Eur. J. Lipid Sci. Technol.

108 (2006) 3342

Vronique Fourniera
Frdric Destaillatsb
Pierre Juandaa
Fabiola Dionisib
Pierre Lambeletb
Jean-Louis Sbdioc
Olivier Berdeauxa
a

INRA, UMR FLAVIC,

Dijon, France
b
Nestl Research Center,
Vers-chez-les-Blancs,
Switzerland
c
INRA,
Clermont-Ferrand, France

DOI 10.1002/ejlt.200500290

33

Thermal degradation of long-chain polyunsaturated

fatty acids during deodorization of fish oil
Long-chain polyunsaturated fatty acids (LC-PUFA) of the n-3 series, particularly eicosapentaenoic (EPA) and docosahexaenoic (DHA) acid, have specific activities especially in the functionality of the central nervous system. Due to the occurrence of
numerous methylene-interrupted ethylenic double bonds, these fatty acids are very
sensitive to air (oxygen) and temperature. Non-volatile degradation products, which
include polymers, cyclic fatty acid monomers (CFAM) and geometrical isomers of EPA
and DHA, were evaluated in fish oil samples obtained by deodorization under vacuum
of semi-refined fish oil at 180, 220 and 250 7C. Polymers are the major degradation
products generated at high deodorization temperatures, with 19.5% oligomers being
formed in oil deodorized at 250 7C. A significant amount of CFAM was produced during
deodorization at temperatures above or equal to 220 7C. In fact, 23.9 and 66.3 mg/g of
C20 and C22 CFAM were found in samples deodorized at 220 and 250 7C, respectively. Only minor changes were observed in the EPA and DHA trans isomer content
and composition after deodorization at 180 7C. At this temperature, the formation of
polar compounds and CFAM was also low. However, the oil deodorized at 220 and
250 7C contained 4.2% and 7.6% geometrical isomers, respectively. Even after a
deodorization at 250 7C, the majority of geometrical isomers were mono- and di-trans.
These results indicate that deodorization of fish oils should be conducted at a maximal
temperature of 180 7C. This temperature seems to be lower than the activation energy
required for polymerization (intra and inter) and geometrical isomerization.
Keywords: Deodorization, fish oil, geometrical isomerization, long-chain polyunsaturated fatty acids, thermal degradation.

The nutritional importance of long-chain polyunsaturated

fatty acids (LC-PUFA) has been well established. PUFA of
the n-3 series, and especially eicosapentaenoic (EPA,
20:5 n-3) and docosahexaenoic acid (DHA, 22:6 n-3), have
specific roles particularly in blood clotting [1], in the inflammatory systems [2], in the functionality of the retina [3] and in
the central nervous system [4]. Fish oils and marine products are the major food sources of n-3 LC-PUFA.
Fish oils have to undergo refining steps before their consumption or their utilization as food supplements. Refining is usually divided into four steps: degumming, neutralization, bleaching and deodorization. The degumming
step is usually applied only to vegetable oils. The last step
is critical as it involves high temperatures (180270 7C)
that could give rise to side reactions [5]. Deodorization
primarily removes undesirable volatile substances and
converts the oil into a bland-tasting, odorless and colorless liquid. Therefore, this process improves the oils
Correspondence: Olivier Berdeaux, UMR FLAVIC, INRA, 17 rue
Sully BP 86510, 21065 Dijon Cedex, France. Phone: 133 3
80693540, Fax: 133 3 80693223, e-mail: berdeaux@dijon.inra.fr

quality and stability. Less unsaturated fats and oils were

deodorized successfully, but then this method was
applied to polyunsaturated oil [6]. Nowadays, steam
refining is the only large-scale practicable method used in
the industry [6] and has been used for fish oil deodorization. However, its repercussion on the production of degradation products from LC-PUFA still needs to be evaluated.
Due to the occurrence of numerous methylene-interrupted ethylenic double bonds, LC-PUFA are unstable
and heat treatment induces a number of chemical transformations (oxidation, polymerization, cyclization, geometrical isomerization and/or double bond migration) [7].
Although deodorization aims to remove undesirable
compounds that affect the taste and the smell of fish oil, a
simultaneous loss of valuable components could occur.
For this reason, it is essential to be able to quantify degradation products in refined fish oils and to find deodorization conditions to prevent their formation during processing. Among the degradation products formed during
heat treatment in the absence of air, polar compounds
(mostly oligomers), cyclic fatty acid monomers (CFAM)
and geometrical isomers (trans fatty acid isomers) are
more likely to be produced.

