Food Chemistry 122 (2010) 285–291

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Food Chemistry
journal homepage: www.elsevier.com/locate/foodchem

Analytical Methods

Optimization of the GC method for routine analysis of the fatty acid profile in
several food samples
Marinko Petrović *, Nataša Kezić, Vesna Bolanča
Faculty of Food Technology and Biotechnology, Food Control Center, Jagićeva 31, Zagreb, Croatia

a r t i c l e i n f o a b s t r a c t

Article history: The fatty acids profile of food samples was determined by gas chromatography (GC). The fat was
Received 12 February 2009 extracted from different food samples using Soxhlet technique. Extracted triglycerides were converted
Received in revised form 22 October 2009 to corresponding methyl esters using methanolic solution of potassium hydroxide (trans-esterification).
Accepted 9 February 2010
GC method for the analysis of obtained methyl esters was optimized on two different cyanopropyl cap-
illary columns. Good resolution of all fatty acids commonly found in foodstuffs was achieved. After opti-
mization, the method was validated and the results for linearity, precision, limit of quantitation (LOQ),
Keywords:
limit of detection (LOD), robustness and stability were presented. The method has been applied to the
Fatty acids
Gas chromatography (GC)
quantitative determination of the fatty acid content in different food samples: edible oil, dairy products
Method optimization rich with omega-3 fatty acids, different food supplements, baby food, etc.
! 2010 Elsevier Ltd. All rights reserved.

1. Introduction cholesterol and lower level of high-density cholesterol in plasma

The fatty acids (FAs) composition of food is very important
because lipids are one of the three major constituents of food. Their
roles in biological tissues are: (1) source of energy, (2) components
of biological membranes, (3) precursor for many different mole-
cules and (4) transport vehicle for vitamin A, D, E and K. The com-
position of fatty acids plays an important role in the transport of
substances in and out of the cell because of their impact on the flu-
idity of the cell membrane (Chow, 2000; O’Keefe, 2000). Also each
essential fatty acid has been subjected to its own metabolic path.
The elongation and desaturation of linoleic acid leads to arachi-
donic acid, which is the precursor of prostaglandines while linole-
nic acid is metabolized eventually to provide eicosapentaenoic acid
(EPA) and docosahexaenoic acid (DHA).
Major causes of death in Western societies such as degenerative
cardiovascular diseases and cancer are linked to aspects of dietary
fat intake (Kritchevsky, 2002). A lot of attention is given to dietary
fats as an important factor human health. Due to that, the content
of different kinds of FAs having bad effects on health is significant
even to the everyday consumer. The content of FAs with potential
benefits on health also attracts the attention of dietary supplement
consumers. Nutritional labeling laws in many countries require all
processed foods to be analyzed for various kinds of saturated,
mono and polyunsaturated fatty acids (PUFA) and reporting the
results to the consumers. Consumption of trans FAs is linked with
a higher level of cholesterol and low-density-lipoprotein (LDL)
* Corresponding author. Tel.: +385 1 3740 090; fax: +385 1 3744 044.
E-mail address: mpetrovic@ckn.pbf.hr (M. Petrović).
(Kritchevsky, 2002; Vijver et al., 2000).
The main sources of lipids in our diet are vegetable oils and dif-
ferent foodstuffs of animal source e.g. meat, eggs, milk and milk
products. The authenticity of vegetable oil has been analyzed for
over 50 years. Lessening the quality of oil with proven health ben-
efit by adding certain oils of poor quality can make significant
financial gain (Aparicio & Aparicio-Ruiz, 2000; Hajimahmoodi
et al., 2005). The ratio between omega-6 and omega-3 FAs in the
western diet is in the range of 10–30:1, which is recognized by
many nutritionists as insufficient and responsible for health disor-
ders (Chapkin, 2000; McKenzie, 2001). It is possible to distinguish
the FA profile in foodstuffs of animal source by changing the diet
(Bell et al., 1997; McKenzie, 2001). Enriching animal diet with
omega-3 FAs resulted in a higher amount of omega-3 FAs in lipids
in comparison with the control group of animals, which were fed
the classical diet. Also, many omega-3 products of animal origin
with different content and types of omega-3 FA have been mar-
keted the few last years. That emphasizes the importance of deter-
mining FA profile of food for correct nutrition labeling and also for
control of labeling authenticity.
Prior to the analysis, lipids were extracted by the standard
procedure (ISO 1443, 1973). The lipid mixture obtained by solvent
extraction consists of a variety of lipid classes of different polarity:
triacylglycerols, phospholipids, free fatty acids, free sterols, steryl
esters, etc. All of them are extracted when using chloroform/meth-
anol mixture. In the case of petrol ether only non-polar lipids are
extracted. The obtained lipids are then converted into fatty acid
methyl esters (FAMEs) for GC analysis. Many different methylation
methods are described in literature and four of them are

