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Vittorio M. Moretti and Fabio Caprino
21.1 Introduction .................................................................................................................377
21.2 Analysis of Total Fatty Acids .........................................................................................379
21.2.1 Lipid Extraction ...............................................................................................379
21.2.2 Preparation of Fatty Acid Derivatives ..............................................................380
18.104.22.168 Acid-Catalyzed Esterifcation and Transesterifcation .....................380
22.214.171.124 Diazomethane .................................................................................382
126.96.36.199 Base-Catalyzed Transesterifcation ..................................................382
21.2.3 Gas Chromatographic Analysis .......................................................................382
21.2.4 Column ...........................................................................................................383
21.2.5 Mass Spectrometry of Fatty Acid Derivatives ..................................................384
Te last two decades have seen an exponential increase of interest in the health efects of n-3 fatty
acids from fsh oils. Several reviews are available on their physiological roles and functions,1,2
on their tissue distribution in humans,3
on their structural and functional roles in cellular
on their role in gestation and parturition,5
immune response and
cortical and retinal developments,8–10
cellular life span.17
378 ◾ Handbook of Seafood and Seafood Products Analysis
Clinical and epidemiological studies indicated that the consumption of fsh and fsh oils can
prevent certain human cardiovascular conditions.18
Tese potential healthy properties of marine
lipids renewed interest in investigating the lipid content and the fatty acid composition of fsh and
seafood products around the world. Studies were especially concentrated on the content and the
availability of long-chain polyunsaturated fatty acids (PUFAs) that are typical of the aquatic habi-
tats. Examples of PUFA include eicosapentanoic acid (EPA), and docosahexaenoic acid (DHA).
Te fatty acid composition of fsh, shellfsh, seafood products, and encapsulated fsh oils products
have been extensively treated and discussed.19,20
Fish oils and marine lipids are considered to be the main source of n-3 PUFAs in the human
diet. Tey are composed of complex fatty acids, typically an even-carbon chain length from C14
to C24 with 0–6 methylene interrupted double bonds. Marine lipids also contain minor amounts
of less common fatty acids with nonmethylene interrupted and branched-chain fatty acids.21
According to the popular shorthand system PUFAs of interest to biochemists, food scientists,
and nutritionists can be indicated with the chain length (e.g., 22): number of cis ethylenic bonds
(e.g., 6) and number of carbon atoms counted from the terminal methyl group (e.g., n-3). For
example, DHA is equivalent to 22:6n-3.
n-3 and n-6 fatty acids, in particular linoleic acid (18:2n-6), a-linolenic acid (18:3n-3), EPA
(20:5n-3), and DHA (22:6n-3), are generally known as essential fatty acids (EFAs) in verte-
Tese fatty acids cannot be completely synthesized de novo in the body and consequently
Table 21.1 Pathways of the Biosynthesis of C20 and C22 PUFA from
n-3 and n-6 C18 Precursors
18:3n-3 (α-linolenic acid)
18:2n-6 (linoleic acid)
↓ Δ6 desaturase
↓ Δ6 desaturase
18:4n-3 (stearidonic acid)
18:3n-6 (γ-linolenic acid)
20:3n-6 (dihomo-γ-linolenic acid)
↓ Δ5 desaturase
↓ Δ5 desaturase
20:4n-6 (arachidonic acid)
22:5n-3 (docosapentaenoic acid)
22:4n-6 (adrenic acid)
↓ Δ6 desaturase
↓ Δ6 desaturase
↓ Peroxisomal oxidation
↓ Peroxisomal oxidation
22:6n-3 (docosaesahexaenoic acid)
Analysis of n-3 and n-6 Fatty Acids ◾ 379
must be supplied in the diet. Humans and animals cannot interconvert n-3 and n-6 fatty acids. In
some, but not in all (e.g., lions and cats23
), vertebrate species the long chain–polyene acids of the
n-3 and n-6 families are biosynthesized through a combination of elongation and desaturation
reactions, starting from a-linolenic and linoleic acids, respectively. Tese processes are particu-
larly important in animal systems and are required for the production of C20 and C22 PUFAs,
which are often termed highly unsaturated fatty acids (HUFAs), as the precursors of biologically
Although these statements also apply to fsh, the extent to which they apply
quantitatively to a given fsh species varies widely. In fsh species that cannot perform these con-
versions, the C20 and C22 HUFAs are dietary EFAs and their C18 homologues do not satisfy
EFA requirements in the fsh diet.25
Te elongation and the desaturation pathways, whereby
the C18 is converted to their C20 and C22 counterparts in fsh, are reasonably understood and
are presented in Table 21.1. For a more extensive information about the classifcation and the
nomenclature of fatty acids and lipids the reader is referred to Fahy et al.,26
Lobb and Chow,27
21.2 Analysis of Total Fatty Acids
Usually, the analysis of fatty acids in a fsh tissue involves mainly three steps: lipid extraction,
preparation of fatty acid derivatives, and gas chromatographic (GC) analysis. For decades, GC
has been the most applied method for fatty acids analysis.29–32
Te success of GC with fame
ionization detector (FID) for the analysis of fatty acids is based on the ability of this technique
to separate dozens of fatty acids depending on the type and the length of the column, and on the
economical accessibility of the GC instrumentation that is actually present in all analytical labo-
ratories. Te advent of wall-coated open tubular (WCOT) capillary column, available in a wide
range of diferent stationary phases, has led to an excellent resolution capability of this technique.
