X

GRANADA UNIVERSITY
FACULTY OF SCIENCE
Department of Analytical Chemistry
Research Group FQM-297 Environmental, Biochemical and Nutritional
Analytical Control
DOCTORAL THESIS
CHARACTERIZATION OF BIOACTIVE COMPUNDS IN FOOD PRODUCTS AND

SUB PRODUCTS USING ADVANCED SEPARATIVES TECHNIQUES
Submitted for the degree of Doctor of Chemistry

by
SALEH M. S. SAWALHA
GRANADA, 2009
Editor: Editorial de la Universidad de Granada
Autor: Saleh M.S. Sawalha
D.L.: GR 2681-2010
ISBN: 978-84-693-2011-2
This doctoral thesis has been conduced through a pre-doctoral fellowship
granted by the Spanish Agency of International Cooperation (AECI) and
financing from funds of the group FQM-297 Environmental, Biochemical
and Nutritional Analytical Control from different projects and contracts
coming from the Spanish Ministry of Education and Science and Andalusia
Regional Government.
3
CHARACTERIZATION OF BIOACTIVE COMPUNDS IN FOOD PRODUCTS AND SUB
PRODUCTS USING ADVANCED SEPARATIVES TECHNIQUES
by
SALEH M. S. SAWALHA
Granada, November 2009
Signed: Dr. Alberto Fernndez Gutirrez

Professor of the Department of Analytical Chemistry
Faculty of Sciences. University of Granada
Signed: Dr. Antonio Segura Carretero

Professor of the Department of Analytical Chemistry
Signed: Dr. David Arrez Romn

Post-doctoral researcher of the Department of Analytical Chemistry
Research work submitted to get the Doctor in chemistry degree
Signed: Saleh M. S. Sawalha
5
D. ALBERTO FERNNDEZ GUTIRREZ, Professor, Department of Analytical
Chemistry, Faculty of Sciences of the Granada University and Head of
Research Group FQM-297 Environmental, Biochemical and Nutritional
Analytical Control.
CERTIFY:
That the work presented in this DOCTORAL THESIS with the title
CHARACTERIZATION OF BIOACTIVE COMPOUNDS IN FOOD PRODUCTS AND SUB
PRODUCTS USING ADVANCED SEPARATIVES TECHNIQUES, have been
developed under my direction and of the doctors Dr. Antonio Segura Carretero
and Dr. David Arrez Romn in the laboratories of the Department of
Analytical Chemistry and Research Group FQM-297 and shows all requirements
for eligibility to the Degree of Doctor in Chemistry.
In Granada, first of December of two thousand and nine.
7
Acknowledgments
9
Acknowledgments
Acknowledgments
This study was carried out at the University of Granada, Department of

Analytical Chemistry, into the research group FQM-297 Environmental,
biochemical and nutritional analytical-control.
I wish to express my deepest gratitude to my two principal supervisors Dr.

Alberto Fernndez Gutirrez and Dr. Antonio Segura Carretero, for them
encouragement to start this work and for the opportunity to be a member
of the inspiring research group. Them endless support and constructive
criticism have been precious during these years. I am greatly indebted to
my third supervisor Dr. David Arrez Romn. I thank David for his
continuous support during my Ph.D. studies.
Also to all of my colleagues and friends in the research group (FQM-297)
deserve warm thanks, for making my work easier during these years, for
giving hand in solving problems, and for providing a pleasant working
atmosphere.
My warmest thanks belong to my parents (Abu al Amin and Om Al amin)

for their confidence in me and for being always so supportive and
interested in my work and well-being.
Finally, my dearest thanks are addressed to my family, my wife Athar for

her love and tireless support, and our wonderful and active son
Mohammed Al Habib for being the sunshine of my life.
Saleh, December 2009
11
Table of contents
13
Table of contents
Table of contents
Objectives 17
Introduction 21
1. Functional food 23
1.1. Bioactive compounds 29
1.2. Phenolic compounds 30

1.2.1. Phenolic acids 33
1.2.2. Flavonoids 34
1.2.3. Lignans 38
1.2.4. Stilbenes. 40
2. Analytical determination of polyphenols in food sample 42
2.1. Introduction 42
2.2. Sample preparation 43
2.2.1. Liquid extraction (LE) 44
2.2.2. Solid-phase extraction (SPE) 45
2.3. Analytical techniques 46
2.3.1. Liquid Chromatography (LC) 46
A) Instrumentation LC system 47
B) Types of LC 48
2.3.2. Capillary Electrophoresis (CE) 50
2.3.3. Mass Spectrometry (MS) 53
2.3.3.1. Mass Analyzer 54
A. Ion-trap (IT) 54
b. Time-of-Flight (TOF) 55
2.3.3.2. Ion source 57
2.3.3.3. The Interfaces for coupling CE/MS and LC/MS 59
15
Table of contents
A) Coupling of LC/MS 59
B) Coupling of CE-MS 61
2.4. Phenolic compounds by HPLC and CE 64
2.4.1. Phenolic compounds by HPLC 64

2.4.2. Phenolic compounds by CE 66
3. Samples: Importance, main phenolic compounds and health properties 70

3.1. Orange skin 70
3.2. Diatomaceous earth using in olive oil industry 72
3.3. Olive leaves 77
3.4. Almond skin 79
3.5. Flaxseed oil 81
Experimental Part, Results and dissection 85
Chapter I Quantification of main phenolic compounds in sweet and bitter

Orange peel using CEMS/MS 87
Chapter II Characterization of phenolic compounds in diatomaceous earth used

in the filtration process of olive oil by HPLC-ESI-TOF (MS) 97
Chapter III Identification of phenolic compounds in olive leaves using CE-ESI-

TOF-MS 104
Chapter IV HPLC/CE-ESI-TOF (MS) methods for the characterization of

polyphenols in almond skin extracts 111
Chapter V Characterization of phenolic and other polar compounds in Flaxseed

oil using HPLC-ESI-TOF (MS) 137
Conclusions 159
Conclusiones 164
Abstract 170
Resumen 175
16
Objectives
17
Objective
Objective of PHD Thesis:
Functional foods are those that provide some health benefits, for this reason the
chemical characterization of its bioactive compounds is very important. Among the
bioactive compounds are phenolic. These compounds have great interest due to its
antioxidant properties, chemo preventive effect in humans, influence on the
oxidation stability that presented food and effect in the organoleptic properties. On
other hand, food processing industries create large quantities of by-products and
some plant material wastes from these industries can contain high levels of phenolic
compounds and the isolation of these bioactive compounds from these by-products
can be of interest to the food industry.
For this reason, the aim of the present PhD thesis is to characterize the phenolic
composition from different by-product generated by the food industry, such as
orange skin, olive leaves, diatomaceous earth used in the filtration process of olive
oil and almond skin and one product such as flaxseed oil. To carry out the chemical
characterization, the use of advanced analytical techniques to develop rapid, robust
and reliable methods for the determination of these compounds is proposed. The
combination of separative techniques such as capillary electrophoresis (CE) or high
performance liquid chromatography (HPLC) coupled to mass spectrometry (MS)
detectors such as time-of-flight (TOF) and ion-trap (IT) permits the development of
potent analytical methods to carry out a detailed characterization of phenolic
compounds in the different samples selected.
19

Introduction
21
Functional food
1. Functional food.
Traditionally, the healthiness of food has been linked to a nutritionally healthy diet
recommended by nutrition specialists and the role of diet as a whole has been
emphasised instead of emphasising individual food items. Lately, new kinds of foods,
so-called functional foods, have been developed and launched. They provide a novel
approach to the idea of healthy eating by linking a single component with a certain
health effect in a single product1.
Conventionally, food healthiness has been associated with nutritional factors such as
fat, fibre, salt and vitamin content. In addition to this conventional or traditional
healthiness, food may contain single components that may have a positive impact on
our well-being1. Products that are claimed to have special beneficial physiological
effects in the body have been called nutraceuticals, pharma foods, designer foods,
nutritional foods, medical foods or super foods 2 . More usually they are named as
functional foods.
The concept of functional foods is often considered to have emerged in Japan in the
late 1980s. However, functional foods actually have a quite long history. Belief in the
medicine power of foods is not a recent event but has been a widely accepted
philosophy for generations. Although Hippocrates may not have started the functional
foods movement, he stated Let food be the medicine and medicine be the food3.
The realization that attention to diet as part of a healthy lifestyle can reduce
considerably the risk of disease and promote health has created a lucrative market
for a whole range of new products called functional foods, nutraceuticals, etc...
Nutraceuticals are natural, bioactive chemical compounds that are characterized by

health promoting, disease-preventing and medicinal properties. The scope of
nutraceuticals is substantially different from that of functional foods. Although the
prevention and treatment of disease (i.e. medical claims) are related to
nutraceuticals, only the reduction of disease is involved with functional foods. In
contrast to nutraceuticals, including dietary supplement as well as other type of
foods, functional foods are expected to be in the form of ordinary food4. Dietary
supplement stands for a food, not in its conventional form, providing a component
1. Lhteenmki, L. (2003). Consumers and Functional Foods. In: T. Mattila-Sandholm & M. Saarela
(Eds.). Functional Dairy Products. Cambridge: Woodhead Publication Ltd.
2. Childs, N.M., Poryzees, G.H. (1998). Foods that help prevent disease: consumer attitudes and
public policy implications. British Food Journal, 9, 419.426.
3. Milner, J.A. (1999). Functional Foods and Health Promotion, Journal of Nutrition, 129:1395S1397S.
4. Arvanitoyannis I.S., Van Houwelingen-Koukaliaroglou M. (2005). Functional Foods: A Survey of
Health Claims, Pros and Cons, and Current Legislation, Critical Reviews in Food Science and
Nutrition, 45:385404.
23
Introduction
to supplement the diet by increasing the total dietary intake of that component.
The term functional food is surfacing as a generic descriptor of the benefits that
accompany ingesting foods that go beyond those accounted for merely by the
nutritive provided (Milner 1998)5.
As a result of a long decision-making process to establish a category of foods for

potential enhancing benefits as part of a national effort to reduce the escalating cost
of health care, the concept of foods for specified health use (FOSHU) was established
in 1991.
In the 1994 the Institute of Medicine of the National Academy of Sciences has
expanded this definition to include any food or food ingredient that may provide a
health benefit beyond the traditional nutrients it contains3.
The target of functional foods is seen as clearly different from that of drugs, which
are aimed at preventing or curing diseases.
Functional foods have been broadly defined as foods similar in appearance to

conventional foods that are consumed as part of a normal diet and have
demonstrated physiological benefits and/or reduce the risk of chronic disease beyond
basic nutritional functions6. In 2006 several authors, such as Spence7 and Kotilainen
and co-worker8, have reported the prominent types of functional foods:
Fortified product. A food fortified with additional nutrients.
Enriched products. A food with added new nutrients or components not

normally found in a particular food.
Altered products. A food, from which a deleterious component has been

removed, reduced or replaced with another substance with beneficial
effects.
Enhanced commodities. A food in which one of the components has been

naturally enhanced through special growing conditions, new feed
composition, genetic manipulation, or otherwise.
5. Milner J.A. (1998). Do functional foods offer opportunities to optimize nutrition and health?
Food Technology, 52: 24.
6. Clydesdale, F.M. 1997. A proposal for the establishment of scientific criteria for health claims for
functional foods. Nutr. Rev., 55:413422.
7. Spence, J.T. (2006). Challenges related to the composition of functional foods. Journal of Food
Composition and Analysis, 19: S4S6.
8. Kotilainen L., Rajalahti R., Ragasa C., Pehu E. (2006). Health enhancing foods: Opportunities for
strengthening the sector in developing countries. Agriculture and Rural Development Discussion
Paper 30.
24
Functional food
Research on food and nutrition has been an important topic in the EU Framework
Programmes for Research and Technology Development of the European Commission9.
In the 1990s a significant number of EU projects addressed issues such as fibres and
pro- and prebiotics, whereas more recent EU programmes focus on areas such as
antioxidants, vitamins and phytoestrogens, as well as the socio-economic aspects of
nutrition and health 10 . With regard to biological benefits in functional foods, the
International Life Sciences Institutes concerted action on Functional Food Science in
Europe (FUFOSE) has proposed six broad groups that are considered relevant from a
scientific perspective. These are growth, development and differentiation; substrate
metabolism; defence against reactive oxidative species; the cardiovascular system;
gastrointestinal physiology and function; and behaviour and psychological functions11.
In the United States, functional attributes can be communicated through health

claims, structurefunction claims, and nutrient content claims. The Food and Drug
Administration must approve health claims that describe the relationship between a
food component and a disease or health-related condition. The approval of claims
has been based on an extensive review of existing scientific literature, in the form of
an authoritative statement of a scientific body of the US government or the National
Academy of Sciences. Nutrient content and structurefunction claims are clearly
defined in the regulations and do not need to be approved by the Food and Drug
Administration12. The Codex Alimentarius is of great importance for world trade and,
although advisory, has defined three types of health claims (Table 1): nutrient
function claims; enhanced function claims and reduction of disease risk13. At present
there are no Europe-wide regulations in place to regulate health claims; this includes
not only European Union directives but also domestic legislations of the member
states. The scientific concepts of the European Community Concerted Action on
Functional Food Science (FUFOSE), which has been coordinated by the International
Life Science Institute Europe, defined the same nutrient function claims as that of
the Codex Alimentarius.
9. Lucas, J. (2002). EU-funded research on functional foods, British Journal of Nutrition, 88, Suppl. 2:
S131 S132.
10. Verschuren P.M. (2002). Functional Foods: Scientific and Global Perspectives, British Journal of
Nutrition, 88, Suppl. 2: S125S130.
11. Weststrate J.A., van Poppel G., Verschuren P.M. (2002). Functional foods, trends and future,
British Journal of Nutrition, 88, Suppl. 2:S233S235
12. Milner J.A. (2002). Functional foods and health: a US perspective, British Journal of Nutrition, 88,
Suppl. 2: S151S158.
13. Shimizu T. (2003). Health claims on functional foods: the Japanese regulations and an
international comparison, Nutrition Research Reviews, 16: 241252.
25
Introduction
Table 1. Codex Alimentarius Definitions
Term Definition
Functional Food that has physiological functions, including regulation of

food biorhythms, the nervous system, the immune system, and bodily defence
beyond nutrient functions, as defined by the Japanese ad hoc national
project in 1984.
Health claims Presentation that states, suggests, or implies that a relationship exists
between a food or the constituents of a food and health. Health claims
include nutrientfunction claims, enhanced function claims, and
reduction of disease risk claims. This definition is the same as that
included in the Proposed Draft Guidelines for Use of Health and Nutrition
Claims of the Codex Alimentarius in 1999 (Codex Alimentarius
Committee on Food Labelling 28 Session).
Generic Claims based on well-established, generally accepted knowledge derived

from evidence in the scientific literature and/or on recommendations
health claims
from national or international public health bodies.
Product- Claims that concern certain physiological effects other than a generic
specific claims health claim, which requires demonstrations based on scientific
evidence for individual products.
Enhanced Claims that concern specific beneficial effects regarding the

function claims consumption of foods and their constituents in the context of the total
diet regarding physical or psychological functions or biological activities
but that do not include nutrient function claims.
Structure/ Any statements regarding the effects of dietary supplementation on the

structure or function of the body, that is defined by the Dietary
function claims
Supplement, Health and Education Act in the USA in 1994. These claims
are generally similar to the enhanced function (or other) claims.
Dietary A product intended to supplement the diet, which contains one or more
supplement of dietary ingredients such as vitamins, minerals, amino acids, etc,
which is in a dosage form such as capsules, tablets, etc.
Though an official definition of functional foods is lacking in both the US 14 (ADA

Reports, 2004) and Europe 15 (ILSI Europe, 2002), the influence of the Japanese
legislation on EU and US views of functional foods is apparent. According to an EU
14. ADA Reports (2004) Position of the American Dietetic Association: Functional foods. Journal of the
American Dietetic Association, 104, 814.826.
15. ILSI Europe (2002) Concepts of functional foods. ILSI Europe Concise Monograph Series. Belgium.
26
Functional food
concerted action project FUFOSE (Functional Food Science in Europe) coordinated by

ILSI (International Life Sciences Institute),
"a food can be regarded as functional if it has been satisfactorily
demonstrated to affect beneficially one or more target functions in
the body beyond adequate nutritional effects in a way that is
relevant to either an improved state of health and well-being and/or
a reduction of risk of disease".
Besides providing scientifically proven health effects, functional foods have to

maintain a food-like nature and they have to be easily incorporated into the daily
diet:
"a functional food must remain food and it must demonstrates its
effects in amounts that can normally be expected to be consumed in
the diet: it is not a pill or a capsule, but part of the normal food
pattern16".
The unique features of a functional food are17,18: "A conventional or everyday food,
consumed as part of the normal/usual diet, composed of naturally occurring (as
opposed to synthetic) components, perhaps in unnatural concentrations or present in
foods that would not normally supply them, having a positive effect on target
function(s) beyond nutritive value/basic nutrition, that may enhance well-being and
health and/or reduce the risk of disease or provide health benefit so as to improve
the quality of life including physical, psychological and behavioural performances and
have authorized and scientifically based claims.
A functional food component can be a macronutrient if it has specific physiologic

effects (eg, resistant starch or n-3 fatty acids) or an essential micronutrient if its
intake is more than the daily recommendations. It can also be a food component that,
even though of some nutritive value, is not essential (eg, some oligosaccharides) or is
even of no nutritive value (eg, live microorganisms or plant chemicals). Indeed,
beyond its nutritional (metabolic requirements) value and function of providing
pleasure, a diet provides consumers with components able to both modulate body
16. Diplock, A.T., Agget, P.J., Ashwell, M., Bornet, F., Fern, E.B. & Roberfroid, M.B. (1999) Scientific
concepts of functional foods in Europe: Consensus Document. British Journal of Nutrition, 81, 1.27.
17. Bellisle F., Diplock A.T., Hornstra G., Koletzko B., Roberfroid M., Salminen S., Saris W.H.M. (1998).
Functional food science in Europe, British Journal of Nutrition, 80, Suppl. 1: S1S193.
18. Knorr D. (1998). Functional food science in Europe, Trends in Food Science and Technology, 9:
295340.
27
Introduction
functions and reduce the risk of some diseases 19 . The International Life Sciences
Institute of North America (ILSI) has defined functional foods as foods that by virtue
of physiologically active food components provide health benefits beyond basic
nutrition. Health Canada defines functional foods as similar in appearance to a
conventional food, consumed as part of the usual diet, with demonstrated
physiological benefits, and/or to reduce the risk of chronic disease beyond basic
nutritional function. Most early developments of functional foods were those of
fortified with vitamins and/or minerals such as vitamin C, vitamin E, folic acid, zinc,
iron, and calcium. Subsequently, the focus shifted to foods fortified with various
micronutrients such as omega-3 fatty acid, phytosterol, and soluble fibre to promote
good health or to prevent diseases such as cancer20. More recently, food companies
have taken further steps to develop food products that offer multiple health benefits
in a single food 21 . Schematically speaking, the combination of "market pull. and
"science push. in functional foods research will result in a research funnel starting
from consumer needs and narrowing down to the final functional foods products by
the following stepwise approach:
1. Consumer understanding: what kind of health benefits in foods or
technology solutions do consumers really want?
2. Bio-informatics: what molecules could do the job?
3. In cursive screening testing: which molecules work best in model systems?
4. Bioavailability: are the bioactive compounds digested and absorbed?
5. Functional food technology: can we source the ingredient and make an

attractive food?
6. Biomarkers: can we measure relevant effects in human?
7. Human intervention studies: does it really work?
8. Communication: how do we explain the benefits?
Briefly, the functional foods are endowed with specific physiological benefits that
discriminate them from traditional foods. The functionality of functional foods is
derived from bioactive ingredients and depends on several technological factors.
Bioactive ingredients in functional foods may, e.g., help in the prevention of (chronic)
diseases or the enhancement of performance and well-being of the individual beyond
19. Roberfroid M.B. (2000). Concepts and strategy of functional food science: the European
perspective, The American Journal of Clinical Nutrition; 71(suppl):1660S1664S
20. Sloan A.E. (2000). The top ten functional food trends. Food Technology, 54, 3362
21. Sloan A.E. (2004). The top ten functional food trends. Food Technology, 58, 2851
28
Functional food
their established role in nutritional function. Bioactive ingredients may, therefore,

be considered as potentially health enhancing components of our diet.
1.1. Bioactive compounds.
The interest in functional foods continues to grow, powered by progressive research

efforts to identify properties and potential applications of bioactive substances, and
coupled with public interest and consumer demand. In the past decade, substantial
progress has been made concerning our knowledge of bioactive components in plant
foods and their links to health. Human diets of plant origin contain many hundreds of
compounds which cannot be considered as nutrients, but appear to play a role in the
maintenance of health22. Evidence for the existence of bioactive compounds is based
primarily on observational studies that demonstrate the beneficial effects of certain
dietary patterns that include vegetarianism, high whole-grain consumption, the
prudent diet, the Mediterranean diet, and the traditional Japanese diet. The
traditional Japanese diet has a high content of soybean products and vegetables. The
Mediterranean diet has a high content of olive oil, fruits and vegetables, and whole-
grain breads. The prudent diet is characterized by high intakes of fruits and
vegetables, fish, poultry, whole-grain products, and legumes 23 . Many of the
characteristic components of the traditional Mediterranean diet are known to have
positive effects on health, capacity and well-being, and can be used to design
functional foods. Vegetables, fruits and nuts are all rich in flavonoids, isoflavonoids,
phytosterols and essential bioactive compounds providing health benefits. The
polyunsaturated fatty acids found in fish effectively regulate haemostatic factors,
protect against cardiac arrhythmias, cancer and hypertension, and play a vital role in
the maintenance of neural functions and the prevention of certain psychiatric
disorders.
Bioactive components include a range of chemical compounds with varying structures

such as carotenoids, flavonoids, phytosterols, omega-3 fatty acids (n-3), allyl and
diallyl sulfides, indoles (benzopyrroles), and polyphenols (Figure.1). Data and
databases on the levels of bioactive components in foods are needed so that
22. Orzechowski A., Ostaszewski P., Jank M., Berwid S.J. (2002). Bioactive substances of plant origin
in food impact on genomics, Reproduction Nutrition Development, 42: 461477.
23. Kris-Etherton P.M., Lefevre M., Beecher G.R., Gross M.D., Keen C.L., Etherton T.D. (2004).
Bioactive compounds in nutrition and health-research methodologies for establishing biological
function: The antioxidant and anti-inflammatory effects of flavonoids on atherosclerosis. Annual
Review of Nutrition, 24: 511538.
29
Introduction
researchers may accurately assess their dietary intake, investigate their physiological
functions, and determine their relationships to health and disease24.
Bioactive compounds
Isoprenoids Phenolic Protein/ Carbohydrate Lipidic Minerals Microbial

(Terpenoids) compound Amino Acid & derivatives compounds
Carotenoids Phenolic acids Amino acids Ascorbic acid n-3 PUFA Ca Probiotics
Saponins Flavonoids Allyl -S-Compds Oligosaccharides CLA Se
Tocotrienols Secoiridoids Capsaicinoids Non starch PS MUFA K
Tocopherols Lignin Isothiocyanates Sphingolipids Cu
Simple terpenes Coumarins Indoles Lecithin Zn
Tannins Folate Sterols
Choline
Figure 1: Some bioactive compounds in foods
1.2. Phenolic compounds.
Polyphenolic occur throughout foods of plant origin with over 4000 different
structures identified. They have been shown to have a range of health related effects
including anti-oxidant, anti-viral, anti-allergic, anti-inflammatory anti-proliferative
and anticarcinogenic. Most interest has centred on a possible role in cancer and
heart disease but recently their role in brain functions such as learning and memory
have received attention with a number of studies being undertaken with herbals such
as ginko and ginseng. Other polyphenols such as epicatechin and catechin (found in
tea) have all been shown to have some beneficial effects in animal models.
In broad terms the polyphenols are important for:
Their antioxidant properties, i.e. their ability to scavenge naturally

occurring free radicals before they can damage macromolecules
directly or indirectly involved in either cell proliferation (relevant to
carcinogenesis) or lipid metabolism (relevant to cardiovascular
disease).
24. Pennington J.A.T. (2002). Food composition databases for bioactive food components, Journal of
Food Composition and Analysis, 15: 419434.
30
Functional food
Blocking the formation of carcinogenic nitrosamines arising from the

reaction of dietary nitrates/nitrites with secondary amines and amides
in the stomach.
Their capacity to act as electrophile traps. In much the same manner

in which they can scavenge nucleophilic free radicals, many plant
phenols can also absorb highly reactive electrophiles thereby
preventing damage to cellular components
Inhibiting the generation of prostaglandins from arachidonic acid, and

thereby retarding a promotional phase of carcinogenesis.
The term plant phenols encompasses a wide variety of naturally occurring compounds
which are structurally related to the extent that they all contain one or more
benzene rings each with one or more hydroxyl group substitutions.
Several thousand different polyphenols exist and can be subdivided into different
subclasses. Polyphenols represent awide variety of compounds,which are divided into
several classes, ie, hydroxybenzoic acids, hydroxycinnamic acids, anthocyanins,
proanthocyanidins, flavonols, flavones, flavanols, flavanones, isoflavones, stilbenes,
and lignans. The main subclasses that are important from a human health
perspective are the phenolic acid, flavones, flavonols, flavan-3-ols, isoflavones,
flavanones, anthocyanidins and lignans 25 , 26 (Figure 2). Distinctions are thus made
between the phenolic acids (hydroxybenzoic acids and hydroxycinnamic acids),
flavonoids, stilbenes, and lignans (Figure 3).
25. Hooper L., Cassidy A. (2006). A review of the health care potential of bioactive compounds.
Journal of the Science of Food and Agriculture, 86:18051813.
26. Manach, C. et al., (2004), Polyphenols: food sources and bioavailability, Am. J. Clin. Nutr., 79,
727.
31
Introduction
Figure 2: classification scheme for polyphenols
Figure 3: Chemical structures of major classes of polyphenols.
32
Functional food
1.2.1. Phenolic acids.
Phenolic acids can be distinguished into two main classes: derivatives of benzoic acid
and derivatives of cinnamic acid. The hydroxybenzoic acid content of edible plants is
generally very low, with the exception of certain red fruits, black radish, and onions,
which can have concentrations of several tens of milligrams per kilogram fresh
weight27. Tea leaves are an important source of gallic acid: they may contain up to
4.5 g/kg fresh weight 28 . Additionally, hydroxybenzoic acids are components of
complex structures such as hydrolyzable tannins (gallotannins in mangoes and
ellagitannins in red fruit such as strawberries, raspberries, and blackberries) 29 .
Because these hydroxybenzoic acids, both free and esterified, are found in only a
few plants eaten by humans, they have not been extensively studied and are not
currently considered to be of great nutritional interest.
The occurrences of hydroxycinnamic acids in human food are more common than
hydroxybenzoic acids and consist mainly of p-oumaric, caffeic and ferulic acids.
These acids are rarely found in the free form, except in processed food that has
undergone freezing, sterilization, or fermentation26. The types of fruit having the
highest concentrations (blueberries, kiwis, plums, cherries, apples) contain 0.52 g
hydroxycinnamic acids/kg fresh weight30. p-Coumaric acid can be found in a wide
variety of edible plants such as peanuts, tomatoes, carrots, and garlic. It has
antioxidant properties and is believed to lower the risk of stomach cancer by
reducing the formation of carcinogenic nitrosamines31,32.
Caffeic acid frequently occurs in fruits, grains and vegetables as simple esters with
quinic acid (forming chlorogenic acid) or saccharides, and are also found in
traditional Chinese herbs33.
Chlorogenic acid is found in particularly high concentrations in coffee: the green

coffee beans typically contain 6-7% of this component (range: 4-10%) and a cup of
instant coffee (200 ml) contains 50150 mg of chlorogenic acid34.
27. Shahidi, F. and Naczk, M., (1995). Food phenolics, sources, chemistry, effects, applications,
TechnomicPublishing Co Inc, Lancaster, PA,
28. Tomas-Barberan, F.A., Clifford, M.N. (2000). Dietary hydroxybenzoic acid derivatives and their
possible role in health protection, J. Sci. Food Agric., 80, 1024.
29. Clifford, M.N. and Scalbert, A. (2000). Ellagitanninsoccurrence in food, bioavailability and cancer
prevention, J. Food Sci. Agric., 80, 1118,.
30. Macheix, J-J., Fleuriet, A. and Billot, J. (1990).Fruit phenolics, CRC Press, Boca Raton, FL,.
31. Ferguson, L.R., Zhu, S. and Philip, H.J. (2005). Antioxidant and antigenotoxic effects of plant cell
wall hydroxycinnamic acids in cultured HT-29 cells, Mol. Nutr.Food Res., 49, 585.
32. Kikugawa, K. et al. (1983). Reaction of p-hydroxycinnamic acid derivatives with nitrite and its
relevance to nitrosamine formation, J. Agric. Food Chem., 31, 780.
33. Jiang, R.W. et al.(2005). Chemistry and biological activities of caffeic acid derivatives from Salvia
miltiorrhiza, Curr. Med. Chem., 12, 237.
33
Introduction
This compound, long known as an antioxidant, also slows the release of glucose into
the blood stream after a meal35 . Ferulic acid is the most abundant phenolic acid
found in cereal grains. The main food source of ferulic acid is wheat bran (5 g/kg)
and it may represent up to 90% of total polyphenols 36,37 . As ferulic acid is found
predominantly in the outer parts of the grain, the ferulic acid content of different
wheat flours is directly related to levels of sieving38 . Rice and oat flours contain
approximately the same quantity of phenolic acids as wheat flour (63 mg/kg),
although the content in maize flour is about 3 times as high.
1.2.2. Flavonoids.
Flavonoids are a widely distributed group of polyphenolics, which have been reported
to act as antioxidants in various biological systems. They are particularly abundant in
citrus plants. Four types of flavonoids (flavanones, flavones, flavanols and
anthocyanins) occur in citrus and more than 60 individual flavonoids have been
identified. Flavanone glycosides and the polymethoxylated flavones are two
flavonoid compound families. The common citrus glycosides include narirutin,
naringin, hesperidin, neohesperidin, didymin and poncirin and the common citrus
polymethoxylated flavones include sinessetin, hexamethoxyflavone, nobiletin,
scutellarein, heptamethoxyflavone and tangeretin.
Flavanones are the most abundant. The highly methoxylated flavones have higher
biological activity even if in lower concentrations. The antioxidant properties of
these substances give them anticancer, antiviral and antiinflammatory capabilities.
They can also affect capillary fragility and platelet aggregation39,40.
The antioxidant activity can express itself as:
Antiradical activity
Antilipoperoxidant activity
34. Clifford, M,N., (1999) Chlorogenic acids and other cinnamatesnature, occurence and dietary
burden, J. Sci. Food. Agric., 79, 362.
35. Hemmerle, H. et al. 1997, Chlorogenic Acid and Synthetic Chlorogenic Acid Derivatives: Novel
Inhibitors of Hepatic Glucose-6-phosphate Translocase J. Med. Chem., 40, 137.
36. Kroon, P. A. et al. (1997), Release of covalently bound ferulic acid from fiber in the human colon,
J. Agric. Food Chem., 45, 661.
37. Lempereur, I., Rouau, X. and Abecassis, J.(1997).Genetic and agronomic variation in arabinoxylan
and ferulic acid contents of durum wheat (Triticum durum L.) grain and its milling fractions, J.
Cereal Sci., 25, 103.
38. Hatcher, D.W. and Kruger, J.E., (1997) Simple phenolic acids in flours prepared from Canadian
wheat: relationship to ash content, color, and polyphenol oxidase activity, Cereal Chem., 74, 337.
39. Hertog M., Feskens E., Hollman P., Katan M., Kromhout D. (1993). Dietary antioxidant flavonoids
and risk of coronary heart disease: the Zutphen elderly study. Lancet, 342:10071011.
40. Linseisen J., Radtke J., Wolfram G. (1997). Flavonoid intake of adults in a Bavarian subgroup of
the national food consumption survey. Z. Ernhrungswiss, 36:403412.
34
Functional food
Antioxygen activity and/or
Metal chelating activity
Flavonoids are polyphenolic compounds sharing a common structure consisting of 2

aromatic rings (A and B) that are bound together by 3 carbon atoms that form an
oxygenated heterocycle (ring C) (Figure 3). They may be divided, according to the
oxidation level of the C ring, into 14 subclasses the most common being flavonols,
flavones, isoflavonoids (isoflavones, coumestans), flavanones, anthocyanidins, and
flavanols (catechins and proanthocyanidins)41,42 (Figure 4).
Figure 4: Chemical structures of flavonoids.
41. Dinelli,G., et al., (2006) Biosynthsis of polyphenol phytoestrogens in plants, in Phytoestrogens in

