GRANADA UNIVERSITY Department of Analytical …hera.ugr.es/tesisugr/1858679x.pdfAnalytical Chemistry...
Transcript of GRANADA UNIVERSITY Department of Analytical …hera.ugr.es/tesisugr/1858679x.pdfAnalytical Chemistry...
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 GranadaAutor: Saleh M.S. SawalhaD.L.: GR 2681-2010ISBN: 978-84-693-2011-2
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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.
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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 Fernández Gutiérrez
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
Faculty of Sciences. University of Granada
Signed: Dr. David Arráez Román
Post-doctoral researcher of the Department of Analytical Chemistry
Faculty of Sciences. University of Granada
Research work submitted to get the Doctor in chemistry degree
Signed: Saleh M. S. Sawalha
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D. ALBERTO FERNÁNDEZ GUTIÉRREZ, 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 Arráez Román 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.
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Acknowledgments
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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 Fernández Gutiérrez 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 Arráez Román. 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 parent’s (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
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Table of contents
Table of contents
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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
Table of contents
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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 CE–MS/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
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Objectives
Objective
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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.
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Introduction
Functional food
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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 foods2. 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 food’’3.
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. Lähteenmäki, 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, 129:1395S–1397S. 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, 45:385–404.
Introduction
24
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 contains’’3.
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 functions”6. 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., 55:413–422. 7. Spence, J.T. (2006). Challenges related to the composition of functional foods. Journal of Food
Composition and Analysis, 19: S4–S6. 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.
Functional food
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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 health10. With regard to biological benefits in functional foods, the
International Life Sciences Institute’s 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, structure–function 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 structure–function 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: S125–S130. 11. Weststrate J.A., van Poppel G., Verschuren P.M. (2002). Functional foods, trends and future,
British Journal of Nutrition, 88, Suppl. 2:S233–S235 12. Milner J.A. (2002). Functional foods and health: a US perspective, British Journal of Nutrition, 88,
Suppl. 2: S151–S158. 13. Shimizu T. (2003). Health claims on functional foods: the Japanese regulations and an
international comparison, Nutrition Research Reviews, 16: 241–252.
Introduction
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Table 1. Codex Alimentarius Definitions
Term Definition
Functional food
Food that has physiological functions, including regulation of 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 nutrient–function 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
health claims
Claims based on well-established, generally accepted knowledge derived from evidence in the scientific literature and/or on recommendations from national or international public health bodies.
Product-specific claims
Claims that concern certain physiological effects other than a generic health claim, which requires demonstrations based on scientific evidence for individual products.
Enhanced function claims
Claims that concern specific beneficial effects regarding the 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/
function claims
Any statements regarding the effects of dietary supplementation on the structure or function of the body, that is defined by the Dietary Supplement, Health and Education Act in the USA in 1994. These claims are generally similar to the enhanced function (or other) claims.
Dietary supplement
A product intended to supplement the diet, which contains one or more 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 US14 (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.
Functional food
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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: S1–S193. 18. Knorr D. (1998). Functional food science in Europe, Trends in Food Science and Technology, 9:
295–340.
Introduction
28
functions and reduce the risk of some diseases19. 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 food21. 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):1660S–1664S 20. Sloan A.E. (2000). The top ten functional food trends. Food Technology, 54, 33–62 21. Sloan A.E. (2004). The top ten functional food trends. Food Technology, 58, 28–51
Functional food
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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: 461–477. 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: 511–538.
Introduction
30
researchers may accurately assess their dietary intake, investigate their physiological
functions, and determine their relationships to health and disease24.
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 anti- carcinogenic. 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: 419–434.
Bioactive compounds
Microbial
Minerals Lipidic
compounds
Carbohydrate
& derivatives
Protein/
Amino Acid
Phenolic
compound
Isoprenoids
(Terpenoids)
Carotenoids
Saponins
Tocotrienols
Tocopherols
Simple terpenes
Phenolic acids
Flavonoids
Secoiridoids
Lignin
Coumarins
Tannins
Amino acids
Allyl -S-Compds
Capsaicinoids
Isothiocyanates
Indoles
Folate
Choline
Ascorbic acid
Oligosaccharides
Non starch PS
n-3 PUFA
CLA
MUFA
Sphingolipids
Lecithin
Sterols
Ca
Se
K
Cu
Zn
Probiotics
Functional food
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• 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 lignans25,26(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, 86:1805–1813. 26. Manach, C. et al., (2004), Polyphenols: food sources and bioavailability, Am. J. Clin. Nutr., 79,
727.
Introduction
32
Figure 2: classification scheme for polyphenols
Figure 3: Chemical structures of major classes of polyphenols.
Functional food
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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 0.5–2 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 50–150 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). Ellagitannins—occurrence 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.
Introduction
34
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 polyphenols36,37. 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 cinnamates—nature, 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, 342:1007–1011.
40. Linseisen J., Radtke J., Wolfram G. (1997). Flavonoid intake of adults in a Bavarian subgroup of the national food consumption survey. Z. Ernährungswiss, 36:403–412.
Functional food
35
• 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.
Introduction
36
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) 46Cereals such as millet and wheat contain C-glycosides of
flavones47–49 .
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 15–85
mg narirutin/L, and a single glass of orange juice may contain between 40 and 140
mg flavanone glycosides50. 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.
Functional food
37
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 diseases56–59. 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. 371–542.
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.
Introduction
38
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
that dietary flavonoids exert health benefits through antioxidant mechanisms 65–67.
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.
Functional food
39
Figure 5: Structures of plant and mammalian lignans.
They may occur glycosidically bound to various sugar residues, esterified or as
structural subunits of biooligomers69–71. 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
lariciresinol) were reported to be relatively high 72 . 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.
Introduction
40
(Figure 6)74. After the conversion, END is oxidized to ENL75 . 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.
Functional food
41
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.
Introduction
42
2. Analytical determination of polyphenols in food sample.
2.1. Introduction.
Food quality control and food nutritional value have become major topics of public
interest82. 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 science83 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. Ibañez , E. Cifuentes. A. (2001) "New analytical techniques in food science". Crit. Rev. Food Sci.
41 413-450. 83. Señorans, F.J., Ibañez, 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., YRJÖNEN, 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.
Analytical determination of polyphenols
43
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
flavonoids90–92, tannins93 , and phenolic acids94–96 .
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., SIRÉN, 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 SØRENSEN, 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.
Introduction
44
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: 95–111.
Analytical determination of polyphenols
45
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
methanol/water (with different quantities of water ranging between 0% and 40% 101
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 centrifugation103 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., García, A., García, 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.
Introduction
46
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.
Analytical determination of polyphenols
47
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), Cromatografía líquida en columna en Técnicas de separación en Química Analítica. Ed. Síntesis S. A. Madrid. P: 399-498.
Introduction
48
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. Valcárcel Cases, M.; Gómas Hens, A, (1990); Cromatografía líquida en columna (II). Técnicas de
absorción y partición, en Técnicas analíticas de separación,. Ed. Reverté S.A, 485-531.
Analytical determination of polyphenols
49
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 µm) 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;
Introduction
50
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.
Analytical determination of polyphenols
51
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.
Anode
Buffer Sample Buffer
High voltage supplier
Detector
Capillary
Introduction
52
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
Analytical determination of polyphenols
53
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).
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).
Ionizer
Sample
+ _
Mass Analyzer Detector
Introduction
54
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, 1015–1026.
Analytical determination of polyphenols
55
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 Guilhaus118, 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.
1. Pump stage
2. Pump stage
3. Pump stage
4. Pump stage
Glass Capillary
Drying Gas
Dual Octopole
Skimmer
Partition
Lenses
Ion Trap
Detector
Ion Source
Introduction
56
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, 951–962.
Analytical determination of polyphenols
57
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.
Introduction
58
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 µm in diameter) droplets125. This spray was described in greater detail and
photographs presented in Zeleny’s 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
Analytical determination of polyphenols
59
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 Abian129, while Gelpí has updated this work, with attention
paid to LC techniques only 130 . 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. Gelpí, E. (2002) Interfaces for coupled liquid-phase separation/mass spectrometry techniques. An
update on recent developments, J. Mass Spectrom., 37, 241-253.
Introduction
60
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 µL 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 µL 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 droplets132. 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.; Neusüß, C.; Pelzing, M.; Segura-Carretero, A.; Fernández-Gutiérrez, A.
(2007) CE- and HPLC-TOF-MS for the characterization of phenolic compounds in olive oil. Electrophoresis, 28, 806-821.
Analytical determination of polyphenols
61
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;37:241–253. 136. Schmitt-Kopplin, P., Frommberger, M. (2003) Capillary electrophoresis mass spectrometry: 15
years of developments and applications. Electrophoresis, 24, 3837–3867. 137. Aline Staub et el, (2009) CE-TOF/MS: Fundamental concepts, instrumental considerations and
applications., Electrophoresis, 30, 1610–1623
Introduction
62
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 liquid–junction 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; 37:241–253.
Analytical determination of polyphenols
63
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
outlet139. 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, 81–109. 140. Zamfir, A. D. (2007) Recent advances in sheathless interfacing of capillary electrophoresis and
electrospray ionization mass spectrometry. J. Chromatogr. A, 1159, 2–13.
Introduction
64
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 (989–1009. 142. Deshpande, S.S. Cheryan, M. Salunkhe, D.K. (1986) Tannin analysis food-products .CRC Crit. Rev.
Food Sci. Nutr. 24 401–449. 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. 209–222.
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. 11 279–308.
145. Makkar, H.P.S. (1989) Relation of rumen degradability with microbial colonization. J. Agric. Food Chem. 37 1197–1202.
146. Naczk, M. Shahidi, F. (2004) Extraction and analysis of phenolics in food J. Chromatogr. A 1054 95–111.
147. Porter, L.J. Harborne,J.B. (1989) Methods in Plant Biochemistry, vol. 1, Academic Press, San Diego, CA, pp. 389–420.
148. Scalbert, A. Monties, B. Janin, G. (1989) Tannins in wood: comparison of different estimation methods, J. Agric. Food Chem. 37 1324–1329.
149. Scalbert, A. in: R.W. Hemingway, Laks P.S. (1992) (Eds.), Plant Polyphenols: Synthesis, Properties Significance, Plenum Press, New York, NY, pp. 259–280.
150. Tempel, A.S. (1982) Tannin-measuring techniques, J. Chem. Ecol. 8 1289–1298. 151. Tsao, R. Deng, Z. (2004) separation procedures for naturally occurring antioxidant phytochemical,
J. Chromatogr. B 812, 85–99. 152. Shahidi, F. , Naczk, M., in: Otles S. (2005) Methods of Analysis of Food Components Additives, CRC
Press, Boca Raton, FL, pp. 199–259. 153. Robards, K. (2003) the determination of bioactive phenols in plants, fruit and vegetables J.
Chromatogr. A 1000 657–691. 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 890 251–259.
Analytical determination of polyphenols
65
chromatograms. Shoji et al. 155 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
phenolics159–162.
Polyphenols are commonly detected using UV–vis and photodiode array detectors163–
165 . Other methods used for the detection of phenolics include electrochemical
coulometric array detector 166 , chemical reaction detection technique 167 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 HPLC–MS 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, 206−213.
156. Merken H.M., Beecher G.R., (2000) Measurement of food flavonoids by high-performance liquid chromatography: A review, J. Agric. Food Chem. 48 577–599.
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 1259–1260, 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. 1–18.
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. 6 105–118. 161. Daigle D.J., Conkerton E.J., (1988) Analysis of flavonoids by HPLC – an update, J. Liq. Chromatogr.
11 309–325. 162. Robards K., Antolovitch M., (1997) Analytical chemistry of fruit bioflavonoids– A review, Analyst
122 11R–34R. 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 4748–4760.
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, 26–31.
165. Barnes S., Coward L., Kirk M., Sfakianos J., (1998) HPLC-mass spectrometry analysis of isoflavones, J. Proc. Soc. Exp. Biol. Med. 217 254–262.
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 5834–5841.
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, 4209–4213.
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, 3693–3701.
169. Lunte C.E., Wheeler J.F., Heineman W.R., (1988) Determination of selective phenolic-acids in beer Extract by LC, Analyst 113 94–95.
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, 946–951.
Introduction
66
been employed for structural confirmation of phenolics in plums, peaches,
nectarines171, grapeseeds164, soyfoods172, cocoa173 and olive oil174–176 Satterfield and
Brodbelt177 demonstrated that complexation of flavonoids with Cu2+ enhanced the
detection of flavonoids by ESI-MS. Mass spectra obtained for metal–flavonoids
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 (EI–MS)179 .Matrix-assisted laser desorption/ionization mass spectrometry
(MALDI–MS) 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, 4748–4760.
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, 3651–3655.
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, 490–496.
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, 4331–4340.
175. Bianco, A., Buiarelli, F., Cartón, 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, 417–424.
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. 49,2767–2773.
180. Wang J., Sporns P., (1999) , Analysis of Anthocyanins in Red Wine and Fruit Juice using MALDI-MS. J. Agric. Food Chem. 47 2009–2015.
Analytical determination of polyphenols
67
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
amperometric detection. Chu et al. 188 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. 93 417–423. 182. Cifuentes A., (2006) "Recent advances in the application of capillary electromigration methods for
food analysis". Electrophoresis 27, 283–303. 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, 919–921. 184. Andrade P, Seabra R, Ferreira M, Ferreres F, Garciá-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: 161–164.
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, 153–156.
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, 1519–1523.
188. Chu Q., O’Dwyer M., Zeece M.G., (1998) Direct analysis of resveratrol in wine by Micel capillary electrophoresis. J. Agric. Food Chem. 46, 509–513.
Introduction
68
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
electrophoresis. Later Zeece 194 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
teas. Subsequently, Bonoli et al. 197 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 4649–4662. 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, 187−193 191. Cancalon, P. F.(1999) Analytical monitoring of citrus juices by using capillary electrophoresis. J.
AOAC Int., 82 (1), 95−106. 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., Ibáñez E., García E., Rodríguez de Pablos R., Señoráns 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 758 332–335.
196. Larger P.J., Jones A.D., Dacombe C., (1998) Separation of polyphenols using micellar electrokinetic chromatography with diode array detection. J. Chromatogr. A 799 309–320.
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, 1141–1147.
Analytical determination of polyphenols
69
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/MS201-206. 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, 1561–1567.
199. Arraez-Roman, G. Zurek, C. Babmann, N. Almaraz-Abarca, et al., (2007) Identification of phenolic compounds from pollen extracts using capillary electrophoresis–electrospray time of flight mass spectrometry, Anal. Bioanal. Chem. 389, pp. 1909–1917.
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, 806–821.
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, 2135–2147.
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, 2852–2861.
203. Petersson, E. V., Puerta, A., Bergquist, J., Turner, C., (2008) Electrophoresis, 29, 2723–2730. 204. Arra´ez-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, 2112–2116.
205. Garcia-Villalba, R., Leon, C., Dinelli, G., Segura-Carretero, A., Fernandez-Gutierrez, A., Garcia- Canas, V., Cifuentes, A., J. Chromatogr. A 2008, 1195, 164–173.
206. Simó, 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.
Introduction
70
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 5–15
m tall, with spiny shoots and alternately arranged evergreen leaves with an entire
margin. The flowers are solitary or in small corymbs, each flower 2–4 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, 4–30 cm long and 4–20 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.
Samples: Importance, main phenolic compounds and health properties
71
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 anticancer210, 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: 709–712.
Introduction
72
platelet aggregation 212 and another potential beneficial biological actions in
humans213-216.
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: 91–100. 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, 3198–3203.
Samples: Importance, main phenolic compounds and health properties
73
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. Gómez-Caravaca A.M., Cerretani L., Bendini A., Segura-Carretero A., Fernández-Gutiérrez 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.
Introduction
74
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 quality227. 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 species228 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, 3869–3874.
229. Vázquez-Roncero, A., Janer Del Valle, L., Janer Del Valle, C. (1976). Componentes feno�licos de la aceituna. III. Polifenoles del aceite. Grasas Aceites, 27, 185-191.
230. Carrasco-Pancorbo, A., Gómez-Caravaca, A. M., Cerretani, L., Bendini, A., Segura-Carretero, A., Fernández-Gutiérrez, 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., García, A., García, P., Ríos, 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.
Samples: Importance, main phenolic compounds and health properties
75
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)
Gentisic acid 2,5-OH (154)
Vanillic acid 3-OCH3, 4-OH (168)
Gallic acid 3,4,5-OH (170)
Syringic acid 3,5-OCH3, 4-OH (198)
COOH1
23
4
5 6
Cinnamic acids and derivatives
o-Coumaric acid 2-OH (164)
p-Coumaric acid 4-OH (164)
Caffeic Acid 3,4-OH (180)
Ferulic Acid 3-OCH3, 4-OH (194)
Sinapinic Acid 3,5-OCH3, 4-OH (224)
COOH
1
23
4
5 6
Phenyl ethyl alcohols
Tyrosol [(p-hydroxyphenyl)ethanol] or
p-HPEA 4-OH (138)
Hydroxytyrosol [(3,4-
dihydroxyphenyl)ethanol] or 3,4-DHPEA
3,4-OH (154)
OH
1
23
4
5 6
Other phenolic acids and derivatives
p-Hydroxyphenylacetic acid 4-OH (152)
3,4-Dihydroxyphenylacetic acid 3,4-OH (168) COOH
1
23
4
5 6
Introduction
76
4-Hydroxy-3-methoxyphenylacetic acid 3-OCH3, 4-OH (182)
3-(3,4-Dihydroxyphenyl)propanoic acid (182)
OH
OH COOH
Dialdehydic forms of secoiridoids
Decarboxymethyloleuropein aglycon
(3,4-DHPEA-EDA) R1-OH (304)
Decarboxymethyl ligstroside aglycon
(p-HPEA-EDA) R1-H (320)
O
CHO
CHO
O dialdehydic form of Elenolic Acid (EDA)
R*
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
Elenolic Acid (EA)
OR1
OH
O
O
C
OCH3
O
OH
p-HPEA or 3,4-DHPEA
Elenolic Acid (EA)
R*O
O CH3aldehydic form of
Compound Substituent (MW) Structure
Flavonols
(+)-taxifolin (304)
O
OOH
HO
OH
OH
OH
Flavones
R2
OH
Samples: Importance, main phenolic compounds and health properties
77
Apigenin R1-OH, R2-H (270)
Luteolin R1-OH, R2-OH
(286)
Lignans
(+)-Pinoresinol R-H (358)
(+)-1-Acetoxypinoresinol R-OCOCH3 (416)
(+)-1-Hydroxypinoresinol R-OH (374)
O
O
RH
OCH3
H3CO
HO
OH
Hydroxyisochromans
1-phenyl-6,7-dihydroxyisochroman R1,R2-H (242)
1-(3’-methoxy-4’-hydroxy)phenyl-6,7-
dihydroxy-isochroman
R1-OH,R2-OCH3
(288)
O
HO
OH
R1
R2
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, 107–112. 235. Servili, M., Baldioli, M., Montedoro, G. F., (1994) ISHS Acta Horticulturae: II International
Symposium on Olive Growing, 356, 331–336.