Specifc separation problems of fatty acids connected to specifc applications could be solved by
the alternative methodologies of sample preparation.33,34
21.2.1 Lipid Extraction
Fish lipids are prone to oxidation, and should be analyzed immediately after sampling to mini-
mize the changes occurring in lipid components. When the immediate extraction is not feasible,
the sample should be frozen as soon as possible, possibly in liquid nitrogen or dry ice, and stored
in glass containers under nitrogen at −80°C. Both wet fsh tissues and organic solvents must not
come in contact with any plastic ware, since plasticizers are very easily leached out and could be
co-chromatographed with fatty acids causing severe interferences in chromatograms. Such com-
pounds (usually the esters of phthalic acid) are characterized by an abundant base peak at m/z 149
in their mass spectra. Also any source of contamination by mineral oils and detergents should be
avoided. It is usually advisable to add an appropriate antioxidant, such as butylated hydroxy tolu-
ene (BHT), at 50–100 mg L−1
level to the solvent in which lipids are dissolved. Optimal conditions
for sample handling and lipid storage were extensively reviewed by Christie.35
Te most cited methods for lipid extraction in research papers are the Bligh and Dyer36
the Folch et al.37
methods. Tese methods are based on the use of a chloroform/methanol mixture,
with the water content of the matrix as a tertiary component, or with an appropriate addition of
water in order to obtain a tertiary system. Te food tissue is usually homogenized in the presence
of such mixtures using an Ultra-Turrax or a Waring blender. Te main diferences between the
380 ◾ Handbook of Seafood and Seafood Products Analysis
two methods are in the solvent/sample ratio and in the chloroform/methanol ratio. In the Bligh
and Dyer method, the solvent/sample ratio is 1 part sample to 3 parts 1:2 chloroform/methanol,
followed by 1 part chloroform. In contrast, the Folch method employs a ratio of 1 part sample to
20 parts 2:1 chloroform/methanol mixture, followed by the washing of the extract with a weak
Many modifcations of the original procedures have been published38–41
in order to improve
lipid extraction in certain food matrices or for particular applications. Iverson et al.42
the two methods for total lipid extraction in a broad range of fsh and seafood tissues, and found
that the Bligh and Dyer method produced lower amount of lipid content in fsh samples contain-
ing >2% lipid when compared to the Folch method. Te authors explained this incomplete lipid
yield from several standpoints, mainly with the limited solubility of triacylglycerols in the chloro-
form/methanol mixture (1:2, v/v). To overcome this problem, Christie35
suggested, in the presence
of fatty samples, to add a preliminary extraction step with a nonpolar solvent, such as chloroform
or diethyl ether, prior to the Bligh and Dyer procedure.
