Citrus oils composition, advanced analytical techniques, contaminants, and biological activity

Citrus Oils
Composition, Advanced Analytical Techniques,
Contaminants, and Biological Activity

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Medicinal and Aromatic Plants — Industrial Profiles
Individual volumes in this series provide both industry and academia with in-depth
coverage of one major genus of industrial importance.

Series Edited by Dr. Roland Hardman
Volume 1
Valerian, edited by Peter J. Houghton
Volume 2
Perilla, edited by He-ci Yu,
  Kenichi Kosuna and Megumi Haga
Volume 3
Poppy, edited by Jenö Bernáth
Volume 4
Cannabis, edited by David T. Brown
Volume 5
Neem, edited by H.S. Puri
Volume 6
Ergot, edited by Vladimír Kˇren and
  Ladislav Cvak
Volume 7
Caraway, edited by Éva Németh

Volume 8
Saffron, edited by Moshe Negbi
Volume 9
Tea Tree, edited by Ian Southwell and
  Robert Lowe
Volume 10
Basil, edited by Raimo Hiltunen and
  Yvonne Holm
Volume 11
Fenugreek, edited by
  Georgios Petropoulos
Volume 12
Ginkgo biloba, edited by
  Teris A. Van Beek
Volume 13
Black Pepper, edited by P.N. Ravindran
Volume 14
Sage, edited by Spiridon E. Kintzios
Volume 15
Ginseng, edited by W.E. Court

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Volume 16
Mistletoe, edited by Arndt Büssing
Volume 17
Tea, edited by Yong-su Zhen
Volume 18
Artemisia, edited by Colin W. Wright
Volume 19

Stevia, edited by A. Douglas Kinghorn
Volume 20
Vetiveria, edited by Massimo Maffei
Volume 21
Narcissus and Daffodil, edited by
  Gordon R. Hanks
Volume 22
Eucalyptus, edited by John J.W. Coppen
Volume 23
Pueraria, edited by Wing Ming Keung
Volume 24
Thyme, edited by E. Stahl-Biskup and
  F. Sáez
Volume 25
Oregano, edited by Spiridon E. Kintzios
Volume 26
Citrus, edited by Giovanni Dugo and
  Angelo Di Giacomo
Volume 27
Geranium and Pelargonium, edited by
  Maria Lis-Balchin
Volume 28
Magnolia, edited by Satyajit D. Sarker
and Yuji Maruyama
Volume 29
Lavender, edited by Maria Lis-Balchin
Volume 30
Cardamom, edited by P.N. Ravindran
and K.J. Madhusoodanan

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Volume 31
Hypericum, edited by Edzard Ernst
Volume 32
Taxus, edited by H. Itokawa and
  K.H. Lee
Volume 33
Capsicum, edited by Amit Krish De
Volume 34
Flax, edited by Alister Muir and
  Niel Westcott
Volume 35
Urtica, edited by Gulsel Kavalali
Volume 36
Cinnamon and Cassia, edited by
  P.N. Ravindran, K. Nirmal Babu
  and M. Shylaja
Volume 37
Kava, edited by Yadhu N. Singh
Volume 38
Aloes, edited by Tom Reynolds
Volume 39
Echinacea, edited by
  Sandra Carol Miller
  Assistant Editor: He-ci Yu
Volume 40
Illicium, Pimpinella and Foeniculum,   
  edited by Manuel Miró Jodral

Volume 41
Ginger, edited by P.N. Ravindran
  and K. Nirmal Babu
Volume 42
Chamomile: Industrial Profiles,
  edited by Rolf Franke and
  Heinz Schilcher
Volume 43
Pomegranates: Ancient Roots to
  Modern Medicine, edited by
  Navindra P. Seeram,
  Risa N. Schulman and David Heber

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Volume 44
Mint, edited by Brian M. Lawrence
Volume 45
Turmeric, edited by P. N. Ravindran,
  K. Nirmal Babu, and K. Sivaraman
Volume 46
Essential Oil-Bearing Grasses,
edited by Anand Akhila
Volume 47
Vanilla, edited by Eric Odoux
  and Michel Grisoni
Volume 48
Sesame, edited by Dorothea Bedigian
Volume 49
Citrus Oils, edited by Giovanni Dugo

and Luigi Mondello

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Citrus Oils
Composition, Advanced Analytical Techniques,
Contaminants, and Biological Activity

Edited by

Giovanni Dugo and Luigi Mondello

Medicinal and Aromatic Plants — Industrial Profiles

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CRC Press
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Library of Congress Cataloging-in-Publication Data
Citrus oils : composition, advanced analytical techniques, contaminants, and biological activity /
edited by Giovanni Dugo and Luigi Mondello.
p. cm. -- (Medicinal and aromatic plants--industrial profiles ; 49)
Includes bibliographical references and index.
ISBN 978-1-4398-0028-7
1. Citrus oils--Composition. 2. Citrus oils--Analysis. 3. Citrus oils--Therapeutic use. I. Dugo,
Giovanni. II. Mondello, Luigi. III. Title: Composition, analysis, contamination and properties of citrus
oils. IV. Series: Medicinal and aromatic plants--industrial profiles ; v. 49.
TP959.C54C575 2011
665’.3--dc22