Introduction
78
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.
Samples: Importance, main phenolic compounds and health properties
79
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.
Introduction
80
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 removed242.
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
world’s 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 70–100% 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) Árbolesproductores de frutos secos. Origen, descripción, distribucióny
producción. 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.
Samples: Importance, main phenolic compounds and health properties
81
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. 59–63. 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): 27–33.
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): 285–90. 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.
Introduction
82
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 chromatography−mass spectrometry. Biomed. Mass Spectrom., 10, 227−35.
Samples: Importance, main phenolic compounds and health properties
83
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
responsible for disease prevention have been reviewed 264.
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, 37−43. 258. Thompson, L. U.; Robb, P.; Serraino, M.; Cheung, F. (1991) Mammalian lignan production from
various foods. Nutr. Cancer, 16, 43−52. 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, 353−358.
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 219−236.
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 281−294.
262. McCord, H., Rao, L., (1997) Top seed: with its healing powers, flax is the next nutritional star. Prevention, 49, 81−85.
263. Caragay A.B., (1992) Cancer Preventive Foods and Ingredients. Food Technol. 46, pp. 65–68.
Introduction
84
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 disease269–273.
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. 91–138.
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. 244–260.
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.
85
Experimental part.
Results and Discussion.
87
CHAPTER I: Quantification of main phenolic compounds in sweet and
bitter Orange peel using CE–MS/MS
88
This work was published in Food Chemistry Journal. Quantification of main phenolic compounds in sweet and bitter Orange peel using CE–MS/MS. (Journal of Food Chemistry 116 (2009) 567–574) Saleh M.S. Sawalha, David Arráez-Román, Antonio Segura-Carretero, Alberto Fernández-Gutiérrez. Department of Analytical Chemistry, Granada University.
Food Chemistry 116 (2009) 567–574
Contents lists available at ScienceDirect
Food Chemistry
journal homepage: www.elsevier .com/locate / foodchem
Analytical Methods
Quantification of main phenolic compounds in sweet and bitter orange peelusing CE–MS/MS
Saleh M.S. Sawalha a, David Arráez-Román b, Antonio Segura-Carretero a,*, Alberto Fernández-Gutiérrez a,*
a Research Group FQM-297, Department of Analytical Chemistry, Faculty of Sciences, University of Granada, C/Fuentenueva s/n, E-18071 Granada, Spainb Verbionat S.C.A, C/Santa Fé de Bogotá 45, 18320 Santa Fé, Granada, Spain
a r t i c l e i n f o
Article history:Received 11 September 2008Received in revised form 9 January 2009Accepted 1 March 2009
Keywords:Phenolic compoundsOrange peelCapillary electrophoresisElectrospray ionisation–mass spectrometrydetection
0308-8146/$ - see front matter � 2009 Elsevier Ltd. Adoi:10.1016/j.foodchem.2009.03.003
* Corresponding authors. Fax: +34 958249510.E-mail addresses: [email protected] (A. Segura
(A. Fernández-Gutiérrez).
a b s t r a c t
The food and agricultural products processing industries generate substantial quantities of phenolics-richsubproducts, which could be valuable natural sources of polyphenols. In oranges, the peel representsroughly 30% of the fruit mass and the highest concentrations of flavonoids in citrus fruit occur in peel.In this work we have carried out the characterisation and quantification of citrus flavonoids in methanolicextracts of bitter and sweet orange peels using CE–ESI–IT–MS. 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, usingMS/MS experiments, and also the quantification of naringin (5.1 ± 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. CE cou-pled to MS detection can provides structure-selective information about the analytes. In this work wehave developed a CE–ESI–IT–MS method for the analysis and quantification of main phenolic compoundsin orange peels.
� 2009 Elsevier Ltd. All rights reserved.
1. Introduction
Polyphenols are amongst the most popular antioxidants andmany natural sources are being suggested for their recovery (Tura,2002). Crud extract of fruits, herbs, vegetables, cereals, nuts andother plant material rich in phenolics are increasingly of interestin the food industry (Sang et al., 2002). Citrus is a common termand genus of flowering plants in the family Rutaceae, originatingin tropical and subtropical areas in southeast Asia. Citrus fruitsare notable for their fragrance, partly due to flavonoids and limo-noids (a kind of terpenes) contained in the peel, they are also goodsources of vitamin C and flavonoids. Cultivated Citrus may be de-rived from as few as four ancestral species. Numerous naturaland cultivated origin hybrids include commercially important fruitsuch as the orange, grapefruit, lemon, some limes, and some tan-gerines. Oranges are one of the most popular fruits in the world.Orange processing in the United States produces �700.000 tonsof peel as byproduct solids annually (Winter, 1995). Plant materialwastes from these industries contain high levels of phenolic com-pounds. Importantly, most of this phytonutrient is found in the or-ange peel and inner white pulp, rather than in its liquid orangecentre, so this beneficial compound is too often removed by theprocessing of oranges into juice. Polyphenols compounds have
ll rights reserved.
-Carretero), [email protected]
health-related properties, which are based on their antioxidantactivity including anticancer, antiviral and antiinflammatory activ-ities (Bouskela, Cyrino, & Lerond, 1997; Tanaka et al., 1997). Thegroup of flavonoids is a widely distributed group of polyphenoliccompounds according to the above fact. Flavonoids in orange peelare comprised primarily of flavanone glycosides (narirutin 40-O-glucoside, eriocitrin, narirutin, hesperidin, isosakuranetin rutino-side), polymethoxylated flavone aglycons (sinensetin, hexa-O-methylquercetagetin, nobiletin, hexa-O-methylgossypetin, 3,5,6,7,8,30,40-heptamethoxyflavone, tetra-Omethylscutellarein, tangeritinand 5-hydroxy-3,7,8,30,40-pentamethoxyflavone) (Horowitz & Gen-tili, 1977), flavone glycosides (diosmin, isorhoifolin, rutin) (Kanes,Tisserat, Berhow, & Vandercook, 1993) and C-glycosylated flavones(6,8-di-C-glucosylapigenin) (Manthley & Grohmann, 2001). Naritu-tin, hesperidin, naringin and neohesperidin (Fig. 1) are the mostabundant flavonoids in the edible part of many species of citrusfruits (Kawai, Tomono, Katase, Ogawa, & Yano, 1999). As is welldocumented naritutin and hesperidin have been determined incommon sweet orange (Ooghe, Ooghe, Detavernier, & Huygheba-ert, 1994), and it is worthwhile referring to the recovery of hesper-idin and naringin from orange peel (El-Nawawi, 1995), which isconsidered to be the most popular source, recovery of naringinfrom bitter orange (Calvarano, 1996).
Even though the characterisation of phenolic compounds in or-ange has been successfully carried out using HPLC (Anagnostopou-lou, Kefalas, Kokkalou, Assimopoulou1, & Papageorgiou1, 2005;Belajová & Suhaj, 2004; Justesen, Knuthsen, & Leth, 1998; Kanaze,
Fig. 1. Chemical structures of: (a) naringin, (b) neohesperidin, (c) hesperidin and (d) narirutin.
568 S.M.S. Sawalha et al. / Food Chemistry 116 (2009) 567–574
Gabrieli, Kokkalou, Georgarakis, & Niopas, 2003; Theodoridis et al.,2006). Capillary electrophoresis (CE) has become an alternative orcomplement to chromatographic separations because it needs noderivatization step, requires only small amounts of sample andbuffer and has proved to be a high-resolution technique (Arráez-Román, Gómez-Caravaca, Gómez-Romero, Segura-Carretero, &Fernández-Gutiérrez, 2006). The hyphenation of CE as analyticalseparation technique coupled to mass spectrometry (MS) as detec-
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Fig. 2A. (a) MS/MS naringin (m/z 579.2) standard, (b) MS/
tion system can provide important advantages in food analysis be-cause of the combination of the high separation capabilities of CEand the power of MS as identification and confirmation method(Arráez-Román et al., 2007; Gómez-Romero et al., 2007; Simó, Bar-bas, & Cifuentes, 2005). In general, if a separation technique is cou-pled with MS the interpretation of the analytical results can bemore straightforward (Brocke, Nicholson, & Bayer, 2001; Macià,Borrull, Calull, & Aguilar, 2004; Schmitt-Kopplin & Frommberger,
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MS naringin (m/z 579.2) in bitter orange peel sample.
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2003). Furthermore, MS/MS experiments using a ion trap (IT) canbe used to obtain fragment ions of structural relevance for identi-fying target compounds in a highly complex matrix. In this sense,electrospray ionisation (ESI) has emerged as a highly useful tech-nique which allows direct coupling with electrophoretic separationtechniques (Smith & Udseth, 1996).
The aim of this present work has been to develop a simple CE–ESI–IT–MS method for the identification and quantification of mainphenolic compounds in orange peel due to these compounds arethe most abundant components in all the orange parts and presenta high concentration (El-Nawawi, 1995; Horowitz & Gentili, 1977).
2. Material and methods
2.1. Chemicals and reagents
All chemicals were of analytical reagent grade and used as re-ceived. Boric acid, purchased from Sigma–Aldrich (St. Louis, MO),and ammonium hydroxide from Merck (Darmstadt, Germany)were used for preparing the CE running buffers at different concen-trations and pH values. Buffers were prepared by weighing theappropriate amount of boric acid at the concentrations indicatedand adding ammonium hydroxide (0.5 M) to adjust the pH. Thebuffers were prepared with doubly deionized water, stored at4 �C and brought to room temperature before use. Doubly deion-ized water was obtained with a Milli-Q water purification system(Millipore, Bedford, MA). 2-Propanol HPLC grade used in the sheathflow, methanol, ethanol, hexane, DMSO and sodium hydroxide,used for capillary cleaning procedures before each analysis, wereobtained from Panreac (Barcelona, Spain) and triethylamine fromAldrich (Steinheim, Germany). All solutions were filtered through
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Fig. 2B. (a) MS/MS neohesperidin (m/z 609.2) standard (b) MS/M
a 0.45 lm Millipore (Bedford, MA, USA) membrane filters beforeinjection into the capillary. Naringin, neohesperidin, narirutinand hesperidin standards used for MS/MS experiments and calibra-tion curves were obtained from Extrasynthese (Lyon, France).
2.2. CE–ESI–IT–MS apparatus
The analyses were made in a P/ACETM System MDQ (BeckmanInstruments, Fullerton, CA, USA), CE apparatus equipped with anUV–Vis detector and coupled to the MS detector by an orthogonalelectrospray interface (ESI). The system comprises a 0–30 kV high-voltage built in power supplier.
All capillaries (fused-silica) used were obtained from BeckmanCoulter Inc. (Fullerton, CA, USA) and had an inner diameter (i.d.)of 50 lm. A detection window was created at 10 cm for the UVdetector and 100 cm was the total length (corresponding to theMS detection length). The instrument was controlled by a PC run-ning the 32 Karat System software from Beckman.
MS and MS/MS experiments were performed on a Bruker Dal-tonics Esquire 2000TM ion-trap mass spectrometer (Bruker DaltonikGmgH, Bremen, Germany) equipped with an orthogonal coaxialsheath-flow electrospray interface (model G1607A from AgilentTechnologies, Palo Alto, CA, USA). This triple tube ESI–MS interfaceprovides both a coaxial sheath liquid make-up flow and a nebuliza-tion gas to assist droplet formation. The drying gas and the nebu-lization gas were both nitrogen. The coaxial sheath liquid and theelectrical contact at the electrospray needle tip were delivered bya 74900-00-05 Cole Palmer syringe pump (Vernon Hills, Illinois,USA).
For the connection between the CE system and the electrosprayion source of the mass spectrometer, the outlet of the separation
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S neohesperidin (m/z 609.2) in bitter orange peel sample.
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Fig. 3A. (a) MS/MS narirutin (m/z 579.2) standard, (b) MS/MS narirutin (m/z 579.2) in sweet orange peel sample.
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capillary was fitted into the electrospray needle of the ion sourceand a flow of conductive sheath liquid established electrical con-tact between the capillary effluent and water for the electrosprayneedle. The instrument was controlled by a PC running the EsquireNT software from Bruker Daltonics.
2.3. Extraction procedures
Five extraction procedures were prepared in order to choose thebest conditions for the extraction of naringin, neohesperidin, nari-rutin and hesperidin from the orange peel samples. Basically, theextraction procedures are very similar but some modificationshave been carried out. The conditions of each extraction procedurewere as follows.
2.3.1. Extraction A0.2 g of the dried sample were weighted and extracted with
10 ml of methanol, the solution was shaken on vortex for 5 minand then centrifuged at 4500 rpm for 10 min. The solution was fil-tered through 0.2 lm filter and collected in a round bottom flask.The concentrated methanol was evaporated by rotary pump at40 �C, and the sample re-dissolved using 2 ml of MeOH:DMSO(50:50, v/v). Finally the extract was kept in the freezer until theanalysis. The samples were diluted 1:1 in water before analysis.
2.3.2. Extraction BThe same as extraction procedure A but the solution was shaken
with magnetic stirrer for 2 h.
2.3.3. Extraction CThe same as extraction procedure A but the dry residue was re-
solved in 2 ml of MeOH:H2O (50:50, v/v).
2.3.4. Extraction D0.2 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-perature for 2 h and then centrifuged at 4500 rpm for 10 min. Thesolution was filtered through 0.2 lm filter. Finally the sampleswere kept in the freezer until analysis. The samples were diluted1:1 in water before analysis.
2.3.5. Extraction EThe same as extraction procedure D but the solution was sha-
ken on vortex for 5 min.
2.4. CE–ESI–IT–MS procedure
In order to develop the CE–ESI–IT–MS method, to obtain thebest selectivity, sensitivity and resolution, the extract C previouslydescribed was used.
CE separation was carried out on a fused-silica capillary of50 lm i.d. with a total length of 100 cm (corresponding to theMS detection length).
Before first use, the bare capillaries were conditioned by rinsingwith 0.5 M sodium hydroxide for 20 min, followed by a 10 minrinse with water. Capillary conditioning was done by flushing for2 min sodium hydroxide, 4 min with water, and then for 10 min
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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 1Analytical 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 5–50 y = 505738x + 2E + 06 0.9858Neohesperidin 2.62 0.23 0.72 5–50 y = 640452x + 1E + 06 0.9886Narirutin 2.71 0.38 1.58 25–80 y = 532136x � 9E + 06 0.9974Hesperidin 3.50 1.15 3.85 25–80 y = 152140x � 0.821550 0.9996
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with the separation buffer. During all the capillary conditioningwas used a pressure of 20 w (1 w = 6895 Pa). At the end of theday the capillary was rinsed for 10 min water and 5 min flushair. The CE conditions used in the method were a buffer solutionof 200 mM boric acid adjusted with ammonium hydroxide at pH9.5. Samples were injected hydrodynamically in the anodic endin low pressure mode (0.5 w) for 5 s. Electrophoretic separationswere performed at 25 kV which caused a current intensity of40 lA.
The optimum ESI–IT–MS parameters were a sheath liquid iso-propanol/water 60:40 with 0.1% (v/v) TEA delivered at a flow rateof 0.28 ml/h, a drying gas flow rate of 5 l/min at 300 �C, compoundstability 25% and a nebulizer gas pressure of 6 w was supplied forESI formation.
The mass spectrometer was run in the negative ion mode andthe capillary voltage was set at 4000 V. The ion trap scanned at100–800 m/z range at 13,000 u/s during the separation and detec-tion. The maximum accumulation time for the ion trap was set at5.00 ms, the target count at 20,000 and the trap drive level at100%.
3. Results and discussion
3.1. Selection of extraction procedure
The CE–ESI–IT–MS method was applied to the analysis of mainpolyphenols in bitter and sweet orange peel extracts (see Section2.3). Under the optimised CE–ESI–IT–MS conditions describedabove it is possible to analyse main compounds in the differenttypes of extraction procedures and to carry out a comparativestudy of the extraction capacity. The compounds with m/z 579.2,from sweet and bitter orange peels, were extracted using the pro-cedures A–C; the compounds with m/z 609.2, from sweet and bit-ter orange peels, were extracted using the procedures C–E.Therefore, the extraction procedure C has been selected due topresence of the target compounds in the extract.
3.2. Identification of main polyphenols by MS/MS analysis
The peaks of the main phenolic compounds in orange peel wereeasily identified by comparing both migration time and MS/MS
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Fig. 4A. Extracted ion electropherograms of: (a) naringin and (b) neohesperidin in bitter orange peel sample.
572 S.M.S. Sawalha et al. / Food Chemistry 116 (2009) 567–574
data obtained from bitter and sweet orange peel samples withstandards. MS/MS can be used to obtain fragment ions of structuralrelevance for identifying target compounds in a highly complexmatrix. As these compounds had the same (m/z): naringin andnarirutin (m/z 579.2), neoheredin and hesperedin (m/z 609.2),MS/MS experiments of both kinds of samples comparing with theMS/MS of standards were useful in order to identify these com-pounds. Figs. 2A and 2B show the MS/MS spectra of naringin andneohesperidin standards and in the bitter orange peel sample. Be-sides, Figs. 3A and 3B show the MS/MS spectra of narirutin andhesperidin standards and in the sweet orange peel sample. Thus,using the MS/MS spectra it is possible to prove that the compoundsunder the current study correspond with the assignment proposed.