0308-8146/$ - see front matter ! 2010 Elsevier Ltd. All rights reserved.
doi:10.1016/j.foodchem.2010.02.018
286 M. Petrović et al. / Food Chemistry 122 (2010) 285–291
commonly used: acid or base-catalyzed methylation, borontrifluo-
ride methylation, methylation with diazomethane and silylation
(ISO 5509, 2000; Mendez Antolin, Marrero Delange, & Gonzalez
Canavaciolo, 2008; Seppanen-Laakso, Laakso, & Hiltunen, 2002).
The first method is more acceptable then other methods because
it uses less aggressive reagents then other methods.
GC is the most widely used technique for determining the FA
profile (AOAC, 2000; ISO 5508, 1999; Lehotay & Hajšlova, 2002;
Mendez Antolin et al., 2008; Seppanen-Laakso et al., 2002). A wide
range of data is available on the fatty acid composition of vegetable
oils (Hajimahmoodi et al., 2005; Ramadan, Sharanabasappa,
Seetharam, Seshagiri, & Moersel, 2006; Stransky, Zarevucka, &
Wimmer, 2005) and animal origin food products (Dayhuff & Wells,
2005; Huang, Wang, & Crenshow, 2006; Jalali-Heravi & Vosough,
2004; Maduko & Park, 2007). The flame ionization detector is suf-
ficient for a majority of analytical applications of this kind. GC/MS
has been used for the analysis of trace amounts of FAs especially
for identifying FAs when no standards are available. Electron
impact ionization may give us useful information on a number of
double bonds in analyzed FAs but for isomer determination various
deconvolution software has been used (Ramadan et al., 2006).
GC ! GC technique is the most useful technique for identification
of odd carbon number FAs (de Geus, Aidos, de Boer, Luten, & Brink-
man, 2001; Mondello, Tranchida, Dugo, & Dugo, 2006).
The aim of this work was to optimize a simple and reliable
method for the determination of FAMEs and to prove its applicabil-
ity for determining the fatty acid profile in different food samples.
Different stationary phases were used for this purpose and we
chose two cyanopropyl stationary phases: DB-23 column with
50%-cyanopropyl-methylpolysiloxane and DB-225ms column with
50%-cyanopropylphenyl-dimethylpolysiloxane stationary phase.
Their main advantage is the ability to separate cis and trans
isomers. The resolution of elaidic and oleic acid was used as crite-
rion during method optimization. The method validation included
linearity, precision, limit of quantification (LOQ), limit of detection
(LOD), robustness and stability of 37 FAMEs included in analytical
standard for food industry.
2. Materials and methods
2.1. Chemicals and reagents
Isooctane and methanol gradient grade was purchased from J.T.
Backer, (Devanter, Holland). Petrol ether, p.a., hydrochloric acid,
p.a., fluted filter paper, quantitative, potassium hydroxide, p.a.,
and sodium hydrogen sulfate monohydrate, p.a., were purchased
from Kemika (Zagreb, Croatia). Analytical standard of fatty acid
methyl esters were: Food Industry FAME, 37 components in meth-
ylene chloride, cis/trans FAME Mix, 8 components in methylene
chloride and AOCS#1, 6 components, all purchased from Restek
(Bellefonte, PA, USA) and Linolenic acid, 94% mixture of isomers,
obtained from Roth (Karlsruhe, Germany).
2.2. Samples description
Central Science Laboratory, Sand Hutton, York, UK provided the
test material and calculate the results for FAPAS proficiency test
1455 and 1467. Test material for test 1455 was vegetable (rape-
seeds) oil and for test 1467 was breakfast cereals. Pumpkin seeds
oil, olive oil, sunflower oil samples were supplied by local producer
in Croatia. Butter cake sample was obtained for nutritional labeling
from Koestlin, Bjelovar, Croatia and canned pilchard sample was