Functional Foods, Yildiz, F., Ed., CRC Press, Boca Raton, FL, 19.
42. Claudine M.,et el., (2004) Polyphenols: food sources and bioavailability., Am J Clin Nutr;79:727
47.
35
Introduction
Flavonols are the most ubiquitous flavonoids in foods, and the main representatives
are quercetin and kaempferol. The richest sources are onions (up to 1.2 g/kg fresh
weight), curly kale, leeks, broccoli, and blueberries43 .
These flavonols accumulate in the outer and aerial tissues (skin and leaves) because
their biosynthesis is stimulated by light. Marked differences in concentration exist
between pieces of fruit on the same tree and even between different sides of a
single piece of fruit, depending on exposure to sunlight44.
Similarly, in leafy vegetables such as lettuce and cabbage, the glycoside

concentration is 10 times higher in the green outer leaves than in the inner light-
colored leaves45. This phenomenon also accounts for the higher flavonol content of
cherry tomatoes than of standard tomatoes, because they have different proportions
of skin to whole fruit.
Flavones are much less common and were identified in sweet red pepper (luteolin)
and celery (apigenin) 46 Cereals such as millet and wheat contain C-glycosides of
flavones4749 .
Citrus fruits are the main food source of flavanones. The main aglycones are
naringenin in grapefruit, hesperetin in oranges, and eriodictyol in lemons. Flavanones
are generally glycosylated by a disaccharide at position 7, either a neohesperidose,
which imparts a bitter taste (such as to naringin in grapefruit), or a rutinose, which is
flavorless. Orange juice contains between 200 and 19600 mg hesperidin/L and 1585
mg narirutin/L, and a single glass of orange juice may contain between 40 and 140
mg flavanone glycosides 50 . Because the solid parts of citrus fruit, particularly the
albedo (the white spongy portion) and the membranes separating the segments, have
a very high flavanone content, the whole fruit may contain up to 5 times as much as
a glass of orange juice.
43. Manach, C. et al.,(1995) Polyphenols: food sources and bioavailability, Am. J. Clin. Nutr., 79,
727,2004.
44. Price, S.F. et al., Cluster sun exposure and quercetin in Pinot noir grapes and wine, Am. J. Enol.
Vitic., 46, 187.
45. Mojca skerget. et el: (2005), Phenols, proanthocyanidins, flavones and flavonols in some plant
materials and their antioxidant activities , J. Food chemistry., Vol, 89, Issue 2,191-198.
46. Hertog, M.G.L., Hollman, P.C.H. and Katan, M. B.(1992). Content of potentially anticarcinogenic
flavonoids of 28 vegetables and 9 fruits commonly consumed in the Netherlands, J. Agric. Food
Chem., 40, 2379.
47. King, H.G.C., (1962). Phenolic compounds of commercial wheat germ, J. Food Sci., 27, 446.
48. Feng, Y., McDonald, C.E. and Vick, B.A. 1988, C-glycosylflavones from hard red spring wheat bran,
Cereal Chem., 65, 452.
49. Sartelet, H. et al. (1996), Flavonoids extracted from Fonio millet (Digitaria exilis) reveal potent
antithyroid properties, Nutrition,12, 100.
50. Tomas-Barberan, F.A. and Clifford, M.N. (2000), Flavanones, chalcones and dihydrochalcones
nature, occurence and dietary burden, J. Sci. Food Agric., 80, 1073.
36
Functional food
Isoflavonoids are a large and very distinctive subclass of the flavonoids. These
compounds differ structurally from other classes of the flavonoids in having the
phenyl ring (B-ring) attached at the 3-rather than at 2-position of the heterocyclic
ring. In addition, the isoflavonoids differ by their greater structural variation and the
greater frequency of isoprenoid substitution 51 . Isoflavones constitute the largest
group of natural isoflavonoids and the most investigated for their structural
similarities to estrogens. Although they are not steroids, they have hydroxyl groups in
positions 7 and 4 in a configuration analogous to that of the hydroxyls in the estradiol
molecule. This confers them pseudohormonal properties, including the ability to bind
to estrogen receptors, and they are consequently classified as phytoestrogens. The
most interesting compounds with regard to oestrogenicity are genistein, daidzein,
glycitein, biochanin A and formononetin .
Isoflavones are found almost exclusively in leguminous plants. Legumes, particularly

soybean (Glycine max L.) and its processed products, are the richest sources of
isoflavones, mainly genistein, daidzein and glycitein, in the human diet52.
Flavanols exist in both the monomer form (catechins) and the polymer form
(proanthocyanidins). In contrast to other classes of flavonoids, flavanols are not
glycosylated in foods. Catechins are found in many types of fruit, especially in
apricots (250 mg/kg fresh weight). They are also present in red wine (up to 300 mg/L)
and chocolate53,54. However, tea is by far the richest source: young shoots contain
200-340 mg of catechin, gallocatechin and their galloylated derivatives/g of dry
leaves55.
Consumption of flavonoid-rich foods is associated with a lower incidence of heart

disease, ischemic stroke, cancer, and other chronic diseases5659. For example, 7 of
51. Mazur, W. and Adlercreutz, H., (1998) Naturally occurring estrogens in food, Pure Appl. Chem., 70,
1759.
52. Reinli, K. and Block, G., (1996) Phytoestrogen content of foods: a compendium of literature values,
Nutr. Cancer Int. J., 26, 123.
53. Frankel, E. N., Waterhouse, A.L. and Teissedre, P.L., (1995) Principal phenolic phytochemicals in
selected California wines and their antioxidant activity in inhibiting oxidation of human lowdensity
lipoproteins, J. Agric. Food Chem., 43, 890.
54. Arts, I.C., Hollman, P.C. and Kromhout, D., (1999) Chocolate as a source of tea flavonoids. Lancet,
354, 488.
55. Y. Hara, S.J. Luo, R.L. Wickremasinghe and T. Yamanishi , (1995) Special issue on tea. Food Rev.
Int. 11, pp. 371542.
56. Lee, M.-J. et al., (1995) Analysis of plasma and urinary tea polyphenols in human subjects, Cancer
Epidemiol. Biomark. Prev., 4, 393.
57. Verlangieri, A.J. et al., (1985) Fruit and vegetable consumption and cardiovascular mortality, Med.
Hypotheses, 16, 7,.
58. Joshipura, K.J. et al., (1999) Fruit and vegetable intake in relation to risk of ischemic stroke,
JAMA, 282, 1233,.
59. Riboli, E. and Norat, T., (2003) Epidemiologic evidence of the protective effect of fruit and
vegetables on cancer risk, Am. J. Clin. Nutr., 78, 559S.
37
Introduction
12 epidemiological studies evaluating the risk of coronary heart disease reported

protective effects of dietary flavonoids 60 . Additional studies also found inverse
associations between flavonoid intake and the risk of stroke 61 , 62 and lung and
colorectal cancer63,64 . Because these chronic diseases are associated with increased
oxidative stress and flavonoids are strong antioxidants in vitro, it has been suggested
6567
that dietary flavonoids exert health benefits through antioxidant mechanisms .
However, a recent study reported that many of the biological effects of flavonoids
appear to be related to their ability to modulate cell signaling pathways, rather than
their antioxidant activity 68 . Unlike in the controlled conditions of a test tube,
flavonoids are poorly absorbed by the human body (less than 5%), and most of what is
absorbed is quickly metabolized and excreted.
The huge increase in antioxidant capacity of blood seen after the consumption of
flavonoid-rich foods is not caused directly by the flavonoids themselves, but most
likely is due to the fact that the body seen flavonoids as foreign compounds and
through different mechanisms, they could play a role in preventing cancer or heart
disease.
1.2.3. Lignans.
Lignans are polyphenolic compounds derived from the combination of two

phenylpropanoid (C6- C3 units) (Figure 5).
60. Bosetti, C. et al., (2005) Flavonoids and breast cancer risk in Italy, Cancer Epidemiol. Biomarkers
Prev., 14, 805.
61. Arts, I.C. and Hollman, P.C., (2005) Polyphenols and disease risk in epidemiologic studies, Am. J.
Clin. Nutr., 81, 317S.
62. Knekt, P. et al., (2002) Flavonoid intake and risk of chronic diseases, Am. J. Clin. Nutr., 76, 560.
63. Keli, S.O. et al.,(1996) Dietary flavonoids, antioxidant vitamins, and incidence of stroke: the
Zutphen study, Arch. Intern. Med., 156, 637.
64. Hirvonen, T. et al.,(2001) Flavonol and flavone intake and the risk of cancer in male smokers
(Finland), Cancer Causes Control, 12, 789.
65. Arts, I.C. et al.,(2002) Dietary catechins and cancer incidence among postmenopausal women: the
Iowa Women's Health Study (United States), Cancer Causes Control, 13, 373.
66. Aviram, M., Fuhrman, B.,(2002) Wine flavonoids protect against LDL oxidation and atherosclerosis,
Ann. N. Y. Acad. Sci., 957, 146.
67. Rietveld, A. and Wiseman, S.,(2003) Antioxidant effects of tea: evidence from human clinical trials,
J. Nutr., 133, 3285S.
68. Serafini, M.J.A. et al.,(2000) Inhibition of human LDL lipid peroxidation by phenol-rich beverages
and their impact on plasma total antioxidant capacity in humans, J. Nutr. Biochem., 11, 585.
38
Functional food
Figure 5: Structures of plant and mammalian lignans.
They may occur glycosidically bound to various sugar residues, esterified or as

structural subunits of biooligomers6971. Flaxseed (Linum usitatissimum L.) is known
as the richest dietary source of lignans, with glycosides of secoisolariciresinol and
matairesinol as the major compounds (370 mg/100 g and 1 mg/100 g, respectively).
Also lignan concentrations in sesame seeds (29 mg/100 g, mainly pinoresinol and
72
lariciresinol) were reported to be relatively high . Significant amounts of
secosisolariciresinol (21 mg/100 g of dry weight) were found in pumpkin seeds. Other
cereals (triticale and wheat), leguminous plants (lentils, soybeans), fruits (pears,
prunes) and certain vegetables (garlic, asparagus, carrots) also contain traces of
these same lignans, but concentrations in flaxseed are about 1000 times as high as
concentrations in these other food sources73. When ingested, secoisolariciresinol and
matairesinol are metabolized by bacteria in the gastrointestinal tract and converted
into the mammalian lignans enterodiol (END) and enterolactone (ENL), respectively
69. Silvina-Lotito, B. and Frei, B.,(2006) Consumption of flavonoid-rich foods and increased plasma
antioxidant capacity in humans: Cause, consequence, or epiphenomenon? Free Radic. Biol. Med.,
41, 1727.
70. Kamal-Eldin,A. et al.(2001), An oligomer from flaxseed composed of secoisolariciresinoldiglucoside
and 3-hydroxy-3-methyl glutaric acid residues, Phytochemistry, 58, 587.
71. Bambagiotti-Alberti,M. et al.(1994), Revealing the mammalian lignan precursor secoisolariciresinol
diglucoside in flax seed by ionspray mass spectrometry, Rapid Commun. Mass Spectrom. 8, 595.
72. Coran, S.A., Giannellini, V. and Bambagiotti-Alberti, M.(1996), A novel monitoring approach for
mammalian lignan precursors in flaxseed, Pharm. Sci., 2, 529.
73. Milder, I.E.J. et al. (2005), Lignan contents of Dutch plant foods: a database including lariciresinol,
pinoresinol, secoisolariciresinol and matairesinol, British J. Nutr., 93, 393.
39
Introduction
(Figure 6) 74 . After the conversion, END is oxidized to ENL 75 . END and ENL are
hormone-like compounds that have the ability to bind to estrogen receptors with low
affinity and with weak estrogen activity.
Figure 6: Secoisolariciresinol and matairesinol and their metabolites in humans.
Lignans possess several biological activities, such as antioxidant and (anti)estrogenic

properties, and thus reduce the risk of certain hormone-related cancers as well as
cardiovascular diseases76,77.
1.2.4. Stilbenes.
Stilbenes are mainly constituents of the heartwood of the genera Pinus (Pinaceae),
Eucalyptus (Myrtaceae), and Maclura (Moraceae). Although stilbene aglycones are
common in heartwood, plant tissues may contain stilbene glycosides. One of these,
resveratrol (3,4',5-trihydroxystilbene) , is found largely in the skins of red grapes and
its amount in red wine range between 0.3 and 7 mg/L 78 . Resveratrol came to
scientific attention few years ago as a possible explanation for the French Paradox,
which is the low incidence of heart disease amongst French people, who eat a
74. Adlercreutz, H. and Mazur, W. (1997), Phyto-oestrogens and Western diseases, Ann. Med., 29, 95 -
120.
75. Mazur, W. et al., Isotope dilution gas chromatographic-mass spectrometric method for the
determination of isoflavonoids, coumestrol, and lignans in food samples, Anal. Biochem., 233, 169,
1996.
76. Borriello, S.P. et al. (1985), Production and metabolism of lignans by the human faecal flora, J.
Appl. Bacteriol. 58,37.
77. Heinonen, S., et al. (2001), In vitro metabolism of plant lignans: new precursors of mammalian
lignans enterolactone and enterodiol, J. Agr. Food Chem., 49, 3178.
78. Adlercreutz, H. et al. (1992), Dietary phytoestrogens and cancer in vitro and in vivo studies, J.
Steroid Biochem. Mol. Biol., 41, 331.
40
Functional food
relatively high fat diet79. More recently, reports on the potential for resveratrol to
inhibit the development of cancer and extend lifespan in cell culture and animal
models have continued to generate scientific interest80,81.
79. Arts, I.C.W. and Hollman, P.C.H. (2005), Polyphenols and disease risk in epidemiological studies,
Am. J. Clin. Nutr., 81, 5317.
80. Jang, M. et al. (1997), Cancer chemopreventive activity of resveratrol, a natural product derived
from grapes, Science, 275, 218.
81. Howitz K.T. et al. (2003), Small molecule activators of sirtuins extend Saccharomyces cerevisiae
lifespan, Nature, 425, 191.
41
Introduction
2. Analytical determination of polyphenols in food sample.
2.1. Introduction.
Food quality control and food nutritional value have become major topics of public
interest 82 . Effects of growing conditions, processing, transport, storage, genetics,
and other factors on the levels of chemical and biochemical components are also
important issues in food science 83 and because food processing industries create
large quantities of by-products, plant material wastes from these industries contain
high levels of phenolic compounds.
In the evaluation of the quality of any kind of food sample the quantity of phenolic
compounds is an important parameter to bear in mind.
The analysis of phenolic compounds is very challenging due to the great variety and
reactivity of these compounds 84 . On the other hand, polyphenols are suitable
compounds for analysis using modern separation and detection methods, such as
hyphenated techniques of high performance liquid chromatography (HPLC) with mass
spectrometry (MS), ultraviolet-visible light (UV/Vis), or nuclear magnetic resonance
(NMR) spectroscopy.
Group-selective chemical reactions, thin layer chromatography (TLC), and gas

chromatography (GC) have been important methods in the qualitative analysis of
phenolics 85 , 86 , however, the latter only after derivatisation 87 . TLC has its own
advantages (e.g. rapidity and inexpensiveness), and modern densitometric and video-
camera detection techniques have further increased its versatility as a widely used
analysis method for phenolic compounds88,89.
82. Ibaez , E. Cifuentes. A. (2001) "New analytical techniques in food science". Crit. Rev. Food Sci.
41 413-450.
83. Seorans, F.J., Ibaez, E. Cifuentes A. (2003) "New trends in food processing" Crit. Rev. Food Sci.,
43 507-526.
84. Bronze, M.R. and BOAS, L.F.V. (1998): Characterisation of brandies and wood extracts by capillary
electrophoresis. Analusis 26(1): 40.47.
85. Bhatia, I.S. and BAJAJ, K.L. (1975): Chemical constituents of the seeds and bark of Syzygium
cumini. Planta Med. 28(4): 346.352.
86. Harborne, J.B. (1975): Chromatography of phenolic compounds, pp. 759.780. In: Chromatography .
A laboratory handbook of chromatographic and electrophoretic methods, HEFTMANN, E. Ed. Van
Nostrand Reinhold Company, New York.
87. Robards, K., LI, X., ANTOLOVICH, M. and BOYD, S. (1997): Characterisation of citrus by
chromatographic analysis of flavonoids. J. Sci. Food Agric. 75(1): 87.101.
88. Summanen, J., YRJNEN, T., HILTUNEN, R. and VUORELA, H. (1998): Influence of densitometer
and videodocumentation settings in the detection of plant phenolics by TLC. J. Planar Chromatogr.
11(6): 421.427.
89. Summanen, J.O. (1999): A chemical and ethnopharmacological study on Phyllantus emblica L.
(Euphorbiaceae),Dissertation book. Yliopistopaino, Helsinki.
42
Analytical determination of polyphenols
The phenolic fraction of food sample is very complex and despite having been
studied for decades and excellent progress having been made, it must be admitted
that a considerable number of compounds present in it have still not been
completely characterized and many problems remain to be resolved. The reason lying
behind these difficulties is the complexity of the chemical nature of these
compounds and the similar complexity of the matrix in which they are found. One of
the current difficulties hindering rapid and reproducible analyses of phenolic
compounds is the scarcity of suitable pure standards, in particular of secoiridoid and
lignan compounds. Phenolic acids of natural origin are weak acids and, owing to their
phenolic hydroxyl groups, flavonoids and tannins also have a slightly acidic nature.
They are therefore ionisable in alkaline conditions, which have led to successful
applications of different types of capillary electrophoresis (CE) in the analysis of
flavonoids9092, tannins93 , and phenolic acids9496 .
In general, any analytical procedure for the determination of individual phenolic

compounds in food samples involves three basic steps: extraction from the food
sample, analytical separation and identification and quantification.
2.2. Sample preparation.
Preparation of the sample is often one of the most important steps in any method to
analyze a fraction of compounds or a family of analytes from any matrix. It may be
said that the isolation of phenolic compounds from the sample matrix is generally a
prerequisite to any comprehensive analytic scheme although enhanced selectivity in
the subsequent quantification step may reduce the need for sample manipulation.
90. PIETTA, P.G., MAURI, P.L., RAVA, A. and SABBATINI, G. (1991): Application of micellar
electrokinetic capillary chromatography to the determination of flavonoid drugs. J. Chromatogr.
549(1.2): 367.373.
91. MARKHAM, K.R. and McGHIE, T.M. (1996): Separation of flavones by capillary electrophoresis: The
influence of pKa on electrophoretic mobility. Phytochem. Anal. 7(6): 300.304.
92. LIANG, H.-R., SIRN, H., JYSKE, P., RIEKKOLA, M.-L., VUORELA, P., VUORELA, H. and HILTUNEN, R.
(1997):Characterization of flavonoids in extracts from four species of Epimedium by micellar
electrokinetic capillary chromatography with diode-array detection. J. Chromatogr. Sci. 35(3):
117.125.
93. BRONZE, M.R., BOAS, L.F.V. and BELCHIOR, A.P. (1997): Analysis of old brandy and oak extracts by
capillary electrophoresis. J. Chromatogr. 768(1): 143.152.
94. SEITZ, U., BONN, G., OEFNER, P. and POPP, M. (1991): Isotachophoretic analysis of flavonoids and
phenolcarboxylic acids of relevance to phytopharmaceutical industry. J. Chromatogr. 559(1.2),
499.504.
95. BJERGEGAARD, C., MICHAELSEN, S. and SRENSEN, H. (1992): Determination of phenolic carboxylic
acids by micellar electrokinetic capillary chromatography and evaluation of factors affecting the
method. J. Chromatogr. 608(1.2): 403.411.
96. HIERMANN, A. and RADL, B. (1998): Analysis of aromatic plant acids by capillary zone
electrophoresis. J. Chromatogr. A 803(1+2): 311.314.
43
Introduction
Extraction of phenolic compounds in plant materials is influenced by their chemical

nature, the extraction method employed, sample particle size, storage time and
conditions, as well as presence of interfering substances. The chemical nature of
plant phenolics compounds varies from simple to highly polymerized substances that
include varying proportions of phenolic acids, phenylpropanoids, anthocyanins and
tannins, among others. They may also exist as complexes with carbohydrates,
proteins and other plant components; some high-molecular weight phenolics and
their complexes may be quite insoluble. Therefore, phenolic extracts of plant
materials are always a mixture of different classes of phenolics that are soluble in
the solvent system used. Additional steps may be required for the removal of
unwanted phenolics and non-phenolic substances such as waxes, fats, terpenes and
chlorophylls.
A great number of procedures for the isolation of the polar phenolic fraction of food
sample using two basic extraction techniques, LE (liquid extraction) and SPE (Solid-
phase extraction), have been published in the literature. The systems not only vary
in the solvents and/or solid-phase cartridges used but also in the quantities of sample
needed for analysis, volumes of the solvents and other such details97.
Even though this section will mainly described liquid-liquid protocols and solid-phase
extraction methods, it is worth emphasizing that sometimes a hydrolysis step has
been introduced to minimize interference in the subsequent analysis. New
developments are widening our future possibilities with regard to the extraction of
phenols from food samples with techniques such as supercritical fluid extraction,
microwave-assisted extraction, simultaneous microwave-assisted solid-liquid
extraction, solid-phase microextraction and pressurized liquid or fluid extraction,
among others.
Extraction periods, usually varying from 1 min to 24 h, have been reported. Longer
extraction times increase the chance of oxidation of phenolics unless reducing agents
are added to the solvent system98. The recovery of polyphenols from food products is
also influenced by the ratio of sample-to-solvent.
2.2.1. Liquid extraction (LE).
Solubility of phenolic compounds is governed by the type of solvent (polarity) used,

degree of polymerization of phenolics, as well as interaction of phenolics with other
97. Hrncirik, K., Fritsche, S. (2004) Comparability and reliability of different techniques for the
determination of phenolic compounds in virgin olive. Eur. J. Lipid Sci. Technol. 106, 540-549.
98. Naczk M., Shahidi F. (2004). Extraction and analysis of phenolics in food. Journal of
Chromatography A, 1054: 95111.
44
food constituents and formation of insoluble complexes. Therefore, there is no

uniform or completely satisfactory procedure that is suitable for extraction of all
phenolics or a specific class of phenolic substances in plant materials. Methanol,
ethanol, acetone, water, ethyl acetate and, to a lesser extent, propanol,
dimethylformamide, and their combinations are frequently used for the extraction of
phenolics99.
Phenolic compounds of food sample have traditionally been isolated by extracting

the sample (dry weight) in a lipophilic solvent with several portions of methanol100 or
101
methanol/water (with different quantities of water ranging between 0% and 40%
followed by evaporation of the solvent from the aqueous extract and a cleanup of
the residue by solvent partition 102 . The most widely used solvent to clean the
sample has been hexane (petroleum ether and chloroform have also been proposed),
although the addition of hexane or other organic solvents in the sample before
extraction do not result in any significant differences in the efficiency of phenol
recovery. Extraction with tetrahydrofuran/water followed by centrifugation 103 and
extraction with N,N-dimethylformamide has also been assayed. For example on olive
oil sample, the best results were obtained by using methanol/water (80:20 v/v),
which is in accordance with data in literature104.
2.2.2 Solid-phase extraction (SPE).
Solid-phase extractions (SPE) can use the same type of stationary phases that are
used in LC columns and so the versatility of this kind of extraction has been taken
advantage for the recovery of phenolics from food sample and various other systems
employing SPE, either as an isolation or a clean-up step before using a
chromatographic or other analytical method to quantify the analyte(s) in the sample.
Some of the suitable adsorbents are alkylsilicas, such as C8 105or C18. In principle; C18-
99. Antolovich M., Prenzler P., Robards K., Ryan D. (2000). Sample preparation in the determination of
phenolic compounds in foods. Analyst, 125: 989-1009.
100. Owen, R. W., Mier, W., Giacosa, A., Hull, W. E., Spiegelhalder, B., Bartsch, H. (2000) Phenolic
compounds and squalene in olive oils: the concentration and antioxidant potential of total phenols,
simple phenols, secoiridoids, lignans and squalene. Food Chem. Toxicol. 38, 647-659.
101. Tsimidou, M., Lytridou, M., Boskou, D., Pappa-Louis, A., Kotsifaki, F., Petrakis, C. (1996) On
determination of minor phenolic acids of virgin olive oil by RP-HPLC. Grasas y Aceites. 47, 151-157.
102. Tasioula-Margari, M., Okogeri, O. (2001) Isolation and characterization of virgin olive oil phenolic
compounds by HPLC/UV and GC-MS. J. Food. Sci. 66, 530-538.
103. Cortesi,, N., Azzolini, M., Rovellini, P., Fedeli, E. (1995) I componenti minori polari degli oli
vergini di oliva: Ipotesi di struttura mediante LC-MS. Riv. Ital. Sost. Grasse 72, 241-251.
104. Brenes, M., Garca, A., Garca, P., Garrido, A. (2000) Rapid and complete extraction of phenols
from olive oil and determination by means of a coulometric electrode array system. J. Agric. Food
Chem. 48, 5178-5183.
105. Pirisi, F. M., Cabras, P., Falqui Cao, C., Migliorini, M., Muggelli, M. (2000) Phenolic compounds in
virgin olive oil. 2. Reappraisal of the extraction, HPLC separation, and quantification procedures. J.
Agric. Food Chem. 48, 1191-1196.
45
Introduction
phase is less suitable for the isolation of polar components from a non-polar matrix
than normal-phase SPE, although C18-cartridges have often been tested for isolating
phenolics from food sample106.
SPE techniques and fractionation based on acidity are commonly used to remove
unwanted phenolics and non-phenolic substances107.
SPE is an increasingly useful sample preparation technique. Using SPE, many of the
problems associated with liquid/liquid extraction can be prevented, such as
incomplete phase separations, less-than-quantitative recoveries, use of expensive,
breakable specialty glassware, and disposal of large quantities of organic solvents.
SPE is more efficient than liquid/liquid extraction, yields quantitative extractions
that are easy to perform, is rapid, and can be automated. Also solvent used and
laboratory time are reduced.
SPE is used most often to prepare liquid samples and extract semivolatile or non-
volatile analytes, but also can be used with solids that are pre-extracted into
solvents. SPE products are excellent for sample extraction, concentration, and
cleanup. They are available in a wide variety of chemistries, adsorbents, and sizes.
Selecting the most suitable product for each application and sample is a very
important issue.
2.3 Analytical techniques.
In order to carry out the separation procedures of polyphenols compounds, different

analytical techniques that are based on the existing differences in the physical-
chemical properties can be used.
The modern separation and detection methods, such as hyphenated techniques of

high performance liquid chromatography (HPLC) and capillary electrophoresis (CE)
with mass spectrometry (MS) are the most popular technique to separate and
characterize phenolic compounds in plant matrices.
2.3.1 Liquid Chromatography (LC).
Liquid Chromatography (LC) is the most used analytical chromatographic technique

for the analysis of polyphenols. Chromatographic process can be defined as
separation technique involving mass-transfer between stationary and mobile phase.
106. Favati, F., Carporale, G., Bertuccioli, M. (1994) Rapid determination of phenol content in extra
virgin olive oil , Grasas Aceites 45, 68-70.
107. Robbins R. (2003), Phenolic acids in foods: an overview of analytical methodology. Journal of
Agricultural and Food Chemistry, 51: 2866-2887.
46
LC use a liquid mobile phase to separate the components of a mixture. The

stationary phase can be a porous solid. Those components are first dissolved in a
solvent, and then forced to flow through a chromatographic column under a high
pressure. In the column, the mixture separates into its components. The resolution is
important, and is dependent upon the extent of interaction between the solute
components and the stationary phase. The stationary phase is defined as the
immobile packing material in the column. The interaction of the solute with mobile
and stationary phases can be manipulated through different choices of both solvents
and stationary phases. As a result, LC acquires a high degree of versatility not found
in other chromatographic systems and it has the ability to easily separate a wide
variety of chemical mixtures108,109.
A) Instrumentation LC system.
LC instrumentation includes a pump, injector, column, detector and data system.

The heart of the system is the column where separation occurs. Since the stationary
phase is composed of micrometer size porous particles, a high pressure pump is
required to move the mobile phase through the column. The chromatographic
process begins by injecting the solute onto the top of the column. Separation of
components occurs as the analytes and mobile phase are pumped through the column.
Eventually, each component elutes from the column as a narrow band (or peak) on
the recorder.
Detection of the eluting components is important, and this can be either selective or
universal, depending upon the detector used. The response of the detector to each
component is displayed on a chart recorder or computer screen and is known as a
chromatogram. To collect, store and analyse the chromatographic data, computer,
integrator, and other data processing equipment are frequently used110,111. The basic
components of this equipment can be seen in (Figure 7).
108. Robards, K., Haddad, P. R., and Jackson, P. E. (1994), "Principles and Practice of Modern
Chromatographic Methods." Academic Press, San Diego.
109. P.L. Zhu, J.W. Dolan, L.R.Snyder, N.M. Djordjevic, D.W. Hill, J.-T. Lin, L.C. Sander and L. Van
Heukelem. (1996), Combined use of temperature and solvent strength in reversed-phase gradient
elution IV. Selectivity for neutral (non-ionized) samples as a function of sample type and other
separation conditions .J. Chromatogr. A 756, p. 63 -72.
110. Thorsten T, (2009) Potential of high temperature liquid chromatography for the improvement of
separation efficiency, Analytica Chimica Acta, Volume 643, Issues 1-2, 8, P: 1-12.
111. Cela R., Lorenzo R.A., Casais M.C. (2002), Cromatografa lquida en columna en Tcnicas de
separacin en Qumica Analtica. Ed. Sntesis S. A. Madrid. P: 399-498.
47
Introduction
Figure 7. Diagram of HPLC.
B) Types of LC.
There are many ways to classify liquid column chromatography. If this classification
is based on the nature of the stationary phase and the separation process, three
modes can be specified112.
a. Adsorption chromatography: the stationary phase is an adsorbent (like

silica gel or any other silica based packing) and the separation is based on
repeated adsorption-desorption steps.
b. Ion-exchange chromatography: the stationary bed has an ionically charged

surface of opposite charge to the sample ions. This technique is used almost
exclusively with ionic or ionizable samples. The stronger the charge on the
sample, the stronger it will be attracted to the ionic surface and thus, the
longer it will take to elute. The mobile phase is an aqueous buffer, where
both pH and ionic strength are used to control elution time.
c. Size exclusion chromatography: the column is filled with material having

precisely controlled pore sizes, and the sample is simply screened or filtered
according to its solvated molecular size. Larger molecules are rapidly washed
through the column; smaller molecules penetrate inside the porous of the
112. Valcrcel Cases, M.; Gmas Hens, A, (1990); Cromatografa lquida en columna (II). Tcnicas de
absorcin y particin, en Tcnicas analticas de separacin,. Ed. Revert S.A, 485-531.
48
packing particles and elute later. This technique is also called gel filtration or
gel permeation chromatography.
Concerning the first type, two modes are defined depending on the relative polarity
of the two phases: normal (NP) and reversed-phase (RP) chromatography. In normal
phase chromatography, the stationary bed is strongly polar in nature (e.g. silica gel),
and the mobile phase is nonpolar (such as n-hexane). Polar samples are thus retained
on the polar surface of the column packing for longer than less polar materials.
Reversed-phase chromatography is the inverse of this. The stationary bed is

(nonpolar) in nature, while the mobile phase is a polar liquid, such as mixtures of
water and methanol or acetonitrile. Here the more nonpolar the material is, the
longer it will be retained. Reverse phase chromatography is used for almost 90% of all
chromatographic applications.
Eluent polarity plays the major role in all types of LC. There are two elution types:
isocratic and gradient. In the first type, constant eluent composition is pumped
through the column during the whole analysis. In the second type, eluent
composition (and strength) is steadily changed during the run.
Initially, pressure was selected as the principal criterion of modern liquid

chromatography and thus the name was "high pressure liquid chromatography" or
HPLC. This was, however, an unfortunate term because it seems to indicate that the
improved performance is primarily due to the high pressure. This is, however, not
true. In fact, high performance is the result of many factors: very small particles of
narrow distribution range and uniform pore size and distribution, high pressure
column slurry packing techniques, accurate low volume sample injectors, and
sensitive low volume detectors and, of course, good pumping systems. Naturally,
pressure is needed to permit a given flow rate of the mobile phase.
HPLC technique is characterised by:
High resolution
a) Small diameter (4.6 mm), stainless steel, glass or titanium columns;
b) Column packing with very small (1,8 and 10 m) particles;
c) Relatively high inlet pressures and controlled flow of the mobile phase;
d) Continuous flow detectors capable of handling small flow rates and

detecting very small amounts;
e) Rapid analysis;
49
Introduction
HPLC is a dynamic adsorption process. Analyte molecules, while moving through the
porous packing beads, tend to interact with the surface adsorption sites. Depending
on the HPLC mode, the different types of the adsorption forces may be included in
the retention process: Hydrophobic (non-specific) interactions are the main ones in
RP separations. Dipole-dipole (polar) interactions are dominant in NP mode.
Ionic interactions are responsible for the retention in ion-exchange chromatography.
All these interactions are competitive. Analyte molecules are competing with the
eluent molecules for the adsorption sites. So, the stronger analyte molecules interact
with the surface. The weaker the eluent interaction, the longer the analyte will be
retained on the surface. SEC is another case. It is the separation of the mixture
based on the molecular size of its components. The basic principle of SEC separation
is that the bigger the molecule, the less possibility there is for it to penetrate into
the adsorbent pore space. So, the bigger the molecule the less it will be retained113.
2.3.2 Capillary Electrophoresis (CE).
Among the different separation techniques employed for polyphenols analysis, CE has
emerged as a good alternative since this technique provides fast, efficient and low-
cost separations in this type of analysis. CE is based on the different electrophoretic
mobility of substances in solution under the action of an electric field.
CE is based on a quite simple design; the basic components of this equipment can be
seen in (Figure 8).
113. Hermansson, J., and Schill, G. (1989) "High Performance Liquid Chromatography" (P. R. Brown and
R. A. Hartwick, eds.), Chemical Analysis, Vol. 98, 337-374.
50
Capillary
Detector
Anode
Buffer Sample Buffer
High voltage supplier
Figure 8: Diagram of capillary electrophoresis system.
Electrophoresis is the differential movement of ions by attraction or repulsion in an

electric field. In a CE system, the ends of the capillary are connected to electrodes,
which are connected to a high voltage power supply. The capillary ends are placed
into buffer reservoirs, and the capillary is filled with a buffer identical to those in
the reservoirs. The sample is introduced into the capillary by replacing one of the
buffer reservoirs with a sample reservoir (usually at the anode end); the sample may
be injected either electrokinetically or hydraulically. After the buffer reservoir is
replaced, the electric field is applied and the separation is performed. Either on-line
or off-line optical detection can be made at the cathode end of the capillary.
The velocity of migration of an analyte in capillary electrophoresis will also depend

upon the rate of electroosmotic flow (EOF) of the buffer solution. In a typical system,
the electroosmotic flow, normally, is directed toward the negatively charged
cathode, so that the buffer flows through the capillary from the source vial to the
destination vial. Separated by differing electrophoretic mobilities, analytes migrate
toward the electrode of opposite charge114. As a result, negatively charged analytes
114. Skoog, D.A., oller, F.J., Crouch, S.R. (2007) "Principles of Instrumental Analysis" 6th ed. Thomson
Brooks/Cole Publishing: Belmont, CA.
51
Introduction
are attracted to the positively charged anode, counter to the EOF, while positively
charged analytes are attracted to the cathode, in agreement with the EOF as
depicted in (Figure 9).
Figure 9: Diagram of the separation of charged and neutral analytes according to

their respective electrophoretic and electroosmotic flow mobilities
The advantages of capillary electrophoresis are:
a) It has very high efficiencies, meaning hundreds of components can be

separated at the same time
b) Requires minimum amounts of sample
c) It is easily automated
d) It can be used quantitatively
e) It consumes limited amounts of reagents
All these characteristics have contributed to the rapid development of CE.
Several modes of CE are available: (a) capillary zone electrophoresis (CZE), (b)
micellar electrokinetic chromatography (MEKC), (c) capillary gel electrophoresis
(CGE), (d) capillary isoelectric focusing, (e) capillary isotachophoresis, (f) capillary
electrochromatography (CEC), and (g) nonaqueous CE. The simplest and most
versatile CE mode is CZE, in which the separation is based on differences in the
charge-to-mass ratio and analytes migrate into discrete zones at different velocities.
Anions and cations are separated in CZE by electrophoretic migration and electro-
osmotic flow (EOF), while neutral species coelute with the EOF. In MEKC, surfactants
are added to the electrolyte to form micelles. During MEKC separations, nonpolar
portions of neutral solutes are incorporated into the micelles and migrate at the
52
same velocity as the micelles, while the polar portions are free and migrate at the
EOF velocity115.
2.3.3 Mass Spectrometry (MS).
Mass Spectrometry is a powerful technique for identifying unknowns and studying

molecular mass data.
All mass spectrometers consist of three basic parts: an ion source, a mass analyzer,
and a detector system (Figure 10).
Sample
+
_
Ionizer Mass Analyzer Detector

