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

3

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.

5

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

7

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.

9

Acknowledgments

11

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

13

Table of contents

Table of contents

15

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

16

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

17

Objectives

Objective

19

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.

 

21

Introduction

Functional food

23

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

25

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

26

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

27

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

29

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

31

• 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

33

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: ansegura@ugr.es (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), albertof@ugr.es

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.

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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: albertof@ugr.es 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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32.

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: ansegura@ugr.es (A. Segura-Carretero), albertof@ugr.es (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.