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Research Paper

1 Introduction

34

V. Fournier et al.

In 1974, Ackman et al. found that ordinary non-hydrogenated vegetable oils contained small amounts of PUFA
(linoleic and a-linolenic) with trans bonds instead of ordinary
cis bonds, and the author showed that these trans fatty
acids were produced during deodorization of oils at elevated temperatures [8]. Investigations on heat treatment
effects on lipids have already been done for mono-, di- and
tri-unsaturated fatty acids [5, 6, 819]. Only few papers
evaluate the effect of deodorization on LC-PUFA [7, 2022].
In the present study, effects of deodorization temperature
were evaluated based on three degradation products of LCPUFA: polar compounds, CFAM and geometrical isomers.

Eur. J. Lipid Sci. Technol. 108 (2006) 3342

ether/diethyl ether 87 : 13 (vol/vol). Then, the polar fraction
was recovered by elution with 150 mL diethyl ether. The
two fractions (polar and apolar) were further submitted to
fractionation by high-performance size-exclusion chromatography (HPSEC) for the separation of oligomers by
molecular size [9]. The separation was achieved with a
SpectraSystem P1000XR pump (ThermoElectron, Courtaboeuf, France) using two columns (Waters, Milford,
USA): an Ultrastyragel column (500 ) and a GPC KF-8025
column having 8 mm internal diameter and 300 mm length,
and a Shimadzu RID-10A differential refractometric
detector (Kyoto, Japan). An isocratic program using tetrahydrofuran at 1 mL/min was employed for elution.

2 Material and methods

2.3 Fatty acid methyl ester preparation

2.1 Samples and reagents

Prior to gas chromatography (GC) analysis, the acylglycerols were transesterified using a basic catalyst (0.5 M
sodium methanolate in methanol). About 20 mg of fish oil
was weighed accurately and diluted in 1 mL toluene. Of the
sodium methanolate solution, 2 mL was added and the tube
held at 50 7C for 5 min. The transesterification was stopped
by adding 0.1 mL acetic acid. Ethyl arachidate (1.8 mg) was
added as internal standard and the esters were washed
with 5 mL distilled water, then extracted successively with 5
and 3 mL hexane. The solvents were evaporated and the
esters diluted in hexane to a concentration of 0.1 mg/mL.

The semi-refined fish oil (Nissui Fine Chemical Dept., Nippon

Suisan Kaisha, Ltd., Tokyo, Japan) was deodorized with a
lab deodorizer. The semi-refined fish oil had a free fatty acid
(FFA) content of 0.01 (expressed as oleic acid) and a peroxide value of ,0.20 meq O2/kg oil. Briefly, fish oil was
heated at either 180, 220 or 250 7C for 3 h under a pressure of
1.5 mbar and with 2%/h (based on oil) direct steam injection.
The control sample was not submitted to any deodorization.
Hexane, petroleum ether (b.p. 4065 7C), chloroform,
dichloromethane, methanol, acetone, tetrahydrofuran, and
acetonitrile were purchased from SDS (Peypin, France).
Platinum oxide was purchased from Merck (Munich, Germany). Standards of fatty acid ethyl and methyl esters and 2amino-2-methyl-1-propanol were purchased from SigmaAldrich (Saint-Quentin Fallavier, France).