As a general consideration, both Bligh and Dyer, and Folch methods produce reliable results
when applied to fsh samples, if performed respecting the solvent/sample ratio. Unfortunately, in
many research papers these methods are modifed, but modifcations to the procedures are neither
described nor validated. Christie has reasonably argued that these extraction methods are often
misunderstood and therefore misused.35
21.2.2 Preparation of Fatty Acid Derivatives
Prior to GC analysis, the lipid sample has to be hydrolyzed and fatty acids converted into nonpolar
derivatives, usually methyl esters (FAMEs). Te formation of FAMEs is normally performed on
the lipid sample in the presence of a catalyst dissolved in or mixed with methanol. A heat treat-
ment may be applied to the sample, that may last from few minutes to several hours, depending
on the temperature and on which catalyst is used. Several types of acid- or base-catalyzed reactions
are suitable for the transesterifcation of lipids. Tese reactions have been extensively described by
188.8.131.52 Acid-Catalyzed Esterifcation and Transesterifcation
Tree common used acid reagents are hydrogen chloride, sulfuric acid, and boron trifuoride, all
mixed or dissolved in methanol. Tese acidic reagents not only transesterify triacylglycerols and
other complex lipids but also esterify free fatty acids (FFAs) present in the lipid sample. Heat-
ing of the sample is required to quicken the reaction. Among them, the most frequently used
reagent for FAME preparation is 5% anhydrous hydrogen chloride in methanol, which is usu-
ally prepared by bubbling dry gaseous hydrogen chloride into dry methanol. Te reagent may
also be prepared by adding acetyl chloride slowly to a large excess of dry methanol (1:10, v/v)
under ice. As a by-product methyl acetate is formed, but it does not interfere with the reaction.43
Tis reagent is reported to have limited stability49
and should not be used if stored for more
than 1 week. In a typical procedure, to the fsh oil (5–10 mg) tricosanoic acid (23:0) is added
as internal standard, the sample is dissolved in 1 mL of toluene in a test tube and methanolic
hydrogen chloride (2 mL) is added. Te sample is then left overnight at 50°C in a stoppered tube
or refuxed for about 2 h. After the addition of a potassium carbonate solution, the FAMEs are
extracted thoroughly with hexane. Te hexane phase is then dried over anhydrous sodium sulfate.
Analysis of n-3 and n-6 Fatty Acids ◾ 381
With this reagent, all fatty acids are transesterifed at the same reaction rate. An inert solvent has
to be added to the reaction mixture (toluene) to facilitate the formation of FAMEs, as triacyl-
glycerols, which often represent more than 80%–90% of the total lipid sample, are not soluble
in methanolic hydrogen chloride. Among solvents used, toluene, tetrahydrofuran, and methyl-
tert-butyl ether are quite efective. Under given reaction conditions, the efect of added
solvent type has been studied.50
Te main disadvantages of this derivatization procedure are the relatively long time needed to
complete the reaction and that it is not suited for some sensitive functional groups, such as epoxyl,
cyclopropane, or cyclopropene rings.43
An alternative acid catalyst is sulfuric acid in methanol. Te reagent could be obtained by
adding concentrated sulfuric acid to methanol at the concentration of 1%–2%. Te reaction times
recommended are the same used with methanolic hydrogen chloride, 2 h under refux or overnight
at 50°C. When using sulfuric acid as the catalyst, care should be taken to avoid concentration
and temperature higher than those commonly recommended. As sulfuric acid is a strong oxidiz-
ing agent, excessive concentration or high temperatures could lead to the destruction of PUFAs
of fsh oils.48
It is worth noting that PUFAs must always be handled with care and should not be
subjected to more vigorous conditions than are necessary.
In general, methanolic hydrogen chloride (5%) or methanol–sulfuric acid (1%) is consid-
ered to be the best general purpose transesterifying reagent. Tey transesterify O-acyl lipids
ef ciently under refux for 2 h or overnight in a stoppered tube at 50°C. Te derivatization
procedure has not been reported to cause any isomerization of monounsaturated fatty acids.
However, there is some controversy over whether derivatization causes the isomerization of
geometrical isomers of conjugated linoleic acid. Kramer et al.51
have evaluated acid and base
catalysts in the esterifcation of milk fat. Tey concluded that acid-catalyzed methods caused
extensive isomerization of conjugated dienes and formed allylic methoxy artifacts, and there-
fore they did not recommend the use of these reagents for the determination of conjugated
linoleic acids in milk fat.