also obtained for nutritional labeling from Mirna, Rovinj, Croatia.
Flaxseed, evening primrose and borage oil capsules together with
CLA food supplements were provided by dm-drogerie market,
Karlsruhe, Germany and Biofarm, Zagreb Croatia. Sample of infant
formula was provided by Vivera, Glina, Croatia and tuna fish sam-
ples were obtained from Tuna-Kali, Kali, Croatia.
2.3. Extraction of lipids
Lipids were extracted by standard procedure (ISO 1443, 1973).
Approximately 5 g of homogenized sample was weighted into a
conical flask and dried for 1 h at 105 "C. The flask was cooled to
room temperature, and 50 mL of 4 M hydrochloric acid was added.
The solution was boiled for 1 h. Then 150 mL of water was added,
the solution was filtered through fluted filter paper and washed
until neutral reaction on litmus paper. Filter paper was dried for
1 h at 105 "C and inserted in extraction thimble of Soxhlet appara-
tus. Lipids were extracted with petrol ether into weighted round
bottom flask for 4 h on the sand bath. After extraction, petrol ether
was evaporated, the flask was dried at 105 "C and weighted.
2.4. Preparation of fatty acid methyl esters (FAMEs)
Lipids obtained after extraction of vegetable oil and fat samples
were converted to corresponding FAMEs by trans-esterification
with potassium hydroxide (ISO 5509, 2000). Approximately
60 mg of sample was dissolved in 4 mL of isooctane in test tube
and 200 lL of methanolic potassium hydroxide solution (2 mol/L)
was added. Solution was shaken vigorously for about 30 s. The
solution was neutralized by addition of 1 g of sodium hydrogen
sulfate monohydrate. After the salt has settled, 1 mL of upper
phase was transferred into 2 mL vial and analyzed.
2.5. GC analysis
GC analyses were performed on CP-3800 (Varian, Palo Alto,
USA) equipped with flame ionization detector and split/splitless
injector. Injector temperature was at 250 "C and samples were
injected manually (1 lL) with split ratio of 1:30. In the case of
small amounts of lipids split injections were performed with a split
ratio of 1:5. Two different cyanopropyl silicone capillary columns
were used: DB-225ms 30 m ! 0.25 mm, with film thickness of
0.25 lm and DB-23 60 m ! 0.25 mm, with film thickness of
0.25 lm. The temperature program 60 "C rising to 220 "C at rate
of 7 "C/min was the same for both columns. Helium was used as
carrier gas at a flow rate of 1 mL/min in case of DB-225ms column
and at 1.5 mL/min in case of DB-23 column. Detector temperature
was at 280 "C. Star GC Workstation Version 6.4 chromatographic
software was used for data collection, and calculation of all
parameters.
2.6. Validation of test procedure
GC method for determining fatty acid methyl esters was sub-
jected to validation following recommendations of the Interna-
tional Conference on Harmonization (ICH, 1996). Quantification
of individual fatty acids was based on the obtained peak area,
results were normalized and no correction factor was used (ISO
5508, 1999). Criteria used for evaluation of obtained results were
established according to literature (AOAC, 2000; European Phar-
macopoeia, 2005) in two different concentration ranges: for major
constituents present in excess if 5% and for minor constituent pres-
ent in smaller quantities. Relative standard deviation (RSD%) of 3%
with a maximum mass deviation of 1% (m/m) for major constitu-
ents and 0.2% (m/m) for minor constituents were used as criteria
for evaluation of linearity, precision (repeatability and reproduc-
ibility), stability of prepared test sample. Accuracy of the method
was tested by participation in proficiency tests on vegetable oils
 M. Petrović et al. / Food Chemistry 122 (2010) 285–291 287

