical, superoxide, etc.) which can be extremely dangerous in biological systems.

ORAC Assay
The Oxygen Radical Absorbance Capacity assay was first proposed by Glazer et al. [18],
then improved by Cao et al. [19]. The method was developed to study the radical chain
breaking properties of antioxidants and provides an indirect measure of the inhibition of
peroxyl radicals induced oxidation. In food chemistry this method is frequently used to study
the inhibition of lipid oxidation operated by several natural antioxidants [20, 21]. ORAC is an
HAT-based method that measures the hydrogen atom donating ability of antioxidants. The
ORAC method employs as a marker of oxidative damage the fluorescence decay caused by
the reaction of a fluorescent probe with the peroxyl radicals. The inhibition of the
fluorescence decay in presence of an antioxidant is a measure of the antioxidant activity. The
fluorescence intensity (485 nm (ex)/525 nm (em)) is measured for 35 minutes at room
temperature. In the first experiments the outcomes of the peroxyl radical scavenging activity
were provided by the measure of lag time before the active decay of the probe. The longer the
lag time the higher the radical quenching effect. However, in many cases the use of the lag
time to estimate the antioxidant activity may be problematic for those antioxidants without a
clear lag phase or for those with no lag phase at all. In order to overcome these limitations

Angela Fadda and Daniele Sanna

74

ORAC values are now calculated as the difference between the net integrated area under the
fluorescence decay curves (AUC) of the sample compared to that of the blank. The AUC
approach is particularly useful for food or plant extracts which are mixtures of antioxidants
with different reaction kinetics. Typically ORAC values are expressed as trolox equivalents
(TE) following the formula:
ORAC (U/mL) = [(AUCsample AUCblank)/(AUCTrolox AUCblank)]xMTrolox/Msample =
M TE
where M is the concentration in mol L1 for pure phytochemicals or ORAC (U/mL) =
(AUCsample AUCblank)/(AUCTrolox AUCblank) in the case of plant or food extracts when
molarity is unknown.
The most commonly used peroxyl radical generator in ORAC assay is the hydrophilic
AAPH (2,2'-azobis(2-amidinopropane) dihydrochloride). At 37 C AAPH undergoes thermal
decomposition loosing nitrogen (N2) and generating AAPH radical which rapidly reacts with
O2 giving the peroxyl radical ROO. As highlighted by Shahidi et al. [22], the maintenance of
the proper temperature is one of the main concerns that may influence the results of the
ORAC assay as the generation of the peroxyl radicals is temperature dependent.
In the original assay the fluorescent probe was -phycoerythrin (B-PE) a protein isolated
from Porphyridium cruentum, however it was found to suffer from some limitations
associated with a large lot variability, photobleaching by excitation light and interactions with
polyphenols. In the further developments of the method -phycoerythrin was replaced by
fluorescine, a synthetic non protein probe.
Fluorescine is pH sensitive and its use should be carefully followed, moreover the use of
fluorescine is limited to hydrophilic antioxidants since this fluorescent probe is lipid insoluble
and its fluorescence intensity decreases in non polar organic solvents.
In its original designation and even after the employment of fluorescine the ORAC assay
was limited to hydrophilic chain breaking antioxidants. The ORAC assay has been modified
to measure lipophylic antioxidants by using cyclodextrins to enhance water solubility. As
alternative a new fluorescent probe BODIPY 665/676 ((E,E)-3,5-bis-(4-phenyl-1,3butadienyl)-4,4-difluoro-4-bora-3a,4a-diaza-s-indacene) and AMVN (2,2azobis(2,4dimethylvaleronitrile)) as new peroxyl radical generators were developed to extend the assay
to lipophylic antioxidants. In this way the assay could be carried out in non polar solvents and
in liposome matrix. The only disadvantage is its low sensitiveness as compared to the classic
ORAC assay likely due to the low efficiency of the radical generator [23].
By changing the radical initiator the ORAC assay could be used for the detection of OH
radical. Ou et al. [24] employed the fluorimetric basics of the ORAC assay to study the
hydroxyl radical scavenging activity of several antioxidants by using Co(II) as a radical
generator and fluorescine as a probe.

NBT Assay
It has been reported [25] that the superoxide radical, O2, can be generated in a system
containing xanthine oxidase (XO) and hypoxanthine (or xanthine) where this latter is oxidized
to xanthine and then to uric acid (see Figure 5).

Advantages and Pitfalls of the Methods for the Antioxidant Activity Evaluation

75

Figure 5. Oxidation of hypoxanthine to xanthine and to uric acid by Xanthine Oxidase (XO).

The superoxide thus generated reduces the yellow, water soluble nitroblue tetrazolium
(NBT) to blue, water insoluble formazan (see Figure 6).

Figure 6. Nitroblue tetrazolium and reduction of the tetrazole ring with formation a radical
intermediate.

As can be seen in Figure 5 the enzymatic reaction normally generates peroxide starting
from water and oxygen, but in some circumstances could produce superoxide.
This is a very complicated system because a series of chemical reactions give, at the end,
a color change which can be detected with a spectrophotometer (reading at 560 nm). It
implies that: (i) oxygen is present in the system as a reactant, since it must accept one electron
to be reduced to superoxide; (ii) an enzyme, apparently very sensitive to inhibition by a large
number of substances, between others flavonoids [1] and metal ions [26], should catalyze the
oxidation of hypoxanthine or xanthine to uric acid, using O2 as the electron acceptor; (iii) a
substrate, nitroblue tetrazolium, should be finally reduced to blue formazan by the superoxide
eventually formed.
The reduction of nitroblue tetrazolium to blue formazan is a stepwise four electron (two
for each tetrazole ring) reaction which involves the opening of two tetrazole rings of NBT
(see Figure 6). This process generates, as intermediates, two radicals and one stable

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intermediate, when only one of the two tetrazolium rings is reduced. Due to this complex
chemistry, further complicated, in aqueous solutions, by its acid-base properties, the NBT is
not suitable for the quantitative evaluation of superoxide radical concentrations [27], in fact
the intermediate species could be relatively stable leading to erroneous evaluation of the
stoichiometry of the reaction.
Moreover nitroblue tetrazolium could be reduced by other reducing agents present in the
system, as recently reported in the case of folic acid [28], and therefore in these cases the
supposed production of superoxide would be overrated or the activity of antioxidants,
eventually present, underestimated.
In the literature modifications of the original method were proposed but maintaining the
use of NBT.
A chemical method to produce superoxide based on the use of NADH and phenazine
methosulfate was reported [29]. In this system NADH should reduce phenazine to
dihydrophenazine being oxidized to NAD+; in its turn dihydrophenazine should reduce
molecular oxygen to superoxide. However it was demonstrated that in this system the
production of superoxide is mediated by tetrazolium, not by phenazine [30].
The riboflavinlightNBT system is a photochemical method for the production of
superoxide radical [31]; photoreduced riboflavin can interact directly with NBT or can reduce
it with superoxide as an electron carrying intermediate: in the presence of oxygen the latter
pathway is preferred. However this introduces an important variable, which is the oxygen
concentration or its partial pressure, rendering impossible the exclusion of a direct interaction
between riboflavin and NBT.
The employment of the spectrophotometric methods based on the use of nitroblue
tetrazolium are strongly discouraged, because in most of the cases the supposed formation of
superoxide is far from being unambiguously demonstrated. For the quantification of
superoxide radical concentration spin trapping methods are better suited and reliable (see
below, Radical generating systems); in fact with these methods it is possible to establish
without a doubt if superoxide is effectively formed in the system under examination and the
complicated chemistry of nitroblue tetrazolium is overcome since its use is not necessary.

-Carotene Bleaching Assay


The -carotene bleaching assay is a popular method for the estimation of the antioxidant
activity in food samples. It analyses the ability of food or plant extracts to react with peroxyl
radicals which are the major cause of food quality deterioration. The -carotene bleaching
assay is classified as HAT method and estimates the antioxidant activity in oil-in-water
emulsions, so is particularly suitable for lipophylic antioxidants.
In this method the peroxyl radicals generated from the oxidation of linoleic acid in the oil
water emulsion causes the discoloration of the -carotene which is followed
spectrophotometrically at 470 nm for 180 minutes. The antioxidant present in the emulsion
reacts with the peroxyl radical and prevents the -carotene bleaching. In this method the
antioxidant activity is measured in terms of kinetics and is the result of the competition
between the antioxidant activity of the sample and that of the -carotene. Very recently
Dawidowicz and Olszowy [32, 33] demonstrated that type and amount of solvent in which the
antioxidants are dissolved, the type and the concentration of transition metal ions and the

Advantages and Pitfalls of the Methods for the Antioxidant Activity Evaluation

77

hydrogen ion concentration in the mixture influence the estimation of the antioxidant activity.
The peroxyl radical generation is inhibited by the presence of hydrogen ions whereas alkaline
metal ions accelerated the formation of peroxyl radical thus increasing the bleaching rate of
-carotene. This aspect is particularly important for food or plant extracts which may have
different concentrations of organic acids that influence the estimation of the antioxidant
activity even if they do not exhibit antioxidant activity.

Electron Spin Resonance methods


Electron spin resonance (ESR) spin trapping is a powerful technique for the detection of
stable and transient radicals formed during redox processes in vivo and in vitro. Free radicals
are high reactive and generally short-lived molecules with one or more unpaired electrons.
The majority of the free radicals, due to their very short lifetime, are undetectable with most
of the analytical techniques.

DPPH Assay
DPPH is a relatively stable radical species; it exhibits a five line spectrum when is
dissolved in organic solvents (EtOH, MeOH and their aqueous mixtures), while it shows a
narrow single line in the solid state or when forms aggregates. This is due to exchange
narrowing, a phenomenon first described by Van Vleck [34].
Aggregation phenomena, which usually anticipate and accompany precipitation, are due
to the insolubility of DPPH in aqueous media or in solvent mixtures containing high amounts
of water; aggregation and precipitation are however difficult to be distinguished because of
the intense color of the solutions containing DPPH.
ESR measurements should be performed whenever possible in order to be sure that there
are not interfering substances or their interferences were correctly taken into account, that
DPPH radical is effectively reduced and that there arent aggregation phenomena.
All the advantages and pitfalls of the DPPH assay were previously reported when
describing this assay in the spectrophotometric methods.

Spin Trapping of Reactive Radicals


Spin Traps Used in Food Analysis
The spin trapping method coupled with ESR spectroscopy is one of the most specific and
reliable techniques for the detection of high reactive radicals in chemical and biological
systems [35]. Spin traps are diamagnetic radical stabilizers that convert transient radicals into
stable, detectable and long-lived radical adducts [36]. ESR spectroscopy provides a specific
identification and quantification of the spin trap radical adducts from the multiplet structure
of the spectrum. In food chemistry the spin trapping method coupled with ESR spectroscopy
is used to evaluate the antioxidant activity of food, beverages and plant extracts [37-42]. The
method is based on the competition between the trapping agent and the antioxidant. The

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Angela Fadda and Daniele Sanna

relative intensity of the ESR radical adduct signal is used to estimate the radical scavenging
activity against biologically relevant radicals (O2 and OH). The spin trapping method is also
used in food analysis to study the oxidative stability of alcoholic beverages [40, 41].
Typically two major classes of spin traps are available: nitrone and nitroso probes. In
food analysis the nitrone spin traps are the most widely used, with these including the cyclic
nitrones 5,5-dimethyl-1-pyrroline N-oxide (DMPO), 5-diethoxyphosphoryl-5-methyl-1pyrroline N-oxide (DEPMPO), 5-tert-butoxycarbonyl-5-methyl-1-pyrroline N-oxide (BMPO)
and acyclic species N-tert-butyl--phenylnitrone (PBN) and -(4-pyridyl-1-oxide)-N-tertbutylnitrone (POBN) (see Figures 7 and 8).

Figure 7. DMPO adducts of hydroxyl (DMPO-OH) and superoxide radical (DMPO-OOH) and the
corresponding spectra.

The performance of a spin trap depends on its concentration, the concentration of the
transient radicals produced, the kinetics of the adduct formation and its stability [43]. The
choice of the proper spin trap and the best experimental conditions play a pivotal role in the
success of the experiment as inappropriate choices may result in a low or absent signal even
though high radical concentration is present.
In food biochemistry DMPO is the most frequently used spin trap [39, 44]. It is employed
to trap oxygen centered radicals O2 and OH yielding DMPO hydroxyl (DMPO-OH) and
DMPO superoxide (DMPO-OOH) adducts (see Figure 7).
In aqueous solution DMPO-OH adduct has a rather short half-life [45]. The decay rates
are strongly influenced by the reaction conditions. In particular pH, temperature and the
presence of metal ions may have dramatic effects on the decay rates of the adduct. The
presence of transition metal ions makes the detection of superoxide radical O2 with DMPO
quite difficult since the radical adduct DMPO-OOH may undergo rapid decay due to a
reduction to the corresponding alcohol giving the same spectra of the radical adduct DMPOOH. Decomposition of DMPO-OOH can occur through different reaction pathways
(heterolytic, hemolytic and redox cleavage of the OO bond) [46]. Even the purity of the

Advantages and Pitfalls of the Methods for the Antioxidant Activity Evaluation

79

spin trap is a factor to be taken into account. DMPO is often supplied with impurities which
can lead to ESR-detectable radicals [47]. As pointed out by Hawkins and Davies [35] even
spin traps with a purity higher than 99% may have excessive impurities due to the high
concentration of spin traps used in many ESR experiments.

Figure 8. Nitrone spin traps DEPMPO, BMPO, PBN and POBN.

The presence of other free radicals in the testing systems may also determine an
underestimation of the free radical scavenging properties of an antioxidant sample.
Some organic solvents like ethanol and DMSO may act as OH scavengers producing
carbon centered radicals [48]. Determining the antioxidant properties of Tokay wines Stako
et al. [41] observed as a minor byproduct the sextet characteristic spectrum of carboncentered radicals added to DMPO. Similarly in aprotic solvents like DMSO the formation of
the DMPO-OOCH3 prevails [49].
In order to overcome the described limitations of DMPO, other spin trap agents with
longer lifetime, less decay of the spin adducts and a faster reaction kinetic were developed. 5Diethoxyphosphoryl-5-methyl-1-pyrroline N-oxide (DEPMPO) and 5-tert-butoxycarbonyl 5methyl-1-pyrroline N-oxide (BMPO) have been frequently used to detect free radicals in
different biological systems [35, 50].
DEPMPO is a phosphorilated analog of DMPO with a lifetime of the OH adduct nearly
ten times longer than DMPO-OH [45]. In food biochemistry DEPMPO is frequently used to
study the superoxide (O2) radical scavenging activities of food and plant extracts [51-54]. Its
advantage over DMPO is the longer half-life of the adduct [55, 56] and the possibility to
distinguish, from the spectrum characteristics, the DEPMPO-OH adduct formed via

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Angela Fadda and Daniele Sanna

DEPMPO-OOH/DEPMPO-OH transformation from the DEPMPO-OH adduct in OH radical


generating systems [43].
In food analysis the use of BMPO as trapping agent is not yet widespread [57, 58].
BMPO was first synthesized by Zhao et al. [59] to trap both O2 and OH radicals. In radical
generating systems BMPO superoxide adduct is much more persistent than DMPO-OOH as it
does not readily decay non enzymatically into the corresponding hydroxyl adduct. Results
indicate that the ESR spectrum of BMPO-OOH could be detected even up 35 minutes [59].
Another advantage of this spin trap agent is that aqueous solutions of BMPO may be
conveniently stored at 20 C without any artificial signal formation [59].
Moreover BMPO adducts have relatively simple spectra that make this trap more suitable
for quantitative than qualitative analysis in radical generating systems [43].

Radical Generating Systems


ESR has been used to explore the radical scavenging properties of dietary antioxidants
against O2 and OH radicals. Hydroxyl and superoxide radicals are biologically relevant
radicals that can be produced in vitro using different radical generating systems.
For ESR determination radical generating systems should provide a constant radical flux
throughout the assay time frame, moreover a good radical generating system should generate
radicals without interference from other ROS.
Moore et al. [48] classified the OH generating systems into 5 categories, based on the
reactions used for radical generation. OH is commonly produced using the Fenton reaction,
however some alternatives including the Fenton like Co(II)/H2O2, Cu(I)/H2O2 reactions [48]
and the thermal decomposition of K2S2O8 [60] were used.
In the Fenton reaction iron(II) is oxidized by hydrogen peroxide to iron(III), forming a
hydroxyl radical and a hydroxide ion in the process.
Fe2+ + H2O2 Fe3+ + OH + OH
In the Fenton Fe2+/H2O2 radical generating system the OH production is quite stable,
however some experimental conditions may influence the Fe2+/H2O2 OH production. In the
literature, most of the reactions are performed in phosphate buffer at physiological pH. Fe(II)
is readily oxidized at neutral pH, whereas is relatively stable in acid solutions [61]. At neutral
pH a buffer with coordinating properties is required to keep Fe(II) and Fe(III) in solution thus
avoiding the hydrolysis and precipitation of the corresponding hydroxides. Phosphate is the
most frequently used buffer to simulate the biological conditions, however it was
demonstrated that the ESR signal of the DMPO-OH adduct in phosphate buffer solution was
remarkably lower than in other buffers (tris-HCl, sodium acetate, sodium trifluoroacetate) at
the same experimental conditions [62]. The ESR detection of the DMPO-OH adduct in
phosphate buffer is possible only when a high concentration of spin trap is used [63] due to
the quenching effect of Fe(II) enhanced by phosphate ions.
Cheng et al. [64] discussed the effects of the most commonly used organic solvents on
the concentration of OH radicals. Hexane, cyclohexane and chloroform at the final
concentration of 5% in the Fenton reaction system slightly reduced the DMPO-OH signal

Advantages and Pitfalls of the Methods for the Antioxidant Activity Evaluation

81

intensity. By contrast under the same experimental conditions benzene, ethyl acetate and
toluene significantly decreased the ESR signal intensity [64].
In food analysis the Fenton Fe2+/H2O2 OH generating system has some limitations when
measuring food matrices containing L-ascorbic acid (or vitamin C). In this case an
overestimation of the OH radical scavenging activity often occurs due to the pro-oxidant
properties of L-ascorbic acid. Vitamin C reduces oxidizing substances like hydrogen peroxide
but can also reduce metal ion thus producing free radicals. In the Fenton system, L-ascorbic
acid can recycle Fe(III) to Fe(II) facilitating further generation of reactive oxygen species by
subsequent Fenton cycles:
2 Fe2+ + 2H2O2 2 Fe3+ + 2 OH + 2 OH
2 Fe3+ + L-Ascorbate 2 Fe2+ + L-Dehydroascorbate
A similar pro-oxidant activity was observed for trolox (6-hydroxy-2,5,7,8tetramethychroman-2-carboxylic acid) the water soluble analog of vitamin E, thus preventing
the expression of results as trolox equivalents.
The main disadvantage of the Fenton Fe2+/H2O2 OH radical generating system is that is
not suitable for lipophilic antioxidants. Sometimes systems containing H2O2 and Fe(III)
instead of Fe(II) are referred as Fenton-like reactions [64].
However in these cases, if a reductant is not present to reduce Fe3+ to Fe2+ and allow a
normal Fenton reaction to take place, the main reaction is the disproportionation of hydrogen
peroxide into water and dioxygen (2 H2O2 H2O + O2) catalyzed by iron(III). The eventual
spin trapping of hydroxyl radicals observed in these cases is due to the participation of this
radical in the mechanism and not to a Fenton-like reaction [65].
The thermal decomposition of potassium peroxydisulfate, K2S2O8, was used as OH
generating system to study the antioxidant properties of red and white wines and coffee [38,
41, 66]. S2O82 ions thermally decompose at 60 C to produce SO4 radicals. In the presence
of DMPO and in aqueous media the adduct of the primary SO4 radical formed is very
unstable (half life of 21 s) [60] and hydrolyses forming dominant DMPO-OH adduct.
A strong solvent effect was observed studying the thermal decomposition of K2S2O8 in
various solvent mixtures. In ethanol/water mixtures the highest DMPO-OH adduct yield was
detected in pure water solutions and decreased with increasing ethanol concentrations. An
opposite trend was observed in DMSO/water solutions showing a dramatic decrease of
DMPO-OH adduct when small amounts of water were added to the mixture [49]. The
advantage of the use of K2S2O8/DMPO/DMSO system is the applicability to both hydrophilic
and hydrophobic types of antioxidant.
Superoxide (O2) is a physiologically relevant radical used to study the radical
scavenging activities of several plant extracts [67]. Typically O2 is generated with the
xanthine/Xanthine Oxidase system and DMPO or BMPO are the main spin trap used. The
reaction is based on the oxidation of hypoxanthine to uric acid operated by the enzyme
Xanthine Oxidase.
The electrons from this oxidation are accepted by dioxygen to produce both H2O2 and
O2 (see Figure 9).

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Angela Fadda and Daniele Sanna

This system is the most frequently used for the generation of superoxide radical, however
is not devoid of pitfalls such as: suitability for hydrophilic antioxidants only, interference of
transition metals, and presence in the food extract of enzyme inhibitors.

Figure 9. Mechanism of generation of superoxide radical with Xanthine Oxidase (XO). Adapted from
ref. 68.

For instance flavonoids are known to inhibit the activity of xanthine oxidase [69, 70],
therefore is not clear whether the reduction of the ESR signal arises from the antioxidant O2
quenching or to the inhibition of the enzyme that suppresses the radical generation.
Non-enzymatic generation systems of O2 have been developed to assess the superoxide
radical scavenging of flavonoid rich food. Non-enzymatic generation systems of O2 includes
the hydrogen peroxide degradation system in alkaline dimethylsulfoxide [42], the NADH
oxidation by phenazine methosulfate (PMS) [71], the riboflavin irradiation system [72].

Voltammetric Methods
Antioxidant compounds can act as reducing agents being, more or less easily, oxidized. If
an inert electrode is used then this oxidation process can take place on the electrode surface
and the produced current can be measured. Cyclic voltammetry (CV) is the most commonly
used electrochemical technique for the determination of antioxidant activity [22, 73]. During
a CV experiment the voltage of the working electrode (usually a glassy carbon electrode is
used for the determination of antioxidant capacity) is scanned between two extremes while
recording the anodic current produced by the antioxidants which oxidize on the electrode
surface. Therefore one or more peaks are obtained, each corresponding to a well defined
oxidation process and characterized by its oxidation potential, with the anodic peak current
being proportional to the concentration of the antioxidants present in the mixture.

Advantages and Pitfalls of the Methods for the Antioxidant Activity Evaluation

83

There is an interesting relationship between electrochemical behavior of compounds with


antioxidant activity and their antioxidant capacity, because a low oxidation potential
corresponds to high antioxidant capacity.
For example considering polyphenolic compounds, those having a catechol moiety are
easily oxidized below 0.4 V, while compounds with one or more isolated phenolic group
show an oxidation potential between 0.45 and 0.8 V [74]. Cyclic voltammetric measurements
of antioxidant activity method have many advantages over conventional chemical assays, as
their speed and simplicity and can be used for a first characterization of antioxidants based on
their reducing power. The method does not require complicated chemical reagents or solvents
while sample preparation it is not necessary, and can be used also on cloudy samples where
spectrophotometric methods are not applicable. These methods are similar to
spectrophotometric/chemical methods previously examined as FRAP, CUPRAC, ABTS
because also in those cases a redox reaction was considered, which involves the oxidation of
the antioxidants. However this method is more versatile because here the potential reduction
of the couple is not fixed but changes during the experiments between two fixed limits.

CONCLUSION
This review provides an overview of the most frequently used methods for the
determination of the antioxidant activity. Simple spectrophotometric methods, as DPPH,
ABTS and FRAP assays, can be conveniently applied for their simplicity and experimental
ease. The only precaution which should be used is to verify the presence of interfering
substances and eventually take into account their effects. These methods have also the
advantage of involving relatively simple instrumentation, in fact only a spectrophotometer is
needed.
The determination of the antioxidant activity with two independent methods is
recommended because, as shown above, each method focuses only on a specific reactivity
and its results could be both under- or over-estimated.
By the biological point of view the most significant methods are the spin trapping ones,
since in these cases reactive species are generated and quantified after they trapping as
relatively stable species; the effects of the antioxidants measured in these systems mimic and
simulate the role carried out in biological ones. The limitation of spin trapping methods is that
the instrumentation that they use is not very common; moreover several factors may affect the
efficiency of the method. Nevertheless spin trapping is the only technique, so far available,
able to detect unequivocally radicals of biological importance.

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In: Advances in Food Analysis Research


Editor: Anita Haynes

ISBN: 978-1-63483-783-5
2015 Nova Science Publishers, Inc.

Chapter 4

RECENT APPLICATIONS OF CHIRAL CAPILLARY


ELECTROPHORESIS IN FOOD ANALYSIS
Raquel Prez-Miguez, Mara Castro-Puyana
and Mara Luisa Marina*
Department of Analytical Chemistry, Physical Chemistry and Chemical Engineering.
Faculty of Biology, Environmental Sciences and Chemistry. University of Alcal,
Madrid, Spain

ABSTRACT
The determination of the enantiomers of chiral food components, additives or
contaminants presents a high interest in Food Science since it can provide information
related to food quality, authenticity and safety, also enabling to evaluate the effect of
processing or manufacturing procedures and possible contaminations. Capillary
Electrophoresis (CE) has demonstrated in the last years a very high potential to achieve
enantiomeric separations due to its high efficiency, versatility, feasibility and the low
consumption of solvents, reagents and samples which makes it an environmentally
friendly analytical technique. In addition, the fact that the chiral selector in CE can be
added to the electrolytic solution makes very easy the screening of different chiral
selectors in order to select the most adequate for the enantiomeric separation of a given
compound which is the most difficult step in the development of a chiral analytical
methodology.
This chapter highlights the interesting role that chirality plays when evaluating the
quality and safety of foods and points out the main practical aspects that should be
considered to carry out the chiral analysis of food components, additives and pesticides
by CE. The characteristics of the CE separation modes mostly employed in chiral food
analysis (Electrokinetic Chromatography, EKC and Capillary Electrochromatography,
CEC) are summarized together with the main detectors employed to achieve the
determination of enantiomers. Moreover, a critical overview on the main applications of
chiral CE methodologies developed during the last ten years including the chiral analysis
of protein and non-protein amino acids, organic acids, phenolic compounds, sweeteners,
and pesticides in foodstuffs is presented. Experimental conditions enabling the chiral
*

Corresponding author: Telephone/Fax: 34-918854935/34-918854971, E-mail: mluisa.marina@uah.es.

90

Raquel Prez-Miguez, Mara Castro-Puyana and Mara Luisa Marina


separations are presented and some representative examples are illustrated. Finally, the
main advantages and drawbacks of these procedures are discussed with the aim of
showing the potential of CE to solve specific problems in the Food Science research
field.

INTRODUCTION
Research in the field of Food Science is continuously demanding the development of
robust, efficient, selective, and sensitive analytical methodologies able to provide information
about chemical composition of foods and to guarantee their safety, quality and traceability.
These methodologies are also crucial in order to meet the requirements from official
laboratories, consumers and legal regulations [1]. The interest is focused on a big number of
nutritional ingredients, additives, adulterants or pollutants [2], many of them being chiral
molecules. Food components such as amino acids, organic acids or phenolic compounds,
additives such as sweeteners, or pesticides used in agriculture to control pest, are examples of
chiral molecules that can exist as, at least, one pair of enantiomers (depending on the presence
of one or more chiral centers). Enantiomers are non-superimposable mirror images of a
molecule (they have identical atomic composition and bonds but different three-dimensional
arrangement). The intrinsic properties of nature (which is chiral because mainly uses one of
the two enantiomers of a chiral compound) make that the receptors of cell machinery, for
example enzymes, are chiral so that they prefer to bind one of the enantiomers of a chiral
compound [3]. As a consequence, when chiral molecules present in foods are consumed, their
enantiomers may interact with the chiral environment present in the human body (enzymes,
proteins, receptors) in a different way originating different effects. In addition, diverse
activities have been shown for the enantiomers of food components that can exhibit, for
instance, different taste or different aromas. For all these reasons, the interest in chiral food
analysis has increased considerably in the last years.
In 1990, Armstrong had already established different areas in which enantiomeric
separations may be relevant when achieving studies on food and beverages characterization
and analysis [4]. Figure 1 shows different topics in which chiral analysis can provide a broad
range of useful and significant information in Food Science; from the identification of
adulterated food and beverages, to the control and monitoring of fermentation processes and
products or the evaluation of age, storage and treatment effects, among others. As a result,
there is a growing interest in the development of analytical methodologies enabling to
increase the knowledge about the enantiomeric composition of foodstuffs in order to evaluate
its effect on both quality and genuineness.
Traditionally, the analytical techniques mainly used for chiral separations have been Gas
Chromatography (GC), High performance Liquid Chromatography (HPLC), and Supercritical
Fluid Chromatography (SFC). Among them, HPLC is used in an important part of the
publications devoted to chiral analysis due to different reasons such as (i) a larger number of
laboratories possess the instrumentation, (ii) many data are available in the literature, (iii) new
research work focused on instrumentation and stationary phases has been carried out, and (iv)
the high interest from companies involved in the market [5]. However, even though HPLC is
widely implemented, Capillary Electrophoresis (CE) has emerged in the last decades as a
powerful tool in chiral analysis. From the first enantiomeric separation by CE published 30

Recent Applications of Chiral Capillary Electrophoresis in Food Analysis

91

years ago by Gassmann et al. [6], the use of CE in chiral separations has continuously grown
until be established, in the last decade, as one of the most employed and powerful techniques
for analytical enantioseparations [7]. This fact is mainly due to different reasons concerning
its short analysis times, high separation efficiency, versatility and feasibility to incorporate a
great variety of chiral selectors to obtain high resolutions. In addition, the fact that the chiral
selector in CE can be added to the electrolytic solution makes very easy the screening of
different chiral selectors in order to select the most adequate to achieve the enantiomeric
separation of a given compound which is the most difficult step in the development of a chiral
analytical methodology. Moreover, CE has certain advantages in terms of green chemistry,
mainly related to the low consumption of solvents and reagents. All these facts explain that
during the last years (2005-2015), more than 700 articles (including research works and
reviews) can be found in the database ISI Web of Knowledge using as keywords capillary
electrophoresis and chiral or enantio* combined with food, beverages,
environmental, agrochemical, pharmaceutical or clinical. Even though
pharmaceutical and clinical fields continue being the fields in which chiral CE has the highest
number of applications, the number of works dealing with enantioseparations by CE in food
analysis (including food and beverage samples) has been increasing in the last years [5, 8-15].
In this chapter, the fundamentals of the enantiomeric separations by CE are summarized
and the main practical aspects that should be considered to carry out the chiral analysis of
food components, additives and contaminants by CE are described.

Figure 1. Areas in which enantioselective separations are relevant in Food Science. (Figure made
following the table described by Armstrong et al. in [4] with permission from the American Chemical
Society).

Moreover, a critical overview on the main applications of chiral CE methodologies to


food and beverage analysis developed during the last ten years including the chiral analysis of
protein and non-protein amino acids, organic acids, phenolic compounds, sweeteners, and
pesticides is presented. Experimental conditions enabling the chiral separations are detailed

92

Raquel Prez-Miguez, Mara Castro-Puyana and Mara Luisa Marina

and some representative examples are illustrated. Finally, the main advantages and drawbacks
of these procedures are discussed with the aim of showing the potential of CE to solve
specific problems in the Food Science research field.

PRINCIPLES OF CHIRAL ANALYSIS BY CE


An enantiomeric separation in CE is not based on an electrophoretic mechanism because
the electrophoretic mobilities of the enantiomers of a chiral compound are equal and
nonselective [3]. Indeed, the enantioselective recognition of enantiomers is due to a
chromatographic mechanism based on their different interaction with a chiral selector. It
implies the formation of either stable diastereoisomers by using an enantiopure chiral
derivatization reagent (indirect mode in which the enantiomers are separated based on their
different physicochemical properties in an achiral environment) or reversible diatereoisomeric
complexes with a chiral selector (direct method where enantiomers-selector complexes are
separated due to their different mobility). Although it is possible to find in the literature
examples of indirect separation of chiral compounds into the food field [16], the truth is that
the use of the indirect approach is much less widespread because of the practical
disadvantages associated with the derivatization reactions (time-consuming) employing
optically pure reagents (expensive and with limited availability). For this reason, the present
chapter is focused on those chiral CE methodologies developed by employing direct
separation methods. In these strategies, the presence of a chiral selector in the environment of
the separation system (either bounded to an immobile support (stationary phase) or as
additive in the background electrolyte (mobile phase)) gives rise to the formation of
enantiomers-selector complexes with different mobility. Therefore, in the two main CE
modes used in chiral analysis, electrokinetic chromatography (EKC) and capillary
electrochromatography (CEC), the enantioseparation is achieved by the combination of
electrophoretic and chromatographic principles. The difference between both modes is related
to the way in which the chiral selector is present (immobilized or in solution). While in EKC
the chiral selector acts as a chiral pseudostationary phase in the electrolytic solution, in CEC
it is immobilized inside the capillary forming a true stationary phase (like in HPLC). From
these two separation modes, EKC is without doubt the most widely used in chiral CE. In
1989, it was defined by Terabe as an analytical separation method which utilizes the
experimental technique of CZE in combination with the principle of chromatography [17].
From a mechanistic point of view, both the enantioseparation of a charged chiral analyte can
been performed using a neutral chiral selector and the enantiomers of a neutral chiral analyte
can be separated by a charged chiral selector [18]. For this reason, regardless of the selector
charge, all chiral separations carried out by using a chiral selector in the electrolytic solution
will be included within the EKC mode. Figure 2 shows three different separation schemes
that can be applied in EKC depending on the analyte and selector charge. When neutral
selectors are used to perform the chiral separation, only charged analytes can be separated. On
the contrary, charged selectors have a high potential not only for the separation of neutral
compounds but also for analytes possessing an opposite charge to that of the chiral selector.
In this case, the enantioresolution can be increased due to the large mobility difference
between the analyte and the selector and to the strength of the analyte-selector interactions.

Recent Applications of Chiral Capillary Electrophoresis in Food Analysis

93

From a practical point of view, at low pH and using a high concentration of negatively
charged selector (or when the binding analyte-selector is strong) it is necessary to reverse the
polarity (detector set at the anode) to carry out the chiral separation of basic or neutral
compounds (using normal polarity they could not reach the detector) (see Figure 2B). On the
contrary, basic or neutral compounds can be separated using a charged selector and normal
polarity when alkaline pH is used as separation medium, since at high pH values, basic
compounds are uncharged so that they are as neutral compounds and they are transported by
the EOF to the detector (Figure 2 C) [7].
Before 1990, ligand-exchange compounds (LE) and cyclodextrins (CDs) were the two
kinds of chiral selectors used to carry out chiral separations by CE. Nowadays, there are a
great variety of compounds that can act as chiral selectors (monomeric and polymeric
surfactants, antibiotics, chiral crown ether, polysaccharides, etc.), but among all of them, CDs
continue being the most widely employed due to their excellent properties related to
availability, diversity, universality, price and safety. Usually, EKC modes are named by
adding the term of the compound used as chiral selector. Whether ionic surfactants are added
at a concentration exceeding their critical micelle concentration, the acronym MEKC
(micellar electrokinetic chromatography) is used, whereas when using CDs the mode is
called CD-EKC (cyclodextrin electrokinetic chromatography).
CEC mode combines the separation efficiency of CE with the high selectivity of HPLC
showing a great potential for enantiomeric separations. In CEC, the capillaries are filled,
packed or coated with a chiral stationary phase, the mobile phases are pushed by the EOF
(which may be assisted by pressure) and an electrical field is applied to perform the
separations. In this way, compounds are separated due to the combination of partitioning
between the stationary and mobile phase, and if they are charged, also by their different
electrophoretic mobilities. Taking into account the different filling methods of stationary
phase, the columns employed to carry out chiral separations by CE can be divided into 3
groups: open tubular columns (OTCs), packed columns (PCs), and monolithic columns [19,
20, 21, 22].
OTCs are based on the use of an open capillary whose inner wall is coated or bonded
with chiral stationary phases (cyclodextrins, celluloses, proteins, molecularly imprinted
polymers (MIPs), or polymeric surfactants). Although chemically bonded phases are
preferred to physically adsorbed phases due to their better stability and longer lifetimes,
physical coating is easier to fabricate, less expensive, and can show as good stability and
separation performance as covalent coating [21]. PCs are the most used columns in chiral
CEC. Here, the capillaries are packed with stationary phases (CDs, macrocyclic antibiotics,
proteins, LE compounds and polysaccharides such as cellulose tris (3-chloro-4methylphenylcarbamate), cellulose tris(4-chloro-3-methylphenylcarbamate) and amylose
tris(5-chloro-2-methylphenylcarbamate)). The main drawback of these columns is that they
are not commercially available and their homemade preparation is relatively complicated due
to bubble formation that can occur at the end-frit what may result in current instabilities and
even current breakdown. Regarding monolithic columns, they are prepared by in situ
polymerization or solidification of monolithic stationary phases with homogeneous and
porous structures. In general, these columns may be divided in monolithic silica-based
columns, polymer monolithic columns, particle-fixed monolithic columns and molecular
imprinted polymers (MIP) columns [7]. In the last years, molecular imprinting technique and
inorganic-organic hybrid monoliths have experienced a high development being an interesting

94

Raquel Prez-Miguez, Mara Castro-Puyana and Mara Luisa Marina

research area in separation science [23, 24, 25]. Even though CEC has evolved during the last
years as separation technique for chiral separations, the lack of commercially available
columns or their complicated homemade fabrication makes that this CE mode has not yet
reached its full potential for practical applications.
In the following section, an in-depth description of the practical aspects directly related to
the application of CE in chiral food analysis is provided.

CHIRAL ANALYSIS OF FOODS AND BEVERAGES BY CE


Different articles dealing with the application of chiral CE to food analysis have been
published during the last decade (2005-2015). Table 1 summarizes the characteristics of the
enantioselective CE methodologies developed to achieve the enantiomeric determination of
different chiral compounds (protein and non-protein amino acids, organic acids, phenolic
compounds, sweeteners, and pesticides) in a great variety of food matrices, including fruit
juices, vinegars, genetically modified organisms, microalgaes, beers, soymilk, wine, food
supplements, infant formulas, cacao beans, fruits, tea, or tap water.

Figure 2. Some chiral separation schemes in EKC. A) A positively charged analyte with a neutral chiral
selector at basic pH. B) A positively charged analyte with a negatively charged chiral selector at low
pH and reversed polarity. C) A basic or neutral analyte with a negatively charged chiral selector at basic
pH.

Recent Applications of Chiral Capillary Electrophoresis in Food Analysis

95

As shown in Table 1, the most employed chiral selectors to carry out the enantioselective
separation of food components, additives and pesticides found in food matrices are CDs, both
as unique selector and combined with anionic achiral micelles where the micelles act as
carriers of the chiral compound and the neutral CD is responsible for the enantiomeric
discrimination. Moreover, other selectors such as ligand-exchange compounds [27, 28, 45,
46, 49], or to a lesser extent, macrocyclic antibiotics [48] have also demonstrated their
potential for the chiral separation of the compounds of interest in the food field. In this sense,
as in other scientific disciplines (such as the pharmaceutical or the environmental ones), EKC
is undoubtedly the most extensively used separation mode in chiral food analysis. In fact,
even though it is possible to find some applications by CEC [37, 58] showing the potential of
different stationary phases to carry out the enantioseparation of standard samples by CEC,
this CE mode seems to still lack of the robustness required for practical applications.
With respect to the detection systems, UV and laser-induced fluorescence (LIF) are the
most popular in chiral food analysis by CE. In the case of the enantiorecognition of protein
and non protein amino acids, it is necessary to include an additional analytical step
(derivatization) to add a chromophore or fluorophore group into the molecule [59]. Among
the different labeling agents available, fluorescein isothiocyanate (FITC), 9fluoroenylmethylchloroformate (FMOC), dansyl chloride DNS, 6-aminoquinolyl-Nhydroxysuccinimidyl carbamate (AQC), and naphthalene-2, 3-dicarboaldehyde (NDA) have
been employed for the derivatization of protein and non protein amino acids. To a lesser
extent, mass spectrometry (MS) and electrochemical and capacitively coupled contactless
conductivity (C4D) have also been reported as detection systems coupled to CE for
enantiomerically determining chiral compounds in food samples. When EKC is hyphenated to
MS to carry out chiral separations, the entry of the (nonvolatile) chiral selector into the MS
ion source should be avoided because it may cause ion suppression and contamination of the
ionization source decreasing the detection sensitivity [60], although in certain cases in which
low concentrated chiral selectors are employed (< 5 mM), it is possible that both analyte and
selector reach the MS detector without a significant detriment on the sensitivity [41, 43, 61].
Basically, the two approaches employed to avoid the entry of the chiral selector in the MS
source are the partial filling technique (PFT) and the counter current migration
technique (CMT) being the former the most used [60, 62]. In PFT, the CE capillary is first
flushed with the buffer without the chiral selector and, before the sample is injected, a band of
the separation medium containing the chiral selector is introduced into the capillary. In the
case of CMT, a charged chiral selector migrates away from the detection system in opposite
direction to the analytes.
A relevant challenge that chiral analysis in food samples has to face is the fact that the
food samples are complex matrices which comprise a great variety of components that can be
found in a very wide dynamic concentration range, from millimolar to femtomolar
concentrations. For these reason, in many cases, the use of different off-line sample
treatments or in-capillary pre-concentration techniques prior to chiral CE analysis are of
paramount importance. As it can be seen in Table 1, the application of different off-line
sample treatments and/or in-capillary preconcentration strategies has enabled to enhance, in
some cases, the detection sensitivity up to 100/110-fold times [35, 36]. Off-line sample
treatments of food matrices include dilution, filtration, ultrasonication, centrifugation, acid
hydrolysis, protein precipitation, liquid extraction, solid-phase extraction or pressurized liquid
extraction (Table 1). The use of these treatments sometimes allows the elimination of

96

Raquel Prez-Miguez, Mara Castro-Puyana and Mara Luisa Marina

interferences from the sample matrix or even the preconcentration of analytes by several
orders of magnitude. The selection and optimization of the sample treatment steps is a crucial
point since errors during sample preparation cannot be corrected even by the best separation
or detection technique so that this process cannot be underestimated.
Regarding in-capillary strategies to improve CE sensitivity by increasing the amount of
sample injected into the capillary, different approaches based on electrophoretic principles
have been developed during the past decades. Usually, these strategies are employed in
combination with off-line sample treatments to eliminate possible matrix constituents because
they also lead to the concentration of these interferences. As Table 1 shows, strategies based
on stacking and sweeping have been reported to increase the sensitivity in chiral food analysis
by CE. In the former the sample is dissolved in a matrix of higher resistance (minor
conductivity) than the separation buffer. In this way, when the injected analyte reachs the
boundary between the sample zone and the buffer zone, it is staked. Therefore, the stacking
phenomenon is based on the analyte concentration because of the difference in conductivity
between the buffer and the sample. In normal sample stacking (NSS) and large volume
sample stacking (LVSS) the sample is injected into the capillary by a hydrodynamic injection,
whereas in field-amplified sample stacking (FASS), and anion or cation selective exhaustive
injection (ASEI or CSEI), an electrokinetic injection is used [63]. On the other hand,
sweeping phenomenon is based on the interactions between the sample and an additive of the
buffer (usually micelles) that acts as a pseudostationary phase (component that originates the
preconcentration). In this case, the key factor is that the sample matrix does not contain the
pseudostationary phase so that sample matrix may have different or the same conductivity
than the buffer [63].