3.3. Analytical parameters of the method proposed
We carried out a study to check the repeatability of the pro-posed method, as well as to establish the calibration curves toquantify naringin and neohesperidin in bitter orange peel and nari-rutin and hesperidin in sweet orange peel.
3.4. Repeatability study
Repeatability of the CE–ESI–IT–MS analysis was studied by per-forming series of separations using the optimised method on theextracts in the same day (intraday precision, n = 5) and on threeconsecutive days (interday precision, n = 15). The relative standarddeviations (RSDs) of analysis time and peak area were determined.The intraday repeatability of the analysis time (expressed as RSD)was 0.22%, whilst the interday repeatability was 0.89%. The intra-day repeatability of the peak area (expressed as RSD) was 6.5%,
whilst the interday repeatability was 6.9% adequate for the aimof this work.
3.5. Calibration curves
In order to quantify the amount of each compound in the bitterorange peel, naringin and neohesperidin, a calibration curve wasprepared with the standards between the ranges from 5 to50 mg/l including five replicated of each point. In the same way,in order to quantify the amount of the sweet orange peel com-pounds, hesperidin and narirutin, a calibration curve was preparedwith the standards between the ranges from 25 to 80 mg/l includ-ing five replicated of each point. All calibration curves showedgood linearity in the studied range of concentration. Regressioncoefficients were higher than 0.985 for narigin and neohesperidinand higher than 0.997 for narirutin and hesperidin. All the featuresof the proposed method are summarised in Table 1.
3.6. Quantification of the main polyphenols in bitter and sweet orangesamples
The proposed method was applied to the quantification ofnaringin, neohesperidin, narirutin and hesperidin in bitter andsweet orange peel real samples. In Figs. 4A and 4B the extractedion electropherogram for each target compound of bitter andsweet orange peel are shown. The studied compounds were dilutedin order to fix them in the calibration range. Finally, the results ex-pressed in mg analyte/g of dry weight peel (n = 5; value = X ± SD)were 5.1 ± 0.2 and 7.9 ± 0.7 mg/g of naringin and neohesperidinin bitter orange peel and 26.9 ± 2.1 and 35.2 ± 3.6 mg/g of narirutinand hesperidin in sweet orange peel, respectively.
b
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Fig. 4B. Extracted ion electropherograms of: (a) narirutin and (b) hesperidin in sweet orange peel sample.
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4. Conclusions
The food and agricultural processing industries generate sub-stantial quantities of phenolics-rich by-products, which could bevaluable natural sources of antioxidants. In oranges, the peel repre-sents roughly half of the fruit mass. The highest concentrations offlavonoids in citrus fruit occur in peel. In this work we propose thecharacterisation, using MS/MS experiments, and quantification ofthe distinctive phenolic compounds (naringin, neohesperidin, nari-rutin and hesperidin) from the peel of sweet and bitter oranges.The CE–ESI–IT–MS allowed to differentiate naringin from narirutinand hesperidin from neohesperidin and it showed to be suitable forthe analysis of this type of natural compounds.
Acknowledgements
The author DAR gratefully acknowledges a ‘‘Torres Quevedo”contract from Ministerio de Educación y Ciencia in VerbionatS.C.A. The authors also gratefully acknowledge the financial sup-port of Projects CTQ2005-01914/BQU and AGL2008-05108-C03-03/ALI from Ministerio de Educación y Ciencia and the ExcellentProyect AGR-02619 from Consejería de Innovación, Ciencia yEmpresa de la Junta de Andalucía.
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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).
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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) Saleh M.S. Sawalha, David Arráez-Román, Antonio Segura-Carretero, Alberto Fernández-Gutiérrez. Department of Analytical Chemistry, Granada University.
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esCharacterization of phenolic compounds in diatomaceous earth used in the filtration process of olive oil by hplC-esi-toF (ms)sAleh sAWAlhA, dAVid ARRÁeZ-RomÁN, ANtoNio seGURA-CARReteRo*, AlBeRto FeRNÁNdeZ-GUtiÉRReZ**Corresponding authorsUniversity of Granada, Faculty of sciences, department of Analytical ChemistryC/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 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.
introduction
there is a rising interest in natural antioxidants as bioactive components of foods. the importance of the antioxidant constituents of plant material in the maintenance of health and protection from coronary heart disease and cancer is also raising interest among scientists, food manufacturers and consumers (1). in recent years, interest in natural antioxidants from vegetable substances has been related to their therapeutic properties (2). Among the different vegetable oils, Virgin olive oil (Voo), which is the juice of the olive obtained by pressing, is one of the few oils that are consumed without any further refining process. the antioxidant potential of phenolic compounds in olive oil has been a subject of great interest, because of i ts chemoprotective effect in human beings (3-5). phenolic compounds are of fundamental importance for their nutritional properties, sensory characteristics, and the shelf life of Voo (6, 7). they also play an important role in human
nutrition as preventive agents against several diseases (8, 9).olive oil production is an important agricultural and alimentary sector in europe. the european Union is the main world producer (10). 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. some of these factors had been studied to see how the polyphenolic content of olive Voo was affected: the cultivation (11-13), climate (14). the production processes are: collecting, washing, pressing, decantation, centrifuging, storage, filtration and packaging, some of those eight steps of Voo production processes (pressing, centrifuging and storage) had been studied about polyphenolic contents in Voo (15-20), but there is a lack of information and studies available about the filtration step (21, 22), 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 fossil ized 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. Considering the fact that polyphenols are polar compounds which can found in the olive fruit; however many of these compounds are modified or lost during the production process of Voo (23). therefore, the qualitative study of phenolic compounds in the diatomaceous earth used in the filtration process of Voo is very important.thus, in the present study hplC coupled with mass spectrometry (ms) detection was used, since this 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 information (24). the on-line coupling of hplC with ms using electrospray ionization (esi) as an interface yields a powerful method because esi–ms allows the determination
ABBReViAtioNshYtY: hydroxytyrosoltY: tyrosolhYtY-Ac: 2-(4-hydroxyphenyl) ethyl acetate or hydroxytyrosol acetatehYtY-Glu: hydroxytyrosol glucoside eA: elenolic aciddoA: decarboxylated derivatives of ol Aglol Agl: oleuropein aglycone10-h-ol Agl: 10-hydroxy-oleuropein aglyconedeacetoxy 10-h-ol Agl: deacetoxi 10-hydroxy-oleuropein aglyconedecarbox-lig Agl: decarboximethylated derivatives of ligstroside aglyconelig Agl: ligstroside aglyconepin: (+)-pinoresinolAc pin: (+)-1-acetoxypinoresinolh-pin: hydroxy-pinoresinollut: luteolin
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Food technologiesB in 10 minutes; 30 percent B to 33 percent B in 2 minutes; 33 percent B to 38 percent B in 5 minutes; 38 percent B to 50 percent B in 3 minutes; 50 percent to 95 percent in 3 minutes. the initial conditions were re-established in 2 minutes and held for 10 minutes more. the total run time, including the conditioning of the column to the initial conditions, was 35 min. the flow rate used was set at 0.80 ml/min throughout the gradient. the effluent from the hplC column was split using a “t” before being introduced into the mass spectrometer (split ratio 1:3). thus in the current paper the flow which arrived to the esi-toF-ms detector was 0.2 ml/min. the column temperature was maintained at 25°C and the injection volume was 10 μL.
esi-tof (ms) esi-toF (ms) conditions were optimized in order to provide 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 optimum esi parameters were as follows: nebulizing gas pressure, 2 bars; drying gas flow, 9 l/min; drying gas temperature, 190ºC. ms was performed using the microtoF (Bruker daltonik, Bremen, Germany), an orthogonal-accelerated toF mass spectrometer (oatoF-ms). transfer parameters were optimized by direct infusion experiments with tuning mix (Agilent technologies) in the range of 50-800 m/z looking for the best conditions regarding sensitivity and resolution. thus, the endplate offset was -500 V; capillary voltage 4500 V, the trigger time was set to 50 µs, 49 µs for set transfer time and 1 µs pre-puls storage time, corresponding to a mass range of 50–800 m/z. spectra were acquired by summarizing 20,000 single spectra, defining the spectra rate to 1 hz. the accurate mass data of the molecular ions were processed through the software data Analysis 3.4 (Bruker daltonik), which provided with a list of possible elemental formulas by using the Generate molecular Formula™ editor. the Generate Formula ™ editor uses a ChNo algorithm, which provides with standard functionalities such as minimum/maximum elemental range, electron configuration, and ring-plus double bonds equivalents, as well as a sophisticated comparison of the theoretical with the measured isotope pattern (sigma Value) for increased confidence in the suggested molecular formula (Bruker daltonics technical Note #008, molecular formula determination under automation). the widely accepted accuracy threshold for confirmation of elemental composit ions has been 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
of a wide range of polar compounds. esi is one of the most versatile ionization methods, and is the method of choice for the detection of ions separated by liquid chromatography. Although hplC can be coupled to different ms analyzers (quadrupole, ion trap (it), time-of-flight (toF), etc) (25), toF (ms) provides excellent mass accuracy over a wide dynamic of range if modern detector technology is used (26). the latter allows also measurements of the isotopic pattern (27, 28), providing with an important additional information for the determination of the elemental composition (29), in this article we have used hplC-esi-toF (ms) to analyze the phenolic compounds present in diatomaceous earth.the aim of this work was the separation and the characterization of a broad series of phenolic compounds present in the diatomaceous earth used in the filtration process of Voo by hplC-esi-toF (ms), which was achieved for the first time.
ExpErimEntal sEction
reagents and materialsAll chemicals were of analytical reagent grade and used as received. the organic solvents, hexane, methanol and ACN, used in the extraction procedure and as hplC mobile phase were purchased from lab-scan (dublin, ireland). Acetic acid used in hplC phase A was purchased from Fluka (switzerland). deionised water was obtained from a water purifier system (millipore, Bedford, mA). All the solvents used in the hplC system were filtered through a 0.20 μm Millipore (Bedford, MA, UsA) membrane filters.
apparatushplc the separation of the phenolic compounds from extra-virgin olive oil was performed using an Agilent 1200 series Rapid Resolution lC (Agilent technologies, palo Alto, CA, UsA), which was equipped with a vacuum degasser, an autosampler, a diode-array detector (dAd), a binary pump, and a thermostated column department. the samples were separated using a reversed-phase C18 analytical column (4.6×150 mm, 1.8 μm particle size, Agilent ZoBRAx eclipse plus). the mobile phase A and B consisted of water with 0.5 percent acetic acid, and ACN. the chromatographic
m e t h o d w a s a s following: gradient
from 5 percent B to 30 percent
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through normal filtration using Whatman filter paper No.4. then it was separated into 5 centrifuge tubes and centrifuged at 1.9 g for 10 min. the solution was collected in a round
complete run without the need for a dual sprayer setup for internal mass c a l i b r a t i o n . t h e s e c a l i b r a t i o n s w e r e performed in quadratic + high precision calibration (hpC) regression mode.
samplethe diatomaceous earth filter used in the olive oil industry was composed of 75 percent Celite®545 a n d 2 5 p e r c e n t Kenite®700. this filter was used in the last step of Voo production in order to improve its quality.
Extraction procedurethe extraction procedure w a s : 2 0 g o f t h e 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 from the f i lter paper carefully another time in beaker 500 ml, 120 ml of methanol were added and the solution was shaken by magnetic stir 2 h at 35ºC. the solution was left overnight. After this time the solution was filtered
Figure 1. eies of the well-known phenolic compounds detected in diatomaceous earth extract containing information about the m/z experimental.
table 1. Well-known phenolic compounds determined by hplC-esi-toF (ms) in diatomaceous earth.
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and migration time. Figure 1 shows the extracted ion electropherograms (eie) of the several major compounds present in diatomaceous earth sample.
thus, the proposed method is able to detect nineteen phenolic compounds in the same run. Furthermore, all detected compounds observed in table 1 exhibited good sigma values smaller than 0.05 and mass accuracy (ppm and mda) as indicated by the error values, even a low tolerance was chosen (5 ppm), except in two cases Vanillin and o-Coumaric acid the tolerance was 10 and 12 ppm respectively, meanwhile the sigma values were below 0.05. We could detect four phenyl alcohols (hYtY, tY, hYtY-Ac and hYtY-Glu), several compounds from secoiridoid family (eA,
bottom flask. the concentrated methanol was evaporated by rotary pump below 40ºC, and the dry residue was resolved by 4 ml of methanol. Finally, the solution was filtered through a 0.2 µm filter before the hplC analysis.
rEsults and discussion
Well-known phenolic compounds Under the proposed hplC-esi-toF (ms) method, a large number of well-known phenolic compounds present in diatomaceous earth were detected. these are summarized in table 1, with their formula, selected ion, experimental and calculated m/z, error (ppm and mda), sigma value, tolerance
table 2. Unknown phenolic compounds determined by hplC-esi-toF (ms) in diatomaceous earth.
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1. J. löliger, taylor et al., london, UK, p. 129, (1991).2. p.C.h. hollman, m.G. l.hertog et al., Food Chem., 57(1), pp.
43-46 (1996).3. F. Caponio, t. Gomes et al., Eur. Food Res. Technol., 212, pp. 329-
333 (2001).4. R. Briante, F. la Cara et al., J. Agric. Food Chem., 49, pp. 3198-
3203 (2001).5. l. Cerretani, A. Bendini et al., AgroFood Ind Hi Tec., 19, pp. 64-66
(2008).6. G. F. montedoro, m. Baldioli et al., Nutr. Clin.Prevent., 1, pp.
19-31(1991).7. m. tsimisou., J. Food Sci., 10, pp. 99-116 (1998).8. A. Romani, N. mulinacci et al., J. Agric. Food Chem.,47, pp. 964-
967 (1999).9. R.W. owen, A. Giacosa et al., Lancet Oncol., 1, pp. 107-112
(2000).10. Anonymous, Faostst, database, www.fao.org (last access on:
19.11.2004).11. m.J. tovar, m.J. motilva et al., J. Agric. Food Chem., 49(11), pp.
5502-5508 (2001).12. m.J. tovar, m.p. Romero et al., J. Sci. Food Agric., 82(15), pp.
1755-1763 (2002).13. m. servili, s. esposto et al., J. Agric. Food Chem., 55(16), pp. 6609-
6618 (2007).14. (m. Bonoli, A. Bendini et al., J. Agric. Food Chem., 52(23), pp.
7026-7032 (2004).15. A. parenti, p. spugnoli et al., J. Lipid. Scien and Tech., 110(8), pp.
753-741(2008).16. m. servili, R. selvaggini et al., J. Agric. Food Chem., 51(27), pp.
7980-7988 (2003).17. m. servili, R. selvaggini et al., J. Amer. Oil Chem Society, 80(7),
pp. 685-695 (2003).18. F. Angerosa, l. di Giovacchino et al., Grasas y Aceites ., 47(4),
pp. 247-254 (1996).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.
5609-5614 (2001).21. A. Bottino, A. Capannelli et al., J. Food. Engin., 65(2), pp. 303-309
(2004).22. A. m. Gómez-Caravaca, l. Cerretani et al., Am. J. Food.
Technol., 2(7), pp. 671-678 (2007).23. m. Brenes et al., J. Agric. Food Chem., 43, pp. 2702-2706 (1995).24. A. Carrasco-pancorbo, C. Neusüß et al., Electrophoresis, 28, pp.
806-821(2007).25. C. simó, m. herrero et al., Electrophoresis, 26, pp. 2674-2683
(2005).26. A.W.t. Bristow, K.s. Webb., J Am Soc Mass Spectrom., 24, pp.
1086-1098 (2003).27. m. pelzing, J. decker et al., talk A042670. in: 52nd Asms Conf on
mass spectrometry and Allied topics, 23–27 may, Nashville, tN (2004).
28. d. Arráez-Román, s. sawalha et al., AgroFood Ind Hi Tec., 19, pp. 18-22 (2008).
29. G. Bringmann, i. Kajahn et al., Electrophoresis, 26, pp. 1513-1522 (2005).
30. i. Ferrer, J.F. García-Reyes et al., J. Chromatogr. A, 1082, pp. 81-89 (2005).
31. m. ibáñez, J.V. sancho et al, Rapid Commun. Mass Spectrom., 19, pp. 169-178 (2005).
32. t. Kina, o. Fiehn, bNC bioinformatics, 7, pp. 234-243 (2006).
doA, ol Agl, 10-h-oi Agi, deacetoxy 10-h-ol Agl, decarbox-lig Agl and lig Agl), three lignans (pin, Ac pin and h-pin), three phenolic acids (vanillin, vanillic acid and o-Coumaric acid) and also the present method allowed the determination of two flavonoids (lut and Apig). All the compounds detected in this work have been described in the previous studies on Voo, which means that the diatomaceous earth used in the filtration process of Voo can affect the phenolic composition of the final product.
unknown phenolic compounds Besides the previously mentioned phenolic compounds detected with the above described method, it was also possible to study other compounds present the diatomaceous earth fraction, which had not been described before in the literature. table 2 summarizes all the results for 17 unknown compounds including migration time, experimental m/z, selected ion, tolerance (ppm), list of possible molecular formulas, error and sigma value for the compound with molecular formula CxhYoZ. these compounds have been included since they suppose a significant fraction of the extract from diatomaceous earth sample. A reduced number of possible elemental compositions are obtained from the accurate mass of the suspected peak. these elemental compositions can then be matched against available databases (the merck index, Chemindex, commercial e-catalogues) using the deduced molecular formula as a search criterion (31, 32). it has to be mentioned that some of the possible elemental compositions calculated within a certain mass accuracy does not seem to be chemically coherent. this fact helps in the unequivocal identification of the “unknown” species and the assignment of its correct elemental composition since it reduces the number of possibilities.
conclusion
in this work a large number of well-known phenolic compounds present in diatomaceous earth extracts can be separated and identified. due to 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), the described hplC-esi-toF (ms) method represents a valuable tool and a good alternative for simultaneous characterization of phenolic components in diatomaceous earth.
acknoWlEdgEmEnts
the authors are grateful to the spanish ministry of education and science for the project (AGl2008-05108-C03-03) and to Andalusian Regional Government Council of innovation and science for the project p07-AGR-02619. the authors also thank Aceites maeva, s.l. for providing the samples. the author ss gratefully acknowledges the Agencia española de Cooperación internacional (AeCi).