2.2 Quantification and composition of polar

compounds
During oil refining or frying processes, a complex mixture
of degradation products, e.g. polar compounds, can be
formed [9]. These degradation products not only differ by
their polarity, but also by their molecular weight. They are
principally due to the action of atmospheric oxygen and
the water content of foodstuffs in the case of frying, and the
high temperatures in the case of the degradations that
occur during refining. Thus, polar compound determination stands out as the most commonly used methodology
to evaluate oil degradation and has been included to
establish the limits of alteration acceptable for the oils
intended for human consumption [9]. Fish oils were fractionated by column chromatography according to a
standard method [23] using a column filled with a homogenized blend of 20 g silica with water at a ratio of 95 : 5.
Briefly, the apolar fraction was eluted by 150 mL petroleum

2006 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim

2.4 GC analysis
Fatty acid methyl esters (FAME) were analyzed on a Hewlett Packard Model 4890 capillary gas chromatograph
(Palo Alto, CA, USA). Most GC analyses were performed
using a CP-Sil88 capillary column (100 m60.25 mm ID,
0.2 mm film; Varian, Courtaboeuf, France) except for GCmass spectrometry (GC-MS) analysis of CFAM which was
done using a BPX70 (120 m60.25 mm ID capillary column, 0.25 mm film; SGE, Melbourne, Australia). The
instrument was equipped with a split/splitless injector
(splitless for 0.5 min). Linear velocity of hydrogen was
37.0 cm/s at 60 7C. The temperature was held at 60 7C for
5 min, programmed to 165 7C at 15 7C/min and held for
1 min, and then to 225 7C at 2 7C/min and finally held at
225 7C for 17 min [24]. The injection port was held at 250 7C
and a flame ionization detector (FID) was used at 250 7C
(hydrogen at 35 mL/min and air at 350 mL/min).

2.5 Quantification of C20 and C22 CFAM

CFAM were quantified according to literature procedure
[18]. About 100 mg of the oil samples were weighed and
the FAME prepared as described previously. The resultwww.ejlst.com

Eur. J. Lipid Sci. Technol. 108 (2006) 3342

ing FAME were weighed and a known amount of internal standard was added [0.125% of two standards,
stearic and eicosanoic fatty acid ethyl esters (FAEE)].
Catalytic hydrogenation using platinum oxide under a
stream of hydrogen was carried out on FAME of deodorized fish oils. Platinum oxide, around 5 mg, was
added along with a magnetic stirrer before hermetically
closing the tube and opening the hydrogen supply. After
3 h, the reduction reaction was completed. The hydrogenated FAME were dried under a stream of nitrogen
and then solubilized in 700 mL acetone. The totally
hydrogenated FAME were fractionated by reversephase high-performance liquid chromatography (RPHPLC) (Ultrabase C18 column 250610 mm, particle
size 5 mm; Shandon HPLC, Cheshire) as described by
Sebedio et al. [25]. The analysis was run in isocratic
mode with a flow rate of 4 mL/min using a mixture of
acetonitrile and acetone (90 : 10, vol/vol). A differential
refractometric detector was used for detection. Solvent,
column and detector were maintained at 58 7C to prevent crystallization of the sample. Fractions were further
analyzed by GC-FID and GC-MS.

2.6 Separation of geometrical isomers by

argentation thin-layer chromatography
FAME fractions containing isomers of EPA and DHA were
separated according to their number of trans double
bonds by argentation thin-layer chromatography (AgTLC) [26]. Briefly, TLC plates (Silica gel, 20620 cm;
Merck, Darmstadt, Germany) were impregnated by
immersion in a silver nitrate solution (10% wt/vol in acetonitrile) for 30 min. Plates were eluted with toluene/
methanol (85 : 15, vol/vol). Bands containing mono-, di-,
tri-, tetra-, penta-trans isomers for EPA and DHA and
hexa-trans isomer for DHA were scraped off the plate,
and the FAME were recovered by adding 5 mL of a 1%
NaCl methanol/water 90 : 10 (vol/vol) blend, then extracted twice with 2 mL hexane.

Degradation of LC-PUFA during deodorization

35

2.8 Preparation of 4,4-dimethyloxazoline

derivatives
4,4-Dimethyloxazoline (DMOX) derivatives were prepared
as described by Dobson and Christie [28]. Briefly, 250 mL
of 2-amino-2-methyl-1-propanol was added to 1 mg
FAME. The mixture was flushed with nitrogen, then held
for 8 h at 170 7C. The DMOX derivatives were extracted
with 3 mL dichloromethane and washed with distilled
water until neutrality. The solvent was evaporated and the
DMOX derivatives were diluted in hexane to a final concentration of 0.1 mg/mL.