2010012530

Visit the Taylor & Francis Web site at

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To the wonderful women of my family
To my grandmother, Francesca, who cheered and protected me. To my mother,
Paola, who was always excessively proud of my limited success. To my wife,
Anna, who loved and assisted me, always indulgent and understanding. To my
daughters, Paola, Monica, and Laura, who showed to me how lucky a father
can be. To the sweetest daughters of my daughters, Alice, Viola, and Laura,
who, more than anything else, give a sense to my life and sweeten my old age.
Giovanni Dugo

To my wife, Paola, and to my children, Alice and Viola,
for their understanding and patience while I spent seemingly endless
evenings and weekends working in my research laboratory.
To my parents
for their love and for believing in me and encouraging me in my career.
Luigi Mondello

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Contents
Series Preface ....................................................................................................................................xi
Preface............................................................................................................................................ xiii
Editors .............................................................................................................................................. xv
Contributors ...................................................................................................................................xvii
Chapter 1

Composition of the Volatile Fraction of Citrus Peel Oils.............................................1
Giovanni Dugo, Antonella Cotroneo, Ivana Bonaccorsi, Alessandra Trozzi

Chapter 2

Volatile Components in Less Common Citrus Species............................................ 163
Estelle Delort, Regula Naef

Chapter 3

Composition of Distilled Oils .................................................................................. 193
Luis Haro-Guzmán

Chapter 4

Concentrated Citrus Oils .......................................................................................... 219
Herta Ziegler

Chapter 5

Composition of Petitgrain Oils ................................................................................. 253
Giovanni Dugo, Antonella Cotroneo, Ivana Bonaccorsi

Chapter 6

Extracts from the Bitter Orange Flowers (Citrus aurantium L.): Composition
and Adulteration ....................................................................................................... 333
Giovanni Dugo, Louis Peyron, Ivana Bonaccorsi

Chapter 7

The Chiral Compound of Citrus Oils....................................................................... 349
Luigi Mondello, Rosaria Costa, Danilo Sciarrone, Giovanni Dugo

Chapter 8

The Oxygen Heterocyclic Components of Citrus Essential Oils .............................405
Paola Dugo, Marina Russo

Chapter 9

Carotenoids of Citrus Oils........................................................................................ 445
Paola Dugo, Daniele Giuffrida

Chapter 10 Minor Components in Extracts of Citrus Fruits....................................................... 463

Regula Naef

ix

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x

Contents

Chapter 11 Advanced Analytical Techniques for the Analysis of Citrus Oils ........................... 477
Peter Quinto Tranchida, Paola Dugo, Luigi Mondello, Giovanni Dugo
Chapter 12 Contaminants in Citrus Essential Oils: State of the Art (2000–2009) .................... 513
Giacomo Dugo, Giuseppa Di Bella, Marcello Saitta
Chapter 13 Biological Activities of Citrus Essential Oils ........................................................... 529
Giuseppe Bisignano, Francesco Cimino, Antonella Saija
Index .............................................................................................................................................. 549

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Series Preface
There is increasing interest in industry, academia, and the health sciences in medicinal and aromatic
plants. In passing from plant production to the eventual product used by the public, many sciences
are involved. This series brings together information that is currently scattered through an ever

increasing number of journals. Each volume gives an in-depth look at one plant genus about which
an area specialist has assembled information ranging from the production of the plant to market
trends and quality control.
Many industries are involved, such as forestry, agriculture, chemical, food, flavor, beverage,
pharmaceutical, cosmetic, and fragrance. The plant raw materials are roots, rhizomes, bulbs, leaves,
stems, barks, wood, flowers, fruits, and seeds. These yield gums, resins, essential (volatile) oils,
fixed oils, waxes, juices, extracts, and spices for medicinal and aromatic purposes. All these commodities are traded worldwide. A dealer’s market report for an item may say “drought in the country
of origin has forced up prices.”
Natural products do not mean safe products, and account of this has to be taken by the above
industries, which are subject to regulation. For example, a number of plants that are approved for
use in medicine must not be used in cosmetic products.
The assessment of “safe to use” starts with the harvested plant material, which has to comply
with an official monograph. This may require absence of, or prescribed limits of, radioactive material, heavy metals, aflatoxin, pesticide residue, as well as the required level of active principle. This
analytical control is costly and tends to exclude small batches of plant material. Large-scale, contracted, mechanized cultivation with designated seed or plantlets is now preferable.
Today, plant selection is not only for the yield of active principle, but also for the plant’s ability
to overcome disease, climatic stress, and the hazards caused by mankind. Such methods as in vitro
fertilization, meristem cultures, and somatic embryogenesis are used. The transfer of sections of
DNA is giving rise to controversy in the case of some end uses of the plant material.
Some suppliers of plant raw material are now able to certify that they are supplying organically
farmed medicinal plants, herbs, and spices. The Economic Union directive CVO/EU No. 2092/91
details the specifications for the obligatory quality controls to be carried out at all stages of production and processing of organic products.
Fascinating plant folklore and ethnopharmacology lead to medicinal potential. Examples are
the muscle relaxants based on the arrow poison curare from species of Chondrodendron, and the
antimalarials derived from species of Cinchona and Artemisia. The methods of detection of pharmacological activity have become increasingly reliable and specific, frequently involving enzymes
in bioassays and avoiding the use of laboratory animals. By using bioassay-linked fractionation of
crude plant juices or extracts, compounds can be specifically targeted, which, for example, inhibit
blood platelet aggregation, or have antitumor, antiviral, or any other required activity. With the
assistance of robotic devices, all the members of a genus may be readily screened. However, the
plant material must be fully authenticated by a specialist.
The medicinal traditions of ancient civilizations such as those of China and India have a large