Also boron trifuoride in methanol is a powerful transesterifying reagent for fatty acids.52,53
Tis reagent could be used to transesterify most lipid classes at the concentration of 6%–14%, at
reaction temperature from 80°C to 100°C, and time taken for transesterifcation ranges from 2
to 60 min. A procedure using alkali transesterifcation and boron trifuoride/methanol has been
adopted both by the Association of Of cial Analytical Chemists (AOAC) and by the American
Oil Chemists’ Society (AOCS), and are the recommended of cial methods for the determination
of fatty acids in fsh oils.54,55
Tese methods have two steps. Te frst step is a brief treatment of the lipid sample with alkali
(commonly NaOH in methanol). Te second step is the heating of the sample in BF3–methanol
for 2 min.
Some criticism on these of cial methods has been reported by Ackman.56
He suggested to omit
the alkali transesterifcation step, and used equal volumes of BF3–methanol at 7% concentration
and of n-hexane in order to reduce the concentration of the catalyst to 3.5% of the total volume,
and heated for 1 h at 100°C in a screw-cap tube fushed with nitrogen.
According to Christie,43
the BF3 method has serious drawbacks. Te main one is the formation
of methoxy artifacts from unsaturated fatty acids by the addition of methanol across the double
when high concentrations of BF3 in methanol are used. In addition, there is some evidence
that the formation of artifacts is more likely when aged reagents are used. Christie, in view of the
many side reactions and the high acid content in comparison with other reagents, did not consider
to use this method in his own laboratory.43
382 ◾ Handbook of Seafood and Seafood Products Analysis
Diazomethane is another methylation reagent.58
Compared to acid catalysts, diazomethane
esterifes fatty acids more rapidly, but it has no ability to catalyze the transesterifcation of
lipids. As a consequence, when diazomethane is used for the preparation of FAMEs from lip-
ids, an alkaline hydrolysis step has to precede. Diazomethane is generally prepared in ethereal
solution by the action of alkali on nitrosamide in the presence of an alcohol.59
It is potentially
explosive and great care must be taken during its preparation, and apparatus with ground-glass
joints and strong light must be avoided. Tis reagent has a short life and must be prepared
fresh daily. Suitable microapparatus are commercially available for small-scale diazomethane
184.108.40.206 Base-Catalyzed Transesterifcation
Compared with acid catalysts, base catalysts transesterify neutral lipids in anhydrous methanol
at a much faster speed. However, they are unable to esterify FFAs. Furthermore, the reaction
requires more rigid anhydrous conditions because the presence of water leads to the irreversible
hydrolysis of lipids. A comprehensive review on this group of reagents has been reported by Ban-
non et al.60
Te most popular, basic transesterifying reagent is 0.5–2.0 M sodium methoxide
in anhydrous methanol, which is prepared by dissolving fresh clean sodium in methanol.
Other reagents include potassium hydroxide or sodium hydroxide in methanol. Te reaction
is very rapid. For example using 0.5 M sodium methoxide in dry methanol, phosphoglycerides
could be completely transesterifed in a few minutes at an ambient temperature.48
backs associated with alkaline catalysts are their inability to esterify FFAs and their require-
ment of more anhydrous condition during the reaction. Terefore, this method cannot be used
for any oil if much FFAs are present, often a problem with raw fsh oils, because these FFAs
remain as FFAs.
A comparison of the features of the common methods used for transesterifcation/esterifca-
tion of fatty acids is presented in Table 21.2.
21.2.3 Gas Chromatographic Analysis
Te GC analysis of fsh and marine oils generally requires simultaneous separation and quan-
titation of 25 or more fatty acids. Obviously, the number of fatty acids detected depends on
many factors; mainly the type of column used, separation conditions, sample loading, avail-
ability of authentic standards, objective of the study, and on the skill of the analyst, who is
required to investigate a larger number of minor and novel fatty acids.21
For routine use, split
injection is by far the most practical means of sample introduction. Te essential problem of
this type of injection is that of achieving a linear split of the injected sample at the column
Bayer and Liu have listed many factors that have been claimed to give rise to nonlinear
and have studied these factors. In a particularly readable report on this subject
has described various injection techniques, and reviewed various existing problems
Te commonly used carrier gases in capillary GC are helium and hydrogen.
recommends the use of hydrogen, which is less expensive than helium and at opti-
mum fow rates hydrogen takes 1.5 times less for an analysis.