Figure 10: Basic parts of the mass spectrometer.
The inlet transfers the sample into the vacuum of the mass spectrometer. In the
source region, neutral sample molecules are ionized and then accelerated into the
mass analyzer. The mass analyzer is the heart of the mass spectrometer. This section
separates ions, either in space or in time, according to their mass to charge ratio.
After the ions are separated, they are detected and the signal is transferred to a
data system for analysis. All mass spectrometers also have a vacuum system to
maintain the low pressure, which is also called high vacuum, required for operation.
High vacuum minimizes ion-molecule reactions, scattering, and neutralization of the
ions. In some experiments, the pressure in the source region or a part of the mass
spectrometer is intentionally increased to study these ion-molecule reactions. Under
normal operation, however, any collisions will interfere with the analysis.
115. Shahidi F., Naczk M. (2004). Methods of Analysis and Quantification of Phenolic Compounds. In:
Phenolics in food and nutraceuticals. Edited by Shahidi F., Naczk M. CRC Press (Boca Raton,
FL,USA).
53
Introduction
2.3.3.1 Mass Analyzer.
Mass analyzers detect the ions according to their mass-to-charge ratio. All mass
spectrometers are based on dynamics of charged particles in electric and magnetic
fields in vacuum.
There are many types of mass analyzers; each analyzer type has its strengths and
weaknesses. Many mass spectrometers use two or more mass analyzers for tandem
mass spectrometry (MS/MS).
The most MS analyzers which have been used in analytical chemistry profiling are
triple quadrupole (QqQ), ion trap (IT), and time of flight (TOF). Regarding to the
analysers, IT and TOF systems are the two most common mass analyzer to be found
in food laboratory. IT allows structure elucidation of compounds by MSn. Orthogonal
acceleration TOF provides much better accuracy and precision of mass information
generated. These accurately measured mass values with a mass error less than 5 ppm
can be used to produce candidate empirical formulae and identify the potential
substance with elemental composition analysis116.
A. Ion-trap (IT).
In the IT, the ions are trapped and sequentially ejected. Ions are created and
trapped in a mainly quadrupole radio frequency (RF) potential and separated by m/q,
non-destructively or destructively. The ion-trap mass spectrometer uses three
electrodes to trap ions in a small volume. The mass analyzer consists of a ring
electrode separating two hemispherical electrodes (Figure 11). A mass spectrum is
obtained by changing the electrode voltages to eject the ions from the trap. The
advantages of the ion-trap mass spectrometer include compact size, and the ability
to trap and accumulate ions to increase the signal-to-noise ratio of a measurement
and MSn.
There are many mass/charge separation and isolation methods but the most
commonly used is the mass instability mode in which the RF potential is ramped so
that the orbit of ions with a mass a > b are stable while ions with mass b become
unstable and are ejected on the z-axis onto a detector.
116. Xie, G. X., Plumb, R., Su, M. M., Xu, Z. H., Zhao, A. H., Qiu, M. F., et al. (2008). Ultra-
performance LC/TOF MS analysis of medicinal Panax herbs for metabolomic research. Journal of
Separation Science, 10151026.
54
Ion Source
Skimmer Ion Trap
Glass Dual
Capillary Octopole
Lenses
Detector
Drying Gas Partition
1. Pump 2. Pump 3. Pump 4. Pump

stage stage stage stage
Figure 11: Diagram show the Ion Trap connected with Ion source and Detector.
Ions may also be ejected by the resonance excitation method, whereby a

supplemental oscillatory excitation voltage is applied to the endcap electrodes, and
the trapping voltage amplitude and/or excitation voltage frequency is varied to bring
ions into a resonance condition in order of their mass/charge ratio 117 . The
cylindrical ion trap mass spectrometer is a derivative of the quadrupole ion trap mass
spectrometer.
b. Time-of-Flight (TOF).
TOF is characterised by being a pulsed rather than a continuous technique. TOF

systems can record thousands of mass spectra per second, which is far in excess of
most other mass analysers. A good introduction to TOF instrumentation and theory
has been provided by Guilhaus 118 , with an account focused more on applications
117. March, R. E. (2000). "Quadrupole ion trap mass spectrometry: a view at the turn of the century".
International Journal of Mass Spectrometry 200 (1-3): 285-312.
118. Guilhaus, M. (1995) Special feature: Tutorial. Principles and instrumentation in time-of-flight mass
spectrometry. Physical and instrumental concepts J. Mass Spectrom., 30, 1519-1532.
55
Introduction
being provided by Cotter119. Mamyrin has also recently reviewed the development of
TOF instrumentation120.
TOF-MS is method of mass spectrometry in which ions are accelerated by an electric

field of known strength. This acceleration results in an ion having the same kinetic
energy as any other ion that has the same charge. The velocity of the ion depends on
the mass-to-charge ratio. The time that it subsequently takes for the particle to
reach a detector at a known distance is measured. This time will depend on the
mass-to-charge ratio of the particle (heavier particles reach lower speeds). From this
time and the known experimental parameters one can find the mass-to-charge ratio
of the ion. Therefore, all of the ions will reach the detector at different times.
Because the velocity of the ions is proportional to the mass, the mass-to-charge ratio
(m/z) can be calculated by knowing the time that an ion reaches the detector. Also,
because no scanning is involved, all ions reach the detector, giving the instrument a
theoretically limitless mass range. The key features enabling accurate mass
measurement include high efficiency in gating ions from an external continuous
source, simultaneous correction of velocity and spatial dispersion, and increased
mass resolving power121.
TOF technology presents numerous advantages over other analyzers, such as high
mass resolution, high mass accuracy, theoretically unlimited mass range and
relatively low cost. Moreover, TOF/MS is ideal for pulsed or spatially confined
ionization, and a complete mass spectrum for each ionization event can be obtained,
as well as spectra from extremely small sample amounts122. A schematic diagram of
TOF-MS is shown in (Figure 12).
119. Cotter, R. J. (1992) Time-of-flight mass spectrometry for the structural analysis of biological
molecules. Anal. Chem. 64, 1027A-1039A
120. Mamyrin, BA, (2001)Time-of-flight mass spectrometry (concepts, achievements, and prospects),
Int. J. Mass Spectrom., , 206, 251-266.
121. Hayashida, M. Takino, M. Terada, M.. Kurisaki, E Kudo, K. Ohno , Y. (2000) Time-of-flight mass
spectrometry (TOF-MS) exact mass database for benzodiazepine screening. Legal Medicine, 11, P.
S423-S425
122. Guilhaus, M., Mlynski, V., Selby, D., (1997) "Perfect Timing: Time-of-flight Mass Spectrometry." ,
Rapid Commun. Mass Spectrom., 11, 951962.
56
Figure 12: Schematic diagram of Time of Flight Mass Spectrometer (TOF-MS)
2.3.3.2 Ion source.
The ion source is the part of the mass spectrometer that ionizes the analyte. The
ions are then transported by magnetic or electric fields to the mass analyzer.
Techniques for ionization have been key to determining what types of samples can be
analyzed by mass spectrometry. Electrospray ionization (ESI) and atmospheric
pressure chemical ionization (APCI) are used for gases and vapors and also commonly
used for detection of low molecular weight polar and non-polar compounds. In APCI
sources, the analyte is ionized by chemical ion-molecule reactions during collisions in
the source. Two techniques often used with liquid and solid biological samples
include ESI and matrix-assisted laser desorption/ionization (MALDI)123.
In spite of the variety of interphases developed for connection CE or HPLC with MS,
the most used at the moment, it is interphase ESI. This interphase allows the direct
transformation of compounds from liquid to gas phase to the mass spectrometer. ESI
is one of the most versatile ionization techniques and offers the biggest possibilities
for the analysis of polar compounds (100-200,000 Dalton range) or charged species.
So, ESI becomes the preferred choice for detection of polar compounds separated by
123. Lin H., Nathan, M. J. Keating, C. D. (2000) Surface-enhanced Raman scattering: A structure-
specific detection method for capillary electrophoresis, Anal. Chem. 72, no. 21, pp. 5348-5355.
57
Introduction
LC and CE in food analysis. (Figure 13) shows the different ion source techniques for
the analysis of polar compounds124.
Figure 13. The different ion source techniques used for analysis of polar compounds
In ESI, the process of formation of electrospray, a slight pressure was applied to a

conductive liquid in a glass tube, which had at one end a fine needle, so that a
droplet was produced at the needle tip. An electrical potential was then applied
between the liquid and a large planar electrode. It was observed that under certain
conditions a spray was produced that resulted in the production of very small (less
than 1 m in diameter) droplets125. This spray was described in greater detail and
photographs presented in Zelenys 1917 paper126. More detailed descriptions of the
electrospray process can be found elsewhere124,127, but an outline is provided here.
Applying a potential of 2 3 kV to the tip of narrow steel capillary that contains an
electrolyte solution, which is 1 3 cm from a large planar electrode, results in
electrospray. Considering the case where the capillary tip is held at a positive
potential, the meniscus of the solution at the metal capillary tip will become
enriched in positive electrolyte ions. This accumulated charge is pulled downfield
124. Cole, R. B. (1997), Electrospray Ionisation Mass Spectrometry: Fundamentals, Instrumentation

and Applications, John Wiley and Sons, New York,.
125. Zeleny, J. (1915) On the Conditions of Instability of Liquid Drops with Applications to the Electrical
Discharge from Liquid Point. Camb. Philos. Soc., 18, 71-83.
126. Zeleny, J. (1917) Instability of electrified liquid Surfaces. Phys. Rev. 10, 1-6.
127. Cole, R. B. (2000) "Some tenets pertaining to electrospray ionization Mass Spectrometry" J. Mass
Spectrom . 35 :763-772
58
towards the planar electrode, expanding the meniscus into a cone (the Taylor cone)
as illustrated in (Figure 14).
Figure 14: Diagram to explain the electrospray process.
2.3.3.3 The Interfaces for coupling CE/MS and LC/MS.
A. Coupling of LC/MS.
When analytes are both volatile and thermally stable, it is the technique of choice.
Most analytes, however, are both involatile and thermally instable, requiring the use
of LC techniques. Mass spectrometry is used for detection in chromatography because
of the extra information available than with other techniques.
Over the last 30 years, a great deal of attention has naturally been paid to the on-
line interfacing of chromatographic techniques to mass spectrometry. A wide and
varied body of literature is available, with early development having been well
reviewed by McFadden128. A more recent review and a good starting point for reading
has been provided by Abian 129, while Gelp has updated this work, with attention
130
paid to LC techniques only . Development of liquid interfaces for mass
spectrometry has largely been accomplished with the use of pressurised flow systems
only, due to their reliability and ease of use. The major types of LC/MS interfaces
were reviewed such as, automated off-line interfaces, electron impact ionization,
128. McFadden, W. H. (1979) "Interfacing Chromatography and Mass Spectrometry", Journal of

Chromatographic Science, vol. 17, pp. 2-16.
129. J. Abian, (1999)"The coupling of gas and liquid chromatography with mass spectrometry", J. Mass
Spectrom. 34, 157-168.
130. Gelp, E. (2002) Interfaces for coupled liquid-phase separation/mass spectrometry techniques. An
update on recent developments, J. Mass Spectrom., 37, 241-253.
59
Introduction
chemical ionization source , atmospheric pressure ionization, mechanical transfer,

thermospray interface and electrospray ionization (ESI).
The development of ESI mass spectrometry has resulted in LC/MS becoming a routine
technique in many analytical science laboratories. The greatest advantage of
electrospray is that ions are produced with very little excess energy, meaning that
very large and very involatile molecules can be analysed. Electrospray is currently
the most universal interface between LC and mass spectrometry and is able to accept
flow rates up to approximately 10 L min-1, requiring flow splitting when interfaced
to many LC techniques. ESI/MS sensitivity, however, is largely concentration
dependent, removing the disadvantage of flow splitting. Pneumatically assisted
electrospray or ion spray was developed to increase the flow rate capable of being
accepted into an electrospray source up to approximately 200 L min-1131 , and is
currently the most widely used form of ESI/MS. Essentially ion spray simply combines
gas-assisted nebulization with electrospray to accommodate higher flow rates.
A novel recent extension of ESI/MS is droplet electrospray. Here, a piezoelectric

buzzer is used to make a capillary vibrate such that the liquid held is emitted in the
form of small droplets 132 . This work was originally performed to investigate the
method of action of the electrospray process, but was later used for mass
spectrometry133 . Most LC/MS systems began their development as continuous flow
liquid interfaces and were only later adapted for chromatography, so an interface
based on a droplet dispenser may be developed in time. A key disadvantage of all
electrospray systems, is that there is preferential ionisation of polar analytes that
migrate to the droplet surface, leaving non-polar analytes in the centre of the
droplet.
ESI is one of the most accurate analytical techniques used for the analysis of phenolic
compounds. Moreover the advantages of MS detection include the capability to both
determine molecular weights and to obtain structural information134. Thus, the on-
line coupling of HPLC with MS using ESI as an interface yields a powerful method
because ESI MS allows the determination of a wide range of polar compounds.
131. Bruins A.P, Covey T.R, Henion J.D. (1987) Ion spray interface for combined liquid
chromatography/atmospheric pressure ionization mass spectrometry. Anal. Chem. 59: 2642-2646.
132. Hager D. B., Dovichi N. J. (1994) Behavior of Micro- scopic Liquid Droplets near A Strong
electrostatic field: Droplet Electrospray. Anal. Chem. 66(9): 1593-1594.
133. Hager D. B., Dovichi N. J., Klassen J. and Kebarle, P. (1994) "Droplet Electrospray Mass
Spectrometry", Anal. Chem., 66, 3944-3949.
134. Carrasco-Pancorbo, A.; Neus, C.; Pelzing, M.; Segura-Carretero, A.; Fernndez-Gutirrez, A.
(2007) CE- and HPLC-TOF-MS for the characterization of phenolic compounds in olive oil.
Electrophoresis, 28, 806-821.
60
B. Coupling of CE-MS.
Over the past 15 years, considerable advances have been introduced in CE-MS
interfaces to facilitate the transfer of the analytes from the liquid phase (from the
CE capillary) to the gas phase for MS detection. In spite of the large number of
ionization techniques available135 , the principal interface used for direct coupling of
CE to MS has been electrospray (ESI). ESI is a soft ionization method that produces
gaseous ions from highly charged evaporating liquid droplets. ESI is a continuous
source, and selecting packets of ions from a continuous stream is by no means
straightforward.
The aim of the developed interfaces for CE-ESI-MS is to achieve both a stable CE
current and high efficiency of ionization. Unfortunately, many of the running buffers
and other additives used in CE are non-volatile substances and, therefore, they are
not suitable for CE-ESI-MS coupling. They can suppress the ionization of the analyte,
yielding poor mass spectral sensitivity or, can even clog the system. This limitation
has to be taken into account when using CE in its different modes (e.g., MEKC or
capillary gel electrophoresis (CGE), in which frequently non-ESI-MS-compatible
substances have to be used in order to achieve adequate CE separation of analytes.
Nevertheless, CE-ESI-MS seems well-suited for a large number of applications.
Many processes occur during electrospray: the production of charged droplets at the
nebulizer tip, shrinkage of the charged droplet by solvent evaporation, disintegration
of the drops resulting from the highly charged droplets and the formation of gas-
phase ions136.
Interfacing CE with MS via an ESI source can roughly be performed in two different
ways, with or without an additional liquid. The first approach, known as the sheath-
flow interface, is the most common one due to its robustness and ease of
implementation, while the second one the sheathless approach should feature a
higher sensitivity137.
1. Sheath-flow interface.
The voltage is applied to the CE buffer via a supportive contact liquid. There are two
groups of liquid-supported systems: the sheath liquid interface and the liquid
junction.
135. Gelpi E, (2002) Interfaces for coupled liquid-phase separation/mass spectrometry techniques. An
update on recent developments. J Mass Spectrom;37:241253.
136. Schmitt-Kopplin, P., Frommberger, M. (2003) Capillary electrophoresis mass spectrometry: 15
years of developments and applications. Electrophoresis, 24, 38373867.
137. Aline Staub et el, (2009) CE-TOF/MS: Fundamental concepts, instrumental considerations and
applications., Electrophoresis, 30, 16101623
61
Introduction
A. Sheath liquid interface.
In the sheath liquid interface, the separation capillary is surrounded by a second

tube of larger diameter in a coaxial arrangement. The supportive liquid is guided
through this outer tube and mixed with the CE buffer directly at the exit end of
the capillary. This arrangement may be surrounded by a third tube, through
which a stream of gas can be pumped to support droplet formation136. The
sheath-liquid systems are relatively easy to implement and use, although they are
demanding terms of optimization of operational parameters, such as capillary tip
position, flow-rate and sheath liquid composition (Figure 15).
Figure 15: Schematic diagram of the sheath liquid interface.
B. Liquid junction interface
In liquid junction systems, the CE column is partially disconnected from the ESI
emitter. In fact, the liquidjunction interface provides the electrical connection
to close the CE circuit via a liquid reservoir. Post-capillary liquid introduction
shows flexibility because the make-up liquid can be selected with an appropriate
pH, flow-rate and composition for optimized ESI operation.
This arrangement decouples the CE separation process from the ESI, allowing the
individual optimization of each of the two systems138. It is worth mentioning that
dilution of the CE effluent by the sheath liquid flow rate may reduce sensitivity,
but does not significantly affect it since the sheath liquid is also evaporated
during the spray process (Figure 16).
138. Gelpi E, (2002): Interfaces for coupled liquid-phase separation/mass spectrometry techniques. An
update on recent developments. J Mass Spectrom; 37:241253.
62
Figure 16:. Schematic diagram of the liquid junction interface.
2. The sheathless interface
In this approach, the voltage is directly applied to the CE buffer. The main difficulty
is to close the electrical circuit required for any CE separation. This can be achieved
by applying a metal coating to the end of a tapered separation capillary or by
connecting a metal-coated, full metal or conductive polymeric sprayer tip to the CE
outlet 139 . A recent review written by Zamfir describes advances in the sheathless
interfacing of CE and ESI-MS140 (Figure 17).
Figure 17: Schematic diagram of the sheathless interface.
139. Haselberg, R., de Jong, G. J., Somsen, G. W. (2007) Capillary electrophoresis-mass spectrometry
for the analysis of intact proteins. J. Chromatogr. A 2007, 1159, 81109.
140. Zamfir, A. D. (2007) Recent advances in sheathless interfacing of capillary electrophoresis and
electrospray ionization mass spectrometry. J. Chromatogr. A, 1159, 213.
63
Introduction
2.4 Phenolic compounds by HPLC and CE.
A number of reviews on the analysis of polyphenols have been published 141 152 .
According to Robards153 selection of proper analytical strategy for studying bioactive
phenolics in plant materials depends on the purpose of the study as well as the
nature of the sample and the analyte. The assays used for the analysis of phenolics
can be classified as either those which determine total phenolics content, or those
quantifying a specific group or class of phenolic compounds. Quantification of
phenolic compounds in plant materials is influenced by their chemical nature, the
extraction method employed, sample particle size, storage time and conditions, as
well as assay method, selection of standards and presence of interfering substances
such as waxes, fats, terpenes and chlorophylls.
2.4.1. Phenolic compounds by HPLC.
HPLC techniques are now most widely used for both separation and quantitation of
phenolic compounds. Numerous studies suggest that the consumption of plant foods
containing dietary phenolics may significantly contribute to human health. Hundreds
of publications on the analysis of food phenolics have already appeared over the past
two decades.
According to Yanagida et al. 154 the elution order does not follow the degree of
polymerization and the peaks of highly polymerized oligomers tend to overlap on
141. Antolovich, M. Prenzler, P. Robards, K. Ryan, D. (2000) Sample preparation in the determination of
phenolic compounds in fruits. Analyst 125 (9891009.
142. Deshpande, S.S. Cheryan, M. Salunkhe, D.K. (1986) Tannin analysis food-products .CRC Crit. Rev.
Food Sci. Nutr. 24 401449.
143. Hagerman, A.E. Zhao, Y. Johnson, S. Shahidi. F. (1997) Methods for determination of condensed
and hydrolyzable tannins, ACS Symposium Series, vol. 662, American Chemical Society, Washington,
DC, pp. 209222.
144. Jackman, R.L. Yada, R.Y. Tung, M.A. (1987) A Review- Separation and chemical-properties of
anthocyanins used for their qualitative and quantitative-analysis, J. Food Biochem. 11 279308.
145. Makkar, H.P.S. (1989) Relation of rumen degradability with microbial colonization. J. Agric. Food
Chem. 37 11971202.
146. Naczk, M. Shahidi, F. (2004) Extraction and analysis of phenolics in food J. Chromatogr. A 1054
95111.
147. Porter, L.J. Harborne,J.B. (1989) Methods in Plant Biochemistry, vol. 1, Academic Press, San
Diego, CA, pp. 389420.
148. Scalbert, A. Monties, B. Janin, G. (1989) Tannins in wood: comparison of different estimation
methods, J. Agric. Food Chem. 37 13241329.
149. Scalbert, A. in: R.W. Hemingway, Laks P.S. (1992) (Eds.), Plant Polyphenols: Synthesis, Properties
Significance, Plenum Press, New York, NY, pp. 259280.
150. Tempel, A.S. (1982) Tannin-measuring techniques, J. Chem. Ecol. 8 12891298.
151. Tsao, R. Deng, Z. (2004) separation procedures for naturally occurring antioxidant phytochemical,
J. Chromatogr. B 812, 8599.
152. Shahidi, F. , Naczk, M., in: Otles S. (2005) Methods of Analysis of Food Components Additives, CRC
Press, Boca Raton, FL, pp. 199259.
153. Robards, K. (2003) the determination of bioactive phenols in plants, fruit and vegetables J.
Chromatogr. A 1000 657691.
154. Yanagida A. Kanda T. Takahashi T. Kamimura A. Hamazono T. Honda S. (2000) Fractionation of
apple procyanidins according to their degree of polymerization by normal-phase high-performance
liquid chromatography. J. Chromatogr. A 890 251259.
64
155
chromatograms. Shoji et al. applied a combination of normal phase
chromatography and HPLC for separation and identification of apple procyanidins up
to decamers. Various supports and mobile phases are available for the analysis of
anthocyanins, procyanidins, flavonones, flavonols, flavan-3-ols, flavones and phenolic
acids156,157. Introduction of reversed phase columns has considerably enhanced the
HPLC separation of different classes of phenolic compounds158. Several reviews have
been published on the application of HPLC methodology for the analysis of
phenolics159162.
Polyphenols are commonly detected using UVvis and photodiode array detectors163
165
. Other methods used for the detection of phenolics include electrochemical
166 167
coulometric array detector , chemical reaction detection technique and
fluorimetric detector168. A combination of HPLC technique and voltammetry has been
successfully employed for detection, identification and quantification of flavonoid
and non-flavonoid phenolics in wine169,170 . MS detectors coupled to HPLCMS have
been commonly employed for structural characterization of phenolics. ESI-MS has
155. Shoji, T.; Masumoto, S.; Moriichi, N.; Kanda, T.; Ohtake, Y. (2006) Apple (Malus pumila)
procyanidins fractionated according to the degree of polymerization using normal-phase
chromatography and characterized by HPLC-MS and MALDI-TOF/MS. J. Chromatogr. A, 1102,
206213.
156. Merken H.M., Beecher G.R., (2000) Measurement of food flavonoids by high-performance liquid
chromatography: A review, J. Agric. Food Chem. 48 577599.
157. Senter S.D., Robertson J.A., Meredith F.I.,(1989) Phenolic Compounds of the Mesocarp of
Cresthaven Peaches during Storage and Ripening ,J. Food Sci. 54 12591260, 1268.
158. Hostettmann K., Hostettman M., in: Harborne J.B., Mabry T.J. (1982)Eds. The flavonoids:
Advances in Research, Chapman and Hall, New York, NY, , pp. 118.
159. Karchesy J.J., Bae Y., Chalker-Scott L., Helm R.F., Foo L.Y., in: Hemingway R.W., Karchesy J.J.,
(1989) Chemistry and Significance of Condensed Tannins, Plenum Press, New York, NY, , pp. 139
152.
160. Daigle D.J., Conkerton E.J., (1983) analysis of flavonoids by HPLC, J. Liq. Chromatogr. 6 105118.
161. Daigle D.J., Conkerton E.J., (1988) Analysis of flavonoids by HPLC an update, J. Liq. Chromatogr.
11 309325.
162. Robards K., Antolovitch M., (1997) Analytical chemistry of fruit bioflavonoids A review, Analyst
122 11R34R.
163. Tomas-Barberan F.A., Gil M.I., Cremin P., Waterhouse A.L., Hess- Pierce B., Kader A.L., (2001)
HPLC-DAD-ESIMS analysis of phenolic compounds in nectarines, peaches, and plums, J. Agric. Food
Chem. 49 47484760.
164. Peng Z., Hayasaka Y., Iland P.G., Sefton M., Hoj P., Waters E.J., (2001) Quantitative Analysis of
Polymeric Procyanidins (Tannins) from Grape (Vitis vinifera) Seeds by Reverse Phase High-
Performance Liquid Chromatography, J. Agric. Food Chem. 49, 2631.
165. Barnes S., Coward L., Kirk M., Sfakianos J., (1998) HPLC-mass spectrometry analysis of isoflavones,
J. Proc. Soc. Exp. Biol. Med. 217 254262.
166. Mattila P., Astola J., Kumpulainen J., (2000) Determination of flavonoids in plant material by
HPLC with diode-array and electro-array detections J. Agric. Food Chem. 48 58345841.
167. de Pascual-Teresa S., Treutter D., Rivas-Gonzalo J.C., Santos-Buelga C., (1998) Analysis of
flavanols in beverages by high-performance liquid chromatography with chemical reaction
detection J. Agric. Food Chem. 46, 42094213.
168. Lazarus S.A., Adamson G.E., Hammerstone J.F., Schmitz H.H., (1999) High-performance liquid
chromatography/mass spectrometry analysis of proanthocyanidins in foods and beverages, J. Agric.
Food Chem. 47, 36933701.
169. Lunte C.E., Wheeler J.F., Heineman W.R., (1988) Determination of selective phenolic-acids in beer
Extract by LC, Analyst 113 9495.
170. Mahler S., Edwards P.A., Chisholm M.G., (1988) HPLC identification of phenols in Vidal blanc wine
using electrochemical detection J. Agric. Food Chem. 36, 946951.
65
Introduction
been employed for structural confirmation of phenolics in plums, peaches,

nectarines171, grapeseeds164, soyfoods172, cocoa173 and olive oil174176 Satterfield and
Brodbelt 177 demonstrated that complexation of flavonoids with Cu2+ enhanced the
detection of flavonoids by ESI-MS. Mass spectra obtained for metalflavonoids
complexes were more intense and simpler for interpretation than those of
corresponding flavonoids.
Identification of phenolics collected after HPLC analysis was also carried out using
fast atom bombardment mass spectrometry (FAB-MS) 178 and electron impact mass
spectrometr (EIMS)179 .Matrix-assisted laser desorption/ionization mass spectrometry
(MALDIMS) has also been employed for qualitative and quantitative analysis of
anthocyanins in foods180.
2.4.2. Phenolic compounds by CE.
Even when the phenolic compounds from food samples have been successfully
characterized and quantified by HPLC with different detectors and without any
previous separation, the use of faster analytical techniques and screening tools to
allow a rapid screening of these compounds is strongly recommended.
CE can achieve the aims traditionally achieved by HPLC, providing an alternative way
of characterizing phenolic compounds from food samples, and proved that in
instances in which none of the HPLC methods provides enough resolution CE, with its
171. Tomas-Barberan F.A., Gil M.I., Cremin P.,. Waterhouse A.L, Hess- Pierce B.,. Kader A.L, (2001)
HPLC-DAD-ESIMS analysis of phenolic compounds in nectarines, peaches, and plums, J. Agric. Food
Chem. 49, 47484760.
172. Zafrilla P., Ferreres F., Tomas-Barberan F.A., (2001) Effect of processing and storage on the
antioxidant ellagic acid derivatives and flavonoids of red raspberry (Rubus idaeus) jams, J. Agric.
Food Chem. 49, 36513655.
173. Hammerstone J.F., Lazarus S.A., Mitchell A.E., Rucker R., Schmitz H.H., (1999) Identification of
procyanidins in cocoa (Theobroma cacao) and chocolate using high-performance liquid
chromatography mass spectrometry, J. Agric. Food Chem. 47, 490496.
174. De la Torre-Carbot, K., Jauregui, O., Gimeno, E., Castellote, A. I. et al., (2005) Characterization
and quantification of phenolic compounds in olive oils by solid-phase extraction, HPLC-DAD, and
HPLC-MS/MS, J. Agric. Food Chem., 53, 43314340.
175. Bianco, A., Buiarelli, F., Cartn, G., Coccioli, F. et al., (2003) Analysis by liquid chromatography-
tandem mass spectrometry of biophenolic compounds in virgin olive oil, Part II, J. Sep. Sci., 26,
417424.
176. Carrasco-Pancorbo, A., Cerretani, L., Bendini, A., Segura- Carretero, A. et al., (2005) Evaluation
of the antioxidant capacity of individual phenolic compounds in virgin olive oil , J. Agric. Food
Chem., 53, 8918 8925.
177. Satterfield, M., Brodbelt, J., (2000) Enhanced detection of flavonoids by metal complexation and
electrospray ionization-mass spectrometry, Anal. Chem., , 72, 5898-5906.
178. Bakker J, Bridle P, Koopman A (1992) Strawberry juice colour: the effect of some processing
variables on the stability of anthocyanins. J Sci Food Agric 60 471-476.
179. Edenharder R., Keller G., Platt K.L., Unger K.K., (2001) Isolation and characterization of
structurally novel antimutagenic flavonoids from spinach (Spinacia oleracea). J. Agric. Food Chem.
49,27672773.
180. Wang J., Sporns P., (1999) , Analysis of Anthocyanins in Red Wine and Fruit Juice using MALDI-MS.
J. Agric. Food Chem. 47 20092015.
66
flexible experimental conditions, should be assayed as a complementary second-

choice technique.
According to Herrero et al. 181 capillary techniques have a great potential for a
broader application in separation of natural multicomponent mixtures after solving
such issues as reproducibility and sensitivity. During the last 8 years more than 20
reviews on advances in the application of electromigration methods for analysis of
natural antioxidants, foods and food components have been published182. Phenolics
present in grapes, wines, olives, spices, medicinal herbs, tea, fruits and oilseeds
have been studied using electromigration methods181,182.
Hall et al.183 used capillary electrophoresis for separation of food antioxidants such
as butylated hydroxyanisole (BHA) and butylated hydroxytoluene (BHT). Later,
Andrade et al. 184 utilized capillary zone electrophoresis to evaluate the effect of
grape varieties and wine ageing on the composition of non-colored phenolics in port
wine. Non-colored phenolics were extracted from wine into diethyl ether, then
concentrated to dryness and redissolved in methanol.
Peng et al. 185 utilized capillary electropheresis with electrochemical detection for
simultaneous determination of catechin, epicatechin and trans-resveratrol in red
wine.
Moane et al. 186 utilized capillary electrophoresis for direct detection of phenolic
acids in beer. Recently, Pan et al. 187 developed a method for determination of
protocatechuic aldehyde and protocatechuic acid by capillary electrophoresis with
188
amperometric detection. Chu et al. separated pure forms of cis- and
transresveratrol isomers from wine using capillary electrophoresis in micellar mode.
181. Herrero M, Martin-Alvarez PJ, Senorans FJ, et al. (2005) Optimization of accelerated solvent
extraction of antioxidants from Spirulina platensis microalga. J.Food Chem. 93 417423.
182. Cifuentes A., (2006) "Recent advances in the application of capillary electromigration methods for
food analysis". Electrophoresis 27, 283303.
183. Hall C.A., Zhu A., Zeece M.G., (1994) comparison between CE and HPLC separation of food grade
antioxidants. J. Agric. Food Chem. 42, 919921.
184. Andrade P, Seabra R, Ferreira M, Ferreres F, Garci-Viguera C. (1998) Analysis of non-coloured
phenolics in port wines by capillary zone electrophoresis. Influence of grape variety and ageing. Z
Lebensm- Untersuch Forsch; 206: 161164.
185. Peng Y., Chu Q., Liu F., Ye J., (2004) Determination of phenolic constituents of biological interest
in red wine by capillary electrophoresis with electrochemical detection. J. Agric. Food Chem. 52,
153156.
186. Moane, S.; Park, S.; Lunte, C. E.; Smyth, M. R. (1998) Detection of phenolic acids in beverages by
capillary electrophoresis with electrochemical detection. Analyst, 123, 1931-1936.
187. Pan Y., Zhang L., Chen G., (2001) Determination of protocatechuic aldehyde and protocatechuic
acid by capillary electrophoresis with amperometric detection. Analyst 126, 15191523.
188. Chu Q., ODwyer M., Zeece M.G., (1998) Direct analysis of resveratrol in wine by Micel capillary
electrophoresis. J. Agric. Food Chem. 46, 509513.
67
Introduction
On the other hand, Kreft et al. 189 utilized capillary electrophoresis with a UV
detector for determination of rutin content in different fractions of buckwheat flour
and bran.
Capillary electrophoresis has been used for separation of limonoid glucosides in citrus
seeds 190 as well as limonoid glucosides and phlorin in citrus juices 191 Recently,
Braddock and Bryan192 applied capillary electrophoresis for quantification of limonin
glucoside and phlorin in extracts from citrus byproducts. Crego et al.193 optimized
conditions for separation of complex mixture of rosemary phenolics by capillary
194
electrophoresis. Later Zeece quantitatively characterized rosemary phenolics
using capillary electrophoresis coupled with orthogonal electrospray to mass
spectrometry. Six phenolics, namely isoquercitrin, carnosic acid, rosmarinic acid,
homoplantaginin and gallocatechin were detected in this study.
Horie et al.195 reported a separation of five catechins together with ascorbic acid,
caffeine and theanine in green tea infusions by capillary zone electrophoresis
techniques. Later, Larger et al.196 utilized micellar electrochromatography with UV
detection for separation detection of flavonoids in green and black tea infusions. ()-
Epicatechin gallate and (+)-catechin were only detected in green tea, but ()-
epicatechin, ()-epigallocatechin gallate, and ()-epicatechin were found in both
197
teas. Subsequently, Bonoli et al. successfully applied micellar
electrochromatography for detection of catechins in green tea, namely (+)-catechin,
()-epigallocatechin,()-gallocatechin, ()-gallocatechingallate, ()-epigallocatechin-
3-gallate, ()-epicatechingallate, and ()-epigallocatechin gallate.
189. Kreft S., Knapp M., Kreft I., (1999) Extraction of rutin from buckwheat (Fagopyrum esculentum
Moench) seeds and determination by capillary electrophoresis. J. Agric. Food Chem. 47 46494662.
190. Moodley, V. E.; Mulholland, D. A.; Raynor, M. W. (1995) Micellar electrokinetic capillary
chromatography of limonoid glucosides from citrus seeds. J. Chromatogr. A, 718, 187193
191. Cancalon, P. F.(1999) Analytical monitoring of citrus juices by using capillary electrophoresis. J.
AOAC Int., 82 (1), 95106.
192. Braddock R.J., Bryan C.R.,(2001) Extraction parameters and capillary electrophoresis analysis of
limonin glucoside and phlorin in citrus byproducts. J Agric Food Chem 49: 5982-5988.
193. Crego A.L., Ibez E., Garca E., Rodrguez de Pablos R., Seorns F.J., Reglero G., Cifuentes A.,
(2004) Capillary lectrophoresis separation of rosemary antioxidants from subcritical water extracts.
European Food Research and Technology 219, 549-555.
194. Zeece, M. (1992). Capillary electrophoresis: a new analytical tool for food science. Trends in food
science and technology. 3, pp. 6 10.
195. Horie H., Mukai T., Kohata K., (1997) quality and contains higher amounts of theanine. J.
Chromatogr. A 758 332335.
196. Larger P.J., Jones A.D., Dacombe C., (1998) Separation of polyphenols using micellar
electrokinetic chromatography with diode array detection. J. Chromatogr. A 799 309320.
197. Bonoli M, Colabufalo P, Pelillo M, et al., (2003) Fast determination of catechins and xanthines in
tea beverages by micellar electrokinetic chromatography. J. Agric. Food Chem. 51, 11411147.
68
Futhermore, Cifuentes et al. 198 demonstrated that the separation of complex

mixtures of procyanidin B3, procyanidin B2, procyanidin B1, (+)-catechin, ()-
epicatechin, cis- and trans-p-coumaric acids can be achieved in less than 5 min with
the application of micellar electrochromatography technique.
Arraez-Roman et al. and Carrasco-Pancorbo et al. worked on the analysis of phenolic

