Table Of Contentfrontmatter 3/16/06 6:22 AM Page 1
Lipid Analysis and Lipidomics
New Techniques and Applications
Copyright (c) 2006 by AOCS Press(cid:9)
frontmatter 3/16/06 6:22 AM Page 3
Lipid Analysis and Lipidomics
New Techniques and Applications
Editors
Magdi M. Mossoba
John K.G. Kramer
J. Thomas Brenna
Richard E. McDonald
Champaign, Illinois
Copyright (c) 2006 by AOCS Press(cid:9)
frontmatter 3/16/06 6:22 AM Page 4
AOCS Mission Statement
To be the global forum for professionals interested in lipids and related materials through
the exchange of ideas, information science, and technology.
AOCS Books and Special Publications Committee
M. Mossoba, Chairperson, U.S. Food and Drug Administration, College Park, Maryland
R. Adlof, USDA, ARS, NCAUR, Peoria, Illinois
P. Dutta, Swedish University of Agricultural Sciences, Uppsala, Sweden
T. Foglia, ARS, USDA, ERRC, Wyndmoor, Pennsylvania
V. Huang, Yuanpei University of Science and Technology,Taiwan
L. Johnson, Iowa State University,Ames, Iowa
H. Knapp, Billings Clinic Research Center, Billings, Montana
D. Kodali, Global Agritech Inc, Minneapolis, Minnesota
T. McKeon, USDA, ARS, WRRC, Albany, California
R. Moreau, USDA, ARS, ERRC, Wyndoor, Pennsylvania
A. Sinclair, RMITUniversity, Melbourne, Victoria, Australia
P.White, Iowa State University,Ames, Iowa
R. Wilson, USDA, REE, ARS, NPS, CPPVS, Beltsville, Maryland
Copyright (c) 2006 by AOCS Press. All rights reserved. No part of this book may be repro-
duced or transmitted in any form or by any means without written permission of the pub-
lisher.
The paper used in this book is acid-free and falls within the guidelines established to ensure
permanence and durability.
Library of Congress Cataloging-in-Publication Data
Lipid analysis and lipidomics : new techniques and applications / editors,
Magdi M. Mossoba ... [et al.].
p. cm.
Includes bibliographical references.
ISBN-13: 978-1-893997-85-1 (alk. paper)
1. Lipids—Analysis. 2. Chromatographic analysis. I. Mossoba,
Magdi M.
[DNLM: 1. Lipids—analysis. 2. Chromatography—methods.
3. Spectrum Analysis—methods. QU 85 L76115 2006]
QP751.L542 2006
612’.01577—dc22
2005035465
Printed in the United States of America.
10 09 08 07 06 5 4 3 2 1
Copyright (c) 2006 by AOCS Press(cid:9)
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Contents
P re f a c e . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . v i i
Part I. Overview
1 Techniques and Applications in Lipid Analysis
Nils Hinrichsen and Hans Steinhart . . . . . . . . . . . . . . . . . . . . . . . . 3
Part II. Mass Spectral Techniques/Lipidomics
2 An Overview of Modern Mass Spectrometry Methods
in the Toolbox of Lipid Chemists and Biochemists
R. Moreau . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2 9
3 Global CellularLipidome Analyses by Shotgun Lipidomics Using
Multidimensional Mass Spectrometry
X. Han and R.W. Gross . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5 1
4 LC/MS and Chiral Separation
A. Kuksis and Y. Itabashi . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7 3
5 LC/MS and Lipid Oxidation
A. Kuksis and O. Sjovall . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 0 9
6 Structural Analysis of Unsaturated Fatty Acid Methyl
EsterIsomers with Acetonitrile Covalent Adduct
Chemical Ionization (CACI)
J.T. Brenna . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 5 7
Part III. Chromatographic Techniques
7 Recent Advances in Silver-Ion HPLC Utilizing Acetonitrile in
Hexane as Solvent: Temperature Effects on Lipid Elution
Orders/Resolution
R.O. Adlof . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 7 5
8 Analysis of trans-18:1 Fatty Acids by SilverIon HPLC
P. Delmonte, J.K.G. Kramer and M.P. Yurawecz . . . . . . . . . . . . . 1 9 1
9 High-Performance Size-Exclusion Chromatography forLipid
Analysis in Organic Media
G. Márquez-Ruiz and M.C. Dobarganes . . . . . . . . . . . . . . . . . . . . 2 0 5
1 0 Lipid Separations Using Packed-Column Supercritical Fluid
Chromatography
D.G. Hayes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2 3 9
v
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vi Contents
11 TLC-FID with Special Reference to Marine Lipids and Other
High-Molecular-Weight Organic Compounds
R.G. Ackman and A. Timmins . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2 6 1
1 2 Fast GC forCellularFAME Analysis of Bacteria
J.S. Buyer . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2 7 1
Part IV. Vibrational Spectroscopic Techniques
1 3 Use of CellularFatty Acids to Identify Food-Borne Pathogens by
Infrared Spectroscopy and Capillary Gas Chromatography
M.M. Mossoba and S.F.Al-Khaldi . . . . . . . . . . . . . . . . . . . . . . . . . 2 8 7
1 4 Application of FT-NIR forRapid Determination of the Trans
Fatty Acid Composition in Fats and Oils
H. Azizian and J.K.G. Kramer . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3 0 3