APPLICATIONS
The purpose of this section is to provide the reader an in-depth overview about the
usefulness of chiral CE to solve specific problems into the Food Science field through the
enantioselective determination of different chiral compounds such as protein and non-protein
amino acids, organic acids, phenolic compounds, sweeteners, and pesticides in a variety of
food matrices. The main characteristics (CE mode and detection system, BGE, sample
treatment, sample analyzed, application and LOD) of the most relevant CE methodologies
applied to the analysis of these chiral compounds are summarized in Table 1.

Enantiomeric Determination of Protein and Non Protein Amino Acids


In addition to the 20 L-amino acids universally distributed as protein constituents in
living organisms, there are hundreds of other amino acids of non-protein origin. These have
been defined as those amino acids that are not found in the protein main chain either for lack
of a specific transfer RNA and codon triplet or because they do not arise from protein
amino acids by post-translational modification [64]. In nature, the free amino acids (both
protein and non protein) are generally composed by their L-form whereas foodstuffs are the
main source of D-amino acids. In fact, different processing conditions/treatments or

Recent Applications of Chiral Capillary Electrophoresis in Food Analysis

97

technologies employed by the food industry to improve flavour, consistency or shelf life
among other food characteristics, may produce the racemization of L-amino acids in their Damino acids. In addition, D-forms can be even synthesized by microorganisms in several
enzymatic pathways or can be found as a consequence of the fraudulent
addition of racemic mixtures in supplemented foodstuffs where regulations establish the use
of pure L-enantiomers [8, 59]. All these facts make that, in general terms, the chiral analysis
of protein and non protein amino acids is very useful to obtain relevant information about
food quality and food adulteration, to evaluate the effects of processing, fermentation,
microbiological activity and storage, or to characterize the main L- and D-amino acids found
in foods [5, 10, 65, 66].
Different CE methodologies based mainly on the use of MEKC and EKC separation
modes have been developed for the chiral separation of protein and non protein amino acids
in foods (see Table 1). CEC mode has been employed to a lesser extent; only one work
employed this mode for the chiral separation of amino acids during the period of time covered
in this chapter. From the high number of references published, it is possible to infer that this
is the most frequent application of chiral CE in food analysis. This fact is probably due both
to the well-known structure and character of amino acids and to the large amount of
information than can be obtained from their chiral analysis.
As shown in Table 1, neutral CDs, such as -CD, 2-hydroxypropyl--CD, -CD or
succinyl--CD, have been the preferred selectors to carry out the chiral separation of different
protein and non protein amino acids, although other charged CDs have also been employed
successfully. With respect to the detection systems, UV, LIF and MS are the most frequently
employed. In both UV and LIF detection, a derivatization step is required to add a
chromophore or fluorophore group into the amino acid structure. Among the reagents used for
derivatization of amino acids, FITC and FMOC have been the most popular labeling reagents.
Derivatization is also employed when using a MS detector in order to obtain larger molecules
providing a better detection sensitivity.
The chiral analysis of protein amino acids by CE has been applied in the last years to
obtain valuable information about fermentation processes [26, 28], thermal treatments [29],
fruit juices adulterations [30], genetically modified organisms [32, 34], or just to determine
the L and D-amino acids content to assess if legal regulations are accomplished [35, 36].
The formation of D-amino acids in fermented foods depends mainly on the fermentation
conditions and the action and autolysis of the microorganism involved. For this reason, the
chiral determination of the amino acidic profile can be used as a potent marker of these
processes. For instance, the application of a MEKC-LIF methodology based on the use of CD as chiral selector enabled to find interesting differences in the L and D- amino acids
profiles of different vinegars samples (balsamic, sherry, white wine, cava, and wine vinegar
with extract of herbs). The detection and quantification of D-amino acids present in
fermentation processes made possible to distinguish the type of vinegar and the degree of
maturation of balsamic vinegars [26]. Along with MEKC, EKC methods based on the
formation of diastereomeric ternary-mixed metal complexes between the chiral ligand (Zn
(II)-L-arginine complex) and amino acids have also been employed to establish a relationship
between aromatic amino acids enantiomers and the brand of fermented rice-brewed
suspension (called Laozao in Chinese) [27]. Also, these methods were applied to the
separation and quantification of some pairs of enantiomers which were proposed as markers

98

Raquel Prez-Miguez, Mara Castro-Puyana and Mara Luisa Marina

for the quality control of rice vinegar samples because their concentration depends on the
time and activity of fermentation [28]. Chiral analysis of protein amino acids has also been
very useful to characterize different microalgaes and to study the effect of different
microalgae drying process (hot air and lyophilization) on their L-and D- amino acid contents
[29]. Quantitation of five different chiral amino acids (namely Arg, Lys, Ala, Glu, and Asp)
in three microalgae species (Spirulina platensis, Dunaliella salina, and Tetraselmis suecica)
was carried out using a MEKC-LIF strategy with -CD as chiral selector which provided a
separation in less than 25 min and LODs down to 8.9 nM (see Table 1). Different percentages
of D-amino acids were measured depending on the microalgaes species, extraction protocol
(classical and pressurized liquid extraction) and drying process which demonstrated the
potential of the developed methodology to differentiate among microalgaes and to investigate
the effect of thermal processing on the amino acid content [29].

(A)

(B)
Figure 3. A) CE-MS extracted ion electropherogram (EIE) obtained from an orange juice sample
derivatized with FITC. B) CE-MS EIE from an orange juice sample adulterated with 0.8% of D/L-Asp.
CE conditions: 5 mM -CD in 100 mM ammonium acetate buffer (pH 6.0); polymer-coated capillary,
50 m x 87 cm; voltage, -15 kV; injection, 0.5 psi, 18 s. ESI conditions: positive ion mode; sheath
liquid methanol-water (1:1 v/v) with 25% BGE without -CD; flow rate, 3.5 mL/min; dry gas flow, 3
L/min; temperature, 350C; mass scan, 150700. Reprinted from [31], copyright (2005) with
permission from John Wiley and Sons.

Table 1. Most relevant works published in the period 2005-2015 on the chiral analysis of food samples by CE
Group
Protein
amino
acids

Compounds

CE Mode/
Detection

BGE

Off-line sample treatment


and/or in-capillary
preconcentration
Filtration, FITC-derivatization

Sample
Vinegars

Pro, Ala, Arg,


Glu, Asp.

CDMEKC/LIF

20 mM -CD + 30 mM
SDS in 100 mM borate (pH
9.7)

Tyr, Trp, Phe

LEEKC/UV

3 mM Zn(II)/6 mM L-Arg
in 5 mM acetate, 100 mM
boric acid (pH 8.20)

Centrifugation, filtration,
ultrasonication, FMOCderivatization

Rice-brewed
suspensions

17 pair of
enantiomers

LEEKC/UV

Centrifugation, filtration, (10fold) dilution, DNS-derivatization

Rice vinegar

Arg, Lys, Ala,


Glu, Asp

CDMEKC/LIF

3 mM Zn(II)/6 mM L-Arg
in 5 mM acetate, 100 mM
boric acid (pH 8.0)
20 mM -CD + 30 mM
SDS in 100 mM borate (pH
9.7)

Classical extraction or PLE,


FITC-derivatization

Arg, Pro, Ala,


Asp

CDMEKC/LIF

FITC-derivatization

Asp, Glu, Ser,


Asn, Ala, Pro,
Arg.

CDEKC/MS

10 mM -CD + 5 mM
SDBS in 50 mM borate (pH
9.6)
5 mM -CD in 100 mM
acetate (pH 6.0)

Microalgaes
(Spirulina
platensis,
Dunaliella salina,
and Tetraselmis
suecica)
Fruit juices

Squeeze, Filtration,
centrifugation, FITCderivatization

Orange juice

Arg, Ser, Ala,


Glu, Asp

CDMEKC/
LIF

20 mM -CD + 80 mM
SDS in 100 mM borate (pH
10.0)

Acid hydrolysis, protein


precipitation, centrifugation,
FITC derivatization

Transgenic and
nontransgenic
maize

Arg, Asn, Ala,


Glu, Asp

CDMEKC/LIF
CDEKC/MS

Acid hydrolysis, protein


precipitation, centrifugation,
FITC derivatization
Soybean: Acid hydrolysis, protein
precipitation, centrifugation,
FITC derivatization
Vinegar: Filtration, FITC
derivatization

Transgenic and
wild yeast

Arg, Asn, Ala,


Glu, Asp

20 mM -CD + 30 mM
SDS in 100 mM borate (pH
10.0)
0.5 mM 3-monodeoxy-3monoamino--CD in 50
mM carbonate (pH 8.0)

Transgenic and
wild soybean and
vinegar

Application

LOD*

Ref.

< 16.6 nM

[26]

0.15 g/mL
(Trp) 0.5 g/mL
(Phe) 1.25
g/mL (Tyr)
0.5-1.0 g/mL

[27]

Characterization of microalgae
species as well as different
microalgae drying processes.

< 8.9 nM

[29]

Proposal of L-Asn as marker for the


adulteration of pomegranate juices
with apple juice
Analysis of the main amino acids in
fresh orange juice and detection of
fraudulent addition of synthetic
amino acids used to mask water
dilution
Chiral analysis of amino acids for
assessing the existence (or not) of
modifications in metabolic pathways
linked to the amino acid profile
within genetically modified
organisms
Analysis of amino acids profile to
differentiate transgenic and wild
yeast
Analysis of amino acids profile in
vinegar and soya (transgenic and wild
soybean).

0.85-3.99 nM

[30]

16-29.4 M

[31]

160-790 nM

[32]

LODs in nM
range

[33]

7.19 x 10-9-1.09
x 10-6 M

[34]

Determination of amino acids as


markers of maturation of balsamic
vinegars or production of vinegars by
microbial fermentation
Relationship of the type and content
of aromatic amino acids enantiomers
with the brand of these fermented
products
Monitoring of D,L-amino acids as
markers for quality control

[28]

Table 1. (Continued)
Group

Nonprotein
amino
acids

Compounds

CE Mode/
Detection

BGE

Asp

CDEKC/LIF

60 mM Hp--CD +150
mM SDS in 150 mM Trisborate (pH 9.0)

Trp, Phe, Glu

CDMEKC/UV

citrulline
(among others
20 pairs of
amino acids
enantiomers)
Orn
(along with
18 pairs of
amino acids
enantiomers)
Orn, Arg, Lys

CEC/UV

35 mM -CD + 35 mM
STDC + 12.5% (v/v) IPA in
1.5 mM tris borate (pH 8.5)
Sepapak-2,
0.5 M formate/H2O/ACN
(1/19/80, v/v/v) (pH 2.5)

Off-line sample treatment


and/or in-capillary
preconcentration
water dilution (50-fold), NDA
derivatization and in-capillary
preconcentration by sweeping and
stacking (LVSS)
FMOC-derivatization and ijncapillary preconcentration by
sweeping and stacking (LVSS)
Water solution and FMOC
derivatization

Sample

Application

LOD*

Ref.

2.5 x 10-10 M
(D-Asp)
2.4 x 10-10
M ( L-Asp)
40-60 nM

[35]

Determination of L-citrulline and its


enantiomeric impurity in food
supplements

7.5 x 10-7 M

[37]

Soymilk and beer

Sensitive determination of Asp


enantiomers in beer and soymilk
samples

Beers

Sensitive determination of Trp, Phe,


and Glu enantiomers in beers

Food supplements

[36]

CDEKC/UV

5% (m/v) HS- -CD + 2%


(m/v) Ac--CD in 50 mM
phosphate (pH 2.0)

Filtration (beer was previously


degassed in a ultrasonic bath)
followed by AQC derivatization

Beer, wine and


vinegar (fermented
foods)

Analysis of Orn enantiomers in


fermented foods (beer, wine, vinegar)

9 x 10-6 M

[38]

CDEKC/UV

5% (m/v) HS- -CD + 2%


(m/v) Ac--CD in 50 mM
phosphate (pH 2.0)

Dietary
supplements and
wine

Determination of Orn, Arg and Lys


enantiomers in dietary supplements
and wines

6.4 x 10-6 M

[39]

Orn, Lys, Arg,


Asp

CD-EKC/U
V

1 mM -CD in 100 mM
borate (pH 10.0)

Dietary supplements: water


solution, filtration, and incapillary derivatization with AQC
Wine: Filtration and in-capillary
derivatization with AQC
Water solution, filtration,
dissolution in borate buffer and
accelerated derivatization with
FITC using a ultrasound probe

Food supplements

Enantiomeric determination of Orn in


food supplements

1.6 x 10-7 M

[40]

Orn

CDEKC/MS2

0.75 mM -CD in 50 mM
ammonium carbonate (pH
10.0)

Beers

Enantiomeric determination of beers


submitted to different fermentation
processes

2.5 x 10-9 M

[41]

Carnitine

CDEKC/MS2

10 mM Succ--CD in 0.5
M ammonium formate (pH
2.5)

Degasification (ultrasonic bath),


filtration, pH adjustment (pH 10),
and filtration prior to FITC
derivatization
Water solution (sonication)
followed by ultrafiltration (5 kDa
cut-off filter) and water
dissolution before FMOC
derivatization

Infant formulas

Quantitative analysis of D/L-carnitine


in infant formulas. Detection of Dcarnitine up to 8%.

0.1M

[42]

Group

Organic
acids

Compounds

CE Mode/
Detection

BGE

Carnitine

CDEKC/MS2

0.2% (m/v) Succ--CD in


0.5 M formate (pH 2.5)

S-Adenosyl-Lmethionine

EKC-UV

300 mM glycine in 50 mM
phosphate (pH 2.5)

Isocitric acid
(citric acid)

LEEKC/UV

20 mM Ni(II)/ 80 mM Dquinic acid in 30% ACN,


20 mM acetic acid (pH 5.0)

Malic, tartaric
and isocitric
acid (citric acid)

LEEKC/UV

Lipoic acid

lactic acid
(hydroxybutyric
acid, 2hydroxycaproic
acid, 2hydroxyoctanoic
acid, 2hydroxydecanoi
c acid,
+ Asp and Glu
Tartaric acid

Off-line sample treatment


and/or in-capillary
preconcentration
Drinks: Water solution, FMOC
derivatization
Biscuits, tablets, and capsules:
Water extraction, centrifugation,
dilution 1/10 in water, FMOC
derivatization

Sample
Dietary food
supplements
(drinks, biscuits,
tablets)

Freezing, Liquid-liquid extraction


(5% TCA (w/v)), centrifugation
and filtration
Water dilution (2-fold),
centrifugation, filtration

Fruits and Fruit


juices

100 mM D-quinic acid +


10 mM CuSO4 + 1.8 mM
ScCl3 in 20 mM acetate (pH
5.0)

Apple, grape, pineapple juice:


water dilution, centrifugation,
filtration
Orange and mixed fruit juice:
centrifugation, SPE

Fruit juices

CDEKC/UV

8 mM TM--CD in 100
mM phosphate (pH 7.0)

Extraction with 70% MeOH

Dietary
supplements

EKC-C4D

5 mM vancomycin + 0.03
mM CTAB in 10 mM Tris,
4.4 mM maleic acid (pH
7.35)

ACN solution, centrifugation,


drying, redissolution in buffer

Milk and yogurt

LEEKC/C4D

7 mM CuCl2 + 14 mM
trans-4-hydroxy-L-proline
in 100 mM
-aminocaproic acid (pH
5.0)

Wine: water dilution, filtration


Grapes: Squeeze, filtration, water
dilution

Wine and grapes

Fruit juices

Application
Identification and quantification of
D/L-carnitine in dietary food
supplements (drinks, biscuits,
capsules and tablets). The use of
racemic carnitine (not allowed by the
legislation) was corroborated in one
of the food sample analyzed
Quantification of SAM in fruits and
fruit juices
Detection of adulterations in fruit
juices through the determination of
the ratio of citric acid to D-isocitric
acid
Detection of fruit juice adulteration
through the content of -hydroxy
acids and their enantiomers.

Determination of R/S lipoic acid in


dietary supplements to control the use
of racemic mixture or pure
enantiomers
Determination of lactic acid
enantiomers in milk and yogurt. The
presence of D-lactate in dairy
products may indicate a microbial
contamination

Enantiomeric separation of tartaric


acid as a food additive for quality
control and safety of food products.

LOD*

Ref.

10 ng/mL (Lcarnitine)

[43]

0.5 M

[44]

[45]

3 mg/L ( DLmalic, -isocitric)


2 mg/L ( DLtartaric,-citric)

[46]

1.5 mg/l

[47]

2.8 M ( LLactic)
2.4 M ( DLactic)

[48]

19 M ( DTartarate)
21 M ( LTartarate)

[49]

Table 1. (Continued)
Group

Compounds

Phenolic
compounds

()-catechin
(along with
other catechins
and
methylxanthine)
()-catechin,
()gallocatechin
(along with
other catechins
and
methylxanthine)
()gallocatechin
(along with
other catechins
and
methylxanthine)
()-catechin,
()-epicatechin

CE Mode/
Detection

BGE

Off-line sample treatment


and/or in-capillary
preconcentration
Extraction with 29% ethanol
(assisted by ultrasonication),
centrifugation, filtration

Sample

Application

LOD*

Ref.

Theobroma cacao
beans

Separation of phytomarkers of T.
cacao and proposal of (-)-catechin as
marker of epimerization occurring in
T. cacao during manufacturing
processes
Study of thermal epimerisation of C
and GC to demonstrate the relevance
of non-epistructured (-)-C and (-)-GC
as markers of manufacturing
processes involving heating

0.03 g/mL ((-)EC/ (+)-C)


1.0 g/mL (TB)

[50]

0.05-0.7 g/mL

[51]

CDMEKC/UV

12 mM HP--CD + 12
mM SDS in 50 mM
BrittonRobinson (pH 2.50)

CDMEKC/UV

25 mM HP--CD in 25
mM boratephosphate + 90
mM SDS (pH 2.5)

Extraction with water, filtration

Tea

CDMEKC/UV

25 mM HP--CD + 90
mM SDS in 25 mM boratephosphate (pH 2.5)

Extraction with water, filtration

Green tea

Evaluation of quality changes in


commercial green tea leaves during a
long-term storage. Proposal of
Epigallocatechin as a marker of
uncorrected storage conditions

[52]

CDEKC/UV

12 mM HP- -CD in 0.1


M borate (pH 8.5)

Apple juice,
guaran seeds and
guaran extracts

Authenticity of guaran through the


analysis of the catechin and
epicatechin enantiomers

[53]

Isoxanthohumol

CDMEKC/UV

45 mM HP--CD + 100
mM SDS in 20 mM
phosphate (pH 7.0)

Guaran seeds: water solution,


filtration
Apple juice: SPE
Guaran extracts: SPE
Filtration (Sep pack),
centrifugation, concentration
(rotary vacumm evaporation),
ethanol solution

Beer

17 ng/L

[54]

Sweeteners

Neotame

CDEKC/UV

30 mM TM--CD in 50
mM phosphate (pH 5.5)

Extraction with methanol/water


(50/50)

Mango juice, cola


soft drink, orange
soft drink

The racemization of IX in beer could


be attributed to the production of a
racemic mixture from XN during
boiling and to the fact that IX
enantiomers were easily
interconverted.
Enantiomeric separation of neotame
in spiked beverages

[55]

Pesticides

Malathion,
naled,
isomalation,
phenthoate,
phenamiphos

CDEKC/UV

10 mM CM--CD in 25
mM Tris (pH 7.0)

SPE extraction (ethylacetatodiethyle ether (50/50)) +


redissolution in methanol.

Tap water

0.018 nM (L,Lneotame)
0.082 nM (D,Dneotame)
200 ng/mL

Enantiomeric determination of
malathion in tap water.

[56]

Group

Compounds

CE Mode/
Detection

Propiconazole
Tebuconazole
Fenbuconazole

CDMEKC/UV

Metalaxyl
tebuconazole,
diniconazole,
uniconazole,
hexaconazole,
benalaxyl,
myclobutayl,
futriafol

CEC-UV

BGE
30 mM HP--CD + 50
mM SDS + methanol-ACN
10/5 (v/v) in 25 mM
phosphate (pH 3.0)
Sepapak-4, 90/9/1 (v/v/v)
ACN/H2O/ formate (pH 2.5)

Off-line sample treatment


and/or in-capillary
preconcentration
Filtration, SPE, drying,
redissolution in buffer solution
and in-capillary preconcentration
by sweeping
SPE, drying, redissolution in
80/20 (v/v) ACN/H2O dissolution

Sample

Application

LOD*

Ref.

Grapes

Enantiomeric determination of
triazole fungicides in grapes samples

0.09-0.1 g/mL

[57]

Tap water

Enantiomeric separation of metalaxyl


in spiked tap water

1.4 mg/L (Smetalaxyl )


1.6 mg/L (Rmetalaxyl)

[58]

ACN, acetonitrile; Ac--CD, acetylated--CD; Ala, alanine; AQC, 6-aminoquinolyl-N-hydroxysuccinimidyl carbamate; Arg, arginine; Asn, asparagine; Asp,
aspartic acid; (+)-C, catechine; C4D, capacitively coupled contactless conductivity; CD, cyclodextrin; CEC, capillary electrochromatography; CTAB,
cetyltrimethylammonium bromide; CZE, capillary zone electrophoresis; DNS, dansyl chloride; (-)-EC, epicatechine; EKC, electrokinetic chromatography;
FICT, fluorescein isothiocyanate; FMOC, 9-fluorenyl-methyloxycarbonyl chloride; GC, gallocatechin; Glu, glutamic acid; GMO, genetically modified
organism; HP--CD, hydroxypropyl- -cyclodextrin; HS--CD, highly sulfated--cyclodextrin; IPA, isopropanol; LE: ligand exchange; LIF, laser-induced
fluorescence; LLE, liquid-liquid extraction; Lys, lysine; LVSS, Large volume sample stacking; MEKC, micellar electrokinetic chromatography; MS2,
tandem mass spectrometry; NDA; naphthalene-2,3-dicarboxaldehyde; OPs, organohosphorus pesticides; Orn, ornithine; PEO, ethylene oxide; Phe,
phenylalanine; PLE, pressurized liquid extraction, Pro, proline; SDBS, sodium dodecylbenzene sulphonate; SDS, sodium dodecyl sulfate; SEPAPAK-2,
cellulose tris(3-chloro-4-methylphenylcarbamate); SEPAPAK-4, cellulose tris(4-chloro-3-methylphenylcarbamate); Ser, serine; SPE, solid phase
extraction; STDC, sodium taurodeoxycholate; Succ--CD, Succinyl--cyclodextrin; TB, theobromine; Thr, threonine; TM--CD, trimethyl--cyclodextrin;
Trp, tryptophan; Tyr, tyrosine; XN, xanthohumol.

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Raquel Prez-Miguez, Mara Castro-Puyana and Mara Luisa Marina

Fruit juice industry is one of the most important agricultural businesses in the world so
that, due to its economic impact, the adulteration of fruit juices is a relevant topic which
requires the development of analytical methodologies able to detect sophisticated adulteration
processes. As it can be observed in Table 1, different chiral CE methodologies have been
employed with success for the recognition of adulterated fruit juices. On the one hand, the
application of a MEKC-LIF method based on the use of sodium dodecylbenzene sulphonate
as surfactant, -CD as chiral selector, and FITC as derivatization reagent, enabled to compare
the amino acid profile of pomegranate and apple juices and propose L-Asn as marker for the
adulteration of pomegranate juices with apple juices [30]. On the other hand, an EKC-MS
methodology, in which a polymer coating protocol was used to reduce the EOF and minimize
the entrance of the -CD into the MS ion source, was developed to detect fraudulent additions
of synthetic amino acids to mask water dilution of orange juices [31]. As the developed
methodology enabled the determination of 2% of the D-Asp enantiomer (derivatized with
FITC), it could be used as marker for controlling possible adulterations with synthetic amino
acids (containing L and D enantiomers). Figure 3 shows the EKC-MS extracted ion
electropherograms obtained from a fresh orange juice and from the same juice adulterated
with 0.8% of D/L-Asp.

Figure 4. Chiral-MEKC-LIF electrophoregrams of free D/L-amino acids from different maize varieties:
(1) Aristis; (2) Aristis-Bt; (3) Tietar; (4) Tietar-Bt; (5) PR33P66; (6) PR33P66-Bt. Peaks marked with
an asterisk correspond to FITC. CE conditions: 20 mM -CD in 100 mM sodium tetraborate, 80 mM
SDS (pH 10.0); uncoated fused-silica capillary, 50 m x 57 cm; voltage, 20 kV; LIF detection, 488
(excitation) and 520 nm (emission). Reprinted from [32], copyright (2007) with permission from
American Chemical Society.

Recent Applications of Chiral Capillary Electrophoresis in Food Analysis

105

The safety of the use of genetically modified organisms in food science needs to be
corroborated through the equivalence between transgenic and non-transgenic foods. In this
regard, the investigation of the protein amino acidic profile through the application of
enantioselective CE procedures has opened new perspectives in the study of transgenic foods
since it can provide information about unexpected modifications in other metabolic pathways
linked to the amino acids profile. Two similar procedures including amino acids extraction,
FITC derivatization and MEKC separation with -CD as chiral selector and LIF detection
have been applied to quantify L and D-amino acids in different lines of conventional and
transgenic maize [32], and yeasts used for sparkling wine production [33]. In maize samples,
very similar L and D-enantiomer composition was observed for one of the maize variety
studied (Tietar) whereas significant differences were obtained for the other maize lines
(Aristis, and PR33P66) [32]. Figure 4 depicts the D/L amino acids profile obtained from the
different maize varieties studied. Regarding the study of yeasts, it can be concluded that a
faster autolysis (which is the main source of molecules that contribute to the quality of
sparkling wines made by the traditional method) takes place in the genetically modified yeast
releasing a higher amount of L-amino acids to the medium in a short time [33]. Not only
MEKC has been applied to distinguish conventional and transgenic varieties of foods but also
EKC-MS has demonstrated to be a potent strategy in this field. Using as chiral selector a
modified CD (namely 3-monodeoxy-3-monoamino--CD), five different D/L-amino acids
(Arg, Asn, Ala, Glu, and Asp) were separated in 20 min reaching LODs in the nM range [34].
This methodology was useful for the determination of these amino acids in wild and
transgenic soybeans (different quantities of amino acids were determined in both varieties)
and vinegar samples (in which only L-amino acids were detected).
Interesting methodologies are those employing an in line preconcentration technique to
carry out the sensitive enantiomeric determination of amino acids in food samples. For
instance, the sensitive determination of Asp enantiomers (derivatized with NDA) in beers and
soymilk has been carried out using a preconcentration by stacking prior to EKC separation
with LIF detection [35]. The separation was accomplished using a discontinuous system, i.e.,
buffer vials contained poly(ethylene oxide) (PEO, which acts as concentrating media), SDS
(acting as pseudostationary phase) and HP--CD (acts as chiral selector) whereas the
capillary was filled with SDS and HP--CD. The stacking mechanism is mainly based on the
difference in viscosity between the sample zone and PEO whereas the sweeping is due to the
interaction between Asp enantiomers and SDS. Using this system, it was possible to achieve
the stacking of the derivatized amino acid without the loss of chiral resolution and to obtain a
100 and 110-fold enhancement in the sensitivity of D and L-Asp enantiomers, respectively
(LODs of 2.4 and 2.5 x 10-10 M). Following the idea of a discontinuous system and modifying
slightly the components of the buffer vial and the capillary (in this case using
taurodeoxycholate and -CD instead of SDS and HP--CD), it was also possible to
enantioseparate Trp, Phe and Glu (derivatized with FMOC) with high sensitivity in various
types of beer samples (LODs in the nM levels) [36].
Regarding the chiral analysis of non protein amino acids by CE, the most relevant works
published during the last years have been focused on the separation of the L-enantiomer from
the D-enantiomer which can have in some cases toxic properties and whose addition during
the elaboration of foods is forbidden. Thus, the enantiomeric separation allows guaranteeing a
good quality control of food products. Samples analyzed have been mainly food supplements,

106

Raquel Prez-Miguez, Mara Castro-Puyana and Mara Luisa Marina

infant formulas, and fermented foods (such as wine, beer etc.) as described in detail in Table
1. In all the research works grouped in this table, EKC was employed as CE mode to carry out
the separation of different non protein amino acid enantiomers, except in one article in which
CEC was used to achieve the enaniomeric determination of citrulline in food supplements
with cellulose tris (3-chloro-4-methylphenylcarbamate chiral stationary phase (CSPs) as
chiral selector, FMOC as labeling reagent and formate as running buffer [37]. This
methodology enabled to achieve LODs of 7.5 x 10-7 M for citrulline and it demonstrated to be
more efficient than nano-LC when comparing both techniques under similar experimental
conditions.

(A)
Figure 5. (Continued).

Recent Applications of Chiral Capillary Electrophoresis in Food Analysis

107

(B)
Figure 5. A) Enantiomeric separation by EKC of a mixture of the 20 protein amino acids, Orn, and
GABA divided in two migration zones: (a) first-migrating zone, and (b) second-migrating zone. B)
Electropherogram corresponding to different fermented foods derivatized off-line with AQC (a) a rose
wine (uncoated fused-silica, 50 m 72.5 cm) and (b) a beer (uncoated fused-silica, 50 m 48.5 cm);
injection by pressure, 5066.25 Pa, 5 s of sample followed of 5 s of BGE; non spiked sample and sample
spiked with 5104 M racemic Orn. CE conditions: 5% (m/v) HS--CD and 2% (m/v) acetylated--CD
in 50 mM phosphate buffer (pH 2.0); uncoated fused-silica, 50 m 72.5 cm; voltage, -25 KV;
temperature, 15C; UV detection, 260 nm. (*) Unknown peaks. Reprinted from [38], copyright (2008)
with permission from Elsevier.

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Raquel Prez-Miguez, Mara Castro-Puyana and Mara Luisa Marina

Ornithine and carnitine are the non protein amino acids whose chiral separation by CE
has been the most studied during the last decade. The former has received huge attention due
to the beneficial properties of the L-enantiomer and the harmful effects of the D-enantiomer
which may be originated during food processing or fermentation processes and can produce
depletion in the urea synthesis giving toxic consequences. Carnitine has also shown to have
different biological activities depending on its enantiomeric form; L-carnitine plays an
important role in the long chain fatty acids metabolism while D-carnitine possesses toxic
properties.
Three different EKC-UV procedures under both basic and acidic conditions have been
proposed to analyze ornithine enantiomers in fermented foods (beers, wine, vinegars) and
food supplements [38, 39, 40]. An EKC-UV methodology was developed using AQC as offline derivatizing reagent, and phosphate buffer (pH 2.0) containing HS--CD and acetylated-CD as BGE, which allowed to reach LODs of 9 x 10-6 M for ornithine [38]. As shown in
Figure 5, this method made possible the chiral separation of ornithine from the enantiomers of
twenty protein amino acids and GABA (Figure 5A), as well as their enantioseparation in
fermented foods (Figure 5B). Using this methodology as starting point, a second procedure
was developed including an in-capillary derivatization with AQC which enabled to increase
CE automatization and decrease the analysis time. In this case, the ECK-UV method was
successfully applied to the determination of ornithine, Arg and Lys enantiomers in wine
samples (these compounds demonstrated to be responsible of wines organoleptic properties)
and dietary supplements [39]. FITC was also used as labeling reagent in the chiral
determination of ornithine enantiomers in food supplements. In fact, an interesting precapillary derivatization with FITC using an ultrasound probe was proposed to reduce
derivatization time from 16 h to 10 min. The method was employed in combination with an
EKC-UV separation method based on the use of -CD as chiral selector and the LOD for
ornithine was 1.6 x 10-7 M [40]. Using this fast FITC derivativation procedure, an EKC-MS2
methodology was also developed to enhance the sensitivity and selectivity in the chiral
determination of ornithine in beers submitted to different fermentation processes [41]. The
method, based on the use of carbonate buffer (pH 10.0) containing -CD as BGE, enabled to
achieve LODs of 2.5 x 10-9 M. The percentages for D-ornithine ranged from 1.5% to 10% in
the analyzed samples (the lowest value corresponding to a dietetic beer and the maximum to a
double fermentation beer).
Regarding carnitine, its chiral determination by EKC-MS2 was carried out in infant
formulas [42] and dietary supplements [43] to assess that the content of the D-enantiomer was
not higher than the limits established by regulations. With this purpose, an EKC-MS2
methodology was developed using FMOC as labeling reagent and formate containing Succ-CD as running buffer. Due to the high CD concentration needed to achieve the enantiomeric
separation, a partial filling technique was employed to avoid the contamination of the MS ion
source. LODs for carnitine enantiomers in the M range were obtained and the application of
the method to the analysis of supplemented infant formulas revealed amounts as high as 8%
of D-carnitine (which exceeded by far the limits established by the European Pharmacopeia)
in some of the samples analyzed [42]. In order to avoid the use of the partial filling technique,
the method was subsequently optimized without this step by using a low amount of Succ-CD (0.2% (m/v)) with a longer length capillary which improved the precision and sensitivity
(LODs of 10 ng/mL) [43]. The application of the optimized method to the analysis of twenty
two dietary food supplements demonstrated the use of racemic carnitine (not allowed by the

Recent Applications of Chiral Capillary Electrophoresis in Food Analysis

109

legislation) in one of the food supplements whereas in the other samples L-carnitine was the
enantiomer employed with enantiomeric impurities (D-carnitine) up to 6%.
Other non protein amino acid whose enantiomeric separation by CE has also been
reported in the last years is S-adenosyl-L-methionine. Using an EKC-UV procedure, it was
possible to establish the relationship between heat treatment and S-adenosyl-L-methionine
content in tomato samples which demonstrated that the content of this non protein amino acid
varies depending on food processing [44].

Enantiomeric Determination of Organic Acids


The enantiomeric separation of organic acids is also a field of huge interest in Food
Science. In general, they naturally occur as single enantiomers so that the presence of racemic
mixtures in foods may indicate their use as additives which must be controlled because it is
not always allowed. Moreover, taking into account that different organic acid enantiomers can
provide different flavor or taste to beverages and food products, their analysis is of great
interest for quality control.
EKC using buffers at a pH ranging from 5.0 to 7.0 and different chiral selectors, from
ligand-exchange complexes or CDs to macrocyclic antibiotics enabled the chiral
determination of organic acids in foods (see Table 1). UV detection is, without doubt, the first
choice to carry out the detection of these compounds, while C4D seems to be a good
alternative in those cases in which a chromophore group does not exist in the organic acid
structure.
The development of chiral analytical methodologies for the determination of organic
acids provides interesting tools to fight against fruit juice adulteration. With this aim,
different strategies based on the use of ligand exchange EKC with UV detection have been
proposed in the literature. Taking into account that the ratio of citric acid to D-isocitric acid is
one of the most relevant parameters used to distinguish authentic and adulterated fruit juices,
D- and L-isocitric acid enantiomers were separated in different fruit juices using D-quinic
acid as chiral selector and Ni(II) as central ion system (see Figure 6) [45]. The contents of
DL-isocitric and citric acids estimated using the developed methodology were in agreement
with the fruit juice industry Code of Practice. However, not only the chiral determination of
DL-isocitric acid can serve to detect fruit juice adulteration; the enantiomeric determination
of other -hydroxy acids, such as DL-malic acid or DL-tartaric acid can also be used as
indicators of adulteration [46].
Lipoic acid (a powerful antioxidant with multiple beneficial effects in the therapy of
different diseases such as diabetes, atherosclerosis, or degenerative processes in neurons,
among others) naturally occurs as the (R)-enantiomer, while synthetic lipoic acid is racemic.
Since the quality control of dietary supplements should include not only the evaluation of
total lipoic acid but also the quantification of each enantiomer, it is of interest to know the
(R)-enantiomer content of lipoic acid (the potency of (S)-lipoic acid is not known exactly). To
do that, a simple sample treatment combined with a rapid EKC-UV methodology with TM-CD as chiral selector was developed to quantity both enantiomers in different dietary
supplements demonstrating that in most samples analyzed the racemic mixture was present in

110

Raquel Prez-Miguez, Mara Castro-Puyana and Mara Luisa Marina

spite of some of these samples claimed to use only the naturally occurring enantiomer ((R)lipoic acid) [47].
As above-mentioned, C4D has demonstrated to be a potent alternative to UV detection to
determine organic acids without chromophore groups in their structure. As it can be seen in
Table 1, this detection system coupled to EKC methodologies has been used in the
determination of the enantiomers of -hydroxy acids and two -amino acids (Asp. Glu) [48]
or the enantioseparation of tartaric acid [49]. In the first case, the determination of the lactic
acid enantiomers in yogurt and milk demonstrated to be relevant since the presence of Denantiomer acts as an indicator of natural fermentation process or microbiological
contamination in dairy products [48]. In the last case, the analysis of tartaric acid enantiomers
in wine and grapes by EKC-C4D using vancomycin as chiral selector was employed to
guarantee the quality and safety of both products, since the presence of D-tartaric acid could
come from bacterial contamination (D-tartatic was not found in any of the samples analyzed
proving that they were not contaminated) [49].

Figure 6. EKC electropherograms of (A) a standard solution (containing 400 mg DL-isocitric acid +
400 mg/L citric acid), (B) an apple juice sample, and (C) an orange juice sample (C). CE conditions:
30% ACN, 20 mM acetic acid, 20 mM NiSO4, and 80 mM D-quinic acid (pH 5.0); FunCap-CE/Type S
capillary (sulfonated capillary), 50 m x 64.5 cm; voltage, 15 KV; temperature, 15C; UV detection,
200 nm. The inset in (B) was the electropherogram obtained when 5mL of a mixture of citric acid (100
mg/L) and DL-isocitric acid (200 mg/L) was added to 5mL of an apple juice sample. The insets in (C)
show part of the same data with an expanded y-axis. *represents a peak of malic acid. Reprinted from
[45], copyright (2010) with permission from John Wiley and Sons.

Recent Applications of Chiral Capillary Electrophoresis in Food Analysis

111

Figure 7. CD-MEKC electrophoregram of extract from Kokeicha Green tea. CE conditions; 25 mM


borate-phosphate buffer (pH 2.5) containing 90 mM SDS and 25 mM HP--CD; uncoated fused silica
capillary, 50 m x 38.5 cm (used in the short-end mode (effective length 8.5 cm)); voltage, 15 kV;
temperature, 25C; hydrodynamic injection, 3 s (pressure at 25 mbar); UV detection, 200 nm. Peak
assignment: EC, epicatechin; IS, Internal standard; ECG, epicatechin gallate; EGC, epigallocatechin;
CF, caffeine; EGCG, epigallocatechin gallate; (+)-C, (+)-catechin; (-)-C, (-)-catechin; (+)-GC; (+)gallocatechin; (-)-GC; (-)-gallocatechin. Reprinted from [51], copyright (2009) with permission from
John Wiley and Sons.

Enantiomeric Determination of Phenolic Compounds


Phenolic compounds have demonstrated to have healthy properties associated to
antioxidant, antimicrobial and antiviral activities, but the main interest they present in the
food field is because they are a powerful tool for the characterization of food quality and
safety. Flavonoids (group comprising flavonones, isoflavones, flavonols, flavanonoes,
catechines, anthocyanidins and chalcones) are one of the largest natural occurring phenolic
compounds in food and beverages, and many of them are responsible for the flavor and color
of foods [8]. The chiral determination of flavonoids by CE has mainly been focused on the
analysis of catechines, as it can be seen in Table 1, since their enantiomeric profile can be
used for food characterization including the effects of processing and storage. Most of the
developed methodologies described in the literature are aimed to study the epimerization of
catechines to evaluate their potential as markers of manufacturing processes [50, 51], storage
conditions [52] or food authenticity [53]. Thus, similar MEKC-UV methodologies using HP-CD as chiral selector (see Table 1 for specific details) have been developed to propose (-)catechin as a phytomarker of epimerization occurring during manufacturing processes in
Theobroma cacao (since this enantiomer appears after processing) [50], (-)-catechin and (-)-

112

Raquel Prez-Miguez, Mara Castro-Puyana and Mara Luisa Marina

gallocatechin as markers of manufacturing processes involving heating in tea (Figure 7) [51]


and epigallocatechin as a marker of uncorrected storage conditions in green tea sample [52].
In addition, using an EKC-UV methodology with HP--CD as chiral selector it was possible
to investigate the authenticity of guaran through the analysis of catechin and epicatechin
enantiomers [53]. By using this methodology it was possible to demonstrate that the presence
of all catechin and epicatechin enantiomers ((+)- and (-)-catechin and (+)- and (-)epicatechin) in guaran seeds was not due to pulping and drying by roasting. Therefore, they
are natural compounds that can be used to evaluate guaran authenticity.
Other flavonoid not included in catechines group whose enantiomeric separation in foods
has also been reported is isoxanthohumol. This prenylflavanone is produced from the cycling
reaction of xanthohumol during brewing process of beer samples. Both compounds have
received much attention as cancer chemopreventive and/or estrogenic agents [54]. In this
case, the enantiomeric analysis of isoxanthohumol in beer samples was performed using a
MEKC-UV method with HP--CD as chiral selector which enabled to achieve LODs of
17 ng/L. The levels of both enantiomers were almost the same showing that the
isoxanthohumol was racemic in the beer samples analyzed. This result suggests that the
racemic mixture is produced during boiling and the isoxanthohumol enantiomers are easily
interconverted [54].