2.9 GC-MS analysis

GC-MS analysis was performed using an Agilent technologies gas chromatograph 6890A Network coupled to a
selective mass detector 5973 (Palo Alto, CA, USA) using
the same temperature program as described before,
except that the final temperature was kept for a longer
period of time to allow heavier derivatives to elute. The
column outlet was directly connected to the ion source of
the mass spectrometer operated at 230 7C and using an
ionization energy of 70 eV. Spectral data was acquired
over a mass range of 50450 amu.

3 Results and discussion

FAME prepared from deodorized oil were separated using

a Kromasil-C18 25 cm610 mm ID 5 mm (Thermo Quest,
Courtaboeuf, France) column. FAME were dissolved in
100 mL acetone and eluted with an isocratic program
using distilled and filtrated acetonitrile at a flow rate of
4 mL/min, as described by Juaneda and Sebedio [27].
Fractions containing EPA, DHA and their geometrical isomers were collected to recover enough material (seven
times, at around 3 mg per run) for further separation by
Ag-TLC.

The deodorization process was found to affect the concentration of LC-PUFA in fish oil. Deodorization of fish oil
at 220 and 250 7C led to critical losses of LC-PUFA.
Actually more than 60% of LC-PUFA was lost after deodorization at 250 7C (Fig. 1). It could be noticed (Fig. 1)
that at 220 and 250 7C, n-6 LC-PUFA, as arachidonic
(20:4 n-6) and 22:5 n-6, are less prone to thermal degradation than all other n-3 LC-PUFA reported. Degradation
of fish oil during deodorization was investigated by determining the total amount of the non-volatile compounds. Three classes of degradation products are predominantly formed during heat treatment in the absence
of air: polar compounds (mostly polymers), CFAM and
trans LC-PUFA isomers. This study was done to evaluate
the effect of the deodorization temperature on the degradation of a fish oil containing 5.8% EPA and 19.5%
DHA, and to find the optimal experimental conditions
that could be used to prevent the degradation of LCPUFA during deodorization. Fatty acid compositions of
control and heated oils are reported in Tab. 1. Three
deodorization temperatures were selected: 180 7C;
220 7C, which appears to be the mean temperature
recommended for industrial deodorization of vegetable
oils in France [19]; and 250 7C, which is a positive control
for degradation.

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2.7 Purification of 20:5 and 22:6 FAME by

RP-HPLC

36

V. Fournier et al.

Eur. J. Lipid Sci. Technol. 108 (2006) 3342

Tab. 1. The fatty acid composition (g/100 g of oil, n = 3) of deodorized fish oil as a function of deodorization temperature.
Fatty acids

Control

Deodorization temperature [ 7C]

180

14:0
16:0
16:1
18:0
18:1
18:2
20:1
22:1
all-cis 20:4 n-6
all-cis 20:5 n-3
all-cis 22:4 n-6
all-cis 22:5 n-6
all-cis 22:5 n-3
all-cis 22:6 n-3
Other

220

250

MV

SD

MV

SD

MV

SD

MV

SD

2.77
15.79
4.57
3.96
16.63
1.30
2.55
1.04
1.76
5.81
0.23
1.11
1.25
19.54
21.68

0.03
0.26
0.07
0.06
0.26
0.02
0.02
0.01
0.01
0.07
0.00
0.01
0.01
0.06
0.88

2.88
16.02
4.65
3.96
16.65
1.29
2.53
1.03
1.74
5.64
0.22
1.06
1.20
18.70
22.42

0.08
0.24
0.04
0.03
0.16
0.00
0.01
0.01
0.01
0.03
0.00
0.01
0.00
0.14
0.73

2.81
15.67
4.54
3.90
16.41
1.24
2.54
1.03
1.52
4.18
0.19
0.85
0.92
12.68
31.53

0.04
0.30
0.11
0.07
0.30
0.02
0.04
0.01
0.01
0.07
0.00
0.01
0.01
0.10
1.06

2.85
16.02
4.61
3.98
16.60
1.15
2.62
1.05
0.62
0.90
0.06
0.24
0.22
2.15
46.92

0.06
0.16
0.04
0.04
0.16
0.01
0.02
0.01
0.02
0.06
0.02
0.01
0.00
0.01
0.54

We note an accumulation of polar compounds already in

oil deodorized at 180 7C, but more significantly at 220 and
250 7C.