compounds by CE-ESI-TOF/MS from pollen extracts and olive oil, respectively199,200.
More recently, many groups have worked on the analysis of natural compounds by CE-
ESI-TOF/MS 201 - 206 . The major phenolic compounds, previously observed in several
studies, they belong to several important families polyphenols (phenyl alcohols,
phenyl acids, lignans, flavonoids and secoiridoids)....
198. Cifuentes A, Bartolome B, Gomez-Cordoves C, (2001) Fast determination of procyanidins and other
phenolic compounds in food samples by micellar electrokinetic chromatography using acidic
buffers. J. Electro. 22, 15611567.
199. Arraez-Roman, G. Zurek, C. Babmann, N. Almaraz-Abarca, et al., (2007) Identification of phenolic
compounds from pollen extracts using capillary electrophoresiselectrospray time of flight mass
spectrometry, Anal. Bioanal. Chem. 389, pp. 19091917.
200. Carrasco-Pancorbo, A., Neususs, C., Pelzing, M., Segura-Carretero, A., Fernandez-Gutierrez, A.,
(2007) CE- and HPLC-TOF-MS for the characterization of phenolic compounds in olive oil.
Electrophoresis, 28, 806821.
201. Chen, J., Zhao, H., Wang, X., Lee, F. S.-C., Yang, H., Zheng, L., (2008) Analysis of major alkaloids
in Rhizoma coptidis by capillary electrophoresis-electrospray-time of flight mass spectrometry with
different background electrolytes. Electrophoresis, 29, 21352147.
202. Segura-Carretero, A., Puertas-Mejia, M. A., Cortacero- Ramirez, S.,et al., (2008) Selective
extraction, separation, and identification of anthocyanins from Hibiscus sabdariffa L. using solid
phase extraction-capillary electrophoresis-mass spectrometry (time-of-flight /ion trap) J.
Electrophoresis, 29, 28522861.
203. Petersson, E. V., Puerta, A., Bergquist, J., Turner, C., (2008) Electrophoresis, 29, 27232730.
204. Arraez-Roman, D., Zurek, G., Bassmann, C., Segura- Carretero, A., Fernandez-Gutierrez, A.,
(2008) Characterization of Atropa belladonna L. compounds by capillary electrophoresis-
electrospray ionization-time of flight-mass spectrometry and capillary electrophoresis-electrospray
ionization-ion trap-mass spectrometry. Electrophoresis, 29, 21122116.
205. Garcia-Villalba, R., Leon, C., Dinelli, G., Segura-Carretero, A., Fernandez-Gutierrez, A., Garcia-
Canas, V., Cifuentes, A., J. Chromatogr. A 2008, 1195, 164173.
206. Sim, C., Moreno Arribas, M.V., Cifuentes, A., (2008) Ion-trap versus time-of-flight mass
spectrometry coupled to capillary electrophoresis to analyze biogenic amines in wine. Journal of
Chromatography A, , 1195, 150-156.
69
Introduction
3. Samples: Importance, main phenolic compounds and health properties.
3.1. Orange skin.
Cultivated Citrus may be derived from as few as four ancestral species. Numerous
natural and cultivated origin hybrids include commercially important fruit such as the
orange, grapefruit, lemon, some limes and some tangerines. Citrus is a common term
and genus of flowering plants in the family Rutaceae, originating in tropical and
subtropical Southeast Asia. The plants are large shrubs or small trees, reaching 515
m tall, with spiny shoots and alternately arranged evergreen leaves with an entire
margin. The flowers are solitary or in small corymbs, each flower 24 cm diameter,
with five (rarely four) white petals and numerous stamens. They are often very
strongly scented. The fruit is a hesperidium, a specialized berry, globose to
elongated, 430 cm long and 420 cm diameter (Figure 18), with a leathery rind
surrounding segments or "liths" filled with pulp vesicles.
The orange fruit is commercially important and usually is eaten fresh or pressed for
juice. Orange processing in the United States produces ~700000 tons of peel by-
products solids annually207, because the majority (96%) of citrus fruits in major citrus
producing converted into juice. In Spain the citrus fruits represented 12 percent, of
the country's agricultural production. Therefore, food processing industries create
large quantities of by-products. Citrus peel is also known to be rich in phenolic
compounds, so, the isolation of these compounds from citrus peel can be of interest
to the food industry.
Figure 18: Exterior peel and inner white pulp of the orange
207. Florida Citrus Processors Association. Statistical Summary, 1993-1994 Season; Winter Haven, FL,
1995; p 1D.
70
Samples: Importance, main phenolic compounds and health properties
The main phenolic constituents of citrus peel are flavanone and flavone glycosides
(Figure 19)208,209.
Figure 19: Structures of the main flavonoids in orange peel.
The phenolic compounds from orange peel have health-related properties due to
their antioxidant and radical scavenging activity. These properties have been
reported to manifest anticancer 210 , anti-cardiovascular disease, antiviral and anti-
inflammatory activities211, effects on capillary fragility, an ability to inhibit human
208. Kanes, K.; Tisserat, B.; Berhow, M.; Vandercook, C. (1993) Phenolic composition of various tissues
of Rutaceae species. Phytochemistry, 32, 967-974.
209. Peleg, H.; Naim, M.; Rouseff, R. L.; Zehavi, U. (1991) Distribution of bound and free phenolic acids
in oranges and grapefruit. J. Sci. Food Agric., 417-426.
210. Kandaswami C, Perkins E, Soloniuk DS, Drzewiecki G and Middleton E. (1991) Antiproliferative
effects of citrus flavonoids on a human squamous cell carcinoma in vitro. Cancer Letters; 56: 147
152.
211. Galati EM, Monforte MT, Kirjavainen S, Forestieri AM, Trovato A and Tripodo MM. (1994) Biological
effects of hesperidin, a citrus flavonoid (Note I): Antiinflammatory and analgesic activity. Farmaco;
40: 709712.
71
Introduction
212
platelet aggregation and another potential beneficial biological actions in
213-216
humans .
3.2. Diatomaceous earth using in olive oil industry.
Olive oil is a fruit oil obtained from the olive (Olea europaea; family Oleaceae), a
traditional tree crop of the Mediterranean Basin. The wild olive tree originated in
Asia Minor and spread from there as far as southern Africa, Australia, Japan and
China217. It is commonly used in cooking, cosmetics, pharmaceuticals, and soaps and
as a fuel for traditional oil lamps. Olive oil is used throughout the world, but
especially in the Mediterranean area. Olive and olive oil industry is an important
employer in the agro-food-sector with over 800.000 employees, and olive oil
production is an important agricultural and alimentary sector in Europe. The
European Union is the main world producer. In fact during the season 2003/2004,
2.282.650 tons were produced in several thousand of olive oil mills218.
Many olive oil producers consider several factors on the effective oil quality. These
factors are the soil condition, climate and altitude of the olive tree, time and system
of harvest, pruning of the tree, fertiliser usage, the cultivation, and the production
process.
These factors affected the characterization of virgin olive oil, in particular, oxidative
stability, water content, and the presence of each phenolic compounds. These
compunds are polar compounds that can found in the olive fruit; however many of
these compounds are modified or lost during the production process of virgin olive
oil 219 . The production processes of olive oil are: collecting, washing, pressing,
decantation, centrifuging, storage, filtration and packaging, there is a lack of
212. Tzeng S.H., Teng C.M. (1991) Inhibition of platelet aggregation by some flavonoids. Thrombosis
Research; 64: 91100.
213. Manthey, J. A.; Guthrie, N.; Grohmann, K. (2001) Biological properties of citrus flavonoids
pertaining to cancer and inflammation. Curr. Med. Chem., 8, 135-153.
214. Benavente-Garcia, O.; Castillo, J.; Marin, F. R.; Ortuno, A.; Del Rio, J. A. (1997) Uses and
properties of citrus flavonoids. J. Agric. Food Chem., 45, 4505-4515.
215. Hasegawa, S.; Miyake, M.; Ozaki, Y. (1994)Biochemistry of citrus liminoids and their
anticarcinogenic activity. In Food Phytochemicals for Cancer Prevention I, Fruits and Vegetables;
Huang, M. T., Osawa, T., Ho, C. T., Rosen, R. T., Eds.; American Chemical Society: Washington,
DC, pp 198-208.
216. Widmer, W. W.; Montanari, A. (1996) The potential for citrus phytochemicals in hypernutritious
foods. In Hypernutritious Foods; Finley, J. W., Armstrong, D. J., Nagy, S., Robinson, S. F., Eds.;
AgScience: Auburndale, FL, pp 75-90.
217. International Olive Oil Council. "The Olive Tree, The Origin and Expansion of the Olive Tree".
http://www.internationaloliveoil.org/web/aa-ingles/oliveWorld/olivo.html. Retrieved on 2008
218. Anonymous, Faostst, Database, www.fao.org.
219. Briante, R., La Cara, F., Tonziello, M. P., Frebbraio, F., Nucci, R., (2001) Antioxidant activity of
the main bioactive derivatives from oleuropein hydrolysis by hyperthermophilic beta-glycosidase.J.
Agric. Food Chem., 49, 31983203.
72
information and studies available about the filtration step220,221 , which is the last
step just before packaging. At this step, the filter used for olive oil filtration from
several years ago is diathomite, which is the fossilized remains of microscope algae,
also called diatomaceous earth.
This filtration process affects the characteristics of VOO, in particular, oxidative

stability, water content, and the presence of each phenolic compound. Polyphenols
are polar compounds which can found in the olive fruit. Many of these compounds are
modified or lost during the production process of olive oil 222 . Therefore, the
qualitative study of phenolic compounds in the diatomaceous earth used in the
filtration process of olive oil is very important.
Oleuropein belongs to a specific group of coumarin-like compounds, the secoiridoids,

which are abundant in Oleaceae. Secoiridoids are compounds that are usually bound
to glycosides and produced from the secondary metabolism of terpenes. The
secoiridoids, found only in plants belonging to the family of Oleaceae which includes
Olea europaea L., are characterised by the presence of elenolic acid in its glucosidic
or aglyconic form in their molecular structure. In particular, they are formed from a
phenyl ethyl alcohol (hydroxytyrosol and tyrosol), elenolic acid and, eventually, a
glucosidic residue. Oleuropein is an ester of hydroxytyrosol (3,4-DHPEA) and the
elenolic acid (EA) glucoside (oleosidic skeleton common to the secoiridoid glucosides
of Oleaceae) 223 225 . Olive-oil secoiridoids in aglyconic forms derive from the
glycosides in olives via the hydrolysis of endogenous glucosidases during crushing and
malaxation. These newly formed amphiphilic substances are to be found both in the
oily layer and the water although they are more concentrated in the latter fraction
because of their polar functional groups. During the storage of VOO hydrolytic
mechanisms may be involved in the release of simple phenols such as hydroxytyrosol
and tyrosol from the more complex secoiridoids. The most abundant secoiridoids in
VOO are the dialdehyde form of elenolic acid linked to hydroxytyrosol or tyrosol (p-
220. A. Bottino, A. Capannelli et al., (2004) application of membrane processes for the filtration of
extra virgin olive oil. J. Food. Engin., 65(2), pp. 303-309.
221. Gmez-Caravaca A.M., Cerretani L., Bendini A., Segura-Carretero A., Fernndez-Gutirrez A.,
Lercker G. (2007) "Effect of filtration systems on the phenolic content in virgin olive oil by HPLC-
DAD-MSD" Am. J. Food Technol. 2: 671-678.
222. Brenes M. et al., (1995) Biochemical-changes in phenolic-compunds during olive processing. J.
Agric. Food Chem., 43, pp. 2702-2706.
223. Montedoro, G.F., Servili, M., Baldioli, M., Miniati, E. (1992). Simple and Hydrolyzable Phenolic
Compounds in Virgin Olive Oil. 2. Initial Characterization of the Hydrolyzable Fraction., 40, 1577
1580. J. Agric. Food. Chem.
224. Montedoro, G.F., Servili, M., Baldioli, M., Selvaggini, R., Miniati, E., Macchioni, A. (1993). Simple
and Hydrolyzable Compounds in Virgin Olive Oil. 3. Spectroscopic Characterizations of the
Secoiridoid Derivatives., J. Agric. Food Chem.41, 2228-2234.
225. Amiot, M.J., Fleuriet, A., Macheix, J.J. (1986). Importance and evolution of phenolic compounds
in olive during growth and maturation. J. Agric. Food Chem. 34, 823-825.
73
Introduction
HPEA), known respectively as 3,4-DHPEA-EDA and p-HPEA-EDA, and an isomer of the

oleuropein aglycon (3,4-DHPEA-EA) (Table 2). In 1999 another hydroxytyrosol
derivative, hydroxytyrosol acetate (3,4-DHPEA-AC), was found in VOO.
Phenolic acids are naturally occurring secondary aromatic plant metabolites found
widely throughout the plant kingdom226. They contain two distinguishing constitutive
carbon frameworks, the hydroxycinnamic and hydroxybenzoic structures. Their
various contributions to plant life are currently being subject to intense scrutiny, one
aspect of which deals specifically with their role in food quality 227 . In particular,
several phenolic acids such as gallic, protocatechuic, p-hydroxybenzoic, vanillic,
caffeic, syringic, p- and o-coumaric, ferulic and cinnamic have been identified and
quantified in VOO (in quantities lower than 1 mg of analyte kg-1 of olive oil). (+)-
Pinoresinol is a common component of the lignan fraction of several plants such as
Forsythia species 228 and Sesamum indicum seeds, whereas (+)-1-acetoxypinoresinol
and (+)-1-hydroxy-pinoresinol and their respective glucosides have been detected in
the bark of Olea europaea L.. Flavonoids are widespread secondary plant metabolites.
Flavonoids are largely planar molecules and their structural variation comes in part
from the pattern of modification by hydroxylation, methoxylation, prenylation, or
glycosylation. Flavonoid aglycones are subdivided into flavones, flavonols, flavanones,
and flavanols depending upon the presence of a carbonyl carbon at C-4, an OH group
at C-3, a saturated single bond between C-2 and C-3 or a combination of a non-
carbonyl at C-4 with an OH group at C-3 respectively. Several authors have reported
that flavonoids such as luteolin and apigenin are also present in VOO229-232. Luteolin
may originate from rutin or luteolin-7-glucoside, and apigenin from apigenin
glucosides. Several interesting studies have also been published describing how
several flavonoids have been found in olive leaves and fruit.
226. Exarchou, V., Godejohann, M., van Beek, T. A., Gerothanassis, I. P., Vervoort, J. (2003). LC-UV-
Solid-Phase Extraction-NMR-MS Combined with a Cryogenic Flow Probe and Its Application to the
Identification of Compounds Present in Greek Oregano. Anal. Chem., 75, 6288-6294.
227. Hakkinen, S., Heinonen, M., Karenlampi, S., Mykkanen, H., Ruuskanen, J., Torronen, R. (1999).
Screening of selected flavonoids and phenolic acids in 19 berries. Food Res. Int., 32, 345-353.
228. Davin, B.D., Bedgar, D.L., Katayama, T., Lewis, N.G. (1992). On the stereoselective synthesis of
(1)-pinoresinol in Forsythia suspensa from its achiral precursor, coniferyl alcohol. Phytochemistry,
31, 38693874.
229. Vzquez-Roncero, A., Janer Del Valle, L., Janer Del Valle, C. (1976). Componentes fenolicos de la
aceituna. III. Polifenoles del aceite. Grasas Aceites, 27, 185-191.
230. Carrasco-Pancorbo, A., Gmez-Caravaca, A. M., Cerretani, L., Bendini, A., Segura-Carretero, A.,
Fernndez-Gutirrez, A. (2006). Rapid quantification of the phenolic fraction of Spanish virgin
olive oils by capillary electrophoresis with uv detection. J. Agric. Food Chem. 54, 7984-7991.
231. Brenes, M., Garca, A., Garca, P., Ros, J. J., Garrido, A. (1999). Phenolic compounds in Spanish
olive oils. J. Agric. Food Chem., 47, 3535-3540.
232. Murkovic, M., Lechner, S., Pietzka, A., Bratacos, M., Katzogiannos, E. (2004). Analysis of minor
components in olive oil. J. Biochem. Methods, 61, 155-160.
74
Table 2. Phenolic compounds in virgin olive oil: compound name, general chemical
structure and molecular weight.
Compound Substituent (MW) Structure
Benzoic and derivative acids
3-Hydroxybenzoic acid 3-OH (138)
p-Hydroxybenzoic acid 4-OH (138)
3,4-Dihydroxybenzoic acid 3,4-OH (154)

5 6
Gentisic acid 2,5-OH (154)
4 COOH
1
Vanillic acid 3-OCH3, 4-OH (168) 3 2
Gallic acid 3,4,5-OH (170)
Syringic acid 3,5-OCH3, 4-OH (198)
Cinnamic acids and derivatives
o-Coumaric acid 2-OH (164)

5 6
p-Coumaric acid 4-OH (164) COOH
4
1
Caffeic Acid 3,4-OH (180)
3 2
Ferulic Acid 3-OCH3, 4-OH (194)
Sinapinic Acid 3,5-OCH3, 4-OH (224)
Phenyl ethyl alcohols
Tyrosol [(p-hydroxyphenyl)ethanol] or
4-OH (138) 5 6
p-HPEA OH
4
1
Hydroxytyrosol [(3,4-
3 2
dihydroxyphenyl)ethanol] or 3,4-DHPEA 3,4-OH (154)
Other phenolic acids and derivatives

5 6
p-Hydroxyphenylacetic acid 4-OH (152)
4 COOH
1
3,4-Dihydroxyphenylacetic acid 3,4-OH (168)
3 2
75
Introduction
4-Hydroxy-3-methoxyphenylacetic acid 3-OCH3, 4-OH (182)
HO COOH
3-(3,4-Dihydroxyphenyl)propanoic acid (182)

HO
Dialdehydic forms of secoiridoids

R* O O dialdehydic form of
Decarboxymethyloleuropein aglycon Elenolic Acid (EDA)
R1-OH (304)
(3,4-DHPEA-EDA) CHO
CHO
Decarboxymethyl ligstroside aglycon
R1-H (320)
(p-HPEA-EDA)
Compound Substituent (MW)
Secoiridoid Aglycons
Oleuropein aglycon or 3,4-DHPEA-EA R1-OH (378)
Ligstroside aglycon or p-HPEA-EA R1-H (362)
Aldehydic form of oleuropein aglycon R1-OH (378)
Aldehydic form ligstroside aglycon R1-H (362)
Structure
R1 OCH 3
O O
C O
HO
p-HPEA or 3,4-DHPEA O O
R*
OH O CH3
Elenolic Acid (EA) aldehydic form of
Elenolic Acid (EA)
Compound Substituent (MW) Structure
Flavonols OH
OH
HO O
(+)-taxifolin (304)
OH
OH O
Flavones
OH
R2
76
Apigenin R1-OH, R2-H (270)
R1-OH, R2-OH
Luteolin
(286)
Lignans OCH 3
OH
O
(+)-Pinoresinol R-H (358)
H R
(+)-1-Acetoxypinoresinol R-OCOCH3 (416) O

HO
H 3CO
(+)-1-Hydroxypinoresinol R-OH (374)
Hydroxyisochromans
O
1-phenyl-6,7-dihydroxyisochroman R1,R2-H (242) R2
1-(3-methoxy-4-hydroxy)phenyl-6,7- R1-OH,R2-OCH3 HO R1
OH
dihydroxy-isochroman (288)
The antioxidant potential of phenolic compounds in olive oil has been a subject of
great interest, because of its chemoprotective effect in human beings219. Phenolic
compounds are of fundamental importance for their nutritional properties, sensory
characteristics, and the shelf life of virgin olive oil233. They also play an important
role in human nutrition as preventative agents against several diseases 234 . The
composition of phenolic compounds in virgin olive oil is related to agronomic and
technological aspects235.
3.3. Olive leaves.
Olive leaf is the leaf of the olive tree (Olea europaea) (Figure 20). While olive oil is
well known for its flavour and health benefits, the leaf has been used medicinally in
various times and places. Olive leaves were chosen as the plant model because they
are by-products of olive farming, one of the most important agricultural activities in
the Mediterranean region.
233. Tsimisou, M. (1998). Polyphenols and quality of virgin olive oil in retrospect. J. Food Sci., 10, 99
116.
234. Owen, R. W., Giacosa, A., Hull, W. E., Haubner, R. et al., (2000) Olive-oil consumption and health:
the possible role of antioxidants. Lancet Oncol. 2000, 1, 107112.
235. Servili, M., Baldioli, M., Montedoro, G. F., (1994) ISHS Acta Horticulturae: II International
Symposium on Olive Growing, 356, 331336.
77
Introduction
Figure 20: Olive tree leaves: Top side and under side
In the olive fruits, phenyl acids, flavonoids and secoiridoids have been reported, the
phenolic compounds representing 1-3 % (w/v). In the leaves, 19 % (w/w) is oleuropein
and 1.8 % flavonoids236. There are many antioxidants available in olive leaves, the
most active identified so for include: Oleuropein, Hydroxytyrosol and Tyrosol (Figure
21).
Oleuropein Hydroxytyrosol Tyrosol
Figure 21:. The most important phenolic compounds in olive leaves
Olive leaves have been used by ancient Egyptian and Mediterranean cultures to treat
a variety of health conditions. Olive leaves are utilized in the complementary and
236. LE Tutour B. et al., (1992): Antioxidative activities of Olea europaea leaves and related phenolic
compounds. Phytochem 31 (4), 1173-1178.
78
alternative medicine community for its perceived ability to act as a natural

pathogens killer by inhibiting the replication process of many pathogens. Olive leaves
recently gained international attention when it was shown to have an antioxidant
capacity almost double green tea extract and 400% higher than vitamin C 237. It is
known that free radical- mediated events are involved in several pathological
processes, such as cancer and coronary heart disease. This fact has increased the
interest in natural antioxidants. It has proven to be useful in cases of yeast and
fungal infections, herpes, chronic fatigue, allergies, psoriasis and many other
pathogens. Since it works like a broad-spectrum antibiotic, it is useful against colds,
flu, and upper respiratory and sinus infections. In addition, it has been shown to
lower blood sugar, normalize arrhythmias, inhibit oxidation of LDL (the bad
cholesterol), and relax arterial walls, thereby helping to lower blood pressure. Other
benefits are that it boosts energy and eases pain. Several compounds from olive
leaves, oleuropein among them, have shown a variety of biological activities as an
anti-microbial antioxidant238,239. Some recent research on the olive leaf has shown its
antioxidants to be effective in treating some tumors and cancers such as liver,
prostate, and breast cancer. However the research on this is preliminary240,241.
3.4. Almond skin.
Almond (Prunus dulcis) is a species of tree of the genus Prunus, belonging to the
subfamily Prunoideae of the family Rosaceae and native to the Middle East. Within
Prunus, it is classified in the subgenus Amygdalus, distinguished from the other
subgenera by the corrugated seed shell. Almond is also the name of the edible and
widely cultivated nut. Although popularly referred to as a nut, the almond fruit's
seed is botanically not a true nut, but the seed of a drupe (a botanic name for a type
of fruit).
237. Stevenson, L., et al. (2005) Oxygen Radical Absorbance Capacity (ORAC) Report on Olive Leaf
Australia's Olive Leaf Extracts, Southern Cross University,.
238. Soler- Rivas, C. et al., (2000): Oleuropein and related compounds. J. Sci.Food Agric. 80, p. 1013-
1023.
239. Servill M. et al., (1996): Antioxidant I activity of tocopherols and phenolic compounds of virgin
olive oil. J. Am. Oil Chem. Soc. 1996, 73, 1589-1593.
240. Hamdi et al. (2005) Oleuropein, a non-toxic olive iridoid, is an anti-tumor agent and cytoskeleton
disruptor.
241. Dr Stevenson, L,. et al. (2006) In vitro Biological Activities of Pure Olive Leaf Extract & High
Strength Olive Leaf Extract.
79
Introduction
Figure 22:. Almond fruit with its brown skins.
The whole natural almonds have had their shells removed but still retain their brown
skins; blanched whole almonds have had both their shells and skins removed 242 .
Usually, during some industrial processing of almonds, the skin (seed coat) is
removed from the kernel by blanching and then discarded243. Around 12 % of the
worlds almond production is grown in Spain. This leads to the accumulation of large
amounts of by-products and subsequent environmental problems due to their difficult
degradation. The skin, which has very low economic value, represents 4% of the
total almond weight but contains 70100% of total phenols244.
Many studies have shown that almond skins are a rich source of phenolic
compounds 245 -244. The main flavonoids found in almond skins team up with the
vitamin E found in almond meat to more than double the antioxidant punch either
delivers when administered separately (Figure 23).
242. Menninger E.A., Hoticultural Books: Stuart, FL (1997) 175.

243. Vargas, F. J. (2005) rbolesproductores de frutos secos. Origen, descripcin, distribuciny
produccin. In Frutos secos, saludy culturas mediterraneas. J. Eds.; EditorialGlosa: Barcelona, p
21.
244. Milbury, P. E.; Chen, C. Y.; Dolnikowski, G. G.; Blumberg, J. B. (2006) Determination of flavonoids
and phenolics and their distribution in almonds J. Agric. Food Chem. 54, 5027 5033.
245. Sang, S., Lapsley, K., Jeong, W.S., Lachance, P.A., Ho, C.T. and Rosen, R.T. (2002) Antioxidative
phenolic compounds isolated from almond skins (Prunus amygdalus. Batsch.). J. Agric. Food Chem.
50:2459-2463.
246. Wijeratne S.S.K, Abou-Zaid M.M, Shahidi F. (2006) Antioxidants polyphenols in almond and its
coproducts. J. Agric. Food Chem. 54, 312-318.
80
Figure 23. Structures of the major almond flavonoids.
New research on almonds adds to the growing evidence that eating whole foods is
the best way to promote optimal health. Recent studies have shown that the
constituents of almond have anti-inflammatory, immunity boosting, and anti-
hepatotoxicity effects 247 . Claimed health benefits of almonds include improved
complexion, improved movement of food through the colon (feces) and the
prevention of cancer248. Recent research associates the inclusion of almonds in the
diet with elevating the blood levels of high density lipoproteins and of lowering the
levels of low density lipoproteins249,250.
A controlled trial showed that 73 g of almonds in the daily diet reduced LDL
cholesterol by as much as 9.4%, reduced the LDL:HDL ratio by 12.0%, and increased
HDL-cholesterol (i.e., the good cholesterol) by 4.6% 251.
3.5. Flaxseed oil
Flax also known as common flax or linseed (binomial name: Linum usitatissimum) is a
member of the genus Linum in the family Linaceae. It is native to the region
247. Puri, Har Sharnjit Singh (2002). "Badam (Prunus amygdalus)". Rasayana: Ayurvedic Herbs for
Longevity and Rejuvenation (Traditional Herbal Medicines for Modern Times, 2). Boca Raton: CRC.
pp. 5963. ISBN 0-415-28489-9.
248. Davis P.A., Iwahashi C.K. (2001) "Whole almonds and almond fractions reduce aberrant crypt foci
in a rat model of colon carcinogenesis". Cancer Lett. 165 (1): 2733.
249. Porter Novelli (2002) Almonds: Cholesterol lowering, heart-healthy snack. Press rele.
250. Spiller GA, Jenkins DA, Bosello O, Gates JE, Cragen LN, Bruce B (1998). "Nuts and plasma lipids: an
almond-based diet lowers LDL-C while preserving HDL-C". J Am Coll Nutr 17 (3): 28590.
251. Jenkins DJ, Kendall CW, Marchie A, et al. (2002). "Dose response of almonds on coronary heart
disease risk factors: blood lipids, oxidized low-density lipoproteins, lipoprotein(a), homocysteine,
and pulmonary nitric oxide: a randomized, controlled, crossover trial". Circulation 106 (11): 1327
32.
81
Introduction
extending from the eastern Mediterranean to India and was probably first
domesticated in the Fertile Crescent252. Flax was extensively cultivated in ancient
Egypt.
Originally bred thousands of years ago for its fibre (linen) and for the medicinal
properties of the seed, it is now cultivated mainly for its oil (Figure 24). The flax
seed is composed of approximately 41% oil 253 , Canada is a leading producer of
flaxseed in the world, producing 1 M t/year which accounts for 30-40% of total
world production254.
Figure 24: Flax seed shape.
The lignans secoisolariciresinol and matairesinol (Figure 25) are found in a variety of
foods and are at their highest levels in flaxseed255. They are believed to be the plant
precursors of the lignan metabolites enterolactone and enterodiol (Figure 25)
referred to as the mammalian lignans, first discovered in human urine by Setchell et
al. (1983)256.
252. Alister D. Muir, Neil D. Westcot, (2003) Flax: The Genus Linum. P. 3.
253. Flax Council of Canada. (2008) www.flaxcouncil.ca.
254. Bhatty R.S. (1995) Nutrient compostion of whole flaxseed and flaxseed meal in flaxseed in human
nutrition. S. Cunnane and L. Thompson Editors. AOCS Press, Champaign, Ill. p 22-42.
255. Mazur W, Fotsis T, Wahala K, et al. (1996) Isotope dilution gas chromatographic mass
spectrometric method for the determination of isoflavonoids, coumestrol, and lignans in food
samples. J. anal. Bichem. 233(2) 169-180.
256. Setchell, K. D. R.; Lawson, A. M.; McLaughlin, L. M.; Patel, S.; Kirk, D. N.; Axelson, M. (1983)
Measurement of enterolactone and enterodiol, the first mammalian lignans, using stable isotope
dilution and gas chromatographymass spectrometry. Biomed. Mass Spectrom., 10, 22735.
82
The mammalian lignans are produced from plant lignans by in vitro human fecal flora
metabolism257. Fecal inoculum has been utilized to analyze the mammalian lignan
production from plant precursors in various foods258. This incubation has shown that
flaxseed contains higher levels of total lignans (enterolactone and enterodiol) than
other plant foods. There is increasing interest in flaxseed in human nutrition259,260 as
it gains popularity as a health food, a dietary supplement, and an ingredient in bread,
muffins, and breakfast cereals261,262.
Figure 25: Structures of the lignans secoisolariciresinol, matairesinol, enterodiol,

and enterolactone.
These components of flaxseed are of great interest both for the food and
pharmaceutical industries 263 . The physiological aspects of flaxseed components
264
responsible for disease prevention have been reviewed .
257. Borriello, S. P.; Setchell, K. D. R.; Axelson, M.; Lawson, A. M. (1985) Production and metabolism of
lignans by the human fecal flora. J. Appl. Bacteriol. 58, 3743.
258. Thompson, L. U.; Robb, P.; Serraino, M.; Cheung, F. (1991) Mammalian lignan production from
various foods. Nutr. Cancer, 16, 4352.
259. Kurzer, M. S.; Lampe, J. W.; Martini, M. C.; Adlercreutz, H. (1995) Fecal lignan and isoflavonoid
excretion in premenopausal women consuming flaxseed powder. Cancer Epidemiol. Biomarkers
Prev., 4, 353358.
260. Thompson, L. U. (1995) Flaxseed, Lignans and Cancer. In Flaxseed in Human Nutrition; Cunnane, S.,
Thompson, L. U., Eds.; AOAC Press: Champaign, IL,; pp 219236.
261. Jenkins, D. J. A. (1995) Incorporation of Flaxseed or Flaxseed Components into Cereal Foods. In
Flaxseed in Human Nutrition; Cunnane, S., Thompson, L. U., Eds.; AOAC Press: Champaign, IL, pp
281294.
262. McCord, H., Rao, L., (1997) Top seed: with its healing powers, flax is the next nutritional star.
Prevention, 49, 8185.
263. Caragay A.B., (1992) Cancer Preventive Foods and Ingredients. Food Technol. 46, pp. 6568.
83
Introduction
Flax seed is the richest known source of lignan precursors265,266. The reported health
benefits of flaxseed are mostly related to its three main components: fat, mostly in
the form of alpha linolenic acid, lignans and fiber267. Lignans have been shown to
play a role in lowering total and LDL cholesterol, and may possess anti-cancer
properties as well. In addition, they have been shown to reduce tumor formation and
growth in animals268. It is proposed that SDG may prevent LDL oxidation, which is a
precursor for atherosclerosis (plaque), giving the lignans antioxidant properties14.
Clinical studies suggest that flaxseed oil and other omega-3 fatty acids may be
helpful in treating a variety of conditions. The evidence is strongest for heart disease
and problems that contribute to heart disease269273.
264. Oomah B.D., Mazza G., (1998) Flaxseed Products for Disease Prevention. In: G. Mazza Editor,
Functional Foods, Biochemical and Processing Aspects Technomic Publ. Co. Inc, Lancaster, PA, pp.
91138.
265. Bloedon, L.T. and Szapary P.O. (2004) Flaxseed and Cardiovascular Risk. Nutrition Reviews. 62-
1:18 27
266. Peirce, Andrea. (1999) Practical Guide to Natural Medicines: The American Pharmaceutical
Association. The Stonesong Press. 269-270.
267. Brown, L., Rosner, B., Willett W. and Sacks F.M. (1999) Cholesterol-Lowering Effects of Dietary
Fiber: a Meta-Analysis. Amer J of Clin Nutr. 69: 30-42
268. Wang L., Chen J. and Thompson L.U. (2005) The inhibitory effect of flaxseed on the growth and
metastasis of estrogen receptor negative human breast cancer xenografts is attributed to both its
lignan and oil components. International Journal of Cancer. 116: 793-798
269. Nesbitt P.D., Lam Y., and Thompson L.U. (1999) Human Metabolism of Mammalian Lignan
Precursors in Raw and Processed Flaxseed. American Journal of Clinical Nutrition. 69: 549-555.
270. Jenkins D. J., Kendall C., Vidgen E., Agarwal S., Rao A.V., Rosenburg R., Diamandis E., Novokmet
R., Mehling C., Perera T., Griffin L. and Cunnane S.C. (1999) Health aspects of partially defatted
flaxseed, including effects on serum lipids, oxidative measures, and ex vivo androgen and
progestin activity: a Controlled Crossover Trial. American Journal of Clinical Nutrition. 69: 395-402.
271. Mayo Clinic. Flaxseed and flaxseed Oil. Feb 2008.
www.mayoclinic.com/health/flaxseed/NS_patient-flaxseed.
272. Parbtani A., Clark W.F., (1995) Flaxseed and its Components in Renal Disease. In: S.C. Cunnane
and L.U. Thompson Editors, Flaxseed in Human Nutrition AOCS Press, Champaign, IL pp. 244260.
273. Thompson L.U., Rickard S.E., Siedl M.M., (1996) Flaxseed and its Lignan and Oil Components
Reduce Mammary Tumor Growth at a Late Stage of Carcinogenesis. Carcinogenesis 17, pp. 1373
1376.
84
Experimental part.
Results and Discussion.
85
CHAPTER I: Quantification of main phenolic compounds in sweet and
bitter Orange peel using CEMS/MS
87
This work was published in Food Chemistry Journal.
Quantification of main phenolic compounds in sweet and bitter Orange peel using
CEMS/MS.
(Journal of Food Chemistry 116 (2009) 567574)
Saleh M.S. Sawalha, David Arrez-Romn, Antonio Segura-Carretero, Alberto