Enantiomeric Determination of Sweeteners


To a lesser extent, chiral CE methodologies have also been developed for the
stereoselective analysis of sweeteners in food samples. Neotame is a new high intensity
artificial sweetener that has two chiral centers, and hence, four diastereomers (L, L; L, D; D,
D; and D, L) and its sweetness is attributed to the presence of well-oriented hydrophobic
groups in the L, L-diastereomer [55]. In a racemic mixture, the L, L-diastereomer is present
as the sweetener, while the D, D-diastereomer is not sweet. In any case, the presence of amino
acids and organic groups in its structure makes that this compound exhibits high sweetness
(nearly 10,000 times sweeter than sugar and 40 times more sweeter than aspartame) [55]. As
it can be seen in Table 1, the chiral separation of neotame has been achieved by EKC with
UV detection using heptakis-2,3,6-tri-o-methyl--CD as chiral selector. The main purpose of
this study was to provide a better understanding of the interaction established between
neotame and the chiral selector. Using molecular docking studies it was possible to show that
both electrostatic and hydrophobic interactions were relevant in the stabilization of the
inclusion complexes and in the elution order (a stronger interaction with the chiral selector
was observed for the D, D-neotame than for L, L-neotame). Moreover, the developed EKC
methodology was successfully applied to the determination of neotame in different spiked
beverages, such as mango juice, cola soft drink, and orange soft drink with LODs of 0.018
and 0.082 nM for L,L- and D,D-neotame, respectively.

Enantiomeric Determination of Pesticides

Recent Applications of Chiral Capillary Electrophoresis in Food Analysis

113

Pesticides are active compounds used in agriculture to control pest. Approximately a 25%
of pesticides employed in the world are chiral compounds and they are introduced in the
environment as racemates. However, one of the enantiomers can present the desired pesticide
activity while the other can present different activity or be inactive. For this reason, the
evaluation of their real toxicity in food samples and the study of their enantioselective
degradation require the use of enantioselective methodologies.

Figure 8. CEC electrochromatogram obtained for tap water spiked with the commercial product
containing only metalaxyl-M at a concentration of approximately 75 mg/L (according to its label). CEC
conditions: 90/9/1 (v/v/v) ACN/H2O/ formate (pH 2.5); capillary, 100 m x 24 cm packed with
Sepapak-4 and 32.5 cm total length; voltage, -10 kV with 12 bar pressure in both inlet and outlet vials;
temperature, 25C; injection 10 bar for 0.2 min (followed by a plug of mobile phase at 10 bar x 0.2
min); UV detection, 210 nm. Figure slightly modified from [58], copyright (2012) with permission
from Elsevier.

Three different examples of CE methodologies developed to perform the chiral


determination of pesticides in food samples are included among the most relevant works
published in the last decade (see Table 1). In the case of the determination of contaminants in
food samples it has to be taken into account that it is imperative to have the necessary
sensitivity to detect minor amounts of each enantiomer. For this reason, the analytical
strategies developed included off-line sample treatments and in capillary preconcentration
techniques to achieve the maximum sensitivity. For instance, to carry out the determination of
malathion enantiomers in water samples spiked at g/mL level (one of the most widely used
organophosphorus pesticides), it was necessary to combine an off-line disk solid-phase
extraction treatment as preconcentration step with an EKC-UV methodology consisting in the

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Raquel Prez-Miguez, Mara Castro-Puyana and Mara Luisa Marina

use of a Tris buffer at pH 7.0 containing CM--CD as BGE [56]. In this way, a LOD around
200 ng/mL was obtained for each malathion enantiomer. In addition to off-line
preconcentration by SPE, it is also interesting to employ in capillary preconcentration
techniques to improve the method sensitivity. A clear example which demonstrated this fact is
the determination of chiral triazole fungicides (namely propiconazole, fenbuconazole and
tebuconazole) by combining SPE pretreatment and sweeping-CD-MEKC procedure [57]. An
enhancement factor about 100-fold in the detection sensitivity was achieved by sweepingCD-MEKC at acidic pH compared to conventional CD-MEKC.
In addition to EKC and CD-MEKC separation modes, CEC has also been applied to
analyze chiral pesticides. For instance, a polysaccharide-based chiral stationary phase known
as Sepapak-4 (cellulose tris(4-chloro-3- methylphenylcarbamate)) demonstrated its potential
for the chiral separation of sixteen pesticides, including herbicides, insecticides, and
fungicides by CEC-UV using ACN/H2O/phosphate at pH 2.5 as running buffer [58]. The
excellent analytical characteristics of this methodology allowed its application to the
separation of metalaxyl and its enantiomeric impurity in tap water samples spiked with the
commercial product containing metalaxyl-M (R-enantiomer) (see Figure 8). The developed
CEC methodology was compared with the chiral separation of the same pesticides by nanoLC under similar experimental conditions observing advantages in the use of the CEC
strategy.

CONCLUSION
The individual determination of the enantiomers of chiral compounds in food samples has
demonstrated to be an interesting tool to assess food quality, safety and traceability. The high
potential of CE in chiral analysis has enabled the development of a broad range of
enantiomeric methodologies that have successfully been applied to the determination of food
components, additives and pesticides. Features of CE such as high efficiency, short analysis
times and low consumption of solvents, reagents and samples confer this technique important
advantages for chiral analysis. Moreover, the high versatility of EKC in which the chiral
selector is added to the electrolytic solution has originated that this is the preferred separation
mode in CE to achieve the chiral analysis of food samples although CEC using chiral
stationary phases has also been employed. The most successful chiral selectors in EKC and
MEKC methodologies developed have been cyclodextrins although the use of ligand
exchange complexes and macrocyclic antibiotics have also been reported. The limited
concentration sensitivity obtained in CE with UV detection can be overcome by using off-line
sample treatment techniques and in capillary preconcentration strategies and/or LIF or mass
spectrometry detection. Bearing in mind the different applications shown in this chapter, it
can be deduced that employing an adequate sample treatment sometimes combined with in
capillary preconcentration strategies (stacking or sweeping) it is possible to reach LODs at
nM levels.
The enantioselective analysis of different chiral compounds (protein and non-protein
amino acids, organic acids, phenolic compounds, sweeteners, and pesticides) in a broad range
of food matrices including fruit juices, vinegars, genetically modified organisms, microalgaes,
beers, soymilk, wine, food supplements, infant formulas, cacao beans, fruits, tea, or tap water

Recent Applications of Chiral Capillary Electrophoresis in Food Analysis

115

has enabled to obtain significant and useful information to evaluate food quality, safety and
genuineness. In general terms, through the chiral analysis of all these food components, it is
possible to propose some of the enantiomers of these compounds as potential markers to
obtain valuable information about fruit juices adulterations, food authenticity, thermal
treatments and fermentation processes, the effects of processing and storage, equivalence of
genetically modified organisms, or to evaluate if the enantiomer content of a food ingredient
fulfills the legal regulations. It is clear that all this information is of great relevance in Food
Science not only from a scientific point of view but also due to the increasing concern of
consumers about the quality of food. However, in spite of the work developed in the last
decade and the interesting information provided in the field of food analysis, it can be said
that chiral food analysis is still a quite unexplored field. Undoubtedly, CE is nowadays more
than a promising technique to face the different challenges involved in the research related to
the field of chiral Food Analysis.

ACKNOWLEDGMENTS
Authors thank the Comunidad of Madrid (Spain) and European funding from FEDER
program for research project (S2013/ABI-3028, AVANSECAL-CM). M. Castro-Puyana also
thanks the Ministry of Economy and Competitiveness (Spain) for her Ramn y Cajal
research contract (RYC-2013-12688) and R. Prez-Miguez thanks the University of Alcal
for her pre-doctoral contract.

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In: Advances in Food Analysis Research


Editor: Anita Haynes

ISBN: 978-1-63483-783-5
2015 Nova Science Publishers, Inc.

Chapter 5

CHROMATOGRAPHIC AND CHEMOMETRIC METHODS


FOR THE AUTHENTICATION OF OLIVE OIL
GEOGRAPHICAL ORIGIN
Raffaella Preti*, Simone Vieri and Giuliana Vinci
Department of Management, Sapienza University of Rome,
Rome, Italy

ABSTRACT
In recent years, the issues of food quality and safety have received a special attention
both from the producers and the consumers, in particular the geographical origin is
gaining importance as guarantee of quality.
On 2009 the European Union (EU) Member States agreed to require origin labeling
for virgin and extra virgin olive oils (EC Regulation 182/2009) to defend consumers need
about true characteristics and origin. This regulation, together with the well established
laws regarding denominations and protected indications of origin (POD PGI and STG),
has led to a new olive oil market where geographic origin labels are a big market
competitive advantage.
To enforce these laws, a measure of the authenticity of samples must be made, most
often in the form of proving the presence/absence of adulterants, or verifying
geographical or cultivar origin by comparison with known and reliable samples. Many
analytical methods (NMR; DNA fingerprint, isotopic ratio, elemental analysis etc.) have
been employed in the last decades in order to verify the real compliance to the
geographical origin declared in the label.
Among them, chromatographic techniques are the most utilized for their widespread
availability, low cost and reliability, and their data have been successfully used for olive
oil traceability models building. These methods often include the use of multivariate
statistical techniques such as principal components analysis, linear discriminant analysis,
canonical variance analysis and partial least squares regression.
In view of the growing interest in this topic, this paper reviews the literature of the
application of unsupervised/supervised chemometric methods on chromatographic data
for the characterization and authentication of the geographical origin of olive oil.
*

Corresponding author: raffaella.preti@uniroma1.it.

122

Raffaella Preti, Simone Vieri and Giuliana Vinci

Keywords: Olive oil, geographical origin, chemometric methods, chromatographic methods,


authenticity

INTRODUCTION
Olive oil (OO) is produced from the fruit of the olive tree (Olea europea L.). Virgin olive
oil (VOO) is obtained by the sole use of mechanic or other physical means, under conditions
that do not lead to any alteration of the oil. The VOO has undergone no other treatment other
than fashing, decanting centrifugation and filtration.
The olive tree is a perennial, long-lived and evergreen tree, which is probably among the
oldest domesticated trees in the Mediterranean region. About 90% of the olive fruit world
production is used for obtaining OO; according to the International Olive Council (IOC), in
2014, the olive oil world production reached 3,271,000 tonnes (IOC, 2014), placing the olive
tree as the sixth most relevant oil crop in the world. The Mediterranean countries are still
producing more than 90% of the worlds OO, with Spain (1,775,000 tonnes) the leading
country, followed by Italy (461,000 tonnes) and Turkey (190,000 tonnes), altought olive
cultivation is also widespread in Marocco, Argentina, Chile and South Africa. Its chemical
compositions is mainly constituted by triacylglycerols (TAG, more than 98%), but there is a
growing evidence of the healthy bioactive properties of some of the minor components of
VOO, such as vitamin E, pigments, phytosterols and phenolic compounds (Lpez-Miranda et
al., 2010). Among its several minor constituents, phenolic compounds, are attracting
considerable attention because they contribute to the stability of VOO against auto-oxidation
(Gallina-Toschi, Cerretani, Bendini, Bonoli- Carbognin, & Lercker, 2005; Tura et al., 2007),
they have certain influence on the organoleptic characteristics of VOO by conferring
bitterness, pungency and astringency (Bendini et al., 2007; Servili et al., 2004), and they are
also considered as components with an important health promoting properties (Boskou et al.,
2005).
Different epidemiological and clinical studies carried out to assess the implications of
VOO phenolic compounds in prevention and treatment against many diseases, have shown a
broad spectrum of bioactive properties that characterise these compounds, including antiinflammatory (Cicerale, Lucas, & Keast, 2012), antioxidant (Carrasco- Pancorbo et al., 2005)
and antimicrobial properties (Medina, de Castro, Romero, & Brenes, 2006), among others.
Indeed, VOO phenolic compounds have shown beneficial effects on diseases associated with
oxidative damage, such as coronary heart disease and cancer (Covas et al., 2006; LpezMiranda et al., 2010).
Thus, as a result of the high nutritional value and the unique flavor characteristics of olive
oil, it represents a high quality and value-added product, whose price can be even 67 times
higher than that of other edible vegetable oils. Given the specificities of its production, VOO
is one of the foods whose quality is most closely related to the terroir, or provenance; oils
from certain regions are perceived by consumer as superior in quality to others (Tres et al.,
2013). The specific cultivars used to produce OO, the pedoclimatic conditions of each area
and the agronomical and technological practices might lead to peculiar compositional
profiles, and consequently peculiar taste and quality.

Chromatographic and Chemometric Methods for the Authentication

123

In this contest, the European legislation has regulated all aspects of oil trade, in
particular, EC Regulation 182/2009 and Commission Implementing Regulation EU No.
29/2012 set out mandatory indication for the origin labelling (a reference to a Member State,
to the Union or to a third country) of extra virgin and virgin olive oil marketed within the
European Union; Commission Regulation EU No. 1151/2012 rules oils with a protected
designation of origin or a protected geographical indication (PDO, PGI).
The indication of origin related to a Member State of the European Union shall concern
the geographical area where the olives were harvested or the oil extraction mill is situated. If
the olives have been harvested in a Member State or a third country different from that where
the oil extraction mill is situated, or in the case of blends of olive oils originating from more
than one Member State or third country, the label must report whether the mixture is
composed exclusively of olive oils originating from European Union or outside the Union or
rather if the blend contains both types of products.
The verification of the geographical origin of OO is therefore an important issue in order
to protect honest producers from unfair market competition by products of inferior quality
and to ensure consumers about their purchases. However the traceability system enacted so
far by European institutions do not suggest any analytical procedure to verify the information
provided on the label and in most cases the geographical declaration of OO is never
controlled by physic-chemical parameters but its verification relies exclusively on the
inspection of the production registries and of the administrative protocols (Garcia-Gonzalez et
al., 2008).
In literature several analytical approaches are reported both to determine the olive oil
geographical authenticity and to detect possible olive oils adulterations by adding cheaper
vegetable oils (Mannina and Sobolev, 2011.), they can be resumed in three main strategies:

target analysis, when one is interested in a specific metabolite or a class of


metabolites. For the target analysis, separation techniques such as HPLC, UPLC and
GC are frequently used and often the chromatographic techniques are coupled with
detection techniques such as MS, UV and NMR.]. Typical target compounds are
fatty acids and triacylglycerol, sterols, volatiles, phenolic compounds, phospholipids
and trace elements. Their determination is important both for nutritional and control
purposes.
metabolic profiling, that requires the identification and quantification of a number of
selected metabolites, belonging to various classes of compounds, in a given sample
often without a separation procedure.
metabolic fingerprinting, that is used without any preliminary identification of
individual metabolites; in this case, any fingerprinting technique can produce a
spectrum that can be considered the whole trace of the product.

The analytical characterization of the products obtained by the three approaches briefly
described above should be processed by coupling chemometric explorative and classification
methods.
Since its beginning, chemometrics has played an important role in resolving food
chemistry problems, and its application becomes essential for the geographical authentication
of OO, in consideration that the wide natural variability of OO products that implies a large

124

Raffaella Preti, Simone Vieri and Giuliana Vinci

dataset to build and validate classification models. In most application in food authentication,
principal component analysis (PCA) is used for a preliminary inspection of the data structure.
In the next step, various classification methods such as linear discriminant analysis (LDA),
partial least-squares discriminant analysis (PLS-DA), soft independent modelling of class
analogy (SIMCA), or artificial neural networks (ANNs) are the most commonly considered
options (Berrueta et al.,2007).
This review is focused on the application of exploratory and pattern recognition
chemometric methods (unsupervised and supervised) on chromatographic data analyzed by
targeted and untargeted approaches. It describes and discusses papers published in the last ten
years on this emerging analytical challenge.

TARGETED ANALYSIS
This approach provides both nutritional and authentication data on samples analyzed and
has been extensively used in the past decade on several organic compounds determined by
GC or HPLC-based techniques. In this paragraph are discussed the main targeted compounds
and the results achieved on OO geographical origin authentication.

Fatty Acids and Tricyglycerol Compositional Data


Triacylglicerols (TAG) represent 95-98% of olive oil and fatty acids (FA) are mainly
present in this form. The FA composition of OO is genetically determined, but can be
influenced by the pedoclimatic conditions and agronomical practices, and therefore their
determination has been extensively used for the geographical characterization of OO (Tres et
al., 2013).
Principal component analysis (PCA) and linear discriminant analysis have been applied
for the characterization of of Sicilian virgin olive oils (VOO) produced in 10 different crop
years (from 1993 to 2004), and coming from two different geological zones, according to the
cultivar and the geographical origin by FA composition (Di Bella et al., 2007). Another
important zone for Italian OO production, Apulia was subject of the study by Longobardi et
al., 2012, where general discriminant analysis (GDA), partial least squares-discriminant
analysis (PLS-DA) and soft independent modelling of class analogy (SIMCA) applied to FA,
TAG, sterols and other chemical parameters data succeed in achieving a classification ability
of more than 80%. An interesting result has been achieved in three different PDOs of Sicily
(Monte Etna, Valli Trapanesi and Monti Iblei; harvests 2002 and 2003), where SIMCAand
UNEQ succeeded in a classification rate of more than 87 and 77% respectively, as evaluated
by leave-one-out cross-validation. This study highlights also the great difference in the
parameters studied in the two production years (Marini et al., 2006).
The sensory and chemical characteristics (fatty acid and triacylglycerol compositions) of
the five registered designations of origin (RDOs) of French virgin olive oils (Aix-enProvence, Haute-Provence, Nyons, Nice and Valle des Baux de Provence) were determined
over a six year harvest period.

Chromatographic and Chemometric Methods for the Authentication

125

Linear discriminant analysis applied to the samples, described by 37 parameters, allow us


to perfectly differentiate the RDOs: Nyons, Nice and Haute-Provence (Ollivier et al., 2006).
In a study involving OO samples coming from different European countries, Spain, Italy
and Portugal, the highest classification level, focused on the grouping of samples by country,
was achieved through analysis of fatty acids, with 99.9% of samples classified by artificial
neural network (ANN) models on a total of 64 compounds analysed. While to characterize
Spanish oils coming from stricter levels (region, province, PDO) was necessary to take into
consideration FA together with alcohols and sterols to have the same classifications rate
(Garca-Gonzlez et al., 2009).
The specificity of Tunisian VOOs was evaluated by comparing the samples to Algerian,
Moroccan and French Protected Designation of Origin VOOs. Classification in varietal
origins by SIMCA used the FAME compositions and NIR spectra of the most represented
varieties (Chemlali Sfax, Chetoui and Oueslati) showed a high potential to authenticate the
varietal origin of Tunisian VOOs.
Regarding another important producing country, Greece, a study on samples of virgin
olive oils (VOOs) coming from four different Greek Ionian islands, i.e., Zakynthos,
Kefalonia, Lefkada and Kerkyra, was carried out by Longobardi et al., 2012. In this work,
data of free acidity, peroxide content, spectrophotometric parameters, chlorophyll content,
phenol, sterol, fatty acid and triacylglycerol composition, were analyzed by PCA and
discriminant function analysis (DFA) and a good separation of the four geographical groups
was achieved with classification and prediction abilities equal to 97.7% and 95.3%,
respectively. Moreover, the analysis of the standardised coefficients showed that the fatty
acids and triacylglycerols were the most discriminant variables. Also Casale et al., 2010
applied chemometric analysis to data originating from FA determination and NIR spectra on
Ligurian OO from Taggiasca cultivar, an appreciated typical variety. The conclusions of this
study highlight how FA data alone are not able to provide a reliable characterization of the
authentic samples as instead are achieved by NIR spectra with a more affordable analysis.
The results of two studies by Diraman et al., 2010 showed that application of
chemometric methods, PCA and DA, to fatty acid composition is quite successful for the
classification of Turkish virgin olive oil samples produced in 4 zones of the Izmir province
(Diraman et al., 2010) and of the Agean region (Diraman et al., 2011), two important olive
production regions of Turkey, with respect to variety, geographical origin and harvest year
(20012002 and 20022003).

Volatile Compounds Analysis


The distinctive aroma of virgin olive oil is attributed to a large number of chemical
compounds of different chemical classes, i.e., aldehydes, alcohols, esters, hydrocarbons,
ketones and, probably, to other unidentified volatile compounds. It is related to genetic
factors, agronomic practices and climate and soil characteristics (Kalua et al., 2007).For these
reasons has been investigated as a marker of OO geographical origin.
Headspace solid-phase microextraction coupled with GC mass spectrometry (HS-SPMEGC-MS) or electronic nose, is the elective analytical technique for volatiles determination in
OO, it was applied to the analysis of volatile compounds of virgin olive oils from Oueslati
variety cultivated in different geographical areas of Tunisia. Twenty-seven compounds

126

Raffaella Preti, Simone Vieri and Giuliana Vinci

belonging mainly to alcohols, esters, aldehydes, ketones and hydrocarbons chemical classes
characterised the volatile profiles and their quantification permitted a correct discrimination
of each region (Youssef et al., 2011). This determination was successfully applied also to
Moroccan OO where a total of 40 volatile compounds belonging to different chemical classes
were identified and quantified and after the analysis by stepwise linear discriminant analysis
(s-LDA), results revealed a satisfactory classification of the studied oils according their
geographic origin (Bajoub et al., 2015).
Another recent study reported how soil and climate of the Chilean regions have much
more influence than cultivars on the concentration of the compounds responsible of the flavor
of EVOO (volatiles and phenols), providing good classification rates of samples according
their origin (Romero et al., 2015).

Sterols
Phytosterols are one of the major classes of compounds present in the unsaponifiable
fraction of olive oil; they exert appreciable effects on health due to the anticancer and
hypocholesterolemic characteristics (Ostlund, 2002; Pelletier et al., 1995; Pollack &
Kritchevsky, 1981; Rao & Janezic, 1992). In a recent study by Giacalone et al., 2015 on the
differentiation of Italian from non-Italian OO (Spanish, Tunisian, and blends of EU origin),
esterified sterols resulted to be more effective than sterols in characterisation of olive oils
according to their origin; and they can particularly useful in this food science problem since
they appear to be independent of those factors causing the formation of ethyl esters and
related to olive oil production (degree of ripening of olives, storage conditions of oil).

Phenolic Compounds
Phenolic compounds of olive oil have been of major interest to researchers due to their
positive effects for both human health and olive oil itself. Tuck and Hayball reviewed the
metabolism and health effects of major phenolic compounds in olive oil. Perez-Jimenez et al.
(2007) reviewed the antioxidant biological activities of olive and olive oil phenols. Phenolic
compounds are one of the main reasons for the rather high oxidative stability of the olive oil
compared to other edible oils (Gallina-Toshi et al., 2005).
For testing the ability of phenolic profiles for tracing the geographical origin of
Moroccan OO, multivariate analysis tools were used, getting a good rate of correct
classification and prediction by using a cross validation procedure, 25 phenolic compounds
belonging to different chemical families were identified and quantified by two different
platforms (LC-ESI-TOF MS and LC-ESI-IT MS) (Bajoub et al., 2015)
The same research group studied the applicability of multivariate tools to a large dataset
of 55 physico chemical parameters (among them, sterols, FA and alcohols) in OO of
Picholine Marocaine cultivar grown in 7 Moroccan regions were collected, and oils
extracted over two consecutive crop Seasons (2011 and 2012), good rate of correct
classification and prediction in relation of geographic growing origin (Bajoub et al., 2015b).
The phenolic profile of EVOO samples belonging to three different geographical origins
(Croatia, Italy and Spain) obtained by capillary electrochromatography (CEC) with UVVis

Chromatographic and Chemometric Methods for the Authentication

127

detection using lauryl acrylate (LA) ester-based monolithic columns provided a good
classification with an excellent resolution among all the geographical categories (LermaGarca et al., 2009). Also HPLc DAD is a widely used technique for phenolic compound
determination and in combination with PLS-DA provided a good discrimination in OO
coming from different parts of the Aegean coastal region, which is the largest olive oil
producer and exporter of Turkey (Alkan et al., 2012).
Eighteen phenolic compounds present in Arbequina 32 olive-oil samples from different
locations in southern Catalonia (Spain) were analyzed by a rapid and effective HPLCESITOF/MS method.
Phenolic content of extra-virgin olive oils was found to depend highly on geographical
area (Bakhouche et al., 2013).

UNTARGETED ANALYSIS
Among various approaches applicable for untargeted profiling of olive oil characteristics
for the identification of geographical origin, fingerprinting by employing SPME sampling of
head-space volatiles, followed by GC separation and MS detection, represents an effective
analytical option, since the whole process can be fully automated. Even if electronic noses do
not allow identifying the individual volatiles compounds involved in the fingerprint, they
could represent a reliable, cheap and fast screening techniques of OO authentication.
Several examples of this techniques are reported in literature. Chromatographic profiles
obtained by headspace solid-phase microextraction (HS-SPME) coupled with gas
chromatography (GC) were processed as continuous and non-specific signals through
multivariate analysis techniques in order to select and identify the most discriminate volatile
marker compounds related to the geographical origin of extra virgin olive oils from different
Spanish geographical regions considered (La Rioja, Andalusia and Catalonia). The blind
analysis of the chromatographic profiles was carried out on several steps including
preliminary mathematical treatments, explorative analysis, feature selection and
classification.
The results obtained through the application of stepwise linear discriminant analysis
(SLDA) method revealed a perfect discrimination between the samples categories
investigated (Pizarro et al., 2011). A study on the discrimination of OO originating from
Liguria region (Northern Italy) from the other regions has involved HS-SPME coupled both
with gas chromatographyion trap mass spectrometry (GCITMS) and with two-dimensional
gas chromatographytime-of-flight mass spectrometry (GC x GCTOFMS), allowing a
comprehensive analysis of olive oil volatiles with satisfactory level of classification (Cajka et
al., 2010).
Headspace-mass spectrometry methodology was used for simple differentiation among
virgin olive oils coming from several Spanish Protected Designations of Origin, on the basis
of its protected designation of origin, olive variety and geographical origin. K-nearest
neighbor and soft independent modeling of class analogy algorithms were employed to the
classification models building with ca. 87% of samples correctly classified and a specificity
of ca. 97% (Lopez Feria et al., 2008).

128

Raffaella Preti, Simone Vieri and Giuliana Vinci

The electronic nose (head-space mass spectrometry) has been widely used for
authentication purposes alone or together other chimica parameters, such as in the study of
Casale et al., 2010b where its combination with results from UVvisible and NIR
spectroscopy was able to provide a multivariate class model for OO from Liguria.
An un-targeted approach has been recently reported on the phenolic fingerprint ofOO
samples of Sabina PDO (Central Italy), where by selecting specific portions of HPLC-DAD
chromatograms recorded at 280 nm or 340 nm, it was possible to correctly classify about 85%
of samples in external validation. Further identification of these analytes by HPLC-MS
analysis showed that the substances which contributed the most to the discrimination of
Sabina PDO from other oils are all phenols of high nutritional and biological value: vanillic
acid, p-coumaric acid, luteolin, pinoresinol, acetoxypinoresinol, apigenin, and
methoxyluteolin (Nescatelli et al., 2014).

CONCLUSION
Food authentication has been a major issue over the last three decades. Olive oil
adulteration was extensively studied because it is a high added value product and adulteration
employs more sophisticated methods nowadays. Among adulteration practice the most
challenging to reveal, from an analytical point of view, is the counterfeiting of geographical
origin, which is gaining more and more importance in consumer perception. The currently
available detection methodology for olive oil adulteration comprises volatiles, phenolics and
lipidic composition in conjunction with multivariate analysis. It was shown that the
implementation of the abovementioned methodologies can lead to very high detection
effectiveness.
More efforts are needed to exploit new compounds that could be assigned as reliable
adulteration markers able to detect fraudulent practices with high selectivity, sensitivity and
accuracy. Also the analytical methods should be evaluated in light of a cost-benefit approach,
as to widespread the possibility of controls even at small laboratories scale. The construction
of large databases of physico-chemical parameters characterizing olive oils of high
commercial value could be an useful tool for routine analysis of authentication, in order to
ensure consumers of their quality purchases and producers of a fair competition.
Table 1. Principal characteristics of the studies with a targeted approach examined in
this review
Country

Target
compound
FA

Analytical method

Italy (Sicily)

No of
samples
475

Italy (Apulia)

57

FA, TAG,
sterols

GC-FID

France (5
regions)

539

GC-FID, HPLC RID

Spain, Italy
and Portugal

687

FA, TAG,
sensory
analysis
Several
FA

GC-FID

GC-FID, HPLC

Chemometric
method
PCA LDA
PCA,GDA,
SIMCA, PLSDA
LDA
ANN

References
Di Bella et
al., 2007
Longobardi et
al., 2012
Ollivier et al.,
2006
GarcaGonzlez et

Chromatographic and Chemometric Methods for the Authentication

129

Country

No of
samples

Target
compound

Analytical method

Chemometric
method

Spain

259

GC-FID, HPLC

ANN

Italy (Sicily)

200

GC-FID, HPLC

SIMCA,
UNEQ

Tunusia,
Morocco,
France
Greece

247

Several
FA, Alcohols,
sterols
22 compounds Mainly
sterols and FA
FA and NIR
spectra

GC-FID NIR
spectrometer

SIMCA

47

FA,TAG,
phenols,
chlorophyll

PCA, DFA

Turkey
(Izmir
province)
Turkey
(Agean
province)
Italy, Spain,
Tunisia blends
EU countries
Morocco
(7 regions)
Turkey (Agean
region)
Spain

103

FA

GC-FID, HPLC
RID
UV-Vis
spectrometer
GC-FID

PCA and DA

Diraman et
al., 2010

268

FA

GC-FID

PCA and DA

Diraman et
al., 2011

161

GC-FID

PCA and
SIMCA

Giacalone et
al., 2015

LC-ESI-TOF MS
and LC-ESI-IT MS
HPLC-DAD

PCA and
CDA
PLS-DA

279

HPLCESITOF/MS
Various

CDA

Morocco
(7 regions)

CDA

Bajoub et al.,
2015
Alkan et al.,
2012
Bakhouche et
al., 2013
Bajoub et al.,
2015 b

Morocco
(7 regions)

92

Sterols,
esterified
sterols
Phenolic
profile
Phenolic
profile
Phenolic
profile
Several
physicchemical
compounds
Volatiles

HS-SPME-GC-MS

s-LDA

Bajoub et al.,
2015

Croatia, Italy
and Spain
Tunisia

30

CEC

LDA

50

HS-GC-MS

PCA

Chile

180

HPLC-MS
SPME-GC-FID

PCA; S-LDA

Lerma-Garca
et al., 2009
Youseff et
al., 2011
Romero et
al., 2015

156
47
32

Phenolic
profile
Volatile
profile
Volatile and
phenol profile

References
al., 2009
GarcaGonzlez et
al., 2009
Marini et al.,
2006
LaroussiMezghani et
al., 2015
Longobardi et
al., 2012

Table 2. Principal characteristics of the studies with an un-targeted approach examined


in this review
Country
Italy
(Liguria)
Spain

No of
samples
189

102

Fingerprint
Volatiles, UVVIS and NIR
spectra
Volatiles

Analytical
method
HS-GC-MS, UVVIS and NIR
spectrometer
HS-GC-MS

Chemometric
References
method
SIMCA and UNEQ- Casale et al.,
QDA
2010 b
PCA, CA, SIMCA
and KNN

Lopez-Feria et
al., 2008

130

Raffaella Preti, Simone Vieri and Giuliana Vinci


Table 2. (Continued)

Country
Italy
(Center)
Italy
(Liguria)

Spain

No of
samples
77

Fingerprint

914

Volatiles

40

Volatiles

Phenolic fraction

Analytical
method
HPLC-DAD
HS-SPME-GCMS
(GC x GC
TOFMS
HS-SPME-GCMS

Chemometric
method
PLS-DA and biPLSGA
LDA, ANN-MLP

PCA and SLDA

References
Nescatelli et
al., 2014
Cajka et al.,
2010

Pizarro et al.,
2011

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In: Advances in Food Analysis Research


Editor: Anita Haynes

ISBN: 978-1-63483-783-5
2015 Nova Science Publishers, Inc.

Chapter 6

APPLICATION OF ULTRA-PERFORMANCE LIQUID


CHROMATOGRAPHY-PHOTODIODE DETECTORQUADRUPOLE/TIME OF FLIGHT-MASS
SPECTROMETRY (UPLC-PDA-Q/TOF-MS)
METHOD FOR THE CHARACTERIZATION OF
PHENOLIC COMPOUNDS OF PRODUCTS AT THE
MANUFACTURING STAGE OF OLIVE OIL
Joanna Kolniak-Ostek1, Agnieszka Kita2,,
Mamdouh Helmy El-Kalyoubi3,
Mohamed Magdy Mostafa Khallaf3
and Adel Gabr Abdel-Razek4
1

Dept. of Fruit, Vegetable and Cereals Technology


2
Dept. of Food Storage and Technology,
Wroclaw University of Environmental and Life Sciences, Poland
3
Food Sci. Dept., Fac. Agric., Ain Shams Univ.,
Shoubra El-Kheima, Cairo, Egypt
4
Fats and Oils Dept.; National Research Centre, Dokki, Giza, Egypt

ABSTRACT
The aim of the study was to identify and compare phenolic compounds in different
products at the manufacturing stage of olive oil. In addition the content of bioactive
compounds and antioxidant capacity of olive oil manufacturing products were
investigated. Identification of phenolic compounds was carried out using UPLC-PDA

Corresponding author: Current address: Wroclaw University of Environmental and Life Sciences, 37
Chelmonskiego Street, 51-630 Wroclaw, Poland. Tel: +48 71 320 77 62; e-mail address:
agnieszka.kita@up.wroc.pl (A. Kita).

136

J. Kolniak-Ostek, A. Kita, M. Helmy El-Kalyoubi et al.


Q/TOF-MS method. Total phenolics was measured by the Folin-Ciocalteau method, and
the antioxidant properties have been determined by DPPH, ABTS and FRAP methods.
Extraction was conducted using two solvents: 70% methanol and 70% ethanol. Influence
of the solvent type on the extraction of phenolics and antioxidant properties of the olive
extracts was also investigated. Forty two compounds belong to the compound groups of
simple phenols, hydroxycinnamic acids, flavonoids, lignans and secoiridoids were
identified. Total phenolics content of olive products ranged from 24.05 to 235.60 mg of
gallic acid/1 g DW. The highest antioxidant properties were determined in olive leaves.
Obtained results revealed that extracting solvent has a significant influence on the
polyphenolic profile, total phenolic concentration and antioxidant properties of olive
products, and that methanol is the most efficient extraction solvent.

Keywords: Antioxidant capacity, olive mill wastes, olive leaves, phenolic compounds,
UPLC-MS

1. INTRODUCTION
The extraction of olive oil generates huge quantities of wastes that may have a great
impact on land and water environments because of their high phytotoxicity. Olive mill wastes
represent an important environmental problem in Mediterranean areas where they are
generated in huge quantities in short periods of time. Several studies have proven the negative
effects of these wastes on soil microbial populations, on aquatic ecosystems (Della Greca et
al. 2001) and even in air medium (Rana et al. 2003). Therefore, there is a need for guidelines
to manage these wastes through technologies that minimize their environmental impact and
lead to a sustainable use of resources (Riog et al. 2006). Their high phenol, lipid and organic
acid concentrations, turn them into phytotoxic materials, but these wastes also contain
valuable resources such as a large proportion of organic matter and a wide range of nutrients
that could be recycled (Riog et al. 2006).
Since, only 2% of the phenolic compounds are transferred to the oil and as much as 98%
retained in the cake, olive pomace has been considered to be an interesting source of phenolic
compounds (Surez et al. 2009). Biophenols have attracted increasing attention during the
past few years due to their biological activities and natural abundance and are potential targets
for the food and pharmaceutical industries.
Olive leaf is a source of several antioxidants. The major one is oleuropein, which has
been demonstrated to have strong antimicrobial properties with potential applications for the
treatment of intestinal or respiratory-tract infections in humans (Bisignano et al. 2001).
Studies conducted by pharmaceuticals researchers' identified the primary ingredient calcium
elenolate in oleuropein, as the active agent. The second active compound in olive is delenolic acid. This ingredient is becoming widely recognized for the important part it can play
in helping to fight a broad spectrum of disease-causing pathogens and thereby boosting the
immune system (Wellman 2001).
Currently, extra virgin olive oil can be obtained by two main milling processes: the threephase system, which is widely used in Italy, Greece and other Mediterranean countries, and
the two-phase system, which is mainly used in Spain.
In the first case, olive oil is separated from two other by-products olive mill waste
water and a solid olive residue (olive pomace).In the latter system, only a semi-solid waste is

Application of Ultra-Performance Liquid

137

obtained, that contains both water and solid residue. Olive pomace can reach up to 30% of
olive oil manufacturing. Until now, only a few papers in the literature have focused on the
evaluation of the phenolic content of solid olive residues from different milling processes
(Aludatt et al. 2010; Mulinacci et al. 2005).
The disposal of by-products of the olive oil industry creates a major environmental
problem in the main olive-producing countries (Clemente et al. 1997; Lesage-Meessen et al.
2001). Therefore, a suitable use of these olive oil residues could not only improve the
economic status of olive oil producers but could also reduce such an environmental problem.
In this study, we aim to identify and compare phenolic compounds in different products
at the manufacturing stage of olive oil. In addition the content of bioactive compounds and
antioxidant capacity of olive oil manufacturing products were investigated.

2. MATERIALS AND METHODS


2.1. Chemicals
Methanol and ethanol were purchased from Merck (Darmstadt, Germany). Acetonitrile
and acetic acid were purchased from Merck (Darmstadt, Germany). The FolinCiocalteau
phenol reagent, gallic acid, 2,2-diphenyl-1-picryl-hydrazyl radical (DPPH), 2,4,6-tri (2pyridyl) s-triazine (TPTZ) and 2,2-azinobis (3-ethyl-benzothiazoline 6 sulfonate (ABTS)
were purchased from Sigma Chemical Co. (SigmaAldrich Company Ltd, Great Britain). The
phenolic standards were of purity of 9899%, and were purchased from Extrasynthese (Lyon,
France).

2.2. Plant Material


Olive oil mill waste and olive leaves were provided by an olive oil industry (two-phase
and three-phase decanter system), located in Egypt in December of 2011.
The olive oil mill wastes were promptly analyzed for pH, moisture and oil content using
the official methods (AOAC 2005).
The highest moisture content was determined in wastes from 2-phase process (61.13%).
Wastes from 3-phase process and olive leaves were characterized by a similar moisture
content 46.3 and 47.3%, respectively. Wastes from 3-phase process were characterized by a
higher oil content (13.3%) and pH (5.6) in comparison to 2-phase process (12.2% and 5.1,
respectively).
The olive mill wastes were dried in a vacuum oven at 40-50C for 48h to reduce the
moisture content, then the dried wastes were grinded and packed in dark polyethylene bags
and kept in refrigerator for further experimentation.

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J. Kolniak-Ostek, A. Kita, M. Helmy El-Kalyoubi et al.

2.3. Extraction of Phenolic Compounds


The extraction of phenolic compounds was carried as follows: five grams from each olive
wastes was weighted in conical flask 250 mL with stopper, and added 50 mL of solvent (70%
EtOH or 70% MeOH).
Then, extraction was running using ultrasonic water bath for 30 min and shaking for 90
min, after that the samples were storage at -22C for 20 h. Then, the flasks were shaking for
90 min before centrifugation at 10000 rpm for 10 min, after that the solvent was evaporated at
30C and vacuum 100 mbar.
The samples were freeze at -22C, and freeze-dried for 24 h. After that the samples were
stored at -22C until analyses.

2.4. UPLC-MS/MS Analysis of Polyphenols


Identification of olive oil mill waste and olive leaves polyphenols was carried out using
an ACQUITY Ultra Performance LCTM system (UPLCTM) with binary solvent manager
(Waters Corporation, Milford, USA) and a Micromass Q-Tof Micro mass spectrometer
(Waters, Manchester, U.K.) equipped with an electrospray ionization (ESI) source operating
in negative mode. Separations of individual polyphenols were carried out using a UPLC BEH
C18 column (1.7 mm, 2.1 mm 50 mm, Waters Corporation, Milford, USA) at 30C. The
elution solvents were aqueous 0.1% formic acid (A) and 100% acetonitrile (B). Samples (10
L) were eluted according to the linear gradient described previously by Kolniak-Ostek et al.
(2013a). Analysis was carried out using full scan, data-dependent MS scanning from m/z 100
to 2500. The effluent was led directly to an electrospray source with a source block
temperature of 130C, desolvation temperature of 350C, capillary voltage of 2.5 kV and
cone voltage of 30 V. Nitrogen was used as a desolvation gas at flow rate of 300 L/h.

2.5. Total Phenol Content Determination


Total polyphenols content (TPC) were determined by the Folin-Ciocalteu method
(Xianggun et al. 2000). An aliquot (100 l) of extract was mixed with 2000 l of distilled
water and 200 l of Folin-Ciocalteu reagent. Two hundred microlitres of sodium carbonate
solution (200 g l1) was added to the mixture. The mixture was incubated at 20C for 1 hour
in darkness. The absorbance was read at 765 nm on a UVvis spectrophotometer (Shimadzu
UV-2401 PC, Kyoto, Japan). Solutions of gallic acid from 0 to 500 mg l1 were measured
with the same procedure, for the creation of the calibration curve. Total polyphenolics were
expressed as milligrams of gallic acid equivalents (GAE) per 1 g dry weight (DW).

2.6. Analysis of Antioxidant Activity


The total antioxidant potential of samples was determined using a ferric reducing ability
of plasma (FRAP) assay by Benzie and Strain (1996) as a measure of antioxidant power. The

Application of Ultra-Performance Liquid

139

DPPH radical scavenging activity of samples was determined according to the method of Yen
& Chen (1995). The ABTS+ activity of samples was determined according to the method of
Re et al. (1999). For all analyses, a standard curve was prepared using different
concentrations of Trolox. All determinations were performed in triplicate using a Shimadzu
UV-2401 PC spectrophotometer (Kyoto, Japan). The results were corrected for dilution and
expressed in M Trolox/1 g DW.