Fig. 1. Degradation of LC-PUFA relative to deodorization

temperature. ARA, arachidonic acid; EPA, eicosapentaenoic acid; DPA, docosapentaenoic acid; DHA, docosahexaenoic acid.

The application of size-exclusion chromatography (SEC)

to the direct separation of polar compounds produced
during heating of fats has been explored by Marquez-Ruiz
et al. [29]. With this method, it is only necessary to dilute
the fat in the appropriate solvent before the chromatographic determination, but the resolution and detection of
minor compounds are very poor owing to the presence of
unaltered TAG as major components having molecular
weights similar to oxidized compounds. For this reason,
in a second step, we separated the samples into total
polar and nonpolar fractions using adsorption chromatography. Therefore, a IUPAC column chromatography
method was used prior to HPSEC to investigate the
composition of the fractions.

As a first step to assess the effect of the deodorization

temperature on polar compound appearance, HPSEC
was directly used for oil fractionation. Fig. 2 illustrates the
size-exclusion chromatogram of undeodorized and deodorized fish oils giving triacylglycerol (TAG) polymers and
dimers as a function of the deodorization temperature.

Then, the two fractions were separated by HPSEC. An

apolar fraction always gave one peak in HPSEC, corresponding to TAG. Tab. 2 shows the evolution of the
polar compounds separated by HPSEC, including total
levels and their composition, during deodorization.
Control oil contains 2.6% of polar compounds. A part of
these polar compounds in control oil are partial acylglycerols which naturally occur in crude oil. Monoacylglycerols are removed by deodorization at a temperature of 220 and 250 7C. An explanation for the
presence of other polar compounds is that the control
oil has already been semi-refined. The polar fraction

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3.1 Polymerization of LC-PUFA during

deodorization of fish oil

Eur. J. Lipid Sci. Technol. 108 (2006) 3342

Degradation of LC-PUFA during deodorization

37

Fig. 2. Size-exclusion chromatogram of fish oil

showing formation of TAG polymers and dimer
apparition as a function of deodorization temperature. 1: Triacylglycerol polymers (TAGP), 2: triacylglycerol dimers (TAGD), 3: triacylglycerols (TAG)
and oxidized triacylglycerol monomers (oxTAGM),
4: diacylglycerols
(DAG),
5: monoacylglycerols
(MAG), and 6: free fatty acids (FFA).

Tab. 2. Total polar compounds (mg/g of oil, n = 2) and polar compounds distribution in deodorized
fish oil as a function of deodorization temperature.
Polar compound

Control

Deodorization temperature [ 7C]

180

Total
Triacylglycerol polymers
Triacylglycerol dimers
Oxidized triacylglycerols
Diacylglycerols
Monoacylglycerols
Free fatty acids

220

250

MV

SD

MV

SD

MV

SD

MV

SD

26.4
1.3
4.6
5.6
6.5
7.0
1.0

1.1
0.3
0.3
0.5
0.4
0.5
0.1

34.1
1.5
5.4
12.7
7.5
6.0
0.9

3.7
0.2
0.6
2.1
0.8
0.5
0.0

51.0
8.1
16.4
11.1
10.0
4.5
0.9

1.3
0.2
0.0
0.7
0.3
0.1
0.1

194.9
104.4
67.7
6.6
13.2
1.7
1.3

1.1
2.9
1.0
0.4
2.3
0.3
0.9

Also, quantification by GC-FID of FAME prepared from

fish oil with an internal standard showed a loss in matter
visible in GC (non-volatile matter), which correlated with