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CHAPTER III: Identification of phenolic compounds in olive leaves using
CE-ESI-TOF-MS
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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 Fernández-Gutiérrez. Department of Analytical Chemistry, Granada University. David Arráez-Román. Verbionat S.C.A, C/ Santa Fé de Bogotá 45 Santa Fé, 18320, Granada, Spain Javier Menedez. Catalan Institute of Oncology (ICO) Health Services Division of Catalonia, Spain.
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Identification of phenolic compounds in olive leaves using CE-ESI-TOF-MSDAVID ARRÁEZ-ROMÁN1, SALEH SAWALHA2, ANTONIO SEGURA-CARRETERO2*, JAVIER MENENDEZ3, ALBERTO FERNÁNDEZ-GUTIÉRREZ2**Corresponding authors1. Verbionat S.C.A, C/ Santa Fé de Bogotá 45Santa Fé, 18320, Granada, Spain2. Department of Analytical Chemistry, Faculty of Sciences, University of GranadaC/ Fuentenueva s/n, Granada, 18071, Spain3. Catalan Institute of Oncology (ICO)Health Services Division of Catalonia, Spain
be a high-resolution technique (14-17). Regarding the advantages of MS detection include the capability of determination of molecular weight and providing structural information (18). Also TOF-MS provides excellent mass accuracy (19) over a wide dynamic range. The latter, moreover, allows measurements of the isotopic pattern (20), providing important additional information for the determination of the elemental composition (21). Thus, the on-line coupling of CE with TOF-MS yields a powerful technique for the analysis of phenolic compounds (22). The goal of this work is to develop a new, rapid and simple CE-ESI-TOF-MS method to identify phenolic compounds in two varieties of olive leaves (Hojiblanca and Manzanilla).
EXPERIMENTAL SECTION
Reagents and materialsAll chemicals were of analytical reagent grade and used as received. Ammonium hydroxide was from Fluka (Buchs, Switzerland) and ammonium acetate and methanol from Merck (Darmstadt, Germany). 2-propanol HPLC grade used in the sheath flow, methanol and sodium hydroxide, used for capillary cleaning procedures before each analysis, were obtained from Panreac (Barcelona, Spain) and triethylamine from Aldrich (Steinheim, Germany). Distilled water was deionised by using a Milli-Q system (Millipore, Bedford, MA). CE buffers were prepared by weighing ammonium acetate at the concentrations indicated and adjusting the pH when necessary by adding ammonium hydroxide. The buffers were stored at 4ºC and warmed to room temperature before use. All solutions were filtered through a 0.45 μm Millipore (Bedford, MA, USA) membrane filters before injection into the capillary.
ApparatusCE experiments were performed using a P/ACETM System MDQ (Beckman Instruments, Fullerton, CA, USA) and fused-silica capillaries of 85 cm in length and 50 μm inner diameters (360 μm outer diameters) coupled to the MS detector by an orthogonal electrospray interface (ESI) with a coaxial sheath-liquid (Agilent Technologies, Palo Alto, CA, USA) delivered by
INTRODUCTION
Natural antioxidants are primarily plant polyphenolic compounds that may be obtained from plant parts. Plant phenolics are multifunctional and can act as reducing agents (free radical terminators), metal chelators, and singlet oxygen quenchers (1). Crude extract of fruits, herbs, vegetables, cereals, nuts and other plant materials rich in phenolics are increasingly of interest in the food industry (2). The importance of the antioxidant constituents of plant material in the maintenance of health and protection from coronary heart disease and cancer is also raising interest among scientists, food manufacturers, and consumers (3).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 were produced in several thousand of olive oil mills (4). From this important industry, both, olive tree culture and the olive oil industry, produce large amounts of by-products. It has been estimated that pruning alone produces 25 kg of by-products (twigs and leaves) per tree annually. It must also be considered that leaves represent 5 percent of the weight of olive oil extraction then this represent a significant by-product in olive oil production process (5). Historically, olive leaf has been used as a folk remedy for combating fevers and other diseases, such as malaria. Several reports have shown that olive leaf extract and also olive oil had the capacity to lower blood pressure in animals (6, 7) and increased blood flow in the coronary arteries (8), relieved arrhythmia and prevented intestinal muscle spasms. This type of by-products (olive leaves) are a rich source of an important number of phenolic compounds (9-13). However, the analysis of these compounds is not an easy work. Because of this, the characterization of individual olive leaves compounds requires the use of separate techniques. Thus, due to its high efficiency, flexibility, very high resolution and rapidity of the method, CE has gained widespread interest as a favourable technique for the analysis of phenolic compounds. It has become an alternative or complementary technique to chromatographic separations for the analysis of phenolic compounds because it needs no derivatization step, requires only small amounts of sample and buffer and has proved to
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 series of phenolic compounds present in olive leaves.
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Spectra were acquired by summarizing 20,000 single spectra, defining the spectra rate to 1 Hz.
Sample preparationThe variety Hojiblanca olive tree is at least 200 years old and is localized in the shadow area of dry lands. The collection is made directly from the tree. Afterwards the leaves were washed using only distilled water, in order to avoid polyphenols degradation. Later, this water was introduced
a 5 mL gas-tight syringe (Hamilton, Reno, NV, USA) using a syringe pump of 74900-00-05 Cole-Parmer (Vernon Hill, IL, USA). MS experiments were performed using the micrOTOFTM (Bruker Daltonik GmbH, Bremen, Germany), an orthogonal-accelerated TOF mass spectrometer (oaTOF-MS). An electrospray potential of +4.1 kV was applied at the inlet of the MS (negative ion polarity). The trigger time was set to 50 μs, 49 μs for set transfer time and 1 μs pre-pulse storage time, corresponding to a mass range of 50–800 m/z.
Figure 1. EIEs of the well-known phenolic compounds detected in Hojiblanca leaves extract containing information about the m/z experimental
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repeated four times. Then, the concentrated methanol was evaporated by rotary pump at 40◦C and the sample was resolved in 4 ml of MeOH:H2O (50:50 v/v). Finally the extract was kept in the freezer until the analysis.
RESULTS AND DISCUSSION
CE-ESI-TOF-MS methodIn order to develop the optimization of CE-ESI-TOF-MS method, the extract of Hojiblanca leaves was used. The CE-ESI-TOF-MS method was developed in order to obtain the best selectivity, sensitivity and resolution. Initially, the electrophoretic conditions were optimized based on the migration behaviour, sensitivity, analysis time and peak
into a stove with forced air at 40ºC during 48 hours for dehydration purposes. The entire leaf was kept in paper envelopes. Once it was grounded, the leaf was introduced in sealed glass jars, wrapped in aluminium foil, and then 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 procedures were identical to those ones described above. In the present study the two varieties of olive leaves samples were characterized. The extraction procedures were as follows: 0.5 g of the dried (powder) sample was weighted in a 10 ml test tube. 5 ml of methanol were added and the solution was shaken on vortex 5 minutes and centrifuged at 4500 r.p.m for 10 minutes. The liquid part was collected in a round bottom flask. These steps were
Figure 2. EIEs of the well-known phenolic compounds detected in Manzanilla leaves extract containing information about the m/z experimental
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compounds present in Hojiblanca and Manzanilla leaves extracts is given in Figure 1 and Figure 2 respectively. The reproducibility of the CE-ESI-TOF-MS analysis, expressed by the RSD percent of five consecutive injections was 1.04 percent for the analysis time and 5.89 percent for the peak area, both measured for each peak.
Use of TOF-MS for the identification of phenolic compounds The accurate mass data of the molecular ions were processed through the software DataAnalysis 3.3 (Bruker Daltonik GmbH), which provided a list of possible elemental formula by using the GenerateMolecularFormulaTM editor. The GenerateFormulaTM editor uses the sigmaFitTM algorithm, which provides standard functionalities such as minimum/maximum elemental range, electron configuration and ring-plus double bonds equivalents, as well as a sophisticated theoretical and measured comparison of the isotope pattern (SigmaValueTM) for increased confidence in the suggested molecular formula (29). An external calibration was performed using sodium formate cluster by switching the sheath liquid to a solution containing 5 mM sodium hydroxide in the sheath liquid of 0.2 percent formic acid in water:isopropanol 1:1 v/v at the end of the analysis. Using this method an exact calibration curve based on numerous cluster masses each differing by 68 Da (NaCHO2) was obtained. This external calibration provided accurate mass values (better 5 ppm) for a complete run without the need for a dual sprayer setup for internal mass calibration. All the detected phenolics compounds in Hojiblanca and Manzanilla leaves extracts are summarized in Tables 1 and 2 respectively, with their formula, selected ion, m/z experimental and calculated error (ppm and mDa), sigma value, tolerance and migration time. Thus, the proposed method is able to detect seventeen phenolic compounds in
shape. First, different buffers compatible with CE-ESI-MS were used (ammonium acetate/NH3 and ammonium borate/NH3) and the best results were obtained using ammonium acetate/NH3. Thus, 50 mM ammonium acetate as running buffer was selected, due to its best performance to achieve a high signal response as well as a good resolution. 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 shape, resolution and analysis time. Under these CE experimental conditions, a voltage of 30 kV shortened the analysis time and yielded good separation and acceptable current. The injections were made at the anodic end using a N2 pressure 0.5 p.s.i. for 20 s (1 p.s.i. = 6894.76 Pa). These conditions were chosen for the subsequent optimization of the ESI-TOF-MS parameters. It is well known that the choice of sheath liquid has significant effects on sensitivity and in the electrical contact between CE and ESI (23, 24). Thus, we optimized the sheath liquid by varying the ratio at different isopropanol/water solutions. The use of an isopropanol/water mixture 60:40 (v/v) resulted in the highest TOF-MS signal. Generally, a small amount of volatile triethylamine (TEA) or ammonium hydroxide is used for ESI-negative detection (25). For that reason 0.1 percent (v/v) TEA was added yielding a better sensitivity. The sheath liquid flow was expected to dilute the CE sample zone as it passed concentrically around the CE column effluent and mixed with it. Finally, 0.20 mL/h was selected as optimum in terms of signal response and stability. Nebulizer gas pressure is a compromise between maintaining an efficient electrophoretic separation and improving the ionization performance (26-28) obtaining the best signal at 0.4 Bar. Finally the dry gas temperature was found to be optimal at 180ºC with the best dry gas flow rate at 4 L/min. Under these conditions, the Extracted Ion Electropherograms (EIEs) for a large number of well-known phenolic
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REFERENCES AND NOTES1. G.R. Takeoka, L.T. Dao, J. Agric. Food Chem., 51, pp. 496-501 (2003).2. S. Sang, K. Lapsley et al., J. Agric. Food Chem., 50, pp. 2459-2463 (2002).3. J. Löliger, Taylor et al., London, U.K. pp. 129 (1991).4. Anonymous, Faostst, Database, www.fao.org [last access on: 19.11.2004].5. E. Molina et al., J. Inter. Biodet. & biodeg., 1, pp. 227-235 (1996).6. G. Samulsson., J. Farma. Revy, 15, pp. 229-239 (1951).7. V. Di Fronzo, R. Gente et al., Agro Food Ind Hi Tec., 18, pp. 4-5 (2007).8. Zarzuelo., J. Plant. Medica., 57, pp. 417-419 (1991).9. D. Ryan, M. Antolovich et al., Scientia Horticulrurae, 92, pp. 147-176
(2002).10. F. Visioli, A Poli et al., J. Med. Res. Rev., 22, pp. 65-75 (2002). 11. P. Gariboldi, G. Jommi et al., Phytochem., 25, pp. 865-869 (1986). 12. B le Tutour, D. Guedon et al., Phytochem., 31, pp. 1173-1178 (1992). 13. H kuwajima, T. Uemura et al., Phytochem., 27, pp. 1757-1759 (1988). 14. D. Arráez-Román, A.M. Gómez-Caravaca et al., J. Pharm. Biomed. Anal.,
41, pp. 1648-1656 (2006).15. M. Gómez-Romero, D. Arráez-Román et al., J. Sep. Sci., 30, pp. 595-603
(2007).16. D. Arráez-Román, S. Cortacero-Ramirez et al., Electrophoresis, 27, pp.
2197-2207 (2006).17. N. Volpi, Electrophoresis, 25, pp. 1872-1878 (2004).18. D. Arráez-Román, G. Zurek et al., Electrophoresis, 29, pp. 2112-2116
(2008).19. A.W.T. Bristow, K.S. Webb et al., J. Am. Soc. Mass Spectrom., 24, pp.
1086-1092 (2003.)20. M. Pelzing, J. Decaer et al., talk A042670, presented on 52nd ASMS
Conference on Mass Spectrometry and Allied Topics, Nashville, TN, May 23 (2004).
21. G. Bringmann, I. Kajahn et al., Electrophoresis, 26, pp. 1513-1522 (2005).22. D. Arráez-Román, G. Zurek et al., Anal. Bioanal. Chem., 389, pp. 1909-
1917 (2007).23. C.W. Klampfl, W. Ahrer, Electrophoresis, 22, pp. 1579-1584 (2001).24. K. Vuorensola, J. Kokkonen et al., Electrophoresis, 22, pp. 4347-4354
(2001).25. R.D. Voyksner, Electrospray Ionization Mass Spectrometry, Wiley, New
York, pp. 323 (1997).26. R. Sheppard, X.Tong et al., Anal. Chem., 67, pp. 2054-2058 (1995).27. Macià, F. Borrull et al., Electrophoresis, 25, pp. 3441-3449 (2004).28. K. Huikko, T. Kotiaho et al., Rapid Comun. Mass Spectrom., 16, pp. 1562-
1565 (2002).29. Bruker Daltonics Technical Note #008, Molecular formula determination
under automation.
Hojiblanca and fourteen in Manzanilla leaves in the same run with an accuracy of 5 mDa. All detected compounds observed in Table 1 and 2 exhibited good sigma values and mass accuracy (ppm and mDa) as indicated by the error values.
CONCLUSION
In this work a large number of well-known phenolic compounds present in Hojiblanca and Manzanilla leaves extract were determined and identified. Hence, the described CE-ESI-TOF-MS method represents a valuable tool for the identification of phenolic compounds and will certainly complement the already existing LC-MS and GC-MS techniques. In comparison to the chromatographic methods, the proposed method is a good alternative for simultaneous characterization of phenolic components in olive leaves since technique provides fast and efficient separations in this type of analysis and uses reduced sample and solvents consumption. Also, the hyphenation of CE to MS combines the advantages of CE with the selectivity, sensitivity and mass accuracy inherent to TOF-MS. Due to TOF-MS provides excellent mass accuracy over a wide dynamic range and allows measurements of the isotopic pattern, it provides important additional information for the determination of the elemental composition.
ACKNOWLEDGEMENTS
The author DAR gratefully acknowledges the “Torres Quevedo” contract from Ministerio de Educación y Ciencia in Verbionat S.C.A and the author SS the Agencia Española de Cooperación Internaciona (AECI). The authors also gratefully acknowledge the financial support of CTQ2005-01914/BQU and AGL2008-05108-CO3-03/ALI from MEC, the P431073
Table 1
Table 2
111
CHAPTER IV: HPLC/CE-ESI-TOF (MS) methods for the characterization of
polyphenols in almond skin extracts
112
This work was submitted to Electrophoresis Journal. HPLC/CE-ESI-TOF (MS) methods for the characterization of polyphenols in almond skin extracts. Saleh M.S. Sawalha, David Arráez-Román, Antonio Segura-Carretero, Alberto Fernández-Gutiérrez. Department of Analytical Chemistry, Granada University.
