Gas-Liquid Chromatography (GLC) methods such as AOAC Fat in foods 966.06 (2005), AOCS Official Methods Ce 1h-05 (2005), Ce 1j-07 (2007), allow for analyzing the fatty acids (FAs) in dietary fats using highly polar liquid phase capillary columns. However, there are still difficulties in completely separating butiric acid from solvent, FA critical pairs with similar polarity, conjugated linoleic acid (CLA) isomers, and long chain-polyunsaturated FAs (LC-PUFAs). Therefore, the selection of the temperature program to be employed is important. This work aimed to improve the AOCS Ce 1j-07 Method for the FA composition of a mixture of soybean and sunflower oil, fish oil, and butterfat, using a modified temperature program, tested among five laboratories. It takes more time, but it allows to completely separate butyric acid from the solvent,
The analysis of fatty acids (FAs) is required for many applications in food science such as nutritional labeling, quality control, composition databases, traceability for international markets, nutrition and health, medical purposes and research. The method for analyzing FAs should include the maximum resolution and identification of FAs, covering from short chain to long chain FAs, saturated (SFAs), monounsaturated (MUFAs), polyunsaturated (PUFAs), long chain-PUFAs (LC-PUFAs), as well as positional and geometrical FA isomers, naturally or industrially produced. The main procedures for FA analysis in different matrixes are based on GLC methods (Seppänen-Laakso
The identification and quantification of SFAs, MUFAs, PUFAs, LC-PUFAs,
The aim of this study was to improve Method AOCS Ce 1j-07, using a modified temperature program, for identifying and quantifying a wide spectrum of FAs present in a sample of soybean and sunflower mixed oil, fish oil and butterfat. It takes more time, but it allows for a complete separation of butyric acid from the solvent,
Internal standard (IS) Tritridecanoine [13:0-triacylglycerol (TAG)], external standards GLC-463 Reference Standard containing 52 FAME -mixture (purity >99%) and
The fat samples were selected according to their different FA compositions and were purchased from local commercial sources: a) Refined mixture of soybean and sunflower oil (80/20), (Santa Fe, Argentina); b) butterfat (Santa Fe, Argentina); c) fish oil soft gel capsules (Santiago, Chile).
The fat samples, the same lots of FAME standards and BCR 163 reference material were distributed among the five participating laboratories from the following institutions: Universidad Nacional del Litoral (UNL), Santa Fe, Argentina; Universidade Federal do Rio de Janeiro (UFRJ), Río de Janeiro, Brazil; Universidade de São Paulo (USP), São Paulo, Brazil; Universidad de Chile (UCH), Santiago, Chile and INCIENSA, Ministerio de Salud, San José, Costa Rica. All these materials were maintained at -23 °C until they were analyzed.
New fused silica capillary columns SP™-2560 0.25 mm i.d. × 100 m length, coated with 100% cyanopropyl polysiloxane stationary phase, film thickness 0.20 µm, were used in four laboratories (Supelco, Inc., Bellefonte, PA, USA, Part N° 24056), one laboratory used CP Sil™88 with the same characteristics (Varian, Walnut Creek, CA, USA, Part N° CP7489). Focus Liner with glass wool (Catalog N°21022-211.5, Restek or equivalent Supelco, Sigma–Aldrich, St. Louis, MO), and capped test tubes Pyrex USA, N° 9826, with teflon liner, 150 mm × 20 mm (Fisher Scientific Corp., USA) were used. The following gases were employed: as carrier gas helium or hydrogen, as make up gas of nitrogen, hydrogen and air for the flame ionization detector (FID) of chromatography quality.
The GLC instruments located at the five laboratories from the participating institutions were the following: UNL/Argentina: Shimadzu GC 2014 with GC Solutions software; UFRJ/Brazil: Agilent 7890 A and EZ Chrom Elite; USP/Brazil: Shimadzu GC 17A and Class GC 10; UCH/Chile: HP 5890 Serie II and Clarity Chromatography SW Data Apex 2006, Waters; INCIENSA/Costa Rica: Agilent 7890 A with Chemstation Agilent, with FID detector (air to hydrogen ratio, 400:40). The injector and detector temperatures were maintained at 250 °C, split ratio 1:100, 1 µL of standard or sample, equivalent to 20 µg of total FAMEs were injected using an autosampler device in each GLC run. The fluxes of hydrogen and helium were 1mL·min−1 and 2 mL·min−1, respectively, the nitrogen flux as make up gas was 25 mL·min−1.