1 5 Infrared Spectroscopy and Partial Least Square Calibration
in the Simultaneous Quantification of Isolated trans
and Conjugated Linoleic Acids
A.A. Christy . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3 3 5
1 6 Investigation of Protein-Lipid Interactions by Vibrational
Spectroscopy
G. Meng, N.K. Howell, and E.C.Y. Li-Chan . . . . . . . . . . . . . . . . . 3 5 5
Part V.Applications
1 7 Fat Replacers: An Overview
W.E. Artz, S.M. Mahungu, and S.L. Hansen . . . . . . . . . . . . . . . . . 3 7 9
1 8 Phospholipids: Structures and Physicochemical Activities
M.C. Erickson . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3 9 9
1 9 Waxes and Sterols: Structures and Chemistry
E.J. Parish and A.D. Bell . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4 2 1
Copyright (c) 2006 by AOCS Press(cid:9)
Copyright (c) 2006 by AOCS Press(cid:9)
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P re f a c e
Lipid Analysis and Lipidomics: New Techniques and Applicationsis a book that will
give to both experienced researches in chemistry, chemical engineering, and food
science in the oils and fats industry as well as advanced students a resource that will
provide an overview of the latest developments in the rapidly changing world of
lipid analysis.
The Chapters were written by an impressive group of internationally recognized
experts. Most authors provide a basic or theoretical background as well as the latest
developments in their areas of expertise. State of the art instrumentation and novel
developments in lipid applications and lipidomics are also discussed in depth. A
comprehensive list of the latest applicable references is provided for each chapter.
Hyphenated techniques as well as the latest developments in several areas
including fast GC, HPLC, LC-MS, SFC, chiral separation, size exclusion chro-
matography,TLC, multidimensional mass spectrometry, mid- and near-infrared and
Raman spectroscopy as well as chemometrics are presented. Techniques to analyze
a wide conglomerate of matrixes are outlined; these include global cellular
lipidomes, trans and conjugated fatty acid isomers, fat replacers, oxidized lipids,
phosholipids, waxes and sterols. Several chromatographic, spectroscopic, and mass
spectral techniques are presented that are applicable to structural lipids, cellular
lipids and/or bacterial lipids.
We would like to thank each author who contributed to this book as well as the
staff of AOCS and members of the Books and Special Publications Committee.
Magdi M. Mossoba
John K.G. Kramer
J. Thomas Brenna
Richard E. McDonald
vii
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Part I. Overview
Copyright (c) 2006 by AOCS Press(cid:9)
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1
Techniques and Applications in
Lipid Analysis
Nils Hinrichsen and Hans Steinhart
Institute of Biochemistry and Food Chemistry, University of Hamburg, 20146
Hamburg, Germany
I n t ro d u c t i o n
The term “lipids” describes a variety of different substances. All of these substances
have in common a distinct hydrophobicity and hence good solubility in nonpolar sol-
vents. The term includes, for example, triacylglycerols, di- and monoacylglycerols,
sterols, and their esters, tocopherols, free fatty acids (FFA), phospholipids, proteo-
lipids, CoA esters, and more. Therefore, there are distinct differences in the molecular
structures of lipids. According to the diversity of the substances, there are numerous
methods and applications in lipid analysis. In addition, the choice of the right method
for the analysis depends not only on the substances to be determined, but also on the
information that is required. The following chapter describes the most frequently used
techniques and applications for the analysis of those compounds.
Extraction Methods
For nearly all lipid analyses, a purified lipid extract is needed. Because lipids normally
do not appear in their free form, but embedded in a matrix, an extraction step is neces-
sary before further analysis. In fact, this step frequently generates mistakes that lead to
a false analytical conclusion. Therefore, a well-chosen method of extraction for the
lipid to be determined is required.
The components obtained in the lipid extract depend on the method of extraction
used, especially on the solvent. Nonpolar solvents (e.g., hexane, ether, or supercritical
carbon dioxide) can be adopted for the extraction of simple neutral lipids, for exam-
ple, esters of fatty acids (FAs) and acylglycerols. More complex and more polar lipids
(e.g., phospholipids, lipoproteins, glycolipids, FFAs) require more polar solvents such
as methanol or acetonitrile. Generally solid phase extraction (SPE) methods are advis-
able for complex polar and nonpolar lipid components (1).