armamentarium of plants in their pharmacopoeias that are used throughout southeast Asia. A similar
situation exists in Africa and South America. Thus, a very high percentage of the world’s population
relies on medicinal and aromatic plants for their medicine. Western medicine is also responding. Already in Germany all medical practitioners have to pass an examination in phytotherapy
before being allowed to practice. It is noticeable that medical, pharmacy, and health-related schools
throughout Europe and the United States are increasingly offering training in phytotherapy.
xi

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xii

Series Preface

Multinational pharmaceutical companies have become less enamored of the single compound,
magic-bullet cure. The high costs of such ventures and the endless competition from “me-too” compounds from rival companies often discourage the attempt. Independent phytomedicine companies
have been very strong in Germany. However, by the end of 1995, 11 (almost all) had been acquired
by the multinational pharmaceutical firms, acknowledging the lay public’s growing demand for
phytomedicines in the Western world.
The business of dietary supplements in the Western world has expanded from the health store to
the pharmacy. Alternative medicine includes plant-based products. Appropriate measures to ensure
their quality, safety, and efficacy either already exist or are being answered by greater legislative
control by such bodies as the US Food and Drug Administration and the recently created European
Agency for the Evaluation of Medicinal Products based in London.
In the United States, the Dietary Supplement and Health Education Act of 1994 recognized the
class of phytotherapeutic agents derived from medicinal and aromatic plants. Furthermore, under
public pressure, the US Congress set up an Office of Alternative Medicine, which in 1994 assisted
the filing of several Investigational New Drug (IND) applications required for clinical trials of some

Chinese herbal preparations. The significance of these applications was that each Chinese preparation involved several plants and yet was handled as a single IND. A demonstration of the contribution to efficacy of each ingredient of each plant was not required. This was a major step toward more
sensible regulations in regard to phytomedicines.
The citrus oils have a large industrial profile in beverages, household products, perfumes, medicines, and so forth. According to David A. Moyler, technical director of Fuerst Day Lawson (FDL)
Ltd, London, UK, from November 2010 citrus oils, such as orange, lemon, and lime will come
under a massive piece of European Union legislation called REACH covering their Registration,
Evaluation, Authentication, and CHemical data. This legislation will apply to all companies handling more than 1000 tons of citrus oils per year. In 2013, this will apply to companies handling more
than 100 tons of these oils and in 2018 the figure will drop to those companies handling 1 tonne. So
this volume, Volume 49 and its accompanying earlier one, Citrus: The Genus Citrus, Volume 26,
edited by Giovanni Dugo and Angelo Di Giacomo, are most relevant.
For Volume 49, I thank its editors Giovanni Dugo and Luigi Mondello for their dedicated work
and the chapter contributors for their authoritative information. My thanks are also due to Barbara
Norwitz, executive editor, life sciences for CRC Press and her staff for their unfailing help.
Roland Hardman, Bpharm, BSc (Chem), PhD (University of London), FRPharmS

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Preface
This book follows a previous volume that deals with the historical, botanical, agronomical, technological, chemical–analytical, and biological–pharmacological aspects of citrus or their derivatives,
mainly essential oils.
Not all of the subjects treated in the first volume are included here. In fact, we present those
topics that have evolved in the last decade, such as composition of essential oils, including possible
contaminants of different origin (agriculture, environment, and industry), and development of the
analytical techniques applied in these fields. The last chapter is dedicated to the new information
available on the pharmacological properties of citrus essential oils and their components.
Many of the authors who participated in the first book contributed again to this volume. Other
scientists of great fame and recognized competence in this field and some young researchers whose
scientific interest also focuses on the chemistry of citrus have also contributed to this volume.