Fernndez-Gutirrez.
Department of Analytical Chemistry, Granada University.
88
Food Chemistry 116 (2009) 567574
Contents lists available at ScienceDirect
Food Chemistry
journal homepage: www.elsevier.com/locate/foodchem
Analytical Methods
Quantication of main phenolic compounds in sweet and bitter orange peel

using CEMS/MS
Saleh M.S. Sawalha a, David Arrez-Romn b, Antonio Segura-Carretero a,*, Alberto Fernndez-Gutirrez a,*
a
Research Group FQM-297, Department of Analytical Chemistry, Faculty of Sciences, University of Granada, C/Fuentenueva s/n, E-18071 Granada, Spain
b
Verbionat S.C.A, C/Santa F de Bogot 45, 18320 Santa F, Granada, Spain
a r t i c l e i n f o a b s t r a c t
Article history: The food and agricultural products processing industries generate substantial quantities of phenolics-rich
Received 11 September 2008 subproducts, which could be valuable natural sources of polyphenols. In oranges, the peel represents
Received in revised form 9 January 2009 roughly 30% of the fruit mass and the highest concentrations of avonoids in citrus fruit occur in peel.
Accepted 1 March 2009
In this work we have carried out the characterisation and quantication of citrus avonoids in methanolic
extracts of bitter and sweet orange peels using CEESIITMS. Naringin (m/z 579.2) and neohesperidin
(m/z 609.2) are the major polyphenols in bitter orange peels and narirutin (m/z 579.2) and hesperidin
Keywords:
(m/z 609.2) in sweet orange peels. The proposed method allowed the unmistakable identication, using
Phenolic compounds
Orange peel
MS/MS experiments, and also the quantication of naringin (5.1 0.4 mg/g), neohesperidin (7.9 0.8 mg/
Capillary electrophoresis g), narirutin (26.9 2.1 mg/g) and hesperidin (35.2 3.6 mg/g) in bitter and sweet orange peels. CE cou-
Electrospray ionisationmass spectrometry pled to MS detection can provides structure-selective information about the analytes. In this work we
detection have developed a CEESIITMS method for the analysis and quantication of main phenolic compounds
in orange peels.
2009 Elsevier Ltd. All rights reserved.
1. Introduction health-related properties, which are based on their antioxidant

activity including anticancer, antiviral and antiinammatory activ-
Polyphenols are amongst the most popular antioxidants and ities (Bouskela, Cyrino, & Lerond, 1997; Tanaka et al., 1997). The
many natural sources are being suggested for their recovery (Tura, group of avonoids is a widely distributed group of polyphenolic
2002). Crud extract of fruits, herbs, vegetables, cereals, nuts and compounds according to the above fact. Flavonoids in orange peel
other plant material rich in phenolics are increasingly of interest are comprised primarily of avanone glycosides (narirutin 40 -O-
in the food industry (Sang et al., 2002). Citrus is a common term glucoside, eriocitrin, narirutin, hesperidin, isosakuranetin rutino-
and genus of owering plants in the family Rutaceae, originating side), polymethoxylated avone aglycons (sinensetin, hexa-O-
in tropical and subtropical areas in southeast Asia. Citrus fruits methylquercetagetin, nobiletin, hexa-O-methylgossypetin, 3,5,6,7,
are notable for their fragrance, partly due to avonoids and limo- 8,30 ,40 -heptamethoxyavone, tetra-Omethylscutellarein, tangeritin
noids (a kind of terpenes) contained in the peel, they are also good and 5-hydroxy-3,7,8,30 ,40 -pentamethoxyavone) (Horowitz & Gen-
sources of vitamin C and avonoids. Cultivated Citrus may be de- tili, 1977), avone glycosides (diosmin, isorhoifolin, rutin) (Kanes,
rived from as few as four ancestral species. Numerous natural Tisserat, Berhow, & Vandercook, 1993) and C-glycosylated avones
and cultivated origin hybrids include commercially important fruit (6,8-di-C-glucosylapigenin) (Manthley & Grohmann, 2001). Naritu-
such as the orange, grapefruit, lemon, some limes, and some tan- tin, hesperidin, naringin and neohesperidin (Fig. 1) are the most
gerines. Oranges are one of the most popular fruits in the world. abundant avonoids in the edible part of many species of citrus
Orange processing in the United States produces 700.000 tons fruits (Kawai, Tomono, Katase, Ogawa, & Yano, 1999). As is well
of peel as byproduct solids annually (Winter, 1995). Plant material documented naritutin and hesperidin have been determined in
wastes from these industries contain high levels of phenolic com- common sweet orange (Ooghe, Ooghe, Detavernier, & Huygheba-
pounds. Importantly, most of this phytonutrient is found in the or- ert, 1994), and it is worthwhile referring to the recovery of hesper-
ange peel and inner white pulp, rather than in its liquid orange idin and naringin from orange peel (El-Nawawi, 1995), which is
centre, so this benecial compound is too often removed by the considered to be the most popular source, recovery of naringin
processing of oranges into juice. Polyphenols compounds have from bitter orange (Calvarano, 1996).
Even though the characterisation of phenolic compounds in or-
ange has been successfully carried out using HPLC (Anagnostopou-
* Corresponding authors. Fax: +34 958249510.
E-mail addresses: ansegura@ugr.es (A. Segura-Carretero), albertof@ugr.es lou, Kefalas, Kokkalou, Assimopoulou1, & Papageorgiou1, 2005;
(A. Fernndez-Gutirrez). Belajov & Suhaj, 2004; Justesen, Knuthsen, & Leth, 1998; Kanaze,
0308-8146/$ - see front matter 2009 Elsevier Ltd. All rights reserved.
doi:10.1016/j.foodchem.2009.03.003
568 S.M.S. Sawalha et al. / Food Chemistry 116 (2009) 567574
Fig. 1. Chemical structures of: (a) naringin, (b) neohesperidin, (c) hesperidin and (d) narirutin.
Gabrieli, Kokkalou, Georgarakis, & Niopas, 2003; Theodoridis et al., tion system can provide important advantages in food analysis be-
2006). Capillary electrophoresis (CE) has become an alternative or cause of the combination of the high separation capabilities of CE
complement to chromatographic separations because it needs no and the power of MS as identication and conrmation method
derivatization step, requires only small amounts of sample and (Arrez-Romn et al., 2007; Gmez-Romero et al., 2007; Sim, Bar-
buffer and has proved to be a high-resolution technique (Arrez- bas, & Cifuentes, 2005). In general, if a separation technique is cou-
Romn, Gmez-Caravaca, Gmez-Romero, Segura-Carretero, & pled with MS the interpretation of the analytical results can be
Fernndez-Gutirrez, 2006). The hyphenation of CE as analytical more straightforward (Brocke, Nicholson, & Bayer, 2001; Maci,
separation technique coupled to mass spectrometry (MS) as detec- Borrull, Calull, & Aguilar, 2004; Schmitt-Kopplin & Frommberger,
Intens.
x10 6
a
1.25 459.1
1.00
0.75
0.50 270.9
0.25 313.0
234.9 357.1
176.7 204.9 339.0 441.0
0.00
100 200 300 400 500 600 700 m/z
Intens.
x10 6
1.0 b
459.1
0.8
0.6
0.4 270.9
0.2
313.0 357.1
234.8
204.8 339.0 441.0
0.0
100 200 300 400 500 600 700 m/z
Fig. 2A. (a) MS/MS naringin (m/z 579.2) standard, (b) MS/MS naringin (m/z 579.2) in bitter orange peel sample.
S.M.S. Sawalha et al. / Food Chemistry 116 (2009) 567574 569
2003). Furthermore, MS/MS experiments using a ion trap (IT) can a 0.45 lm Millipore (Bedford, MA, USA) membrane lters before
be used to obtain fragment ions of structural relevance for identi- injection into the capillary. Naringin, neohesperidin, narirutin
fying target compounds in a highly complex matrix. In this sense, and hesperidin standards used for MS/MS experiments and calibra-
electrospray ionisation (ESI) has emerged as a highly useful tech- tion curves were obtained from Extrasynthese (Lyon, France).
nique which allows direct coupling with electrophoretic separation
techniques (Smith & Udseth, 1996). 2.2. CEESIITMS apparatus
The aim of this present work has been to develop a simple CE
ESIITMS method for the identication and quantication of main The analyses were made in a P/ACETM System MDQ (Beckman
phenolic compounds in orange peel due to these compounds are Instruments, Fullerton, CA, USA), CE apparatus equipped with an
the most abundant components in all the orange parts and present UVVis detector and coupled to the MS detector by an orthogonal
a high concentration (El-Nawawi, 1995; Horowitz & Gentili, 1977). electrospray interface (ESI). The system comprises a 030 kV high-
voltage built in power supplier.
2. Material and methods All capillaries (fused-silica) used were obtained from Beckman
Coulter Inc. (Fullerton, CA, USA) and had an inner diameter (i.d.)
2.1. Chemicals and reagents of 50 lm. A detection window was created at 10 cm for the UV
detector and 100 cm was the total length (corresponding to the
All chemicals were of analytical reagent grade and used as re- MS detection length). The instrument was controlled by a PC run-
ceived. Boric acid, purchased from SigmaAldrich (St. Louis, MO), ning the 32 Karat System software from Beckman.
and ammonium hydroxide from Merck (Darmstadt, Germany) MS and MS/MS experiments were performed on a Bruker Dal-
were used for preparing the CE running buffers at different concen- tonics Esquire 2000TM ion-trap mass spectrometer (Bruker Daltonik
trations and pH values. Buffers were prepared by weighing the GmgH, Bremen, Germany) equipped with an orthogonal coaxial
appropriate amount of boric acid at the concentrations indicated sheath-ow electrospray interface (model G1607A from Agilent
and adding ammonium hydroxide (0.5 M) to adjust the pH. The Technologies, Palo Alto, CA, USA). This triple tube ESIMS interface
buffers were prepared with doubly deionized water, stored at provides both a coaxial sheath liquid make-up ow and a nebuliza-
4 C and brought to room temperature before use. Doubly deion- tion gas to assist droplet formation. The drying gas and the nebu-
ized water was obtained with a Milli-Q water purication system lization gas were both nitrogen. The coaxial sheath liquid and the
(Millipore, Bedford, MA). 2-Propanol HPLC grade used in the sheath electrical contact at the electrospray needle tip were delivered by
ow, methanol, ethanol, hexane, DMSO and sodium hydroxide, a 74900-00-05 Cole Palmer syringe pump (Vernon Hills, Illinois,
used for capillary cleaning procedures before each analysis, were USA).
obtained from Panreac (Barcelona, Spain) and triethylamine from For the connection between the CE system and the electrospray
Aldrich (Steinheim, Germany). All solutions were ltered through ion source of the mass spectrometer, the outlet of the separation
Intens.
x10 5
a
301.0
6
2
489.1
343.0
198.8 403.0 429.1

0
100 200 300 400 500 600 700 m/z
Intens.
x10 5
8 b
6 301.0
343.0 489.1
385.1 447.1
0
100 200 300 400 500 600 700 m/z
Fig. 2B. (a) MS/MS neohesperidin (m/z 609.2) standard (b) MS/MS neohesperidin (m/z 609.2) in bitter orange peel sample.
Intens.
x10 6
1.5
a
271.0
1.0
0.5
0.0
100 200 300 400 500 600 700 m/z
Intens.
x10 5
4
b
270.9
176.7
0
100 200 300 400 500 600 700 m/z
Fig. 3A. (a) MS/MS narirutin (m/z 579.2) standard, (b) MS/MS narirutin (m/z 579.2) in sweet orange peel sample.
capillary was tted into the electrospray needle of the ion source 2.3.3. Extraction C
and a ow of conductive sheath liquid established electrical con- The same as extraction procedure A but the dry residue was re-
tact between the capillary efuent and water for the electrospray solved in 2 ml of MeOH:H2O (50:50, v/v).
needle. The instrument was controlled by a PC running the Esquire
NT software from Bruker Daltonics. 2.3.4. Extraction D
0.2 g of the sample were weighted and extracted with 10 ml of
MeOH:DMSO (50:50, v/v) The solution was shaken at a room tem-
2.3. Extraction procedures
perature for 2 h and then centrifuged at 4500 rpm for 10 min. The
solution was ltered through 0.2 lm lter. Finally the samples
Five extraction procedures were prepared in order to choose the
were kept in the freezer until analysis. The samples were diluted
best conditions for the extraction of naringin, neohesperidin, nari-
1:1 in water before analysis.
rutin and hesperidin from the orange peel samples. Basically, the
extraction procedures are very similar but some modications
have been carried out. The conditions of each extraction procedure 2.3.5. Extraction E
were as follows. The same as extraction procedure D but the solution was sha-
ken on vortex for 5 min.
2.3.1. Extraction A
0.2 g of the dried sample were weighted and extracted with 2.4. CEESIITMS procedure
10 ml of methanol, the solution was shaken on vortex for 5 min
and then centrifuged at 4500 rpm for 10 min. The solution was l- In order to develop the CEESIITMS method, to obtain the
tered through 0.2 lm lter and collected in a round bottom ask. best selectivity, sensitivity and resolution, the extract C previously
The concentrated methanol was evaporated by rotary pump at described was used.
40 C, and the sample re-dissolved using 2 ml of MeOH:DMSO CE separation was carried out on a fused-silica capillary of
(50:50, v/v). Finally the extract was kept in the freezer until the 50 lm i.d. with a total length of 100 cm (corresponding to the
analysis. The samples were diluted 1:1 in water before analysis. MS detection length).
Before rst use, the bare capillaries were conditioned by rinsing
2.3.2. Extraction B with 0.5 M sodium hydroxide for 20 min, followed by a 10 min
The same as extraction procedure A but the solution was shaken rinse with water. Capillary conditioning was done by ushing for
with magnetic stirrer for 2 h. 2 min sodium hydroxide, 4 min with water, and then for 10 min
Intens.
x10 5
a
6 301.0
0
100 200 300 400 500 600 700 m/z
Intens.
x10 6
2.0
b
301.0
1.5
1.0
0.5
0.0
100 200 300 400 500 600 700 m/z
Fig. 3B. (a) MS/MS hesperidin (m/z 609.3) standard, (b)MS/MS hesperidin (m/z 609.3) in sweet orange peel sample.
Table 1
Analytical parameters of the proposed method.
Analyte RSD LOD (mg/l) LOQ (mg/l) Calibration range (mg/l) Calibration equations R2
Naringin 2.35 0.99 3.30 550 y = 505738x + 2E + 06 0.9858
Neohesperidin 2.62 0.23 0.72 550 y = 640452x + 1E + 06 0.9886
Narirutin 2.71 0.38 1.58 2580 y = 532136x 9E + 06 0.9974
Hesperidin 3.50 1.15 3.85 2580 y = 152140x 0.821550 0.9996
with the separation buffer. During all the capillary conditioning 3. Results and discussion
was used a pressure of 20 w (1 w = 6895 Pa). At the end of the
day the capillary was rinsed for 10 min water and 5 min ush 3.1. Selection of extraction procedure
air. The CE conditions used in the method were a buffer solution
of 200 mM boric acid adjusted with ammonium hydroxide at pH The CEESIITMS method was applied to the analysis of main
9.5. Samples were injected hydrodynamically in the anodic end polyphenols in bitter and sweet orange peel extracts (see Section
in low pressure mode (0.5 w) for 5 s. Electrophoretic separations 2.3). Under the optimised CEESIITMS conditions described
were performed at 25 kV which caused a current intensity of above it is possible to analyse main compounds in the different
40 lA. types of extraction procedures and to carry out a comparative
The optimum ESIITMS parameters were a sheath liquid iso- study of the extraction capacity. The compounds with m/z 579.2,
propanol/water 60:40 with 0.1% (v/v) TEA delivered at a ow rate from sweet and bitter orange peels, were extracted using the pro-
of 0.28 ml/h, a drying gas ow rate of 5 l/min at 300 C, compound cedures AC; the compounds with m/z 609.2, from sweet and bit-
stability 25% and a nebulizer gas pressure of 6 w was supplied for ter orange peels, were extracted using the procedures CE.
ESI formation. Therefore, the extraction procedure C has been selected due to
The mass spectrometer was run in the negative ion mode and presence of the target compounds in the extract.
the capillary voltage was set at 4000 V. The ion trap scanned at
100800 m/z range at 13,000 u/s during the separation and detec- 3.2. Identication of main polyphenols by MS/MS analysis
tion. The maximum accumulation time for the ion trap was set at
5.00 ms, the target count at 20,000 and the trap drive level at The peaks of the main phenolic compounds in orange peel were
100%. easily identied by comparing both migration time and MS/MS
Intens.
x106 a
5
0
0 2 4 6 8 10 12 14 Time [min]
Intens.
x106 b
0
0 2 4 6 8 10 12 14 Time [min]
Fig. 4A. Extracted ion electropherograms of: (a) naringin and (b) neohesperidin in bitter orange peel sample.
data obtained from bitter and sweet orange peel samples with whilst the interday repeatability was 6.9% adequate for the aim
standards. MS/MS can be used to obtain fragment ions of structural of this work.
relevance for identifying target compounds in a highly complex
matrix. As these compounds had the same (m/z): naringin and 3.5. Calibration curves
narirutin (m/z 579.2), neoheredin and hesperedin (m/z 609.2),
MS/MS experiments of both kinds of samples comparing with the In order to quantify the amount of each compound in the bitter
MS/MS of standards were useful in order to identify these com- orange peel, naringin and neohesperidin, a calibration curve was
pounds. Figs. 2A and 2B show the MS/MS spectra of naringin and prepared with the standards between the ranges from 5 to
neohesperidin standards and in the bitter orange peel sample. Be- 50 mg/l including ve replicated of each point. In the same way,
sides, Figs. 3A and 3B show the MS/MS spectra of narirutin and in order to quantify the amount of the sweet orange peel com-
hesperidin standards and in the sweet orange peel sample. Thus, pounds, hesperidin and narirutin, a calibration curve was prepared
using the MS/MS spectra it is possible to prove that the compounds with the standards between the ranges from 25 to 80 mg/l includ-
under the current study correspond with the assignment proposed. ing ve replicated of each point. All calibration curves showed
good linearity in the studied range of concentration. Regression
3.3. Analytical parameters of the method proposed coefcients were higher than 0.985 for narigin and neohesperidin
and higher than 0.997 for narirutin and hesperidin. All the features
We carried out a study to check the repeatability of the pro- of the proposed method are summarised in Table 1.
posed method, as well as to establish the calibration curves to
quantify naringin and neohesperidin in bitter orange peel and nari- 3.6. Quantication of the main polyphenols in bitter and sweet orange
rutin and hesperidin in sweet orange peel. samples
3.4. Repeatability study The proposed method was applied to the quantication of
naringin, neohesperidin, narirutin and hesperidin in bitter and
Repeatability of the CEESIITMS analysis was studied by per- sweet orange peel real samples. In Figs. 4A and 4B the extracted
forming series of separations using the optimised method on the ion electropherogram for each target compound of bitter and
extracts in the same day (intraday precision, n = 5) and on three sweet orange peel are shown. The studied compounds were diluted
consecutive days (interday precision, n = 15). The relative standard in order to x them in the calibration range. Finally, the results ex-
deviations (RSDs) of analysis time and peak area were determined. pressed in mg analyte/g of dry weight peel (n = 5; value = X SD)
The intraday repeatability of the analysis time (expressed as RSD) were 5.1 0.2 and 7.9 0.7 mg/g of naringin and neohesperidin
was 0.22%, whilst the interday repeatability was 0.89%. The intra- in bitter orange peel and 26.9 2.1 and 35.2 3.6 mg/g of narirutin
day repeatability of the peak area (expressed as RSD) was 6.5%, and hesperidin in sweet orange peel, respectively.
Intens.
x10 5
a
8
0
0 2 4 6 8 10 12 14 Time [min]
Intens.
x106 b
3
0
0 2 4 6 8 10 12 14 Time [min]
Fig. 4B. Extracted ion electropherograms of: (a) narirutin and (b) hesperidin in sweet orange peel sample.
4. Conclusions Arrez-Romn, D., Gmez-Caravaca, A. M., Gmez-Romero, M., Segura-Carretero, A.,

& Fernndez-Gutirrez, A. (2006). Identication of phenolic compounds in
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CHAPTER II: Characterization of phenolic compounds in diatomaceous
earth used in the filtration process of olive oil by HPLC-ESI-TOF (MS).
97
This work was published in AgroFood industry hi-tech Journal.
Characterization of phenolic compounds in diatomaceous earth used in the filtration

process of olive oil by HPLC-ESI-TOF (MS)
(Journal of AgroFood industry hi-tech (2009) 20, 46-50)

Fernndez-Gutirrez.
98
Characterization of
phenolic compounds in
diatomaceous earth
used in the filtration process of olive
oil by hplc-esi-tof (ms)
SALEH SAWALHA, DAVID ARREZ-ROMN, ANTONIO SEGURA-CARRETERO*, ALBERTO FERNNDEZ-GUTIRREZ*
*Corresponding authors
University of Granada, Faculty of Sciences, Department of Analytical Chemistry
C/Fuentenueva s/n, Granada, 18071, Spain
ABSTRACT: The main producer of olives and olive oil is Europe Union with over 80 percent. Olive oil production processes
produces a large amount of by-products, where the healthy value of olive oil is undervalued. This study has been carried
out to determine the phenolic content in diatomaceous earth used in the filtration step which is the last step in the
production processes of olive oil. We propose an HPLC-ESI-TOF (MS) method for the separation and detection of a broad
Food technologies
series of phenolic compounds present in the diatomaceous earth. Thus, we achieved the characterization of 19 phenolic
compounds from several important families (phenolic alcohols, secoiridoids, lignans, phenolic acids and flavonoids) of the
polar fraction of olive oil. Furthermore, other unknown compounds were also characterized. Thus the results observed in this
study mean that diatomaceous earth used in the filtration step of olive oil production affects the phenolic composition of
olive oil, because an important amount of phenolic compounds are still present at the filtration material, being the most
abundant HYTY, Lig Agl, H-Pin, Vanillic acid, TY, Lut and Apig.
nutrition as preventive agents against several diseases (8, 9).

ABBREVIATIONS
Olive oil production is an important agricultural and
HYTY: hydroxytyrosol
alimentary sector in Europe. The European Union is the main
TY: tyrosol
world producer (10). Many olive oil producers consider
HYTY-Ac: 2-(4-hydroxyphenyl) ethyl acetate or
several factors on the effective oil quality. These factors are
hydroxytyrosol acetate
the soil condition, climate and altitude of the olive tree, time
HYTY-Glu: hydroxytyrosol glucoside
AgroFood industry hi-tech - July/August 2009 - vol 20 n 4
and system of harvest, pruning of the tree, fertiliser usage,

EA: elenolic acid
the cultivation and the production process. Some of these
DOA: decarboxylated derivatives of Ol Agl
factors had been studied to see how the polyphenolic
Ol Agl: oleuropein aglycone
content of olive VOO was affected: the cultivation (11-13),
10-H-Ol Agl: 10-hydroxy-oleuropein aglycone
climate (14). The production processes are: collecting,
Deacetoxy 10-H-Ol Agl: Deacetoxi 10-hydroxy-oleuropein
washing, pressing, decantation, centrifuging, storage,
aglycone
filtration and packaging, some of those eight steps of VOO
Decarbox-Lig Agl: decarboximethylated derivatives of
production processes (pressing, centrifuging and storage)
ligstroside aglycone
had been studied about polyphenolic contents in VOO (15-
Lig Agl: ligstroside aglycone
20), but there is a lack of information and studies available
Pin: (+)-pinoresinol
about the filtration step (21, 22), which is the last step just
Ac Pin: (+)-1-acetoxypinoresinol
before packaging. At this step, the filter used for olive oil
H-Pin: hydroxy-pinoresinol
filtration from several years ago is diathomite, which is the
Lut: luteolin
fossilized remains of microscope algae, also called
diatomaceous earth.
introduction This filtration process affects the characteristics of VOO, in
particular, oxidative stability, water content, and the
There is a rising interest in natural antioxidants as bioactive presence of each phenolic compound. Considering the fact
components of foods. The importance of the antioxidant that polyphenols are polar compounds which can found in
constituents of plant material in the maintenance of health the olive fruit; however many of these compounds are
and protection from coronary heart disease and cancer is modified or lost during the production process of VOO (23).
also raising interest among scientists, food manufacturers Therefore, the qualitative study of phenolic compounds in
and consumers (1). In recent years, interest in natural the diatomaceous earth used in the filtration process of VOO
antioxidants from vegetable substances has been related to is very important.
their therapeutic properties (2). Among the different Thus, in the present study hplc coupled with Mass
vegetable oils, Virgin Olive Oil (VOO), which is the juice of Spectrometry (MS) detection was used, since
the olive obtained by pressing, is one of the few oils that are this is one of the most accurate analytical
consumed without any further refining process. The techniques used for the analysis of phenolic
antioxidant potential of phenolic compounds in olive oil has compounds. Moreover the advantages of
been a subject of great interest, because of its MS detection include the capability to both
chemoprotective effect in human beings (3-5). Phenolic determine molecular weights and to obtain structural
compounds are of fundamental importance for their information (24). The on-line coupling of hplc with MS
nutritional properties, sensory characteristics, and the shelf using Electrospray Ionization (ESI) as an interface yields a
life of VOO (6, 7). They also play an important role in human powerful method because ESIMS allows the determination
46
of a wide range of polar compounds. ESI is one of the most B in 10 minutes; 30 percent B to 33 percent B in 2 minutes; 33
versatile ionization methods, and is the method of choice for percent B to 38 percent B in 5 minutes; 38 percent B to 50
the detection of ions separated by liquid chromatography. percent B in 3 minutes; 50 percent to 95 percent in 3 minutes.
Although hplc can be coupled to different MS analyzers The initial conditions were re-established in 2 minutes and
(quadrupole, ion trap (IT), time-of-flight (TOF), etc) (25), TOF held for 10 minutes more. The total run time, including the
(MS) provides excellent mass accuracy over a wide dynamic conditioning of the column to the initial conditions, was 35
of range if modern detector technology is used (26). The min. The flow rate used was set at 0.80 mL/min throughout
latter allows also measurements of the isotopic pattern (27, the gradient. The effluent from the hplc column was split
28), providing with an important additional information for using a T before being introduced into the mass
the determination of the elemental composition (29), in this spectrometer (split ratio 1:3). Thus in the current paper the
article we have used hplc-ESI-TOF (MS) to analyze the flow which arrived to the ESI-TOF-MS detector was 0.2 mL/
phenolic compounds present in diatomaceous earth. min. The column temperature was maintained at 25C and
The aim of this work was the separation and the the injection volume was 10 L.
characterization of a broad series of phenolic compounds
present in the diatomaceous earth used in the filtration esi-tof (ms)
process of VOO by hplc-ESI-TOF (MS), which was achieved esi-tof (ms) conditions were optimized in order to provide
for the first time. strong mass signals for all the studied phenolic compounds.
The hplc system was coupled to a tof-ms equipped with
an ESI interface operating in negative ion mode. The
Experimental section optimum ESI parameters were as follows: nebulizing gas
pressure, 2 bars; drying gas flow, 9 L/min; drying gas
Food technologies
Reagents and materials temperature, 190C.
All chemicals were of analytical reagent grade and used as MS was performed using the microtof (Bruker Daltonik,
received. The organic solvents, hexane, methanol and ACN, Bremen, Germany), an orthogonal-accelerated TOF mass
used in the extraction procedure and as hplc mobile phase spectrometer (oaTOF-MS). Transfer parameters were
were purchased from Lab-Scan (Dublin, Ireland). Acetic acid optimized by direct infusion experiments with Tuning Mix
used in hplc phase A was purchased from Fluka (Switzerland). (Agilent Technologies) in the range of 50-800 m/z looking for
Deionised water was obtained from a water purifier system the best conditions regarding sensitivity and resolution. Thus,
(Millipore, Bedford, MA). All the solvents used in the HPLC the endplate offset was -500 V; capillary voltage 4500 V, the
system were filtered through a 0.20 m Millipore (Bedford, MA, trigger time was set to 50 s, 49 s for set transfer time and 1
USA) membrane filters. s pre-puls storage time, corresponding to a mass range of
50800 m/z. Spectra were acquired by summarizing 20,000
Apparatus single spectra, defining the spectra rate to 1Hz. The
hplc accurate mass data of the molecular ions were processed
The separation of the phenolic compounds from extra-virgin through the software Data Analysis 3.4 (Bruker Daltonik),

olive oil was performed using an Agilent 1200 series Rapid which provided with a list of possible elemental formulas by
Resolution LC (Agilent Technologies, Palo Alto, CA, USA), using the Generate Molecular Formula Editor. The
which was equipped with a vacuum degasser, an Generate Formula Editor uses a chno algorithm, which
autosampler, a diode-array detector (DAD), a binary pump, provides with standard functionalities such as minimum/
and a thermostated column department. The samples were maximum elemental range, electron configuration, and ring-
separated using a reversed-phase C18 analytical column plus double bonds equivalents, as well as a sophisticated
(4.6150 mm, 1.8 m particle size, Agilent zobrax Eclipse comparison of the theoretical with the measured isotope
plus). The mobile phase A and B consisted of water with 0.5 pattern (Sigma Value) for increased confidence in the
percent acetic acid, and ACN. The chromatographic suggested molecular formula (Bruker Daltonics Technical
method was as Note #008, Molecular formula determination under
following: gradient automation). The widely accepted accuracy threshold for
from 5 percent confirmation of elemental compositions has been
B to 30 percent established at 5 ppm (30). It must be added even with very
high mass accuracy (<1 ppm) many chemically possible
formulae are obtained depending on the mass regions
considered. So, high mass accuracy (<1 ppm) alone is not
enough to exclude enough candidates with complex
elemental compositions. The use of isotopic abundance
patterns as a single further constraint removes >95
percent of false candidates. This orthogonal filter
can condense several thousand candidates down
to only a small number of molecular formulas.
During the development of the hplc method,
external instrument calibration was performed using
a 74900-00-05 Cole Palmer syringe pump (Vernon Hills,
Illinois, USA) directly connected to the interface, passing
a solution of sodium formate cluster containing 5 mM
sodium hydroxide in water/isopropanol 1/1 (v/v), with
0.2 percent (v/v) of formic acid at the end of each run.
Using this method an exact calibration curve based on
numerous cluster masses each differing by 68 Da
(NaCHO2) was obtained. Due to the compensation of
temperature drift in the Microtof, this external calibration
provided with accurate mass values (better 5 ppm) for a
47
Food technologies
Table 1. Well-known phenolic compounds determined by HPLC-ESI-TOF (MS) in Diatomaceous Earth.
complete run without the

need for a dual sprayer
setup for internal mass
calibration. These
calibrations were
performed in quadratic +
high precision calibration
(HPC) regression mode.
Sample
The Diatomaceous earth
filter used in the olive oil
industry was composed
of 75 percent Celite545
and 25 percent
Kenite700. This filter was
used in the last step of
VOO production in order
to improve its quality.
Extraction procedure
The extraction procedure
was: 20 g of the
Diatomaceous earth
were weighted in a
beaker 500 ml, 100 ml of
hexane were added to
clean the sample from
the no polar fraction of
the oil, and then the
solution was shaken by
magnetic stir 2 h, after this
time the solution was
filtered through normal
filtration using Whatman
filter paper No. 4. The
sample was collected Figure 1. EIEs of the well-known phenolic compounds detected in Diatomaceous Earth extract containing information about the
from the filter paper m/z experimental.
carefully another time in
beaker 500 ml, 120 ml of methanol were added and the through normal filtration using Whatman filter paper No.4.
solution was shaken by magnetic stir 2 h at 35C. The solution Then it was separated into 5 centrifuge tubes and centrifuged
was left overnight. After this time the solution was filtered at 1.9 g for 10 min. The solution was collected in a round
48
Table 2. Unknown phenolic compounds determined by HPLC-ESI-TOF (MS) in Diatomaceous Earth.
bottom flask. The concentrated methanol was evaporated by and migration time. Figure 1 shows the Extracted Ion
rotary pump below 40C, and the dry residue was resolved by Electropherograms (EIE) of the several major compounds
Food technologies
4 ml of methanol. Finally, the solution was filtered through a 0.2 present in diatomaceous earth sample.
m filter before the hplc analysis.
Thus, the proposed method is able to detect nineteen
phenolic compounds in the same run. Furthermore, all
Results and discussion detected compounds observed in Table 1 exhibited good
sigma values smaller than 0.05 and mass accuracy (ppm and
Well-known phenolic compounds mDa) as indicated by the error values, even a low tolerance
Under the proposed hplc-esi-tof (MS) method, a large was chosen (5 ppm), except in two cases Vanillin and
number of well-known phenolic compounds present in O-Coumaric acid the tolerance was 10 and 12 ppm
diatomaceous earth were detected. These are summarized in respectively, meanwhile the sigma values were below 0.05.
Table 1, with their formula, selected ion, experimental and We could detect four phenyl alcohols (HYTY, TY, HYTY-Ac and
calculated m/z, error (ppm and mDa), sigma value, tolerance HYTY-Glu), several compounds from secoiridoid family (EA,
49
DOA, Ol Agl, 10-H-OI AgI, Deacetoxy 10-H-Ol Agl, Decarbox- References and notes
lig Agl and Lig Agl), three lignans (Pin, Ac Pin and H-Pin), three
phenolic acids (vanillin, vanillic acid and O-Coumaric acid) 1. J. Lliger, Taylor et al., London, UK, p. 129, (1991).
and also the present method allowed the determination of 2. P.C.H. Hollman, M.G. L.Hertog et al., Food Chem., 57(1), pp.
two flavonoids (Lut and Apig). All the compounds detected in 43-46 (1996).
this work have been described in the previous studies on 3. F. Caponio, T. Gomes et al., Eur. Food Res. Technol., 212, pp. 329-
VOO, which means that the diatomaceous earth used in the 333 (2001).
filtration process of VOO can affect the phenolic composition 4. R. Briante, F. La Cara et al., J. Agric. Food Chem., 49, pp. 3198-
of the final product. 3203 (2001).
5. L. Cerretani, A. Bendini et al., AgroFood Ind Hi Tec., 19, pp. 64-66
(2008).
Unknown phenolic compounds
6. G. F. Montedoro, M. Baldioli et al., Nutr. Clin.Prevent., 1, pp.
Besides the previously mentioned phenolic compounds
19-31(1991).
detected with the above described method, it was also
7. M. Tsimisou., J. Food Sci., 10, pp. 99-116 (1998).
possible to study other compounds present the diatomaceous 8. A. Romani, N. Mulinacci et al., J. Agric. Food Chem.,47, pp. 964-
earth fraction, which had not been described before in the 967 (1999).
literature. Table 2 summarizes all the results for 17 unknown 9. R.W. Owen, A. Giacosa et al., Lancet Oncol., 1, pp. 107-112
compounds including migration time, experimental m/z, (2000).
selected ion, tolerance (ppm), list of possible molecular 10. Anonymous, Faostst, Database, www.fao.org (last access on:
formulas, error and sigma value for the compound with 19.11.2004).
molecular formula CXHYOZ. These compounds have been 11. M.J. Tovar, M.J. Motilva et al., J. Agric. Food Chem., 49(11), pp.
5502-5508 (2001).
included since they suppose a significant fraction of the
Food technologies
12. M.J. Tovar, M.P. Romero et al., J. Sci. Food Agric., 82(15), pp.
extract from diatomaceous earth sample. A reduced number
1755-1763 (2002).
of possible elemental compositions are obtained from the
13. M. Servili, S. Esposto et al., J. Agric. Food Chem., 55(16), pp. 6609-
accurate mass of the suspected peak. These elemental 6618 (2007).
compositions can then be matched against available 14. (M. Bonoli, A. Bendini et al., J. Agric. Food Chem., 52(23), pp.
databases (The Merck Index, ChemIndex, commercial 7026-7032 (2004).
e-catalogues) using the deduced molecular formula as a 15. A. Parenti, P. Spugnoli et al., J. Lipid. Scien and Tech., 110(8), pp.
search criterion (31, 32). It has to be mentioned that some of 753-741(2008).
the possible elemental compositions calculated within a 16. M. Servili, R. Selvaggini et al., J. Agric. Food Chem., 51(27), pp.
certain mass accuracy does not seem to be chemically 7980-7988 (2003).
17. M. Servili, R. Selvaggini et al., J. Amer. Oil Chem Society, 80(7),
coherent. This fact helps in the unequivocal identification of
pp. 685-695 (2003).
the unknown species and the assignment of its correct
18. F. Angerosa, L. di Giovacchino et al., Grasas y Aceites ., 47(4),
elemental composition since it reduces the number of
pp. 247-254 (1996).
possibilities. 19. (F. Angerosa, R. Mostallino et al., J. Scie. Food. Agric., 80(15), pp.
2190-2195 (2000).
20. M. Brenes, M. Gracia et al., J. Agric. Food Chem., 49(11), pp.