results obtained with SEC. This is in agreement with the

results of Grandgirard and Julliard [13] who demonstrated
that the non-volatile fraction was well correlated with
results obtained by HPSEC. Our results are in agreement
with those of Marquez-Ruiz et al. [29] who showed that
polymers and dimers occurred at the highest levels in the
most unsaturated oils. Results provided in Tab. 1 suggest
that LC-PUFA are more prone to thermal degradation
compared to C18 PUFA. It could be hypothesized that
due to the higher number of ethylenic double bonds, LCPUFA are the prevalent substrate of polymerization reactions. On the contrary, opposite results were observed by
Cmolik and Pokorny [6]. It was observed for a-linolenic
acid-rich vegetable oils that the overall content of most
polyunsaturated TAG changed slightly compared to large
variations due to isomerization of linolenic acid. This
result concerned oil deodorized at temperatures from 265
to 269 7C; these temperatures favored isomerization over
polymerization reactions.

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increased slightly with temperature for the 180 and 220 7C

samples and greatly for the oil deodorized at 250 7C, to
reach 19.5 wt-%.
In the polar fraction, six groups of compounds could be
identified by HPSEC, i.e. TAG polymers (TAGP), TAG
dimers (TAGD), oxidized TAG monomers (oxTAGM), diacylglycerols (DAG), monoacylglycerols (MAG) and FFA.
While TAGP, TAGD and oxTAGM are compounds formed
through oxidation and polymerization reactions, DAG,
MAG and FFA are components arising from hydrolysis [9].
oxTAGM is the major polar compound formed at 180 7C.
Polymers are the major degradation products generated
at high deodorization temperatures; 6.8% and 10.4% of
dimers and polymers, respectively, are formed in oil deodorized at 250 7C.

38

V. Fournier et al.

Eur. J. Lipid Sci. Technol. 108 (2006) 3342

3.2 Cyclization of LC-PUFA during

deodorization of fish oil
First evidence for the presence of CFAM in heated fats was
reported in 1953 [30]. Toxicity experiments 3 years later on
fractions isolated from heated fats had shown a possible
toxic effect of some of the CFAM. Then, a great deal of
work has been done on the identification and quantification of linoleic and linolenic cyclic monomers. Sebedio et
al. [18] demonstrated that ten times less CFAM are formed
in an oil rich in C18:2 than in a C18:3-rich oil. This suggests
that PUFA are more prone to intramolecular cyclization
than other, less unsaturated fatty acids. Moreover, Sebedio and De Rasilly [21] demonstrated the occurrence of
cyclic fatty acids in refined fish oil concentrate.
Three independent quantifications of CFAM were performed for each sample (control oil and oils deodorized at
180, 220 and 250 7C) using a previously described method [25]. FAEE of stearic and eicosanoic acids were used
as internal standards instead of odd fatty acids found
naturally in fish oil. Fully hydrogenated fish oil was analyzed by HPLC and two fractions were collected (see
Fig. 3). The fraction collected between C18:0 FAME and
C20:0 FAME contained a mixture of C19:0 FAME, the first
internal standard C18:0 FAEE and C20 CFAM. The fraction collected between C20:0 FAME and C22:0 FAME
contained a mixture of C21:0 FAME, the second internal

Fig. 3. HPLC chromatogram of the hydrogenated FAME

of fish oil deodorized at 250 7C.

standard C20:0 FAEE and a mixture of C22 CFAM. The

two fractions were converted into DMOX derivatives prior
to GC-MS analysis.
GC-MS analyses of the two isolated fractions confirmed
the presence of CFAM of C20 and C22 with the corresponding molecular weights and fragmentation pattern
already observed by Sebedio and De Rasilly [21]. Fragmentation of CFAM DMOX derivatives gives characteristic spectra (see Fig. 4). Electron impact mass spectra
with molecular ion at m/z 363 for C20 species and 391 for
C22 species were obtained for each peak, confirming that
the peaks are DMOX derivatives of C20 and C22 hydrogenated CFAM. Fig. 5A shows the single ion monitoring

Fig. 4. Example of mass spectra of DMOX

derivatives of hydrogenated CFAM; (A) corresponds to a C20 CFAM with a six-membered carbon ring located at position C9
C14 (R1, C12NOH22; R2, C6H13), and (B) corresponds to a C22 CFAM with a five-membered carbon ring located at position C9
C13 (R1, C12NOH22; R2, C9H19).