The chapters relative to the different compositional aspects of the oils first briefly report for each
oil the information given in the former book summarized in tables, and then the more recent information is discussed in detail. Concentrated oils and the composition of oils obtained from minor
citrus species are discussed here in a new format compared to the previous book; two new aspects,
the minor components of citrus essential oils and the carotenoid fraction, are also discussed in this
book. The chapter on the adulteration of citrus essential oil, present in the former volume, is not
included in this new book for the following reasons: the general and specific information on this
topic were brilliantly treated in the former volume, and are still valid; to avoid the reiteration of
information given in chapters focused on the composition of the volatile fraction, on the oxygen heterocyclic compounds, and on the chiral components of volatiles in citrus essential oils; the information given in these chapters can be sufficient for researchers and operators in this field to recognize
contaminations and/or adulterations.
We hope that the results of our work together with that of the contributors to this book will be
appreciated and will be useful to most of those who work in the fascinating field of citrus.

xiii

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Editors
Giovanni Dugo is currently full professor of food chemistry at the University of Messina, Italy. His
research activity is directed toward the development of innovative methods and toward the study
of food matrices by using innovative methodologies such as multidimensional liquid chromatography (comprehensive LC), multidimensional gas chromatography (MDGC and comprehensive GC),
ultrafast-GC and ultrafast-GC/MS, on-line SPME-GC/MS, micro-HPLC and micro-HPLC/API/
MS, multidimensional HPLC and micro-HPLC, superheated HPLC, LC × GC; method validation

by using pure standard compounds and complex food samples; exploitation of the developed methods for the study of food matrices such as essential oils, fruit juices of citrus and noncitrus origin,
food lipids, wines, coffee, cheese, vegetable products; study of the following classes of compounds
contained in the previously reported food products: triglycerides, fatty acids, sterols, tocopherols,
monoterpenes, sesquiterpenes, coumarines, psoralens, polymethoxyflavones, carotenoids, anthocyanins, and other flavonoid-structured compounds, pesticides, paraffins, aromatic hydrocarbons,
pyrazines, and so on; and the correlation of the results attained with the genuineness, quality, and
the nutritional characteristics of the studied food samples.
Prof. Dugo’s scientific activity, focused mainly on the development of separation methods and
on the analysis of complex matrices, is reported in about 300 national and international papers;
approximately 300 congress presentations; several scientific books and encyclopedia chapters; one
book (as editor and author of some chapters) on the chemistry and technology of citrus products,
and one on food toxicology.
Prof. Dugo has participated, chaired, and coordinated numerous committee and congress organization activities and research projects on food chemistry, advanced analytical techniques, and aromatic plants and citrus chemistry and technology. In 2005 he founded The Mediterranean Separation
Science Foundation Research and Training Center in Messina co-chaired with Prof. Mondello. He
was the director of the PhD school on Food Chemistry and Safety, University of Messina, from
2002 to 2004. Prof. Dugo is the coordinator of the Food Chemistry Group of the Italian Chemistry
Society (SCI) and is a member of the board of Journal Essential Oil Research.
Prof. Dugo also held the following academic positions: vice-rector of the University of Messina
and president of the evaluation board “Nucleo di Valutazione” of the University of Messina from
1995 to 1998; and vice-rector and delegate for the Scientific Research Activity of the same university from 2003 to 2007.
Prof. Dugo received the medals awarded by the Food Science Italian Society and the Flavor
Science Italian Society; recently (2009) he was awarded the Liberti Medal by the Italian Society of
Chemistry (SCI) for his contribution to the diffusion of science.
Luigi Mondello is full professor of analytical chemistry at the University of Messina, Italy and
teaches the same course at the “Campus Biomedico” in Rome. He is the author of 200 scientific
papers, 29 book chapters and 2 reviews; he is the co-editor of Multidimensional Chromatography
(John Wiley & Sons); and he has been chairman and invited lecturer in national and international
congresses and meetings. His research interests include chromatography techniques (HRGC, HPLC,
HRGC/MS, HPLC/MS, OPLC) and the development of coupled techniques, such as LC–GC–MS,
GC–GC, GC × GC, LC × LC, LC × GC, and their applications in the study of natural complex
matrices in the fields of food, environmental, and biochemical science. Prof. Mondello has been a

member of the organizing committees of national and international meetings. He is a permanent
member of the scientific committee of the International Symposium on Capillary Chromatography
xv

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xvi

Editors

(ISCC), of the International Symposium on Essential Oils (ISEO), of the International Symposium
on Hyphenated Techniques in Chromatography and Hyphenated Chromatographic Analyzers
(HTC), of the Brazilian Symposium on Chromatography and Related Techniques (SIMCRO),
of the Congresso Latino-Americano de Cromatografia e Técnicas Relacionades (COLACRO), a
member of the Scientific Committee of the Workshop-Symposium on Analytical and Preparative
Enantioseparation (Enantiosep ‘07). He has also been a member of the Scientific Committee of the
1st International Symposium on Separation Sciences, Pardubice, Czech Republic; co-chairman of
the 34th International Symposium on Capillary Chromatography, Riva del Garda, Italy; member of
the Scientific Committee of the 16th International Symposium on Separation Sciences, Rome, Italy;
member of the Scientific Committee of the ExTEch on Advance in Extraction Technologies, Poznan,
Poland; and the chairman of the 20th International Symposium on Solid Phase Microextraction;
member of the steering committee of the Italian Separation Science Group of the Italian Chemical
Society; member of the expert team of “Chromedia” (Chromatography Knowledge Base); editor of the Journal of Separation Science and Flavour and Fragrance Journal, both published by
John Wiley & Sons; member of the Central Technical Committee of the National System for the
Accreditation of the Laboratory (SINAL); member of the Advisory Board of LC-GC, Europe,
Separation Science e Scientia Chromatographica; and reviewer for 42 different journals in the
field of analytical chemistry and food chemistry. In February 2006 (York, U.K.), Prof. Mondello