Conclusion 5609-5614 (2001).
21. A. Bottino, A. Capannelli et al., J. Food. Engin., 65(2), pp. 303-309
In this work a large number of well-known phenolic (2004).
compounds present in diatomaceous earth extracts can be 22. A. M. Gmez-Caravaca, L. Cerretani et al., Am. J. Food.
Technol., 2(7), pp. 671-678 (2007).
separated and identified. Due to the hyphenation of HPLC to
23. M. Brenes et al., J. Agric. Food Chem., 43, pp. 2702-2706 (1995).
MS, which combines the advantages of HPLC with the
24. A. Carrasco-Pancorbo, C. Neus et al., Electrophoresis, 28, pp.
selectivity, sensitivity, mass accuracy and measurements of
806-821(2007).
the isotopic pattern associated with TOF (MS), the described 25. C. Sim, M. Herrero et al., Electrophoresis, 26, pp. 2674-2683
HPLC-ESI-TOF (MS) method represents a valuable tool and a (2005).
good alternative for simultaneous characterization of 26. A.W.T. Bristow, K.S. Webb., J Am Soc Mass Spectrom., 24, pp.
phenolic components in diatomaceous earth. 1086-1098 (2003).
27. M. Pelzing, J. Decker et al., Talk A042670. In: 52nd asms Conf on
Mass Spectrometry and Allied Topics, 2327 May, Nashville, TN
acknowledgements (2004).
28. D. Arrez-Romn, S. Sawalha et al., AgroFood Ind Hi Tec., 19, pp.
18-22 (2008).
The authors are grateful to the Spanish Ministry of Education
29. G. Bringmann, I. Kajahn et al., Electrophoresis, 26, pp. 1513-1522
and Science for the project (AGL2008-05108-C03-03) and to
(2005).
Andalusian Regional Government Council of Innovation and 30. I. Ferrer, J.F. Garca-Reyes et al., J. Chromatogr. A, 1082, pp.
Science for the project P07-AGR-02619. The authors also thank 81-89 (2005).
Aceites Maeva, S.L. for providing the samples. The author SS 31. M. Ibez, J.V. Sancho et al, Rapid Commun. Mass Spectrom.,
gratefully acknowledges the Agencia Espaola de 19, pp. 169-178 (2005).
Cooperacin Internacional (AECI). 32. T. Kina, O. Fiehn, bnc Bioinformatics, 7, pp. 234-243 (2006).
50

CHAPTER III: Identification of phenolic compounds in olive leaves using
CE-ESI-TOF-MS
104
This work was published in AgroFood industry hi-tech Journal.
Identification of phenolic compounds in olive leaves using CE-ESI-TOF-MS.

(Journal of AgroFood industry hi-tech (2008) 20, 18-22)
Saleh M.S. Sawalha, Antonio Segura-Carretero, Alberto Fernndez-Gutirrez.

David Arrez-Romn.
Verbionat S.C.A, C/ Santa F de Bogot 45
Santa F, 18320, Granada, Spain
Javier Menedez.
Catalan Institute of Oncology (ICO)
Health Services Division of Catalonia, Spain.
105
Identification of
phenolic compounds
in olive leaves using CE-ESI-TOF-MS
DAVID ARREZ-ROMN1, SALEH SAWALHA2, ANTONIO SEGURA-CARRETERO2*,
JAVIER MENENDEZ3, ALBERTO FERNNDEZ-GUTIRREZ2*
*Corresponding authors
1. Verbionat S.C.A, C/ Santa F de Bogot 45
Santa F, 18320, Granada, Spain
2. Department of Analytical Chemistry, Faculty of Sciences, University of Granada
C/ Fuentenueva s/n, Granada, 18071, Spain
3. Catalan Institute of Oncology (ICO)
Health Services Division of Catalonia, Spain
ABSTRACT: An easy and rapid method using capillary electrophoresis coupled with electrospray ionization time-of-flight-
mass spectrometry (CE-ESI-TOF-MS) has been developed to analyze phenolic compounds in two varieties of olive
leaves (Hojiblanca and Manzanilla). The separation parameters have been performed in respect to resolution, sensitivity,
analysis time and peak shape. Namely the optimization of both electrophoretic parameters and electrospray conditions
are required for reproducible analyses. The method allows the simultaneous identification of seventeen and fourteen
phenolic compounds in Hojiblanca and Manzanilla leaves extracts respectively. Due to its high efficiency, rapidity, small
sample amounts required and high resolution of CE coupling to the sensitivity, selectivity, mass accuracy and true
isotopic pattern from TOF-MS have revealed an enormous separation potential allowing the identification of a broad
Polyphenols
series of phenolic compounds present in olive leaves.
INTRODUCTION be a high-resolution technique (14-17). Regarding the

advantages of MS detection include the capability of
Natural antioxidants are primarily plant polyphenolic determination of molecular weight and providing structural
compounds that may be obtained from plant parts. Plant information (18). Also TOF-MS provides excellent mass
phenolics are multifunctional and can act as reducing agents accuracy (19) over a wide dynamic range. The latter,
(free radical terminators), metal chelators, and singlet oxygen moreover, allows measurements of the isotopic pattern (20),
quenchers (1). Crude extract of fruits, herbs, vegetables, providing important additional information for the
cereals, nuts and other plant materials rich in phenolics are determination of the elemental composition (21). Thus, the
AgroFOOD industry hi-tech
increasingly of interest in the food industry (2). The on-line coupling of CE with TOF-MS yields a powerful
importance of the antioxidant constituents of plant material in technique for the analysis of phenolic compounds (22). The
the maintenance of health and protection from coronary heart goal of this work is to develop a new, rapid and simple
disease and cancer is also raising interest among scientists, CE-ESI-TOF-MS method to identify phenolic compounds in
food manufacturers, and consumers (3). two varieties of olive leaves (Hojiblanca and Manzanilla).
Olive oil production is an important agricultural and alimentary
sector in Europe. The European Union is the main world
-
producer, and during the season 2003/2004, 2.282.650 tons EXPERIMENTAL SECTION
vol 19 n 6 - November/December 2008
were produced in several thousand of olive oil mills (4). From

this important industry, both, olive tree culture and the olive oil Reagents and materials
industry, produce large amounts of by-products. It has been All chemicals were of analytical reagent grade and used as
estimated that pruning alone produces 25 kg of by-products received. Ammonium hydroxide was from Fluka (Buchs,
(twigs and leaves) per tree annually. It must also be Switzerland) and ammonium acetate and methanol from
considered that leaves represent 5 percent of the weight of Merck (Darmstadt, Germany). 2-propanol HPLC grade used
olive oil extraction then this represent a significant by-product in the sheath flow, methanol and sodium hydroxide, used for
in olive oil production process (5). Historically, olive leaf has capillary cleaning procedures before each analysis, were
been used as a folk remedy for combating fevers and other obtained from Panreac (Barcelona, Spain) and triethylamine
diseases, such as malaria. Several reports have shown that from Aldrich (Steinheim, Germany). Distilled water was
olive leaf extract and also olive oil had the capacity to lower deionised by using a Milli-Q system (Millipore, Bedford, MA).
blood pressure in animals (6, 7) and increased blood flow in CE buffers were prepared by weighing ammonium acetate at
the coronary arteries (8), relieved arrhythmia and prevented the concentrations indicated and adjusting the pH when
intestinal muscle spasms. This type of by-products (olive necessary by adding ammonium hydroxide. The buffers were
leaves) are a rich source of an important number of phenolic stored at 4C and warmed to room temperature before use.
compounds (9-13). However, the analysis of these All solutions were filtered through a 0.45 m Millipore
compounds is not an easy work. Because of this, the (Bedford, MA, USA) membrane filters before injection into the
characterization of individual olive leaves compounds capillary.
requires the use of separate techniques. Thus, due to its high
efficiency, flexibility, very high resolution and rapidity of the Apparatus
method, CE has gained widespread interest as a favourable CE experiments were performed using a P/ACETM System
technique for the analysis of phenolic compounds. It has MDQ (Beckman Instruments, Fullerton, CA, USA) and fused-
become an alternative or complementary technique to silica capillaries of 85 cm in length and 50 m inner diameters
chromatographic separations for the analysis of phenolic (360 m outer diameters) coupled to the MS detector by an
compounds because it needs no derivatization step, requires orthogonal electrospray interface (ESI) with a coaxial sheath-
only small amounts of sample and buffer and has proved to liquid (Agilent Technologies, Palo Alto, CA, USA) delivered by
18
Polyphenols
AgroFOOD industry hi-tech - November/December 2008 - vol 19 n 6
Figure 1. EIEs of the well-known phenolic compounds detected in Hojiblanca leaves extract containing information about the m/z experimental
a 5 mL gas-tight syringe (Hamilton, Reno, NV, USA) using a Spectra were acquired by summarizing 20,000 single spectra,
syringe pump of 74900-00-05 Cole-Parmer (Vernon Hill, IL, defining the spectra rate to 1 Hz.
USA). MS experiments were performed using the
micrOTOFTM (Bruker Daltonik GmbH, Bremen, Germany), an Sample preparation
orthogonal-accelerated TOF mass spectrometer (oaTOF- The variety Hojiblanca olive tree is at least 200 years old and
MS). An electrospray potential of +4.1 kV was applied at the is localized in the shadow area of dry lands. The collection is
inlet of the MS (negative ion polarity). The trigger time was made directly from the tree. Afterwards the leaves were
set to 50 s, 49 s for set transfer time and 1 s pre-pulse washed using only distilled water, in order to avoid
storage time, corresponding to a mass range of 50800 m/z. polyphenols degradation. Later, this water was introduced
19
Polyphenols
-
Figure 2. EIEs of the well-known phenolic compounds detected in Manzanilla leaves extract containing information about the m/z experimental
into a stove with forced air at 40C during 48 hours for repeated four times. Then, the concentrated methanol was
dehydration purposes. The entire leaf was kept in paper evaporated by rotary pump at 40C and the sample was
envelopes. Once it was grounded, the leaf was introduced resolved in 4 ml of MeOH:H 2O (50:50 v/v). Finally the
in sealed glass jars, wrapped in aluminium foil, and then extract was kept in the freezer until the analysis.
kept in the refrigerator. The variety Manzanilla olive tree is
at least 20 years old and is localized in the sunshine area of
dry lands. The collection, washing and conservation RESULTS AND DISCUSSION
procedures were identical to those ones described above.
In the present study the two varieties of olive leaves CE-ESI-TOF-MS method
samples were characterized. The extraction procedures In order to develop the optimization of CE-ESI-TOF-MS
were as follows: 0.5 g of the dried (powder) sample was method, the extract of Hojiblanca leaves was used. The
weighted in a 10 ml test tube. 5 ml of methanol were added CE-ESI-TOF-MS method was developed in order to obtain
and the solution was shaken on vortex 5 minutes and the best selectivity, sensitivity and resolution. Initially, the
centrifuged at 4500 r.p.m for 10 minutes. The liquid part was electrophoretic conditions were optimized based on the
collected in a round bottom flask. These steps were migration behaviour, sensitivity, analysis time and peak
20
shape. First, different buffers compatible with CE-ESI-MS compounds present in Hojiblanca and Manzanilla leaves
were used (ammonium acetate/NH3 and ammonium borate/ extracts is given in Figure 1 and Figure 2 respectively. The
NH3) and the best results were obtained using ammonium reproducibility of the CE-ESI-TOF-MS analysis, expressed
acetate/NH3. Thus, 50 mM ammonium acetate as running by the RSD percent of five consecutive injections was 1.04
buffer was selected, due to its best performance to achieve percent for the analysis time and 5.89 percent for the peak
a high signal response as well as a good resolution. area, both measured for each peak.
Moreover, different pHs were tested in the range of 8 to
10.5. Finally, pH 9.5 gave the best results in term of peak Use of TOF-MS for the identification of phenolic
shape, resolution and analysis time. Under these CE compounds
experimental conditions, a voltage of 30 kV shortened the The accurate mass data of the molecular ions were
analysis time and yielded good separation and acceptable processed through the software DataAnalysis 3.3 (Bruker
current. The injections were made at the anodic end using a Daltonik GmbH), which provided a list of possible elemental
N2 pressure 0.5 p.s.i. for 20 s (1 p.s.i. = 6894.76 Pa). These formula by using the GenerateMolecularFormulaTM editor.
conditions were chosen for the subsequent optimization of The GenerateFormula TM editor uses the sigmaFit TM
the ESI-TOF-MS parameters. It is well known that the algorithm, which provides standard functionalities such as
choice of sheath liquid has significant effects on sensitivity minimum/maximum elemental range, electron configuration
and in the electrical contact between CE and ESI (23, 24). and ring-plus double bonds equivalents, as well as a
Thus, we optimized the sheath liquid by varying the ratio at sophisticated theoretical and measured comparison of the
different isopropanol/water solutions. The use of an isotope pattern (SigmaValueTM) for increased confidence in
isopropanol/water mixture 60:40 (v/v) resulted in the highest the suggested molecular formula (29). An external
TOF-MS signal. Generally, a small amount of volatile calibration was performed using sodium formate cluster by
triethylamine (TEA) or ammonium hydroxide is used for switching the sheath liquid to a solution containing 5 mM
ESI-negative detection (25). For that reason 0.1 percent sodium hydroxide in the sheath liquid of 0.2 percent formic
(v/v) TEA was added yielding a better sensitivity. The sheath acid in water:isopropanol 1:1 v/v at the end of the analysis.
liquid flow was expected to dilute the CE sample zone as it Using this method an exact calibration curve based on
passed concentrically around the CE column effluent and numerous cluster masses each differing by 68 Da (NaCHO2)
Polyphenols
mixed with it. Finally, 0.20 mL/h was selected as optimum in was obtained. This external calibration provided accurate
terms of signal response and stability. Nebulizer gas mass values (better 5 ppm) for a complete run without the
pressure is a compromise between maintaining an efficient need for a dual sprayer setup for internal mass calibration.
electrophoretic separation and improving the ionization All the detected phenolics compounds in Hojiblanca and
performance (26-28) obtaining the best signal at 0.4 Bar. Manzanilla leaves extracts are summarized in Tables 1 and
Finally the dry gas temperature was found to be optimal at 2 respectively, with their formula, selected ion, m/z
180C with the best dry gas flow rate at 4 L/min. Under experimental and calculated error (ppm and mDa), sigma
these conditions, the Extracted Ion Electropherograms value, tolerance and migration time. Thus, the proposed
(EIEs) for a large number of well-known phenolic method is able to detect seventeen phenolic compounds in
AgroFOOD industry hi-tech - November/December 2008 - vol 19 n 6
21
Table 1
Polyphenols
Table 2
Hojiblanca and fourteen in Manzanilla leaves in the same from Consejera de Innovacin, Ciencia y Empresa and
run with an accuracy of 5 mDa. All detected compounds Excellence Proyect AGR 02619, both from Junta de Andaluca.
observed in Table 1 and 2 exhibited good sigma values and

mass accuracy (ppm and mDa) as indicated by the error REFERENCES AND NOTES
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7. V. Di Fronzo, R. Gente et al., Agro Food Ind Hi Tec., 18, pp. 4-5 (2007).
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alternative for simultaneous characterization of phenolic 41, pp. 1648-1656 (2006).
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sensitivity and mass accuracy inherent to TOF-MS. Due to (2008).
TOF-MS provides excellent mass accuracy over a wide 19. A.W.T. Bristow, K.S. Webb et al., J. Am. Soc. Mass Spectrom., 24, pp.
1086-1092 (2003.)
dynamic range and allows measurements of the isotopic 20. M. Pelzing, J. Decaer et al., talk A042670, presented on 52nd ASMS
pattern, it provides important additional information for the Conference on Mass Spectrometry and Allied Topics, Nashville, TN, May
23 (2004).
determination of the elemental composition. 21. G. Bringmann, I. Kajahn et al., Electrophoresis, 26, pp. 1513-1522 (2005).
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(2001).
The author DAR gratefully acknowledges the Torres Quevedo 25. R.D. Voyksner, Electrospray Ionization Mass Spectrometry, Wiley, New
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contract from Ministerio de Educacin y Ciencia in Verbionat 26. R. Sheppard, X.Tong et al., Anal. Chem., 67, pp. 2054-2058 (1995).
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Cooperacin Internaciona (AECI). The authors also gratefully 28. K. Huikko, T. Kotiaho et al., Rapid Comun. Mass Spectrom., 16, pp. 1562-
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acknowledge the financial support of CTQ2005-01914/BQU 29. Bruker Daltonics Technical Note #008, Molecular formula determination
and AGL2008-05108-CO3-03/ALI from MEC, the P431073 under automation.
22

CHAPTER IV: HPLC/CE-ESI-TOF (MS) methods for the characterization of
polyphenols in almond skin extracts
111
This work was submitted to Electrophoresis Journal.
HPLC/CE-ESI-TOF (MS) methods for the characterization of polyphenols in almond

skin extracts.

Fernndez-Gutirrez.
112
1 HPLC/CE-ESI-TOF (MS) methods for the characterization of polyphenols in
2 almond skin extracts
4 Saleh M. S. Sawalha, David Arrez-Romn, Antonio Segura-Carretero, Alberto
5 Fernndez-Gutirrez.
7 Department of Analytical Chemistry, Faculty of Sciences, University of Granada,
8 C/Fuentenueva s/n, E-18071 Granada, Spain
10 Correspondence: Dr. A. Fernndez-Gutirrez, Research Group FQM-297, Department
11 of Analytical Chemistry, Faculty of Sciences, University of Granada, C/Fuentenueva
12 s/n, E-18071 Granada, Spain
13 E-mail: albertof@ugr.es
14 Fax: +34958249510
15
16 Keywords: Capillary electrophoresis / high-performance liquid chromatography /

17 Electrospray ionization-time of flight-mass spectrometry / Phenolics compounds /
18 Almond skin
113
19 ABSTRACT
20
21 In this article, two rapid methods has been developing using, capillary electrophoresis
22 (CE) and high-performance liquid chromatography (HPLC) coupled to electrospray
23 ionization-time of flight-mass spectrometry (ESI-TOF-MS) have been compared for the
24 separation and characterization of antioxidant phenolic compounds in almond skin
25 extract. Under the optimum CE-ESI-TOF-MS conditions we achieved the determination
26 of nine compounds of the polar fraction in 35 min. Furthermore, by using HPLC-ESI-
27 TOF-MS method, a total of twenty-three compounds corresponding to phenolic acids
28 and flavonoids family were identified from almond skin only in 9 min. We have
29 demonstrate that the sensitivity, together with mass accuracy and true isotopic pattern of
30 the TOF-MS, allowed the identification of a broad series of known phenolics
31 compounds present in almond skin extracts using HPLC and CE as separative
32 techniques.
114
33 1. Introduction
34
35 Natural antioxidants are primarily plant polyphenolic compounds that may be obtained
36 from plant parts. Plant phenolics are multifunctional and can act as reducing agents (free
37 radical terminators), metal chelators, and singlet oxygen quenchers [1]. Crude extract of
38 fruits, herbs, vegetables, cereals, nuts and other plant material rich in phenolics are
39 increasingly of interest in the food industry [2]. The importance of the antioxidant
40 constituents of plant material in the maintenance of health and protection from coronary
41 heart disease and cancer is also raising interest among scientists, food manufacturers,
42 and consumers [3,4].
43 The need to identify the phenolic compounds meant that traditional methods should be
44 replaced for more potential methods based on the use of advances chromatographic
45 separatives techniques, such as gas chromatography GC [59] or, specially, high
46 performance liquid chromatography HPLC [1015]. Furthermore, capillary
47 electrophoresis CE has been recently applied for the analysis of phenolic compounds in
48 natural products and has opened up great expectations, especially due to the higher
49 resolution, reduced sample volume and analysis duration [1619].
50
51 At the beginning of 21st century, HPLC with reversed phase and CE are two of the most
52 modern separation techniques frequently used. There are an important number of
53 articles using HPLC with different detectors such as UV (photodiode array) [20,21],
54 fluorescence [22,23], electrochemical [24,25], biosensors [26], NMR [27], and MS [28
55 30] detectors are used. CE has been used with UV as a detection system [16- 18] and,
56 more recently, MS detectors [19,3133]
115
57 Of all the HPLC and CE detection methods reported to date, MS clearly has the greatest
58 potential. The advantages of MS detection include the capability to both determine
59 molecular weight and providing structural information. HPLC and CE can be coupled
60 with different MS analyzers (i.e., with quadrupole, (IT), (TOF), etc.) and use several
61 ionization methods (APCI, ESI, MALDI, etc.). ESI is one of the most versatile
62 ionization methods and is the natural method of choice for the detection of ions
63 separated by CE. Moreover, the coupling with TOF-MS provides excellent mass
64 accuracy [34] over a wide dynamic range if a modern detector technology is chosen.
65 The latter, moreover, allows measurements of the correct isotopic pattern [35],
66 providing important additional information for the determination of the elemental
67 composition [36].
68 Almond, scientifically know as Prunus dulcis, belongs to the family Rosaceae, and is
69 related to stone fruits such as peaches, plums, and cherries [37]. They are typically used
70 as snack foods and as ingredients in a variety of processed foods, especially in bakery
71 and confectionery products. The peach-like almond fruit consists of the edible seed or
72 kernel, the shell, and the outer hull. At maturity the hull splits open. When dry, it may
73 be readily separated from the shell. The almond pit, containing a kernel or edible seed,
74 is the nut of commerce. Shelled almonds may be sold as whole natural almonds or
75 processed into various almond forms. The whole natural almonds have had their shells
76 removed but still retain their brown skins; blanched whole almonds have had both their
77 shells and skins removed [38,39]. Usually, the removed skins will be discarded.
78 However, much study has shown that almond skins are a rich source of phenolic
79 compounds [2, 37,40,41]. In this sense, a few chromatographic methods have been
80 proposed for the identification of phenolic compounds in almond skin [37,42].
116
81 The determination of polyphenols in almonds and almond skin has been studied by
82 several analytical methods. These methods include HPLC-DAD/ESI-MS [41], in which
83 a total of 33 compounds were characterized in 88 minute, and using HPLC-
84 electrochemical detection, UV detection and LC/MS/MS, 20 polyphenols were
85 determined in 90 minute [42]. Liquid chromatography (HPLC) coupled to diode-array
86 UV (DAD-UV) and mass spectrometry (MS) has provided the most comprehensive
87 elucidation of phenolics in food and natural products. A longer LCMS method
88 identified 21 flavonoids and phenolics in almonds in 120 min [42], another shorter
89 method was proposed for the determination of 15 flavonids in almond skin in 9 minute
90 by using capillary LCMS method and determined the same number of flavonids by
91 using LC-UV in 84 minute [43]. In another research, 8 phenolics compound were
92 characterised using LC-UV in 44 minute [44], where Reverse phase HPLC coupled to
93 negative mode electrospray ionization (ESI) mass spectrometry (MS) was used to
94 quantify 16 flavonoids and 2 phenolic acids from almond skin extracts [45].
95
96 In this article we show in the first time the use CE-ESI-TOF-MS method for the
97 determination of polyphenols in almond skin and a comparative study with a new rapid
98 HPLC-ESI- TOF (MS) method.
99
100 2. Experimental
101
102 2.1. Reagents and material
103
104 All chemicals were of analytical reagent grade and used as received. The organic
105 solvents acetonitrile, used in the HPLC mobile phase, 2-propanol used in the sheath
117
106 flow, methanol and hexane were purposed from Lab-Scan (Dublin, Ireland). Formic
107 acid used in HPLC phase A and B was purchased from Fluka (Switzerland).
108 Ammonium hydroxide was from Fluka (Buchs, Switzerland) and boric acid was
109 purchased from Sigma Aldrich (St. Louis, MO). Sodium hydroxide, used for capillary
110 cleaning procedures before each analysis, was obtained from Panreac (Barcelona,
111 Spain) and triethylamine (TEA) from Aldrich (Steinheim, Germany). Distilled water
112 was deionized by using a Milli-Q system (Millipore, Bedford, MA). All solutions were
113 filtered through a 0.45 m Millipore (Bedford, MA, USA) membrane filters before
114 injection.
115
116 2.2. Extraction procedure
117
118 To isolate the phenolic fraction in almond skin we used a Liquid-Liquid Extraction
119 (LLE) procedure: 10 g of the dried sample were weighted in beaker 250 ml, 150 ml of
120 hexane were added then the solution was shaken by magnetic stir 40 min, and then
121 filtered by gravity through Whatman No.4 filter paper. Then the sample was collected in
122 250 ml round bottom flask and was stirred with 100 ml of 70% methanol under reflux
123 condition in a thermostatic water bath at 60 oC for 45 min. The resulting solution was
124 filtered by gravity through Whatman No.4 filter paper, and then the concentrated
125 methanol was evaporated by rotary evaporator under vacuum condition at 40 oC. The
126 dry residue was resolved by 2 ml of methanol: water (50:50, v/v) for analysis by CE,
127 and in 2 ml methanol for analysis by HPLC. Finally the extract was kept in the
128 refrigerator at -4 oC until the analysis.
129
118
130 2.3. CE coupling
131
132 CE experiments were performed using a Prince CE system (Prince Technologies,
133 Emmen, The Netherlands) and fused-silica capillaries of 95 cm in length and 50 m
134 inner diameters (360 m outer diameters) coupled to the MS detector by an orthogonal
135 electrospray interface (ESI) with a coaxial sheath-liquid sprayer was used (Agilent
136 Technologies, Palo Alto, CA, USA). Isopropanol/water (60:40) with 0.1% (v/v) TEA
137 was applied as sheath-liquid at a flow rate of 0.20 mL/min delivered by a 5 mL gas-tight
138 syringe (Hamilton, Reno, NV, USA) using a syringe pump Cole-Parmer (Vernon Hill,
139 IL, USA). The ESI-voltage of the TOF is applied at the end cap of the transfer capillary
140 to the MS with the spray needle being grounded. A nebulizer gas (N2) pressure of 0.4
141 bar was applied to assist the spraying. Dry gas temperature was set to 190C at a dry gas
142 flow of 4 L/min operating in negative ion mode.
143 Before first use, the bare capillaries were conditioned with 0.1 M sodium hydroxide
144 during 20 min followed by a water rinse for another 10 min. between runs the capillary
145 was flushed with water and separation buffer for 5 min. At the end of the day the
146 capillary was flushed with water for 10 min (all rinses during capillary conditioning
147 have been done using N2 at a pressure of 20 psi).
148 CE buffers were prepared by weighing boric acid and adjusting the pH when necessary
149 by adding ammonium hydroxide. The buffers were stored at 4C and warmed to room
150 temperature before use. After optimization, a running buffer 200 mM ammonium borate
151 at pH 10 was used. The separation voltage was set to 30 kV at the inlet of the capillary.
152 Injection was performed hydrodynamically at 50 mBar during 15 s, corresponding to
153 about 15 nL injected (0.9 % of the capillary).
119
154 All conditions were optimized in order to provide high resolution and strong mass
155 signals for all the studied phenolic compounds.
156
157 2.4. HPLC coupling
158
159 The separation of the phenolic compounds from almond skin was performed also by
160 using an Agilent 1100 series HPLC instrument (Agilent Technologies, Palo Alto, CA,
161 USA) was equipped with a vacuum degasser, an autosampler, a binary pump, and a
162 thermostated column department. The standards and samples were separated using a
163 reversed-phase C18 analytical column (50 x 2 mm, 2.5 m particle size; Phenomenex
164 Synergi Fusion-RP100A) with a SecuityGuardTM C18 guard column (4 x 2 mm;
165 Phenomenex Fusion-RP) maintained at 35C. The injection volume of standards and
166 samples was 5 L. The mobile phase consisted of deionised water (A) and acetonitrile
167 (B), each containing 0.1% (v/v) formic acid. The chromatographic method consisted of
168 a linear gradient from 1 to 100% B during 9.5 min. The total run time, including the
169 conditioning of the column to the initial conditions, was 13 min. The flow rate was set
170 at 0.5 mL/min throughout the gradient. The effluent from the HPLC column was split
171 using a T before being introduced into the mass spectrometer (split ratio 1:3). Thus in
172 the current paper the flow which arrived to the ESI-TOF (MS) detector was 0.2
173 mL/min. The HPLC system was coupled to a TOF (MS) by an orthogonal electrospray
174 (ESI) interface (Agilent Technologies, Palo Alto, CA, USA). A nebulizer gas (N2)
175 pressure of 2 bar was applied to assist the spraying. Dry gas temperature was set to 190
176 C at a dry gas flow of 7 L/min operating in negative ion mode.
177 All conditions were optimized in order to provide high resolution and strong mass
178 signals for all the studied phenolic compounds.
120
179
180 2.5. TOF-MS
181
182 MS was performed using the microTOF (Bruker Daltonik, Bremen, Germany), an
183 orthogonal-accelerated TOF mass spectrometer (oaTOF-MS). Transfer parameters were
184 optimized by direct infusion experiments with Tuning Mix (Agilent Technologies) in
185 the range of 50-800 m/z looking for the best conditions regarding sensitivity and
186 resolution. Thus, the endplate offset was -500 V; capillary voltage 4500 V, the trigger
187 time was set to 50 s, 49 s for set transfer time and 1s pre-puls storage time,
188 corresponding to a mass range of 50800 m/z. Spectra were acquired by summarizing
189 20,000 single spectra, defining the spectra rate to 1 Hz. The accurate mass data of the
190 molecular ions were processed through the software Data Analysis 3.4 (Bruker
191 Daltonik), which provided a list of possible elemental formulas by using the Generate
192 Molecular Formula Editor. The Generate Formula Editor uses a CHNO algorithm,
193 which provides standard functionalities such as minimum/maximum elemental range,
194 electron configuration, and ring-plus double bonds equivalents, as well as a
195 sophisticated comparison of the theoretical with the measured isotope pattern (Sigma
196 Value) for increased confidence in the suggested molecular formula (Bruker Daltonics
197 Technical Note #008, Molecular formula determination under automation). The widely
198 accepted accuracy threshold for confirmation of elemental compositions has been
199 established at 5 ppm.
200 We also have to say that even with very high mass accuracy (<1 ppm) many chemically
201 possible formulae are obtained depending on the mass regions considered. So, high
202 mass accuracy (<1 ppm) alone is not enough to exclude enough candidates with
203 complex elemental compositions. The use of isotopic abundance patterns as a single
121
204 further constraint removes > 95% of false candidates. This orthogonal filter can
205 condense several thousand candidates down to only a small number of molecular
206 formulas.
207 During the development of the CE method external instrument calibration was
208 performed using a 74900-00-05 Cole Palmer syringe pump (Vernon Hills, Illinois,
209 USA) directly connected to the interface, passing a solution of sodium formate cluster
210 by switching the sheath liquid to a solution containing 5 mM sodium hydroxide in the
211 sheath liquid of 0.2% formic acid in water:isopropanol 1:1 v/v at the end of the analysis.
212 Regarding the HPLC method, external instrument calibration was also performed using
213 a 74900-00-05 Cole Palmer syringe pump (Vernon Hills, Illinois, USA) directly
214 connected to the interface, passing a solution of sodium formate cluster at the end of
215 each run.
216 Using this method an exact calibration curve based on numerous cluster masses each
217 differing by 68 Da (NaCHO2) was obtained. Due to the compensation of temperature
218 drift in the MicroTOF, this external calibration provided accurate mass values (better 5
219 ppm) for a complete run without the need for a dual sprayer setup for internal mass
220 calibration.
221 These calibrations were performed in quadratic + high precision calibration (HPC)
222 regression mode.
223 3. Results and discussion
224
225 3.1. CE-ESI-TOF (MS) for the identification of phenolic compounds
226
122
227 Under the optimized CE-ESI-TOF (MS) method previously described above, in order to
228 obtain the best selectivity, sensitivity and resolution, the optimized CE-ESI-TOF
229 method was applied to the identification of the phenolic compounds present in the
230 almond skin extract.
231 Fig. 1 shows the extracted ion electropherograms (EIEs) for a nine of well-known
232 phenolic compounds present in the extract. These compounds are summarized in Table
233 1, with their formula, selected ion, m/z experimental and calculated, error (ppm and
234 mDa), sigma value, tolerance, migration time and the list of possibilities.
235 Thus, the proposed method is able to detect nine phenolic compounds in the same run.
236 These compounds are: (1) Quercetin-3-O-glucoside or galactoside ([M-H]-exp. 463.0906
237 m/z), (2) Isorhamnetin-3-rutinoside ([M-H]-exp. 623.1618 m/z), (3) Kampferol-3-
238 rutinoside ([M-H]-exp. 593.1508 m/z), (4) Naringenin-7-O-glucoside ([M-H]-exp.
239 433.1145 m/z), (5) Isorhamnetin-3-glucoside or galactoside ([M-H]-exp. 477.1028 m/z),
240 (6) p-Hydroxybenzoic acid ([M-H]-exp. 137.0248 m/z), (7) Naringenin ([M-H]-exp.
241 271.0601 m/z), (8) Protocatechuic acid ([M-H]-exp. 153.0185 m/z) and (9) Vanillic acid
242 ([M-H]-exp. 167.0352 m/z) all of them with an accuracy of 3 mDa.
243 As TOF-MS provides excellent mass accuracy over a wide dynamic range and allows
244 measurements of the isotopic pattern, providing important additional information for the
245 determination of the elemental composition. The identification by TOF (MS) was
246 carried out using the Generate Molecular Formula Editor. In this sense a low tolerance
247 was chosen (5 ppm) and options with a low sigma value (<5ppm) were taken into
248 account in the most cases. Therefore all detected compounds observed in Table 1
249 exhibit good sigma values smaller 0.05 and mass accuracy (ppm and mDa) as indicated
250 by the error values, except compound number 4 (Naringenin-7-O-glucoside) which
251 present a sigma value of 0.0959, but nevertheless it present a good mass accuracy.
123
252 Whereas compound number 1 (Quercetin-3-O-glucoside or galactoside) has a little bit
253 high error (5.3 ppm), but present a good sigma value (0.0311).
254 Therefore, using this method, nine phenolic compounds can be determined and
255 identified in 35 minute in an almond skin extract. These detected compounds can be
256 classified as phenolic acids and flavonoids family. Thus, we can charaterized three
257 phenolic acids (p-Hydroxybenzoic acid, Protocatechuic acid and Vanillic acid) between
258 21.3 - 35 min. These three compounds were the first hits in the list of possibilities (see
259 table 1). Furthermore, the method allows the determination 6 flavonids, 3 of them are
260 flavonids with sugar bond (e.g. glucose, rutinose) detected in the same sequence
261 between 19.3 - 20.2 min (Isorhamnetin-3-rutinoside, Kampferol-3-rutinoside,
262 Naringenin-7-O-glucoside). Unfortunately, two of the detected compounds are
263 (Quercetin-3-O-glucoside or galactoside and Isorhamnetin-3-glucoside or galactoside),
264 which they are glycosides with glucose or galactose bond being mass isomer. These
265 compounds have the same aglycone and cannot be differentiated in this method as they
266 have identical molecular weights. The sixth one of flavonids is the aglycone Naringenin
267 detected at 22.5 min. Naringenin and the three phenolic acids detected by this method
268 did not yielded fragmentation patterns while the other five compounds yielded
269 fragmentation as we have seen in Table 1.
270 Most of the compounds found in this work using CE method, have been previously
271 described in almond skin using HPLC [42,43], but this is the first time that these
272 compounds have been characterized by CE method.
273
274 3.2. HPLC-ESI-TOF (MS) for the identification of phenolic compounds
275
124
276 All the well-known compounds which were detected and identified by CE-ESI-TOF
277 (MS) were also identified using HPLC-ESI-TOF (MS). Fig. 2 shows the extracted ion
278 electropherograms (EIEs) of the major phenolic compounds in almond skin. These
279 compounds are summarized in Table 2, with their formula, selected ion, m/z
280 experimental and calculated, error (ppm and mDa), sigma value, tolerance, migration
281 time and the list of possibilities.
282 These compounds were characterised by TOF (MS) and carried out using the Generate
283 Molecular Formula Editor. First of all, a low tolerance was chosen (5 ppm). After that,
284 options with a low sigma value (<0.05) and a low error (<5ppm) were taken into
285 account in most cases, most of them were the first hits in the list of possibilities and
286 presents a good sigma and error values (see table 2).
287 Thus, first five phenolic compounds regarding to phenolic acids family were
288 characterised such as; Protocatechuic acid, Trans - p- coumaric acid, p- hydroxybenzoic
289 acid, Chlorogenic acid and Vanillic acid. These five compounds were the first hits in the
290 list of possibilities (see table 2) and were detected between 2.2 3.4 min, they presents
291 a very good error (>5 ppm) and sigma values (>0.05), except two compounds, Trans -
292 p- coumaric acid and Chlorogenic acid which presents sigma values of 0.0576 and
293 0.090 respectively.
294 If we consider the flavonids family, we can characterized and detected the following
295 eighteen compounds: Catechin, Dihydrokaempferol-3-O-glucoside , Epicatechin ,
296 Eriodictiol-7-O-glucoside, Quercetin-3-O-rutinoside (rutin), Quercetin-3-O-galactoside,
297 Dihydroquercetin, Quercetin-3-O-glucoside, Kaempferol-3-O-rutinoside, Naringenin-7-
298 O-glucoside, Isorhamnetin-3-O-rutinoside, Quercetin-3-O-rhamnoside, Isorhamnetin-3-
299 O-glucoside/galactoside, Dihdrokaempherol or Eriodictiol, Quercetin, Naringenin,
300 Isorhamnetin and Kaempferol using this method.
125
301 Therefore, using this method 9 flavonids with sugar bond ( glucose, galactose, rutinose
302 and rhamnose) were detected in the same sequence between 3.0 4.1 min, and one
303 compound of these group (Isorhamnetin-3-O-glucoside/galactoside), which are mass
304 isomer with the same aglycone and cannot be differentiated in this method. The rest of
305 compounds of Flavonids, are 7 aglycones (Catechin, Epicatechin, Dihydroquercetin,
306 Quercetin, Naringenin, Isorhamnetin and Kaempferol). Besides, there is a further
307 Flavonids with two possibilities (Dihydrokaempherol or Eriodictiol) due to they have
308 the similar molecular weight. The 8 aglycones and the 5 phenolic acids detected by this
309 method did not yielded fragmentation patterns while all the Flavonids yielded
310 fragmentation (see Table 2).
311 Thus, these 23 compounds using HPLC-ESI-TOF (MS) method have been previously
312 described in almond skin by Paul E. Milbury et al [42] using HPLC- UV detector in 90
313 minute and by Christine A Hughey et al [43] using capillary LC-MS in 9 minute.
314
315 3.3 Comparison between the results obtained by CE-ESI-TOF (MS) and HPLC-
316 ESI-TOF (MS) methods
317
318 The observed mass values are identical for these separation techniques within the 5 ppm
319 mass accuracy, giving confidence in the identification. If we consider phenolic acid, we
320 can observe that the three phenolic acids (p-Hydroxybenzoic acid, Protocatechuic acid
321 and Vanillic acid) detected by CE method, were also detected by HPLC method.
322 Besides another two phenolic acid compounds (Trans - p- coumaric acid and
323 Chlorogenic acid) were not observed in the CE profiles. Although, difference between
324 both methods is really clear in the case of the three phenolic acids detected by both that
325 the sigma and error value are better in HPLC method. In addition to previous point, the
126
326 number of compounds that identified in HPLC (5 Compounds) more than CE method (3
327 compounds).
328 Concerning flavonoids, it is possible to say that the HPLC method provides better
329 results. The CE method was able to detect six flavonids compounds, while using HPLC
330 is possible to detect 12 flavonids, even all of them had good sigma and error values in
331 shorter time than CE method. Further at the optimum electrophoretic conditions, the
332 peak shape of the Flavonids was modest. However, most of the peaks had a good shape
333 and intensity in HPLC.
334 In general, the HPLC method was more appropriate for studying the flavonids family,
335 since all of the flavonids represented peaks of significant intensity in the central zone of
336 the chromatogram. Moreover, using the optimum electrophoretic conditions, we could
337 not observe so many isomeric forms as in the chromatograms obtained in HPLC.
338 Eriodictiol, Quercetin, Naringenin, Isorhamnetin and Kaempferol were the major
339 almond flavonids [42, 43] detected by HPLC method whereas just Naringenin was
340 detected with CE with a very low intensity.
341 Both methods can be successfully applied to the analysis of phenolic compounds in
342 almond skin and both techniques are reliable enough for determining this class of
343 compounds. However, if we understand them as complementary techniques to improve
344 the characterization of this polar fraction, the results will be more complete.
345
346 Conclusions
347
348 The separation by HPLC/CE with on-line detection by ESI-TOF-MS is successfully
349 applied to the analysis of the phenolic compounds present in almond skin samples. In
350 this work the CE was used for the first time in this type of samples. The two
127
351 methodologies are able to determine known phenolic compounds present in almond skin
352 and provide information about the presence and relative concentration of minor
353 phenolic compounds. HPLC can detect 23 compounds in 9 min, but only 9 compounds
354 were detected by CE method in 35 min.
355
356 4. References
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130
Table 1. Well-known phenolic compounds determined by CE-ESI-TOF-MS in an almond skin extract.
Classification Tolerance
order (ppm) in
Error considering Generate Migration
Selected m/z m/z Sigma m/z
Compound Formula other Molecular time CE
ion experimental calculated Value Fragments
possibilities Formula (min)
# (ppm) (mDa)
Quercetin-3-O-
1 glucoside/galactosi C21H19O12 [M-H] 463.0906 463.0882 5.3 -2.44 0.0311 1st (5) 10 14.2 301.1225
de
Isorhamnetin-3-
2 C28H31O16 [M-H]- 623.1618 623.1617 0.2 -0.14 0.0212 2st (8) 5 19.3 315.0245
rutinoside
Kampferol-3-
3 C27H29O15 [M-H]- 593.1508 593.1511 0.6 0.33 0.0114 2st (7) 5 19.7 285.1155
rutinoside
Naringenin-7-O-
4 C21H21O10 [M-H]- 433.1145 433.1140 1.3 -0.57 0.0959 5st (10) 5 20.2 271.1122
glucoside
Isorhamnetin-3-
5 glucoside/galactosi C22H21O12 [M-H]- 477.1028 477.1038 2.1 1.02 0.0403 2st (5) 5 20.7 315.0541
de
p-Hydroxybenzoic 21.3
6 C7H5O3 [M-H]- 137.0248 137.0244 3.9 -0.42 0.0227 1st (1) 5
acid
7 Naringenin C15H11O5 [M-H]- 271.0601 271.0611 3.8 1.04 0.0348 1st (1) 5 22.5
Protocatechuic
8 C7H5O4 [M-H]- 153.0185 153.0182 2.3 -0.35 0.0454 1st (1) 5 26.7
acid
9 Vanillic acid C8H7O4 [M-H]- 167.0352 167.0349 1.9 -0.31 0.0112 1st (3) 5 34.5
131
Table 2. Well-known phenolic compounds determined by HPLC-ESI-TOF-MS in an almond skin extract.
Classification Tolerance
order (ppm) in Migration
m/z
Selecte m/z m/z Error Sigma considering Generate time HPLC
Compound Formula Fragments
d ion experimental calculated Value other Molecular (min)
possibilities Formula
# (ppm) (mDa)
-
1 Protocatechuic acid C7H5O4 [M-H] 153.0191 153.0193 1.4 0.21 0.0066 1st (1) 5 2.281
Trans-p-coumaric
2 C9H7O3 [M-H]- 163.0405 163.0401 2.5 -0.40 0.0576 1st (1) 5 2.536
acid
p-Hydroxybenzoic
3 C7H5O3 [M-H]- 137.0244 137.0244 0.1 -0.02 0.0051 1st (1) 5 2.587
acid
4 Catechin C15H14O6 [M-H]- 289.0718 289.0711 2.4 0.68 0.0075 1st (2) 5 3.004
- st
5 Chlorogenic acid C16H17O9 [M-H] 353.0865 353.0878 3.7 1.31 0.0900 1 (3) 5 3.029
Dihydrokaempferol
6 C21H21O11 [M-H]- 449.1090 449.1089 0.2 -0.11 0.0288 2st (5) 5 3.148 287.0581
3-O-glucoside
7 Epicatechin C15H14O6 [M-H]- 289.0716 289.0711 0.6 0.17 0.0057 1st (1) 5 3.216
8 Vanillic acid C8H7O4 [M-H] 167.0356 167.0350 3.5 -0.58 0.0253 1st (1) 5 3.420
Eriodictiol-7-O-
9 C21H21O11 [M-H]- 449.1099 449.1089 2.1 -0.93 0.0372 1st (5) 5 3.658 287.1082
glucoside
Quercetin-3-O-
10 C27H29O16 [M-H]- 609.1460 609.1461 0.2 0.15 0.0245 1st (4) 5 3.709 301.1107
rutinoside
Quercetin-3-O-
11 C21H19O12 [M-H]- 463.0897 463.0882 3.2 -1.48 0.0766 3st (3) 5 3.726 301.1253
galactoside
12 Dihydroquercetin C15H11O7 [M-H]- 303.0514 303.0510 1.2 -0.35 0.0173 1st (2) 5 3.794
Quercetin-3-O-
13 C21H19O12 [M-H]- 463.0891 463.0882 1.9 -0.90 0.0298 2st (5) 5 3.811 301.1225
glucoside
Kaempferol-3-O-
14 C27H29O15 [M-H]- 593.1518 593.1512 1.0 -0.61 0.0117 1st (8) 5 3.896 285.1285
rutinoside
Naringenin-7-O-
15 C21H21O10 [M-H]- 433.1133 433.1140 1.6 0.71 0.0278 2st (3) 5 3.930 271.0722
glucoside
Isorhamnetin-3-O-
16 C28H31O16 [M-H]- 623.1612 623.1618 0.9 0.57 0.0191 1st (5) 5 3.947 315.0541
rutinoside
17 Quercetin-3-O- C21H19O11 [M-H]- 447.0929 447.0933 0.9 0.40 0.0545 2st (5) 5 4.015 301.1283
132
rhamnoside
Isorhamnetin-3-O-
18 glucoside/galactosid C22H21O12 [M-H]- 477.1023 477.1038 3.3 1.58 0.0064 1st (6) 5 4.049 315.0553
e
Dihydrokaempherol
19 C15H11O6 [M-H]- 287.0561 287.0561 0.0 0.01 0.0181 2st (3) 5 4.167
or Eriodictiol
20 Quercetin C15H9O7 [M-H]- 301.0355 301.0354 0.3 -0.10 0.0178 2st (3) 5 4.695
- st
21 Naringenin C15H11O5 [M-H] 271.0607 271.0612 1.9 0.51 0.0209 1 (1) 5 4.950
- st
22 Isorhamnetin C16H11O7 [M-H] 315.0526 315.0510 5.0 -1.56 0.1036 2 (3) 5 5.154
- st
23 Kaempherol C15H9O6 [M-H] 285.0416 285.0405 3.9 -1.12 0.0967 2 (3) 5 5.137
133
CAPTION FIGURES
Figure 1
EICs of characterized compounds in almond skin by CE-ESI-TOF (MS)
Figure 2
EICs of the major characterized compounds in almond skin by HPLC-ESI-TOF (MS)
134
Intens. Intens.
x104
EIE 463.090 0.005 EIE 623.161 0.005
6000
1 6 2
4
4000
2
2000
0
0
0 5 10 15 20 25 30 Time [min]
0 5 10 15 20 25 30 Time [min]
Intens. Intens.
x104 EIE 593.150 0.005 EIE 433.114 0.005
4
3 4000
4
3 3000
2 2000
1 1000
0 0
0 5 10 15 20 25 30 Time [min] 0 5 10 15 20 25 30 Time [min]
Intens. Intens.
x10 4
x10 4
EIE 477.102 0.005 EIE 137.024 0.005
1.25
5 6
3
1.00
2
0.75
0.50
0.25
0.00 0
0 5 10 15 20 25 30 Time [min] 0 5 10 15 20 25 30 Time [min]
Intens. Intens.
x10 4
EIE 271.060 0.005 EIE 153.018 0.005
7 8
1.5 6000
1.0 4000
0.5 2000
0.0 0
0 5 10 15 20 25 30 Time [min] 0 5 10 15 20 25 30 Time [min]
Intens.
x104
EIE 167.025 0.005
8 9
6
0
0 5 10 15 20 25 30 Time [min]
Fig. 1
Intens. Intens.
x10 4 EIC 153.0180.005 x10 4 3 EIC 137.0250.005
3
1 1.25
1.00
2
0.75
0.50
1
0.25
0 0.00
0 1 2 3 4 5 6 7 8 Time [min] 0 1 2 3 4 5 6 7 8 Time [min]
Intens. Intens.
x10 4 x10 4 EIC 593.1510.005
6 4 EIC 289.0720.005 14
5 6
4
4
3
2
2
1
0 0
0 1 2 3 4 5 6 7 8 Time [min] 0 1 2 3 4 5 6 7 8 Time [min]
Intens. Intens.
x10 4 EIC 433.1140.005 x10 5 EIC 623.1620.005
15 1.25
16
0.8
1.00
0.6
0.75
0.4
0.50
0.2 0.25
0.0 0.00
0 1 2 3 4 5 6 7 8 Time [min] 0 1 2 3 4 5 6 7 8 Time [min]
Intens. Intens.
x10 4 EIC 477.1030.005 EIC 287.0560.005
1.5 18 4000 19
3000
1.0
2000
0.5
1000
0.0 0
0 1 2 3 4 5 6 7 8 Time [min] 0 1 2 3 4 5 6 7 8 Time [min]
Intens. Intens.
EIC 301.0350.005 EIC 271.0610.005
2000 20 6000
21
1500
4000
1000
2000
500
0 0
0 1 2 3 4 5 6 7 8 Time [min] 0 1 2 3 4 5 6 7 8 Time [min]
Fig. 2
CHAPTER V: Characterization of phenolic and other polar compounds in
Flaxseed oil using HPLC-ESI-TOF (MS)
137
This work was submitted to Food chemistry Journal.
Characterization of phenolic and other polar compounds in Flaxseed oil using