The theoretical correction factor (TCFr) relative to the IS (13:0-TAG) was used to correct the FID response for the quantitative expression of each FAME (g FAME·100 g−1 FAME). The TCFr for short chain FAs and LC-PUFAs did not show a proper quantitative response by FID, with the real possibility of underestimating short chain FAs or to overestimate LC-PUFAs. Then, each laboratory experimentally determined the empirical correction factor (ECF) for each one of the 52 FAMEs present in GLC 463, from butyric acid to DHA, using g % and % purity indicated in the Certificate of Analysis. These calculated ECFs were used for the FAMEs quantification in the Reference Certified Material BCR 163, and in the commercial samples. Even the Reference Standard GLC 714 is recommended by the AOCS Official Method Ce 1j-07 (2007); Reference Standard GLC 463 has been used for its important comparative advantages: it contains 52 FAMEs with a wide spectrum of FAs from 4:0 to 24:1, most of them present in the fat matrixes analyzed in this study. The FAMEs are in different percentages, 1%, 2% and 4%, including critical pairs with close polarity, which permits a good separation, clear identification according to their respective relative retention times to 18:0 in SP-2560 and CP Sil 88 columns and their quantification. Standard mixture GLC 714 has only 24 FAMEs, and is missing key FAMEs, such as 4:0, 5:0, 6:0, which are fundamental for the butterfat analysis.
In order to check the quantitative performance of the modified temperature program, the seven certified FAMEs were quantified and compared, including as appropriate their positional and geometric isomers present in the BCR-163 certified reference material using the calculated ECF and tabulated TCFr. The percentage of recovery for each certified FAME was calculated by each laboratory considering the analytical value obtained and the certified value declared by the Institute for Reference Materials and Measurements (Geel, Belgium).
In accordance with the procedure of the AOCS Official Method Ce 1j-07 (2007), 1 mL of the IS TAG 13:0 solution containing 5 mg was added to 0.100 ± 0.001 g of BCR-163 and weighed at least in triplicate. For identification, the relative retention time of each FAME to 18:0 was used, and compared with those obtained for the FAMEs present in GLC 463, and literature references.
To 0.100 ± 0.001 g of each anhydrous fat samples, soybean and sunflower oil mixture l (80/20), fish oil and butterfat, 1 mL of the IS solution containing 5 mg of 13:0-TAG was added, followed by the FAME derivatization procedure using BF3 14% in methanol according to International Standards – ISO 5509 (2000), indicated by the AOCS Method Ce 1j-07. The FAMEs in the fat samples were identified by the GLC procedure using the modified temperature program, by comparison of their relative retention times calculated to 18:0 with the respective relative retention times of the 52 FAMEs in the GLC-463 standard, other reference FAME materials mentioned in 2.1 and in the literature. The results for the soybean/sunflower oil mixture were determined using its respective calculated ECF and tabulated TCFr values. For fish oil and anhydrous butterfat only ECF values were used for the quantitative procedure. The final results were expressed in g FAME·100 g−1 FAMEs, g FA/100 g FAs and TAG equivalents% (TAGe), according to the AOCS Method Ce 1j-07 (2007).
In the five laboratories, the z score was calculated for each one of the seven certified FAMEs of the reference certified material BCR 163, using their respective calculated ECF, according to IUPAC (Thompson
The AOCS Ce 1j-07 (2007) initial isotherm temperature program, i.e. 180 °C (held for 32 min), icreased at a rate of 20 °C·min−1 to 215 °C (held for 31.25 min), for a total time 50 min was assayed running the GLC 463 standard.
A. Representative chromatogram of GLC 463 Certified Reference Standard using the AOCS Official Method Ce 1j-07.
B. Representative chromatogram of GLC 463 Certified Reference Standard using the proposed temperature program (see paragraph 3.1 for operating conditions).
Even considering this longer chromatographic run, the positive results obtained with this modified temperature program permitted a clear resolution for very short chain FAs. Butyric acid became quite separated from the peak of solvent, and the overlapping of critical pairs was properly resolved, such as 14:1 with 15:0, 15:1 with 16:0,
The analytical performance of the capillary columns SP™-2560 or CP Sil™ 88 used in this study yielded similar analytical results with hydrogen or helium as carrier gas.