2.7. Statistical Analysis


Results were presented as the mean standard deviation of three technical replications.
All statistical analyses were performed with Statistica version 10.0 (StatSoft, Tulsa, OK,
USA). One-way analysis of variance (ANOVA) by Duncans test was used to compare the
mean values. Differences were considered to be significant at p < 0.05.

3. RESULTS AND DISCUSSION


3.1. Qualitative Analysis of Polyphenols. General
Table 1 shows the list of forty two compounds identified through UPLC-MS/MS (with
PDA and Q/TOF detectors) experiments along with their retention times (tR), UV-Vis
spectral profiles at 200-600 nm and by comparing them with those of corresponding authentic
standards, when available. The molecules that were certainly or putatively identified in
negative ion mode belong to the compound groups of simple phenols, hydroxycinnamic
acids, flavonoids, lignans and secoiridoids.
Two simple phenols (peaks 1 and 16), five hydroxycinnamic acid derivatives (3, 5, 12, 24
and 31), eleven flavonoids (8, 9, 10, 17, 22, 25, 28, 33, 36, 40 and 42), four lignans (13, 14,
19 and 35), and twenty secoiridoids (peaks 2, 4, 6, 7, 11, 15, 18, 20, 21, 23, 26, 27, 29, 30, 32,
34, 37, 38, 39 and 41) were identified in olive oil mill wastes.
Twenty compounds, mainly from the group of flavonoids and secoiridoids, was identified
in the olive leaves. In addition compounds 2, 23, 33 and 42 were found only in those samples
(Table 1). In samples obtained by 2-phase extraction, twenty nine compounds, mainly from
the group of secoiridoids were identified. Peaks 12, 15, 16, 31 and 39, were identified only in
2-phase samples. Twenty seven compounds, mainly from the group of secoiridoids, were
identified in samples obtained during 3-phase extraction. Compounds 4, 6, 11, 13 and 19
were identified only in those samples.
Analysis revealed, that samples extracted with methanol were distinguished by a greater
variety of polyphenolic compounds compared with samples extracted with ethanol (Table 1).

3.1.1. Simple Phenols


The examination of the chromatograms in TOF-MS mode revealed the presence of two
simple phenols (Table 1). As previously stated, the identification of simple phenols was
performed by using the complementary information of chromatographic behavior and mass
fragmentation, together with retention times and UV-Vis profiles.

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J. Kolniak-Ostek, A. Kita, M. Helmy El-Kalyoubi et al.

The examination of the UPLC chromatograms in TOF-MS mode of olive oil mill waste
revealed the presence of two hydroxytyrosol derivatives (peaks 1 and 16). Peak 1 has a
pseudomolecular ion at m/z 481.2 and fragments at m/z 265 and 163, and was tentatively
identified as hydroxytyrosolrhamnoside. Peak 16 has a pseudomolecular ion at m/z 315.1 and
MS/MS fragments at m/z 153 and 123, and was identified as hydroxytyrosolhexoside (Obied
et al. 2007).

3.1.2. Cinnamic Acid Derivatives


The examination of the chromatograms in TOF-MS mode revealed the presence of four
hydroxycinnamic acid derivatives. Hydroxycinnamic acid (peak 3) yielded [M-H]- at m/z
341.1, which was fragmented to generate m/z at 179 (caffeate ion). This component was
identified as caffeic acid hexoside by comparing it with retention times and UV-Vis profile of
authentic standards of caffeic, chlorogenic and p-coumaric acid. The MS/MS fragmentation
demonstrated that the pseudomolecular anions of identified derivatives (peaks 5, 12 and 24)
were the precursors of verbascoside ([M-H]- at m/z 623.2) (Innocenti et al. 2006). Peak 31 had
pseudomolecular ion at m/z 461.2 which fragmented in m/z 315, owing to the loss of a
rhamnose residue (146 Da). This compound was therefore tentatively identified as
verbasoside (Sanz et al. 2012).
3.1.3. Flavonoids
The examination revealed the presence of free luteolin (peak 40, m/z 285.1; based on its
retention time and UV-Vis spectra, compared with those of corresponding authentic
standards) and 5 luteolin derivatives (peaks 8, 10, 17, 22 and 36).
Peak 8 has a pseudomolecular ion at m/z 593.2 and MS/MS fragment at m/z 285; and was
tentatively identified as luteolin rutinoside (Herrero et al. 2011).
The analysis in the TOF-MS revealed the presence of luteolindihexoside (peak 10, m/z
609.1). The MS/MS mass spectrum showed peaks at m/z 447 and 285 corresponding to the
loss of two hexose residues (162 and 162 Da) (Herrero et al. 2011). Peaks 17, 22 and 36, with
an identical pseudomolecular anion at m/z 447.1 that fragmented in m/z 285 owing the loss of
a hexose residue (162 Da), were identified as luteolinhexoside isomers, based on their
retention times, UV-Vis spectral profiles and accurate standards (Yorulmaz et al. 2011).
The analysis in the TOF-MS revealed the presence of free apigenin (peak 42, m/z 269.1;
based on its retention time and UV-Vis spectra, compared with those of corresponding
authentic standards) and one apigenin derivative. Peak 25 had a [M-H]- at m/z 577.2 that
fragmented in the MS/MS at m/z 269 corresponding to the loss of a rutin residue (308 Da).
This compound was identified as apigenin rutinoside (Romero et al. 2002). The analysis in
the TOF-MS revealed the presence of three quercetin derivatives (peaks 9 with m/z at 435.1,
28 with m/z at 607.2 and 33 with m/z at 463.2). Those compounds were identified as
dihydroquercetin xyloside, quercetin glucoside-piranoside and quercetin hexoside,
respectively (Bouaziz et al. 2005).

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141

3.1.4. Lignans
The examination of the chromatograms revealed the presence of two fraxiresinol
derivatives. Peaks 13 and 35 have a pseudomolecular ions at m/z 565.2 and MS/MS
fragments at m/z 403, corresponding to the loss of a hexose residue.
These peaks were identified as fraxiresinolhexosides (Sanz et al. 2012). Peak 14, which
showed [M-H]- at m/z 579.2 which fragmented to m/z 417 owing to the loss of a hexose
residue (162 Da), was tentatively identified as syringaresinolhexoside (Sanzet al. 2012).
Peak 19 had a [M-H]- at m/z 415.2. This compound was identified as acetoxypinoresinol
(Surez et al. 2010).
3.1.5. Secoiridoids
The examination revealed the presence of elenolic acid diglucose (peak 37, m/z 565.2).
The MS/MS mass spectrum showed peaks at m/z 403 and 241 corresponding to the loss of
two hexosides (162 and 162 Da) (Obied et al. 2008). Peaks 38 and 41 had a pseudomolecular
ion at m/z 403.1 and MS/MS fragment at m/z 241, corresponding to the loss of a hexose
residue. These peaks were identified as elenolic acid hexosides (Kanakis et al. 2013). Peaks 2,
7, 15, 18, 21, 23 and 26 were identified as oleuropein derivatives. Peak 2 has a
pseudomolecular ion at m/z 701.2 and MS/MS fragments at m/z 539 and 377, corresponding
to the loss of two hexosides. This peak was identified as oleuropeindihexoside (Silva et al.
2006). The examination of the UPLC chromatograms in TOF-MS mode of olive oil mill
waste revealed the presence of three oleuropein isomers (peaks 18, 21 and 26). These peaks
have a [M-H]- at m/z 539.1 with fragment at m/z 377, owing the loss of a hexose residue (162
Da) (Surez et al. 2010). Peak 7 had a pseudomolecular ion at m/z 555.2 and fragmented at
m/z 403. This peak was characterized as 10-hydroxy-oleuropein (Herrero et al. 2011). Peak 15
with m/z at 543.2 and fragment with m/z 539 was identified as dihydro-oleuropein (PeralboMolina et al. 2012). The examination of the UPLC chromatograms in TOF-MS mode
revealed the presence of free oleoside (peak 39, m/z at 389.1) and three oleoside derivatives
(peaks 11, 32 and 34) (Silva et al. 2006; Peralbo-Molina et al. 2012; Obied et al. 2008). Peaks
11 and 34 had a pseudomolecular ion at m/z 551.3 and fragment with m/z 389, owing the loss
of a hexose residue. These peaks were identified as oleosidehexosides. Peak 32 had a
pseudomolecular ion at m/z 565.1. This peak was tentatively identified as 7--1-Dglucopyranosyl-11-methyloleoside.
The examination of the UPLC chromatograms in TOF-MS mode revealed the presence of
three comselogoside isomers (peaks 20, 27 and 30) with [M-H]- at m/z 535.1 (Obied et al.
2008). The analysis in the TOF-MS revealed also the presence of nzhenide (peak 4, m/z at
685.2) (Bouaziz et al. 2010), loganinhexoside (peak 6, m/z at 569.2) (Peralbo-Molina et al.
2012) and ligstroside (peak 29, m/z at 523.2) (DeMarco 2007).

3.2. Total Yields of Polyphenols


Total yields of polyphenols in different olive mill wastes (OMW), varied significantly (p
0.05). The highest yields was obtained in case of leaves (15.2% in methanol and 15.5% in
ethanol solution), whereas the lowest yields were found in samples after 3-phase extraction
(9.17 in both solvents) (Table 2).

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J. Kolniak-Ostek, A. Kita, M. Helmy El-Kalyoubi et al.

Total yields of polyphenolic substances in samples extracted with 70% methanol was
about 5% higher in comparison to ethanolic solvents. Solvents could significantly affect
phenolics due to differences in solvent polarities, which might influence the solubility of
various constituents present in OW. Hence, the selection of the appropriate solvent is one of
the most relevant steps in optimizing the recovery of plant phenolics (Theodora-Ioanna et al.
2011).

3.3. Total Phenols Content (TPC)


The amount of total phenolic substances in the annualized mill wastes, varies
significantly (p 0.05) among the different materials (Table 2). The highest concentration of
phenolic substances was determined in leaves (235.60 mg/g in methanol and 204.87 mg/g in
ethanol solution), whereas the lowest concentration was found in 2-phase samples (28.75
mg/g in methanol and 24.06 mg/g in ethanol).
In addition analysis revealed significant differences (p 0.05) of TPC in samples
obtained with use of different solvents. Samples extracted with 70% methanol had about 10%
higher concentration of phenolic substances compared to ethanol samples (Table 2). Also
Kalogerakis et al. (2013) in their studies of olive mills wastewaters revealed significant
differences in content of antioxidant substances, in samples extracted with use of different
solvents.
Concentration of total phenolic substances was quite similar to those reported by other
authors. Theodora-Ioanna et al. (2011) presented that olive leaves had polyphenolic content,
varying from 206 mg/g to 273 mg/g. At the same time Borja, Raposo and Rincn (2006) in
liquid and solid wastes from two-phase olive oil mills reported from 0.08 mg/g to 0.4 mg/g of
total phenolics, which was 100-fold times lower than our results.
Research conducted by Wang and Lin, (2000) shows that the leaves from thornless
blackberry, black and red raspberry, and strawberry plants had higher total phenolic content
compared to their fruit tissues. Also Kolniak-Ostek et al. (2013) in their studies of apple
beverages found, that addition of apple leaves increase total phenolic content and antioxidant
capacity.

3.4. Antioxidant Activities


The highest antioxidant capacity was measured in leaves samples extracted with
methanol (Table 2). DPPH, ABTS and FRAP activities reached 1122.50 Mol, 77.54
Moland 525.87 Mol/g, respectively. The lowest antioxidant capacities were determined in
3-phase samples, extracted with ethanol. DPPH, ABTS and FRAP activities were 677.44
Mol, 19.54 Mol and 101.22 Mol/g, respectively. In general, samples extracted with 70%
methanol, were characterized by significantly higher (p 0.05) antioxidant capacities,
compared to samples with ethanol (Table 2).

Table 1. Identification of phenolic compounds in olive leaves and mills wastesa


Rt

max

[M-H]-

MS/MS

(min)

(nm)

(m/z)b

(m/z)b

1.

2.45

236/279 481.2

265 / 163

Hyroxytyrosolrhamnoside

2.

3.32

236/280 701.2

539/377/275/307

Oleuropeindihexoside

3.

3.69

324

341.1

179 / 161 / 135

Caffeicacidhexosidec

4.

4.36

250

685.2

523/ 421/ 453/ 299

Nzhenide

5.

4.46

247/331 639.2

621

-OH-verbascoside

6.

4.58

235

389

Loganinhexosideisomer

7.

4.96

238/280 555.2

403/393

10-hydroxy-oleuropein

8.

5.09

254/343 593.2

285

Luteolin rutinoside

9.

5.24

340

435.1

303/301

Dihydroquercetinxyloside

10.

5.39

269/338 609.1

285/447

Luteolin dihexoside

11.

5.48

220/266 551.3

507/389

Oleoside glucoside
-ethyl-OH-verbascoside

569.2

Tentative identification

EtOH

12.

6.14

246/329 667.2

667/621/ 487/ 459/


179/ 161

13.

6.42

236/279 565.2

505 / 439

Fraxiresinolhexoside

14.

6.50

276

579.2

417

Syringaresinolhexoside

15.

6.53

240/280 543.2

539

Dihydrooleuropein

16.

7.02

235/276 315.1

153 / 123

Hydroxytyrosolhexoside

17.

7.18

254/349 447.1

285

Luteolin hexosidec

18.

7.39

236/280 539.1

377/275/307

Oleuropeinisomer

19.

7.56

238/279 415.2

461

Acetoxypinoresinol
Comselogosideisomer
Oleuropeinisomer

20.

7.92

238/314 535.1

535/ 491/ 389/ 345/


265/ 163

21.

8.17

236/280 539.2

377/275/307

Leaves
MeOH

2-phase
mill waste

3-phase
mill waste

EtOH

EtOH

MeOH

MeOH

22.

8.62

268/339 447.1

285

Luteolin hexoside

23.

8.63

236/280 809.3

539/371/307

Unidentifiedoleuropeinderivative

Table 1. (Continued)

Rt

max

[M-H]-

MS/MS

(min)

(nm)

(m/z)b

(m/z)b

24.

8.80

247/330 623.2

461/315

Verbascoside

25.

8.81

335

269

Apigenin rutinosidec

26.

8.94

236/280 539.2

377/275/307

Oleuropeinisomer

Comselogosideisomer

577.2

Tentative identification

Leaves
EtOH

MeOH

27.

9.04

238/314 535.1

28.

9.05

340

463/301/ 178/ 151

Quercetin glucoside-piranoside

29.

9.20

236/280 523.2

361/291/ 259

Ligstroside

Comselogoside

30.

9.36

238/314 535.1

535/ 491/ 389/ 345/


265/ 163

31.

9.42

222/280 461.2

315 / 297 / 135

Verbasoside

3-phase
mill waste

EtOH

MeOH

EtOH

535/ 491/ 389/ 345/


265/ 163

607.2

2-phase
mill waste

32.

9.53

240

565.1

539

33.

9.83

340

463.2

301/ 178/ 151

Quercetin hexoside

34.

10.16

220/266 551.1

507/389

Oleoside glucoside

35.

10.35

236/270 565.2

461

Fraxiresinolhexoside

36.

10.60

269/340 447.1

285

Luteolin hexosidec

37.

11.00

340

565.2

403 / 241

Elenolicaciddiglucoside

38.

11.21

345

403.1

241/179

Elenolicacidhexoside

39.

11.39

240

389.1

226 /182 / 121

Oleoside

40.

11.41

254/350 285

Luteolinc

41.

11.68

345

42.

12.00

266/337 269.1

241/179

7--1-D-glucopyranosyl-11methyloleoside

403.1

MeOH

Elenolicacidhexoside

Apigeninc

Abbreviations: Rt retention time; EtOH 70% Ethanol; MeOH 70% Methanol; bExperimental data; cIdentification confirmed by commercial
standards.

Table 2. Total yield, total phenolic content and antioxidant activity of different olive mill waste (OW) extracts*$
Leaves

2-phase mill waste

3-phase mill waste

EtOH

MeOH

EtOH

MeOH

EtOH

MeOH

Total yield [%]

15.53 0.24a

15.20 0.43a

11.07 0.44 c

12.96 0.16b

9.17 0.67d

9.17 0.24d

TPC [mg/g]

204.87 0.056b

235.60 0.011a

24.05 0.053e

28.74 0.057d

31.68 0.036c

31.84 0.465c

DPPH [Mol/g]

988.96 0.02 b

1122.50 0.02a

921.00 0.03c

860.25 0.08d

677.44 0.03f

748.26 0.01e

ABTS [Mol/g]

66.15 1.30b

77.54 3.40a

34.71 3.80d

40.99 1.40c

19.54 0.60f

22.87 2.70e

FRAP [Mol/g]

451.28 0.02b

525.87 0.06a

136.16 0.02d

154.12 0.03c

101.22 0.02e

109.64 0.01e

Abbreviations: EtOH 70% Ethanol; MeOH 70% Methanol; TPC total phenolic content; Values are means standard deviation,
n = 3; Mean values with different letters are significantly different at p 0.05; Values are presented as mg of gallic acid/g.

146

J. Kolniak-Ostek, A. Kita, M. Helmy El-Kalyoubi et al.

Minioti and Georgiou (2010) in their studies on Greek virgin olive oil obtained similar
ABTS activity results (5.42-22.5 Mol/g but much lower DPPH activity (only 1.29-9.95
Mol/g). Abaza et al. (2011) in their studies on different extraction solvents obtained similar
results. DPPH activity in samples extracted with 70% methanol was about 20% higher in
comparison with ethanol extracts.
The antioxidant activity is positively correlated with the content of polyphenolic
compounds (Kolniak-Ostek et al. 2013). Tabart et al. (2006) demonstrated that black currant
leaves had higher content of phenolics and antioxidants than fully ripened berries, where the
total phenolic level was correlated with antioxidant activity. Increased content of
polyphenolic compounds present in the apple beverages enriched with leaves, caused an
increase in antioxidant capacity values. The data presented by Rice-Evans et al. (1997) shows,
that the compounds from the group of flavan-3-ols have strong antioxidative properties, while
Horubaa (1999) argues that some polyphenols, such as quercetin and tannins, have
antioxidant activity several times higher than ascorbic acid. Quercetin is 4.7-fold and tannins
are as much as 3-30-fold more active.

CONCLUSION
This study suggest, that olive oil solid wastes are a low-cost, renewable and abundant
source of polyphenolic compounds, and possess potent antioxidant capacity.
Obtained result shows, that leaves are a great source of bioactive compounds and may
have relevance in the prevention of diseases in which free radicals are implicated. Results
demonstrated also, that methanol is the most efficient extraction solvent. Phenolic extracts
from olive oil mill waste can be used as an alternative to synthetic antioxidants in order to
increase the stability of foods by preventing lipid peroxidation, and protect living systems
from oxidative damage by scavenging oxygen radicals.

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In: Advances in Food Analysis Research


Editor: Anita Haynes

ISBN: 978-1-63483-783-5
2015 Nova Science Publishers, Inc.

Chapter 7

FROZEN MINCED FISH FROM LOWER-VALUE


SPECIES: EVALUATION OF FATTY ACIDS
VARIABILITY BY CHEMOMETRICS
Maria Joo Ramalhosa1,2, Simone Morais1, Susana Casal 2,
Eullia Mendes2, M. Rui Alves2,3,Cristina Delerue-Matos1
and Maria Beatriz Prior Pinto Oliveira2
1

REQUIMTE/LAQV, Instituto Superior de Engenharia doInstituto Politcnico do Porto,


Rua Dr. Antnio Bernardino de Almeida, Porto, Portugal
2
REQUIMTE/LAQV, Departamento de Cincias Qumicas, Faculdade de Farmcia da
Universidade do Porto, Rua Jorge Viterbo Ferreira, Porto, Portugal
3
ESTG, Instituto Politcnico de Viana do Castelo, Av. Atlntico,
Viana do Castelo, Portugal

ABSTRACT
Sardine (Sardine pilchardus), chub mackerel (Scomber japonicus) and horse
mackerel (Trachurus trachurus) are widely distributed in Atlantic Ocean, and represent
some of the most important pelagic groups captured. They are classified according to
their fat content as medium (horse mackerel) and high (sardine and chub mackerel) fat
fish. These fish species are usually consumed fresh, due mostly to their high local
availability, with little industrial applications rather than caning. Effective preservations
techniques are needed to reduce fish losses by deterioration. Also, innovative strategies
are necessary to enhance their acceptability among the youngers. Studies determining the
fatty acid profiles of minced frozen fillets of these three species, in particular, are scarce.
This aspect is very important since minced fish is a valuable commercial product, which
has a high number of possible applications. However, it is less stable than whole fish
requiring effective knowledge of the variation during frozen storage to determine its shelf
life and define complementary preservation techniques, if necessary.

Correspondence: S. Morais *REQUIMTE/LAQV, Instituto Superior de Engenharia do Instituto Politcnico do


Porto, Rua Dr. Antnio Bernardino de Almeida, 431, 4200-072 Porto, Portugal Email:sbm@isep.ipp.pt;
Phone: +351-228340500; Fax: +351-228321159

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Maria Joo Ramalhosa, Simone Morais, Susana Casal et al.


Thus, the main goal of this study was to characterize the long-chain omega-3 fatty
acids stability of these three fish species during frozen storage of minced fillets, for up to
six months. Fish gender and catching season were also taken into account, as these
factors are known to influence the lipid amounts and composition.
High variability on the lipid content was observed between seasons, particularly in
female specimens. All species were characterized by high omega-3 polyunsaturated fatty
acids (PUFA) contents (0.53 g/100 g - 2.37 g/100 g), particularly eicosapentaenoic acid
(EPA) and docosahexaenoic acid (DHA). Sardine fillets presented higher relative
amounts of EPA, while horse mackerel samples exhibited higher levels of DHA, and
chub mackerel had the highest nutritional scores regarding potential benefits on
cardiovascular health. Reduced differences were detected for up to six months of frozen
storage, using principal components and canonical variates analyses, indicating a good
frozen stability.
These results highlight the potential use of these underutilized species for the
preparation of frozen fish alternatives, with high nutritional value. These preliminary
findings will support the evolution into industrial applications to reduce fish losses.

Keywords: Minced fish, fatty acid profiles, frozen storage, sardine, horse mackerel, chub
mackerel, food composition, chemometry

INTRODUCTION
Eating habits are among the major health concerns as the quality and amount of nutrients
ingested are regarded as fundamental for disease prevention and wellbeing. Fish, as well as
other marine species, represents an important source of diverse nutrients, including essential
minerals, fatty acids, aminoacids and vitamins. Marine lipids, in particular, have been
intensively investigated over the years due to their known richness in long-chain omega-3
fatty acids polyunsaturated fatty acids (PUFA). PUFA ingestion is increasingly associated
with positive health effects, particularly in the prevention and modulation of certain diseases,
including a reduced risk for heart disease, hypertension, blood clots formation, protection
against cancer, and even alleviating depression and certain neurological diseases (Chow,
2008; Sahena et al., 2009; Kastoriniet al., 2010; Opara and Al-Ani, 2010; Selmi et al., 2011;
Pregoet al., 2012). The worlds apparent fish consumption has grown dramatically in the last
five decades, from an average of 9.9 kg per capita in the 1960s to 18.9 kg in 2010, and
preliminary estimates for 2012 point to a further increase in fish consumption to 19.2 kg
(European Commission, 2014; FAO, 2014). In 2012, 39.6% (63 million tonnes) of world fish
production was marketed as fresh, 46.4% (73 million tonnes) of fish was frozen, cured or
otherwise prepared for direct human consumption, while 14% was destined for non-food uses
(FAO, 2014). Indeed, fresh fish is among the highest perishable food products and effectible
preservations techniques are mandatory for nutrients preservation and worldwide distribution
(Sarmaet al., 2000; Vanhonacker et al., 2013). Also, there is a great potential for the fishing
industry to utilize a greater part of the total catch for higher-value products, fuelled by
changing consumer tastes and advances in technology, packaging, logistics and transport
(FAO, 2010; Selmi and Sadok, 2010). Minced fish, besides providing the raw material for a
multitude of food applications, is also more easily accepted by the youngers, whose
appetence for fish is usually reduced (Turan and Snmez, 2007).

Frozen Minced Fish From Lower-Value Species

153

Freezing storage is an important method for fish preservation. This technique offers some
distinct advantages over other preservation methods, such as chilled, smoked and canned,
providing products of consistently high quality (Tironiet al.,2010; Indergrd et al., 2014).
Another challenge for frozen fish producers is to make a wider use of fish species, using
sustainable sources that are currently under-utilized for human consumption (Hedges, 2002).
However, some deteriorative changes and modifications may occur during freezing, namely
lipid hydrolysis and oxidation in a clear dependency of the lipid composition and amounts,
which is highly variable among species, gender and season (Hedges, 2002; Nazemroaya et al.,
2009; Prez-Palacios et al., 2014). In this regard, it has been reported by some authors that
whole fish may deteriorate less rapidly than fillets. Mincing fillets, despite increasing texture
and flavour, enhances chemical changes during frozen storage, particularly on its lipids due to
the disruption of cellular membranes (Pastoriza et al., 1994; Siddaiah et al., 2001; Tironi et
al., 2010).
Sardine (Sardine pilchardus), chub mackerel (Scomber japonicus) and horse mackerel
(Trachurus trachurus) are widely distributed in Atlantic Ocean, and represent some of the
most important pelagic groups captured (chub mackerel was the sixth species that contributed
most to global catches in 2012; FAO, 2014). Still, according to FAO (2014) horse mackerel is
an underutilised species. Fish is highly appreciated in Portugal, reaching a per capita
consumption of 56.7 kg/year in 2010 (European Commission, 2014), and these species are the
most important species in quantitative terms. They are classified according to their fat content
as medium (horse mackerel) and high (sardine and chub mackerel) fat fish (Hedges, 2002).
These fish species are usually consumed fresh, due mostly to their high local availability, but
effective preservations techniques are needed to reduce fish losses by deterioration. Also,
innovative strategies are necessary to enhance their acceptability among the youngers. The
fatty acid composition of these selected species has been studied (Aubourg et al., 2002;
Aubourg and Ugliano, 2002; Caponio et al., 2004; Celik, 2008; Garca-Arias et al., 2003;
Losada et al., 2005; Osako et al., 2003; Passiet al., 2002; Zlatanos and Laskaridis, 2007;
Orban et al., 2011). However, studies determining the fatty acid profiles of minced frozen
fillets of these three species are scarce. This aspect is very important since minced fish is a
valuable commercial product, which has a high number of possible applications, but it is less
stable than whole fish, requiring effective knowledge of the variation during frozen storage.
Only the study of Serdarolu and Felekollu (2005) was found concerning the effect of frozen
storage in sardine mince from the Aegean Sea.
Thus, the main goal of this study was to characterize the long-chain omega-3 fatty acids
stability of these three fish species during frozen storage of minced fillets, for up to six
months. Fish gender and catching season were also taken into account, as these factors are
known to influence the lipid amounts and composition.

MATERIAL AND METHODS


Reagents and Materials
Dicloromethane, methanol, petroleum ether, acetone, butylated hydroxytoluene (BHT),
glyceryl trinonadecanoate (99%, Sigma-Aldrich, USA; used as internal standard), and the

154

Maria Joo Ramalhosa, Simone Morais, Susana Casal et al.

BF3 solution (14% in methanol) were supplied by Sigma-Aldrich (Steinheim, Germany,


purity > 99.9%). Sodium chloride was obtained from Riedel-de Han (Seelze, Germany,
99.8%), and both heptane and potassium hydroxide were from Merck (Darmstadt, Germany).
Anhydrous sodium sulphate (purity 99%) was purchased from Panreac (Barcelona, Spain)
and before use, it was previously dried for 4 h at 400C in a muffle furnace.

Sample Preparation
Fresh individuals, from the Atlantic Ocean, were purchased randomly from different
local markets in Porto region (NW Portugal) during 2009. The species collected were sardine
(n=26) (71.3-78.0 g weight; 19.9-21.1cm length; 14 females; 12 males), chub mackerel
(n=26) (155.0-379.0 g weight; 27.6-35.6 cm length; 12 females; 14 males) and horse
mackerel (n=12) (283.0-295.0 g weight; 31.5 cm length; 5 females; 7 males), in a total of 64
samples (Ramalhosa et al., 2012). They were sampled seasonally (autumn - winter and spring
- summer) on a regular basis (Table 1). Sample collection, biometric characterization and
preparation were performed in accordance with EPA Guide No 823-B-00-07 (USEPA, 2000)
and EC Regulation N 333/2007 (European Commission, 2007). Specimens were carefully
identified (gender, weight and length), and manually headed, eviscerated, skinned and
filleted. Spines, still present in tissues after filleting, were carefully removed. Each sample for
further analysis (composite) consisted of an equal amount of the edible parts of, at least, four
individuals and had a minimum mass of 200 g. Male and female samples were kept separated.
Fillet mincing was performed with a mechanical blender (Ufesa, Brio 400 WMAX, PD-5310,
Spain) until a smooth paste was obtained. Composites were immediately analysed (time zero,
T0) and placed in six 50 ml polycarbonate containers for storage at -20C until further
analysis. The composites were analysed monthly during the six months storage, named from
T1 to T6.

Moisture Determination
As fresh and after being defrosted, each batch was evaluated for moisture content, by
oven drying at 100C until constant weight, in accordance with the Portuguese standard
procedure NP 2282-1991 and the official AOAC method (AOAC, 2007). Analyses were
performed in triplicate and results were expressed as g water per g muscle (wet weight, ww).

Lipid Extraction
Total lipids were extracted according to the Folch method (Folch et al., 1957), with slight
adaptations (Ramalhosa et al., 2012). Fresh (T0) and defrosted samples were homogenised.
Then, a 0.5 g sample portion was extracted twice with 20 mL of a mixture of dichloromethane
: methanol (2:1, v/v) with 0.01% of BHT and the internal standard (glyceryl trinonadecanoate,
5.0 mg from a dicholoromethane solution), by using a vortex mixer (Nahita 681/5, Navarra,
Spain) during 10 minutes.

Genderc

Total Lipids

SFAd

MUFAe

PUFAf

mega-3

n-3/n-6

AIg

TIh

HHi

EPA
Tj=0

EPA
Tj=6

DHA
Tj=0

DHA
Tj=6

Sardine

Capture
season

Species

Table 1. Lipid content and fatty acids composition (g/100 g wet weight of fish) of fresh sardine, chub mackerel and horse mackerel
(mean values standard deviations),
a
and the EPA and DHAb amounts after 6 month freeze storage

winter

2.040.12

0.690.05

0.330.04

0.950.04

0.760.04

4.8

0.58

0.26

2.17

0.290.02

0.260.02

0.420.03

0.470.06

1.870.05

0.660.02

0.290.03

0.870.05

0.700.05

5.0

0.60

0.27

2.11

0.250.01

0.200.01

0.390.04

0.440.05

0.26

2.18

spring
winter

Chub
mackerel

Horse
mackerel
a

spring
winter

3.660.02

1.210.07

0.680.02

1.760.02

1.360.04

3.6

0.63

0.700.03

0.750.02

0.530.01

0.450.01

3.850.11

1.200.08

0.740.06

1.910.05

1.430.04

3.6

0.61

0.24

2.26

0.740.06

0.650.15

0.560.03

0.530.09

0.23

3.36

1.660.04

0.560.03

0.300.03

0.810.03

0.650.03

4.6

0.38

0.130.01

0.090.01

0.460.03

0.460.07

1.270.22

0.430.06

0.210.05

0.630.12

0.530.09

5.7

0.37

0.25

3.31

0.100.02

0.080.01

0.390.07

0.380.08

8.480.45

1.750.06

1.520.07

5.210.35

2.370.21

2.0

0.32

0.21

2.47

0.400.04

0.280.02

1.730.15

1.580.33

0.18

3.14

0.110.01

0.080.01

0.700.01

0.600.01

1.94

0.260.03

0.180.02

0.760.08

0.480.06

2.01

0.190.01

0.140.03

0.610.01

0.410.10

2.570.20

0.680.05

0.550.01

1.630.06

0.880.01

2.5

0.42

3.750.30

1.470.09

1.380.11

1.810.18

1.170.11

4.5

0.55

0.31

4.670.37

1.230.02

1.080.01

1.440.02

0.900.01

4.2

0.59

0.33

EPA- eicosapentaenoic acid; 20:5n-3; DHA- docosahexaenoic acid; 22:6n-3; F- female and M- male; SFA- saturated fatty acids; eMUFA- monounsaturated
fatty acids; fPUFA- polyunsaturated fatty acids; gAI atherogenic index; hTI thrombogenic index; iHH - hypocholesterolaemic / hypercholesterolaemic
ratio; jT- time of freeze storage in months.

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Maria Joo Ramalhosa, Simone Morais, Susana Casal et al.

After centrifugation (2.16 Sartorius centrifuge, Sigma, Goettingen, Germany) during 5


minutes at 3400 rpm, the extract was filtered through a Whatman No. 4 filter paper (Whatman
International Ltd., Maidstone, UK.). 8 mL portion of aqueous NaCl solution (1%) was added
to the total extract, and the lower phase was filtered through anhydrous Na2SO4 (to remove
residual water) and collected into an evaporator tube.
The extract was evaporated in a rotary evaporator (BchiRotavapor, R-200, Switzerland)
at 45C, and the residue was further redissolved with 2 mL of dichloromethane and kept at 20C until analysis. Inter-day extraction reproducibility was controlled regularly by internal
standard recovered amounts. All extractions were performed in duplicate.

Gas Chromatography Analysis


Fatty acid composition was determined by gas chromatography, after conversion to their
fatty acid methyl esters (FAMEs). Methanolic KOH solution was added to the lipid extracts
(0.5 M; 1.5 mL). The mixture was thoroughly capped, slightly mixed and heated for 10
minutes at 100C (dry-oven, WTC-Binder). After a 5 minutes cooling period on ice, 1.5 mL
of BF3 solution (14% in methanol, Sigma, Spain) was added, capped and again heated for 30
minutes. After complete cooling, 2.5 mL of aqueous NaCl solution (1%) and 3 mL of heptane
(pesticide grade, Fluka, Spain) were added. After careful homogenisation the mixture was
centrifuged (3; 3000 rpm). A portion of the superior phase was removed, dehumidified with
a small portion of anhydrous sodium sulphate, and transferred to microvials with PTFE septa
(Supelco, Bellefonte, USA).
The fatty acid methyl esters were analysed on a CP-9001 gas-chromatograph
(Chrompack, Middelburg, The Netherlands), equipped with a FID detector and an auto
sampler (CP-9050). Separation was achieved on a CP-SIL 88 chromatographic column (50 m
0.25 mm; 0.2 m;Varian, Palo Alto, USA) with a temperature programme between 140C
and 220C, using helium as carrier gas. The fatty acids were identified by their retention times
using individual FAME standard solutions and mixtures, all from Supelco (Bellefonte, USA).
A commercial qualitative FAME mix was also used (PUFA N 1, marine source, analytical
standard grade, Supelco, Bellefonte, USA). A quantitative FAME mixture with 37 fatty acids
was used for FID calibration purposes (Supelco 37 Component FAME Mix; analytical
standard grade,Supelco, Bellefonte, USA).
The response factors were used to correct the individual fatty acid areas before
calculating their amounts. The fatty acids were initially quantified individually on a g/100 g
fatty acids basis, and then converted to 100 g of fresh minced fish using the internal standard
(19:0).
The saturated fatty acids (SFA) included 14:0, 15:0, 16:0, 17:0, 18:0, 20:0, and 22:0;
monounsatured fatty acids (MUFA) included 16:1 isomers(n-7 and n-9), 18:1 (n-7 and n-9),
20:1n-9, 22:1n-9 and 24:1n-9, while polyunsaturated fatty acids (PUFA) included 18:2n-6,
20:2n-6, 20:3n-6, 20:4n-6, 22:4n-6, 18:3n-3, 20:5n-3, 22:2n-6, 22:5n-3 and 22:6n-3.
Atherogenic index (AI) and thrombogenic index (TI) were calculated as suggested by
Ulbricht and Southgate (1991), and the hypocholesterolaemic / hypercholesterolaemic ratio
(HH) as detailed in Santos-Silvaet al.(2002).

Frozen Minced Fish From Lower-Value Species

157

Statistical Analysis
All statistical analyses were carried out using the Statistica for Windows, version 7
(StatSoft, Inc., 2004) and the SPSS software, version 21.0 (IBM Corporation, New York,
USA), following standard procedures (Barbosa et al., 2011). All univariate statistical analyses
were performed at a 5% significance level. For principal components analysis of specimen vs
fatty acids, averages of replicates were used. Canonical variates analyses were used as the
main discriminant analysis technique, using initial data (fish specimen vs fatty acids) or
transformed data (fish specimen vs principal components).

RESULTS AND DISCUSSION


Fatty Acids Composition
Moisture content ranged from 74.3 to 82.4 g/100 g wwin sardine; 75.5 to 77.3 g/100 g
wwin horse mackerel, and 68.6 to 79.5 g/100 g ww in chub mackerel (Ramalhosa et al.,
2012).Atlantic chub mackerel exhibited the highest relative proportion of PUFA in the fat,
varying between 48.5 2.0% (winter male) and 61.5 1.0% (spring females), followed by
sardine samples ranging from 46.3 2.7% (winter males) to 49.7 1.1% (spring males), and
horse mackerel with only PUFA levels of 29.7 0.6% (males) and 38.7 0.6% (females).
Saturated fatty acids (SFA) content was quite constant among species, gender, and season,
ranging from 31.1 1.7 to 35.5 1.2%, except for spring chub mackerel, varying from 20.7
0.7% (males) to 23.9 1.7% (female). The monounsaturated fatty acids (MUFA) amounts
presented small variations between sardine and chub mackerel, ranging between 15.5 1.4%
and 19.2 1.4%, except for horse mackerel (28.8 0.3% for male to 29.7 0.6% for female).
These values are in total agreement with Orban et al. (2011), for Mediterranean horse
mackerel, as well as with Celik (2008) for both mackerel species, and with Zlatanos and
Laskaridis (2007) for sardine.
Despite this apparent homogeneity among the fatty acids composition, only the mass
amount of lipids per minced fish can give their true nutritional value. Therefore, the main
fatty acids classes are presented in Table 1 on a fresh fish basis. As it is easily perceived, the
total lipid amount is highly variable, not only between species, but particularly between
gender and season. Indeed, lower lipid amounts were determined in winter chub mackerel, on
both genders, while the highest lipid amounts were found on the same species, in spring
females. The true amounts of fatty acids ingested are therefore conditioned by this lipid mass
variation, and the comparisons made previously are slightly altered. Chub mackerel PUFA
amounts were highly variable between season and gender, ranging between 0.63 g/100 g
(winter male) to 5.21 g/100 g (spring female) on a fresh fish basis, which corresponds to
almost a tenfold increase of nutritional significance. Sardine PUFA amounts were quite
constant between gender but doubled in the spring season.
Concerning omega-3 PUFAs, and in particular eicosapentaenoic acid (EPA; 20:5n-3) and
docosahexaenoic acid (DHA; 22:6n-3), spring sardine presents the higher EPA amounts
(0.7 g/100 g ww), followed by spring female chub mackerel (0.4 g/100 g ww), with the
remaining samples varying between 0.11 and 0.29 g/100 g ww. DHA content was higher in

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Maria Joo Ramalhosa, Simone Morais, Susana Casal et al.

spring chub mackerel female (1.73 g/100 g), followed by the equivalent male and winter
horse mackerel, all with around 0.7 g/100 g, while sardine presented always the lower
amounts 0.39-0.56 g/100 g.
All selected species had a n-3/n-6 ratio clearly high; 3.6 (spring males and females) 5.0
(winter males) for sardine; 2.0 (spring females) 5.7 (winter males) for chub mackerel and
4.2 (winter females) 4.5 (winter males) for horse mackerel, confirming the importance of
these species for a balanced ingestion of n-3 PUFAs. These results are in agreement with
previous studies on the fatty acids composition of sardine, chub mackerel and horse mackerel
from the Mediterranean Sea (Celik, 2008; Serdarolu and Felekollu, 2005; Zlatanos and
Laskaridis, 2007). The important nutritional lipid quality indexes, AI and TI, were also
estimated, as well as the influence on cholesterol synthesis estimated by the HH index. The
best results were consistently presented by chub mackerel, independently of the gender or
season (Table 1). Moreover, the attained mean AI values (0.32 for chub mackerel to 0.63 for
sardine) and TI (0.18 for chub mackerel to 0.33 for horse mackerel) (Table 1) can be
considered favorable for the consumption of all species, due to the cardioprotective effect of
their PUFA concentrations. The AI and TI are indicative of potential cardiovascular disease
risk factors and therefore must be kept low. Concerning the obtained HH index (1.94 for
horse mackerel to 3.36 for chub mackerel) high values are desired from a nutritional
standpoint (Guimares et al., 2013) since it estimates specific effects of fatty acids on
cholesterol metabolism. The high levels of EPA and DHA constitute an added value for these
species, which also have low to moderate prices being affordable for most of the Portuguese
population.

Influence of Species, Gender and Season on the Fatty Acids Composition


Despite being aware that other factors (water temperature and salinity, sexual maturity,
and fatty acid composition of their food) might influence the final lipids content and
composition, gender and season are the main contributors for lipid variability within a
species. Above all, they are those more easily perceived by those acquiring fish samples,
either for domestic consumption or industrial processing. In order to perform a broad
comparison of the fatty acid profiles of the minced fillets, and possible differences between
species, gender and the effect of season, a principal components analysis (PCA) was carried
out on the relative fatty acid amounts. The results are presented in figures 1 (a-b) and in Table
2. Since PCA looks for groups of correlated variables along which the variance is maximized,
each principal component (PC) is interpreted as a group of correlated variables. The
correlated fatty acids important to attribute a meaning to each component are highlighted in
Table 2 and are displayed in Figure 1 at the edges of the respective PC axes.
Figure 1 shows the planes of principal components 1 vs 2 (Figure 1a), and 2 vs 3 (Figure
1b). PC1 is mainly separating sardines (loaded on the negative, left side of PC1) from the
other fish species, showing higher amounts of 16:1 isomers, 14:0, 18:2n-6, 20:5n-3 and
20:2n-6.
These fatty acids tend to increase in spring, with no differences between males and
females. Projecting all points onto the horizontal axis, winter Atlantic chub mackerel is
displaced towards the right hand-side of the graph (positive side of PC1), followed by spring
Atlantic chub mackerel.