HPLC-ESI-TOF (MS)

Fernndez-Gutirrez.
Wahid Herchi, Habib Kallel.

Laboratoire de Biochimie des lipides, Dpartement de Biologie, Facult des
sciences de Tunis, 2092 ELmanar-Tunisie.
138
1 Characterization of phenolic and other polar compounds in
2 Flaxseed oil using HPLC-ESI-TOF (MS)
3
4 Saleh Sawalhaa, Wahid Herchib, David Arrez-Romna, Antonio Segura-Carreteroa,*,
5 Habib Kallelb, Alberto Fernndez-Gutierreza,*
6
a
7 Department of Analytical Chemistry, Faculty of Sciences, University of Granada,
8 C/Fuentenueva s/n, 18071 Granada, Spain.
b
9 Laboratoire de Biochimie des lipides, Dpartement de Biologie, Facult des sciences de
10 Tunis, 2092 ELmanar-Tunisie.
11
12 Abstract
13 A sensitive method based on high-performance liquid chromatography coupled with
14 electrospray ionization time-of-flight-mass spectrometry (HPLCESITOF (MS)) has been
15 used to analyze phenolic compounds in Flaxseed oil. Several important phenolic compounds
16 such as secoisolariciresnol, ferulic acid and its methyl ester, coumaric acid methyl ester,
17 diphylin, pinoresinol, matairesinol, p-hydroxybenzoic acid, vanillin and vanillic acid have
18 been detected from Flaxseed oil. The efficiency, rapidity and high resolution of HPLC
19 coupled to the sensitivity, selectivity, mass accuracy and true isotopic pattern from TOF (MS)
20 have revealed an enormous separation potential allowing the characterization of a broad series
21 of phenolic compounds present in Flaxseed oil for the first time.
22
23 Keywords:
24 Phenolic compounds, Flaxseed oil, HPLC, ESI-TOF (MS)
25
*
26 Corresponding author. Fax: +34 958249510
27 E-mail addresses: ansegura@ugr.es (A. Segura-Carretero), albertof@ugr.es (A. Fernndez-
28 Gutirrez)
139
1
3 1. Introduction
5 Flaxseed has been gaining popularity in the health food market because of its reported health
6 benefits and disease preventive properties on coronary heart disease (Oomah & Mazza,
7 2000), some kinds of cancer and neurological and hormonal disorders (Huang & Ziboh, 2001;
8 Simopoulos, 2002). During the last decade, there has been an increasing interest in the use of
9 Flaxseed in the diet in order to improve the nutritional and health status (Oomah, 2001).
10 Flaxseed is rich in lignans and the embryo is rich in oil with a high omega-3 fatty acid content
11 (Westcott and Muir, 2003; Wiesenborn, Tostenson & Kangas, 2003). The beneficial effects of
12 lignans on human health are well recognised (Westcott and Muir, 2003, McCann, Gill,
13 McGlynn, & Rowland, 2005). Other phenolic compounds of interest that are accumulated in
14 flaxseed include ferulic and vanillic acid. The qualitative and quantitative determination of the
15 phenolic compounds in oils is very important and several methods have been already used in
16 recent years. Various methods have been reported for the identification of these substances in
17 Flaxseed starting from the early days, non-specific analytical methods, such as paper, thin
18 layer (Coran, Giannellini & Bambagiotti-Alberti ,2004), and column chromatography as well
19 as UV spectroscopy , were applied to polyphenols analysis (Christophoridou, Dais, Tseng, &
20 Spraul,2005). The need identify individual phenolic compounds meant that traditional
21 methods were replaced and significant progress was achieved when more specific analytical
22 techniques were used, such as Gas Chromatography (GC) (Penalvo, Haajanen, Botting &
23 Adlercreutz,2005) or High-Performance Liquid Chromatography (HPLC) (Charlet et al,
24 2002). The results obtained by using GC are very reliable and interesting, but the use of this
140
1 technique is less common because the derivatization step is essential and the use of high
2 temperature which could damage this kind of analytes.
3 HPLC hyphenated to Mass Spectrometry (MS) detection is one of the most important
4 analytical techniques used for the analysis of phenolic compounds (Carrasco-Pancorbo et al,
5 2005; Morales & Tsimidou, 2000). The advantages of MS detection include the ability to
6 determine molecular weights and to obtain structural information (Carrasco-Pancorbo et al,
7 2007).
8 The on-line coupling of HPLC with MS using Electrospray Ionization (ESI) as an interface
9 yields a powerful method because ESIMS allows the determination of a wide range of polar
10 compounds. ESI is one of the most versatile ionization methods, and is the method of choice
11 for the detection of ions separated by liquid chromatography. Although HPLC can be coupled
12 to different MS analyzers (quadrupole, ion trap (IT), time-of-flight (TOF), etc (Simo et al,
13 2005), in this paper we have used HPLCESITOF (MS) to characterize phenolic compounds
14 in Flaxseed oil. TOF (MS) provides excellent mass accuracy (Bristow & Webb, 2003) over a
15 wide dynamic of range if modern detector technology is used. The latter, moreover, allows
16 measurements of the isotopic pattern (Pelzing, Decker, Neus & Rther , 2004), providing
17 important additional information for the determination of the elemental composition
18 (Bringmann et al, 2005). To our knowledge, the present work represents the first time that a
19 HPLCESITOF (MS) method has been applied to the characterization of phenolic
20 compounds in Flaxseed oils.
21
22 2. Materials and methods
23 2.1. Chemicals and reagents
24 All chemicals were of analytical reagent grade and used as received. The organic solvents,
25 hexane, methanol and ACN, used in the extraction procedure and as HPLC mobile phase were
141
1 purposed from Lab-Scan (Dublin, Ireland). Acetic acid used in HPLC phase A was purchased
2 from Fluka (Switzerland). Deionised water was obtained from a water purifier system
3 (Millipore, Bedford, MA). All the solvents used in the HPLC system were filtered through a
4 0.20 m Millipore (Bedford, MA, USA) membrane filters.
6 2.2. Plant materials
7 The seeds of the three varieties H52, O116 and P129 were purchased from INRAT (Institut
8 National Recherche Agronomie Tunis, North of Tunisia).
10 2.3. Oil extraction from Flaxseed
11 The total lipids were extracted by the method of Folch, Lees, & Sloane Stanley (1957)
12 modified by Bligh & Dyer (1959). Seeds (5 g) were washed with boiling water for 5 min to
13 denature the phospholipases and then crushed in a mortar with a mixture of CH3Cl-MeOH
14 (2:1, v/v). The water of fixation was added and the homogenate was centrifuged at 3000 g for
15 15 min. The lower chloroformic phase containing the total lipids was kept and dried in a
16 rotary evaporator at 40C.
17
18 2.4. Solid-phase Extraction (SPE) Procedure
19 100 mg of DSC-Diol (Supelco, Bellefonte, PS, USA) as powder was added in a test tube of 10
20 mL and it was conditioned as follows: 1) 100 L of methanol were added, shaken on vortex
21 for 5 minutes, centrifuged at 4500 rpm for 10 minutes and the liquid part was then discarded.
22 2) 100 L ml of hexane was added, shaken on vortex for 5 minutes, centrifuged at 4500 rpm
23 for 10 minutes and then the liquid part was discarded.
24 Flaxseed oil (1 g) was dissolved in 1200 L hexane in a test tube of 10 mL, shaken on vortex
25 for 5 minutes and the solution was added into the test tube with the conditioned DSC-Diol.
142
1 All was shaken on vortex for 5 minutes, centrifuged at 4500 rpm for 10 minutes and the liquid
2 part was discarded. Then, the DSC-Diol was washed with 1200 L of hexane, shaken on
3 vortex 5 minutes, centrifuged at 1000 rpm for 10 minutes and the hexane were then discarded
4 in order to remove the non-polar fraction of Flaxseed oil. The polar fraction was recovered by
5 addition of 1200 L of methanol; the solution was shaken on vortex 5 minutes and
6 centrifuged at 1000 rpm for 10 minutes. Finally, the methanolic part was removed into an
7 eppendorf 2 mL tube and evaporated by a rotary evaporator (Concentrator plus, Eppendorf
8 AG, Hamburg, Germany) under reduced pressure at 30C. The sample was resolved in 20 l
9 of methanol and filtered through a 0.2 m.
10
11 2.5. HPLC
12 The separation of the phenolic compounds from Flaxseed oil was performed using an Agilent
13 1200 series Rapid Resolution LC (Agilent Technologies, Palo Alto, CA, USA) was equipped
14 with a vacuum degasser, an autosampler, a binary pump, and a thermostated column
15 department. The standards and samples were separated using a reversed-phase C18 analytical
16 column (4.6150 mm, 1.8 m particle size, Agilent ZORBAX Eclipse plus). The mobile
17 phase A and B consisted of water with 0.5% acetic acid, and ACN. The chromatographic
18 method was as following: gradient from 5% B to 30% B in 10 minutes; 30% B to 33% B in 2
19 minutes; 33% B to 38% B in 5 minutes; 38% B to 50% B in 3 minutes; 50% to 95% in 3
20 minutes. The initial conditions were re-established in 2 minutes and held for 10 minutes. The
21 total run time, including the conditioning of the column to the initial conditions, was 35 min.
22 The flow rate used was set at 0.80 mL/min throughout the gradient. The effluent from the
23 HPLC column was split using a T before being introduced into the mass spectrometer (split
24 ratio 1:3). Thus in the current paper the flow which arrived to the ESI-TOF (MS) detector was
143
1 0.2 mL/min. The column temperature was maintained at 25 C and the injection volume was
2 10 L.
4 2.6. ESI-TOF (MS)
5 ESI-TOF (MS) conditions were optimized in order to provide strong mass signals for all the
6 studied phenolic compounds. The HPLC system was coupled to a TOF (MS) equipped with
7 an ESI interface operating in negative ion mode. The optimum ESI parameters were as
8 follows: nebulizing gas pressure, 2 bar; drying gas flow, 9 L/min; drying gas temperature, 190
9 C.
10 MS was performed using the microTOF (Bruker Daltonik, Bremen, Germany), an orthogonal-
11 accelerated TOF mass spectrometer (oaTOF-MS). Transfer parameters were optimized by
12 direct infusion experiments with Tuning Mix (Agilent Technologies) in the range of 50-800
13 m/z looking for the best conditions regarding sensitivity and resolution. Thus, the endplate
14 offset was -500 V; capillary voltage 4500 V, the trigger time was set to 50 s, 49 s for set
15 transfer time and 1s pre-puls storage time, corresponding to a mass range of 50800 m/z.
16 Spectra were acquired by summarizing 20,000 single spectra, defining the spectra rate to
17 1 Hz. The accurate mass data of the molecular ions were processed through the software Data
18 Analysis 3.4 (Bruker Daltonik), which provided a list of possible elemental formulas by using
19 the Generate Molecular Formula Editor. The Generate Formula Editor uses a CHNO
20 algorithm, which provides standard functionalities such as minimum/maximum elemental
21 range, electron configuration, and ring-plus double bonds equivalents, as well as a
22 sophisticated comparison of the theoretical with the measured isotope pattern (Sigma Value)
23 for increased confidence in the suggested molecular formula (Bruker Daltonics Technical
24 Note #008, Molecular formula determination under automation). The widely accepted
144
1 accuracy threshold for confirmation of elemental compositions has been established at 5 ppm
2 (Fereer et al. 2005).
3 We also have to say that even with very high mass accuracy (<1 ppm) many chemically
4 possible formulae are obtained depending on the mass regions considered. So, high mass
5 accuracy (<1 ppm) alone is not enough to exclude enough candidates with complex elemental
6 compositions. The use of isotopic abundance patterns as a single further constraint removes >
7 95% of false candidates. This orthogonal filter can condense several thousand candidates
8 down to only a small number of molecular formulas.
9 During the development of the HPLC method, external instrument calibration was performed
10 using a 74900-00-05 Cole Palmer syringe pump (Vernon Hills, Illinois, USA) directly
11 connected to the interface, passing a solution of sodium formate cluster containing 5 mM
12 sodium hydroxide in water/isopropanol 1/1 (v/v), with 0.2% (v/v) of formic acid at the end of
13 each run. Using this method an exact calibration curve based on numerous cluster masses
14 each differing by 68 Da (NaCHO2) was obtained. Due to the compensation of temperature
15 drift in the MicroTOF, this external calibration provided accurate mass values (better 5 ppm)
16 for a complete run without the need for a dual sprayer setup for internal mass calibration.
17 These calibrations were performed in quadratic + high precision calibration (HPC) regression
18 mode.
19
20 3. Results and discussion
21 3.1. Repeatability study
22 Repeatability of the HPLC-ESI-TOF (MS) analysis was studied by performing a series of
23 separations using the optimized method by the analysis of methanol extracts on the same day
24 (intraday precision, n=5) and on three consecutive days (interday precision, n=15). The
25 relative standard deviations (RSDs) of analysis time and peak area were determined. The
145
1 intraday repeatability on the migration time (expressed as RSD) was 0.5%, whilst the interday
2 repeatability was 0.9%. The intraday repeatability of the peak area (expressed as RSD) was
3 1.2%, whilst the interday repeatability was 4.3%.
5 3.2. Identification of phenolics compounds in Flaxseed oil
6 The HPLCESITOF (MS) method was applied to the identification of the phenolic
7 compounds present in Flaxseed oil. The identification of phenolics compounds was carried
8 out comparing their migration times and mass spectra provided by TOF (MS) with those of
9 authentic standards when available. Thus, Fig. 1 shows the chemical structures of the
10 characterized phenolic compounds and Fig. 2 shows the extracted ion chromatograms (EIC)
11 according to the elution order: (1) diphylin ([M-H]exp m/z 379.0835), (2) vanillic acid
12 ([M-H]exp m/z 167.0350), (3) vanillin ([M-H]exp m/z 151.0400), (4) p-hydroxybenzoic acid
13 ([M-H]exp m/z 137.0244), (5) methyl ester coumaric acid ([M-H]exp m/z 177.0557). (6)
14 secoisolariciresinol ([M-H]exp m/z 361.1656), (7) methyl ester ferulic acid ([M-H]exp m/z
15 207.0657), (8) ferulic acid ([M-H]exp m/z 193.0506), (9) pinoresinol ([M-H] exp m/z
16 357.1343), (10) matairesinol ([M-H]exp m/z 357.1341). These compounds are also
17 summarized in Table 1 (ten phenolic compounds were characterized in H52 and P129
18 varieties and nine compounds in O116 variety) along with their molecular formula, selected
19 ions, experimental and calculated m/z values, errors (ppm and mDa), sigma values, tolerances
20 in generated molecular formula and migration times. The identification by TOF (MS) was
21 carried out using the Generate Molecular Formula Editor. First of all, a low tolerance was
22 chosen (5 ppm). After that, options with a low sigma value (<0.05) and a low error (<5ppm)
23 were taken into account in most cases.
24 Most of the compounds found in this work have been previously described in Flaxseed.
146
1 Lignans and their derivatives have been characterized before in Flaxseed (Kamal-Eldin et al.,
2 2001) as well as phenolics acids (Johnsson et al., 2002) but this is the first time in Flaxseed
3 oil.
4 These detected compounds can be classified as lignans, phenolic acids, simple phenols and
5 diphylin family. Regarding the lignan family, it is possible to study the following compounds
6 (in elution order) with the HPLC-ESI-TOF (MS) method: secoisolariciresinol, pinoresinol and
7 matairesinol. Pinoresinol was found in Flaxseed oil in higher concentration but matairesinol
8 and secoisolariciresinol were found in low concentrations in all varieties. The main lignan
9 secoisolariciresinol diglucoside (SDG) is not present in these varieties of Flaxseed oil
10 probably because of its high solubility (Lavelli and Bondesan, 2005).
11 These compounds were detected in H52 and P129 varieties, while secoisolariciresinol was
12 absent in O116 variety. Considering in fact, that pinoresinol lariciresinol reductase
13 catalyses the conversion of pinoresinol to secoisolariciresinol (Xia, 2000), we suggested that
14 this enzyme was present in low amount and inactive in O116 variety than in H52 and P129
15 varieties. Other reason, it was presumed that in Flaxseed oil oligomers under a large amount
16 of methanol, the glycosidic unit SDG was esterified by p-coumaric acid glucoside or ferulic
17 acid glucoside which was easily released by alkaline hydrolysis to form the methyl ester of p-
18 coumaric acid glucoside or ferulic acid glucoside in a reaction medium containing a large
19 amount of methanol (Li et al, 2008), these forms will be converted by deglycosylation
20 products respectively in p-coumaric acid and ferulic acid.
21 Recent studies have described in a little more details this family of lignan using HPLC-NMR;
22 HPLC-MS (Hosseinian, Muir, Westcott, and Krol; Strandas, Kamal-Eldin, Andersson, Aman,
23 2006).
24 Regarding the family of phenolics acids, the method allows the determination of ferulic acid
25 and its methyl ester, vanillic acid, p-hydroxybenzoic acid and methyl ester coumaric acid.
147
1 In all varieties, p-coumaric acid was not detected while ferulic acid was detected in small
2 amount. This indicates that, in a reaction medium containing large amount of methanol, p-
3 coumaric and ferulic acid have been almost completely esterified. A methyl ester ferulic acid
4 ([M-H] 207.0662 m/z) and methyl ester coumaric acid ([M-H] 177.0557 m/z) respectively,
5 were characterized by mass spectra and using the Generate Molecular Formula Editor. Li et
6 al. (2008) reported that p-coumaric acid or ferulic acid standard dissolved in an aqueous
7 methanol solution could be esterified by methanol to produce p-coumaric acid methyl ester or
8 ferulic acid methyl ester. Kozlowska, Zadernowski, & Sosulski (1983) showed that the
9 highest proportion of phenolic acids in Flaxseed oil and other oil seeds were ester bound. In
10 addition to the previously mentioned phenolics compounds, a further two families of
11 phenolics compounds was detected in the three varities of Flaxseed oil using HPLC method
12 namely simple phenols presented by vanillin compound and diphyllin family presented by
13 diphyllin compound.
14
15 3.3. Unknown phenolic compounds
16 Besides the previously mentioned phenolic compounds detected, it was also possible to study
17 others compounds present in Flaxseed oil which have so far not been described in the
18 literature. These unknown compounds have been included in Table 2 as they are an important
19 part of the polar fraction of Flaxseed oil. Even though the characterization of these
20 unidentified compounds was not possible with the generated data by TOF analysis, it is
21 possible to observe the experimental m/z, selected ion, tolerance (ppm), a list of possibilities,
22 the mass deviation, and the sigma value. 14 unknown compounds were detected in H52
23 variety, 16 in P129 and 20 were detected in O116 variety. This difference appeared in 6
24 compounds which are (m/z 197.0452; 131.0721; 216.9996; 369.1037; 177.0188 and
25 179.0339). Two compounds of them were detected in P129 and O116 while were not detected
148
1 in H52 (m/z 197.0457; 131.0739). The proposed HPLC method allowed the determination of
2 four compounds in O116 and not determined in P129 and H52 (m/z 216.9996; 369.1037;
3 177.0188; 179.0339). In general, O116 had the greatest possibilities of unknown compounds,
4 probably due to the hydrolysis of others compounds, also this difference could be linked to
5 the differences in relatives activities and abundances of the complex of enzymes responsible
6 for phenolic compounds biosynthesis.
7 A reduced number of possible elemental compositions are obtained from the accurate mass of
8 the suspected peak. These elemental compositions can then be matched against available
9 databases (The Merck Index, ChemIndex, commercial e-catalogues) using the deduced
10 molecular formula as a search criterion (Ibanez et al 2005; Kina & Fiehn, 2006).
11
12 4. Conclusions
13 The sensitive HPLC-ESI-TOF (MS) method allows the characterization of many well-known
14 and the detection of unknown phenolic compounds present in Flaxseed oil. Furthermore, the
15 use of TOF (MS) provides excellent resolving power and mass accuracy over a wide dynamic
16 range if modern detector technology is chosen. Moreover, it allows the measurement of the
17 correct isotopic pattern, providing important additional information for the determination of
18 the elemental composition. Thus, significant differences were found between the three
19 varieties using the proposed method. This fact could be used in future to find potential
20 markers for the geographical origin of the oil or the flaxseed variety.
21
22 Acknowledgements
23 The authors are grateful to the Spanish Ministry of Education and Science for the project
24 (AGL2008-05108-C03-03) and to Andalusian Regional Government Council of Innovation
25 and Science for the project P07-AGR-02619.
149
1 The author SS gratefully acknowledges the Agencia Espaola de Cooperacin Internacional
2 (AECI).
3
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153
Table 1. Well-known phenolic compounds characterized by HPLC- ESI-TOF (MS) in Flaxseed oil.
Tolerance
HPLC
(ppm) in
Selected Experimental Error Sigma Migration
Compound Formula Generated H52 P129 O116
Ion m/z (ppm) value Time
Molecular
(min)
formula
Lignans
Secoisolariciresinol C20H25O6 [M-H] 361.1656 0.0 0.1295 5 12.9 + + -
Pinoresinol C20H21O6 [M-H] 357.1343 -1.6 0.0274 5 15.6 + + +
Matairesinol C20H21O6 [M-H] 357.1375 -4.2 0.0217 5 17.3 + + +
Phenolic acids
Vanillic acid C8H7O4 [M-H] 167.0350 -0.5 0.0492 5 8.8 + + +
p-hydroxybenzoic
C7H5O3 [M-H] 137.0247 -2.4 0.0450 5 10.7 + + +
acid
Methyl Ester
C11H11O4 [M-H] 177.0557 0.0 0.0138 5 12.6 + + +
Coumaric acid
Methyl Ester
C10H9O3 [M-H] 207.0657 2.7 0.0870 5 13.2 + + +
Ferulic acid
Ferulic acid C10H9O4 [M-H] 193.0508 1.3 0.0607 5 15.1 + + +
Simple phenols
Vanillin C8H7O3 [M-H] 151.0400 -1.7 0.0091 5 9.0 + + +
Diphyllin
Diphyllin C21H15O7 [M-H] 379.0835 -3.2 0.084 5 2.1 + + +
154
Table 2. Unknown phenolic compounds detected by HPLC-ESI-TOF (MS) in Flaxseed oil
Tolerance List of
(ppm) in possibilities
Experimental
Selected Ion Generated in Generate Error (ppm) Sigma value H52 P129 O116
m/z
Molecular Molecular
Formula Formula
187.0965 [M-H] 10 C9H15O4 5.5 0.0031 + + +
173.1182 [M-H] 5 C9H17O3 0.3 0.0121 + + +
145.0870 [M-H] 5 C7H13O3 -0.1 0.0086 + + +
225.1115 [M-H] 10 C12H17O4 7.6 0.0208 + + +
199.1337 [M-H] 5 C11H19O3 1.3 0.0203 + + +
227.1283 [M-H] 5 C12H19O4 2.4 0.0230 + + +
171.1031 [M-H] 5 C9H15O3 -3.1 0.0070 + + +
307.1916 [M-H] 5 C18H27O4 -0.6 0.0362 + + +
329.2341 [M-H] 5 C18H33O5 -2.5 0.0050 + + +
157.0874 [M-H] 5 C8H13O3 -3.0 0.0198 + + +
169.0871 [M-H] 5 C9H13O3 -1.0 0.0122 + + +
211.1325 [M-H] 10 C12H19O3 -4.0 0.0085 + + +
327.2174 [M-H] 5 C18H31O5 -0.5 0.0158 + + +
159.1036 [M-H] 10 C8H15O3 -6.1 0.0056 + + +
197.0452 [M-H] 5 C9H9O5 1.3 0.0416 - + +
131.0721 [M-H] 10 C6H11O3 -6-0 0.0179 - + +
216.9996 [M-H] 5 C7H5O8 -2.9 0.0236 - - +
369.1037 [M-H] 5 C13H21O12 0.2 0.0511 - - +
177.0188 [M-H] 5 C9H5O4 2.5 0.1607 - - +
179.0339 [M-H] 10 C9H7O4 5.6 0.0386 - - +
155
Caption Figures
Fig. 1
Structures of the characterized compounds in the phenolic fraction of Flaxseed oil.