and dietary products provided by FAPAS, Central Science 3.2.2. Linearity

Laboratory, York, UK. Linearity of the method was verified in range of 50–200% of
injection volume prescribed by the method. Pumpkin seed oil
was analyzed by injecting different amounts of prepared sample:
3. Results and discussion 0.5 lL, 0.8 lL, 1.0 lL, 1.2 lL, 1.5 lL and 2.0 lL.
Obtained results showed acceptable RSD% and mass deviation
3.1. Method optimization of these six analyses for major and minor constituents and con-
firms the linearity of the method.
Samples other than vegetable oils were extracted using Soxhlet
apparatus with petrol ether as a solvent prior to esterification. In
the case of direct extraction without prior treatment with diluted 3.2.3. Precision
hydrochloric acid some tissue samples gave lower yield in total Precision of the method was checked through the repeatability
fat content, but there was no significant difference in fatty acid and reproducibility experiment. Repeatability of the method was
profile. The separation of FAMEs in the chromatogram was opti- evaluated by three preparations of pumpkin seed oil samples,
mized by adjusting the starting temperature of the column oven and each preparation was analyzed in duplicate according to the
and the rate of the temperature program. Table 1 shows the opti- method. Reproducibility was evaluated by comparing the results
mization of the method on DB-225ms column due to the resolution obtained for repeatability with the results of the same sample pre-
of elaidic and oleic acid. pared by different analysts during two different days. The differ-
The first temperature program was used before optimization of ence between two analysts was determined using t-test.
vegetable oil analysis. It was noticed that a faster temperature rate RSD% and mass differences of these experiments are acceptable.
gave better resolution between critical pair elaidic and oleic acid Obtained results confirmed precision of the method due to multi-
therefore the starting temperature was lowered to 60 "C and the ple preparation of the sample and preparation by different
temperature rate of 7 "C/min was applied. Such conditions are con- analysts.
sidered as an optimum because, in contrast to analytical standards,
the samples are comprised of two or three major peaks and their
high concentrations reduce resolution. To speed up the analysis 3.2.4. Limit of quantitation and detection
on DB-23 the column carrier gas flow was raised to 1.5 mL/min. Limit of quantitation (LOQ) and limit of detection (LOD) of the
An even faster flow rate may be applied if the resolution of cis/trans method were calculated according to literature (European Pharma-
isomers is not obligatory. copoeia, 2005). LOQ was determined as the signal to noise ratio of
10:1 and LOD was determined as signal to noise ratio of 3:1 of
averaged value of methyl myristate peak obtained during precision
3.2. Method validation determination experiments. The calculated value for LOQ was
0.03% and for LOD was 0.01% but values were corrected based on
3.2.1. Specificity established criteria for mass deviation to 0.1% for LOQ and 0.05%
Specificity of the method was evaluated by recording chro- for LOD. The obtained results for LOQ and LOD proved that the
matograms of analytical standard Food Industry FAME, 37 com- method is sensitive enough for its purpose.
ponents in methylene chloride on both columns using the same
chromatographic condition except for the carrier gas flow which
was 1 mL/min for DB-225ms column and 1.5 mL/min for DB-23 3.2.5. Robustness
column. The obtained chromatograms are shown below. Fig. 1 Robustness of the method was evaluated in four different
is the chromatogram obtained after injecting the analytical stan- experiments. For the first experiment sample weight was varied
dard on DB-225ms column and Fig. 2 is the chromatogram ob- in the range of 50–70 mg (80–120% of prescribed sample weight).
tained on DB-23 column. Obtained results for fatty acid composition in the tested pumpkin
The resolution between elaidic and oleic acid was critical. The oil sample confirmed that a slight change of sample weight does
obtained resolution of trans and cis oleic acid using DB-225ms col- not have any influence on the results. In the next experiment the
umn was 1.3 and more than 2 for all other peaks. Analysis on DB- starting temperature of analysis was changed to 55 "C and 65 "C.
23 column gave a resolution of 2.9 for elaidic and oleic acid after Then carrier gas flow was different (80–120% of prescribed flow
optimization. Different retention times and elution orders of rate). At the end the temperature program was varied to 6.5 "C/
FAMEs on two tested columns gave us confirmation of the identity min and 7.5 "C/min. Resolution of all peaks was slightly different
of those peaks. Routine samples of pumpkin seed oil and analytical in experiments then in chromatograms obtained by the prescribed
standards AOCS#1 were analyzed according to the method on both conditions but in the acceptable range. The Tailing factor for all
columns. There was no significant difference between the obtained peaks was in the range of 0.8–1.2 and remained stable during
results for major and for minor constituents. robustness experiments. The Robustness experiments prove that
it is possible to speed up the analysis by speeding up the carrier
flow rate and temperature program without significantly disrupt-
Table 1 ing peak resolution although this was not done by reason of rou-
Optimization of elaidic and oleic acid resolution on DB-225ms column. tine sample analysis.
Starting Temperature Final temperature Resolution of
temperature rate ("C/min) ( "C)/time (min) trans/cis C18:1n9
("C) 3.2.6. Stability of prepared test sample
150 2 200/20 N/A Stability of prepared test samples was evaluated during a 48 h
120 2 200/14 <0.7 period that should be enough for testing prepared samples in rou-
100 2 200/10 0.7 tine analysis. Samples prepared for the evaluation of precision was
80 4 210/15 1.0
injected 24 h after preparation and 48 h after. The obtained results
80 5 210/25 1.1
80 7 210/25 1.2 met acceptable criteria for mass difference and RSD% of results for
60 7 210/30 1.3 major and minor constituents. Due to that, prepared test samples
are stabile at least for 48 h.
288 M. Petrović et al. / Food Chemistry 122 (2010) 285–291