Analysis of n-3 and n-6 Fatty Acids ◾ 383
Commercially available WCOT capillary columns are made of capillary tubing (0.05–0.53 mm
internal diameter) with a flm (thickness 0.1–8.0 mm) of stationary phase uniformly applied to
the inside of the capillary wall.65
Basically, the separation of fatty acid methyl esters can be per-
formed on three types of WCOT capillary columns coated with nonpolar, polar, and very polar
stationary phases depending on the type of lipid sample to be analyzed and on the objectives of
Te choice of the stationary phase obviously afects the retention time and the
resolution of the analytical method. Te use of apolar column, such as DB-5 (5% phenyl 95%
dimethyl polysiloxane), leads to a separation profle that is rather diferent from that obtained with
polar columns, with unsaturated fatty acids eluted ahead of saturated fatty acids of the same chain
length. Te main disadvantage of these columns is the partial overlapping of some unsaturated
fatty acids. In fact, 18:2n-6 is not fully resolved from 18:1n-9 and coelutes with 18:3n-3, and this
is also true for the corresponding C20 and C22 fatty acids.66
For these reasons, these columns
are practically not used for the separation of fsh oil fatty acids, although they could have some
advantages in GC–mass spectrometry applications (GC–MS), due to their low grade of bleed and
their stability at high temperatures.
Table 21.2 A General Comparison among Commonly Used Transesterifying/Esterifying
Acid in MeOH
Alkali in MeOH
30 min–2 h30 min–2 h2–10 minSeconds–1 h
Sensitive to water
Source: Modifed from Liu, K.-S., J. Am. Oil Chem. Soc., 71(11), 1179, 1994. With permission.
384 ◾ Handbook of Seafood and Seafood Products Analysis
Among polar columns, mainly two types of stationary phases of progressive polarity can be
chosen for the analysis of FAMEs. In the polyethylene glycol stationary phases (i.e., DB-Wax,
Supelcowax-10, Omegawax, AT-FAME), a broad range of fatty acids from C4 to C24 can be
separated according to the number of carbon atoms and to the degree of unsaturation. Te use
of polyethylene glycol columns is widely accepted and these columns are used for the analysis of
a wide range of samples, such as vegetable oils, animal fats, and fsh and marine oils,67–72
excellent separation of n-3 and n-6 fatty acids.
On these columns, the separation of geometrical cis and trans isomers cannot be obtained.
Terefore, for the separation of complex mixtures of unsaturated FAMEs, containing many
positional and geometrical isomers of monoenoic, dienoic, and trienoic fatty acids, as in the
partially hydrogenated vegetable or marine oils, an additional resolution is needed. Better
resolution and separative performance are obtained using capillary columns coated with
50% (DB-23) to 100% of cyanopropyl polysiloxane phase, such as SP2340,73–75
(see Table 21.3). It is gen-
erally recognized that columns coated with cyanopropyl phase are mandatory for the GC
analysis of cis and trans isomers of fatty acids. Both the AOCS Of cial Method Ce 1h-05,89
developed for the determination of cis, trans, saturated, and monounsaturated PUFAs in veg-
etable or nonruminant animal oils and fats76
and the AOAC International Of cial Method of
developed for the determination of total, saturated, and monounsaturated
fats in foodstufs91
recommend the use of this type of high polarity columns for the analysis
of FAMEs isomers.
As a general rule, an optimal separation of a classic fsh oil can be obtained ef ciently with
polyethylene glycol columns 30 m length × 0.25 mm i.d., as already mentioned. A medium-polarity
cyanopropyl column, such as DB-23, is suitable for the analysis of complex mixtures of PUFAs,
including n-3 fatty acids, and the partial separation of 18:1 isomers could be obtained. When
detailed information on positional and geometrical isomers has to be acquired, the use of a 100-m
column coated with 100% of a cyanopropyl phase is recommended.