Frozen Minced Fish From Lower-Value Species

159

(a)

(b)
Legend: asm (Atlantic chub mackerel, spring, male), asf (Atlantic chub mackerel, spring, female), awm
(Atlantic chub mackerel, winter, male), awf (Atlantic chub mackerel, winter, female), ssm (sardine,
spring, male), ssf (sardine, spring, female), swm (sardine, winter, male), swf (sardine, winter,
female), hm (horse mackerel, winter, male), hf (horse mackerel, winter, female).
Figure 1. Principal components analysis (a) Plane of PC1 vs PC2, (b) Plane of PC2 vs PC3.

Therefore, although having lower levels of the above mentioned fatty acids, the same
seasonal pattern is observed as for sardines. Also, females of the Atlantic chub mackerel
species show a higher difference between spring and winter than males. PC2 is mainly
characterizing the spring Atlantic chub mackerel (displaced towards the upper, positive side
of PC2), with high levels of 20:1n-9, 20:3n-3 and 24:1n-9, and low levels of 20:4n-6 and
18:1n-7, the opposite being true for the same species during winter. Again, a small difference
is observed between males and females in spring. Horse mackerel is represented in the
negative, lower part of PC3. As it is clear in Figure 1b, this species (data available for winter
only) has higher levels of 18:1n-9 and 16:0 than the other species. It is important to note that

Maria Joo Ramalhosa, Simone Morais, Susana Casal et al.

160

clear significative differences between males and females were only observed for Atlantic
chub mackerel during spring.
Table 2. Correlationsa between fatty acids and principal components (PC)

Fatty acid
14:0
15:0
16:0
17:0
18:0
20:0
22:0
16:1 sum
18:1n-9
18:1n-7
20:1n-9
22:1n-9
24:1n-9
18:2n-6
18:3n-3
20:2n-6
20:3n-3
20:4n-6
20:5n-3
22:2n-6
22:4n-6
22:5n-3
22:6n-3

PC1
-0.893127
0.404648
-0.239048
0.666523
0.525028
0.165573
0.205897
-0.902759
0.429238
0.194365
0.184767
0.221300
0.312132
-0.704721
0.201505
-0.810337
0.212624
0.302475
-0.847286
-0.645404
-0.352307
-0.289168
0.606059

PC2
0.206054
-0.314582
-0.515725
-0.481520
-0.643064
-0.431846
-0.517691
-0.128887
0.105311
-0.758274
0.936532
-0.024838
0.717917
-0.412951
-0.301824
0.287057
0.947719
-0.708918
-0.423416
0.028926
-0.206340
-0.598301
-0.141258

PC3
-0.160548
-0.560479
-0.664385
-0.052862
0.234570
0.449341
0.408327
-0.348477
-0.787056
-0.291833
0.090133
0.173291
0.156510
0.199034
0.501086
0.403851
0.071344
0.388416
0.208328
0.378693
0.299105
-0.462392
0.548558

Correlations presented in italic are important for the attribution of a meaning to components.

Effect of Frozen Time on the Fatty Acid Composition


As this work is particularly concerned with the effects from storage on minced fish flesh,
it is important to evaluate, in broad terms, which are the effects of these treatments on the
fatty acid profiles, particularly the long-chain omega-3. Despite having been analysed
monthly, to avoid excessive data detail, we report the results achieved for EPA and DHA
amounts after the 6 months freezing storage, due to their increased nutritional importance
(Table 1). On a comparative basis, most losses are apparently negligible, except for the spring
female chub mackerel. The same was observed in the nutritional indexes, with similar values
after 6 months of frozen storage (data not shown), except for spring female chub mackerel. In
this species, an increase on both AI (to 0.46) and TI (to 0.36) was observed, accompanied by
a reduction in the HH ratio to 2.08 (Table 1). The higher PUFA amounts of chub mackerel
make it prone to oxidative lipid degradation, being therefore less advised for prolonged
freezing storage.

Frozen Minced Fish From Lower-Value Species

161

In terms of frozen storage, it is important to use available data to evaluate if these


treatments affect the fatty composition of minced fish flesh. For a sustained discussion,
canonical variates analyses (CVA) were performed. In contrast to principal component
analysis, CVA is a type of discriminant analysis that looks for differences between groups.
Therefore, since we are now interested in the evaluation of broad changes in fatty acids due to
the effects of freezing and frozen storage, each group was defined as being all the results
relative to a given period of frozen storage, independently of species, gender or season. CVA
was therefore carried out with seven groups: T0 (fresh minced fillets) and T1 to T6
(corresponding to the six months of frozen storage).
CVA is somewhat similar to PCA in the sense that each canonical variate (CV) may be
interpreted as a group of correlated variables. However, in the case of CVA, these
"correlated" variables reflect variations along which the previously defined groups are most
separated, i.e., along which the differences between group means are higher in relation to
within-group variabilities.
An important feature of CVA is that it magnifies small differences between groups in
detriment of main variations, and may lead to erroneous conclusions. For this reason, the
experimenter needs to be cautious in order to verify if such small differences correspond to
real differences in chemical composition, or if they are due to random information, errors in
data values, etc. As such problems exist (Alves et al., 2005; Naes and Indhal, 1998), CVA can
be carried out based on individual fatty acids (with possible over-estimations if random
information exists), or based on groups of correlated fatty acids, i.e., based on principal
components (and small, but important data details may be lost). Taking these aspects into
consideration, both approaches were carried as follows.
Table 3 shows general results of a CVA carried out with all fatty acids as explanatory
variables. Only canonical variates CV1 and CV2 seem really important, mainly the former,
with a canonical R of 0.91. The other canonical variates (CV3 to CV6), with eigen values
equal to unity or less, show little discriminatory power.
Figure 2a is a display of canonical variates1 vs 2. It is seen that group T0, i.e., minced
fish flesh without freezing (on the right, positive side of CV1) seems clearly different from
the others, with higher levels of 22:5n-3 and 22:2n-6. After one month in frozen storage, a
slight decrease in PUFA (22:5n-3 and 22:2n-6) is observed (loaded in the lower, negative part
of CV2), which is a feature characterizing samples of periodsT1, T2, and the majority of T3.
Table 3. General results for canonical variate analysis with time of freezing
as the grouping variable and fatty acids as explanatory variables

Canonical
Dimension
1

Eigen- value Canonical R

Wilks' Lambda Chi-Sqr.

dfa

p-level

4.846412

0.910470

0.017820

777.2982

150

0.000000

1.301201

0.751960

0.104181

436.4933

120

0.000000

1.034351

0.713052

0.239742

275.6411

92

0.000000

0.432935

0.549665

0.487719

138.5769

66

0.000000

0.284435

0.470582

0.698870

69.1501

42

0.005215

0.114016

0.319917

0.897653

20.8386

20

0.406688

df- degree of freedom.

162

Maria Joo Ramalhosa, Simone Morais, Susana Casal et al.

(a)

(b)
Figure 2. Canonical variates analysis with time of freezing as the grouping variable and: (a) Fatty acids
as the explanatory variables (plane of the first two canonical variables); (b) Principal components as the
explanatory variables. T0- analysis of fresh minced fillets; T1, T2, T3, T4, T5 and T6 - analysis of minced
fillets after one, two, three, four, five and six months of freezing storage, respectively.

The other groups (periods T4, T5 and T6) are displaced towards the left and upper parts
of the plot, representing a decrease in PUFA. Based on chemical knowledge, it can be
concluded that these observations are not due to random information, and that the fatty acid
profiles of minced fish fillets changed during the studied freezing times. These findings are in
agreement with observations made by other researchers. Tironi et al. (2010) studied the lipid
changes during the frozen storage of minced sea salmon (Pseudopercis semifasciata) and
concluded that the main PUFA affected were also the 22:6n-3, 22:5n-3 and 20:4n-6 fatty
acids. Nazemroaya et al. (2009) reported that contents of PUFA n-3, n-3/ n-6, PUFA/SFA and
EPA + DHA/16:0 decreased during frozen storage of Spanish mackerel (Scomberomorus
commersoni) and white cheek shark (Carcharhinus dussumieri).

Frozen Minced Fish From Lower-Value Species

163

Table 4. General results of canonical variate analysis with time of freezing as the
grouping variable and principal components as the explanatory variables

Canonical Dimension

Eigen- value

1
2
3

0.304739
0.006708
0.002629

Canonical R Wilks'
Lambda
0.483284
0.759333
0.081632
0.990732
0.051207
0.997378

Chi-Sqr.

dfa

p-level

56.16418
1.89956
0.53562

18
10
4

0.000008
0.997054
0.969942

df- degree of freedom.

As explained before, a second CVA was carried out, this time based on principal
components instead of individual fatty acids, therefore taking into account only the main data
structures (main groups of correlated fatty acids) (Naes and Indhal, 1998). The principal
components used were those reported in Table 2. The general results are presented in Table 4.
It can be seen that all eigen values are inferior to unity, which demonstrates that canonical
variates have no real meaning and that there are no significant differences between the groups
taken in consideration. Consequently, as it is shown in figure 2b, no groups are evident and
no interpretation of canonical variables is possible. As a matter of fact groups are
intermingled (figure 2b), showing no differences due to main groups of fatty acids.
Simultaneously, because differences do not exist, it is not possible to describe canonical
variates in terms of original principal components. These results are interesting since they
show that the bulk of the fish fat is not altered by mincing and frozen storage, while the CVA
carried out with individual fatty acids showed that minor differences exist in terms of PUFA.

CONCLUSION
The three fish species analyzed have high omega 3 PUFA contents, favorable nutritional
lipid indexes and medium to high lipid amounts, being therefore recommended on a healthy
diet. The highest PUFA and lipid amounts were observed in Atlantic chub mackerel, followed
by sardine, with a clear increase of PUFA in spring. Gender influenced lipid amounts but not
lipid profile. Horse mackerel, an underutilized species, is characterized by medium lipid
content and an equilibrated fatty acid profile, being also recommended as a source of healthy
fatty acids in the PUFA fraction. Minced chub mackerel, however, is the less appropriate for
the development of frozen minced products. Therefore, further studies will now be designed
to determine other important chemical (thiobarbituric acid reactive substances formation;
carbonyl compounds) and sensorial attributes. These conclusions are of great importance for
the fish industry, providing the support for the preparation of an increased variety of
commercial frozen and minced products.

ACKNOWLEDGMENTS
Maria Joo Ramalhosa thanks Instituto Politcnico do Porto and Instituto Superior de
Engenharia do Porto for herfellowship. This work received financial support from the
European Union (FEDER funds through COMPETE) and National Funds (FCT) through

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Maria Joo Ramalhosa, Simone Morais, Susana Casal et al.

project UID/QUI/50006/2013. To all financing sources the authors are greatly indebted. The
authors declare no conflicts of interests.

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In: Advances in Food Analysis Research


Editor: Anita Haynes

ISBN: 978-1-63483-783-5
2015 Nova Science Publishers, Inc.

Chapter 8

VALIDATION OF A MATRIX SOLID PHASE DISPERSION


METHODOLOGY FOR THE DETERMINATION OF
TRIAZINES HERBICIDES IN FISH
M. J. Gonzlez-Castro*, V. Castro-Bustelo,
N. Rodrguez-Gonzlez and E. Beceiro-Gonzlez
Grupo Qumica Analtica Aplicada, Instituto Universitario de Medio Ambiente, Centro
de Investigaciones Cientficas Avanzadas, Departamento de Qumica Analtica, Facultade
de Ciencias, Universidade da Corua, A Corua, Spain

ABSTRACT
A method using dual process columns of Matrix Solid Phase Dispersion (MSPD) and
Solid Phase Extraction (SPE) has been validated for extracting and cleaning-up of nine
triazine herbicides (ametryn, atrazine, cyanazine, prometryn, propazine, simazine,
simetryn, terbuthylazine and terbutryn) in trout samples. For this purpose, freeze dried
samples (0.2 g) were blended with 2 g of octadecylsilyl-derivatized silica (EnviTM-18)
and transferred into a SPE cartridge containing ENVITM-Carb II/SAX/PSA (0.5/0.5/0.5 g)
as clean up co-sorbent. Then the dispersed sample was washed with 10 mL of n-hexane
and triazines were eluted with 20 mL ethyl acetate and 5 mL acetonitrile. Finally the
extract was concentrated to dryness, re-constituted with 1 mL methanol and injected into
the HPLC-DAD system. Recoveries varied between 88 and 105% with associate standard
deviations below 8.6%, meeting the requirements stipulated by European Union
legislation. The main advantages of this methodology when compared with conventional
methods of sample preparation to screen herbicides in fish matrices are easy of work-up,
fast, cheap, avoidance of clean-up procedure, as well as the reduction of organic solvents
and energy requirements in agreement with the principles of Green Chemistry.

Corresponding author: e-mail: mjgc@udc.es.

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1. INTRODUCTION
Fish can be harvested from waters that are contaminated by environmental chemicals,
which may accumulate in fish at levels that can cause human health problems. Concern for
these contaminants primarily focuses on fish harvested from aquaculture ponds, freshwater
bodies, estuaries, and near-shore coastal waters (areas subject to shoreside contaminant
discharges), rather than from the open ocean [1]. Environmental contaminants, such as
pesticides, may also accumulate in aquacultured fish through contaminated feed ingredients
[2]; furthermore, certain pesticides are applied directly to the water in aquaculture ponds to
control weeds and algae and to eliminate invertebrates.
Nowadays there is a wide public interest in aquaculture. In fact, world aquaculture
production is increasing much more rapidly than animal husbandry and capture fisheries, the
other two sources of animal protein for the world population. There is widespread recognition
that seafood production from capture fisheries is at or near its peak, and that aquaculture will
become increasingly important as a source of seafood production [3]. Therefore the control of
chemical groups which can originate important problems when reaching fish in farms is of a
great interest.
Triazines are a group of herbicides that are present in the ten most-used herbicide
formulations in Europe. Because of their relatively low soil adsorption and high solubility in
water, these compounds migrate from soil to water [4] and due to the water cycle, fluxes of
them reach seawater and affect the marine biota, having the coastal systems little capacity to
degrade these compounds [5]. Furthermore, aquatic organisms are able to accumulate
pesticide residues in much higher concentration than the surrounding water [6].
Therefore, triazines are considered as an important class of chemical pollutants and the
Environmental Protection Agency (EPA) has proposed simazine and atrazine in the list of 67
pesticide for screening in the Endocrine Disruption Screening Program [7]. European Union
has also included simazine and atrazine in the list of 33 priority substances in the European
Union Water Framework Directive (2000/60/EC) [8], which committees the member states to
achieve good qualitative and quantitative status of all water bodies, by way of Decision
2455/2001/EC [9]. Moreover, the Directive 2008/105/EC sets the Environmental Quality
Standards (EQS) for these compounds in water and also shows the need to set EQS for these
compounds in sediments and/or biota in order to protect the aquatic environment [10]. Since
article 16 of the Water Framework Directive (2000/60/EC), demands a regular review of the
priority substances, in January 2012, fifteen additional substances were proposed by the
Commission (Proposal COM (2011)876) to be added to the list of priority substances [11].
Among these, terbutryn was proposed. European Union has not established yet limit values
for triazines in fish and fishery products; however the U.S. Food and Drug Administration
(FDA) has set a tolerance level of 12 mg kg-1 ww (wet weight) for simazine in the edible
portion of finfish [1].
The sample preparation procedure is one of the most critical steps in analytical methods.
In recent years, many innovations have been developed in the analytical processes applied to
prepare food samples for the extraction and determination of pesticide residues. These
methodologies include matrix solid phase-dispersion (MSPD), which combines aspects of
several analytical techniques allowing sample homogenization, disruption, extraction,
fractionation and clean-up within a single process [12]. In MSDP the solid sample is blended

Validation of a Matrix Solid Phase Dispersion Methodology

169

in a mortar with an appropriate sorbent to obtain complete disruption and dispersion of the
sample on the solid support. The blend is packed into a column from which the analytes are
eluted with a relatively low solvent volume. Often, a co-sorbent material is placed at the
bottom of the column to be filled with the blended sample to assist the extract clean up [13].
The key factors for the success of MSPD are its feasibility, flexibility, versatility, high
throughput, low cost and rapidity. MSPD methods have been developed for the extraction of
triazine residues from different plants and plant materials [14, 15]; however references for the
determination of triazines in animal tissues by MSPD are scarce and to the best of our
knowledge, no studies using MSPD have been done to extract these chemicals residues from
finfish.
Therefore, the aim of this work was the validation of an effective, simple and fast method
for the simultaneous determination of nine triazine herbicides in fish based on Matrix Solid
Phase Dispersion (MSPD) and Solid Phase Extraction (SPE) clean-up followed by High
Performance Liquid Chromatography (HPLC) coupled to Diode array Detection (DAD). In
this study, rainbow trout (Oncorhynchus mykiss) from aquaculture was selected because there
has been a large increase in salmonid production, and unlike many of the major products from
aquaculture, there is substantially more aquaculture production of salmonids in developed
countries than in developing countries [16]. On the other hand, trout is one of the four typical
representative commodities included in the category of fish to carry out procedures for
pesticide residues analysis in food and feed by SANCO guidelines [17]; furthermore, FDA
considers the aquaculture trout as a potential fish-related hazard by environmental chemicals
[1].

2. MATERIAL AND METHODS


2.1. Samples
1 kg of rainbow trout (Oncorhynchus mykiss) from aquaculture were purchased from a
local market in A Corua city (Northwest of Spain). Skin, bones and inner organs were
discarded and muscle tissues were chopped, homogenized and freeze-dried. Then samples
were homogenized again grounding it to a fine powder by an electrical mill and finally they
were stored in glass bottles out of light exposure until analysis. The water content was
determined gravimetrically by weighing before and after lyophilisation.

2.2. Chemicals
(a) Herbicide standards Herbicides (ametryn, atrazine, cyanazine, prometryn,
propazine, simazine, simetryn, terbuthylazine and terbutryn) analytical standards
were supplied by Sigma-Aldrich (Inc. St. Louis, MO, USA). The individual stock
standard solutions of 1000 mg L-1 were prepared in methanol by exact weighing of
high-purity substances and stored at -18 C in the dark. Then a mixture of all the
compounds was prepared in methanol containing 10 mg L-1 each individual triazine

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M. J. Gonzlez-Castro, V. Castro-Bustelo, N. Rodrguez-Gonzlez et al.


and stored at -18 C. All working solutions were daily prepared by appropriate
dilution of the 10 mg L-1 standard solutions with methanol.
(b) Solvents n-hexane 95% and methanol were superpurity Solvents from Romil
(Cambridge, UK). Acetonitrile (ACN) (HPLC grade) and ethyl acetate (PAR,
solvents for analysis of pesticide residues by GC) for instrumental analysis were
from Panreac (Barcelona, Spain). Milli-Q water was obtained from a purification
system from Millipore (Billerica, MA).
(c) Sorbents Bulk packing: SupelcleanTM EnviTM-18 and SPE tubes: SupelcleanTM
Envi-CarbTM II/SAX/PSA (500 mg/500 mg/500 mg) were from Sigma-Aldrich (Inc.
St Louis, MO, USA).
(d) Filters Polytetrafluoroethylene (PTFE) filters of 0.45 m were from Teknocroma
(Barcelona, Spain).

2.3. Materials and Apparatus


A Visiprep vacuum distribution manifold from Supelco (Bellefonte, PA, USA) was
employed in the purification step. A Bchi R-3000 rotary evaporator (Bchi Labortechnic
AG, Flawil, Switzerland) was used in the evaporation step.
Chromatographic analyses were carried out in a high performance liquid
chromatography-diode array detector (HPLC-DAD). The system consisted of a 2695 pump
with a 996 Diode Array Detector from Waters (Milford, MA, USA). The column was a
stainless steel column (150 mm x 4.6 mm ID, particle size 5 m) packed with Hypersil
GOLD C18 chemical bonded phase from Thermo Scientific (Austin, TX, USA).

Figure 1. Scheme of the conditions of MSPD-SPE procedure.


(1) 2.00 g C18; (2) 0.2000 g freeze-dried trout; (3) Homogenization; (4) Transfer to a clean-up co
column with EnviCarb/SAX/PSA (500/500/500mg); (5) Rinsing with 10 mL hexane; (6) Elution with
20 mL ethyl acetate and 5 mL acetonitrile.

Validation of a Matrix Solid Phase Dispersion Methodology

171

2.4. Extraction Procedure


Samples were extracted by following a procedure previously developed in our laboratory
for determination of the target compounds in mussel samples [18], with a modification
regarding the sample amount. Under final conditions, 0.2000 g of freeze-dried trout sample
was homogenised with 2.00 g of EnviTM-18 in a glass mortar with a pestle for 5 min. The
final mixture was transferred into a 20-mL SPE cartridge containing a triple sorbent layer of
1.5g SupelcleanTM Envi-CarbTM-II/SAX/PSA (500/500/500 mg). Once packed, MSPD/SPE
columns were connected to a Visiprep vacuum distribution manifold and washed with 10
mL of hexane. Elution was performed with 20 mL of ethyl acetate and 5 mL of ACN and the
obtained eluate was evaporated to a drop in rotary-evaporator and got to dryness by a gentle
nitrogen stream. The residue was reconstituted in 1 mL methanol and the solution was filtered
through of a 0.45 m syringe filter of PTFE. As an illustration, Figure 1 shows the scheme of
the MSPD-SPE procedure.

2.5. HPLC-DAD Conditions


The chromatographic analysis was carried out using the following ACN:H2O gradient
elution: ACN initial percentage of 30% (8 min), increased linearly to 40% in 5 min; increased
to 50% in 5 min, after which the percentage was returned to the initial conditions in 9 min. A
constant mobile phase flow rate of 1 mL min-1 and 20 L of sample volume were used.
The absorbance was measured continuously in the 200-400 nm range and peaks areas
quantification were carried out at 222.7 nm in order to achieve maximum sensitivity. All
triazine herbicides were identified initially by retention time and then by spectral
identification contrasting the spectrum with a standard library created in the same wavelength
interval.

3. RESULTS AND DISCUSSION


3.1. MSPD Application
Sorbent selection is of utmost importance since it is one of the variables controlling the
selectivity of MSPD processes [19, 20]. In this work, we applied a method which employs
C18 as dispersant, Envi-Carb II/SAX/PSA as clean-up co-sorbent and a mixture of ethyl
acetate and acetonitrile as elution solvent. C18 is by far the most popular sorbent, especially
for analyte extraction from animal. Regarding the tri-layer SPE cartridge, ENVI-Carb II is a
graphitized non porous carbon that has a strong affinity towards planar molecules and can
remove pigments and sterols, PSA is a polymerically bonded ethylenediamine-N-propyl
phase that contains both primary and secondary amines, that retains fatty acids, organic acids,
sugars and some polar pigments, and SAX is a quaternary amine that offers additional ion
exchange capacity for removing matrix components.
During extraction of complex matrices, multiple unwanted compounds from the matrix
are extracted along with the compounds of interest. A major issue when dealing with the

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M. J. Gonzlez-Castro, V. Castro-Bustelo, N. Rodrguez-Gonzlez et al.

chromatographic determination of organic compounds in fish samples is to minimize the lipid


content of the extracts [21, 22]. If lipids are not removed in the sample preparation process,
they may cause retention time shifts, analyte peak distortion, decrease of sensitivity and
reduction of column life, thus compromising quality of analysis. Therefore the determination
the pesticides in food is often complicated by the presence of different fat content [23].
First assays were carried out employing 0.5000 g of trout sample following the procedure
previously described for determination of triazines in mussel samples [18]. After evaporation
of the eluates, it could be seen that the extracts had an appreciable amount of lipid material,
and were not analysed further. This can be explained because of the higher lipid content in
trout (4.8%) than in mussel (2.2%) [24]. Therefore, the following experiments were carried
out using 0.2500 g of trout sample. In this case, after evaporation of eluates, extracts were
clean enough to be processed; however after redissolution on methanol and filtration through
PTFE filters, it could be observed some small particles of fat, thus the extracts were not
analysed. Finally, considering 0.2000 g of trout sample, the extracts obtained were clean
enough to be injected into the HPLC system. As an example, Figure 2 shows the
chromatogram corresponding to a trout sample extracted and purified under the considered
procedure; as it can be seen, none of the triazines under study were detected.

Figure 2. HPLC-UV chromatogram of a trout extract obtained by MSPD.

3.2. Method Validation


The method was validated in terms of accuracy, precision and limits of quantification
according to validation parameters and criteria from SANCO Guidelines for method
validation and quality control procedures for pesticide residue analysis in food and feed [17].
Quantitative results were calculated using matrix matched standards prepared by spiking
the final extracts from blank samples of trout samples with different levels of triazines. The
linearity of the calibration curves was calculated at a concentration range between 0.5-5 mg

Validation of a Matrix Solid Phase Dispersion Methodology

173

kg-1 dried sample by duplicate analysis at five different concentration levels. Excellent
linearities were obtained with coefficients of determination (R2) higher than 0.998 for all
triazines.

(a)

b)
Figure 3. HPLC-UV chromatograms of a spiked trout sample obtained by MSPD. (a) Trout sample
spiked at 0.5 mg kg-1; (b) trout sample spiked at 5 mg kg-1. Target compounds are numbered as follows:
(1) Simazine, (2) Cyanazine, (3) Simetryn, (4) Atrazine, (5) Ametryn, (6) Propazine, (7)
Terbuthylazine, (8) Prometryn, (9) Terbutryn.

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M. J. Gonzlez-Castro, V. Castro-Bustelo, N. Rodrguez-Gonzlez et al.

The accuracy and precision of the proposed method were determined by analysis of five
replicates of uncontaminated trout sample spiked at two concentration levels (0.500 and 5.00
mg kg-1 dried sample, equivalent to 0.125 and 1.25 mg kg-1 fresh sample, respectively). The
obtained results demonstrated that the method achieved satisfactory recoveries for all
compounds in the range of 88-105% for the low level and of 96-100% for the high level with
associate standard deviations below 8.6% and 2.5% respectively (Table 1). These values are
in the acceptance range of the DG SANCO/12571/2013 of the European Quality Control
Guidelines (mean recoveries from 70 to 120% and RSD < 20%), which indicate that the
method meets the requirements stipulated. As an example, Figure 3 shows the chromatograms
corresponding to spiked trout sample, fortified at both levels.
Table 1. Precision and accuracy of target compounds in trout samples with MSDPHPLC-DAD (n=5)

Compound
Simazine
Cyanazine
Simetryn
Atrazine
Ametryn
Propazine
Terbuthylazine
Prometryn
Terbutryn

Analytical recovery (%) RSD (%)


0.5 mg kg-1
5 mg kg-1
90 3.8
97 1.4
105 5.9
98 1.5
90 6.7
97 2.0
101 4.9
99 2.5
96 4.5
96 2.3
101 8.6
98 1.6
88 6.7
100 1.4
95 3.8
100 1.1
100 4.7
100 1.6

The limit of quantification (LOQ) is defined as the lowest concentration of the analyte
which has been validated meeting the method performance acceptability criteria (i.e., the
average recovery was in the range 70-120% with RSD < 20%) and was determined based on
the accuracy and precision data obtained through the recovery studies. As it can be seen on
Table 1, 0.5 mg kg-1 meets the method performance acceptability criteria for trueness and
precision by applying the complete analytical method (DG SANCO/ 12571/2013). On the
other hand, the reduction in the weight of trout samples during freeze-drying accounted for
ca. 75% (0.2 g of freeze-dried sample equivalent to ca.0.8 g of fresh tissue); therefore,
approximately 4-fold lower LOQ values are achieved when referred to fresh sample.

CONCLUSION
The suitability of a procedure based on MSPD and SPE for the extraction of nine
triazines from finfish samples has been demonstrated for the first time. The method uses
EnviTM-18 as dispersant with Envi-CarbTM II/SAX/PSA co-column followed by a
combination of 20 mL ethyl acetate and 5 mL acetonitrile as elution solvent. The developed
method provides satisfactory accuracy and precision for the determination of triazines in
aquaculture trout samples. The main advantages of this methodology when compared with
classical methods of sample preparation to determine triazines in animal tissues are easy of

Validation of a Matrix Solid Phase Dispersion Methodology

175

work-up, fast, low cost, avoidance of clean-up procedure, as well as the significant reduction
of organic solvents and energy requirements in agreement with the principles of the Green
Chemistry.

ACKNOWLEDGMENTS
This work has been supported by Ministerio de Economa y Competitividad (CTM201348194-C3-2-R) and Program of Consolidation and Structuring of Units of Competitive
Investigation of the University System of Galicia (Xunta de Galicia) potentially cofounded by
European Regional Development Fund (ERDF) in the frame of the operative Program of
Galicia 2007-2013 (reference: GRC2013-047). N. Rodrguez-Gonzlez also thanks the
Xunta de Galicia and Campus do Mar International Campus of Excellence for the
concession of her PhD. grant.

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[21] Barriada Pereira, M., Gonzlez Castro, M.J., Muniategui Lorenzo, S., Lpez Maha, P.
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medidas caseras de consumo habitual en Espaa. McGraw-Hill Interamericana.

In: Advances in Food Analysis Research


Editor: Anita Haynes

ISBN: 978-1-63483-783-5
2015 Nova Science Publishers, Inc.

Chapter 9

THE USE OF DNA-BASED METHODS FOR


DETECTION OF PLANT-BASED ADULTERANTS
IN PLANT FOOD PRODUCTS
Nadia Haider, PhD
Department of Molecular Biology and
Biotechnology, AECS, Damascus, Syria

ABSTRACT
Food adulteration is an act of intentionally debasing the quality of food offered for
sale either by the admixture or substitution of inferior substances or by the removal of
some valuable ingredients. In addition to food adulteration, contamination of food
products as a result of inadequate company controls, or due to the nature of the product or
the process has been reported. Food adulteration has been a major contributor to the poor
quality of industrial products, and the number of adulterants used to supplement
expensive ingredients has been continually increasing. A serious problem with the
adulteration of food products with declared or undeclared other foodstuff may cause
severe health problems and may even risk death to people who may have a severe
allergenic response to some adulterants (e.g., nuts and soya). This problem is particularly
acute when the presence of adulterants (e.g., walnuts in bread) cannot be reasonably
anticipated. In consequence, there is a constant and continuing need for improved
methods to detect the presence of adulterants in foodstuffs (e.g., adulteration of single
food commodities). Numerous methods based on DNA analysis have contributed
immensely in the food industry to monitor adulterations of food products of plant or
animal origin. In this chapter, reported examples of adulteration of plant food products
(mainly those which are economically important) with adulterants, of plant origin in
particular, and examples of DNA-based methods and markers used for detection of such
adulteration are presented without making detailed or critical analysis or evaluation of
each of those examples.

Corresponding author: Dr. Nadia Haider. Department of Molecular Biology and Biotechnology, AECS, P.O. Box
6091, Damascus, Syria. E-mail: ascientific@aec.org.sy, tel: 00963-11-2132581,2,3, fax: 00963-11-6112289.

178

Nadia Haider

INTRODUCTION
The act of defrauding buyers of food and food ingredients for economic gain is usually
referred to by the collective term "food fraud" (Johnson 2014). Food industry has been vexed
by food fraud throughout history (Johnson 2014). Criminal gangs target many foodstuffs
because of their high value through mixing them with cheaper alternatives while selling them
at the price of the pure material in order to gain higher profits (Down 2013). During the last
fifty years, there have been high concerns on the safety and quality of the food products, of
the production processes and the relationship between the two (cited in Kartheek et al. 2011).
Busconi et al. (2003) believe that the origin of a food product and the typicality of its
production procedures is a guarantee of the product safety and healthiness. Because people
who commit fraud do not intend to cause physical harm in order to avoid detection, and
consumers usually do not notice a quality problem, most food fraud incidents go undetected.
As a consequence it is not possible to conclusively know how widespread food fraud is
worldwide (Johnson 2014).
Food fraud can be achieved either through 1) intentional omission of a valuable and
authentic constituent without the purchasers knowledge, 2) addition of small amounts of a
non-authentic substance to mask inferior quality ingredient, and 3) partial or complete
replacement of a valuable food ingredient or authentic constituent with a less expensive
substitute (Moore et al. 2012). The majority of food fraud cases, however, involve the
substitution of a high-value product with a lower quality or less expensive alternative
(Johnson 2014).
Food fraud also embodies the deliberate misrepresentation of food, food ingredients or
food packaging. It also includes false or misleading statements made about a product for
economic gain (Lakshmi et al. 2012). The addition of adulterants (additions that are unwanted
by the recipient) is called adulteration. The addition of adulterants to food to increase value
and attractiveness is usually referred to as economic adulteration. An individual or
company adulterate food products to be able to compete, to increase profit or as a result of
cost-cutting pressures (Spice Adulteration online reference).
Food adulteration has been a major contributor to the poor quality of industrial food in
Britain since the early 19th century (Collins 1993), and the number of adulterants used to
supplement expensive ingredients has been continually increasing. According to US
Pharmacopeia (2012), a research of analyses of the first known public database compiling
reports on food fraud and economically motivated adulteration in food has been published in
the April Journal of Food Science. These analyses highlight the most fraud-prone ingredients
in the food supply; analytical detection methods; and the type of fraud reported. The database
provides information that can be useful in evaluating current and emerging risks for food
fraud. It also equips with a baseline understanding of the vulnerability of individual
ingredients and offers information about potential adulterants that could reappear in the
supply chain for particular ingredients. Analytical testing strategies to detect food fraud are
also part of the database (US Pharmacopeia 2012). Johnson (2014) reported that efforts are
ongoing to compile and capture current and historical data on food fraud and economically
motivated adulteration (EMA) incidents through the creation of databases and repositories.
He reviewed available database information.

The Use of DNA -based Methods for Detection of Plant -based Adulterants

179

The consumer has the right to know the species origin of food products for religious or
nutritional reasons. Government or the concern department, however, is not able to fully solve
the food adulterations problem unless the consumer himself becomes conscious of the hazards
to health on such consumption and the law which protects him under such circumstances.
Therefore, consumers must know the provisions of the Food Laws and Regulations and how
they are protected under the said Law (Project Work 2012). Monitoring food adulteration has
a great impact on the quality of food products and ensuring public health (Parvathy et al.
2014). In recent years, increased attention to food safety has stimulated the interest in the
authenticity of food products (Consolandi et al. 2008). The main objective of enforcing
regulation against food adulteration, which represents a major commercial application, is to
detect fraudulent attempts to sell a cheap product (or a mix containing a cheap product) as a
more expensive one (Ashurst and Dennis 1996).
In order to support the authentic composition of food, in relation to religious restriction,
the fraudulent replacement of more expensive food ingredients and the declared presence of
allergens in a food product, correct identification of ingredients in food products is needed
(Burns et al. 2009). There is a large body of reports about adulteration of various types of
food and the necessity for authenticity testing of raw materials and final products (e.g., rice,
meat, flour and milk) (Ibrahim et al. 2011).
Correct and adequate description and/or labeling of food products, particularly if the food
has been processed removing the ability to distinguish one ingredient from another, are also
crucial for ascertaining food authenticity. Control of authenticity parameters dictated by the
legislations in order to protect consumers and prevent fraud requires traceability for which
suitable analytical methods are needed. Traceability is defined as "the ability to track any
food through all stages of production, processing and distribution (including importation and
at retail)" (Food Standards 2012). Such traceability is of fundamental importance for extravirgin olive oils, for example (Consolandi et al. 2008). Traceability in food is a recently
developed concept of control for the entire chain of food production and marketing that
allows food to be traced through every step of its production back to its origin (Testolin and
Lain 2005). It is worth noting here, that governments have different national guidelines for
the production and preservation of food. The demand for reliable food traceability systems
has addressed the scientific research, hence producing different analytical methods to the
problem (Galimberti et al. 2013). Added to that, the need to support food-labeling legislation
has accelerated the development of methods for the analysis of food ingredients (Sawyer et al.
2003). Reid et al. (2006) discussed the relative potential of various technologies for the
confirmation of food authenticity and quality including the polymerase chain reaction (PCR).
Plant food products and agricultural commodities including spices are increasingly
subjected to adulteration. The adulterants range from synthetic chemicals and earthy materials
to products of plant origin (Dhanya and Sasikumar 2010). The need for accurate and reliable
methods for plant species identification in food products in order to protect the producer, the
company and the customer has steadily increased during past decades, particularly with the
recent food scares and the development of trade and technological progress in food
production. Moreover, the development of high added value products based on plants has
raised concerns about adulteration (Madesis et al. 2014). The analytical methodologies/
techniques used for detection of adulterants or authentication of food and agricultural
commodities include physical methods, chemical/biochemical methods, immunoassays and
the most recent DNA-based molecular tools (Dhanya and Sasikumar 2010).

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Though conventional analytical tools have good resolution power to detect the synthetic
adulterants of food and agricultural commodities, these methods are hardly powerful enough
to identify the biological adulterants (Dhanya and Sasikumar 2010).
By genetic traceability, the genetic identity of the plant material from which the
transformed products have been derived is determined. Establishing the genetic origin of food
products allows verification of the authenticity of valuable foods and discourages adulteration
with material of lower cost and value (Testolin and Lain 2005). In their reviews, MartinsLopes et al. (2013) and Scarano and Rao (2014) demonstrated potential and limits of various
DNA markers in food traceability and monitoring the genetic identity of food components in
plant net-chains.
DNA-based methods are more effective than protein-based approaches, which have a low
effectiveness when applied to the analysis of heavily processed foods, because DNA is more
informative, and can be easily extracted from small traces of organic material (Galimberti et
al. 2013). DNA methods take advantage from the fact that DNA sequences do not depend on
environmental factors, and can withstand higher temperatures compared to proteins. This
makes them more efficient in analyzing high-temperature processed foodstuffs, such as pasta
or bread (Pasqualone 2011). Nucleic acid analysis is also quick and easy when compared to
conventional analytical methods like spectrometry, microscopy and Thin Layer
Chromatography for food authentication and the detection of plant-based adulterants
(Parvathy et al. 2014).
Fresh food products without any processing are suitable for many types of analytical or
molecular analyses. Although most of foodstuff samples are processed to some extent and
DNA is usually altered and fragmented into small fragments, extensive research has been,
however, performed and DNA-based methods are becoming the methods of choice for food
authenticity (Madesis et al. 2014).

DETECTION OF FOOD BIOADULTERATION USING DNA


DNA testing can be used in different contexts by different operators including regulatory
authorities, food business operators, and researchers. This methodology helps the regulatory
authorities to assess the safety and authenticity of foodstuffs, and uncover any infringements
of labeling legislation (EUFIC online reference). DNA recovery from food samples might be
of great importance when the raw material used in the production process has to be traced
(Busconi et al. 2003).
Dhanya and Sasikumar (2010) listed examples and referred to studies for each type of
DNA-based methods which are classified into: PCR; sequencing; and hybridization. They
also reviewed molecular marker-based detection of adulteration in traded food and
agricultural commodities of plant origin with special reference to spices. The progress of
DNA -based methods used for authenticity (identification and quantitation) of food products
goes extremely fast. Madesis et al. (2014) reviewed the current state of the art for DNA-based
methods used for species identification and authenticity in foods. They also discussed the
problems, advantages and disadvantages of these methods. Among the DNA-based
techniques, PCR is often the method of choice for identifying the species present in food
products (Drummond et al. 2013).