was awarded with the HTC-Award for the most outstanding and innovative work in the field of
hyphenated chromatographic techniques by the Flemish Chemical Society. In May 2008 (Riva del
Garda, Italy), he was awarded with the Silver Jubilee Medal for his considerable contribution to
the development of separation sciences by the Chromatographic Society. In October 2008, during
the Congresso Latino-Americano de Cromatografia e Técnicas Relacionades held in Florianòpolis,
Brasil, the Instituto Internacional de Cromatografia awarded Prof. Mondello the COLACRO Medal
for his contribution to the development and diffusion of chromatographic techniques.

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Contributors
Giuseppe Bisignano
Dipartimento Farmaco-biologico
University of Messina
Messina, Italy

Giovanni Dugo
Dipartimento Farmaco-chimico
University of Messina
Messina, Italy

Ivana Bonaccorsi
Dipartimento Farmaco-chimico
University of Messina
Messina, Italy

Paola Dugo

Dipartimento Farmaco-chimico
University of Messina
Messina, Italy

Francesco Cimino
Dipartimento Farmaco-biologico
University of Messina
Messina, Italy

Daniele Giuffrida
Dipartimento di Scienze degli Alimenti e dell’
Ambiente
University of Messina
Messina, Italy

Rosaria Costa
Dipartimento Farmaco-chimico
University of Messina
Messina, Italy
Antonella Cotroneo
Dipartimento Farmaco-chimico
University of Messina
Messina, Italy
Estelle Delort
Corporate R&D Division
Firmenich S.A.
Geneva, Switzerland
Giuseppa Di Bella
Dipartimento di Scienze degli Alimenti e dell’
Ambiente

University of Messina
Messina, Italy
Giacomo Dugo
Dipartimento di Scienze degli Alimenti e dell’
Ambiente
University of Messina
Messina, Italy

Luis Haro-Guzmán
Colima, Mexico
Luigi Mondello
Dipartimento Farmaco-chimico
University of Messina
Messina, Italy
Regula Naef
Seewen, Switzerland
Louis Peyron
Grasse, France
Marina Russo
Dipartimento Farmaco-chimico
University of Messina
Messina, Italy
Antonella Saija
Dipartimento Farmaco-biologico
University of Messina
Messina, Italy

xvii

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xviii

Marcello Saitta
Dipartimento di Scienze degli Alimenti e dell’
Ambiente
University of Messina
Messina, Italy
Danilo Sciarrone
Dipartimento Farmaco-chimico
University of Messina
Messina, Italy

Contributors

Alessandra Trozzi
Dipartimento Farmaco-biologico
University of Messina
Messina, Italy
Herta Ziegler
Erich Ziegler GmbH
Aufsess, Germany

Peter Quinto Tranchida
Dipartimento Farmaco-chimico
University of Messina
Messina, Italy

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1

Composition of the Volatile
Fraction of Citrus Peel Oils
Giovanni Dugo, Antonella Cotroneo,
Ivana Bonaccorsi, Alessandra Trozzi

CONTENTS
1.1
1.2

1.3

1.4

1.5

1.6

Introduction ..............................................................................................................................2
Bitter Orange Oil (Citrus aurantium L.) ................................................................................ 10
1.2.1 1979–1999 ................................................................................................................... 10
1.2.2 1998–2009 .................................................................................................................. 10
1.2.2.1 Industrial Oils .............................................................................................. 10