In the GC analysis of fatty acids on a polyethylene glycol column, the key limitation is the
incomplete separation of trans-monoenoic from cis-monoenoic fatty acids. Tese overlaps can be
partially overcome by using a 100-m cyanopropyl column operating at 180°C, as demonstrated
by Ratnayake et al.76
21.2.5 Mass Spectrometry of Fatty Acid Derivatives
GC, coupled to mass spectrometry (GC–MS), is the most powerful technique for the structural
analysis of fatty acid mixtures, in particular for the determination of the position of double bonds
in isomeric fatty acids.92
As reported above, fatty acids are generally analyzed by GC–FID as methyl ester derivatives.
Unfortunately, the structural information obtained from the mass spectra of FAMEs is frequently
of limited value. For example, the position of the double bond in the aliphatic chain cannot be
determined due to the bond migration that occurs during the ionization.93
To overcome this problem, the analyst has two choices. One of them is the preparation of
specifc adducts with the double bonds that give a specifc fragmentation pattern.94
approach is the derivatization of the fatty acid carboxyl group with a reagent containing a nitrogen
When the derivatized fatty acid is ionized in the mass spectrometer, the nitrogen atom
carries the charge and consequently the double bond ionization and the migration are minimized.
Analysis of n-3 and n-6 Fatty Acids ◾ 385
Commonly Used Stationary Phases for the GC Analysis of Fatty Acid M
DB-5; HP-5; Rtx-5 CPSil-8 CB;
−60°C to 325°C/350°C
DB-225; Rtx-225; SPB-225;
CP-Sil 43 CB
40°C to 220°C/240°C
50°C to 220°C
30°C to 220°C
20°C to 250°C/260°C
Long-chain fatty acid
ixture, f sh oils
ax 52 CB; ZEBRO
40°C to 260°C/270°C
50°C to 280°C
Supelcowax 10; ZEBRO
35°C to 280°C
ax 57 CB
20°C to 200°C/225°C
40°C to 250°C/260°C
386 ◾ Handbook of Seafood and Seafood Products Analysis
Table 21.3 (continued)
Commonly Used Stationary Phases for the GC Analysis of Fatty Acid Methyl Esters
40°C to 250°C/260°C
bient to 250°C
0°C to 250°C/260°C
andatory for cis/
trans separation in
hydrogenated f sh or
bient to 275°C
SP-2560; SP-2340; Rt-2560;
CP-Sil 88; AT-SILAR-100
bient to 250°C
Analysis of n-3 and n-6 Fatty Acids ◾ 387
Tus, the formation of characteristic fragments ions permits the localization of unsaturated bonds
and other functional groups in the hydrocarbon chain.
Te most common derivatives of this type used for the analysis of unsaturated fatty acids are
picolinyl (3-hydroxymethylpyridinyl) esters96
and 4,4-dimethyloxazoline (DMOX) derivatives.97
It is worth considering that the mass spectra derived from either picolinyl and DMOX derivatives
do not provide information about the cis/trans confguration of the double bonds. Tese structural
features need to be confrmed by an independent technique.
In the mass spectra of the DMOX derivatives, if a double bond is positioned between carbon
n and carbon n + 1, then a mass interval of 12 amu between ions corresponding to fragments
containing carbon n − 1 and carbon n is usually observed. In the case of the picolinyl esters, when
a double bond is reached along the alkyl chain, a mass interval of 26 amu between ions corre-
sponding to fragments containing n − 1 and n + 1 carbons is observed. Tese methods have been
successfully applied to the analysis of trans-18:1 positional isomers in ruminant fat and in PVHO
by Mossoba et al.98
and Aro et al.80
Recently Van Pelt and Brenna99
presented a GC–MS/MS (ion trap) method for determining
the location of double bonds in PUFA methyl esters. Tis procedure is based on the chemical
ionization of neutral FAMEs in the gas phase with acetonitrile, called by authors “covalent adduct
chemical ionization” (CACI). Briefy, acetonitrile under chemical ionization conditions in an ion
trap mass spectrometer self-reacts to form the (1-methyleneimino)-1-ethenylium (MIE, m/z 54)
that reacts with double bonds of polyunsaturated FAMEs to yield a series of covalent product
ions all appearing at (M + 54)+
. Collisional dissociations of these ions yield diagnostic fragments
permitting unambiguous localization of double bonds.100
Interestingly, this method has been also
used in the analysis of positional and geometrical isomers of conjugated linoleic acids.101
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