Fig. 1. Chromatogram obtained after injecting Food Industry FAME mix on DB-225ms capillary column.

Fig. 2. Chromatogram obtained after injecting Food Industry FAME mix on DB-23 capillary column.

3.3. Sample analysis
Validated method was used for analysis of fatty acid profile in
different samples. Routine analysis of vegetable oils can also be
very demanding due to large differences in the content of various
fatty acids in oils of different origin. The main problem is peak
overlapping caused by peaks of major constituents: oleic acid in
e.g. olive oil and rape seed oil, linoleic acid in e.g. sunflower oil
and soya-bean oil or linolenic acid in flaxseed oil and hempseed
oil. That was the main reason for not using a faster carrier gas flow
 M. Petrović et al. / Food Chemistry 122 (2010) 285–291 289

Table 2
Chromatographic parameters and results obtained after injection of Food Industry FAME mix and samples on DB-23 capillary column.

Peak name Retention time Resolution Pumpkin seeds oil AOCS#1 Olive oil Butter cookies Canned pilchard
C4:0 5.07 – – – – 2.4 –
C6:0 7.07 30.3 – – – 1.5 –
C8:0 9.94 70.0 – – – 0.9 –
C10:0 12.99 72.9 – – – 2.1 –
C11:0 14.45 34.2 – – – – –
C12:0 15.85 33.1 – – – 2.6 0.1
C13:0 17.17 31.7 – – – – –
C14:0 18.44 29.9 0.1 – – 8.6 2.8
C14:1 18.97 12.0 – – – 0.8 –
C15:0 19.64 15.3 – – – – 0.4
C15:1 20.17 12.2 – – – – –
C16:0 20.80 14.0 12.5 6.0 12.8 37.2 12.7
C16:1 21.17 8.5 0.1 – 0.9 1.2 1.8
C17:0 21.89 16.1 – – – 0.3 0.5
C17:1 22.32 9.2 – – – – –
C18:0 23.09 15.1 4.9 3.0 2.7 7.7 3.3
C18:1-trans 23.32 4.1 – – – 0.3 –
C18:1-cis 23.48 2.9 31.1 35.0 72.8 26.6 49.7
C18:2-trans 23.79 5.8 – – – 0.2 –
C18:2-cis 24.20 7.4 50.3 50.0 9.4 6.7 14.7
c-C18:3-cis 24.68 8.3 – – – – –
C18:3-cis 25.19 8.3 0.2 3.1 0.7 0.6 4.4
C20:0 25.97 11.2 0.3 2.9 0.4 0.2 0.7
C20:1 26.52 8.4 0.1 – 0.3 0.1 1.2
C20:2 27.59 13.4 – – – – –
C21:0 27.82 2.7 – – – – –
C20:3n6 28.29 5.4 – – – – –
C20:4n6 28.77 5.3 – – – – 0.3
C20:3n3 29.05 3.0 – – – – –
C22:0 30.11 10.1 0.1 – – – 0.2
C20:5n3 30.42 3.0 – – – – 2.4
C22:1 30.96 4.8 – – – – –
C22:2 32.64 13.3 – – – – –
C23:0 32.92 2.5 – – – – –
C24:0 26.51 22.2 0.3 – – – –
C24:1 37.90 7.5 – – – – –
C22:6n3 38.38 2.6 – – – – 4.8