113
HPLC/CE-ESI-TOF (MS) methods for the characterization of polyphenols in 1
almond skin extracts 2
3
Saleh M. S. Sawalha, David Arráez-Román, Antonio Segura-Carretero, Alberto 4
Fernández-Gutiérrez. 5
6
Department of Analytical Chemistry, Faculty of Sciences, University of Granada, 7
C/Fuentenueva s/n, E-18071 Granada, Spain 8
9
Correspondence: Dr. A. Fernández-Gutiérrez, Research Group FQM-297, Department 10
of Analytical Chemistry, Faculty of Sciences, University of Granada, C/Fuentenueva 11
s/n, E-18071 Granada, Spain 12
E-mail: [email protected] 13
Fax: +34958249510 14
15
Keywords: Capillary electrophoresis / high-performance liquid chromatography / 16
Electrospray ionization-time of flight-mass spectrometry / Phenolics compounds / 17
Almond skin 18
114
ABSTRACT 19
20
In this article, two rapid methods has been developing using, capillary electrophoresis 21
(CE) and high-performance liquid chromatography (HPLC) coupled to electrospray 22
ionization-time of flight-mass spectrometry (ESI-TOF-MS) have been compared for the 23
separation and characterization of antioxidant phenolic compounds in almond skin 24
extract. Under the optimum CE-ESI-TOF-MS conditions we achieved the determination 25
of nine compounds of the polar fraction in 35 min. Furthermore, by using HPLC-ESI-26
TOF-MS method, a total of twenty-three compounds corresponding to phenolic acids 27
and flavonoids family were identified from almond skin only in 9 min. We have 28
demonstrate that the sensitivity, together with mass accuracy and true isotopic pattern of 29
the TOF-MS, allowed the identification of a broad series of known phenolics 30
compounds present in almond skin extracts using HPLC and CE as separative 31
techniques. 32
115
1. Introduction 33
34
Natural antioxidants are primarily plant polyphenolic compounds that may be obtained 35
from plant parts. Plant phenolics are multifunctional and can act as reducing agents (free 36
radical terminators), metal chelators, and singlet oxygen quenchers [1]. Crude extract of 37
fruits, herbs, vegetables, cereals, nuts and other plant material rich in phenolics are 38
increasingly of interest in the food industry [2]. The importance of the antioxidant 39
constituents of plant material in the maintenance of health and protection from coronary 40
heart disease and cancer is also raising interest among scientists, food manufacturers, 41
and consumers [3,4]. 42
The need to identify the phenolic compounds meant that traditional methods should be 43
replaced for more potential methods based on the use of advances chromatographic 44
separatives techniques, such as gas chromatography GC [5–9] or, specially, high 45
performance liquid chromatography HPLC [10–15]. Furthermore, capillary 46
electrophoresis CE has been recently applied for the analysis of phenolic compounds in 47
natural products and has opened up great expectations, especially due to the higher 48
resolution, reduced sample volume and analysis duration [16–19]. 49
50
At the beginning of 21st century, HPLC with reversed phase and CE are two of the most 51
modern separation techniques frequently used. There are an important number of 52
articles using HPLC with different detectors such as UV (photodiode array) [20,21], 53
fluorescence [22,23], electrochemical [24,25], biosensors [26], NMR [27], and MS [28–54
30] detectors are used. CE has been used with UV as a detection system [16- 18] and, 55
more recently, MS detectors [19,31–33] 56
116
Of all the HPLC and CE detection methods reported to date, MS clearly has the greatest 57
potential. The advantages of MS detection include the capability to both determine 58
molecular weight and providing structural information. HPLC and CE can be coupled 59
with different MS analyzers (i.e., with quadrupole, (IT), (TOF), etc.) and use several 60
ionization methods (APCI, ESI, MALDI, etc.). ESI is one of the most versatile 61
ionization methods and is the natural method of choice for the detection of ions 62
separated by CE. Moreover, the coupling with TOF-MS provides excellent mass 63
accuracy [34] over a wide dynamic range if a modern detector technology is chosen. 64
The latter, moreover, allows measurements of the correct isotopic pattern [35], 65
providing important additional information for the determination of the elemental 66
composition [36]. 67
Almond, scientifically know as Prunus dulcis, belongs to the family Rosaceae, and is 68
related to stone fruits such as peaches, plums, and cherries [37]. They are typically used 69
as snack foods and as ingredients in a variety of processed foods, especially in bakery 70
and confectionery products. The peach-like almond fruit consists of the edible seed or 71
kernel, the shell, and the outer hull. At maturity the hull splits open. When dry, it may 72
be readily separated from the shell. The almond pit, containing a kernel or edible seed, 73
is the nut of commerce. Shelled almonds may be sold as whole natural almonds or 74
processed into various almond forms. The whole natural almonds have had their shells 75
removed but still retain their brown skins; blanched whole almonds have had both their 76
shells and skins removed [38,39]. Usually, the removed skins will be discarded. 77
However, much study has shown that almond skins are a rich source of phenolic 78
compounds [2, 37,40,41]. In this sense, a few chromatographic methods have been 79
proposed for the identification of phenolic compounds in almond skin [37,42]. 80
117
The determination of polyphenols in almonds and almond skin has been studied by 81
several analytical methods. These methods include HPLC-DAD/ESI-MS [41], in which 82
a total of 33 compounds were characterized in 88 minute, and using HPLC-83
electrochemical detection, UV detection and LC/MS/MS, 20 polyphenols were 84
determined in 90 minute [42]. Liquid chromatography (HPLC) coupled to diode-array 85
UV (DAD-UV) and mass spectrometry (MS) has provided the most comprehensive 86
elucidation of phenolics in food and natural products. A longer LC–MS method 87
identified 21 flavonoids and phenolics in almonds in 120 min [42], another shorter 88
method was proposed for the determination of 15 flavonids in almond skin in 9 minute 89
by using capillary LC–MS method and determined the same number of flavonids by 90
using LC-UV in 84 minute [43]. In another research, 8 phenolics compound were 91
characterised using LC-UV in 44 minute [44], where Reverse phase HPLC coupled to 92
negative mode electrospray ionization (ESI) mass spectrometry (MS) was used to 93
quantify 16 flavonoids and 2 phenolic acids from almond skin extracts [45]. 94
95
In this article we show in the first time the use CE-ESI-TOF-MS method for the 96
determination of polyphenols in almond skin and a comparative study with a new rapid 97
HPLC-ESI- TOF (MS) method. 98
99
2. Experimental 100
101
2.1. Reagents and material 102
103
All chemicals were of analytical reagent grade and used as received. The organic 104
solvents acetonitrile, used in the HPLC mobile phase, 2-propanol used in the sheath 105
118
flow, methanol and hexane were purposed from Lab-Scan (Dublin, Ireland). Formic 106
acid used in HPLC phase A and B was purchased from Fluka (Switzerland). 107
Ammonium hydroxide was from Fluka (Buchs, Switzerland) and boric acid was 108
purchased from Sigma Aldrich (St. Louis, MO). Sodium hydroxide, used for capillary 109
cleaning procedures before each analysis, was obtained from Panreac (Barcelona, 110
Spain) and triethylamine (TEA) from Aldrich (Steinheim, Germany). Distilled water 111
was deionized by using a Milli-Q system (Millipore, Bedford, MA). All solutions were 112
filtered through a 0.45 µm Millipore (Bedford, MA, USA) membrane filters before 113
injection. 114
115
2.2. Extraction procedure 116
117
To isolate the phenolic fraction in almond skin we used a Liquid-Liquid Extraction 118
(LLE) procedure: 10 g of the dried sample were weighted in beaker 250 ml, 150 ml of 119
hexane were added then the solution was shaken by magnetic stir 40 min, and then 120
filtered by gravity through Whatman No.4 filter paper. Then the sample was collected in 121
250 ml round bottom flask and was stirred with 100 ml of 70% methanol under reflux 122
condition in a thermostatic water bath at 60 oC for 45 min. The resulting solution was 123
filtered by gravity through Whatman No.4 filter paper, and then the concentrated 124
methanol was evaporated by rotary evaporator under vacuum condition at 40 oC. The 125
dry residue was resolved by 2 ml of methanol: water (50:50, v/v) for analysis by CE, 126
and in 2 ml methanol for analysis by HPLC. Finally the extract was kept in the 127
refrigerator at -4 oC until the analysis. 128
129
119
2.3. CE coupling 130
131
CE experiments were performed using a Prince CE system (Prince Technologies, 132
Emmen, The Netherlands) and fused-silica capillaries of 95 cm in length and 50 µm 133
inner diameters (360 µm outer diameters) coupled to the MS detector by an orthogonal 134
electrospray interface (ESI) with a coaxial sheath-liquid sprayer was used (Agilent 135
Technologies, Palo Alto, CA, USA). Isopropanol/water (60:40) with 0.1% (v/v) TEA 136
was applied as sheath-liquid at a flow rate of 0.20 mL/min delivered by a 5 mL gas-tight 137
syringe (Hamilton, Reno, NV, USA) using a syringe pump Cole-Parmer (Vernon Hill, 138
IL, USA). The ESI-voltage of the TOF is applied at the end cap of the transfer capillary 139
to the MS with the spray needle being grounded. A nebulizer gas (N2) pressure of 0.4 140
bar was applied to assist the spraying. Dry gas temperature was set to 190ºC at a dry gas 141
flow of 4 L/min operating in negative ion mode. 142
Before first use, the bare capillaries were conditioned with 0.1 M sodium hydroxide 143
during 20 min followed by a water rinse for another 10 min. between runs the capillary 144
was flushed with water and separation buffer for 5 min. At the end of the day the 145
capillary was flushed with water for 10 min (all rinses during capillary conditioning 146
have been done using N2 at a pressure of 20 psi). 147
CE buffers were prepared by weighing boric acid and adjusting the pH when necessary 148
by adding ammonium hydroxide. The buffers were stored at 4ºC and warmed to room 149
temperature before use. After optimization, a running buffer 200 mM ammonium borate 150
at pH 10 was used. The separation voltage was set to 30 kV at the inlet of the capillary. 151
Injection was performed hydrodynamically at 50 mBar during 15 s, corresponding to 152
about 15 nL injected (0.9 % of the capillary). 153
120
All conditions were optimized in order to provide high resolution and strong mass 154
signals for all the studied phenolic compounds. 155
156
2.4. HPLC coupling 157
158
The separation of the phenolic compounds from almond skin was performed also by 159
using an Agilent 1100 series HPLC instrument (Agilent Technologies, Palo Alto, CA, 160
USA) was equipped with a vacuum degasser, an autosampler, a binary pump, and a 161
thermostated column department. The standards and samples were separated using a 162
reversed-phase C18 analytical column (50 x 2 mm, 2.5 µm particle size; Phenomenex 163
Synergi Fusion-RP100A) with a SecuityGuardTM
C18 guard column (4 x 2 mm; 164
Phenomenex Fusion-RP) maintained at 35ºC. The injection volume of standards and 165
samples was 5 µL. The mobile phase consisted of deionised water (A) and acetonitrile 166
(B), each containing 0.1% (v/v) formic acid. The chromatographic method consisted of 167
a linear gradient from 1 to 100% B during 9.5 min. The total run time, including the 168
conditioning of the column to the initial conditions, was 13 min. The flow rate was set 169
at 0.5 mL/min throughout the gradient. The effluent from the HPLC column was split 170
using a “T” before being introduced into the mass spectrometer (split ratio 1:3). Thus in 171
the current paper the flow which arrived to the ESI-TOF (MS) detector was 0.2 172
mL/min. The HPLC system was coupled to a TOF (MS) by an orthogonal electrospray 173
(ESI) interface (Agilent Technologies, Palo Alto, CA, USA). A nebulizer gas (N2) 174
pressure of 2 bar was applied to assist the spraying. Dry gas temperature was set to 190 175
ºC at a dry gas flow of 7 L/min operating in negative ion mode. 176
All conditions were optimized in order to provide high resolution and strong mass 177
signals for all the studied phenolic compounds. 178
121
179
2.5. TOF-MS 180
181
MS was performed using the microTOF (Bruker Daltonik, Bremen, Germany), an 182
orthogonal-accelerated TOF mass spectrometer (oaTOF-MS). Transfer parameters were 183
optimized by direct infusion experiments with Tuning Mix (Agilent Technologies) in 184
the range of 50-800 m/z looking for the best conditions regarding sensitivity and 185
resolution. Thus, the endplate offset was -500 V; capillary voltage 4500 V, the trigger 186
time was set to 50 µs, 49 µs for set transfer time and 1µs pre-puls storage time, 187
corresponding to a mass range of 50–800 m/z. Spectra were acquired by summarizing 188
20,000 single spectra, defining the spectra rate to 1 Hz. The accurate mass data of the 189
molecular ions were processed through the software Data Analysis 3.4 (Bruker 190
Daltonik), which provided a list of possible elemental formulas by using the Generate 191
Molecular Formula™ Editor. The Generate Formula ™ Editor uses a CHNO algorithm, 192
which provides standard functionalities such as minimum/maximum elemental range, 193
electron configuration, and ring-plus double bonds equivalents, as well as a 194
sophisticated comparison of the theoretical with the measured isotope pattern (Sigma 195
Value) for increased confidence in the suggested molecular formula (Bruker Daltonics 196
Technical Note #008, Molecular formula determination under automation). The widely 197
accepted accuracy threshold for confirmation of elemental compositions has been 198
established at 5 ppm. 199
We also have to say that even with very high mass accuracy (<1 ppm) many chemically 200
possible formulae are obtained depending on the mass regions considered. So, high 201
mass accuracy (<1 ppm) alone is not enough to exclude enough candidates with 202
complex elemental compositions. The use of isotopic abundance patterns as a single 203
122
further constraint removes > 95% of false candidates. This orthogonal filter can 204
condense several thousand candidates down to only a small number of molecular 205
formulas. 206
During the development of the CE method external instrument calibration was 207
performed using a 74900-00-05 Cole Palmer syringe pump (Vernon Hills, Illinois, 208
USA) directly connected to the interface, passing a solution of sodium formate cluster 209
by switching the sheath liquid to a solution containing 5 mM sodium hydroxide in the 210
sheath liquid of 0.2% formic acid in water:isopropanol 1:1 v/v at the end of the analysis. 211
Regarding the HPLC method, external instrument calibration was also performed using 212
a 74900-00-05 Cole Palmer syringe pump (Vernon Hills, Illinois, USA) directly 213
connected to the interface, passing a solution of sodium formate cluster at the end of 214
each run. 215
Using this method an exact calibration curve based on numerous cluster masses each 216
differing by 68 Da (NaCHO2) was obtained. Due to the compensation of temperature 217
drift in the MicroTOF, this external calibration provided accurate mass values (better 5 218
ppm) for a complete run without the need for a dual sprayer setup for internal mass 219
calibration. 220
These calibrations were performed in quadratic + high precision calibration (HPC) 221
regression mode. 222
3. Results and discussion 223
224
3.1. CE-ESI-TOF (MS) for the identification of phenolic compounds 225
226
123
Under the optimized CE-ESI-TOF (MS) method previously described above, in order to 227
obtain the best selectivity, sensitivity and resolution, the optimized CE-ESI-TOF 228
method was applied to the identification of the phenolic compounds present in the 229
almond skin extract. 230
Fig. 1 shows the extracted ion electropherograms (EIEs) for a nine of well-known 231
phenolic compounds present in the extract. These compounds are summarized in Table 232
1, with their formula, selected ion, m/z experimental and calculated, error (ppm and 233
mDa), sigma value, tolerance, migration time and the list of possibilities. 234
Thus, the proposed method is able to detect nine phenolic compounds in the same run. 235
These compounds are: (1) Quercetin-3-O-glucoside or galactoside ([M-H]-exp. 463.0906 236
m/z), (2) Isorhamnetin-3-rutinoside ([M-H]-exp. 623.1618 m/z), (3) Kampferol-3-237
rutinoside ([M-H]-exp. 593.1508 m/z), (4) Naringenin-7-O-glucoside ([M-H]
-exp. 238
433.1145 m/z), (5) Isorhamnetin-3-glucoside or galactoside ([M-H]-exp. 477.1028 m/z), 239
(6) p-Hydroxybenzoic acid ([M-H]-exp. 137.0248 m/z), (7) Naringenin ([M-H]
-exp. 240
271.0601 m/z), (8) Protocatechuic acid ([M-H]-exp. 153.0185 m/z) and (9) Vanillic acid 241
([M-H]-exp. 167.0352 m/z) all of them with an accuracy of 3 mDa. 242
As TOF-MS provides excellent mass accuracy over a wide dynamic range and allows 243
measurements of the isotopic pattern, providing important additional information for the 244
determination of the elemental composition. The identification by TOF (MS) was 245
carried out using the Generate Molecular Formula Editor. In this sense a low tolerance 246
was chosen (5 ppm) and options with a low sigma value (<5ppm) were taken into 247
account in the most cases. Therefore all detected compounds observed in Table 1 248
exhibit good sigma values smaller 0.05 and mass accuracy (ppm and mDa) as indicated 249
by the error values, except compound number 4 (Naringenin-7-O-glucoside) which 250
present a sigma value of 0.0959, but nevertheless it present a good mass accuracy. 251
124
Whereas compound number 1 (Quercetin-3-O-glucoside or galactoside) has a little bit 252
high error (5.3 ppm), but present a good sigma value (0.0311). 253
Therefore, using this method, nine phenolic compounds can be determined and 254
identified in 35 minute in an almond skin extract. These detected compounds can be 255
classified as phenolic acids and flavonoids family. Thus, we can charaterized three 256
phenolic acids (p-Hydroxybenzoic acid, Protocatechuic acid and Vanillic acid) between 257
21.3 - 35 min. These three compounds were the first hits in the list of possibilities (see 258
table 1). Furthermore, the method allows the determination 6 flavonids, 3 of them are 259
flavonids with sugar bond (e.g. glucose, rutinose) detected in the same sequence 260
between 19.3 - 20.2 min (Isorhamnetin-3-rutinoside, Kampferol-3-rutinoside, 261
Naringenin-7-O-glucoside). Unfortunately, two of the detected compounds are 262
(Quercetin-3-O-glucoside or galactoside and Isorhamnetin-3-glucoside or galactoside), 263
which they are glycosides with glucose or galactose bond being mass isomer. These 264
compounds have the same aglycone and cannot be differentiated in this method as they 265
have identical molecular weights. The sixth one of flavonids is the aglycone Naringenin 266
detected at 22.5 min. Naringenin and the three phenolic acids detected by this method 267
did not yielded fragmentation patterns while the other five compounds yielded 268
fragmentation as we have seen in Table 1. 269
Most of the compounds found in this work using CE method, have been previously 270
described in almond skin using HPLC [42,43], but this is the first time that these 271
compounds have been characterized by CE method. 272
273
3.2. HPLC-ESI-TOF (MS) for the identification of phenolic compounds 274
275
125
All the well-known compounds which were detected and identified by CE-ESI-TOF 276
(MS) were also identified using HPLC-ESI-TOF (MS). Fig. 2 shows the extracted ion 277
electropherograms (EIEs) of the major phenolic compounds in almond skin. These 278
compounds are summarized in Table 2, with their formula, selected ion, m/z 279
experimental and calculated, error (ppm and mDa), sigma value, tolerance, migration 280
time and the list of possibilities. 281
These compounds were characterised by TOF (MS) and carried out using the Generate 282
Molecular Formula Editor. First of all, a low tolerance was chosen (5 ppm). After that, 283
options with a low sigma value (<0.05) and a low error (<5ppm) were taken into 284
account in most cases, most of them were the first hits in the list of possibilities and 285
presents a good sigma and error values (see table 2). 286
Thus, first five phenolic compounds regarding to phenolic acids family were 287
characterised such as; Protocatechuic acid, Trans - p- coumaric acid, p- hydroxybenzoic 288
acid, Chlorogenic acid and Vanillic acid. These five compounds were the first hits in the 289
list of possibilities (see table 2) and were detected between 2.2 – 3.4 min, they presents 290
a very good error (>5 ppm) and sigma values (>0.05), except two compounds, Trans - 291
p- coumaric acid and Chlorogenic acid which presents sigma values of 0.0576 and 292
0.090 respectively. 293
If we consider the flavonids family, we can characterized and detected the following 294
eighteen compounds: Catechin, Dihydrokaempferol-3-O-glucoside , Epicatechin , 295
Eriodictiol-7-O-glucoside, Quercetin-3-O-rutinoside (rutin), Quercetin-3-O-galactoside, 296
Dihydroquercetin, Quercetin-3-O-glucoside, Kaempferol-3-O-rutinoside, Naringenin-7-297
O-glucoside, Isorhamnetin-3-O-rutinoside, Quercetin-3-O-rhamnoside, Isorhamnetin-3-298
O-glucoside/galactoside, Dihdrokaempherol or Eriodictiol, Quercetin, Naringenin, 299
Isorhamnetin and Kaempferol using this method. 300
126
Therefore, using this method 9 flavonids with sugar bond ( glucose, galactose, rutinose 301
and rhamnose) were detected in the same sequence between 3.0 – 4.1 min, and one 302
compound of these group (Isorhamnetin-3-O-glucoside/galactoside), which are mass 303
isomer with the same aglycone and cannot be differentiated in this method. The rest of 304
compounds of Flavonids, are 7 aglycones (Catechin, Epicatechin, Dihydroquercetin, 305
Quercetin, Naringenin, Isorhamnetin and Kaempferol). Besides, there is a further 306
Flavonids with two possibilities (Dihydrokaempherol or Eriodictiol) due to they have 307
the similar molecular weight. The 8 aglycones and the 5 phenolic acids detected by this 308
method did not yielded fragmentation patterns while all the Flavonids yielded 309
fragmentation (see Table 2). 310
Thus, these 23 compounds using HPLC-ESI-TOF (MS) method have been previously 311
described in almond skin by Paul E. Milbury et al [42] using HPLC- UV detector in 90 312
minute and by Christine A Hughey et al [43] using capillary LC-MS in 9 minute. 313
314
3.3 Comparison between the results obtained by CE-ESI-TOF (MS) and HPLC-315
ESI-TOF (MS) methods 316
317
The observed mass values are identical for these separation techniques within the 5 ppm 318
mass accuracy, giving confidence in the identification. If we consider phenolic acid, we 319
can observe that the three phenolic acids (p-Hydroxybenzoic acid, Protocatechuic acid 320
and Vanillic acid) detected by CE method, were also detected by HPLC method. 321
Besides another two phenolic acid compounds (Trans - p- coumaric acid and 322
Chlorogenic acid) were not observed in the CE profiles. Although, difference between 323
both methods is really clear in the case of the three phenolic acids detected by both that 324
the sigma and error value are better in HPLC method. In addition to previous point, the 325
127
number of compounds that identified in HPLC (5 Compounds) more than CE method (3 326
compounds). 327
Concerning flavonoids, it is possible to say that the HPLC method provides better 328
results. The CE method was able to detect six flavonids compounds, while using HPLC 329
is possible to detect 12 flavonids, even all of them had good sigma and error values in 330
shorter time than CE method. Further at the optimum electrophoretic conditions, the 331
peak shape of the Flavonids was modest. However, most of the peaks had a good shape 332
and intensity in HPLC. 333
In general, the HPLC method was more appropriate for studying the flavonids family, 334
since all of the flavonids represented peaks of significant intensity in the central zone of 335
the chromatogram. Moreover, using the optimum electrophoretic conditions, we could 336
not observe so many isomeric forms as in the chromatograms obtained in HPLC. 337
Eriodictiol, Quercetin, Naringenin, Isorhamnetin and Kaempferol were the major 338
almond flavonids [42, 43] detected by HPLC method whereas just Naringenin was 339
detected with CE with a very low intensity. 340
Both methods can be successfully applied to the analysis of phenolic compounds in 341
almond skin and both techniques are reliable enough for determining this class of 342
compounds. However, if we understand them as complementary techniques to improve 343
the characterization of this polar fraction, the results will be more complete. 344
345
Conclusions 346
347
The separation by HPLC/CE with on-line detection by ESI-TOF-MS is successfully 348
applied to the analysis of the phenolic compounds present in almond skin samples. In 349
this work the CE was used for the first time in this type of samples. The two 350
128
methodologies are able to determine known phenolic compounds present in almond skin 351
and provide information about the presence and relative concentration of minor 352
phenolic compounds. HPLC can detect 23 compounds in 9 min, but only 9 compounds 353
were detected by CE method in 35 min. 354
355
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131
Tab
le 1. Well-known phenolic compounds determined by CE-ESI-TOF-M
S in an alm
ond skin extract.