1.2.2.2 Laboratory Oils ............................................................................................ 10
Grapefruit Oil (Citrus paradisi Macf.) ...................................................................................20
1.3.1 1979–1999 ...................................................................................................................20
1.3.2 1998–2009 ..................................................................................................................20
1.3.2.1 Industrial Oils ..............................................................................................20
1.3.2.2 Laboratory Oils ............................................................................................ 29
Key Lime Oil (Citrus aurantifolia [Christm.] Swing.) and Persian Lime Oil (Citrus
latifolia Tan.) .......................................................................................................................... 29
1.4.1 1960–1999................................................................................................................... 29
1.4.2 1998–2009 .................................................................................................................. 29
1.4.2.1 Key Lime Industrial Oils ............................................................................. 29
1.4.2.2 Key Lime Laboratory Oils ........................................................................... 43
1.4.2.3 Persian Lime Industrial Oils ........................................................................44
1.4.2.4 Persian Lime Laboratory Oils......................................................................44
1.4.2.5 Other Lime Oils ........................................................................................... 48
Mandarin (Citrus deliciosa Ten.), Tangerine (Citrus tangerina Hort. ex Tan.), and
Clementine (Citrus clementina Hort. ex Tan.) Oils ................................................................ 48
1.5.1 1998–2009 .................................................................................................................. 52
1.5.1.1 Mandarin Industrial Oils ............................................................................. 52
1.5.1.2 Mandarin Laboratory Oils ........................................................................... 58
1.5.1.3 Tangerine Industrial Oils ............................................................................. 65
1.5.1.4 Tangerine Laboratory Oils ........................................................................... 68
1.5.1.5 Industrial and Laboratory-Extracted Clementine Oils (1974–2009) ........... 69
Sweet Orange Oil (Citrus sinensis [L.] Osbeck) .................................................................... 75
1.6.1 1979–1999 ................................................................................................................... 75
1.6.2 1998–2009 ..................................................................................................................80
1.6.2.1 Industrial Oils ..............................................................................................80
1.6.2.2 Laboratory-Extracted Oils ........................................................................... 83

1

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2

Citrus Oils

Bergamot Oil (Citrus bergamia).............................................................................................90
1.7.1 1979–1999 ...................................................................................................................90
1.7.2 1998–2009 .................................................................................................................. 95
1.7.2.1 Industrial Oils .............................................................................................. 95
1.7.2.2 Laboratory Oils .......................................................................................... 109
1.8 Lemon Oil (Citrus limon [L.] Burm) .................................................................................... 115
1.8.1 1979–1999 ................................................................................................................. 115
1.8.2 1998–2009 ................................................................................................................ 115
1.8.2.1 Industrial Oils ............................................................................................ 115
1.8.2.2 Laboratory Oils .......................................................................................... 122
1.9 Final Remarks ....................................................................................................................... 142
References ...................................................................................................................................... 149
1.7

1.1

INTRODUCTION

Citrus essential oils are industrially cold extracted from the peels of sweet orange, lemon, mandarin, tangerine, grapefruit, Key lime, Persian lime, bitter orange, bergamot, and clementine using
mechanical systems. Lime essential oils, however, are most commonly obtained by distillation

(Guenther, 1949).
The cold-extraction process consists of three fundamental steps, regardless of the technology
used:
• Mechanical action on the peel breaks the utricles and releases the oil.
• The oil is carried by streams of water, which in most cases is recycled.
• Separation, by centrifugation, of the essential oil from the aqueous emulsion.
The industrial transformation process of citrus fruit has been described in detail by Di Giacomo
(2002a,b), Di Giacomo and Di Giacomo (2002), and Crupi and Rispoli (2002).
The volatile fraction of citrus essential oils ranges between 85% in Key lime oils and 99%
in some sweet orange oils (Di Giacomo and Mincione, 1994). This fraction mostly consists of
mono- and sesquiterpene hydrocarbons and their oxygenated derivatives, that is, alcohols, aldehydes, esters, ethers, and oxides, and also of linear hydrocarbons, alcohols, aldehydes, esters, and
acids, and of phenolic compounds and their derivatives. In some cases, for example, mandarin,
nitrogen esters such as methyl N-methyl anthranilate are present. These compounds contribute
to the characteristic olfactory note of the oil (Wilson and Shaw, 1981). In citrus essential oil,
volatile fractions also present trace amounts of heterocyclic nitrogen–containing components
(e.g., pyrimidines and pyrazines) (Thomas and Bassols, 1992; Naef and Velluz, 2001) and sulfurcontaining components (Demole et al., 1982). These play a very important role, contributing to
the odor character of the oil. An important role for the olfactory notes is also played by unsaturated aliphatic hydrocarbons, aldehydes, and alcohols, which are mostly present at trace levels
(Naef and Velluz, 2001).
Studies on the volatile fraction of citrus essential oils began as far back as the early nineteenth
century, but it was only in the 1900s that any reliable results were obtained. The reference list is
reported by Guenther (1949). Research in those days, using pioneer techniques, allowed to identify
components that were mostly confirmed later on.
It was the advent of gas chromatography (GC), first on packed columns and then on capillary columns made first of stainless steel then glass, and finally of fused silica, that allowed the study of the
composition of complex mixtures of volatile components, which is what the essential oils are. The

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Composition of the Volatile Fraction of Citrus Peel Oils

3

Limonene

mV

d-Canfene

b-Fellandrene
g-Terpinene

b-Pinene

0

30

60

90

Temperature 100Њ C

FIGURE 1.1 Gas chromatogram of a lemon essential oil. Column: tricresyl phosphate on celite; column
temperature 100°C. (From Liberti, A., and Conte, G., 1956. Possibilita di applicazione della cromatografi a
in fase gassosa allo studio delle essenze. Atti I° Congresso Internazionale di Studi e Ricerche sulle Essenze,
Reggio Calabria, Italy, Marzo 1956.)