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The high specificity and sensitivity of PCR in amplification allow the detection of very
small amounts of DNA and hence accurately verifying low levels of adulteration (Pasqualone
2011). The most important prerequisite for a successful and reliable PCR is the isolation of
amplifiable DNA from the sample (e.g., coffee or honey). Therefore, DNA recovered should
be of sufficient amount and of adequate purity for subsequent PCR analysis. Currently, many
commercial kits are available for the extraction of DNA from a range of food samples in an
easy and quick manner. It should be noted here that DNA extracted from foodstuffs can be
highly fragmented due to the industrial processing conditions. Therefore, targeted region of
DNA should be short somehow (Pasqualone 2011).
Of PCR-based methods, PCR-RFLP, which is an essential component of DNA
fingerprinting, is a classic forensic technique in food authentication (Royal Society of
Chemistry online reference). The development of quantitative detection strategies using PCR
such as quantitative competitive PCR (QC-PCR) (Archak et al. 2007) and real-time PCR
(e.g., Mustorp et al. 2008) tremendously increased the number of PCR applications to
adulterant analysis in food because they allow the confirmation and quantification of
adulterants studied (Dhanya and Sasikumar 2010).
Real-time PCR is highly sensitive and is useful for situations in which occasional
contamination is acceptable (Drummond et al. 2013). Druml and Cichna-Markl (2014)
introduced the development of high resolution melting (HRM) analysis method and described
how HRM data are analyzed and interpreted. The authors discussed the potential use of HRM
analysis in food analysis for the identification of closely related species and cultivars. HRM
involves PCR amplification of the targeted DNA region in the presence of a saturation dye
and subsequent melting of the amplicons generated by gradually increasing the temperature.
HRM analysis was proved highly suitable for the detection of single-base variants and small
insertions or deletions since the melting profile depends on the GC content, length, sequence
and strand complementarity of the amplicon.
The amplification of specific DNA fragments requires the previous knowledge of target
sequences in order to deign appropriate primers (Pasqualone 2011). However, most food
products are made up of a pool of plant species (e.g., multiflower honeys), major and minor
crops, and spontaneous species. Therefore, in order to establish traceability, universal tools
which are able to univocally identify each plant species are needed (Galimberti et al. 2014).
Because such complex matrices usually contain mixtures of DNA from several individuals
belonging to a certain taxonomic group (e.g., angiosperms), DNA amplification of a certain
locus (e.g., a plant DNA barcoding region) may generate amplicons of the same size for this
locus. This impedes direct sequencing with the Sanger approach. The adoption of a
preliminary cloning step to separate single DNA templates may solve this problem. This
strategy, however, has its own limitations (e.g., high costs) and can introduce biases (e.g., low
representation of the sequenced colonies in the case of highly complex matrices) (Galimberti
et al. (2014).
Galimberti et al. (2013) believe that DNA barcoding technique can be used as a universal
tool for food traceability. In their review, they summarized the state of the art in this issue.
They reported that DNA barcoding is a fast, sensitive, cheap, and reliable method for
identifying and tracking a wide panel of raw materials and derived food commodities (even in
the case of strongly processed food products), and for detecting poisonous components or
allergens potentially occurring in food matrices. In their review, they discussed 1) molecularbased approaches to DNA barcoding, 2) DNA barcoding to identify and certify food raw

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material, 3) DNA barcoding as a traceability tool during food industrial processing, and 4)
food safety and commercial fraud. In addition to that, DNA barcoding has a definite
advantage on the already available DNA-based techniques such as inter-simple sequence
repeats (ISSRs), random amplified polymorphic DNAs (RAPDs), sequence characterized
amplified region (SCAR) etc. for detection of adulteration in traded spices and other agri
food commodities because these techniques have some limitations such as less abundance,
low level of polymorphism, poor reproducibility, genotypic specificity, and prior sequence
knowledge (Parvathy et al. 2014).
DNA barcoding is currently being employed in detecting plant-based adulteration in
many traded commodities. For example, the technique was proved to be effective in
identification of ancient Olea europaea L. and Cornus mas L. seeds (Gismondi et al. 2012).
DNA barcoding was also proposed as a useful tool for discrimination and certification of
saffron authenticity (Gismondi et al. 2013). Using plant DNA barcoding regions (trnL and
rpoC) coupled with High Resolution Melting (Bar-HRM), Ganopoulos et al. (2012) have
developed a method allowing us to detect and authenticate Protected Designation of Origin
(PDO) Fava Santorinis, which is often subjected to adulterations from other legume
products coming from other Lathyrus, Vicia or Pisum species.
DNA barcoding was also employed for: (1) discriminating the medicinal plant Scutellaria
baicalensis (Lamiaceae) and its adulterants (S. amoena, S. rehderiana, and S. viscidula) that
may cause a series of inconsistent therapeutic effects and quality control problems in the
herbal medicine industry (Guo et al. 2011); (2) species-level identification of the genus
Ocimum, which comprises of several medicinally important species which are frequently
adulterated due to misidentification (Christina and Annamalai 2014); (3) identification of the
species of Tetrastigma and their differentiation from other medicinal plants (Yuan-Miao et al.
2011); and (4) resolving species admixtures in the raw drug trade of Phyllanthus (Srirama et
al. 2010). Very recently, Galimberti et al. (2014) presented the advantages in the use of DNA
barcoding for the characterization and traceability of minor crops (represented by spices)
which are subjected to species substitution or uncontrolled admixture of manufactured plant
products with severe consequences for the health of consumers.
In partnership with DNATrek, Lawrence Livermore National Laboratory (LLNL)
researchers have developed DNATrax, a DNA-based additive for directly tracking food from
producer to consumer (Szondy 2015).
The various DNA-based methods have application in detection of biological adulterants
and authentication of a wide range of food and agricultural commodities (Dhanya and
Sasikumar 2010). Johnson (2014) reported that foods and food ingredients that are commonly
associated with food fraud include olive oil, fish, honey, milk and dairy products, meat
products, grain-based foods, fruit juices, wine and alcoholic beverages, organic foods, spices,
coffee, tea, and some highly processed foods. Among these, olive oil, milk, and honey have
been found to be the most commonly adulterated food products, according to a new study
published in the Journal of Food Science. The study examined 1,504 scholarly records from
1980 to 2010 to tally the results. Olive oil made up 16 percent of the total records, with 167
records. Milk, honey, saffron, orange juice, and coffee trailed close behind (Frasca 2012).
Based on another review of records from scholarly journals, the top seven adulterated
ingredients in the database are olive oil, milk, honey, saffron, orange juice, coffee, and apple
juice (US Pharmacopeia 2012).

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In this chapter, examples of applications of DNA-based methods in authenticity testing


and detection of biological adulterants in some economically important plant food products
and agricultural commodities (spices, pasta and flour, basmati rice, juices, honey, oil, tea, and
coffee) of plant origin are presented.
Detection of adulteration of these products is important for value assessment, to check
unfair competition and to assure consumer protection against fraudulent practices commonly
observed in unscrupulous trade. This is also important due to health reasons, since
consumption of products containing undeclared constituents may cause intoxication or
problems such as allergy in sensitized individuals (Dhanya and Sasikumar 2010).

SPICES
Traded forms of spice and spice powder are highly subjected to substitution with inferior
substances or admixing (Singhal and Kulkarni 2003). Spice powders and paste are more
vulnerable to adulteration compared to whole spices because adulterants are visually
undetected (Parvathy et al. 2014). Therefore, people are advised to buy whole spices and
grind them at home or purchase properly packed spices (with proper informative labeling) of
standard F.P.O. I.S.I or AGMARK certified companies (Mani 2011). The majority of the
substances involved in the adulteration of spices are of a starchy character. Most of the
common spices, however, contain a considerable amount of starch. Cloves, mustard, and
cayenne are three spices that are practically free from starch. Hence, the presence of starch in
the ground article of these spices is proof of adulteration (Chest of Books online reference).
Highly toxic materials such as lead-bearing pigments and other unapproved color
additives which may have serious public health consequences have been used in some
instances to adulterate spices.
In most instances of adulteration, however, spices are adulterated with material that is not
highly toxic or carcinogenic, and therefore does not present a significant, immediate public
health risk, but are still illegally adulterated and subject to regulatory action should a
regulatory agency determine that action is warranted (Spice Adulteration online reference).
For example, ginger powder (Zingiber officinale) has been reported to be adulterated with
chilli (Capsicum annuum) (Dhanya and Sasikumar 2010), oregano with cistus which has a
dark green color, and cloves with dried seeds of volatile oil (Project Work 2012). Other
examples are: (1) adulteration of cumin seed, poppy seed, and black pepper (Piper nigrum L.,
Piperaceae) with artificially colored foreign seeds (Jaiswal online reference); (2) Badi Elaichi
or black cardamom with Choti Elaichi (green cardamom) seeds; (3) coriander powder with
dung powder; (4) cloves with dried seeds of volatile oil; (5) powdered spices with powdered
bran and saw dust; and (6) black pepper with light berries etc. (Jaiswal online reference).
Powdered beechnut husk aromatized with cinnamic aldehyde has been also marketed as
powdered cinnamon (Vasiredd 2013).
The identification of spices might be important because some of them contain harmful
substances, and for the detection of toxic plant-derived contaminants in herbs such as ramsons
(wild garlic) contamination with lily of the valley or meadow saffron and roquette (salad
rocket) with toxic ragwort. Usually spices are identified morphologically using simple
methods like magnifying glasses or microscopic instruments (Focke et al. 2011).

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Since spices usually encompasses strong manufacturing processes such as crushing,


powdering, or aqueous/alcoholic extraction of plant material, morphology could be
ineffective for tracing spices along the supply chain (i.e., from the crop cultivation sites to the
final products) although some species display distinctive morphological traits (Galimberti et
al. 2014). Added to that, there are many critical spice genera such as Thymus, where
differences among closely related taxa are limited to few minor morphological characters.
Molecular biological methods like PCR enable an accurate and specific detection of spices
even in complex matrices. There is a paucity of methods for the detection of plant-based
adulterants and their acceptable levels in powder forms of traded spices (Parvathy et al.
2014). Focke et al. (2011) argued that generally plants with diverse genetic backgrounds and
relationships are the origins of spices. They added that complex and individual processing
methods are used for the production of spices, and hence the development of a reliable DNAbased method for spice analysis is a challenging intention. The authors developed and
evaluated in detail several alternative methods for the isolation of DNA from spices with
regard to its (i) purity (photometric), (ii) yield (fluorimetric methods), and (iii) amplifiability
(PCR). They also designed specific primer sets, optimized the PCR conditions to detect 18
spices selectively, and performed assays of self-made spice mixtures to proof the applicability
of the developed methods. The authors demonstrated that the designed sets of primers
combined with optimized PCR conditions afford a specific amplification for selective
detection of 18 spices targeted via PCR. Dhanya and Sasikumar (2010) stated some of the
adulterants in powdered black pepper, chilli and turmeric (Curcuma longa L.) and their
detection with special reference to selected molecular markers (RAPDs and SCAR).
Galimberti et al. (2014) proposed the DNA barcoding approach as a universal and
suitable tool to characterize and trace aromatic species. In spices, however there are not many
reports for DNA barcoding use barring the following: (1) De Mattia et al. (2011), who
evaluated the efficacy of a DNA barcoding approach (rpoB, rbcL, matK and trnH-psbA) as a
tool for the discrimination of 64 commercial kitchen spices belonging to the Lamiaceae
family that are usually sold as enhancers of food flavor; (2) Guo et al. (2011), who
discriminated the medicinal plant Scutellaria baicalensis (Lamiaceae) and its adulterants; (3)
Swetha et al. (2014) for discrimination of the economically important Cinnamomum verum
(true cinnamon) from its adulterants (C. cassia and C. malabatrum); and (4) Gismondi et al.
(2013) and Jiang et al. (2014) for determination and certification of authenticity of saffron
(Crocus sativus L.), which is one of the most expensive and important medicinal spice
products in the world.
Black pepper, which is considered as the "King of Spices" or "Black Gold, is an
important spice and medicine and has cosmetic uses. Traded black pepper and its value added
forms are reported to be adulterated with wild Piper species viz., P. attenuatum and P.
galeatum and papaya seeds (Carica papaya L.) (Parvathy et al. 2014) which are added by
some manufacturers to add bulk to pepper. Adulteration of black pepper with small quantity
of papaya seeds cause serious liver problems and stomach disorders (Foster 2014). Dhanya et
al. (2009) reported the development of a specific, sensitive, and reproducible SCAR marker to
detect papaya seed powder adulteration in traded black paper powder. They concluded that
this very simple analytical strategy could be used for large scale screening of powdered black
paper market samples intended for domestic uses and export. Parvathy et al. (2014) presented
the first report on adulteration of black pepper powder with chilli. They used a robust DNA
barcoding (psbA-trnH, rbcL and rpoCl)-based molecular technique for detecting chilli

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adulteration in traded black pepper powder. BLAST searches revealed that except in case of
two market samples, all the others showed maximum (100%) similarity to Piper nigrum
sequences available in NCBI for all the three loci tested. The two distinct market samples
showed maximum similarity to Capsicum annuum sequences implying the probable
adulteration of the sample with chilli.
Turmeric powder is another important medicinal spice product traded internationally
which is adulterated by default or design with powders of related curcumin containing wild
species like Curcuma malabarica and Curcuma zedoaria leading to poor quality of the
product and toxicity (Dhanya et al. 2011a). Sasikumar et al. (2005) described an efficient,
relatively original method for the detection of extraneous Curcuma Sp. contamination in the
powdered market samples of turmeric using RAPD markers. These species cannot be easily
discriminated by other analytical techniques routinely used for the identification of
adulterants in powdered market samples of turmeric. Dhanya et al. (2011a) also reported a
sensitive PCR-based method for detecting C. malabarica and C. zedoaria in traded turmeric
powder. The highly carcinogenic Metanil Yellow and Kesari Dal which can cause stomach
disorders are also often added instead of turmeric (Foster 2014). Turmeric powder may be
also adulterated with mustard or any starch-based items like starch of maize, wheat, tapioca,
rice or even industrial starch. All the other adulterants of this spice except flour or rice
powder are health hazardous and cause irreparable damage to our system when eaten at
regular intervals for a long period of time (Project Work 2012).
For the detection of plant-based adulterants in traded ground chilli, Dhanya et al. (2008)
reported a modified CTAB protocol for isolation of good quality DNA from chilli powder
and its probable adulterants (dried red beet pulp, almond shell dust and powdered Z.
nummularia fruits) and the utility of RAPD primers for detection of adulterants in marketed
chilli powder. In a similar study, Dhanya et al. (2011b) discussed the development and
application of SCAR markers for the detection of dried red beet pulp and powdered Ziziphus
nummularia fruits in ground chilli. Generated markers were able to detect the adulterants at a
concentration as low as 10 g adulterant kg-1 of blended sample. One of the commercial
samples showed adulteration with Z. nummularia fruit, and all the market samples tested were
free from red beet pulp adulteration.
For the quality control of Mediterranean oregano and its contribution to pharmacognostic
techniques, Marieschi et al. (2009) used a RAPD-based method. The pharmacognostic survey
of 84 commercial samples of Mediterranean oregano revealed the presence of extraneous
plant material in 90.5% of the samples.

PASTA AND FLOUR


High volume foods such as the cereals have equally been subject to adulteration with
cheaper species. Durum wheat (Triticum turgidum L. var. durum or T. durum) is considered
genetically very hard. Therefore, pasta made from it commands a premium over other
cereals and is subject to legislation in several European countries, including Italy and
Germany (Osborne 1996). Pasta made from durum wheat is considered superior in several
qualitative aspects to that manufactured with bread wheat or a mixture of the two species
(Terzi et al. 2003). The quality of pasta products is considered the highest (aspect, cooking

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quality and nutritional value) if it is made from 100% durum wheat (Kelly and Bhave 2007).
The use of the cheaper soft, common or bread wheat (T. aestivum L.) in pasta production
carries considerable financial incentives and so not surprisingly bread wheat has commonly
featured as an adulterant.
Bread wheat adulteration in pasta has, in the past, been addressed using a variety of
methods based mainly upon the analysis of proteins, the detection of which are problematical
owing to the wide range of processing conditions employed throughout the industry (Bryan et
al. 1998). For instance, Autran et al. (1994) determined the relative amounts of durum wheat
and common wheat through electrophoresis of and quantification of ID--gliadins. Bryan et
al. (1998) presented a PCR-based method for the detection of the adulteration by hexaploid
bread wheat of pasta products described as being manufactured solely from durum wheat. In
this method, the presence of a previously described DNA sequence amplified in the Dgenome of wheat can be detected at low levels. Alary et al. (2002) quantified bread wheat
adulteration of durum wheat pasta by using real-time duplex PCR. The total DNA content of
pasta was determined by amplifying part of a wheat gene encoding a lipid transfer protein,
and common wheat DNA was quantified by amplifying part of the puroindoline-b gene. It
was revealed that pure durum wheat pastas were distinguished from adulterated pastas
without ambiguity. The authors demonstrated the feasibility of using real-time duplex PCR to
quantify common wheat adulteration of pasta dried at high temperature, quantification that
was impossible with the French official peroxidase-marker method. In 2003, qualitative and
quantitative PCR-based methods were proposed by Terzi et al. to detect hexaploid wheat
adulteration in pasta, and the presence of Triticum species in flour and food.
Pasqualone et al. (2007) evaluated the analysis of DNA microsatellites for the detection
of soft wheat in semolina and durum wheat bread. The results enabled selection of an efficient
D-genome-specific repetitive DNA sequence to detect common wheat in semolina and breads
by qualitative PCR with a threshold of 3 and 5%, respectively, lowered to 2.5% by real-time
PCR. In the same year, Kelly and Bhave applied a qualitative test utilizing PCR based on two
important genes which impart a degree of softness to common wheat and are deleted from
durum wheat to DNA extracted from 20 commercially available pasta samples. Results
indicated that common wheat was used in a number of pasta products, some of which did not
show this as a listed ingredient. Ibrahim et al. (2011) used PCR technology to detect possible
adulteration of durum wheat pasta products in the Jordanian market. They used specific
primers to ascertain the presence or absence of Dgas44 sequence in the extracted genomic
DNA from pasta products. This sequence is known to be present in the genome of bread
wheat and absent in the genome of durum wheat. Another DNA sequence, external
transcribing spacer of highly repeating genes encoding ribosomal RNA in locus NorD3,
specific to the D-genome of soft wheat was detected by PCR for assessment of soft wheat
flour impurity in semolina of durum wheat (RussianPatents.com online reference).
Sonnante et al. (2009) described an improved method of a previous qualitative and
quantitative Sybr green-based real-time experiments that analysed a microsatellite sequence
specific to the wheat D-genome. By means of a quantitative real-time PCR using a duallabeled probe, the same soft wheat genomic region was analysed in this method. Standard
curves based on dilutions of 100% soft wheat flour, pasta, or bread were constructed. In order
to verify the accuracy of the method, increasing amounts of soft wheat were used to prepare
durum wheat semolina, pasta, and bread samples. Results revealed that reliable
quantifications were obtained especially for the samples containing a lower amount of soft

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wheat DNA, fulfilling the need to verify labeling of typical durum wheat breads and pasta. In
2012, Casazza et al. described a novel approach for the detection and quantification of soft
wheat adulteration in whole grain durum flours and dried pasta. The assay relies on the
presence of intron-specific DNA length polymorphisms in the plant -tubulin gene family,
which can be highlighted through the PCR-based TBP (Tubulin-based Polymorphism)
method.
Pasqualone (2011) reviewed DNA-based methods that have been used for detection of
soft wheat in durum wheat pasta and bread such as amplification of fragments of gliadin
gene (GAG56D), the low molecular weight glutenin sequence U86029.1, ALMT1 gene, and
the use of padlock ligation probes. They also reviewed markers used for cultivar identification
and tracing since the quality of the end products is related not only to species but also to
cultivar. Examples of these markers are SSRs, ISSRs, RAPDs and AFLPs. Of these, AFLPs
and SSRs are believed to be the most promising.
Owing to its reported ease of digestion, tastiness, and expected greater nutritional value,
the demand for spelt (T. aestivum ssp. spelta (L.) Thell.) products has risen over the past two
decades, with its price often reaching twice that of comparable products of soft wheat (Mayer
et al. 2012). von Buren et al. (2001) presented and evaluated three PCR-based methods to
determine the proportion of wheat in spelt flour and products. A PCR-RFLP system
consisting of a GAG56D-specific PCR system with subsequent restriction was used to
estimate the relative proportions of wheat and spelt in DNA mixtures. More recently, a DNAbased method for the qualitative and quantitative analysis of wheat in spelt products for
routine analysis was developed by Mayer et al. (2012). Wheat is associated with coeliac
disease, causing characteristic damage to the small bowel mucosa, because it contains gluten,
which is a protein that is also found in grains of other cereals such as barley, rye, a cross
between wheat and rye called triticale and some oats (from contamination). A gluten-free diet
is, therefore, primarily used to treat celiac disease. The extensive use of gluten containing
flours in processed foods ranging from baking goods and dairy products (e.g., custard
powder) to jams, spreads and beverages (e.g., drinking chocolate) makes avoidance of gluten
a huge challenge to the consumer (FARRP 2014). It also asserts the need for declaring the
cereal composition of certain foods and labeling foods properly which are of crucial
importance for coeliac consumers regarding the safety and quality of the final product. It is
also important to accurately determine the contamination of different cereal species using new
control methods (Terzi et al. 2003). Dahinden et al. (2001) described the evaluation of a QCPCR system as an indication of contamination of gluten-free food with the coeliac-toxic
cereals. This standard was calibrated to 0.02% and 0.2% wheat DNA, corresponding to 10
ppm and 100 ppm gliadin, respectively. Makov et al. (2011) optimized a PCR-based
method for wheat, barley, and rye determination, and validated it on selected samples of
gluten free foods, shown by ELISA method to contain more than 1 mg gliadin per 100 g of
food. They argued that DNA analysis of the respective cereal by PCR method exceeds ELISA
methods in sensitivity for detecting the presence of toxic protein in a sample.
Because the biochemical composition of chestnut flour is close to that of many cereals, it
can be used instead of wheat or rye flours for people who may suffer from celiac disease or
are allergic to cereals (Alary et al. 2007). Chestnut and chestnut-derived products are,
however, quite expensive and hence a possible target for fraudulent labeling. Chestnut
producers need to clarify the purity of their products in order to obtain a specific label of
origin. Alary et al. (2007) developed a qualitative PCR-based method to detect cereal and

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leguminous species in chestnut flour. Those are common wheat, barley, rye, oat, durum
wheat, rice, maize, chickpea, kidney bean, soybeans, and fava bean. Another flour that is
subjected to adulteration due to its high demand is Besan, the flour of Bengal gram (Cicer
arietinum Linn), which is often adulterated by unscrupulous traders with flour of other
legumes, such as Lathyrus sativus (lathyrus) or Pisum sativum (pea) (Dattatreya et al. 2011).

BASMATI RICE
Basmati rice (Oryza sativa) is a very special type of aromatic rice known world-wide for
its pleasant and distinct aroma and extra long-grains. Traditional basmati rice cultivars,
confined to Indo-Gangetic regions of the Indian subcontinent, are often reported to be
adulterated with crossbred basmati varieties and long-grain non-basmati (NB) varieties in the
export market (Archak et al. 2007). As such, there is a requirement for a method that would
allow the detection of NB long-grain rice within samples of basmati rice to ensure purchasing
high quality products (Osborn 1996).
Woolfe and Primrose (2004) referred to the initial investigation carried out by Bligh et al.
(1999) and Bligh (2000) in which simple sequence length polymorphisms (SSLPs) have been
proved useful to differentiate basmati from other types of long-grain rice but they could not
completely differentiate true-line and hybrid basmati varieties. They also explained how such
differentiation was achieved when a further 39 microsatellite markers from chromosomal
regions linked to the distinctive traits of basmati rice were selected. It was possible to easily
identify each basmati rice variety when three of these microsatellites were combined with the
original 12.
It should be noted that the majority of studies conducted for authentication of basmati
rice varieties employed SSR markers. Nagaraju et al. (2002) demonstrated the potential use of
SSR and ISSR markers they developed for discrimination of traditional and evolved basmati
(EB) and semidwarf non-basmati (NB) rice varieties in authentication of traditional basmati
varieties. In a similar study, Siwach et al. (2004) investigated alelic diversity among basmati
and non-basmati long-grain indica rice varieties using microsatellite markers. A US patent
has been applied by J. Nagaraju for multiplex SSR kit containing 10 loci which can
simultaneously distinguish traditional basmati, evolved basmati and non-basmati varieties and
quantify the adulterants (File No.FPO2559/RB/SM dated 19.04.2006).
Vemireddy et al. (2007) also employed microsatellite markers for genotyping of basmati
varieties and assaying purity of market samples. By comparison of accuracy and consistency
of capillary electrophoresis (CE) to the slab gel and agarose electrophoretic methods, the
former was recommended for basmati rice purity assays. In the same year, Archak et al.
(2007) reported a CE-based multiplex microsatellite marker assay for detection as well as
quantification of adulteration in basmati rice samples. Capillary electrophoresis was proved
essential for microsatellite marker-based detection and quantification of adulteration of
basmati rice (Vemireddy et al. 2007).
Colyer et al. (2008) compared three calibration methods for the quantification of basmati
and NB rice using microsatellite analysis. These are: power regression, linear regression and
hyperbolic regression of the ratio of basmati to NB peak areas. The linear regression gave the
most precise calibration and hence enhanced quantification of the level of adulteration of

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basmati rice with NB rice. In order to genotype basmati and NB varieties for rice products
traceability and accurate quantitation of adulteration of basmati rice products with NB rice
products, Ganopoulos et al. (2011) developed a DNA-based method coupled with HRM
analysis using five different microsatellite markers. A method for a "single-tube assay" to
generate basmati rice variety-specific allele profiles-based on SSRs was described
(standardsproposals.bsigroup.com/home/getpdf/2135). It has been argued that this method
can be used for the unambiguous identification of different basmati rice varieties and for the
detection of adulteration in basmati rice samples.
Of the studies that used molecular markers other than SSRs for the detection of
adulteration in basmati rice: (1) Aggarwal et al. (2002), who characterized some Indian
basmati and other elite rice genotypes using fluorescent-AFLPs; (2) Lopez (2008), who used
TaqMan-based real time PCR method for quantitative detection of basmati rice adulteration
with NB rice; and (3) Steele et al. (2008), who generated InDel markers to distinguish
basmatis from other fragrant rice varieties. Rice authenticity/adulteration methods and results
were reviewed by Vlachos and Arvanitoyannis (2008). Very recently, Vemireddy et al.
(2014) also reviewed methods for the detection and quantification of adulteration of basmati
rice.

JUICES
The production of fruit juices represents an important and rapidly growing branch of the
beverage industry. The economic value and large-scale production of orange juice and other
valuable fruit juices have made them a target for fraud and adulteration (Ebeler et al. 2007).
Adulteration of fruit juices may occur either deliberately or through unintentional processing
errors (via contamination through inefficient washing procedures or coprocessing of fruits)
(Burns et al. 2009). Nagy et al. (1989) argued that it is beneficial to study juice adulteration
due to the existence of extensive database available on diluted juices and beverages. They
added that scientists understand the complexities of juice variability depending on varietal
changes, maturity changes, and the effects of different horticultural practices and processing
on the juice. They explored the problems associated with contending against the problem of
juice adulteration and examined current methods used to prevent this practice, and stated that
computer-assisted pattern recognition programs are important to those seeking to identify
juice adulteration and geographical origin. Clouding agents or food processing aids such as
palm oil and other allowed food ingredients are often used in fruit juices, jams, and other
foods to enhance the appeal or utility of a food or food component (cited in Johnson 2014).
Although fruit is the listed ingredient on the juice label, some juices may be only water or
dye (cited in Johnson 2014). In the early 1980s, a company in the US was found guilty for
adulteration of apple juice to such an extent that no apple juice remained (Hammond 1996).
Addition of less expensive ingredients such as sugars and wash pulp and the extension of
authentic juice with cheaper alternatives (typically juices obtained from apples, pears, grape,
grapefruits, etc.) and labeling the resulting product as pure (Gao 1995, Muntean 2010) are
also common in juice industry. Various recent studies (e.g., Stj and Targonski 2006)
revealed that some fruit juices labeled as containing a particular fruit contain little or no fruit
of that particular species.

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The adulteration of expensive fruit juices such as red/black fruit (e.g., raspberry,
strawberry and blackberry) with cheaper juices like apple, pear and grape has proved
especially popular amongst fraudulent traders. Other examples of juice adulteration are
adulteration of apple with grape or pear juice, sweet orange with sour orange (Hammond
1996), and orange with grapefruit juice (Attaway and Nagy 1988). Hence, using grapefruit
juice in a carton that contains 100% orange juice is misleading, and illegal (Huffington Post
2012).
Vaclavik et al. (2012) mentioned the methods that have been developed to tackle various
aspects of fruit juice authenticity and referred to several studies that employed these methods.
They argued that each of these methods can be successfully applied to monitor one or only
few specific adulteration practices. They also concluded that analytical approaches and
platforms facilitating more comprehensive insight into chemical composition of fruit juices
and its changes, as associated with adulteration, are required.
Apple juice and orange juices are the most widely purchased juices for consumption
nationwide, as well as for the school meal programs (Gao 1995). Apple juice has been shown
to have added unlisted pear juice (Thavarajah and Low 2006), grape juice, pineapple juice, fig
juice, high fructose corn syrup, and raisin sweetener (cited in Johnson 2014). One of the
methods that have been developed to detect the adulteration of apple juice with pears and pear
juice with apples is the examination of the flavonoids by HPLC with respect to their quality
and quantity (Wald and Galensa 1989). As for orange juice, it has been shown to sometimes
contain added unlisted mandarin juice, lemon juice, grapefruit juice, high fructose corn syrup,
beet sugar, paprika extract (cited in Johnson 2014), and marigold flower extract. A PCRbased heteroduplex assay was evaluated for the detection of mandarin juice in processed
orange juice (Mooney et al. 2006). In this assay, PCR amplification of a fragment of the
chloroplast trnT-trnL intergenic spacer derived from mixtures of DNA extracted from orange
and mandarin juice resulted in heteroduplex formation. Aldeguer et al. (2014) carried out a
dual-probe real time PCR (qPCR) DNA-based analysis for the identification of mandarin in
orange juice based on a single nucleotide polymorphism at the trnLtrnF intergenic region of
the chloroplast DNA. In 2015, Pardo evaluated a dual-probe real time PCR assay, based on
the simultaneous detection of two TaqMan probes, for the detection of mandarin in orange
juice. A single conserved polymorphism, located at the 314 position of intron belongs to
chloroplast trnL gene, was confirmed by sequencing in 30 mandarin, 28 orange cultivars and
13 hybrids. The addition of grapefruit juice to orange juice was also detected by Ammari et
al. (2015) at percentages as low as 1%. Another citrus juice whose adulteration has been
investigated is shiikuwasha (Citrus depressa Hayata) juice with calamondin (C. madurensis
Lour.) juice (Yamamoto et al. 2012).
Other juices such as those obtained from pomegranate and various types of berries have
also become popular because of high levels of antioxidants that have positive effects on
health (Vaclavik et al. 2012). However, owing to the high price of fresh pomegranates and
limited production, adulteration of pomegranate juice (e.g., with grape juice) has been
reported (Nuncio-Juregui et al. 2014). Adulteration of blackberry juice concentrates and
wines with juice of sorbitol-containing fruits has also been detected (Wrolstad et al. 1982).
Burns et al. (2009) reported a novel application of the Agilent 2100 CE system to differentiate
between DNA derived from strawberry and raspberry fruits. The approach uses microsatellite
markers that allow differentiation of strawberry and raspberry DNA based on

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presence/absence or size differentiation of PCR products, easily measured using the DNA
1000 chip.
Concord grape juice is another juice that is associated with many health benefits, and
therefore it is sold at a premium price. However, there is currently no method to verify the
percent composition of Concord grape juice in grape juice blends (Snyder et al. 2014).

HONEY
Honey is produced by honeypees from nectar and from secretions of living plants
(Valentini et al. 2010). It can be processed for direct consumption or used as ingredient in
various processed food products. The price of natural bee honey is much higher than that of
the other sweeteners (refined cane sugar, beet sugar, rice syrup, starch syrup and corn syrup)
due to its nutritional value and unique flavor. This makes honey subjected to be adulterated
with such cheaper sweeteners (Kartheek et al. 2011) without being disclosed on the label. For
example, adulteration of Asian honeys is often based on artificially added cheap rice syrup.
Lander et al. (online reference) amplified a specific rice DNA sequence (the 121 bp cyc
fragment of the rice cytochrome C gene) to detect this fragment in honeys that were
artificially adulterated with at least 10% rice syrup. Some of this honey might also contain
unapproved antibiotics or other additives and heavy metals. Maple syrup is sometimes
thinned out with sugar or corn syrup (cited in Johnoson 2014).
The most common economic adulteration of honey is from the addition of sugar (cane,
corn, beet, wheat, rice, etc.). Other types of fraud such as the false declaration of botanical or
geographical origin also exist.
The major proteins in honey have different molecular weights depending upon the
honeybee species. The measurement of these proteins in honey is a useful method to
discriminate the honey that is produced from different honeybee species (Won et al. 2008).
Using MALDI-TOF, Won et al. (2008) purified and analyzed honey protein produced by Apis
cerana or Apis mellifera in order to confirm the origin of major honey proteins. Kartheek et
al. (2011) referred to the other different analytical techniques that can be used for the
detection of adulteration of honey such as isotopic, chromatographic, thermal analysis and
trace element techniques. In addition to detection of adulteration or chemical residues, the
analytical methods applied to honey also deal with quality control according to the current
standards, and determination of botanical or geographical origin because honey from a nonauthentic geographic origin is also common (Pilizota and Tiban 2009).
There are various methods available for the identification of botanical and geographical
origins of honey. Since pollen reflects the vegetation type from where the nectar has been
collected by the bees, pollen analysis is traditionally used to determine the geographical
origin of honey (Jain et al. 2013). Jain et al. (2013) referred to other methods available for the
determination of botanical and geographical origins of honey like chemical analysis using
chromatographic techniques and analysis of the physical and sensory characteristics of the
honey. They mentioned the disadvantages of these methods and concluded that techniques
based on DNA have become the method of choice since they tend to be precise, quick and
more reliable. Therefore, DNA-based analytical methods used for detection of honey
adulteration were accumulated mostly for determination of botanical or geographical origin of

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honey. Compared to light microscopy which is the traditional method for the determination of
the floral composition of honey, the application of molecular techniques allows the detection
of a much greater range of plant species in honey, overcoming the limitations of the
morphological identification of plant pollen and spores (Lalhmangaihi et al. 2014).
Based on the fact that DNA from the plant(s) may be available in honey, the sources of
DNA which can be used to identify the host plants and determine the botanical origin of
honey using DNA technology are pollen and other plant-derived components present in honey
(Jain et al. 2013). Schone-Michling (2005) described a method for preparation of DNA from
honey and pollen with an optimized sample preparation step for amplification with PCR. Jain
et al. (2013) presented the first report of a CTAB-based DNA extraction method from honey,
and demonstrated that the extracted DNA is enough for PCR amplification and can be used
for botanical identification.
Since honey rarely comes from a single plant species, even if it is attributed to a single
species, and taking into account the health risk from plants producing toxins, Lalhmangaihi et
al. (2014) argued that it is crucial to assess correctly the identity of the plants from which
pollen comes. Based on conventional phenol-chloroform methods, Lalhmangaihi et al. (2014)
also developed an easy, efficient and simple approach for the extraction of PCR amplifiable
cpDNA with a reduced sample amount of 3 ml honey compared with other studies. They
showed that the isolated DNA is of sufficient quantity and quality to allow PCR amplification
and sequencing of genetic markers routinely used for plant barcoding, and proved that
investigation of the plant source of origin of the honey samples can be achieved using
extracted DNA.
Laube et al. (2010) developed plant species specific real-time PCR systems for
identification of PCR markers for the detection of plant species related to honey. Laube et al.
(2014) also identified TAqMan PCR plant specific markers for profiling of honey (origin).
Using a DNA barcoding approach that combines universal primers and massive parallel
pyrosequencing, Valentini et al. (2010) proposed a new method for studying the plant
diversity and the geographical origin of honey. The authors concluded that the method they
proposed is suitable for analyzing plant diversity in honey because it is simple to implement,
fast, and more robust than classical methods.
Since cotton is one of the major honey-source plants, development of detection methods
for bee honey associated with Bt cotton has become urgently needed due to the concern it
raises in the international trade about the presence of any alien DNA sequences in honey
(Cheng et al. 2007). Therefore, Cheng et al. (2007) have developed a DNA extraction
procedure and a PCR protocol for the detection of foreign DNA sequences in bee honey.
Using the PCR protocol developed, it was possible to specifically amplify alien and cotton
endogenous DNA sequences with a length of 125-550 bp.

OIL
Adulteration of edible oil is the biggest source of food fraud all over the world (Hu et al.
2014). Such adulteration poses a serious threat to the trade of edible oil and negatively affects
consumers (Zhang et al. 2009). Therefore, the detection of adulteration or mislabeling of high
priced oils is a particular concern in food safety and quality. Krist et al. (2006) detected the

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adulteration of poppy seed oil with sunflower oil. Adulteration of argan oil with vegetable
oils such as sunflower, soy bean, and olive oil has been also reported (Salghi et al. 2014).
Maize oil and pumpkin seed oil which are rather expensive compared to other vegetable oils
are other targets for adulteration with cheaper vegetable oils (Wenzl et al. 2002; Mottram et
al. 2003). Mustard edible oils and fats maybe also adulterated with Argemone oil (Jaiswal
online reference). Hazelnut oil has been reported to be adulterated with sunflower oil (Ozen
and Mauer 2002), and ground nut oil with castor oil. Distinction between camelina oil (an
edible oil that comes from seeds of Camelina sativa, commonly known as false flax) with
other less expensive oils has been documented by Hrastar et al. (2009). Very recently,
Marikkar and Rana (2014) detected canola oil adulteration with lard stearin.
Methods (e.g., fatty acid profiles) available to discriminate plant oils facilitate the
detection of either accidental or deliberate adulteration (Spaniolas et al. 2008). However,
conventional analytical methods for measuring the oxidation and adulteration of oil are
expensive, destructive, time consuming, laborious, and require chemical reagents (Kartheek et
al. 2011). DNA markers that are specific to the different oils are more appropriate. Zhang et
al. (2009) determined the presence of cheap palm oil as an additive in more expensive oils
using conventional and real-time PCR assays that generated a 109 bp amplicon of the MT3-B
sequence. Recently, Ganopoulos et al. (2013) described a high-resolution melting analysisbased method using chloroplast barcoding regions as targets (Bar-HRM) to obtain barcoding
information for the major vegetable oil species and to quantitatively identify the botanical
origin of plant oils. The authors proved that the proposed method was capable of
distinguishing among different vegetable oil species, and that it is more accurate, faster and
less costly alternative method to authenticate vegetable oils and to detect mixtures of oils
(Ganopoulos et al. 2013). In the same year, Vietina et al. (2013) extracted DNA from
different oils made of hazelnut, maize, sunflower, peanut, sesame, soybean, rice and
pumpkin. By comparing the DNA melting profiles in the oils and in reference plant materials,
it was possible to identify any plant components in oils and mixtures of oils.
Olive oil has a pleasant taste and a fine aroma, and is internationally appreciated for its
health benefits and nutritional value (Harwood and Yaqoob 2002). This makes it of a higher
price than most other edible plant oils, which means that there is a great temptation to
adulterate it with cheaper oils (de la Torre et al. 2004). Most common adulterants found in
virgin olive oil are sunflower, soybean, corn, rapeseed (canola) (Alves et al. 2013), hazelnut
(De Ceglie et al. 2014), peanut (Firestone 2001), almond, walnut (Christy et al. 2004), palm,
sesame, avocado, cotton, vegetable and safflower oils. It is worth noting here that the use of
nut or legume oils could pose a problem for those with certain food allergies (cited in
Johnoson 2014).
Extra-virgin olive oil is considered of the highest quality, regarding both organoleptic and
health aspects (Consolandi et al. 2008). Foster (2014) argued that with less protective
measures to detect frauds in this product, many companies mix it with much cheaper
hazelnut, soy, or sunflower seed oil, and label it as extra virgin as well as mislabeling its
country of origin. Some combinations even have been reported to contain no olive oil. For
example, investigations in 2007 found that ten thousand tons of Turkish hazelnut oil and
Argentinean sunflower-seed oil were delivered as olive oil to the Italian refinery Riolio, one
of Italys leading importers of olive oil (Rosanne 2009). In other olive oil adulteration
instances, low-grade sunflower, canola or other oil may be mixed with olives industrial

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chlorophylls, and flavored with beta-carotene and sold as olive oil (Rabiei and Tahmasebi
Enferadi online reference).
Tahmasebi Enferadi and Rabiei (2013) argued that blending premium olive oil with low
quality oils or with other plant oils and the fraudulent labeling of oil products define an urgent
action in confirming olive oil authenticity. They discussed conventional identification tools of
olive oil and achievements in olive oil genetic traceability between 2000-2012.
The vast majority of studies that employed DNA-based markers for traceability of plant
oils species centered on olive oil. Tahmasebi Enferadi and Rabiei (2013) believe that a
consensus protocol has not been accepted in trade markets yet although significant progress
has been made in the last decade on DNA extraction from olive oil and the choice of
appropriate molecular markers. They added that DNA extraction from oil is no longer
problematical at present, and the critical point remains the choice of markers.
Spaniolas et al. (2008) exploited the length variability of the chloroplast trnL intron
among plant species for the authentication of edible and cosmetic plant oils, with an extra
emphasis on olive oil. The methodology was based on the combinatorial use of a PCR assay
with a capillary electrophoresis system such as the lab-on-a-chip technology. It was
concluded that the developed assay can be useful for the detection of adulteration of olive oil
with other plant oils, with the exception of avocado and sesame oil. The development of
molecular markers for olive oil authentication can be also achieved through qRT-PCR which
can be used for the optimization of critical parameters such as the amplicon size and the DNA
isolation method (Gimnez et al. 2010). A cpDNA-based technology (SNPs variation in
noncoding spacer region between psbA-trnH and partial coding region of matK) has been
proposed as a rapid method for the detection (up to 5%) of adulterants in olive oil such as
canola and sunflower oils (Kumar et al. 2011). Real-Time PCR (RT-PCR) and HRM were
used to analyze olive oils mixed with different percentage of other oils. Results showed HRM
a cost effective method for efficient detection of adulterations in olive oils (Vietina et al.
2013). A PCR-based technology was developed by Agricultural Research Service (ARS)
researchers in Albany, California to detect undisclosed oils mixed with olive oil. The test,
which can be done in several hours with equipment at DNA labs and that was created by
chemist Talwinder Kahlon and co-investigators, distinguishes the DNA of several genes
found in olive oil from the genes in other vegetable oils (Flagg 2013). The development of
DNA markers that are specific to olive and other oil species including sunflower, canola,
cotton, corn, soybean and sesame is in progress (IZTECH 2013).
Traceability of olive oils is relevant not only in protecting against frauds, but also in
assessing their origin. Even among extra-virgin oils, quality depends on the cultivars and on
the environmental conditions of growth employed. Rabiei and Tahmasebi Enferadi (online
reference) referred to non-DNA-based technologies used for the identification of the origin of
olive oil (sterols, phenols, fatty acids, triacylglycerols, volatile compounds and tocopherols).
Because chemical analyses per se are not sufficient to verify olive oil origin authenticity
and chemical composition of virgin olive oil is influenced by genetic (variety) and
environmental factors (climatological and edaphologic conditions), unequivocal
determination of a genetic profile for every cultivar using DNA analysis is important
(Consolandi et al. 2008) since markers generated are independent from environmental
conditions (Tahmasebi Enferadi and Rabiei 2013).
DNA markers that have already been used to identify olive cultivars are increasingly
being exploited to solve traceability and provenance issues (e.g., Breton et al. 2004). Busconi

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et al. (2003) defined a reliable DNA extraction method for extra virgin olive oil and
demonstrated the possibility of using this DNA for fingerprinting the original cultivar. The
authors proved that the purified DNA can be used for RAPD and AFLP analyses. de la Torre
et al. (2004) also optimized an extraction method for isolation of DNA from olive oil that
serves as template for PCR reactions. Extracted DNA was used to identify and characterize
olive oils based on SCAR markers. In 2008, Consolandi et al. distinguished forty-nine
frequently grown Mediterranean olive cultivars in order to address DNA extracted from
monovarietal olive oils by a ligation detection reaction (LDR) and, subsequently, detected by
hybridisation onto a universal array (UA).
The applicability of SSRs-based method to trace the identity of single-cultivar virgin
olive oils was approved by Testolin and Lain (2005), who developed a protocol for DNA
extraction from olive oil and showed how DNA microsatellite fragments up to about 200 bp
long can be successfully amplified and used to identify the olive cultivar from which oil was
produced. In a similar study, Raieta et al. (2015) presented a novel, reliable and not expensive
method for extracting the DNA from commercial virgin and extra virgin olive oils. Using this
method, they characterized the genetic profile of monovarietal olive oils based on SSR
marker analysis.
Rabiei and Tahmasebi Enferadi (online reference) believe that DNA-based methods make
an important contribution to protect high-quality olive oils. They referred to studies that used
DNA-based molecular markers for cultivar traceability in olive oil and olive oil genotyping
such as RAPDs, AFLPs, SCARs, SSRs, ISSRs, chloroplast genome sequencing, ESTs, real
time-PCR, and DNA barcode. They believe that: (1) single locus (SSRs and SNPs) are
preferred to multilocus (AFLP, RAPD) markers because they are simpler to perform, more
easily interpretable and can be combined in high-throughput platforms; and (2) RAPDs and
AFLPs give complex profiles that can be applicable to monovarietal oils but not to mixtures
of three to four cultivars. Although DNA markers have been proved useful for solving
traceability and provenance issue of olive oil, Doveri et al. (2006) concluded that care needs
to be taken in the interpretation of DNA profiles obtained from DNA extracted from oil after
they found that the DNA profile obtained from olive oil is likely to represent a composite
profile of the maternal alleles accumulated with alleles contributed by various pollen donors.