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Eur. J. Lipid Sci. Technol. 108 (2006) 3342

Degradation of LC-PUFA during deodorization

39

Fig. 5. [M]1 and [M15]1 SIM chromatograms of

DMOX derivatives of CFAM. (A) C20 CFAM
(m/z = 348 and 363), and (B) C22 CFAM (m/z =
376 and 391), in fish oils deodorized at 250 7C.

(SIM) chromatogram of C20 CFAM (m/z = 363 [M]1 and

m/z = 348 [M15]1). Fig. 5B shows the SIM chromatogram of C22 CFAM (m/z = 391 [M]1 and m/z = 376
[M15]1). These results confirm the location of CFAM
peaks on the GC-FID chromatograms used for the quantification step. Analysis by GC-MS confirmed the absence
of CFAM in vicinal HPLC fractions. Minor C20 and C22
cyclic fatty acids from arachidonic and 22:5 acids cannot
be distinguished from cyclic fatty acids from EPA and
DHA, respectively, obtained after hydrogenation of oil.
Consequently, cyclized PUFA having 20 or 22 carbons are
all confounded with dominant EPA and DHA CFAM, giving
a total quantification for C20 and C22.

66.3 mg/g of C20 and C22 CFAM were found in samples

deodorized at 220 and 250 7C, respectively. As for polar
compounds, the control (semi-refined) oil already contained CFAM at 1.7 mg/g. The amount of CFAM formed in
oil deodorized at 180 7C (3.8 mg/g) was higher than that
reported by Sebedio and De Rasilly [21]. They measured
only 0.40.6 mg/g of CFAM in encapsulated fish oils, even
if the EPA and DHA concentrations were higher in the supplement as compared to the oil used in this study.

3.3 Geometrical isomerization of EPA (20:5 n-3)

and DHA (22:6 n-3) during deodorization of fish
oil

A good reproducibility was obtained for quantification by

GC-FID of both C20 and C22 species. A significant amount
of CFAM was produced during deodorization at temperatures above or equal to 220 7C (Tab. 3). In fact, 23.9 and

Geometrical isomerization occurs during deodorization,

which is generally conducted at temperatures in the range
of 180270 7C under vacuum in the presence of steam for

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40

V. Fournier et al.

Eur. J. Lipid Sci. Technol. 108 (2006) 3342

Tab. 3. CFAM derived from C20 and C22 unsaturated fatty acids formed during deodorization of fish
oil (mg/g, n = 3)
CFAM

Control

Deodorization temperature [ 7C]

180

C20
C22

220

250

MV

SD

MV

SD

MV

SD

MV

SD

0.2
1.5

0.0
0.1

0.6
3.2

0.0
0.2

4.7
19.2

0.7
1.0

17.0
49.3

1.8
2.3

a few minutes to several hours [5]. These isomers have

been shown to be components in edible vegetable oils
that have been subjected to heat treatment, provided the
temperature was higher than 190 7C [15]. They have been
detected in infant food formulas, either liquid or powdered, from France [5], Canada [31] and the USA [32].
By geometrical isomerization of EPA and DHA, 32 and
64 isomers, respectively, can theoretically be formed.
Geometrical isomerization of PUFA is not a negligible
phenomenon, even if other reactions (polymerization,
oxidation) are quantitatively more important in edible fats
and oils. Fatty acid compositions of the fish oils (Fig. 6)
show that deodorization has an important impact on the
geometrical isomerization of EPA and DHA. Tab. 4 presents the evolution in trans isomers of EPA and DHA as a
function of deodorization temperature. Due to possible
overlapping of trans fatty acids with other degradation
products, e.g. CFAM, the reported values for trans fatty
acids might be slightly overestimated. Only minor
amounts of EPA and DHA trans isomers were formed
during deodorization at 180 7C. However, the oil deodorized at 220 7C contained about 7.7 and 34.1 mg/g of EPA
and DHA geometrical isomers, respectively. Deodorization at 250 7C was so detrimental to PUFA that only 0.9%
EPA and 2.2% DHA remained in the fish oil after such a
treatment (see Tab. 1). Furthermore, 20.0 and 55.6 mg/g
of geometrical isomers of EPA and DHA, respectively,
were detected in the oil deodorized at 250 7C. Our results
indicate that geometrical isomerization of fish oil is minimized when the deodorization temperature does not
exceed 180 7C.
In order to gain information on the nature of these geometrical isomers of LC-PUFA, i.e. the number of trans
ethylenic double bonds, the polyunsaturated FAME purified by RP-HPLC were fractionated according to their
number of trans double bonds by Ag-TLC. The first band
in the Ag-TLC analysis contained all-cis DHA (Rf = 0.2),
the second band mono-trans of DHA and all-cis EPA
(Rf = 0.4), and the third band mono-trans of EPA and ditrans of DHA (Rf = 0.6), and so on.