fascinating relationship between GC and citrus essential oils (a symbol of the southern Italian agroindustrial economy) was first studied in Messina, Sicily, in the mid-1950s, owing to the intuitive
work of Professor Arnaldo Liberti. He was the first to apply the newly developed technique of GC,
described only a few years before by James and Martin (1952), to research in this field. Professor
Liberti presented the first chromatograms relative to bergamot and lemon oils in 1956 during the
First International Congress on Essential Oils held in Reggio Calabria (Liberti and Conte, 1956).
In these chromatograms four and five peaks were reported. Figure 1.1 shows the chromatogram of
lemon oil obtained by Liberti and Conte (1956). Since then, the use of single-column GC in combination with flame ionization and mass selective detection has enabled the qualitative/quantitative
determination of many citrus essential oil volatiles.
Using GC, Bernhard (1957) analyzed five monoterpene hydrocarbons standards occurring in
cold-pressed lemon oil. Later, Bernhard (1958) separated five peaks in Californian lemon oil. Only
two years later, the 5 peaks separated by Bernhard became 22, some of which were representative
of more than one component. However, the 22 different compounds were fully or tentatively identified (Bernhard, 1960).
The preliminary gas chromatographic results obtained gave the information on the complexity
of the essential oils and at the same time indicated the need for some kind of fractionation prior to
the gas chromatographic analysis. In those days, fractionation was particularly necessary, given the
limited efficiency of the columns used.
By separating on silica gel columns the hydrocarbons from the oxygenated compounds, Clark
and Bernhard (1960) found numerous components in lemon oil, and Bernhard (1961) underlined the
presence of about 50 components in sweet orange oil, most of which were also identified.
At the same time, in Great Britain, Slater (1961) reported the presence of 7 monoterpene
hydrocarbons, 9 sesquiterpene hydrocarbons, and 24 oxygenated components in lemon and
lime oils. A few years later, Kovats (1963) and Kugler and Kovats (1963) reported more than
100 components in lime and mandarin oils. Of these, 48 and 44 components were respectively
identified.

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4

Citrus Oils

In the same year Calvarano (1959) and Di Giacomo et al. (1962), in Italy, started research on
the composition of citrus essential oils. Di Giacomo and Rispoli (1962) proposed using GC analysis to differentiate lemon oils extracted by the sponge method from those extracted by mechanical
systems.
Since the 1960s, the capillary columns have been introduced for the analysis of essential oils
(McFadden et al., 1963; Teranishi et al., 1963; MacLeod et al., 1966; Goretti et al., 1967; Di Giacomo
et al., 1971). These columns have gradually replaced packed columns. Figures 1.2 and 1.3 show the
chromatograms of lemon oil obtained on stainless steel capillary column by MacLeod et al. (1966)
and on fused silica capillary column obtained by Dugo et al. (1999).
The availability of the bench-top mass spectrometer, coupled online with high-resolution gas
chromatographs, allowed the identification of numerous minor components in citrus essential

×2.5
×5 ×5
×2.5

×2.5
23

5

2

12

7

6

1

×100
×25
13
16

28

21

17

39

27
31

11
32

3

2
Minutes 0

20

15

10

4

25

30

20
22
18

9
14
35

40

45

50

55

30
29

26

24 25

19
60

65

70

33

75

80

85

90

95

100

34
35
105

Temperature 60°C 80°C 85°C 90°C 95°C 100°C 105°C 110°C 115°C 120°C 125°C 130°C 135°C 140°C 145°C 150°C

38

40
36 37
110

115

150°C

FIGURE 1.2 Gas chromatogram of lemon oil using a stainless steel capillary column. (From MacLeod,
W.D., et al., J. Food. Sci. 31, 591–594, 1966.)

2 3 5 6 8

46

35 38

14 17 20

59

45
50

49
30

16
12
9/

10

22
23

15

27
58

4

57
18

1

4

21

11

8

29 31 34
44
41
33 37
32 36 40 43

52 56
51 55

26
25 28

12

16

20

24

28

32

36

60

48

64
61 62 63

44

65

48

52

FIGURE 1.3 Gas chromatogram of a cold-pressed lemon essential oil. Column: fused silica capillary column (30 m × 0.25 mm × 0.25 µm), coated with SE-52; column temperature 45°C (6 min) to 200°C at 3°C/min.
(From Dugo, G., et al., Essenz. Deriv. Agrum. 69, 79111, 1999.)