TEA
Tea is prepared from leaves of the tea plant, Camellia sinensis (L.) Kuntze, of which
there are two main commercial varieties: small-leafed C. sinensis var. sinensis, and largeleafed C. sinensis var. assamica (J.W. Mast.) Kitam. (Stoeckle et al. 2011).
Tea may be adulterated with used old tea leaves, dried leaves of other plants, black gram
husk, cashew husk (Anacardium occidentale L.) and even with saw dust. Since the dried plant
fragments used to prepare teas cannot be easily identified to species by visual appearance,
accurate labeling is important for consumers, marketers, and regulators (Stoeckle et al. 2011).
Availability of species-specific DNA markers is also crucial to ensure traceability and
authentication of tea products. For example, species-specific PCR primers were developed
from intergenic spacer regions of 5S ribosomal RNA genes and used successfully in the
detection of adulteration of cashew husk in tea samples (Dhiman and Singh 2003).

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Tea infusions are also prepared from a diversity of other plants and plant parts (herbal
teas). Drinking herbal teas may cause serious illnesses and fatalities that may be due to
overdose, mislabeled products, or allergic reactions (Stoeckle et al. 2011). For example,
ingestion of products containing Chinese star anise (Illicium verum) contaminated or
adulterated with Japanese star anise (I. anisatum) or other Illicium species can cause
hallucinations, epilepsy, and nausea due to the rare neurotoxic sesquiterpene dilactone
anisatin that is present in Japanese star anise (Shen et al. 2012). Shen et al. (2012) developed
a method based on Direct Analysis in Real Time (DART) ambient ionisation coupled with
orbitrap high resolution mass spectrometry for distinguishing between the morphologically
similar Chinese star anise and toxic Japanese star anise for food safety issues. Compared with
existing PCR, TLC, GC-MS and HPLC-ESI-MS/MS procedures, the proposed DART-HRMS
procedure is faster and simpler and moreover measures the actual biotoxin.
Stoeckle et al. (2011) tested recovery of standard DNA barcodes for land plants from a
large array of commercial tea products and analyzed their performance in identifying tea
constituents using existing databases for matching commercial tea ingredients to product
labels. They searched a public reference database for the closest match to each barcode
sequence and compared the result to the listed ingredients. About 1/3 of herbal teas generated
DNA identifications not found on labels. Similarly, Long et al. (2014) characterized and
identified commercial non-Camellia teas of original plants and their adulterants based on
DNA barcoding of rbcL, matK, psbA-trnH, and ITS2.
In order to ensure traceability and authentication of premium tea products, accurate
varietal identification is critically important (Fang et al. 2014). Ujihara et al. (2009) used
SSRs, which were analysed by a capillary sequencer, to identify commercial Japanese
monovarietal green tea and imported green tea samples. In 2011, Wu et al. were able to
discriminate between the two commercial taste variants, bitter and sweet, of Gynostemma
pentaphyllum (jiaogulan) herbal tea based on the combination of chemical profiling and
sequencing of ribosomal ITS-1 region. Recently, Fang et al. (2014) unambiguously identified
all 40 tea varieties, including both fresh and processed commercial loose-leaf teas, they tested
using a set of SNP markers developed from the expressed sequence tag (EST) database of C.
senensis.

COFFEE
According to Gregory Tucker at the University of Nottingham, UK, coffee, which is one
of the most important food commodities in world trade, is another classic example of an
expensive food product subjected to fraud. Two coffee species almost entirely dominate the
commercial coffee trade; Arabica (Coffea arabica) and Robusta (Coffea canephora), with
Arabica considered the highest quality and the most expensive (Chemistry World 2007). The
authenticity of Arabica, Robusta or blends of the two species, is an important topic for
producers and customers. The coffee market is regularly developing finished products based
on a single variety (Arabica or Robusta) (Morel et al. 2013). Arabica adulteration with
Robusta coffees can be intentional or not and is carried out at different steps of the coffee
chain; from plantation (one or both species can be cultivated by the same producer) to coffee
beverage (Tornincasa et al. 2010). Green coffee authentication can be very useful for roasters,

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while that of roasted coffee (beans or ground) should be very interesting for retailers and
consumers (Tornincasa et al. 2010). Morel et al. (2013) argued that it is almost impossible to
distinguish coffee varieties at the green coffee stage using physical or chemical analytical
tools. They added that research on coffee in this field is still at the beginning, and referred to
the molecular method which was developed by Spaniolas et al. (2006) and that combined
PCR-RFLP with lab-on-a-chip capillary electrophoresis in order to determine, in this
instance, particular coffee varieties. In this method, a genetic marker in cpDNA was found to
differentiate Arabica from Robusta varieties. Tornincasa et al. (2010) described a Real-time
PCR-based method to perform: (a) a qualitative analysis to evaluate the presence/absence of a
species in a sample; and (b) a quantitative analysis to amplify Robusta samples only, making
possible the detection of less than 5% of Robusta in a mixture. The authors were also able to
identify and certify Arabica varieties sold as specialty coffees by fingerprinting using SSR
markers. Morel et al. (2013) developed a DNA-based method (SSRs and HRM) to allow the
identification of variety purity in Nespresso product through the value chain, from the field to
the finished product. They also mentioned that a new technology based on high throughput
454 DNA sequencing with high reliability and accuracy for DNA traceability of variety purity
in Nespresso product is under test.
Ground coffee might be adulterated with roasted corn, ground roasted barley, and ground
roasted parchment. Instant coffee may include chicory, cereals, caramel, more parchment,
starch, and malt (cited in Johnson 2014). Coffee powder may be also adulterated by mixing it
with cashew husk powder (Nogueira and do Lago 2009) or other cheaper ingredients such as
soybeans, rye, chickpeas, figs, peas, beans, and acai seeds which can spoil the quality and
taste of the final product although they are not harmful (Stoye 2014). Cocoa seeds, wheat
middling, triticale, and Mogdad coffee seeds have also been reported as adulterants for coffee
(Nixdorf 2014; Shaba.Co). Powdered tamarind seeds or ground roasted chicory powder and
roasted acorns, which are made to resemble coffee in color and somewhat in flavor, are very
popular adulterants around the world and are used to add bulk and color to instant coffee.
Tamarind and chicory adulterants can cause diarrhea, stomach disorders, giddiness and severe
joint pains (Foster 2014).
Coffee is one of the main commodities in Brazil and, as such, it has been a target for
fraudulent admixtures with a diversity of cheaper materials (e.g., roasted barley and corn)
(Thiago et al. 2012). This adulteration is considered to be a serious problem affecting
Brazilian coffee quality with corn being considered as the most widely used adulterant (Jham
et al. 2007).
Many techniques have been developed in order to generate appropriate markers for
detection of ground roasted coffee and instant coffee adulteration. However, these methods
usually present low sensitivity and specificity (Thiago et al. 2012). Several genetic markers
are available for assessing coffee origin, but their appropriateness to testing commercial
coffee is limited by the ability to extract DNA from highly processed beans (Martellossi et al.
2005). Martellossi et al. (2005) demonstrated that PCR-grade DNA may be isolated from
roasted beans and even instant coffee. They believe that this would allow analysis of
commercial samples, should suitable markers for species/variety identification are found.
Mishra et al. (2008) developed a simple, efficient and rapid protocol for isolation of DNA
from coffee seeds suitable for molecular studies. In 2012, Thiago et al. used a real-time PCRbased method for detection of two among the main adulterants in commercial coffee: corn
and barley.

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Chicory, the dried root of wild endive plant of the dandelion family, coffee has been used
in times of coffee shortage or economic crisis, like the Civil War and the Great Depression
because it was cheap. It has also been used to stretch supplies in prisons. Though the root has
been cultivated since ancient Egypt, chicory has been roasted, ground and mixed with coffee
in France since the 19th century (Annabelle Smith 2014). Roasted chicory can be adulterated
with roasted ground peas, beans, wheat, carrots or turnips and the dark brown coffee color
was achieved by using 'black jack' (burnt sugar) (Accum 2005).

FOOD ALLERGY
Although the vast majority of food fraud cases do not pose a public health risk and the
adulterants used are safe for human consumption, there have been some cases which have
resulted in actual or potential public health risks (Johnson 2014) when a toxic substance is
added to food as an adulterant.
A serious problem with the adulteration of food products with declared or undeclared
other foodstuff is that it may lead to severe health problems and may even risk death to
people who may have a severe allergenic response to some adulterants. For instance, allergy
to sesame seeds and oil has been identified as one cause of asthma. Peanuts, soybeans, tree
nuts and wheat are among major food allergens of plant origin. A broad spectrum of fruits,
vegetables, seeds, spices, and latex has been reported to possess allergenic potential (Hahn
2009, e.g., Breiteneder and Ebner 2000). Wang et al. (2002) carried out a study to clone
cDNAs encoding cashew food allergens.
The problem of food allergy is particularly acute when the presence of adulterants (e.g.,
walnuts in bread, Gowland 2002) cannot be reasonably anticipated. One such example is the
adulteration of roasted and ground coffee with soya beans and cashew nuts. A second
example is the adulteration of commercial olive oil with soybean oil (Firestone et al. 1988) or
hazelnut oil (Firestone and Reina 1996). Similarly, substitution of olive oil with other types of
seed, legume, or nut oils could have unintended consequences, if consumed by those with
certain food allergies (Johnson 2014).
Fraud involving the addition of clouding agents, whose use in certain highly processed
foods has been increased in recent years, has raised concern given the potential public health
risks (Johnson 2014). Some other common adulterants in food and diseases caused by them
have been reported (http://www.slideshare.net/vibhassingh7/adulteration-of-food). Jaiswal
(online reference) also listed some injurious adulterants/contaminants in foods and their
health effects.
In addition to food adulteration, contamination of food products as a result of inadequate
company controls or due to the nature of the product or the process has been reported
(Gowland 2002). Peanut (50-100 mg of the allergic protein) and tree nuts (almond, hazelnuts,
cashew, pistachio, walnuts and pine nuts) are highly allergic food contaminants (Lessof
1997). Hence, identifying contamination of some food products with nuts like cereal and
confectionary products and contamination of dietary products with soy protein (Kleine-Tabbe
et al. 2001) is crucial (Hourihane 2002).

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199

CONCLUSION
As much as food fraud is a cost-driven economic issue, it is also a public health issue
(Krietsch 2014). To prevent fraud-related food adulteration and health hazards that maybe
associated with it, the development of species authentication techniques that are rapid,
reliable, and reproducible has been a research priority. These include methods based either on
qualitative and quantitative species-specific/multiplex PCR or methods based on post-PCR
analysis such as DNA sequencing, RFLPs, SSCP, SSRs, ISSRs, RAPDs, and AFLPs. Various
chloroplast and mitochondrial genetic markers have also been generated. Combination of
more than one method (e.g., protein- and DNA-based methods) is also possible.
Examination of methods reproducibility and reliability is important. Although genetic
differentiation methods have been extensively researched among certain food products
groups, many challenges still remain. These include the optimization of methods that use
smaller fragments, which can be analyzed in both raw and processed products, and the
identification and quantification of species in mixed samples (Rasmussen and Morrissey
2008). Real-time PCR has now become an accepted analytical tool by the food industry
where the number of applications for the technology is rapidly increasing, despite the
perception that it is costly (Wiseman 2009).
In the near future, DNA barcoding is likely to become a routine test in food quality
control and traceability (Galimberti et al. 2013). Given the rapid standardization and
evolution of next generation sequencing (NGS) advances, a universal approach such as DNA
barcoding combined with NGS can offer a new opportunity for the traceability of food
products such as minor crops from field to table (Galimberti et al. 2014). These and other
emerging technologies have the potential to simplify the methodologies, although their cost
and some other factors limit their uptake by the food industry. Improved methods will assist
in the protection of consumers and producers from frauds (EUFIC online reference). Vaclavik
et al. (2012) noted that as new and more sophisticated adulteration methods are continuously
developed, fraud performers are usually one step ahead of the available testing methods.
Although molecular methods and DNA-based techniques have allowed fast, accurate and
sensitive detection of plant-based adulterants in food products of plants origin, they are
however limited by: (1) cross-over contamination which may occur in the laboratory (in the
absence of good laboratory practices) and/or in the food chain and which can influence
research results (such as the PCR); (2) low quality DNA which may be the result of food
processing where the DNA can be damaged or destroyed due to physical (i.e., heating,
boiling, UV radiation) or chemical treatments (i.e., addition of food preservative, artificial
sweeteners); and (3) genetic hybridization that leads to species with the same DNA profile as
exemplified with the chloroplast and mitochondrial DNAs of the majority of plant species
(EUFIC online reference).

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In: Advances in Food Analysis Research


Editor: Anita Haynes

ISBN: 978-1-63483-783-5
2015 Nova Science Publishers, Inc.

Chapter 10

LC-MS/MS IDENTIFICATION OF UNKNOWN SPECIES


IN BEVERAGES DUE TO INTERACTIONS BETWEEN
DYES AND OTHER ADDITIVES OR DUE TO DYE
DEGRADATION REACTIONS
Fabio Gosetti, Bianca Bolfi, Eleonora Mazzucco,
Marcello Manfredi and Emilio Marengo
University of Piemonte Orientale,
Department of Science and Technological Innovation, Alessandria, Italy

ABSTRACT
The interactions among the ingredients in food and beverages are still an open
problem. Unfortunately, much of these interactions give rise to unknown species that
sometimes are potentially dangerous.
In food safety, particular attention is devoted to food dyes. The risk is not ascribed to
the dye itself, whose use and permitted quantities are rigidly regulated by specific
restrictions, but to the degradation products that may form due to inadequate storage and
management conditions.
Different studies on the toxicity of food dyes have been performed, but very few
information has been collected about their possible degradation reactions in commercial
beverages undergone to sunlight irradiation and their chemical reaction with other
ingredients present in a specific drink. Since many food dyes are characterized by
aromatic aminosulfonate groups, the risk may regards, besides the possible presence in
beverages of impurities deriving from the industrial dye synthesis process, also the
possible formation of carcinogenic aromatic amines in the beverage itself exposed to
sunlight irradiation, which may take place along the different phases of the life cycle of
the specific food product.
After a quick overview about the problem of additive interactions in food, the
possible causes of dye discoloration and their relevance in wastewater pollution, this

Corresponding author: Dr. Fabio Gosetti, tel. +39 0131 360249, fax: +39 0131 360365; e-mail:
fabio.gosetti@uniupo.it

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chapter deals with the potential modifications of permitted dyes taking place in
beverages. These modifications can be due to reactions occurring among different
ingredients, to potential transformations of the ingredients themselves and to
environmental effects. Generally, these interactions take place during the post-production
phases and in particular during the pre-selling storage, conservation and
commercialisation. They are often unpredictable, but they can give rise to different kinds
of contaminations with the formation of new species potentially harmful to the consumer
health.
It is useful to underline that the nature of these contaminations is not due to possible
frauds, adulterations or use of poor quality ingredients, but on a still scarce knowledge
and available documentation concerning these kinds of interactions. The sources of
possible contamination that have been considered can be classified into two types of
reactions, taking into account that some of their effects can also occur simultaneously:
i) unpredictable reactions that potentially take place among food ingredients, both
naturally present or added, ii) degradation reactions induced by not suitable preservation
conditions, as the exposure of the product to sources of intense sun light and/or high
temperature.
This chapter describes the process of identification of these unknown species through
liquid chromatography coupled with tandem mass spectrometry, and in particular,
through the use of high-resolution mass spectrometry.
As a consequence of what described above, the attention to this type of problems is
extremely relevant for the consumers health safety and for the development of
standardized investigation protocols to be applied in the routine control for the
permission of a certain product or to establish management/transport/conservation
procedures of the product in order to avoid the appearance of the problems described.

1. INTRODUCTION
The term food additives refers to natural and synthetic substances, so including many
classes of chemical species. There has been a growth of the demand of processed food, due to
the will of consumers to add specific ingredients to aliments or to substitute fats and sugars
with low-calorie substances [1].
Food dyes are the most interesting group of food additives. Would you drink an orange
drink that is not a nice orange or would you eat a strawberry ice cream that is not red but
grey? The answer is probably no. When you eat and drink, looks also counts. Artificial and
natural dyes make food and beverages more attractive to the eyes because they enlighten
certain properties as the red of an apple or the purple of an aubergine and can restore the
original attractiveness that could be lost during the production steps (for example the mint
essence without no added dyes would be colorless). The addition of dyes to food is not a
recent industrial choice: it is well known that ancient population such as Romans and Greeks
used the extracts of minerals, animals and vegetables to give color to their food.
Colorants play important roles in food industry by providing enhancement, imitation or
masking of the natural color of food products. The use of food colors in soft drinks has gained
over time more and more weight from a commercial and industrial point of view. The number
of food and drinks, in which food dyes are added, is surprisingly high. The addition of these
products is a process widely used in the food industry. These dyes have the primary aim to
replace the natural color of the product, lost during the treatment of raw materials, and to
decorate the drinks themselves. From a commercial point of view, this application is not of

LC-MS/MS Identification of Unknown Species in Beverages due to Interactions 213


secondary importance, especially when the multitude of products on sale is considered. The
color turned out to be an important instrument to attract the consumers attention, in particular
when they are hesitant in front of a shelf crammed with drinks. In fact, the color of the food is
associated with the good quality of the product for sale. Tests carried out in Great Britain
demonstrated how a strawberry jam with the addition of synthetic red dyes was bought, rather
than the same jam with the same taste and equal price, but almost colorless [2].
Natural dyes are unstable and easily undergo degradation during the food processing. On
the contrary, synthetic food dyes guarantee an intensive and permanent color and its
persistence along the industrial treatments. The cost of the production process is much lower
compared to that required for the extraction of natural dyes. These advantages encourage
producers to use synthetic dyes. They can be classified as: synthetic (synthesized by human),
natural (derived from natural sources) or nature identical (the same species found in nature
but synthesized by human) [3]. About 80% of the synthetic food colors are yellow (e.g.,
Tartrazine, Quinoline Yellow, Sunset Yellow FCF,) and red (e.g., Carmoisine, Amaranth,
New Coccine,), and they are divided into different chemical classes such as azo dyes,
triarylmethane, phenylmethane, quinoline, anthraquinone, and phenolic dyes.
However, concerns regarding their harmfulness to human health are rising because of
their potential toxicity. Since few years, food industries showed an increasing trend in
replacing synthetic dyes with the natural ones [3-5]. This led to search for alternatives coming
from plants and microorganisms [6]. From the point of view of a natural perspective, there is
the common belief that natural colors are better than synthetic ones, so consumers demand
their use for foodstuffs [7]. However, the replacement of a synthetic color with natural one,
which is not that straightforward as it might appear, does not guarantee safety. Natural colors
have only been tested to a limited range and their evaluations are based on a number of
assumptions. Taking into account the constant increase of the use of natural colors in food,
there could be the possibility of exceeding the ranges where their use is safe. Recently EFSA,
according to the Commission Regulation (EU) No 257/2010, started a program for the reevaluation of approved food additives. This audit should be completed in 2015 for all color
additives [3].

2. FOOD SAFETY AND LEGISLATION


The safety of food colors has been subject of discussion for several years. The majority of
synthetic dyes have been extensively tested with conventional safety studies. Many methods
for the analysis of dyes in food were focused on the identification of their presence, just to
ensure that their use is allowed. It would be desirable to be able to estimate the initial
concentration of the dye added evaluating the residual concentration of dye and the amount of
its degradation products. The identification and quantification of these degradation products is
important to evaluate the dye potential toxicity, and to see how these products may react with
other components of the food to form undesirable compounds. In food colorant, stability
depends on different factors. The most important is related to the presence of reducing agents
that may lead to split the azo double bond, eventually forming primary amines. Ascorbic acid
(vitamin C) is often added to many beverages as a nutritional supplement and/or as an

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antioxidant, but this gives rise to a reducing environment in which the azo dye can easily be
degraded [8].
For this regards, the attention has been particularly devoted to azo dyes, which are not
only used in the food industry, but also in the textile one. It has been proved that certain azo
dyes used in textiles and leather pose a risk of cancer for consumers and workers. In the
opinion of 18 January 1999, the Scientific Committee on Toxicity, Ecotoxicity and the
Environment (CSTEE) concluded that there is a concrete risk of cancer associated with the
use of certain azo dyes, and also confirmed that azo dye are especially dangerous because
their cleavage form carcinogenic amines like aniline, naphthylamine, benzidine, 4aminobiphenyl, classified with carcinogenicity class 1 or 2.
In order to protect the human health, the Council Directive 76/769 EEC of 1999 prohibits
the use of dangerous azo dyes as well as the marketing of certain textiles and leather articles
colored with these substances. As confirmed by the CSTEE, only the water-soluble azo dyes
are bioavailable and consequently pose a risk to human health; the water solubility is usually
conferred by the presence of one or more sulfonic acid groups or carboxylic acids and basic
groups.
After the breakdown of the azo group, dyes may release toxic products: different
individuals have in fact shown allergic reactions, hives, asthma and rhinitis, after the
ingestion of Tartrazine, so its use in food must be declared on the label [9]. It has also been
proved that the presence or the interaction within the body of some of these dyes (including
Tartrazine itself) with medications such as aspirin, benzoic acid, and some analgesics can
cause allergic reactions and respiratory diseases in the more predisposed people [10].
In addition, several experiments on animals detected the high carcinogenic potential of
azo compounds for humans: in particular it has been proved the toxicity on cultures of
microorganisms such as Bacillus cereus and Escherichia coli [11].
In addition, several dyes are characterized by sulfonate groups: when they undergo
degradation, it can be reasonably assumed that they can give rise to sulfonated aromatics,
whose degradation processes are very slow. When they are poured into the water and spread
unchanged, they become potential pollutants of the local water supply.
The use of food dyes is governed by a set of directives, reporting the list of coloring
agents allowed with the corresponding maximum concentration. The Directive 78/25 EEC
defines that a colorant can be used for food and drugs only if it does not contain more than
0.5% of impurities.
In fact, these species, often carcinogenic or suspected to be toxic, may be present in the
reagents of industrial synthesis, or they may result from incomplete reactions or undesired
side reactions, that occur during the production process. For example, aniline, benzidine, 4amino biphenyl and 4- amino benzene have been identified in Sunset Yellow FCF dye
(E110), widely used for the preparation of food, beverages, pharmaceuticals and cosmetics,
approved by the Food and Drug Administration (FDA).
The stability and inertness of synthetic dyes are still controversial. All the regulations
take into account the dye itself, in other words, they do not evaluate potential toxicity of the
degradation products that might be formed after exposure to light, heat or due to interactions
that occur in the matrix in which they are added to. These products could be more toxic then
their precursors: for example, azo dyes could be destroyed in the human intestine by
azoreductase enzymes, leading to the formation of aromatic amines, which are the cause of
frequent headaches in adults, while children often becomes distractible and hyperactive.

LC-MS/MS Identification of Unknown Species in Beverages due to Interactions 215


However, all natural and artificial colors, flavors and preservatives that are added to food
and drinks must have been previously tested and approved as safe by the regulatory
authorities in the world [5, 12].
In European Union, an identification code consisting of the letter E followed by a
specific number is assigned to each additive. This number contains 3 to 4 digits, which
identify the function performed by the additive (Dyes E1xx; Conservatives E2xx;
Antioxidants and Acidifiers E3xx; Emulsifiers E4xx; Flavors E6xx; modified starch E14xx;
Sweeteners 950968; Food enzymes 11xx). In total 320 additives are authorized to be used in
food products and are listed in 26 categories based on their techno-functional property [13].
The European Union started regulating the use of dyes in food in 1962 with the Council
Directive 62/2645/EEC and then in 1994 with the European Parliament and Council Directive
94/36/EC that established colors for use in foodstuff [14]. In the most recent years, the
European Directive No 1333/2008 of 16 December 2008 on food additives replaces previous
Directives and Decisions concerning food additives permitted for use in foods in general
with the scope to harmonize the use of food additives in the Community. In this context, the
European Directive described colors as substances which add or restore color in a food, and
include natural constituents of foods and natural sources which are normally not consumed
as foods as such and not normally used as characteristic ingredients of food. Preparations
obtained from foods and other edible natural source materials obtained by physical and/or
chemical extraction resulting in a selective extraction of the pigments relative to the nutritive
or aromatic constituents are colors within the meaning of this Regulation" [9]. Moreover, in
Annexes V all the dyes are listed for which the labelling of foods shall report additional
information, since they might have an adverse effect on the activity and attention of children.
The colors reported are Sunset Yellow (E110), Quinoline Yellow (E104), Carmoisine (E122),
Allura Red AC (E129), Tartrazine (E102), and Ponceau 4R (E124) and their effects on
children were reported also by other authors [15, 16]. Currently in the European Union, both
natural and synthetic food colours are regulated by the same legislation and there are the same
restrictions on their use [3].
In the United States the permitted dyes in food are regulated by US Food & Drug
Administration (FDA), in particular in the Title 21 of the Code of Federal Regulations [17].
In the Republic of China, the use of food additives is regulated by the Direct GB 27602011 of the Ministry of Health. According to the Direct, only eleven synthetic dyes can be
added to food products. Permitted synthetic food color additives are: Allura Red, Amaranth,
Azorubine, Brilliant Blue, Erythrosine, Indigotine, Ponceau 4R, New Red, Sunset Yellow,
Quinoline Yellow and Tartrazine [18].
Nowadays food dyes legislation is different according to the Countries and the same food
color can be legal in one Country and illegal in another one [3]. For example, synthetic food
dyes like Acid Green and Patent Blue are not permitted in China, but they can be used in
Europe and Russia. Acid Rose Red B and Rose Bengal are permitted as food additives in
Japan but not in China [19].
In addition, particular attention should be paid to the container. For this purpose, the
European Community established with Commission Regulation No 10/2011 of 14 January
2011 on plastic materials and articles intended to come into contact with food, tests to check
packaging stability. For example, solutions of ethanol 10% or 20% (v/v) and of acetic acid
3% (w/v) were assigned to food that have a hydrophilic character and are able to extract
hydrophilic substances. In particular, acetic acid was used for food presenting a pH below 4.5,

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while ethanol at 20% for alcoholic food and for that containing a relevant amount of organic
ingredients that make the food more lipophilic [20].
As it can be read, all the legislations regard the possible risk associated to the single
species (dyes, sweeteners, preservatives) or packaging while, at our knowledge, only few
studies were performed considering the potential interactions among the ingredients of the
food [21-26].

3. THE PROBLEM OF FOOD ADDITIVE INTERACTIONS


Food safety controversial is of increasing worrying to consumers. In particular, they were
mainly focused on adverse reactions to certain additives such as the artificial food coloring
Tartrazine or the sulfite group of preservatives [1]. Unfortunately, there is still not any
exhaustive study on the possible repercussions of chemical interactions among the permitted
food additives. Some interactions could be investigated by introducing a doorstep of danger,
that is to say a limit imposed by the regulation, related to a limit of no toxicological
significance. For example, if we consider an additive representing 0.1 mg kg-1 in the diet, and
reacts for 1% leading to unknown species, these will correspond to 0.001 mg kg-1 in the diet,
so they could be considered not to be needed for regulations, evaluating its toxicity [1].
When referring to interaction, this is intended for the global effect due to the
simultaneous presence of two or more species. The final effect could be antagonistic, additive
or synergistic [1].
In the last 40 years, many efforts were focused on the investigation of these type of
interactions, but there is still a lot of work to do in this field. Many substances are
intentionally added in order to act as reactive species in the organic matter [1]. Some
examples could be given by the preservatives like sulfur dioxide and nitrite, and the phenolic
antioxidants butylated hydroxyanisole (BHA) and butylated hydroxytoluene (BHT) [1]. Other
additives are incidentally reactive. For example, food colors act on the visual impact of the
product, but they could form other species because of their interaction with the light or heat.
Some of the additives considered chemically inert (i.e., gums and bulk sweeteners), could
lead to non-specific reactions, such as inhibiting the bioavailability of some essential food
components. The connection between the planned and non-planned reactivity, associated to
the possible reactions, is still not very clear, making further research works mandatory.
This kind of research is difficult because of the poor chemical information available not
only on the major food constituents but also on trace nutritional components. To overcome
the problem, preliminary studies on model solutions are necessary, but it has to be pointed out
that the whole comprehension of the possible hazardous effects on the human health has to
include also the effects of beneficial chemicals such as nutrients.
There are numerous cases of food additives interactions. Well-known is the interaction
between lecithin and nitrite ion: lecithin is a source of choline, a quaternary ammonium
compound that can be decomposed by heat, yielding trimethylamine. Then, demethylation
can produce dimethylamine, which subsequently can react with the nitrite ion to form
dimethylnitrosamine, a known carcinogen. On the contrary, ascorbic acid and tocopherols are
particularly useful in inhibiting N-nitrosation, and their simultaneous presence gives a
synergistic effect [1]. It was reported that the antioxidant activity of plant extracts could be

LC-MS/MS Identification of Unknown Species in Beverages due to Interactions 217


modified through interactions with other compounds. For example, a positive effect was
observed when the methanolic extract of rosemary, thyme or oregano are combined with
BHA, BHT or ascorbyl palmitate, whereas a negative effect with -tocopherol was reported
[27]. Some antioxidants may act synergistically, thus showing a much more effective
response against oxidation. The most studied synergism for this category of compounds is
which among -carotene, ascorbic acid and -tocopherol [28].
The problem of the bitterness of ginseng, one of the most popular functional ingredients
found in energy drink formulations, was solved thanks to the interaction between - and cyclodextrins and the triterpenoid peptides, responsible for the bitter taste of ginseng.
Cyclodextrins form inclusion complexes with the bitter compounds, through hydrogen bonds
and van der Waals forces, resulting in reduced bitterness [29].
A study devoted to evaluate the speciation of selenium in some supplements, reported
that selenate originally added is reduced by L-ascorbic acid (another ingredient of the
supplement) to selenium and probably selenide [30].
A negative synergy has been observed between some polyphenols (quercitin, resveratrol,
catechin) used as antioxidant agents in food [31]: the mixture of more polyphenols
unexpectedly leads to a decrease of their antioxidant proprieties. This behavior has been
ascribed by the authors to a series of reactions with other components present in the food
matrix, as metal ions or enzymes, characterized by specific oxidation-reduction properties.
Information on the chemical interactions between intense sweeteners and other food
additives is exiguous [1]. Aspartame is quite reactive towards aldehyde, which represent one
of the principal flavor compounds used in chewing gums and certain soft drinks [32]. A study
reported the possible interaction of saccharin and cyclamate with food ingredients in
particular water-soluble vitamins and essential amino acids among which phenylalanine and
tryptophan [33].

3.1. Interaction between Food Dye and Other Additives


The more recent alarm, supported by only few studies, concerns the behavior of dyes that
are stable in aqueous solution under the action of sunlight, but can undergo degradation when
present in beverages [21-25].
Mixing color additives with oxidizing and reducing agents leads to dye instability. The
color of the dyes is generally due to the presence of conjugated unsaturated system, so any
kind of substance (e.g., oxidizing or reducing agents, sugars, acids and salts) could act on this
system, modifying the aspect of the color [1]. The interaction between sulphur dioxide and
azo dyes is important because of their frequent use together, particularly in fruit drinks, but
also to a minor extent in jams, sauces and other products [34]. Amaranth, Ponceau 4R and
Sunset Yellow produce secondary dyes after reaction with bisulphite [35]. These dyes are
characterized by hydroxyl groups ortho to the azo bond as well as unsubstituted para positions
in the naphthalene nucleus [34]. Such dyes exist predominantly as hydrazone tautomers rather
than strictly as azo compounds [34]. This facilitates the addition of bisulphite ion to the para
position, for the conversion of Sunset Yellow to a lemon yellow compound [36]. In the case
of Carmoisine, a hydrazone tautomer is feasible, but the addition of bisulphite ion to the para
position appears not to take place, probably as a result of charge delocalization via the fused

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aromatic ring of the naphthalene system [34]. Another possible explanation could be that one
molecule of the dye reacts with one molecule of bisulphite to form an unstable complex that
hydrolyses to a colorless hydrazo product [37]. In Tartrazine, the ortho-hydroxy group is a
substituent of the pyrazoline ring that does not possess the pi-electron mobility of a truly
aromatic system [34]. The tautomer of hydrazone is therefore difficult to form, so the dye is
consequently very stable in the presence of bisulphite [34]. Other dyes have a variable
stability to sulfur dioxide: if the triarymethane dye Green S and Quinoline Yellow have a
good and excellent stability, respectively, Violet BNP is unstable as well as the indigoid dye
Indigo carmine [34]. Some studies reported the interactions between amaranth and sulfur
dioxide, where amaranth faded to an orange-yellow compound, which could not be identified
due to its instability [1].
All dyes, especially those characterized by an azo group, will exhibit accelerated fading
rate under both acid and alkaline conditions in the presence of metals, such as zinc, tin,
aluminum, iron and copper, especially at higher temperatures [1]. The added color in canned
products may degrade in the presence of tartaric and citric acids, because the acids can react
with the metal of the container to release hydrogen [1].
Citric acid was also responsible for the degradation of Brilliant Blue FCF in a sealed
beverage that gradually discolored and completely lost its color after 16 days of sun
irradiation [22]. Laboratory experiments also showed that solutions of the standard dye alone
do not undergo any degradation under the same exposure time, but that degradation is assisted
by other components among which citric acid, present in excess in the beverage (100-fold
with respect to the dye concentration) [22]. On the other hand, the degradation products
formed when irradiating the model solutions containing the dye and citric acid in the same
concentration as in the beverage, are different with respect to those formed from the
degradation of the beverage itself. Based on the results obtained using model solutions, the
authors supposed that different ingredients of the drink can give rise to different interactions
in the degradation pathway, making the identification of the degradation species very difficult
and challenging [22].
The dye Black PN was found to be particularly unstable in the presence of ascorbic acid,
and Carmoisine decolorized more rapidly at pH 7 than at pH 3 at 121C in the presence of
this reducing agent, whereas Ponceau 4R was stable at this pH probably thanks to sulfonate
group peri to the azo bond [1]. It was found that the larger concentration of ascorbic acid
leads to the larger rate constants and lower dye half-life. The values of the rate constants of
Brilliant Blue FCF, Quinoline Yellow, Patent Blue V and Tartrazine are 2.504.25 times
larger when the colorant degradation is performed in the presence of 500 mg L1 of ascorbic
acid than in the case of the presence of 100 mg L1 ascorbic acid [38]. From the other side, the
degradation of Brilliant Black BN and Indigotine is very fast with a total degradation for 69
days [38]. The degradation of Amaranth in soft drinks has already been evidenced in 1987,
when no restriction on the admitted quantity was present yet [8]. It was shown that Amaranth
was reduced by ascorbic acid to naphthionic acid and amino-R-salt, which is unstable and is
readily oxidized to naphthoquinone. In addition, a protective effect of the sugars present in
the beverage towards the reductive potentialities of ascorbic acid was observed: the effect was
explained by competitive chelation reactions taking place between sugars and the metal ions
present, which could catalyze the oxidation of ascorbic acid, if non complexed [21]. The
discoloration process was observed also in the case of Sunset Yellow FCF. Its reaction with
ascorbic acid in absence of atmospheric oxygen breaks the azo bond and gives rise to

LC-MS/MS Identification of Unknown Species in Beverages due to Interactions 219


uncolored species, whose structures were identified by LC-MS/MS [21]. These are clear dyefood interaction examples that must be evaluated during the formulation of soft drinks (major
responsible for synthetic dyes introduced in diet and enriched of ascorbic acid due to its
antioxidant and nutritional properties) [1].
Ascorbic acid represents an important way of conservation of natural dyes in beverages
(i.e., anthocyanins) [1]. In the presence of iron or copper ions and oxygen, the oxidation of
ascorbic acid to dehydroascorbic acid is accompanied by the formation of hydrogen peroxide,
which subsequently can oxidize anthocyanins to colorless species; a reaction implicated in the
loss of color from canned strawberries [39]. Ascorbic acid and anthocyanins may react also
under anaerobic conditions in the presence of amino acids, phenols and sugars via a
condensation-type mechanisms, but the products of these interactions, some of which are
complex polymers, have not been well-characterized [40]. Chang reported that to prevent
discoloration of beverages due to ascorbic acid, the addition of vitamin B2 could be useful
[41].
The photodegradation of Allura Red AC contained in soft drink was attributed to the copresence in the beverage of citric acid, ascorbic acid, strawberry juice, aromas, chamomile
flowers extract and sucrose. The latter did not influence the degradation pathway, but
increased the degradation kinetics only when copresent with ascorbic and citric acids [24, 25].
Instability phenomena of synthetic azo-colorants have been observed in presence Arabic
Gum (emulsifier largely used in beverage industries) during the storage of the emulsion
concentrate. Precipitation reactions of the dye induced by the copresence of Ca(II) and Mg(II)
ions caused the instability of the emulsion and the product deterioration [42].
Other interactions are due to the formation of inclusion complexes of -, -cyclodextrin
and some sulphonated azo dyes, such as Orange II, Ponceau SX, Allura Red AC and
Tartrazine [43].
Neurological studies showed a synergistic interaction between the food dye Brilliant Blue
and L-glutamic acid and between Quinoline Yellow and aspartame, species that are often
present together in snacks and beverages, typically consumed by children. While the use of
the individual ingredients can be considered safe for the neurological development of the
infant, their synergism can seriously damage a not yet mature nervous system [44].
A recent study reported the interaction between photoactivated nanoparticles of TiO2
contained in soft drinks or other food products, and Tartrazine. Reactive oxygen species
(ROS) were formed during the process and were responsible for dye discoloration [45].