For the extraction of fats from food and different biological matrices, digestion of
the surrounding material is necessary, especially if a quantitative isolation is required.
There are different “classical” digestion methods that have been used for many years.
The choice of the technique must depend on the surrounding matrix. The Weibull-
3
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4 N. Hinrichsen and H. Steinhart
Stoldt method is used for the extraction of fat from meat, fish, or oilseeds. After the
surrounding proteinogenic material has been decomposed by hot hydrochloric acid,
the fat is extracted in a Soxhlet-apparatus with either ether or hexane. For dairy prod-
ucts, an alkaline decomposition is normally used. Although these methods have
proved to provide exact results regarding the total fat content (2,3), the extreme condi-
tions (high temperature, very high or very low pH value) often lead to unintentional
modifications of the molecular structure of the analytes. Therefore, if further analysis
of certain lipid compounds is required, an extraction method that does not alter the
structure of the analytes must be used (see scheme in Fig. 1).
In 1959 Bligh and Dyer introduced a method for the isolation of total lipid from
fish muscle using chloroform and methanol as solvents (4); the method became very
popular and has often been modified and improved. Disadvantages of this method
include its toxicity and the cost of the solvents. The toxicity can be avoided by using
other, preferentially nonhalogenated solvents (5,6).
Although the operating expense factor remains with these applications, it can be
reduced by using supercritical fluid extraction (SFE) (7). The marginal alteration of
lipid compounds during extraction, the ease of solvent removal from the extract, and
Fig. 1.Approach diagram for the analysis of fat. For the determination of the
total fat content, another way of extraction has to be chosen than for the
analysis of specific compounds (e.g., fatty acids, phospholipids) because
digestion often leads to alterations in individual lipids.
Copyright (c) 2006 by AOCS Press(cid:9)
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Lipid Analysis 5
the lack of toxicity are other advantages of SFE (8). Furthermore, the SFE-equipment
can be coupled with chromatographic systems, thus providing the opportunity to auto-
mate nearly the entire lipid analysis. Although many efforts have successfully been
made to shorten the length of the “classical” lipid extraction, for example, by using an
ultrasonic- (9) or microwave-assisted (10–12) Soxhlet extraction, SFE remains one of
the fastest methods. Modern extraction methods provide reliable results within 30–50
min. However, SFE requires expensive equipment and the savings achieved by avoid-
ing solvents are profitable only after a multitude of extractions. Finally, it should be
emphasized that no single extraction method is applicable to all matrices or all ana-
lytes. The right choice of method applied depends on various factors and is an impor-
tant element of a successful analysis.
Classical Applications
A multitude of classical chemical applications can be used to evaluate the quality and
chemical characteristics of lipids in food.
Chromatographic techniques have reduced the need for identification, but
saponification value (SV), iodine value (IV), acidity, and peroxide value (PV) are
most commonly used to characterize the quality of fats. They are used to determine
the purity (SV), the number of unsaturated compounds (IV), and the FFA content.
The PV is normally obtained by iodometric titration and describes the content of
unsaturated FA hydroperoxides, which result from lipid oxidation. Accordingly,
this measurement displays an indicator for the deterioration of fats in food.
Although these and other classical methods are still in use, they are being replaced
more and more by faster and more modern methods, especially in laboratories con-
trolling foods. Free FAs, formerly evaluated by acidimetric titration, can be mea-
sured rapidly by Fourier transform infrared spectroscopy (FTIR) (13) or by gas
chromatography (GC) and high-performance liquid chromatography (HPLC) (14).
In the same amount of time, the last-mentioned methods not only provide informa-
tion about the quantity of FFAs, but additionally characterize the exact FAs con-
tained in the sample.
For the determination of the IV, different methods are in use. For the Hanus and
Kaufmann methods, the dissolved fat is spiked with a surplus of brome, which leads
to an addition reaction with the ethylenic bonds. The remaining brome, which did not
react with any ethylenic bond, is used to oxidize iodide ions to iodine. This is mea-
sured by titration with a solution of sodium thiosulfate. Both the AOAC and AOCS
suggest the Wijs method for common fats and oils, in which brome is substituted with
iodine, which has a much lower toxicity. In addition to being time consuming, a sub-
stantial disadvantage of the IV is that not only ethylenic bonds from FAs, but also
those from impurities or other lipid compounds, e.g., sterols, are measured. A reason-
able substitute for the IV is the determination of fatty acid methyl esters (FAME) by
GC. This method shows the complete FA composition, so that the content of unsatu-
rated FA can easily be obtained.
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