2006 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim

Tab. 4. Evolution of geometrical isomers from the PUFA

EPA and DHA during deodorization of fish oil (mg/g).
Fatty acid

Total trans-EPA
Relative distribution
[%]
Mono-trans
Di-trans
Other
Total trans-DHA
Relative distribution
[%]
Mono-trans
Di-trans
Other

Deodorization temperature [ 7C]

Control

180

220

250

1.4

1.5

7.7

20.0

100.0

0.7

100.0

1.8

100.0
tr#

34.1

62.1
37.9
tr
55.6

100.0

100.0

96.4
3.6
tr

50.2
39
10.8

Under the limit of detection.

Trace amount, under the limit of quantification.

Methyl tricosanoate (23:0 FAME) was used as internal

standard after recovery of FAME collected by Ag-TLC, to
perform the relative quantification of geometrical isomer
classes by GC.
Results reported in Tab. 4 show that the composition of
trans isomers changes with the deodorization temperature, with di-trans being detected at 220 7C and tri-trans
at 250 7C. Results clearly indicate that DHA is more sensitive than EPA to geometrical isomerization and that
more di-trans geometrical isomers are formed at 250 7C.
Mono-trans fatty acids constitute the majority of the geometrical isomers of EPA and DHA found in the deodorized
fish oil sample analyzed.

4 Conclusion
LC-PUFA degradation products were monitored by determining the amount of the new compounds formed
during heating: polymers, CFAM and LC-PUFA geometwww.ejlst.com

Eur. J. Lipid Sci. Technol. 108 (2006) 3342

Degradation of LC-PUFA during deodorization

41

Fig. 6. GC-FID chromatograms of the

EPA/DHA methyl ester fish oil fractions
obtained by RP-HPLC of (A) non-deodorized and (B) deodorized oil at
180 7C, (C) 220 7C and (D) 250 7C.
(E, F) Enlargement of chromatogram
zones of EPA and DHA methyl esters
purified from fish oil deodorized at
250 7C.

rical isomers. We showed that polymers are the major

degradation products generated at high deodorization
temperatures, with 19.5% oligomers being formed in
the oil deodorized at 250 7C. A significant amount of
CFAM was produced during deodorization at temperatures above or equal to 220 7C. Only minor changes
were observed in the EPA and DHA trans isomer content and distribution after deodorization at 180 7C. At
this temperature, the formation of polar compounds and
CFAM was also low. However, the oil deodorized at 220
and 250 7C contained about 4.2% and 7.6% geometrical isomers, respectively. Even after a deodorization at
250 7C, the majority of geometrical isomers were monoand di-trans. All together, these results indicate that

deodorization of fish oils should be conducted at a

maximal temperature of 180 7C. This temperature
seems to be lower than the activation energy required
for polymerization (intra and inter) and geometrical isomerization. Further studies are in progress in order to
identify geometrical isomers and CFAM from EPA and
DHA.

2006 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim

www.ejlst.com

Acknowledgments
The authors kindly acknowledge Mrs. Bole-Richard for
her technical support and Mr. Semon for GC-MS analysis
(Analytical platform, PPM, UMR-FLAVIC, Dijon, France).

42

V. Fournier et al.

Eur. J. Lipid Sci. Technol. 108 (2006) 3342

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[Received: October 18, 2005; accepted: November 24, 2005]

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