Error
Classification
order
considering
other
possibilities
Tole
rance
(ppm
) in
Gen
erate
Mole
cula
r
Form
ula
#
Com
pound
Form
ula
Sel
ecte
d
ion
m/z
exper
imen
tal
m/z
calc
ula
ted
(ppm
) (m
Da)
Sig
ma
Valu
e
Mig
ration
tim
e CE
(min
)
m/z
Fragments
1
Quer
cetin-3
-O-
glu
coside/
gala
ctosi
de
C21H
19O
12
[M-H
] 463.0906
463.0882
5.3
-2.44
0.0311
1st (5)
10
14.2
301.1225
2
Isorh
am
net
in-3
-
rutinoside
C28H
31O
16
[M-H
]-
623.1618
623.1617
0.2
-0.14
0.0212
2st (8)
5
19.3
315.0245
3
Kam
pfe
rol-3-
rutinoside
C27H
29O
15
[M-H
]-
593.1508
593.1511
0.6
0.33
0.0114
2st (7)
5
19.7
285.1155
4
Naringen
in-7
-O-
glu
coside
C21H
21O
10
[M-H
]-
433.1145
433.1140
1.3
-0.57
0.0959
5st (10)
5
20.2
271.1122
5
Isorh
am
net
in-3
-
glu
coside/
gala
ctosi
de
C22H
21O
12
[M-H
]-
477.1028
477.1038
2.1
1.02
0.0403
2st (5)
5
20.7
315.0541
6
p-H
ydro
xyb
enzo
ic
aci
d
C7H
5O
3
[M-H
]-
137.0248
137.0244
3.9
-0.42
0.0227
1st (1)
5
21.3
7
Naringen
in
C15H
11O
5
[M-H
]-
271.0601
271.0611
3.8
1.04
0.0348
1st (1)
5
22.5
8
Pro
toca
tech
uic
aci
d
C7H
5O
4
[M-H
]-
153.0185
153.0182
2.3
-0.35
0.0454
1st (1)
5
26.7
9
Vanillic
aci
d
C8H
7O
4
[M-H
]-
167.0352
167.0349
1.9
-0.31
0.0112
1st (3)
5
34.5
132
Tab
le 2. Well-known phenolic compounds determined by HPLC-ESI-TOF-M
S in an alm
ond skin extract.
E
rror
Classification
order
considering
other
possibilities
Tole
rance
(ppm
) in
Gen
erate
Mole
cula
r
Form
ula
Mig
ration
tim
e H
PLC
(min
)
m/z
Fragments
#
Com
pound
Form
ula
Sel
ecte
d ion
m/z
exper
imen
tal
m/z
calc
ula
ted
(ppm
) (m
Da)
Sig
ma
Valu
e
1
Pro
toca
tech
uic
aci
d
C7H
5O
4
[M-H
]-
153.0191
153.0193
1.4
0.21
0.0066
1st (1)
5
2.281
2
Tra
ns-
p-c
oum
aric
aci
d
C9H
7O
3
[M-H
]-
163.0405
163.0401
2.5
-0.40
0.0576
1st (1)
5
2.536
3
p-H
ydro
xyb
enzo
ic
aci
d
C7H
5O
3
[M-H
]-
137.0244
137.0244
0.1
-0.02
0.0051
1st (1)
5
2.587
4
Cate
chin
C
15H
14O
6
[M-H
]-
289.0718
289.0711
2.4
0.68
0.0075
1st (2)
5
3.004
5
Chlo
rogen
ic a
cid
C16H
17O
9
[M-H
]-
353.0865
353.0878
3.7
1.31
0.0900
1st (3)
5
3.029
6
Dih
ydro
kaem
pfe
rol
3-O
-glu
coside
C21H
21O
11
[M-H
]-
449.1090
449.1089
0.2
-0.11
0.0288
2st (5)
5
3.148
287.0581
7
Epic
ate
chin
C
15H
14O
6
[M-H
]-
289.0716
289.0711
0.6
0.17
0.0057
1st (1)
5
3.216
8
Vanillic
aci
d
C8H
7O
4
[M-H
] 167.0356
167.0350
3.5
-0.58
0.0253
1st (1)
5
3.420
9
Eriodic
tiol-7-O
-
glu
coside
C21H
21O
11
[M-H
]-
449.1099
449.1089
2.1
-0.93
0.0372
1st (5)
5
3.658
287.1082
10
Quer
cetin-3
-O-
rutinoside
C27H
29O
16
[M-H
]-
609.1460
609.1461
0.2
0.15
0.0245
1st (4)
5
3.709
301.1107
11
Quer
cetin-3
-O-
gala
ctoside
C21H
19O
12
[M-H
]-
463.0897
463.0882
3.2
-1.48
0.0766
3st (3)
5
3.726
301.1253
12
Dih
ydro
quer
cetin
C15H
11O
7
[M-H
]-
303.0514
303.0510
1.2
-0.35
0.0173
1st (2)
5
3.794
13
Quer
cetin-3
-O-
glu
coside
C21H
19O
12
[M-H
]-
463.0891
463.0882
1.9
-0.90
0.0298
2st (5)
5
3.811
301.1225
14
Kaem
pfe
rol-3-O
-
rutinoside
C27H
29O
15
[M-H
]-
593.1518
593.1512
1.0
-0.61
0.0117
1st (8)
5
3.896
285.1285
15
Naringen
in-7
-O-
glu
coside
C21H
21O
10
[M-H
]-
433.1133
433.1140
1.6
0.71
0.0278
2st (3)
5
3.930
271.0722
16
Isorh
am
net
in-3
-O-
rutinoside
C28H
31O
16
[M-H
]-
623.1612
623.1618
0.9
0.57
0.0191
1st (5)
5
3.947
315.0541
17
Quer
cetin-3
-O-
C21H
19O
11
[M-H
]-
447.0929
447.0933
0.9
0.40
0.0545
2st (5)
5
4.015
301.1283
133
rham
noside
18
Isorh
am
net
in-3
-O-
glu
coside/
gala
ctosid
e
C22H
21O
12
[M-H
]-
477.1023
477.1038
3.3
1.58
0.0064
1st (6)
5
4.049
315.0553
19
Dih
ydro
kaem
pher
ol
or Eriodic
tiol
C15H
11O
6
[M-H
]-
287.0561
287.0561
0.0
0.01
0.0181
2st (3)
5
4.167
20
Quer
cetin
C15H
9O
7
[M-H
]-
301.0355
301.0354
0.3
-0.10
0.0178
2st (3)
5
4.695
21
Naringen
in
C15H
11O
5
[M-H
]-
271.0607
271.0612
1.9
0.51
0.0209
1st (1)
5
4.950
22
Isorh
am
net
in
C16H
11O
7
[M-H
]-
315.0526
315.0510
5.0
-1.56
0.1036
2st (3)
5
5.154
23
Kaem
pher
ol
C15H
9O
6
[M-H
]-
285.0416
285.0405
3.9
-1.12
0.0967
2st (3)
5
5.137
134
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)
0 5 10 15 20 25 30 Time [min]
0.0
0.5
1.0
1.5
4x10
Intens.
0 5 10 15 20 25 30 Time [min]0
1
2
3
4
4x10
Intens.
3
6
7
EIE 593.150± 0.005
EIE 271.060± 0.005
0 5 10 15 20 25 30 Time [min]0
2000
4000
6000
Intens.
EIE 463.090± 0.0051
Fig. 1
0
2
4
6
x10
Intens.
2EIE 623.161± 0.005
0 5 10 15 20 25 30 Time [min]
4
0
1000
2000
3000
4000
Intens.
4EIE 433.114± 0.005
0 5 10 15 20 25 30 Time [min]
0 5 10 15 20 25 30 Time [min]
0
1
2
3
4x10
Intens.
0 5 10 15 20 25 30 Time [min]
0.00
0.25
0.50
0.75
1.00
1.25
4x10
Intens.
5EIE 477.102± 0.005
0 5 10 15 20 25 30 Time [min]
0
2000
4000
6000
Intens.
8EIE 153.018± 0.005
EIE 137.024± 0.005
9EIE 167.025± 0.005
0 5 10 15 20 25 30 Time [min]0
2
4
6
8
4x10
Intens.
0 1 2 3 4 5 6 7 8 Time [min]0
1
2
3
4x10Intens.
EIC 153.018±0.005
0 1 2 3 4 5 6 7 8 Time [min]0.00
0.25
0.50
0.75
1.00
1.25
4x10Intens.
EIC 137.025±0.005
0 1 2 3 4 5 6 7 8 Time [min]0
1
2
3
4
5
64x10
Intens.
EIC 289.072±0.005
0 1 2 3 4 5 6 7 8 Time [min]0
2
4
6
4x10Intens.
EIC 593.151±0.005
0 1 2 3 4 5 6 7 8 Time [min]0.0
0.2
0.4
0.6
0.8
4x10Intens.
EIC 433.114±0.005
0 1 2 3 4 5 6 7 8 Time [min]0.00
0.25
0.50
0.75
1.00
1.25
5x10Intens.
EIC 623.162±0.005
0 1 2 3 4 5 6 7 8 Time [min]0.0
0.5
1.0
1.5
4x10Intens. EIC 477.103±0.005
0 1 2 3 4 5 6 7 8 Time [min]0
500
1000
1500
2000
Intens.EIC 301.035±0.005
0 1 2 3 4 5 6 7 8 Time [min]0
2000
4000
6000
Intens.EIC 271.061±0.005
0 1 2 3 4 5 6 7 8 Time [min]0
1000
2000
3000
4000
Intens. EIC 287.056±0.005
Fig. 2
1
20
1918
1615
4 14
3
21
137
CHAPTER V: Characterization of phenolic and other polar compounds in
Flaxseed oil using HPLC-ESI-TOF (MS)
138
This work was submitted to Food chemistry Journal. Characterization of phenolic and other polar compounds in Flaxseed oil using HPLC-ESI-TOF (MS) Saleh M.S. Sawalha, David Arráez-Román, Antonio Segura-Carretero, Alberto Fernández-Gutiérrez. Department of Analytical Chemistry, Granada University. Wahid Herchi, Habib Kallel. Laboratoire de Biochimie des lipides, Département de Biologie, Faculté des sciences de Tunis, 2092 ELmanar-Tunisie.