4. WHY CAN DYES UNDERGO DEGRADATION?


It is possible to describe a dye as a material consisting of two parts [46]: an electron
acceptor (chromogen) and an electron donor (auxochrome) that is responsible for the dye
solubility and color. Furthermore, the first part usually presents an aromatic body, linked to
the responsible for the color: together they constitute the chromophore group [46]. This one,
absorbing light in the visible region, generates a shift (blue shift or red shift) of the
wavelengths, causing a color change [46]. Generally, the main reaction pathways for
hydrophilic compounds such as azo dyes involve the bulk liquid, where they might be
effectively destroyed via oxidative degradation by hydroxyl radicals that attach to the

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chromophore, as well as the aryl rings [47]. The most largely accepted degradation
mechanisms of azo dyes focus on the cleavage of either the N=N bond, resulting in
nitrosoaryl intermediates, or the C-N bond with generation of aniline, sulfanilic acid and
naphthionic acid as the intermediate products [48].
Very often, the mechanisms that give dye discoloration were taken into consideration
owing to the industrial problem of colored wastewaters that have to be discolored before
being depurated [49-57]. However, absence of color does not mean absence of toxicity and, in
addition, the uncolored species formed could be even more toxic than the parent dye.
Nevertheless, the popular motto is if you cannot see, there is nothing, different solutions are
used to discolor the wastewaters: adsorption [58, 59], coagulation [60], oxidation and
electrochemical methods [61, 62]. There is a wide use of catalytic degradation by titanium
dioxide (TiO2). Titania is also used as an ingredient in food as anticaking and brighter agent
(especially in soft drinks, ice creams, gelatins and baked foodstuffs), in cosmetics it plays the
role of UV protectant, finally as a pigment in paints. The use of this material is differently
regulated in world countries. In U.S.A. it could be up to 1% by weight in food [17]; in Japan
it is subjected to particular limitations dealing with specific types of food [63]; in India it is
allowed only in chewing gum and bubble gum up to a maximum of 1%, while it must be less
than 0.01% by weight in powdered concentrate mixtures for juices [64, 65]. In Europe it can
be present in nonalcoholic drinks up to a concentration of 100 mg L-1 (either individually or
mixed with other dyes), 0.015% in desserts and 0.02% in candied fruits and vegetables [14].
The amount of this catalyst, also related to the particle aggregation mainly present at higher
concentrations, influences the degradation of the dye. TiO2 is synthesized as a nanoparticle:
this provides an increase of its surface area, reducing the particle size. When TiO2 is exposed
to light, it can absorb energy for the gap that separates the conduction band from the valence
one (3.0 or 3.2 eV) [45], promoting an electron to the higher energy level, and consequently
generating a valence band hole that can extract other electrons from water or radical ions,
causing the production of hydroxyl radicals OH [66, 67].
The formation of ROS promotes oxidative damages of matrix in which they are
generated, mainly if they are complex as the food one. In fact, ROS are able to destroy
microbial agents [68, 69], generally to degrade [70] the organic part of the product either in
the form of OH or in the form of singlet oxygen, generated by light irradiation [71]. For
example, they are able to cause the degradation of azo dyes, breaking the -N=N- bond. The
more TiO2 is present in foodstuffs, the more their color features fade. This is attributable to
the growing number of active sites on the catalyst surface, that lead to the increase of the OH
radicals responsible for the degradation of the dyes [72]. It has to be noticed that, over a
specific concentration range, this correlation is not maintained because the solution becomes
turbid, reducing the passage of the radiation that provide the photo-catalytic reaction [73].
This is due to the aggregation of free nanoparticles, increasing a light scattering effect within
the solution [74].
Also pH greatly influences the photo-catalytic degradation of the dyes [75, 76]. As the
pH at zero point of charge for TiO2 (pHzpc) is set at 6.8 [77], its surface can be protonated or
deprotonated in acid and alkaline media, respectively [78].
In the former case, the main responsible for the dye degradation are the conduction band
electrons, able to break the azo bonds. It can also be stated that high concentration of H+
could cover the catalyst surface, causing the reduction of free radicals, inhibiting photoexcitation [72]. Because of these reasons, pH has to be higher than 7 [75, 76, 79]. In alkaline

LC-MS/MS Identification of Unknown Species in Beverages due to Interactions 221


conditions, the surface is covered by the OH- adsorbed ions. Hydroxyl radicals can interact
with band valence holes, leading to OH radicals, so favoring the efficiency of the dyes photodegradation [45]. On the other hand, at low pH value the surface is positively charged. This
cause a strong Columbic adsorption of the dyes (usually present in anionic form) on the
surface of TiO2, causing a faster degradation rate of the dyes. In some studies present in the
literature, the maximum of this interaction takes place in neutral conditions [80, 81].
A not less important factor is the concentration of the dye that has to be degraded by
photo-catalysis. It has been found that increasing the concentration of the dye, there is a
decrease in its degradation rate. This inverse proportionality is probably due to the fact that
the solution becomes progressively more impermeable to the UV radiation, taking into
account the high molar extinction of the dyes in general. In other words, at higher
concentration, it is the dye that absorbs the major amount of light and not the catalyst. Even in
this type of dependence, raising a certain value, degradation rate is faster because all the dyes
and their intermediates could be adsorbed on the catalytic surface. When all the sites are
saturated, no more degradation could take place [82, 83].
The intensity of the light that irradiates the samples influences the degradation rate of the
dyes. As the intensity grows, the faster is the degradation process. There is a linear correlation
between the two behaviors. This is due to the fact that the color fading is due to the electronhole pair formation, whose effect is amplified effect by the increase of light intensity. It has to
be underlined that the sun spectral range could be divided into three different portions, all
able to make the dyes fade: the ultraviolet radiation, the visible radiation and the near-infrared
radiation, characterized by different amounts of energy [84].
Ordinary degradation processes could occur in food, beverage, and cosmetics just only by
exposing them to sunlight, for example, on the shelves of the supermarket. Moreover, it is
possible that some side reaction products could be present in foodstuffs, usually resulting
much more hazardous for health than the initial dye, and could follow degradation paths that
lead to toxic products. The stability of natural dyes to pH, temperature and irradiation has
been less analyzed than that regarding synthetic ones [85-87]. It is important to study also
those aspects because these factors determine the common conditions in which degradation
could take place ordinarily. The literature reports that some natural dyes, such as Phycocyanin
are not stable at different temperatures, in relation to their structure and they are also
influenced by the pH of the media in which they are contained [88]. In fact, the increment of
temperature leads to a faster degradation rate, increased furthermore by a neutral pH [88].
There is also a synergistic effect between pH and UV-light intensity. In some beverages
containing iron cation and exposed to irradiation, it is also possible that photo-Fenton process
takes place at low pH values [21, 89-91] and this greatly influences the dye degradation rate
[21].
It has to be evaluated also the combination of the possible effects that can occur in
complex matrices as the edible food are. In other words, the formation of adducts and other
organic species is possible, taking into account the great number of ingredients (i.e.,
sweeteners, acidifying, emulsifiers, acidificants, preservatives, and so on) that are used to
formulate food and be aware of simultaneous degradation of other organic parts of the matrix.
These combinations of effects could either accelerate or decrease the degradation rate of the
dyes. It is clear how the problem is complex and the need of analytical methods to evaluate
those processes, as well as to identify the degradation products possibly present in the food.
To simulate sunlight, for example, it is possible to use a Solarbox (a xenon lamp with

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between 280-800 nm) and reproduce the exact ratio between UV-A and UV-B typical of a
certain season. In order to understand the behavior of the dye in a complex matrix,
preliminary experiments carried out on the solution of standard dye (when commercially
available) are necessary. To obtain this, real samples with all their ingredients have to be
exposed to simulated irradiation in their original and sealed packets. Only by doing, this it is
possible to obtain a wide identification of the synergistic and/or antagonistic effects that
affect food, beverage and cosmetic products [21-26].

5. LC-MS/MS USES IN NON-TARGET ANALYSIS


Identification of trace unknown analytes in complex samples has always represented one
of the biggest challenge for analytical chemistry. Liquid chromatography (LC) and mass
spectrometry (MS) techniques can now be an answer, supporting the so-called non-target
analysis, a new analytical strategy, which aims to guarantee the comprehensive identification
and quantification of all the detectable chemical species present in complex samples.
In recent years, trends in reversed phase LC have been addressed to the use of smaller (<
2 m) stationary phase particles and narrow column diameters, in order to provide an overall
increase of the chromatographic resolution. Thanks to these features, peak widths are
narrower and retention times are shorter than before, being highly suitable for the high
throughput analysis of complex samples. In non-target analysis, the total efficiency of the
separation is the most important parameter for enabling the identification of unknowns, while
sample throughput is less a concern since data analysis is the currently limiting step. LC
efficiency improves both reducing particle size, as in UHPLC, and extending column length.
In most cases, species can be detected in non-target analysis of food samples, as they are
small polar analytes, which are poorly retained in reversed phase separations. Changing the
selectivity of the mobile phase and the column stationary phase, it is possible to achieve a
better separation of highly polar analytes. Hydrophilic interaction liquid chromatography
(HILIC) represents a very suitable technique to reach this aim: it combines a normal phase
mode of separation employing polar functionally modified stationary phases and mobile
phases compatible with the common reversed phase organic solvents.
Coupling liquid chromatography with mass spectrometry (LC-MS), it is possible to
develop an analytical method able to simultaneously identify a very large number of chemical
species in complex sample. This type of approach has been made possible by the
development of high-resolution MS instrumentation, which are characterized by very high
selectivity and that can operate with specificity for a large number of analytes without prior
knowledge of their presence in the sample.
The huge number of recorded data has to be mined by powerful software. Therefore,
multivariate statistical analysis, such as PCA (Principal Component Analysis) or PCA-DA
(PCA coupled with discriminant analysis) is used to determine significant differences
between the groups of samples. There has been a trend in the recent chemical literature
towards the goal of comprehensive non-target analysis using tandem mass spectrometry
(MS/MS). In non-target screening methods, all the compounds eluted from the analytical
column and ionized in the ionization source can be detected without any type of selection

LC-MS/MS Identification of Unknown Species in Beverages due to Interactions 223


(with the obvious limitations derived from the chromatographic and ionization processes in
the LC-MS interfaces used).

5.1. Choice of the Mass Analyser for Non-Target Analysis


Recent technological improvements made many different mass spectrometers suitable for
non-target analysis, each with their specific strengths and drawbacks. Requirements for nontarget MS analysis of complex food samples include the effective ionization of nonvolatile,
thermally labile species, the high or ultra-high resolution mass analysis to provide the
elemental composition of the detected species and the capability for tandem MS experiments
to provide information on their chemical structures. In particular, to elucidate the structures of
unknown compounds, two sets of instruments are suitable. The first one concerns low
resolution instruments, such as triple quadrupole (QqQ) or ion trap (IT) that provide
information concerning the product ion MS/MS spectra. The aptitude to generate detailed
fragment ion data is a desired instrumental feature for structure determination of unknowns.
Typical quadrupole and ion trap mass analyzers exhibit unit resolution; this means that they
are able to resolve only nominal m/z different for at least one unit, but not to distinguish ions
with different elemental composition with the same nominal mass.
Since QqQ instruments generally do not operate in full scan mode because of the lack of
sensitivity, their application in a true non-target analysis is rather limited. Unlike QqQ, the 3D
IT is characterized by high duty cycles because the time to fill an ion trap and generate a mass
spectrum is short. Therefore, ion trap instruments can be also used for non-target analysis
because of their MSn capability that allows the sequential and multistage isolation, in the same
space and as a function of time, of the precursor ions, fragmentation, trapping, and mass
scanning. The full scan sensitivity still increases with the introduction of the linear ion trap
(LIT). With the beginning of the hybrid QqLIT instrument called QTRAPTM [92-95], both the
traditional quadrupole and the LIT scans can be performed in a single LC run. Furthermore,
detailed fragmentation pathways can be elucidated by further dissociations of each of the
fragment ions in the enhanced product ion (EPI) spectrum and using MS3 mode in the same
run.
The second set of instruments concerns high-resolution instruments such as time-of-flight
(TOF), orbitrap, Fourier Transform Ion Cyclotron Resonance (FT-ICR), from which accurate
full-scan MS data can be obtained.
The mass analyzer capable of the highest resolution and most accurate mass measurement
available is the FT-ICR mass spectrometer [96]. This type of mass analyzer takes the
advantage of high magnetic fields to make ions orbit within an ion trap at a frequency related
to their m/z. FT-ICR instruments offer resolutions of up to 2,000,000 with mass accuracies of
less than 0.1 ppm. One of the primary drawbacks limiting the widespread use of FT-ICR is
the high cost of the instrument itself and its upkeep.
Orbitrap technology is based on orbital trapping of ions using electrical fields, but uses a
similar approach of obtaining m/z from the image current of oscillating packets of ions [97].
The use of an electrical field rather than a magnetic one, significantly reduces the cost and
footprint of the instrument compared to FT-ICR in exchange for a moderate decrease of MS
power. Orbitrap instruments report mass resolution of 100,000 at m/z 400 (mass accuracies of
1-2 ppm) operating around cycle time of 1 s, suitable for LC-MS detection. On the other

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hand, their relatively slow data acquisition time represents the main drawback of these two
types of mass analyzers. The sensitivity and resolution of these instruments is proportional to
the window of time in which the image current is detected. Even though this may only be in
the order of few seconds, peak widths in UHPLC separations can also be in the order of few
seconds, limiting their compatibility with ultra-high resolution mass analysis.
The mass analyzer, which possesses the highest spectral acquisition rate, is TOF MS,
which collects spectra about at 20 kHz. TOF is an attractive instrument due to its potentially
unlimited m/z range and high-speed acquisition capabilities [98]. The TOF MS analyzer
provides the selectivity and the sensitivity required for efficient, wide-range screening, as it
combines high, full-spectral sensitivity with high mass resolution that make it able to measure
accurately the mass of any ionizable component in the sample.
Time of flight operates at a lower resolving power of 5,000 to 20,000 and mass accuracy
of 5 - 10 ppm.
The information on the elemental composition of the detected ions is of pivotal
importance for their identification, but further manipulation of the intact ions is required in
order to obtain structural information beyond their mass to charge ratio (m/z). This is easily
overcome by applying additional energy to ions once they are in the gas phase, which leads to
their fragmentation.
This is most often accomplished using a QqLIT or 2D IT instrument, but it is also
possible in the ICR cell of the FT-ICR. For mass analyzers not capable of multiple stages of
MS/MS such as quadrupoles and TOF mass analyzers, hybrid configurations that use ion
transmission optics to couple multiple mass analyzers allow for MS/MS experiments to be
conducted in space. Combining a TOF mass analyzer with a mass selecting quadrupole and
a collision cell yields a hybrid MS/MS instrument called the quadrupole time of flight
(QTOF), which is highly suited to be coupled with fast chromatographic separations. Some
QTOF systems have also the possibility to simultaneously acquire MS/MS spectra at low and
high collision energies, in order to provide useful information on the (de)protonated
molecules and on the main product ions, respectively.
For FT-ICR mass analyzers, which are characterized by a relatively slow MS cycle time,
hybrid configurations are almost exclusively used because of their added flexibility and
robustness.
In Orbitrap, mass selection is carried out by the linear ion trap or quadrupole preceding
the ultra-high resolution mass analyzer. However, operating at higher resolution (e.g.,
100,000 at m/z 400) requires the very long analysis times of several seconds not compatible
with the chromatographic separation.

5.2. Data Dependent Acquisition and Data Independent Acquisition


LC-MS/MS non-target procedures can be divided into two general approaches: datadependent acquisition (DDA), the historic and more commonly applied method, and dataindependent acquisition (DIA), a more recent strategy [99].
When the mass spectrometer operates in DDA mode, usually some precursor ions
detected in an MS survey scan are selected for subsequent isolation and fragmentation in a
serial manner [100]. In selecting the precursor ions, there is a preference towards the ions
having the highest m/z intensity. Other additional selection criteria, such as dynamic

LC-MS/MS Identification of Unknown Species in Beverages due to Interactions 225


exclusion, background subtraction, charge state selection, etc. are also used to prevent
redundant acquisition of the most abundant fragments, or to avoid acquiring product ion
spectra of the interferences [101]. These criteria increase the number of unique precursor ions
selected by preventing the reselection of already fragmented ions in successive dissociation
events [102].
Despite remarkable success, DDA has one principal inherent limitation: the precursor ion
selection is strategically designed to pick ions in order of decreasing relative abundance,
whilst requiring an abundance threshold minimum for each ion selection, and therefore, DDA
methods only sample a restricted part of all possible precursor ions [102] (Figure 1).
Moreover, this technique makes more improbable that a tandem mass spectrum for a
precursor ion will be acquired at the apex of its chromatographic elution profile, which
adversely affects the spectral quality [102]. Overall, DDA performance declines as the sample
complexity increases, because the semi-stochastic selection of the precursor ions aggravates
certain limitations for both identification and quantification: limited reproducibility and
dynamic range, bias toward high abundance compounds, and under-sampling [103, 104].
In contrast to the sequential detection, selection, and analysis of individual ions during
DDA, DIA systematically parallelizes the fragmentation of all detectable ions within a wide
m/z range regardless of intensity, thereby providing broader dynamic range of detected
signals, improving reproducibility for identification, providing a better sensitivity, and
accuracy for quantification of the investigated species (Figure 1) [100].
To accomplish this, predetermined m/z ranges are interrogated either: (i) by fragmenting
all ions entering the mass spectrometer at a single point along the chromatographic time (All
Ion Fragmentation, MSE or MSALL) alternating between low and high collision energy scan or
(ii) by dividing the full m/z range into prefixed smaller m/z isolation windows that are each
independently and consecutively analyzed, such as eXtended Data-Independent Acquisition
(XDIA), Precursor Acquisition Independent From Ion Count (PAcIFIC), Sequential
Windowed Acquisition of All Theoretical Fragment Ion (SWATH), Fourier Transform-All
Reaction Monitoring (FT-ARM), Multiplexed data-independent acquisition (MSX) [101,
102].
Selecting a single isolation window to cover the whole precursor m/z range can reduce
the cycle times, hence generating a large number of data points during elution for accurate
quantification [100]. On the other hand, selecting several narrower isolations increases the
efficiency of fragmentation, reduces the complexity of the acquired composite fragment ion
spectra as well as precursor mass tolerance (which can influence identification results) [100].
In addition, acquisition of high-resolution MS data helps to increase the identification
confidence, as high mass accuracy precursor m/z values are detected and assigned post
acquisition instead of using the center of the isolation window. In principle, large isolation
windows are used to cover a wider precursor mass range with faster cycling rates or with
extended per-spectrum acquisition times [100]. However, large isolation widths increase the
number of precursors concurrently fragmented in the respective window, increasing the
likelihood of overlap of fragment ions from different precursors (fragment ion interference),
which also depends on the resolution and mass accuracy of the fragment ion signals [100].
Techniques employing DIA on selected m/z ranges were successfully developed on IT,
Orbitrap and FT-ICR instruments [105-107]. SWATH performs data-independent
fragmentation of all precursor ions entering the mass spectrometer in 20-30 m/z isolation
windows. The whole m/z range of interest is covered by continuous stepping of the isolation

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window. This allows numerous repeated analyses of each window during the elution of a
single chromatographic peak and results in a complete fragment ion map of the sample [102].
DIA offers several advantages over DDA schemes for a complete non-target characterization
of complex samples [100]. To fully exploit these advantages, composite or multiplexed
fragment ion spectra generated by DIA require more elaborate processing algorithms
compared to DDA [100]. Convoluted or multiplexed MS/MS spectra are generated without
explicit association between each single precursor and its corresponding fragments [100]. As
a result, DIA requires more sophisticated data analysis post acquisition compared to DDA
[100].

Figure 1. Scheme of the difference between the data-dependent acquisition (DDA) and dataindependent acquisition (DIA). In DDA, the selection of precursor ion requires the overcoming of a set
threshold in order to perform successive MS/MS acquisition. On the contrary, in DIA it is possible to
acquire all MS/MS spectrum of every precursor ions without any limitation.

6. SAMPLE PREPARATION FOR NON-TARGET ANALYSIS


The goal of sample preparation in an analytical chemistry workflow is to produce a
sample suitable for analysis by a given method and to eliminate substances that could

LC-MS/MS Identification of Unknown Species in Beverages due to Interactions 227


interfere with the analytical measurement. In complex sample analysis, specific sample
preparation is required if the analysis itself is not selective enough to resolve all mixture
components. In other words, sample preparation is still required to make complex samples
suitable for analysis, particularly when ESI mode is chosen. The biggest challenge of using
ESI for the analysis of complex samples, with or without a liquid separation prior to ESI, is
ionization suppression due to the competition between analytes and matrix interferences to
form a charge on the drop surface area. Hence, the first aim of the sample preparation in ESIMS based non-target analysis is reducing the matrix concentration, which might hinder ESI
ionization, without eliminating components of the mixture that could be of interest. In nontarget analysis, where the goal is to obtain the most realistic fingerprint of the composition of
a sample, sample dilution prior to analysis, when possible, is the most suitable technique to
reduce the concentration of suppressant ions. This approach, often referred to as dilute and
shoot is intended to reduce the total concentration of ESI ionizable species and to minimize
ESI suppression. The main advantage of this method is that it has little impact on the sample
composition, and that the dilution buffer is easily analyzed for background ion composition.
In many cases, ignoring the chemicals to search for, it is preferable not to perform any
pre-treatments in order to avoid possible losses of the same unknown species. When the
amount of sample is sufficiently plentiful, a good rule could be to divide the sample and
address it to different types of pretreatments, analyzing them under different chromatographic
conditions, in order to reduce matrix effects and to explore, as much as possible, the whole
content of the sample.
However, when some sample cleanups are required to enable effective ionization or
chromatographic separation, one of the most suitable techniques in non-target analysis is solid
phase extraction.

7. STRATEGY FOR IDENTIFYING UNKNOWNS


The most difficult task regarding non-target analysis is certainly the identification of
unknowns and their structure elucidation. Just as every good detective intent on solving a
particularly difficult case, would collect all possible clues essential to identify the culprit, so
in the searching for the unknown chemical structure it is important to gather together as much
information as possible.
When the chemical structure of the parent dye is known and its chemical standard is
available, a good point of beginning is to perform a mass spectrometry characterization of the
standard dye solution, in order to identify the major product ions that form in the collisional
sequential fragmentation of the MS/MS analyses. In fact, some of these fragments could be
useful to identify the unknown products.
De novo identification of unknown compounds detected by MS/MS, aims to correlate
observed spectral features in MS/MS to the structures of the investigated species, based on
established reactivity of small organic ions during Collision Induced Dissociation (CID)
experiments. One of the most suitable approaches for non-target identification is the focusing
on the identification of new members of a class of compounds by first analyzing those
standard compounds available for that class, in order to establish their reactivity during CID.
The simplest way of doing this is by comparing fragmentation patterns of analogous species.

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Unfortunately, structural analogues with only minor differences in structure can sometimes
show very different reactivity and consequently fragmentation patterns. A much more
rigorous approach is to determine the impact of any structural modification on reactivity in
terms of the chemical principles of resonance, inductive or steric effects such as the
availability of protons for abstraction. The site of protonation or deprotonation during ESI is
often considered as the starting point for the interpretation of CID reactivity. It is important to
consider that the site of protonation or deprotonation in solution is not necessarily the lowest
energy position for ionization in the gas phase. This is due to the media in which the reaction
takes place. In solution, the charges are stabilized by the participation of solvent molecules.
On the contrary, in gas phase the charged site is stabilized only by intramolecular effects such
as resonance, inductive effects or intramolecular hydrogen bonding. For this reason, in order
to determine the functional group with the greatest proton affinity (in positive ESI), it is not
sufficient to determine the most stable protonation site. Furthermore, ion dissociation during
CID occurs at vibrational excited energy levels, which often induce charge transfer from the
lowest energy structure. A solid fundamental understanding of the gas phase ion chemistry of
small organic ions is currently the best tool available for de novo identification of unknowns
based on MS/MS spectral data.
Often a further help comes from the formation of adducts during the electrospray
ionization. If an adduct forms with the parent dye, it is very probably that it can form also
with the dye degradation products. This is the case of the identification of Sunset Yellow and
Carmoisine degradation products in an aperitif [23]. Evidence in mass spectrometry showed
that both dyes form an adduct with glucose-fructose syrup (contained in the aperitif) in the
ESI source and similarly also the degradation products retained this feature, which permitted
their identification [23].

Figure 2. Total ion current chromatograms of a beverage contained Allura Red dye before (dark grey
line) and after degradation (light grey line) for 48 h under simulated sun light irradiation. The
chromatographic peak intensity of the dye (at retention time of 6.7 min) progressively decreases during
the degradation up to totally disappear. Degradation products formed can be seen by naked observation
and are indicated by the arrows.

LC-MS/MS Identification of Unknown Species in Beverages due to Interactions 229


But how can the unknown species be identified after an LC-MS/MS run? In general in
degradation studies, the chromatogram of a sample not subjected to irradiation (control
sample) is compared with that of the sample subjected to the degradation process (degraded
sample) with the aim of identifying the photodegradation products [21-26, 57, 108, 109].
Figure 2 shows, as an example, the total ion chromatograms of a beverage containing Allura
Red AC dye before and after degradation (48 h of simulated sun light irradiation). It can be
observed that the chromatographic peak intensity of the dye (at retention time of 6.7 min)
disappears along the degradation and other peaks corresponding to the photodegradation
products (pointed out by the arrows) form. The strategy for selecting the non-target peaks
along the chromatogram is carried out in a semi-automated mode by software, after setting
some parameters, such as approximate LC peak width (potential peaks that are less than 10%
of this width are assumed to be noise and are not selected), background subtraction, minimum
intensity threshold (the signals higher than the threshold are taken into account), and so on,
depending on the software used for reprocessing the acquired data [24].
In addition, when all these conditions are satisfied, the software is able to compare the
intensity ratio of two chromatographic profiles, considering as significant peaks only those
that exceed a settled threshold value [24]. In this way only the significant differences between
the two chromatograms (sample vs control) are evidenced and all the significant peaks so
found are automatically included by the software in a list and successively extracted from the
total ion current. A fundamental prerequisite is obviously an excellent chromatographic
reproducibility [24].
When using a low-resolution MS analyzer, the only way to propose chemical structures
of unknowns is the concatenation of more MS experiments, such as full scan, enhanced
resolution or zoom scan to evaluate the isotopic cluster with major accuracy, and MS/MS or
MSn, to obtain the widest information.
The fastest and simplest solution to identify unknowns is the correct matching between
experimental spectral data for a detected compound and the one present in a library database.
It is now routine for instrument manufacturers to provide some sort of spectral databases with
ESI-MS/MS instruments. There are also free spectral databases, which became available
online in recent years. Massbank, which hosts both an online database and a free standalone
program contains more than 20,000 MS/MS spectra for thousands of different compounds
[110]. One of the principal strengths of this database is that in addition to presenting MS/MS
data from a number of different instruments at a variety of collision energies, it also provides
merged spectra from a range of collision energies. This enables database searching using
spectra collected at different collisional energies. Human Metabolome Database [111] and
Metlin [112] are examples of other online free available large databases concerning known
metabolites. The proprietary NIST spectral database contains CID spectra for nearly 8,000
chemicals collected using a variety of ESI-MS techniques and instrumentations [113]. Unlike
GC-MS, where the MS spectra are recorded at fixed energy of 70 eV, in CID no such
universal choice of collision energy exists, because the collision energies required to induce
dissociation depends on the ion stability. Moreover, large differences can be observed
between ion trapping instruments capable of sequential multi-stage MS/MS (MSn)
experiments and radio frequency (RF) of quadrupole collision cells in hybrid instrument
configurations such as quadruple/ion trap (QTRAPTM) and quadrupole time of flight (QTOF)
instruments.

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On the contrary, if the acquired spectrum of the unknown species is not found in a
library, a meticulous interpretation of the spectrum is necessary.
In general, the chemical structures are proposed on the basis of i) the molecular mass
identified by the pseudo molecular ion present in the MS spectrum, ii) the isotopic cluster, iii)
the assignment of even or odd number of N atoms corresponding to an even or odd molecular
mass, iv) the interpretation of the MS/MS or MSn analysis, v) the possibility of the species to
ionize both in positive and negative ion modes, vi) the plausibility of the retention time with
respect to the calculated log P. In most cases, these conditions strongly limit the number of
the possible structures [23, 25]. It is in this way that the aromatic amine or amide
functionalities in the chemical structures of the Allura Red AC photodegradation products
were identified by using a hybrid quadrupole-linear ion trap mass spectrometer [25], and the
photodegradation products of Sunset Yellow FCF and Carmoisine contained in a beverage
were identified by using an ion trap analyzer [23].
On the contrary, the possibility to use high-resolution MS analyzers permits to elucidate
the chemical structures with much less uncertainty. Also in this case, in order to propose the
chemical structures of the unknown species, it is necessary to gather together the largest
amount of information; the most used are i) the elemental compositions within the specific
mass tolerance, ii) the abundance of the isotopic cluster, iii) the number of ring-double bonds
(RDB), iv) the use of accurate MS/MS spectrum, from which it is possible to obtain the
elemental composition for each product ion, and v) the plausibility of the retention time when
correlated to the calculated log P of the unknown species. In particular, the interpretation of
high-resolution product ion MS/MS spectrum is of fundamental importance to propose the
chemical structure of the precursor ion. Figure 3 shows a scheme of the main information
useful for the chemical structure elucidation of unknown species. In addition, some software
permit to compare the MS/MS spectrum of the hypothesized structure with an MS/MS
spectrum derived from in silico fragmentation, and they provide, as result, the percentage of
matching or the number of common MS/MS signals [24, 25].
At this regard, two proprietary software packages, Mass Frontier (High-Chem, Thermo
Scientific, San Jose, USA) and ACD Fragmenter (ACD/Labs, Toronto, Canada) can be used.
They offer highly specific identification of detected ions of very high structural diversity
based on the interpretation of thousands of known reactions and fragmentation rules. On the
contrary, an alternative approach for computational spectral prediction is based on the
generation of a list of all possible masses of fragments formed by combinatorial break of the
chemical bonds in a compound along with a computation of the internal energy of each bond.
The break of bonds with lower internal energy is considered more favorable and weighted at
higher ion abundance for the spectral prediction. It is also likely that this approach could be of
limited help in the prediction of product ions resulting from internal rearrangement or
tautomerization prior to dissociation.
However, it is obvious that the identified chemical structures should be confirmed by
using the chemical standard, when commercially available, or complementary techniques
such as Nuclear Magnetic Resonance, when possible.
Howsoever, when the number of samples to compare is high, the approach above
mentioned, based on direct comparison between sample vs control chromatogram, is
unsuitable. This is the case of a study concerning the photodegradation of sixteen different
beverages, all containing carminic acid or E120 dye [26]. Preliminary experiments, performed
by exposing each beverage in its original sealed bottle to both summer sunlight and solar box

LC-MS/MS Identification of Unknown Species in Beverages due to Interactions 231


irradiation (under the conditions most suitable to simulate sunlight), showed that for each
beverage the brilliant red color intensity decreased as a function of the irradiation time [26].
Naked eye observation determined the irradiation times that gave rise to the complete
disappearance of the red color to obtain the uncolored beverage [26]. Since in this study all
the beverages had different compositions, and presumably also the degradation products
formed were different from drink to drink [21-25], it should performed the comparison
between each pair of beverages before and after degradation. This procedure is not only timeconsuming but the comparison of a degraded sample versus a control sample could show the
presence of degradation products that are not formed from carminic acid degradation but from
other ingredients present in the beverages. To overcome the problem, for the identification of
the carminic acid degradation products in the different beverages, a methodology based on the
use of Principal Component Analysis coupled with Discriminant Analysis (PCA-DA) was
employed [26].

Figure 3. Scheme of the main information useful to the chemical structure elucidation of unknown
species.

PCA-DA was then carried out on a dataset formed by 113 samples (16 beverages before
degradation: each irradiation/analysis replicated 3 times; 16 beverages after degradation:
again each irradiation/analysis replicated 3 times; one carminic acid standard solution and 16
standard solution of carminic acid degraded at different times) and 4332 variables
(corresponding to all the different m/z values obtained in the 113 chromatographic runs),
selected after application of an alignment algorithm [26]. Figure 4 shows the scores plot first
PCs of PCA-DA model. The first PC separated the information about non-irradiated (positive
values) and irradiated (negative values) samples, whereas the second PC gave information on
the difference between the 16 irradiated standard solutions (positive values) and the irradiated
beverages (negative values), demonstrating that different mechanisms were involved in the

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photodegradation of carminic acid dye, depending on whether the dye was present in aqueous
solution or in more complex matrices, like beverages. Therefore, taking into account the
loadings located at negative values of PC1, useful information about the m/z of the carminic
acid degradation products was accordingly obtained [26].
The method is independent of the beverage composition and allows the identification of
only the degradation products originating from the carminic acid dye present in each
beverage.

Figure 4. Scores plot of the first PCs of PCA-DA model (dataset constituted by degraded, non-degraded
beverages, and degraded standard solution of carminic acid). The first PC separates the information
about non-irradiated (positive values) and irradiated (negative values) samples, whereas the second PC
gives information about the difference between the 16 irradiated standard solutions (positive values)
and the irradiated beverages (negative values).

CONCLUSION
It is well established that food dyes (and to a minor extent their degradation products) can
interact with one another and with the food constituents to form a plethora of new species. In
some instances, such products have been identified, whereas, in others they are poorly
characterized or remain unknown structures. Moreover, the results obtained have been
interpreted differently for different dyes and it is not possible to predict a general behavior for
the degradation pathway of every dyes [22]. This is likely also due to the fact that the matrix

LC-MS/MS Identification of Unknown Species in Beverages due to Interactions 233


seems often to play an important role: the beverage recipe often exert its effect on the
formation of degradation products that are generally different from those formed in simplified
model solutions prepared to simulate the beverages. These results strongly show that the
identification of the degradation products must be performed on commercial beverages, in
order to take into account the total composition and all the possible interactions. The impact
of those interactions represents the main topic of this chapter, together with their relation with
the human health. Reactive parameters, such as pH, temperature, light, ROS formation,
oxygen and other effects have been investigated. Furthermore, different effects causing toxic
and mutagenic species after degradation have been studied as well as possible synergistic and
antagonistic properties of the different components of food.
Taking into account the large numbers of food additives permitted for use, many more
interactions dye-additives could occur beyond the ones considered up to now in this chapter,
and they represent an open research area, which is of fundamental importance for the
consumer health.

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INDEX
#

6-aminoquinolyl-N-hydroxysuccinimidyl carbamate,
95, 103, 117
9-fluoroenylmethylchloroformate, 95

cacao beans, 94, 102, 114, 118


Canonical variates analyses, 157
capillary electrochromatography (CEC), ix, 89, 92,
126
Capillary Electrophoresis (CE), ix, 89, 90
carcinogenic aromatic amines, xii, 213
Carmoisine, 215, 217, 219, 220, 230, 232
CD-EKC (cyclodextrin electrokinetic
chromatography), 93
chemical interactions, 218, 219
chemometric methods, vii, x, 121, 122, 124, 125
chemometry, 152
chiral analysis, vii, ix, 89, 90, 91, 92, 95, 97, 99, 105,
114
chiral food analysis, ix, 89, 90, 94, 95, 96, 115
chiral selector, ix, 89, 91, 92, 93, 94, 95, 97, 104,
105, 106, 108, 109, 110, 111, 112, 114, 118
chromatographic methods, 4, 21, 122, 236
chromophore, 95, 97, 109, 110, 221
Citric acid, 220
Collision Induced Dissociation, 229
colorant, 215, 216, 220
confirmatory information, 6, 7
constant neutral loss, 19, 23, 27
cross-contaminated feedstuffs, 17, 33
CUPRAC Assay, 72
cyclodextrins, 74, 93, 114, 117, 219

A
ABTS assay(s), 48, 56, 57, 67, 69
accurate mass measurements, viii, 2, 4, 7, 10, 14, 15,
18, 19, 27
accurate MS/MS spectrum, 232
additive interactions, xii, 213, 237
Allura Red, 217, 221, 230, 231, 232, 236
Antioxidant Capacity, v, 37, 57, 61, 62, 72, 84, 85,
131
aromatic aminosulfonate, xii, 213
ascorbic acid, 50, 52, 55, 56, 58, 68, 81, 146, 147,
218, 219, 220, 221, 237
assay, viii, 19, 37, 42, 48, 49, 50, 51, 52, 53, 54, 56,
57, 58, 59, 60, 61, 62, 63, 68, 69, 70, 71, 72, 73,
74, 76, 77, 80, 84, 85, 86, 138, 146, 148, 189,
190, 191, 192, 196, 202, 208, 209
authenticity, ix, x, 89, 112, 121, 122, 123, 132, 181,
182, 184, 185, 186, 191, 192, 196, 198, 203, 208,
210, 211
azo dye, 215, 216, 219, 221, 222, 235, 236, 238, 240

B
beers, 94, 100, 105, 108, 114
Benzophenone, 11
beverages, vii, ix, xii, 35, 47, 53, 58, 65, 77, 90, 91,
102, 109, 111, 112, 142, 146, 147, 184, 189, 191,
202, 213, 214, 215, 216, 219, 221, 223, 232, 233,
234, 235, 237, 240, 242
Brilliant Blue FCF, 220, 238

D
dansyl chloride, 95, 103
data-dependent (DDA), 20, 27, 226, 227, 228
data-dependent acquisition, 226, 228
data-independent (DIA), 20, 21, 27, 226, 227, 228
data-independent acquisition, 226, 227, 228, 241
De novo identification, 229

Index

242

degradation products, xii, 27, 35, 175, 213, 215, 216,


220, 223, 230, 233, 234, 237, 238, 242
degradation reactions, xii, xiii, 213, 214
desorption electrospray ionization, 17
DPPH, ix, x, 37, 46, 48, 50, 52, 56, 58, 59, 62, 67,
68, 69, 70, 77, 83, 84, 136, 137, 139, 142, 145,
146
DPPH Assay, 68, 77, 84
drink, xii, 102, 112, 213, 214, 219, 220, 221, 233,
235
dye discoloration, xii, 213, 221, 222
dyes, vii, xii, 213, 214, 215, 216, 217, 218, 219, 220,
221, 222, 223, 230, 234, 236, 238, 239, 242
dynamic exclusion, 20, 227

fruit juices, 28, 70, 94, 97, 101, 104, 109, 114, 118,
184, 191, 192, 206, 208, 211
fruit juices adulterations, 97, 115
fruits, tea, 94, 114

G
genetically modified organisms, 94, 97, 99, 105, 114,
206
geographical origin, vii, x, 121, 122, 123, 124, 125,
126, 127, 128, 130, 131, 132, 133, 191, 193, 194
gingerols, 19, 23

H
E
electrochemical and capacitively coupled contactless
conductivity, 95
electrokinetic chromatography (EKC), ix, 89, 92
Electron Spin Resonance (ESR) spectroscopy, ix, 65,
67
equivalence of genetically modified organisms, 115

F
false-negative, viii, 2, 4, 5, 10, 11, 12, 20, 22, 26
false-positive, viii, 2, 4, 5, 10, 11, 22, 26
fatty acid profiles, xi, 151, 152, 153, 158, 160, 162,
195
Fatty Acids and Tricyglycerol Compositional Data,
124
fluorescein isothiocyanate, 95, 103
Folin-Ciocalteu, 48, 56, 60, 138
food additives, 2, 214, 215, 217, 218, 219, 235, 236,
237, 238
food authenticity, 111, 115, 181, 182, 209
food components, additives and pesticides, vii, ix,
89, 95, 114
food contaminant, 5, 6, 7, 8, 10, 12, 15, 17, 18, 27,
200
food quality, safety and genuineness, 114
food quality, safety and traceability, 114
food safety, vii, xii, 3, 5, 28, 34, 181, 184, 194, 198,
203, 213, 235
food supplements, 94, 100, 101, 105, 108, 114, 117,
118
Fourier Transform Ion Cyclotron Resonance, 225
FRAP Assay, 71
frozen storage, xi, 151, 152, 153, 160, 161, 162, 163,
165, 166

heat, 3, 39, 43, 54, 109, 216, 218


high-resolution mass spectrometry, viii, xiii, 2, 4, 27,
31, 32, 34, 35, 214, 237
HILIC, 224
Hydroxyl radicals, 223

I
impurities, xii, 79, 109, 213, 216
in silico fragmentation, 232
infant formulas, 94, 100, 106, 108, 114, 117
in-silico fragmentation, 22, 26
interactions, vii, xii, 74, 92, 96, 112, 213, 214, 216,
218, 220, 221, 235, 238
ion trap, 6, 19, 31, 33, 127, 225, 226, 231, 232, 237,
241
ionization suppression, 229
isotopic cluster, 231, 232

L
ligand-exchange compounds, 93, 95
light, xiii, 54, 55, 73, 74, 76, 116, 117, 128, 166,
169, 185, 194, 209, 214, 216, 218, 221, 222, 223,
230, 231, 235, 239, 240
liquid chromatography, vii, viii, xiii, 1, 4, 5, 12, 26,
28, 29, 30, 31, 32, 33, 34, 35, 116, 119, 147, 170,
209, 214, 224, 236, 237, 238, 241
Liquid chromatography coupled to mass
spectrometry, viii, 1, 4, 5

M
macrocyclic antibiotics, 93, 95, 109, 114

Index
mass spectrometry, 7, 10, 12, 17, 24, 26, 29, 30, 31,
32, 33, 34, 35, 95, 114, 117, 119, 125, 127, 128,
198, 202, 206, 210, 211, 224, 229, 230, 236, 237,
238, 241
matrix interferences, 229
MEKC (micellar electrokinetic chromatography), 93
metabolomics, 22, 211
microalgaes, 94, 98, 114
Minced fish, 152
MS/MS, vi, viii, 2, 4, 5, 6, 7, 8, 9, 10, 11, 12, 17, 19,
23, 26, 27, 29, 30, 31, 32, 35, 63, 138, 139, 140,
141, 143, 144, 148, 198, 213, 221, 224, 225, 226,
228, 229, 231, 232, 237, 241
mycotoxins, vii, 1, 2, 5, 12, 30

N
naphthalene-2, 3-dicarboaldehyde, 95
naphthalene-derived compounds, 7, 31
Natural dyes, 215
NBT Assay, 74
non-target analysis, 17, 18, 19, 26, 27, 224, 225, 229

O
off-line sample treatments or in-capillary preconcentration, 95
Olive oil, 122, 128, 132, 137, 184, 195
omega-3 fatty acids polyunsaturated fatty acids
(PUFA), 152
ORAC Assay, 73
orbitrap, 10, 12, 14, 15, 16, 18, 19, 32, 34, 35, 147,
198, 225, 226, 227
organic acids, x, 2, 56, 77, 89, 90, 91, 94, 96, 109,
110, 114, 171
oxidative damage, vii, viii, 37, 38, 67, 73, 122, 146,
222

P
PCA coupled with discriminant analysis, 224
pesticides, vii, x, 1, 2, 3, 4, 5, 6, 7, 8, 9, 12, 15, 27,
28, 29, 30, 31, 32, 33, 35, 38, 89, 90, 91, 94, 96,
103, 112, 113, 114, 118, 119, 168, 172, 175
phenolic compounds, v, vii, x, 39, 41, 43, 44, 56, 60,
61, 63, 89, 90, 91, 94, 96, 111, 114, 122, 123,
126, 127, 131, 132, 133, 135, 136, 137, 138, 143,
146, 147, 148
photodegradation products, 231, 232, 236
precursor ion, 6, 9, 10, 17, 19, 20, 225, 226, 227,
228, 232
Principal Component Analysis, 224, 233

243

principal components analysis, x, 121, 157, 158


protein and non-protein amino acids, x, 89, 91, 94,
96, 114

Q
QuEChERS, viii, 1, 4, 15, 29, 30

R
reversed energy ramp, 9
ring-double bonds, 232

S
screening methods, 30, 33, 224
simulate sunlight, 223, 233
soymilk, 94, 100, 105, 114
spectral libraries, 22, 25, 26
Sterols, 126, 129
structure elucidation, 229, 232, 233
sunlight irradiation, xii, 213, 239
Sunset Yellow FCF, 215, 216, 220, 232, 236
survey scan, 226
sweeteners, x, 89, 90, 91, 94, 96, 112, 114, 193, 201,
218, 219, 223, 237
synthetic food dyes, 215, 217, 236

T
tandem mass spectrometry, xiii, 5, 28, 29, 30, 31, 32,
33, 34, 35, 103, 118, 119, 148, 214, 224, 236, 241
tap water, 94, 102, 103, 113, 114
TEAC, 48, 50, 52, 56, 57, 70, 71, 85
the effects of processing and storage, 111, 115
thermal treatments and fermentation processes, 115
time-of-flight, 6, 10, 30, 33, 127, 148, 206, 225, 236
titanium dioxide, 222, 239
Triazines, vi, 167, 168
triple quadrupole, viii, 2, 5, 7, 8, 10, 11, 12, 19, 26,
30, 225, 237
Trout, 173

U
UHPLC, viii, 1, 4, 5, 7, 10, 12, 14, 15, 16, 29, 32, 34,
35, 224, 226
ultrahigh performance liquid chromatography, 4
undesirable compounds, 215
unknown analytes, 224

Index

244
unknown species, xii, xiii, 213, 214, 218, 229, 231,
232, 233
unknowns, 20, 24, 34, 224, 225, 229, 230, 231
unpredictable reactions, xii, 214
UV and laser-induced fluorescence, 95
UV ink photoinitiators, 11, 29
UV radiation, 201, 223

V
veterinary drugs, 2, 5, 12, 14, 15, 17, 18, 33, 34, 35
vinegars, 94, 97, 99, 108, 114, 116

Volatile Compounds Analysis, 125

W
wine, 56, 86, 94, 97, 100, 105, 106, 107, 108, 110,
114, 117, 184, 204

-carotene bleaching assay, 76, 85