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Composition of the Volatile Fraction of Citrus Peel Oils

5

oils (Mazza, 1986, 1987a,b). It must be noted, however, that when identifying the components of
a complex matrix, such as essential oils, it is not always possible to compare the commercially
available mass spectra library with the experimental GC-MS data. The spectra interpretation
could be confusing due to peak overlapping and due to the structural similarity of many of the
components, particularly for those present at trace levels. To obtain more reliable information,
mass data along with chromatographic retention data, such as linear retention indices (LRI),
determined on two columns, one polar and one non-polar (Mondello et al., 1995a), were used.
One application reported by these authors on bergamot essential oil proved that the identification
of neryl acetate and α-bisabolol using GC/MS equipped with commercial library could yield
unreliable results, while the use of LRI as interactive filter would simplify the identification of
these components. A more recent application relative to the analysis of lemon oil (Mondello et al.,
2004a) showed that univocal identification of the volatile fraction components can be achieved by

the interactive use of LRI and the mass spectral data given by a conventional MS detector, even if
ultrafast gas chromatography is applied. In this research, in fact, the entire volatile fraction was
separated in 140 seconds.
Sebastiani et al. (1983), after testing five columns with different stationary phases (OV-1, SE-52,
OV-17, UCON, and Carbowax) for the separation of lemon essential oil concluded that, regardless of the efficiency of the chromatographic system used, it was impossible to obtain a complete
resolution of all the components of the essential oil. In particular, on the polysiloxane stationary
phases, such as SE-52, some complete- or partial-peak overlaps were observed between octanal and
α-phellandrene; 1,8-cineole and limonene; and nerol and citronellol. With polar polyglycol stationary phases, coelution was observed between monoterpene alcohols and esters, and sesquiterpene
hydrocarbons. Therefore, in order to obtain satisfactory information on the composition of essential
oils, it would be preferable to perform two different GC analyses, each on a different stationary
phase, or to preseparate the oil, obtaining different fractions that are simpler in composition and
avoiding peak coelution.
Off-line separation performed by open-column liquid chromatography prior to the GC analysis has been largely applied. Chamblee et al. (1985), by high performance liquid chromatography
(HPLC), separated lime essential oil on three different silica columns in tandem, using as mobile
phase an 8% ethyl-acetate solution in hexane in a ratio of 1:1 with methylene chloride. The separation allowed obtaining 18 peaks where the hydrocarbons were concentrated mostly under the first
peak, and some under the second, along with oxygenated components that were also the components of all the other peaks.
Cotroneo et al. (1985, 1986a), and Dugo et al. (1987) used, for the separation of kumquat, lemon
and bergamot oils, open-column liquid chromatography with neutral alumina activity II, with pure
hexane, hexane/diethyl ether mixtures, and pure diethyl ether as mobile phase. These separations
allowed fractionating the oil in classes of compounds, eluting in increasing polarity order: hydrocarbons, esters and ethers, carbonyl compounds and alcohols. Figures 1.4 and 1.5 show examples
of these separations.
Mazza (1986) used open silica column liquid chromatography to separate bergamot oil into four
fractions, eluting with a solvent system similar to that used by Cotroneo et al. (1985, 1986a) and
Dugo et al. (1987).
Off-line methods are laborious, time consuming, and require sample manipulation, and contamination and loss of components can easily occur.
Since the 1990s, it has been possible to perform on-line LC pre-fractionation of the oil for the
analysis of essential oils, so that the fractions can be directly analyzed by GC. This procedure does
not require manipulations, and only sample dilution is necessary, permitting the analysis in a single run (Munari et al., 1990). This technique has been used for the analysis of aldehydes in sweet
orange oil (Mondello et al., 1994a). Coupling the LC-GC system to a mass spectrometer detector
useful information was obtained on bergamot oil (Mondello et al., 1994b), neroli oil (Mondello

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6

Citrus Oils
3
4

A

1
2

3

B

1
C
4

D

2

Signal

E

3.3

6.7

10 13.3 16.7 20

23.3 26.7

30

33.3

36.7 40

FIGURE 1.4 Gas chromatogram on a capillary column coated with SE-52 of a cold-pressed lemon oil and
of the fraction obtained by separation on neutral alumina column: (A) whole oil (B) hydrocarbons (C) ethers
and esters (D) carbonyl compounds (E) alcohols. (1) α-Phellandrene; (2) octanal; (3) limonene; (4) 1,8-cineole.
Octanal and α-phellandrene and limonene and 1,8-cineole are coeluted in chromatogram A. These components are resolved in the chromatograms B, C, and D. (From Cotroneo, A., et al., Flavour Fragr. J. 1, 69–86,
1986a.)

et al., 1994c) and petitgrain oils (Mondello et al., 1996) and on the sesquiterpene hydrocarbons of
different essential oils (Mondello et al., 1995b).
The multidimensional gas chromatography (MDGC) (Mosandl, 1995; Mondello et al., 1998a,b)
with conventional capillary columns, along with Fast GC (David et al., 1999; Mondello et al., 2000),
allowed obtaining reliable and rapid information on the composition of citrus essential oils. The
former technique uses the high resolution of GC during both the preseparation and the final analysis
of the transferred fractions; the latter allows the analysis of the volatile fraction and of some nonvolatile components of citrus essential oils with a speed gain of about five compared with the conventional GC, without resolution loss.

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