139
Characterization of phenolic and other polar compounds in 1
Flaxseed oil using HPLC-ESI-TOF (MS) 2
3
Saleh Sawalhaa, Wahid Herchi
b, David Arráez-Román
a, Antonio Segura-Carretero
a,*, 4
Habib Kallelb, Alberto Fernández-Gutierrez
a,* 5
6
aDepartment of Analytical Chemistry, Faculty of Sciences, University of Granada, 7
C/Fuentenueva s/n, 18071 Granada, Spain. 8
bLaboratoire de Biochimie des lipides, Département de Biologie, Faculté des sciences de 9
Tunis, 2092 ELmanar-Tunisie. 10
11
Abstract 12
A sensitive method based on high-performance liquid chromatography coupled with 13
electrospray ionization time-of-flight-mass spectrometry (HPLC–ESI–TOF (MS)) has been 14
used to analyze phenolic compounds in Flaxseed oil. Several important phenolic compounds 15
such as secoisolariciresnol, ferulic acid and its methyl ester, coumaric acid methyl ester, 16
diphylin, pinoresinol, matairesinol, p-hydroxybenzoic acid, vanillin and vanillic acid have 17
been detected from Flaxseed oil. The efficiency, rapidity and high resolution of HPLC 18
coupled to the sensitivity, selectivity, mass accuracy and true isotopic pattern from TOF (MS) 19
have revealed an enormous separation potential allowing the characterization of a broad series 20
of phenolic compounds present in Flaxseed oil for the first time. 21
22
Keywords: 23
Phenolic compounds, Flaxseed oil, HPLC, ESI-TOF (MS) 24
25
* Corresponding author. Fax: +34 958249510 26
E-mail addresses: [email protected] (A. Segura-Carretero), [email protected] (A. Fernández-27
Gutiérrez) 28
140
1
2
1. Introduction 3
4
Flaxseed has been gaining popularity in the health food market because of its reported health 5
benefits and disease preventive properties on coronary heart disease (Oomah & Mazza, 6
2000), some kinds of cancer and neurological and hormonal disorders (Huang & Ziboh, 2001; 7
Simopoulos, 2002). During the last decade, there has been an increasing interest in the use of 8
Flaxseed in the diet in order to improve the nutritional and health status (Oomah, 2001). 9
Flaxseed is rich in lignans and the embryo is rich in oil with a high omega-3 fatty acid content 10
(Westcott and Muir, 2003; Wiesenborn, Tostenson & Kangas, 2003). The beneficial effects of 11
lignans on human health are well recognised (Westcott and Muir, 2003, McCann, Gill, 12
McGlynn, & Rowland, 2005). Other phenolic compounds of interest that are accumulated in 13
flaxseed include ferulic and vanillic acid. The qualitative and quantitative determination of the 14
phenolic compounds in oils is very important and several methods have been already used in 15
recent years. Various methods have been reported for the identification of these substances in 16
Flaxseed starting from the early days, non-specific analytical methods, such as paper, thin –17
layer (Coran, Giannellini & Bambagiotti-Alberti ,2004), and column chromatography as well 18
as UV spectroscopy , were applied to polyphenols analysis (Christophoridou, Dais, Tseng, & 19
Spraul,2005). The need identify individual phenolic compounds meant that traditional 20
methods were replaced and significant progress was achieved when more specific analytical 21
techniques were used, such as Gas Chromatography (GC) (Penalvo, Haajanen, Botting & 22
Adlercreutz,2005) or High-Performance Liquid Chromatography (HPLC) (Charlet et al, 23
2002). The results obtained by using GC are very reliable and interesting, but the use of this 24
141
technique is less common because the derivatization step is essential and the use of high 1
temperature which could damage this kind of analytes. 2
HPLC hyphenated to Mass Spectrometry (MS) detection is one of the most important 3
analytical techniques used for the analysis of phenolic compounds (Carrasco-Pancorbo et al, 4
2005; Morales & Tsimidou, 2000). The advantages of MS detection include the ability to 5
determine molecular weights and to obtain structural information (Carrasco-Pancorbo et al, 6
2007). 7
The on-line coupling of HPLC with MS using Electrospray Ionization (ESI) as an interface 8
yields a powerful method because ESI–MS allows the determination of a wide range of polar 9
compounds. ESI is one of the most versatile ionization methods, and is the method of choice 10
for the detection of ions separated by liquid chromatography. Although HPLC can be coupled 11
to different MS analyzers (quadrupole, ion trap (IT), time-of-flight (TOF), etc (Simo et al, 12
2005), in this paper we have used HPLC–ESI–TOF (MS) to characterize phenolic compounds 13
in Flaxseed oil. TOF (MS) provides excellent mass accuracy (Bristow & Webb, 2003) over a 14
wide dynamic of range if modern detector technology is used. The latter, moreover, allows 15
measurements of the isotopic pattern (Pelzing, Decker, Neusüß & Räther , 2004), providing 16
important additional information for the determination of the elemental composition 17
(Bringmann et al, 2005). To our knowledge, the present work represents the first time that a 18
HPLC–ESI–TOF (MS) method has been applied to the characterization of phenolic 19
compounds in Flaxseed oils. 20
21
2. Materials and methods 22
2.1. Chemicals and reagents 23
All chemicals were of analytical reagent grade and used as received. The organic solvents, 24
hexane, methanol and ACN, used in the extraction procedure and as HPLC mobile phase were 25
142
purposed from Lab-Scan (Dublin, Ireland). Acetic acid used in HPLC phase A was purchased 1
from Fluka (Switzerland). Deionised water was obtained from a water purifier system 2
(Millipore, Bedford, MA). All the solvents used in the HPLC system were filtered through a 3
0.20 µm Millipore (Bedford, MA, USA) membrane filters. 4
5
2.2. Plant materials 6
The seeds of the three varieties H52, O116 and P129 were purchased from INRAT (Institut 7
National Recherche Agronomie Tunis, North of Tunisia). 8
9
2.3. Oil extraction from Flaxseed 10
The total lipids were extracted by the method of Folch, Lees, & Sloane Stanley (1957) 11
modified by Bligh & Dyer (1959). Seeds (5 g) were washed with boiling water for 5 min to 12
denature the phospholipases and then crushed in a mortar with a mixture of CH3Cl-MeOH 13
(2:1, v/v). The water of fixation was added and the homogenate was centrifuged at 3000 g for 14
15 min. The lower chloroformic phase containing the total lipids was kept and dried in a 15
rotary evaporator at 40°C. 16
17
2.4. Solid-phase Extraction (SPE) Procedure 18
100 mg of DSC-Diol (Supelco, Bellefonte, PS, USA) as powder was added in a test tube of 10 19
mL and it was conditioned as follows: 1) 100 µL of methanol were added, shaken on vortex 20
for 5 minutes, centrifuged at 4500 rpm for 10 minutes and the liquid part was then discarded. 21
2) 100 µL ml of hexane was added, shaken on vortex for 5 minutes, centrifuged at 4500 rpm 22
for 10 minutes and then the liquid part was discarded. 23
Flaxseed oil (1 g) was dissolved in 1200 µL hexane in a test tube of 10 mL, shaken on vortex 24
for 5 minutes and the solution was added into the test tube with the conditioned DSC-Diol. 25
143
All was shaken on vortex for 5 minutes, centrifuged at 4500 rpm for 10 minutes and the liquid 1
part was discarded. Then, the DSC-Diol was washed with 1200 µL of hexane, shaken on 2
vortex 5 minutes, centrifuged at 1000 rpm for 10 minutes and the hexane were then discarded 3
in order to remove the non-polar fraction of Flaxseed oil. The polar fraction was recovered by 4
addition of 1200 µL of methanol; the solution was shaken on vortex 5 minutes and 5
centrifuged at 1000 rpm for 10 minutes. Finally, the methanolic part was removed into an 6
eppendorf 2 mL tube and evaporated by a rotary evaporator (Concentrator plus, Eppendorf 7
AG, Hamburg, Germany) under reduced pressure at 30°C. The sample was resolved in 20 µl 8
of methanol and filtered through a 0.2 µm. 9
10
2.5. HPLC 11
The separation of the phenolic compounds from Flaxseed oil was performed using an Agilent 12
1200 series Rapid Resolution LC (Agilent Technologies, Palo Alto, CA, USA) was equipped 13
with a vacuum degasser, an autosampler, a binary pump, and a thermostated column 14
department. The standards and samples were separated using a reversed-phase C18 analytical 15
column (4.6×150 mm, 1.8 µm particle size, Agilent ZORBAX Eclipse plus). The mobile 16
phase A and B consisted of water with 0.5% acetic acid, and ACN. The chromatographic 17
method was as following: gradient from 5% B to 30% B in 10 minutes; 30% B to 33% B in 2 18
minutes; 33% B to 38% B in 5 minutes; 38% B to 50% B in 3 minutes; 50% to 95% in 3 19
minutes. The initial conditions were re-established in 2 minutes and held for 10 minutes. The 20
total run time, including the conditioning of the column to the initial conditions, was 35 min. 21
The flow rate used was set at 0.80 mL/min throughout the gradient. The effluent from the 22
HPLC column was split using a “T” before being introduced into the mass spectrometer (split 23
ratio 1:3). Thus in the current paper the flow which arrived to the ESI-TOF (MS) detector was 24
144
0.2 mL/min. The column temperature was maintained at 25 °C and the injection volume was 1
10 µL. 2
3
2.6. ESI-TOF (MS) 4
ESI-TOF (MS) conditions were optimized in order to provide strong mass signals for all the 5
studied phenolic compounds. The HPLC system was coupled to a TOF (MS) equipped with 6
an ESI interface operating in negative ion mode. The optimum ESI parameters were as 7
follows: nebulizing gas pressure, 2 bar; drying gas flow, 9 L/min; drying gas temperature, 190 8
ºC. 9
MS was performed using the microTOF (Bruker Daltonik, Bremen, Germany), an orthogonal-10
accelerated TOF mass spectrometer (oaTOF-MS). Transfer parameters were optimized by 11
direct infusion experiments with Tuning Mix (Agilent Technologies) in the range of 50-800 12
m/z looking for the best conditions regarding sensitivity and resolution. Thus, the endplate 13
offset was -500 V; capillary voltage 4500 V, the trigger time was set to 50 µs, 49 µs for set 14
transfer time and 1µs pre-puls storage time, corresponding to a mass range of 50–800 m/z. 15
Spectra were acquired by summarizing 20,000 single spectra, defining the spectra rate to 16
1 Hz. The accurate mass data of the molecular ions were processed through the software Data 17
Analysis 3.4 (Bruker Daltonik), which provided a list of possible elemental formulas by using 18
the Generate Molecular Formula™ Editor. The Generate Formula ™ Editor uses a CHNO 19
algorithm, which provides standard functionalities such as minimum/maximum elemental 20
range, electron configuration, and ring-plus double bonds equivalents, as well as a 21
sophisticated comparison of the theoretical with the measured isotope pattern (Sigma Value) 22
for increased confidence in the suggested molecular formula (Bruker Daltonics Technical 23
Note #008, Molecular formula determination under automation). The widely accepted 24
145
accuracy threshold for confirmation of elemental compositions has been established at 5 ppm 1
(Fereer et al. 2005). 2
We also have to say that even with very high mass accuracy (<1 ppm) many chemically 3
possible formulae are obtained depending on the mass regions considered. So, high mass 4
accuracy (<1 ppm) alone is not enough to exclude enough candidates with complex elemental 5
compositions. The use of isotopic abundance patterns as a single further constraint removes > 6
95% of false candidates. This orthogonal filter can condense several thousand candidates 7
down to only a small number of molecular formulas. 8
During the development of the HPLC method, external instrument calibration was performed 9
using a 74900-00-05 Cole Palmer syringe pump (Vernon Hills, Illinois, USA) directly 10
connected to the interface, passing a solution of sodium formate cluster containing 5 mM 11
sodium hydroxide in water/isopropanol 1/1 (v/v), with 0.2% (v/v) of formic acid at the end of 12
each run. Using this method an exact calibration curve based on numerous cluster masses 13
each differing by 68 Da (NaCHO2) was obtained. Due to the compensation of temperature 14
drift in the MicroTOF, this external calibration provided accurate mass values (better 5 ppm) 15
for a complete run without the need for a dual sprayer setup for internal mass calibration. 16
These calibrations were performed in quadratic + high precision calibration (HPC) regression 17
mode. 18
19
3. Results and discussion 20
3.1. Repeatability study 21
Repeatability of the HPLC-ESI-TOF (MS) analysis was studied by performing a series of 22
separations using the optimized method by the analysis of methanol extracts on the same day 23
(intraday precision, n=5) and on three consecutive days (interday precision, n=15). The 24
relative standard deviations (RSDs) of analysis time and peak area were determined. The 25
146
intraday repeatability on the migration time (expressed as RSD) was 0.5%, whilst the interday 1
repeatability was 0.9%. The intraday repeatability of the peak area (expressed as RSD) was 2
1.2%, whilst the interday repeatability was 4.3%. 3
4
3.2. Identification of phenolics compounds in Flaxseed oil 5
The HPLC–ESI–TOF (MS) method was applied to the identification of the phenolic 6
compounds present in Flaxseed oil. The identification of phenolics compounds was carried 7
out comparing their migration times and mass spectra provided by TOF (MS) with those of 8
authentic standards when available. Thus, Fig. 1 shows the chemical structures of the 9
characterized phenolic compounds and Fig. 2 shows the extracted ion chromatograms (EIC) 10
according to the elution order: (1) diphylin ([M-H]exp m/z 379.0835), (2) vanillic acid 11
([M-H]exp m/z 167.0350), (3) vanillin ([M-H]exp m/z 151.0400), (4) p-hydroxybenzoic acid 12
([M-H]exp m/z 137.0244), (5) methyl ester coumaric acid ([M-H]exp m/z 177.0557). (6) 13
secoisolariciresinol ([M-H]exp m/z 361.1656), (7) methyl ester ferulic acid ([M-H]exp m/z 14
207.0657), (8) ferulic acid ([M-H]exp m/z 193.0506), (9) pinoresinol ([M-H] exp m/z 15
357.1343), (10) matairesinol ([M-H]exp m/z 357.1341). These compounds are also 16
summarized in Table 1 (ten phenolic compounds were characterized in H52 and P129 17
varieties and nine compounds in O116 variety) along with their molecular formula, selected 18
ions, experimental and calculated m/z values, errors (ppm and mDa), sigma values, tolerances 19
in generated molecular formula and migration times. The identification by TOF (MS) was 20
carried out using the Generate Molecular Formula Editor. First of all, a low tolerance was 21
chosen (5 ppm). After that, options with a low sigma value (<0.05) and a low error (<5ppm) 22
were taken into account in most cases. 23
Most of the compounds found in this work have been previously described in Flaxseed. 24
147
Lignans and their derivatives have been characterized before in Flaxseed (Kamal-Eldin et al., 1
2001) as well as phenolics acids (Johnsson et al., 2002) but this is the first time in Flaxseed 2
oil. 3
These detected compounds can be classified as lignans, phenolic acids, simple phenols and 4
diphylin family. Regarding the lignan family, it is possible to study the following compounds 5
(in elution order) with the HPLC-ESI-TOF (MS) method: secoisolariciresinol, pinoresinol and 6
matairesinol. Pinoresinol was found in Flaxseed oil in higher concentration but matairesinol 7
and secoisolariciresinol were found in low concentrations in all varieties. The main lignan 8
secoisolariciresinol diglucoside (SDG) is not present in these varieties of Flaxseed oil 9
probably because of its high solubility (Lavelli and Bondesan, 2005). 10
These compounds were detected in H52 and P129 varieties, while secoisolariciresinol was 11
absent in O116 variety. Considering in fact, that “pinoresinol lariciresinol reductase” 12
catalyses the conversion of pinoresinol to secoisolariciresinol (Xia, 2000), we suggested that 13
this enzyme was present in low amount and inactive in O116 variety than in H52 and P129 14
varieties. Other reason, it was presumed that in Flaxseed oil oligomers under a large amount 15
of methanol, the glycosidic unit SDG was esterified by p-coumaric acid glucoside or ferulic 16
acid glucoside which was easily released by alkaline hydrolysis to form the methyl ester of p-17
coumaric acid glucoside or ferulic acid glucoside in a reaction medium containing a large 18
amount of methanol (Li et al, 2008), these forms will be converted by deglycosylation 19
products respectively in p-coumaric acid and ferulic acid. 20
Recent studies have described in a little more details this family of lignan using HPLC-NMR; 21
HPLC-MS (Hosseinian, Muir, Westcott, and Krol; Strandas, Kamal-Eldin, Andersson, Aman, 22
2006). 23
Regarding the family of phenolics acids, the method allows the determination of ferulic acid 24
and its methyl ester, vanillic acid, p-hydroxybenzoic acid and methyl ester coumaric acid. 25
148
In all varieties, p-coumaric acid was not detected while ferulic acid was detected in small 1
amount. This indicates that, in a reaction medium containing large amount of methanol, p-2
coumaric and ferulic acid have been almost completely esterified. A methyl ester ferulic acid 3
([M-H] 207.0662 m/z) and methyl ester coumaric acid ([M-H] 177.0557 m/z) respectively, 4
were characterized by mass spectra and using the Generate Molecular Formula Editor. Li et 5
al. (2008) reported that p-coumaric acid or ferulic acid standard dissolved in an aqueous 6
methanol solution could be esterified by methanol to produce p-coumaric acid methyl ester or 7
ferulic acid methyl ester. Kozlowska, Zadernowski, & Sosulski (1983) showed that the 8
highest proportion of phenolic acids in Flaxseed oil and other oil seeds were ester bound. In 9
addition to the previously mentioned phenolics compounds, a further two families of 10
phenolics compounds was detected in the three varities of Flaxseed oil using HPLC method 11
namely simple phenols presented by vanillin compound and diphyllin family presented by 12
diphyllin compound. 13
14
3.3. Unknown phenolic compounds 15
Besides the previously mentioned phenolic compounds detected, it was also possible to study 16
others compounds present in Flaxseed oil which have so far not been described in the 17
literature. These unknown compounds have been included in Table 2 as they are an important 18
part of the polar fraction of Flaxseed oil. Even though the characterization of these 19
unidentified compounds was not possible with the generated data by TOF analysis, it is 20
possible to observe the experimental m/z, selected ion, tolerance (ppm), a list of possibilities, 21
the mass deviation, and the sigma value. 14 unknown compounds were detected in H52 22
variety, 16 in P129 and 20 were detected in O116 variety. This difference appeared in 6 23
compounds which are (m/z 197.0452; 131.0721; 216.9996; 369.1037; 177.0188 and 24
179.0339). Two compounds of them were detected in P129 and O116 while were not detected 25
149
in H52 (m/z 197.0457; 131.0739). The proposed HPLC method allowed the determination of 1
four compounds in O116 and not determined in P129 and H52 (m/z 216.9996; 369.1037; 2
177.0188; 179.0339). In general, O116 had the greatest possibilities of unknown compounds, 3
probably due to the hydrolysis of others compounds, also this difference could be linked to 4
the differences in relative’s activities and abundances of the complex of enzymes responsible 5
for phenolic compounds biosynthesis. 6
A reduced number of possible elemental compositions are obtained from the accurate mass of 7
the suspected peak. These elemental compositions can then be matched against available 8
databases (The Merck Index, ChemIndex, commercial e-catalogues) using the deduced 9
molecular formula as a search criterion (Ibanez et al 2005; Kina & Fiehn, 2006). 10
11
4. Conclusions 12
The sensitive HPLC-ESI-TOF (MS) method allows the characterization of many well-known 13
and the detection of unknown phenolic compounds present in Flaxseed oil. Furthermore, the 14
use of TOF (MS) provides excellent resolving power and mass accuracy over a wide dynamic 15
range if modern detector technology is chosen. Moreover, it allows the measurement of the 16
correct isotopic pattern, providing important additional information for the determination of 17
the elemental composition. Thus, significant differences were found between the three 18
varieties using the proposed method. This fact could be used in future to find potential 19
markers for the geographical origin of the oil or the flaxseed variety. 20
21
Acknowledgements 22
The authors are grateful to the Spanish Ministry of Education and Science for the project 23
(AGL2008-05108-C03-03) and to Andalusian Regional Government Council of Innovation 24
and Science for the project P07-AGR-02619. 25
150
The author SS gratefully acknowledges the Agencia Española de Cooperación Internacional 1
(AECI). 2
3
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Table 1. Well-known phenolic compounds characterized by HPLC- ESI-TOF (MS) in Flaxseed oil.