(1) diphylin, (2) vanillic acid, (3) vanillin, (4) p-hydroxybenzoic acid, (5) methyl ester
coumaric acid. (6) secoisolariciresinol, (7) methyl ester ferulic acid, (8) ferulic acid, (9)
pinoresinol, (10) matairesinol.
Fig. 2
EICs of characterized compounds in Flaxseed oil by HPLC-ESI-TOF (MS)
156
1 2 3
O
H3C OH
OH
H3C HO
H3C
O
OH
4 5 6
H3C
7
8
10
9
Fig. 1
Intens Intens
4 4
x10
x10 1 2
4 3
3
2
2
1
1
0
0 0 5 10 15 20 25 30 Time [min]
0 5 10 15 20 25 30 Time [min]
Intens
4 Intens
x10 4
x10
1.25 3 4
1.00 3
0.75 2
0.50
1
0.25
0 0 5 10 15 20 25 30 Time [min]
0.00
0 5 10 15 20 25 30 Time [min]
Intens Intens..
4 4
x10
x10
1.0 5 3
6
0.8
0.6 2
0.4
1
0.2
0.0 0 5 10 15 20 25 30 Time [min] 0 0 5 10 15 20 25 30 Time [min]
Intens.
Intens
5
x10
8000 7 8
1.5
6000
1.0
4000
2000 0.5
0 0.0
0 5 10 15 20 25 30 Time [min] 0 5 10 15 20 25 30 Time [min]
Intens Intens.
4 4
x10
9 x10 10
2.0 1.5
1.5
1.0
1.0
0.5 0.5
0.0 0.0
0 5 10 15 20 25 30 Time [min] 0 5 10 15 20 25 30 Time [min]
Fig. 2
Conclusions
159
Conclusion
CONCLUSIONS
1. In oranges, the peel represents roughly half of the fruit mass. The highest
concentrations of flavonoids in citrus fruit occur in peel. We carried out
five extraction procedures, and from the comparison between them,
procedure C was the best extraction according to the optimum peak
shape, best resolution and efficiency among the phenolic compounds. We
carried out the characterization and quantification of the distinctive
phenolic compounds in these extracts obtained from the peel of sweet
and bitter oranges using CE-ESI-MS with negative-ion electrospray
ionization.
2. CE-MS/MS analysis was done for further characterization of polyphenols in

the samples. This technology was applied on both of the two kinds of the
samples and compared with the MS/MS of each standard. One calibration
curve was prepared for each one: Naringin (m/z 579.2) and neohesperidin
(m/z 609.2) in the peel of bitter oranges, and narirutin (m/z 579.2) and
hesperidin (m/z 609.2) in the peel of sweet oranges. The optimized
method allowed differentiating naringin and narirutin, and hesperidin and
neohesperidin using the IT-MS detection because it provides molecular
weight and structural information. This technique has been shown to be
suitable for the analysis of this type of natural compounds.
3. The filtration process affects the characteristics of VOO, in particular,

oxidative stability, water content, and the presence of phenolic
compounds. The filter used in olive oil is fossilized remain of microscope
algae, also called diatomaceous earth, where it will be a kind of by-
product during olive oil production. A specific and carefully extraction
procedure was developed for isolation of olive oil polyphenols from filter.
4. The hyphenation of HPLC to MS, which combines the advantages of HPLC

with the selectivity, sensitivity, mass accuracy and measurements of the
isotopic pattern associated with TOF (MS), permitted the identification of
19- well known phenolic compounds in filters. Furthermore, 17 unknown
new compounds were determined. The proposed HPLC-ESI-TOF(MS)
161
Conclusion
method represents a valuable tool and a good alternative for simultaneous

characterization of phenolic components in diatomaceous earth.
5. Olive oil production is an important agricultural activity in of the world

and especially in Spain. During olive oil production a large amounts of by-
products were produced, olive leaves represent 5 % of the weight of the
olive oil extraction. This type of by-products (olive leaves) are rich source
of phenolic compounds. In the present study, a simple and rapid
extraction procedure was used to extract the phenolics compounds from
two varieties of olive oil leaves (Hojiblanca and Manzanilla).
6. In this sense, a new CE-ESI-TOF (MS) method was developed to carry out
the determination of 17 and 14 well known phenolic compounds in
Hojiblanca and Manzanilla olive leave extract, respectively. Hence, the
proposed method is a good alternative for simultaneous characterization
of phenolic components in olive leaves.
7. In almond, the skin which has very low economic value, represent 4 % of
the total almond skin weight, but it is very important, due to the high
contents of polyphenols. Thus, a liquid-liquid extraction procedure was
used for the isolation of phenolic compounds from almond skin and they
were subsequently analyzed by applied HPLC and CE coupling to ESI-TOF
(MS). The described extraction procedure was the same in both methods;
it was rapid and has been successfully applied to extract polyphenols from
the almond skin.
8. Subsequently, with respect to analytical method to characterization of

polyphenol, a CE method for the characterization of pholyphenols in
almond skin was developed in the first time and supposes an interesting
alternative tool to the HPLC method. Both methods allow direct and
sensitive characterization of phenolic compounds in almond skin with on-
line detection by ESI-TOF (MS). As is well known, both methods have some
advantages and some drawbacks. However in this kind of samples, HPLC
can detect 23 polyphenols in 9 min, but only 9 compounds were detected
by CE method in 35 min, which mean, HPLC method significantly reduced
analysis time and increased the numbers of polyphenols that they were
characterized, also HPLC can offer performance advantages, such as
162
Conclusion
improved injection precision and detection sensitivity, where as, CE can

offer benefits in term of reduced operating cost.
9. Flax seed are composed of 41 % oil and this oil contains phenolic
compounds that promote good health. Thus, a solid phase extraction
procedure was used for isolation the phenolic compounds in flaxseed oil
and a new HPLC-ESI-TOF (MS) method was developed. The separation by
HPLC with on-line detection by ESI-TOF (MS) is successfully applied to the
analysis of the phenolic compounds present in flaxseed oil samples for the
first time.
10. This method can detect 10 polyphenols in H52 and P129 varieties, these
compounds related for various families of polyphenols, and 9 compounds
in O116 variety, . In the same time and by the described method it is
possible to study others compounds present in samples, 14, 16 and 20
unknown phenolic compounds were detected in H52, P129 and O116
respectively, which they are an important part of the polar fraction of
flaxseed oil. Thus, significant differences were found between the three
varieties using the proposed method. This fact could be used in future to
find potential markers for the geographical origin of the oil or the
flaxseed variety.
11. Therefore, in this doctoral thesis have been developed different

extraction procedures and different methodologies, using CE and HPLC
coupled to MS, to characterize phenolic compounds in a wide variety of
matrices. In this sense, the use of advanced separation techniques
allowed, in most cases, to obtain good results in term of resolution,
efficiency and analysis time. Moreover, these separation techniques were
coupled to a detection system with enormous potential such as MS, whose
prominent features are its sensitivity, selectivity and provide structural
information. In this sense, in this research work has been used the IT
analyzer, whose most prominent feature is the ability to provide real
fragments of a discrete mass (MS/MS) and TOF analyzer, obtain resolution,
exact mass and isotope ratio measures. Thus, we can obtain valuable
information for the characterization of the compounds under study.
163

Conclusiones
164
Conclusiones
CONCLUSIONES
1. Dado que en la naranja, la piel representa aproximadamente la mitad de

su masa y es conocido que en los ctricos la mayor concentracin de
flavonoides se encuentran en la piel, se han estudiado comparativamente
cinco procedimientos de extraccin. Finalmente con el procedimiento C,
en el que se realiz una extraccin usando MeOH, siendo ste
posteriormente evaporado y reconstituido en una mezcla MeOH:H20
(50:50, v/v) adecuada para su anlisis mediante CE, se obtuvieron los
mejores resultados. Posteriormente se llev a cabo la caracterizacin y
cuantificacin de estos compuestos fenlicos caractersticos en extractos
tanto de piel de naranja dulce como amarga empleando CE-ESI-MS
trabajando en modalidad negativa.
2. Se han realizado anlisis mediante CE-MS/MS para los dos tipos de

muestras anteriores y se ha podido confirmar de una forma ms fiable,
comparando con estndares, la presencia de estos compuestos fenlicos
en las muestras objeto de estudio. Para la cuantificacin se realizaron las
curvas de calibrado para cada uno de los compuestos: Naringina y
neohesperidina en piel de naranja amarga, y narirutina y hesperidina en
piel de naranja dulce. As, el mtodo puesto a punto nos permiti
diferenciar naringina y narirutina, y hesperidina y neohesperidina
empleando la deteccin por IT-MS dado que este sistema de deteccin nos
proporciona datos acerca del peso molecular e informacin estructural.
3. Como se sabe, el proceso de filtracin afecta a las caractersticas del

aceite de oliva virgen (VOO), especialmente a la estabilidad oxidativa,
contenido en agua y a la concentracin y presencia de compuestos
fenlicos. Los filtros utilizados en la produccin de aceite de oliva pueden
ser de varios tipos aunque los mas utilizados son algas microscpicas
fosilizadas, denominadas tierras de diatomeas, las cuales despus de la
etapa de filtrado del VOO son desechadas como sub-producto a pesar de
la gran cantidad de polifienoles que se retienen. Para poder recuperarlos
se puso a punto un procedimiento de extraccin utilizando una etapa
previa de extraccin con hexano para separar la fraccin polar de la no
polar. Posteriormente al extracto seco se le aadi MeOH en agitacin a
166
Conclusiones
35 C para de esta manera poder extraer los compuestos polares objeto

de estudio.
4. La caracterizacin de estos extractos se realiz utilizando la tcnica

separativa HPLC acoplada a la deteccin por MS, tcnica que combina los
avances de HPLC con la selectividad, sensibilidad, masa exacta y medidas
de relacin isotpica asociada con el analizador de TOF, permitiendo de
esta manera la caracterizacin de 19 compuestos conocidos de familias
tan importantes como de los alcoholes fenlicos, secoiridoides, lignanos,
cidos fenlicos y flavonoides. Por otro lado, con la informacin
proporcionada por el TOF se pudo determinar la presencia de 17
compuestos desconocidos.
5. Dado que la hoja de olivo es una fuente rica en compuestos fenlicos, y

que se estima que durante la campaa de recogida se producen alrededor
de 25 kg se sub-productos (hojas y ramas) por olivo anualmente en el
captulo 3 se ha puesto a punto un mtodo simple y rpido para la
extraccin de compuestos fenlicos en hojas de olivo, que consisti en
una extraccin usando MeOH, siendo ste posteriormente evaporado y
reconstituido en una mezcla MeOH:H20 (50:50, v/v) adecuada para su
anlisis mediante CE, de dos variedades de hojas de olivo, la Hojiblanca y
la Manzanilla.
6. El anlisis de estos extractos se llev a cabo empleando un nuevo mtodo

mediante CE-ESI-TOF (MS) caracterizando finalmente un total de 17 y 14
compuestos fenlicos en la variedad Hojiblanca y Manzanilla
respectivamente encontrando compuestos tan interesantes como tirosol,
hidroxitirosol, oleuropena y su aglicona, cido cafeico, verbascosido,
apigenina, luteolina, etc.
7. La piel de almendra, la cual presenta un bajo valor econmico,

representa el 4 % del peso total de la almendra, sin embargo en sta
parte contiene una gran cantidad de polifenoles. En este sentido, se
utiliz una extraccin lquido-lquido para extraer los compuestos
fenlicos presentes en la piel de almendra. En este procedimiento de
extraccin se utilizando una etapa previa de extraccin con hexano para
separar la fraccin polar de la no polar. Posteriormente se filtr y se trat
167
Conclusiones
el extracto slido con MeOH al 70 % en condiciones de reflujo a 60 C

durante 45 minutos, se volvi a filtrar y la disolucin resultante se
evapor a vaco, reconstituyendo finalmente en una mezcla MeOH:H2O
para su anlisis mediante CE y en MeOH para el anlisis por HPLC. Estos
extractos fueron posteriormente analizados mediante HPLC y CE
acopladas a ESI-TOF (MS).
8. Ha sido la primera vez que se ha puesto un mtodo a punto mediante CE

para la caracterizacin de stos compuestos fenlicos en piel de
almendra, lo cual supone una alternativa interesante a los mtodos
desarrollados mediante HPLC. Ambos mtodos permitieron una
caracterizacin directa y sensible de estos compuestos fenlicos aunque
ambos presentan algunas ventajas e inconvenientes. As, empleando HPLC
se pudieron caracterizar 23 compuestos en un tiempo de anlisis de 9
minutos y sin embargo solo 9 compuestos mediante CE en un tiempo de 35
minutos. Con lo cual, empleando HPLC se reduce el tiempo de anlisis
significativamente y se logra detectar un mayor numero de compuestos
adems de que HPLC nos ofrece importantes ventajas, como mejoras en
la precisin de la inyeccin y sensibilidad, mientras que CE puede
competir en trminos de costes de funcionamiento reducidos.
9. La semilla de lino se compone de un 41 % de aceite, el cual contiene una

gran cantidad de compuestos fenlicos con propiedades beneficiosas para
la salud. Para caracterizarlos se desarroll previamente un procedimiento
de extraccin de los compuestos fenlicos en el aceite de linaza, que
consisti en una extraccin en fase slida empleando cartuchos DSC-Diol,
y se optimiz un nuevo mtodo utilizando HPLC-ESI-TOF (MS).
10. Con la metodologa propuesta se detectaron 10 polifenoles en la variedad

H52 y P129, y 9 compuestos en la variedad O116, destacando polifenoles
tan caractersticos como secoisolariciresnol, difilin, pinoresinol,
matairesinol, etc. Al mismo tiempo, se pudieron estudiar otros
compuestos desconocidos presentes en las diferentes variedades (H52,
P129 y O116) los cuales son una parte importante de la fraccin polar del
aceite de linaza. En este estudio, se pudieron encontrar diferencias
significativas entre las tres variedades empleando la metodologa
168
Conclusiones
propuesta, de hecho estas diferencias podran ser utilizadas en un futuro

para encontrar posibles biomarcadores del aceite de linaza o de la
variedad de semilla.
11. Finalmente, como conclusin general, se puede afirmar que en la

presente tesis se han puesto a punto diferentes procedimientos de
extraccin y diferentes metodologas, tanto por CE como por HPLC
acopladas a MS, para la caracterizacin de un buen nmero de
compuestos fenlicos en una amplia variedad de matrices de inters. Por
tanto, aparte de estudiar exhaustivamente diferentes procedimientos de
extraccin para el anlisis de los compuestos objeto de estudio en funcin
del tipo de matriz, el empleo de tcnicas separativas avanzadas nos
permiti en la mayora de los casos obtener unos buenos resultados en
cuanto a resolucin, eficiencia y tiempo de anlisis. Por otra parte, stas
tcnicas separativas fueron acopladas a un sistema de deteccin de
enorme potencialidad como es la MS, cuyas caractersticas ms
destacadas son su sensibilidad, selectividad y el proporcionar informacin
estructural. En este sentido, a lo largo de este trabajo de investigacin se
ha utilizado el analizador de IT, cuya caracterstica ms destacada es la
posibilidad proporcionar fragmentos reales de una masa concreta
(MS/MS), y el analizador de TOF, con el que obtenemos resolucin, masa
exacta y medidas de relacin isotpica, obteniendo de esta manera una
informacin muy valiosa y precisa para la caracterizacin los compuestos
fenlicos en las matrices objeto de estudio: piel de naranja, tierras de
diatomeas utilizadas en el proceso de filtracin del aceite de oliva, hoja
de olivo, piel de almendra y aceite de linaza.
169
Abstract
170
Abstract
ABSTRACT
This work is a summary of all the results obtained during the PhD thesis:
Characterization of bioactive compounds in food products and sub-products using

advanced separatives techniques
The current work can be divided in three sections; the first one is the
"INTRODUCTION", which includes outstanding information about functional food,
bioactive compounds, phenolic compounds, the separative techniques (CE and HPLC)
with the different mass spectrometry analyzers (IT and TOF) used and finally
information about the different matrices studied.
Then, we can see the "EXPERIMENTAL SECTION. RESULTS AND DISCUSSION" section,
divided in five Chapters related to every matrix that has been studied: orange skin,
diatomaceous earth used in the filtration process of olive oil, olive leaves, almond
skin and flaxseed oil.
And finally, conclusions of each chapter can be seen in the third section.
Chapter 1: Quantification of main phenolic compounds in sweet and bitter orange

peel using CE-MS/MS
The food and agricultural products processing industries generate substantial

quantities of phenolics-rich subproducts, which could be valuable natural sources of
polyphenols. Thus, the present work describes the development of a method using
CE-ESI-IT (MS) for the analysis and quantification of main phenolic compounds in
orange peels, due to in oranges, the peel represents roughly 30% of the fruit mass
and the highest concentrations of flavonoids in citrus fruit occur in peel. In this sense,
a characterization and quantification of citrus flavonoids in methanolic extracts of
bitter and sweet orange peels using CE-ESI-IT (MS) have carried out due to CE
coupled to MS detection can provides structure-selective information about the
analytes. Naringin (m/z 579.2) and neohesperidin (m/z 609.2) are the major
polyphenols in bitter orange peels and narirutin (m/z 579.2) and hesperidin (m/z
609.2) in sweet orange peels. The proposed method allowed the unmistakable
identification, using MS/MS experiments and also the quantification of naringin (5.1
172
Abstract
0.4 mg/g), neohesperidin (7.9 0.8 mg/g), narirutin (26.9 2.1 mg/g) and
hesperidin (35.2 3.6 mg / g) in bitter and sweet orange peels.
Chapter 2: Characterization of phenolic compounds in diatomaceous earth used in

the filtration process of olive oil by HPLC-ESI-TOF (MS).
This chapter explains the study carried out to determine the phenolic content in
diatomaceous earth used in the filtration step which is the last step in the production
processes of olive oil. Take into account that the main producer of olives and olive
oil is Europe Union with over 80 % and olive oil production processes, there is a large
amount of by-products, in which the healthy value of olive oil is undervalued. Here is
proposed an HPLC-ESI-TOF (MS) method for the separation and detection of a broad
series of phenolic compounds present in the diatomaceous earth. Thus, the
characterization of 19 phenolic compounds from several important families (phenolic
alcohols, secoiridoids, lignans, phenolic acids and flavonoids) of the polar fraction of
olive oil was achieved. Furthermore, other unknown compounds were also
characterized. Thus the results observed in this study mean that diatomaceous earth
used in the filtration step of olive oil production affects the phenolic composition of
olive oil, because an important amount of phenolic compounds are still present at
the filtration material, being the most abundant hydroxytyrosol, ligstroside aglycone,
hydroxy-pinoresinol, vanillic acid, tyrosol and luteolin.
Chapter 3: Identification of phenolic compounds in olive leaves using CE-ESI-TOF

(MS).
This chapter includes an easy and rapid method using CE-ESI-TOF (MS) to analyze
phenolic compounds in two varieties of olive leaves (Hojiblanca and Manzanilla). The
separation parameters have been performed in respect to resolution, sensitivity,
analysis time and peak shape. Namely the optimization of both electrophoretic
parameters and electrospray conditions are required for reproducible analyses. The
method allows the simultaneous identification of seventeen and fourteen phenolic
compounds in Hojiblanca and Manzanilla leaves extracts respectively. Due to its high
efficiency, rapidity, small sample amounts required and high resolution of CE
coupling to the sensitivity, selectivity, mass accuracy and true isotopic pattern from
TOF (MS) have revealed an enormous separation potential allowing the identification
of a broad series of phenolic compounds present in olive leaves.
173
Abstract
Chapter 4: HPLC/CE-ESI-TOF (MS) methods for the characterization of polyphenols in

almond skin extracts.
Chapter 4 includes the development of two rapid methods using CE and HPLC coupled
to ESI-TOF (MS) and both have been compared for the separation and
characterization of antioxidant phenolic compounds in almond skin extracts. Under
the optimum CE-ESI-TOF (MS) conditions we achieved the determination of nine
compounds of the polar fraction in 35 min. Furthermore, by using HPLC-ESI-TOF (MS)
method, a total of twenty-three compounds corresponding to phenolic acids and
flavonoids family were identified from almond skin only in 9 min. We have
demonstrate that the sensitivity, together with mass accuracy and true isotopic
pattern of the TOF (MS), allowed the identification of a broad series of known
phenolics compounds present in almond skin extracts using HPLC and CE as
separative techniques.
Chapter 5: Characterization of phenolic and other polar compounds in flaxseed oil

using HPLC-ESI-TOF (MS).
In this chapter a sensitive method based on HPLC-ESI-TOF (MS) has been used to
analyze phenolic compounds in Flaxseed oil. Several important phenolic compounds
such as secoisolariciresnol, ferulic acid and its methyl ester, methyl ester coumaric
acid, diphylin, pinoresinol, matairesinol, p-hydroxybenzoic acid, vanillin and vanillic
acid have been detected directly from Flaxseed oil. The efficiency, the rapidity and
the high resolution of HPLC coupling to the sensitivity, selectivity, mass accuracy and
true isotopic pattern from TOF (MS) have revealed an enormous separation potential
allowing the characterize of a broad series of phenolic compounds present in
flaxseed oil for the first time.
174

Resumen
175
Resumen
RESUMEN
Aqu se recopilan los principales hitos relativos a los contenidos de la presente

memoria titulada: Caracterizacin de compuestos bioactivos en productos y sub-
productos alimentarios empleando metodologas separativas avanzadas
El trabajo realizado se divide en dos apartados: la "INTRODUCCION" y la "PARTE

EXPERIMENTAL. RESULTADOS Y DISCUSIN.
En el primero de ellos se describe la importancia que tiene hoy da los alimentos

funcionales, debido a que en la actualidad la nutricin est experimentando un veloz
cambio ya que los consumidores buscan aquellos productos en el mercado que,
adems del valor nutritivo, aporten beneficios a las funciones fisiolgicas del
organismo para mantener su salud y bienestar. Los alimentos que ayudan a prevenir
enfermedades y a mantener la salud han sido denominados Alimentos Funcionales,
concepto que nace en Japn en los aos 1980s cuando las autoridades alimentarias
japonesas tomaron conciencia de que para controlar los gastos globales en salud era
necesario desarrollar alimentos que mejoraran la calidad de vida de la poblacin.
Estos alimentos funcionales contienen compuestos bioactivos, que son aquellos que
tiene la capacidad de mermar el efecto daino que puede ocasionar una enfermedad
y entre ellos podemos encontrar diferentes familias como isoflavonas, compuestos
fenlicos, cido ascrbico, carotenos, clorofilas, vitamina E y fitoesteroles, entre
otros, de los cuales algunos se encuentran en pequeas concentraciones. En la
presente memoria doctoral nos centraremos en la caracterizacin de compuestos
fenlicos; estos son compuestos biosintetizados por los vegetales como producto de
su metabolismo secundario normal y algunos de ellos son indispensables para sus
funciones fisiolgicas, mientras que otros son de utilidad para defenderse ante
situaciones de estrs (hdrico, luminoso, etc). stos compuestos fenlicos son un
grupo heterogneo de productos con ms de 10.000 compuestos, por lo que son
clasificado, de acuerdo a su esqueleto bsico, en diferentes familias: cidos
fenlicos, lignanos, estilbenos y flavonoides entre los que destacamos los flavonoles,
flavonas, isoflavonas, flavanonas, etc.
Adems, en esta introduccin se hace una pequea revisin de las diferentes tcnicas
analticas empleadas en la determinacin de compuestos fenlicos as como de los
procedimientos de extraccin utilizados.
177
Resumen
Seguidamente se describen las diferentes tcnicas analticas empleadas en este

trabajo de investigacin. En primer lugar se comenta la cromatografa lquida (LC),
instrumentacin, tipos de LC as como las caractersticas mas destacadas de esta
tcnica separativa y de la misma manera se hace una descripcin del otro tipo de
tcnica separativa utilizada en esta memoria como es la electroforesis capilar (CE),
instrumentacin, modalidades, caractersticas, etc.
Dado que stas tcnicas separativas se acoplarn a la espectrometra de masas (MS)
como sistema de deteccin, en esta introduccin se describe el mecanismo de
funcionamiento de un espectrmetro de masas as como los diferentes tipos de
analizadores que pueden ser utilizados, profundizando en el mecanismo y las
caractersticas de los dos tipos de analizadores que van a ser utilizados en este
trabajo experimental como son la trampa de iones (IT) y el tiempo de vuelo (TOF).
Para poder acoplar la LC y la CE, dos tcnicas separativas que utilizan muestras en
estado lquido, con la MS, que necesita muestras en estado gaseoso, es necesaria la
presencia de interfases que nos solucionen este problema. En este sentido se hace un
pequeo esbozo de las interfases ms usuales para este tipo de acoplamientos,
centrndonos principalmente en el tipo de interfase utilizada en este trabajo, la
ionizacin por electroespray (ESI).
Finalmente se incluye una pequea revisin del anlisis de compuestos fenlicos
utilizando las dos tcnicas separativas que van a ser empleadas, LC y CE.
El segundo apartado est dividido en cinco captulos relacionados con cada matriz
estudiada: 1) Piel de naranja, 2) Tierras de diatomeas utilizadas en el proceso de
filtracin del aceite de oliva, 3) Hoja de olivo, 4) Piel de almendra y 5) Aceite de
linaza.
Captulo 1: Cuantificacin de los compuestos fenlicos principales en piel de naranja

dulce y amarga mediante CE-MS/MS
Los alimentos y las industrias alimentarias generan cantidades importantes de sub-

productos ricos en compuestos fenlicos, que podran ser valiosas fuentes naturales
de polifenoles. Por lo tanto, en el presente trabajo se desarrollo de un mtodo
mediante CE-ESI-IT (MS), puesto que el acoplamiento de CE con la deteccin por MS
nos proporciona datos acerca del peso molecular e informacin estructural de los
analitos objeto de estudio, para el anlisis y cuantificacin de los principales
compuestos fenlicos en piel de naranja dulce y amarga, dado que la piel representa
178
Resumen
aproximadamente el 30% de la masa de fruta y es donde se encuentra la mayor

concentracin de flavonoides. En este sentido, se estudiaron Naringina (m/z 579.2) y
neohesperidina (m/z 609.2) en piel de naranja amarga y narirutina (m/z 579.2) y
hesperidina (m/z 609.2) en piel de naranja dulce. As, el mtodo propuesto permiti
la identificacin inequvoca, mediante experimentos de MS/MS de los analitos objeto
de estudio, adems de la cuantificacin de naringina (5.1 0.4 mg/g),
neohesperidina (7.9 0.8 mg/g), narirutina (26.9 2.1 mg/g) y hesperidina (35.2
3.6 mg / g) en piel de naranja amarga y dulce respectivamente.
Captulo 2: Caracterizacin de compuestos fenlicos en tierras de diatomeas

empleadas en el proceso de filtracin del aceite de oliva mediante HPLC-ESI-TOF
(MS).
Este captulo explica el estudio realizado para determinar el contenido de polifenoles

en la tierra de diatomeas utilizadas en la etapa de filtracin, el cual es el ltimo
paso en el proceso de produccin de aceite de oliva. Teniendo en cuenta que Espaa
es el principal productor de aceite de oliva de la Unin Europea, con ms del 80 %,
podemos concluir que durante todo este proceso se generan una gran cantidad de
sub-productos derivados de la produccin a los cuales se les subestima su valor
saludable. Por tanto en este captulo se ha propuesto un mtodo mediante HPLC-ESI-
TOF (MS) para la separacin y deteccin de un buen nmero de compuestos fenlicos
presentes en las tierras de diatomeas. De esta manera se pudieron caracterizar 19
compuestos fenlicos de diferentes familias (alcoholes fenlicos, secoiridoides,
lignanos, cidos fenlicos y flavonoides) en la fraccin polar. A parte, se
caracterizaron otros compuestos desconocidos pertenecientes a la misma fraccin.
Por tanto de los resultados obtenidos en este estudio podemos decir que la tierra de
diatomeas utilizada en el proceso de filtracin afecta a la composicin fenlica del
aceite de oliva, dado que una importante cantidad de compuestos fenlicos estn
presentes en el material de filtracin, siendo los mas abundantes hydroxitirosol,
ligustrosido aglycona, hydroxipinoresinol, acido vanilico, tirosol and luteolina.
Captulo 3: Identificacin de compuestos fenlicos en hojas de olivo mediante CE-

ESI-TOF (MS).
Se describe un mtodo fcil y rpido empleando CE-ESI-TOF (MS) para el anlisis de

compuestos fenlicos en dos variedades de hojas de olivo, Hojiblanca y Manzanilla.
179
Resumen
Para la eleccin de los parmetros ptimos en la separacin se ha tenido en cuenta la

resolucin, sensibilidad, tiempo de anlisis y forma de pico. As el mtodo propuesto
permiti la identificacin simultnea de 17 y 14 compuestos fenlicos en extractos
de hojas de olivo de la variedad Hojiblanca y Manzanilla respectivamente,
encontrado compuestos tan interesantes como el tirosol, hidroxitirosol, oleuropena y
su aglicona, cido cafeico, verbascosido, apigenina y luteolina entre otros. Por tanto
el mtodo propuesto ha puesto de manifiesto el enorme potencial de la tcnica
empleada para el anlisis de una amplia serie de compuestos fenlicos presentes en
hojas de olivo debido a la alta eficacia, rapidez, pequeo volumen de muestra
requerido y la alta resolucin de CE acoplada con la sensibilidad, selectividad, masa
exacta y relacin isotpica proporcionada por el TOF (MS).
Captulo 4: Caracterizacin de compuestos fenlicos en extractos de piel de almendra

mediante HPLC/CE-ESI-TOF (MS).
En el captulo 4 se describe el desarrollo de dos metodologas empleando CE y HPLC

acopladas a ESI-TOF (MS) las cuales han sido comparadas en la capacidad de
separacin y caracterizacin de compuestos fenlicos en extractos de piel de
almendra. As, bajo las condiciones ptimas de CE-ESI-TOF (MS) se pudo lograr la
determinacin de 9 compuestos de la fraccin polar en un tiempo de 35 minutos. Por
otra parte, empleando HPLC-ESI-TOF (MS) se pudieron determinar un total de 23
compuestos, correspondientes a la familia de los cidos fenlicos y flavonoides, en
solo 9 minutos. Por tanto se puede afirmar que empleando HPLC se reduce el tiempo
de anlisis significativamente y se logra detectar un mayor nmero de compuestos
adems de que el uso HPLC nos ofrece importantes ventajas, como mejoras en la
precisin de la inyeccin y sensibilidad, mientras que CE puede competir en trminos
de costes de funcionamiento reducidos.
Captulo 5: Caracterizacin de fenoles y otros compuestos polares en aceite de

linaza empleando HPLC-ESI-TOF (MS).
En este ltimo captulo se pone a punto una metodologa mediante HPLC-ESI-TOF (MS)
para el anlisis de compuestos fenlicos en aceite de linaza. Un buen numero de
compuestos fenlicos de gran inters, como son secoisolariciresnol, acido ferulico y
su ster metlico, ster metilico del acido cumarico, dilfilin, pinoresinol, matairesinol,
acido p-hidroxibenzoico, vanilina y acido vanilinico, han sido detectados en aceite de
180
Resumen
linaza. Al mismo tiempo, se han estudiado otros compuestos desconocidos presentes

los cuales son una parte importante de la fraccin polar del aceite de linaza. Por
tanto, el mtodo de HPLC-ESI-TOF (MS) desarrollado permiti la caracterizacin de
un buen nmero de compuestos fenlicos presentes en aceite de linaza.
181

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GRANADA UNIVERSITY

CHARACTERIZATION OF BIOACTIVE COMPUNDS IN FOOD PRODUCTS AND

Submitted for the degree of Doctor of Chemistry

Signed: Dr. Alberto Fernndez Gutirrez

Signed: Dr. Antonio Segura Carretero

Signed: Dr. David Arrez Romn

Research work submitted to get the Doctor in chemistry degree

Signed: Saleh M. S. Sawalha

In Granada, first of December of two thousand and nine.

This study was carried out at the University of Granada, Department of

I wish to express my deepest gratitude to my two principal supervisors Dr.

My warmest thanks belong to my parents (Abu al Amin and Om Al amin)

Finally, my dearest thanks are addressed to my family, my wife Athar for

Saleh, December 2009

1.1. Bioactive compounds 29

1.2. Phenolic compounds 30

2. Analytical determination of polyphenols in food sample 42

2.2. Sample preparation 43

2.2.1. Liquid extraction (LE) 44

2.2.2. Solid-phase extraction (SPE) 45

2.3. Analytical techniques 46

2.3.1. Liquid Chromatography (LC) 46

2.3.2. Capillary Electrophoresis (CE) 50

2.3.3. Mass Spectrometry (MS) 53

2.3.3.1. Mass Analyzer 54

2.3.3.2. Ion source 57

2.3.3.3. The Interfaces for coupling CE/MS and LC/MS 59

2.4. Phenolic compounds by HPLC and CE 64

2.4.1. Phenolic compounds by HPLC 64

3. Samples: Importance, main phenolic compounds and health properties 70

Experimental Part, Results and dissection 85

Chapter I Quantification of main phenolic compounds in sweet and bitter

Chapter II Characterization of phenolic compounds in diatomaceous earth used

Chapter III Identification of phenolic compounds in olive leaves using CE-ESI-

Chapter IV HPLC/CE-ESI-TOF (MS) methods for the characterization of

Chapter V Characterization of phenolic and other polar compounds in Flaxseed

Objective of PHD Thesis:

Nutraceuticals are natural, bioactive chemical compounds that are characterized by

As a result of a long decision-making process to establish a category of foods for

Functional foods have been broadly defined as foods similar in appearance to

Fortified product. A food fortified with additional nutrients.

Enriched products. A food with added new nutrients or components not

Altered products. A food, from which a deleterious component has been

Enhanced commodities. A food in which one of the components has been

In the United States, functional attributes can be communicated through health

Table 1. Codex Alimentarius Definitions

Functional Food that has physiological functions, including regulation of

Generic Claims based on well-established, generally accepted knowledge derived

Enhanced Claims that concern specific beneficial effects regarding the

Structure/ Any statements regarding the effects of dietary supplementation on the

Though an official definition of functional foods is lacking in both the US 14 (ADA

concerted action project FUFOSE (Functional Food Science in Europe) coordinated by

"a food can be regarded as functional if it has been satisfactorily

demonstrated to affect beneficially one or more target functions in

the body beyond adequate nutritional effects in a way that is

relevant to either an improved state of health and well-being and/or

a reduction of risk of disease".

Besides providing scientifically proven health effects, functional foods have to

effects in amounts that can normally be expected to be consumed in