rate and temperature program although it is possible do that for
analyzing same type samples. In Table 2, retention time and reso-
lution of peaks obtained on DB-23 column is given together with
results obtained during sample analysis. AOCS #1 standard solu-
tion is used as a verification sample each day before the analysis
of unknown samples to ensure that the system is working prop-
erly. The method was also successfully applied to different food
supplement samples of vegetable origin. It was used for quantita-
tion of a-linolenic acid in flaxseed capsules, for quantitation of
c-linolenic acid in evening primrose and borage oil and for quanti-
tation of conjugated linoleic acid (CLA) in food supplements for
better fat consumption in mussels.
Results obtained in proficiency tests provided by FAPAS are
shown in Table 3. Obtained values in our lab are acceptable since
z-scores were considered satisfactory if |z| 6 2. Result for total
trans fatty acids in test 1467 is also acceptable because assigned
value was below LOQ of our method obtained during method val-
idation. Results for total trans fatty acids in test 1455 were given
only for information as Kernel Density Plot with peak on 0.5 g/
100 g that is in good accordance with our results.
Demand for infant formula, as an alternative to human milk, is
growing because of inability of mothers to breast feed. Therefore,
infant formula must meet the nutritional requirements of growing
infants as close as possible to avoid serious health problems for the
survival of newborn infants. Infant foods should contain a level of
omega-3 fatty acids and arachidonic acid and shouldn’t contain
trans fatty acids (Chapkin, 2000). We have analyzed samples of
baby food enriched with arachidonic acid and docosahexaenoic
acid and the results show that the sample contains 0.2% of arachi-
donic acid and 0.2% of docosahexaenoic acid.
In vegetable oils fatty acids with an even carbon number are
dominant, whereas in oils and fats of animal origin with odd car-
bon number FAs are present. Also, fats and oils of animal origin
contain lot of polyunsaturated fatty acids (PUFA). That makes ana-
lyzing the fatty acid profile even more complex. In Fig. 3 our chro-
matograms of extracted fat from different tuna samples are shown.

Table 3
Overview of results of proficiency tests on fatty acid content.

FAPAS proficiency test 1467, oils and fats, breakfast cereals FAPAS proficiency test 1455, oils and fats, vegetable oil
Result (g/100 g) Assigned value z-score Result (g/100 g) Assigned value z-score
Total fat 7.89 8.00 "0–5 – – –
Saturated 1.67 1.68 "0.2 6.82 7.21 "1.1
Mono-unsaturated 3.28 3.02 1.7 61.03 60.38 0.4
Poly-unsaturated 2.94 2.85 0.6 31.61 31.90 "0.3
Total trans- Not detected 0.01 – 0.54 – –
290 M. Petrović et al. / Food Chemistry 122 (2010) 285–291
Fig. 3. Chromatogram obtained after injection of prepared samples of extracted fat from Tuna fish fed with pilchard (lower line) and control group (upper line).
It is easy to perceive the difference in the content of PUFA, espe-
cially EPA and DHA. Samples of tuna fish fed with pilchard (lower
line) had about 10% of PUFA while control group (upper line) had
only a 1–2% of PUFA and trace amounts of EPA and DHA. The
results show that the method is capable of distinguishing a way
of feeding tuna based only on the fatty acid profile.
4. Conclusions
The method for the determination of fatty acids profile of differ-
ent food samples has been optimized on two different cyanopropyl
columns. Results of the method validation showed that the method
has acceptable selectivity, linearity, precision, detection and quan-
titation limits, robustness and stability. The obtained resolution of
FAMEs are better in comparison with data shown in references for
cis and trans isomers especially for elaidic and oleic acid. The meth-
od is suitable for routine analysis of samples of vegetable and ani-
mal origin with a very distinct fatty acid profile. Results may be
used for various purposes like nutritional labeling, quality control
of different food supplements containing specific fatty acid profiles
and confirmation of diet applied on animals. Better resolution of
trans/cis isomers was obtained with DB-23 column and it will be
used for the determination of trans fatty acid content in food.
DB-225ms column gave us confirmation of peak identity due to
different elution order of FAMEs and it is sufficient for the routine
analysis of vegetable oils.
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