Compound
Formula Selected
Ion
Experimental
m/z
Error
(ppm)
Sigma
value
Tolerance
(ppm) in
Generated
Molecular
formula
HPLC
Migration
Time
(min)
H52
P129
O116
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
acid
C7H5O3
[M-H]
137.0247
-2.4
0.0450
5
10.7
+
+
+
Methyl Ester
Coumaric acid
C11H11O4
[M-H]
177.0557
0.0
0.0138
5
12.6
+
+
+
Methyl Ester
Ferulic acid
C10H9O3
[M-H]
207.0657
2.7
0.0870
5
13.2
+
+
+
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
+
+
+
155
Table
2. Unknown phenolic compounds detected by HPLC-ESI-TOF (MS) in Flaxseed oil
Exper
imen
tal
m/z
Sel
ecte
d Ion
Tole
rance
(ppm
) in
Gen
era
ted
Mole
cula
r
Form
ula
Lis
t of
poss
ibilitie
s
in G
ener
ate
Mole
cula
r
Form
ula
Err
or
(ppm
) Sig
ma v
alu
e H
52
P129
O116
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
- -
+
156
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)
2
4
7
8
3
65
910
Fig. 1
H3C
H3C
1
OHOH
OH3C
OH3C
HO
OH
2
4
7 8
3
65
0 5 10 15 20 25 30 Time [min]0.0
0.2
0.4
0.6
0.8
1.0
4x10
Intens
0 5 10 15 20 25 30 Time [min]0
1
2
3
4x10
Intens
1
0 5 10 15 20 25 30 Time [min]0
1
2
3
4
4x10
Intens
0 5 10 15 20 25 30 Time [min]0.0
0.5
1.0
1.5
5x10
Intens
0.0
Intens
0 5 10 15 20 25 30 Time [min]0
1
2
3
4x10
Intens.
0 5 10 15 20 25 30 Time [min]0
1
2
3
.
0 5 10 15 20 25 30 Time [min]0
1
2
3
4x10
Intens
0 5 10 15 20 25 30 Time [min]0.00
0.25
0.50
0.75
1.00
1.25
4x10
Intens
IntensIntens
0 5 10 15 20 25 30 Time [min]0.0
0.5
1.0
1.5
2.0
4x10
9 10
Fig. 2
0 5 10 15 20 25 30 Time [min]0.0
0.5
1.0
1.5
4x10
Intens.
0 5 10 15 20 25 30 Time [min]0
2000
4000
6000
8000
Intens.
159
Conclusions
Conclusion
161
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)
Conclusion
162
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
Conclusion
163
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.
164
Conclusiones
Conclusiones
166
CONCLUSIONES
1. Dado que en la naranja, la piel representa aproximadamente la mitad de
su masa y es conocido que en los cítricos la mayor concentración de
flavonoides se encuentran en la piel, se han estudiado comparativamente
cinco procedimientos de extracción. Finalmente con el procedimiento C,
en el que se realizó una extracción usando MeOH, siendo éste
posteriormente evaporado y reconstituido en una mezcla MeOH:H20
(50:50, v/v) adecuada para su análisis mediante CE, se obtuvieron los
mejores resultados. Posteriormente se llevó a cabo la caracterización y
cuantificación de estos compuestos fenólicos característicos en extractos
tanto de piel de naranja dulce como amarga empleando CE-ESI-MS
trabajando en modalidad negativa.
2. Se han realizado análisis mediante CE-MS/MS para los dos tipos de
muestras anteriores y se ha podido confirmar de una forma más fiable,
comparando con estándares, la presencia de estos compuestos fenólicos
en las muestras objeto de estudio. Para la cuantificación 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. Así, el método puesto a punto nos permitió
diferenciar naringina y narirutina, y hesperidina y neohesperidina
empleando la detección por IT-MS dado que este sistema de detección nos
proporciona datos acerca del peso molecular e información estructural.
3. Como se sabe, el proceso de filtración afecta a las características del
aceite de oliva virgen (VOO), especialmente a la estabilidad oxidativa,
contenido en agua y a la concentración y presencia de compuestos
fenólicos. Los filtros utilizados en la producción de aceite de oliva pueden
ser de varios tipos aunque los mas utilizados son algas microscópicas
fosilizadas, denominadas tierras de diatomeas, las cuales después 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 extracción utilizando una etapa
previa de extracción con hexano para separar la fracción polar de la no
polar. Posteriormente al extracto seco se le añadió MeOH en agitación a
Conclusiones
167
35 ºC para de esta manera poder extraer los compuestos polares objeto
de estudio.
4. La caracterización de estos extractos se realizó utilizando la técnica
separativa HPLC acoplada a la detección por MS, técnica que combina los
avances de HPLC con la selectividad, sensibilidad, masa exacta y medidas
de relación isotópica asociada con el analizador de TOF, permitiendo de
esta manera la caracterización de 19 compuestos conocidos de familias
tan importantes como de los alcoholes fenólicos, secoiridoides, lignanos,
ácidos fenólicos y flavonoides. Por otro lado, con la información
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 fenólicos, y
que se estima que durante la campaña de recogida se producen alrededor
de 25 kg se sub-productos (hojas y ramas) por olivo anualmente en el
capítulo 3 se ha puesto a punto un método simple y rápido para la
extracción de compuestos fenólicos en hojas de olivo, que consistió en
una extracción usando MeOH, siendo éste posteriormente evaporado y
reconstituido en una mezcla MeOH:H20 (50:50, v/v) adecuada para su
análisis mediante CE, de dos variedades de hojas de olivo, la Hojiblanca y
la Manzanilla.
6. El análisis de estos extractos se llevó a cabo empleando un nuevo método
mediante CE-ESI-TOF (MS) caracterizando finalmente un total de 17 y 14
compuestos fenólicos en la variedad Hojiblanca y Manzanilla
respectivamente encontrando compuestos tan interesantes como tirosol,
hidroxitirosol, oleuropeína y su aglicona, ácido cafeico, verbascosido,
apigenina, luteolina, etc.
7. La piel de almendra, la cual presenta un bajo valor económico,
representa el 4 % del peso total de la almendra, sin embargo en ésta
parte contiene una gran cantidad de polifenoles. En este sentido, se
utilizó una extracción líquido-líquido para extraer los compuestos
fenólicos presentes en la piel de almendra. En este procedimiento de
extracción se utilizando una etapa previa de extracción con hexano para
separar la fracción polar de la no polar. Posteriormente se filtró y se trató
Conclusiones
168
el extracto sólido con MeOH al 70 % en condiciones de reflujo a 60 ºC
durante 45 minutos, se volvió a filtrar y la disolución resultante se
evaporó a vacío, reconstituyendo finalmente en una mezcla MeOH:H2O
para su análisis mediante CE y en MeOH para el análisis 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 método a punto mediante CE
para la caracterización de éstos compuestos fenólicos en piel de
almendra, lo cual supone una alternativa interesante a los métodos
desarrollados mediante HPLC. Ambos métodos permitieron una
caracterización directa y sensible de estos compuestos fenólicos aunque
ambos presentan algunas ventajas e inconvenientes. Así, empleando HPLC
se pudieron caracterizar 23 compuestos en un tiempo de análisis 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 análisis
significativamente y se logra detectar un mayor numero de compuestos
además de que HPLC nos ofrece importantes ventajas, como mejoras en
la precisión de la inyección y sensibilidad, mientras que CE puede
competir en términos 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 fenólicos con propiedades beneficiosas para
la salud. Para caracterizarlos se desarrolló previamente un procedimiento
de extracción de los compuestos fenólicos en el aceite de linaza, que
consistió en una extracción en fase sólida empleando cartuchos DSC-Diol,
y se optimizó un nuevo método utilizando HPLC-ESI-TOF (MS).
10. Con la metodología propuesta se detectaron 10 polifenoles en la variedad
H52 y P129, y 9 compuestos en la variedad O116, destacando polifenoles
tan característicos 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 fracción polar del
aceite de linaza. En este estudio, se pudieron encontrar diferencias
significativas entre las tres variedades empleando la metodología
Conclusiones
169
propuesta, de hecho estas diferencias podrían ser utilizadas en un futuro
para encontrar posibles biomarcadores del aceite de linaza o de la
variedad de semilla.
11. Finalmente, como conclusión general, se puede afirmar que en la
presente tesis se han puesto a punto diferentes procedimientos de
extracción y diferentes metodologías, tanto por CE como por HPLC
acopladas a MS, para la caracterización de un buen número de
compuestos fenólicos en una amplia variedad de matrices de interés. Por
tanto, aparte de estudiar exhaustivamente diferentes procedimientos de
extracción para el análisis de los compuestos objeto de estudio en función
del tipo de matriz, el empleo de técnicas separativas avanzadas nos
permitió en la mayoría de los casos obtener unos buenos resultados en
cuanto a resolución, eficiencia y tiempo de análisis. Por otra parte, éstas
técnicas separativas fueron acopladas a un sistema de detección de
enorme potencialidad como es la MS, cuyas características más
destacadas son su sensibilidad, selectividad y el proporcionar información
estructural. En este sentido, a lo largo de este trabajo de investigación se
ha utilizado el analizador de IT, cuya característica más destacada es la
posibilidad proporcionar fragmentos reales de una masa concreta
(MS/MS), y el analizador de TOF, con el que obtenemos resolución, masa
exacta y medidas de relación isotópica, obteniendo de esta manera una
información muy valiosa y precisa para la caracterización los compuestos
fenólicos en las matrices objeto de estudio: piel de naranja, tierras de
diatomeas utilizadas en el proceso de filtración del aceite de oliva, hoja
de olivo, piel de almendra y aceite de linaza.
170
Abstract
Abstract
172
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 ±
Abstract
173
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.
Abstract
174
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.
175
Resumen
Resumen
177
RESUMEN
Aquí se recopilan los principales hitos relativos a los contenidos de la presente
memoria titulada: “Caracterización de compuestos bioactivos en productos y sub-
productos alimentarios empleando metodologías separativas avanzadas”
El trabajo realizado se divide en dos apartados: la "INTRODUCCION" y la "PARTE
EXPERIMENTAL. RESULTADOS Y DISCUSIÓN”.
En el primero de ellos se describe la importancia que tiene hoy día los alimentos
funcionales, debido a que en la actualidad la nutrición está experimentando un veloz
cambio ya que los consumidores buscan aquellos productos en el mercado que,
además del valor nutritivo, aporten beneficios a las funciones fisiológicas 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 Japón en los años 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 población.
Estos alimentos funcionales contienen compuestos bioactivos, que son aquellos que
tiene la capacidad de mermar el efecto dañino que puede ocasionar una enfermedad
y entre ellos podemos encontrar diferentes familias como isoflavonas, compuestos
fenólicos, ácido ascórbico, carotenos, clorofilas, vitamina E y fitoesteroles, entre
otros, de los cuales algunos se encuentran en pequeñas concentraciones. En la
presente memoria doctoral nos centraremos en la caracterización de compuestos
fenólicos; estos son compuestos biosintetizados por los vegetales como producto de
su metabolismo secundario normal y algunos de ellos son indispensables para sus
funciones fisiológicas, mientras que otros son de utilidad para defenderse ante
situaciones de estrés (hídrico, luminoso, etc). Éstos compuestos fenólicos son un
grupo heterogéneo de productos con más de 10.000 compuestos, por lo que son
clasificado, de acuerdo a su esqueleto básico, en diferentes familias: ácidos
fenólicos, lignanos, estilbenos y flavonoides entre los que destacamos los flavonoles,
flavonas, isoflavonas, flavanonas, etc.
Además, en esta introducción se hace una pequeña revisión de las diferentes técnicas
analíticas empleadas en la determinación de compuestos fenólicos así como de los
procedimientos de extracción utilizados.
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Seguidamente se describen las diferentes técnicas analíticas empleadas en este
trabajo de investigación. En primer lugar se comenta la cromatografía líquida (LC),
instrumentación, tipos de LC así como las características mas destacadas de esta
técnica separativa y de la misma manera se hace una descripción del otro tipo de
técnica separativa utilizada en esta memoria como es la electroforesis capilar (CE),
instrumentación, modalidades, características, etc.
Dado que éstas técnicas separativas se acoplarán a la espectrometría de masas (MS)
como sistema de detección, en esta introducción se describe el mecanismo de
funcionamiento de un espectrómetro de masas así como los diferentes tipos de
analizadores que pueden ser utilizados, profundizando en el mecanismo y las
características 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 técnicas separativas que utilizan muestras en
estado líquido, 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
pequeño esbozo de las interfases más usuales para este tipo de acoplamientos,
centrándonos principalmente en el tipo de interfase utilizada en este trabajo, la
ionización por electroespray (ESI).
Finalmente se incluye una pequeña revisión del análisis de compuestos fenólicos
utilizando las dos técnicas separativas que van a ser empleadas, LC y CE.
El segundo apartado está dividido en cinco capítulos relacionados con cada matriz
estudiada: 1) Piel de naranja, 2) Tierras de diatomeas utilizadas en el proceso de
filtración del aceite de oliva, 3) Hoja de olivo, 4) Piel de almendra y 5) Aceite de
linaza.
Capítulo 1: Cuantificación de los compuestos fenólicos 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 fenólicos, que podrían ser valiosas fuentes naturales
de polifenoles. Por lo tanto, en el presente trabajo se desarrollo de un método
mediante CE-ESI-IT (MS), puesto que el acoplamiento de CE con la detección por MS
nos proporciona datos acerca del peso molecular e información estructural de los
analitos objeto de estudio, para el análisis y cuantificación de los principales
compuestos fenólicos en piel de naranja dulce y amarga, dado que la piel representa
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aproximadamente el 30% de la masa de fruta y es donde se encuentra la mayor
concentración 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. Así, el método propuesto permitió
la identificación inequívoca, mediante experimentos de MS/MS de los analitos objeto
de estudio, además de la cuantificación 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.
Capítulo 2: Caracterización de compuestos fenólicos en tierras de diatomeas
empleadas en el proceso de filtración del aceite de oliva mediante HPLC-ESI-TOF
(MS).
Este capítulo explica el estudio realizado para determinar el contenido de polifenoles
en la tierra de diatomeas utilizadas en la etapa de filtración, el cual es el último
paso en el proceso de producción de aceite de oliva. Teniendo en cuenta que España
es el principal productor de aceite de oliva de la Unión Europea, con más del 80 %,
podemos concluir que durante todo este proceso se generan una gran cantidad de
sub-productos derivados de la producción a los cuales se les subestima su valor
saludable. Por tanto en este capítulo se ha propuesto un método mediante HPLC-ESI-
TOF (MS) para la separación y detección de un buen número de compuestos fenólicos
presentes en las tierras de diatomeas. De esta manera se pudieron caracterizar 19
compuestos fenólicos de diferentes familias (alcoholes fenólicos, secoiridoides,
lignanos, ácidos fenólicos y flavonoides) en la fracción polar. A parte, se
caracterizaron otros compuestos desconocidos pertenecientes a la misma fracción.
Por tanto de los resultados obtenidos en este estudio podemos decir que la tierra de
diatomeas utilizada en el proceso de filtración afecta a la composición fenólica del
aceite de oliva, dado que una importante cantidad de compuestos fenólicos están
presentes en el material de filtración, siendo los mas abundantes hydroxitirosol,
ligustrosido aglycona, hydroxipinoresinol, acido vanilico, tirosol and luteolina.
Capítulo 3: Identificación de compuestos fenólicos en hojas de olivo mediante CE-
ESI-TOF (MS).
Se describe un método fácil y rápido empleando CE-ESI-TOF (MS) para el análisis de
compuestos fenólicos en dos variedades de hojas de olivo, Hojiblanca y Manzanilla.
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Para la elección de los parámetros óptimos en la separación se ha tenido en cuenta la
resolución, sensibilidad, tiempo de análisis y forma de pico. Así el método propuesto
permitió la identificación simultánea de 17 y 14 compuestos fenólicos en extractos
de hojas de olivo de la variedad Hojiblanca y Manzanilla respectivamente,
encontrado compuestos tan interesantes como el tirosol, hidroxitirosol, oleuropeína y
su aglicona, ácido cafeico, verbascosido, apigenina y luteolina entre otros. Por tanto
el método propuesto ha puesto de manifiesto el enorme potencial de la técnica
empleada para el análisis de una amplia serie de compuestos fenólicos presentes en
hojas de olivo debido a la alta eficacia, rapidez, pequeño volumen de muestra
requerido y la alta resolución de CE acoplada con la sensibilidad, selectividad, masa
exacta y relación isotópica proporcionada por el TOF (MS).
Capítulo 4: Caracterización de compuestos fenólicos en extractos de piel de almendra
mediante HPLC/CE-ESI-TOF (MS).
En el capítulo 4 se describe el desarrollo de dos metodologías empleando CE y HPLC
acopladas a ESI-TOF (MS) las cuales han sido comparadas en la capacidad de
separación y caracterización de compuestos fenólicos en extractos de piel de
almendra. Así, bajo las condiciones óptimas de CE-ESI-TOF (MS) se pudo lograr la
determinación de 9 compuestos de la fracción 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 ácidos fenólicos y flavonoides, en
solo 9 minutos. Por tanto se puede afirmar que empleando HPLC se reduce el tiempo
de análisis significativamente y se logra detectar un mayor número de compuestos
además de que el uso HPLC nos ofrece importantes ventajas, como mejoras en la
precisión de la inyección y sensibilidad, mientras que CE puede competir en términos
de costes de funcionamiento reducidos.
Capítulo 5: Caracterización de fenoles y otros compuestos polares en aceite de
linaza empleando HPLC-ESI-TOF (MS).
En este último capítulo se pone a punto una metodología mediante HPLC-ESI-TOF (MS)
para el análisis de compuestos fenólicos en aceite de linaza. Un buen numero de
compuestos fenólicos de gran interés, como son secoisolariciresnol, acido ferulico y
su éster metílico, éster metilico del acido cumarico, dilfilin, pinoresinol, matairesinol,
acido p-hidroxibenzoico, vanilina y acido vanilinico, han sido detectados en aceite de
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linaza. Al mismo tiempo, se han estudiado otros compuestos desconocidos presentes
los cuales son una parte importante de la fracción polar del aceite de linaza. Por
tanto, el método de HPLC-ESI-TOF (MS) desarrollado permitió la caracterización de
un buen número de compuestos fenólicos presentes en aceite de linaza.