The values obtained for the ECF determined experimentally by the five laboratories for each one of the 52 FAMEs in GLC 463 are presented in
Comparison of ECF vs. TCFr values for the 52 FAMEs of the GLC-463 Reference Material
FAME | TCFr | Lab 1 | Lab 2 | Lab 3 | Lab 4 | Lab 5 | |||||
---|---|---|---|---|---|---|---|---|---|---|---|
ECF | Δ (%) | ECF | Δ (%) | ECF | Δ (%) | ECF | Δ (%) | ECF | Δ (%) | ||
4:0 | 1.4534 | 1.6450 | 13.2 | 1.7302 | 19.0 | 2.3807 | 63.8 | 1.9436 | 33.7 | 2.4265 | 67.0 |
5:0 | 1.3224 | 1.4550 | 10.1 | 1.4328 | 8.3 | 1.5614 | 18.1 | 1.6213 | 22.6 | 1.8000 | 36.1 |
6:0 | 1.2351 | 1.4060 | 13.9 | 1.3053 | 5.7 | 1.3169 | 6.6 | 1.4835 | 20.1 | 1.5540 | 25.8 |
7:0 | 1.1727 | 1.3510 | 15.3 | 1.3326 | 13.6 | 1.3621 | 16.1 | 1.4049 | 19.8 | 1.4101 | 20.2 |
8:0 | 1.1259 | 1.1740 | 4.3 | 1.1639 | 3.4 | 1.1696 | 3.9 | 1.1988 | 6.5 | 1.1944 | 6.1 |
9:0 | 1.0896 | 1.1440 | 5.0 | 1.1177 | 2.6 | 1.1329 | 4.0 | 1.1356 | 4.2 | 0.9661 | −11.3 |
10:0 | 1.0604 | 1.0980 | 3.6 | 1.0894 | 2.7 | 1.0872 | 2.5 | 1.1147 | 5.1 | 1.0908 | 2.9 |
11:0 | 1.0366 | 1.1020 | 6.8 | 1.0595 | 2.7 | 1.0636 | 3.1 | 1.1000 | 6.1 | 1.0853 | 5.2 |
5 |
1.0262 | 1.1220 | 9.4 | 1.1136 | 8.5 | 1.1108 | 8.2 | 1.1204 | 9.2 | 1.1095 | 8.1 |
12:0 | 1.0168 | 1.0180 | 0.2 | 1.0444 | 2.7 | 1.0165 | 0.0 | 1.0570 | 3.9 | 1.0240 | 0.7 |
9 |
1.0072 | 1.0620 | 5.5 | 1.0443 | 3.7 | 1.0432 | 3.6 | 1.0707 | 6.3 | 1.0433 | 3.6 |
13:0 | 1.0000 | 1.0000 | 0.0 | 1.0000 | 0.0 | 1.0000 | 0.0 | 1.0000 | 0.0 | 1.0000 | 0.0 |
12 |
0.9912 | 1.0635 | 7.3 | 1.0205 | 3.0 | 1.0425 | 5.2 | 1.0601 | 6.9 | 1.0339 | 4.3 |
14:0 | 0.9856 | 0.9925 | 0.7 | 0.9971 | 1.2 | 0.9814 | −0.4 | 1.0144 | 2.9 | 0.9915 | 0.6 |
9 |
0.9774 | 1.0177 | 4.2 | 1.0046 | 2.8 | 1.0130 | 3.6 | 1.0230 | 4.7 | 1.0097 | 3.3 |
15:0 | 0.9731 | 0.9982 | 2.6 | 0.9675 | −0.6 | 0.9881 | 1.5 | 0.9713 | −0.2 | 0.9783 | 0.5 |
10 |
0.9655 | 1.0148 | 5.1 | 0.9778 | 1.3 | 1.0290 | 6.6 | 0.9918 | 2.7 | 1.0037 | 4.0 |
16:0 | 0.9622 | 0.9654 | 0.4 | 0.9545 | −0.8 | 0.9667 | 0.5 | 0.9685 | 0.7 | 0.9580 | −0.4 |
9 |
0.9550 | 0.9725 | 1.9 | 0.9547 | 0.0 | 1.0072 | 5.5 | 0.9704 | 1.6 | 0.9587 | 0.4 |
9 |
0.9550 | 0.9795 | 2.6 | 0.9604 | 0.6 | 0.9667 | 1.2 | 0.9713 | 1.7 | 0.9768 | 2.3 |
17:0 | 0.9526 | 0.9868 | 3.6 | 0.9462 | −0.7 | 0.9743 | 2.3 | 0.9849 | 1.0 | 0.9558 | 0.3 |
10 |
0.9458 | 0.9852 | 4.2 | 0.9568 | 1.2 | 0.9922 | 4.9 | 1.0030 | 6.0 | 0.9767 | 3.3 |
18:0 | 0.9440 | 0.9621 | 1.9 | 0.9067 | −3.9 | 0.9526 | 0.9 | 0.9546 | 1.1 | 0.9390 | −0.5 |
9 |
0.9377 | 1.0040 | 7.1 | 0.9441 | 0.7 | 0.9812 | 4.6 | 1.0394 | 10.8 | 0.9669 | 3.1 |
11 |
0.9377 | 0.9847 | 5.0 | 0.9360 | −0.2 | 0.9735 | 3.8 | 0.9557 | 1.9 | 0.9364 | −0.1 |
6 |
0.9377 | 0.9921 | 5.8 | 0.9949 | 6.1 | 0.9990 | 6.5 | 1.0229 | 9.1 | 0.9544 | 1.8 |
9 |
0.9377 | 0.9377 | 0.0 | 0.9497 | 1.3 | 0.9419 | 0.4 | 0.9708 | 1.0 | 0.9359 | −0.2 |
11 |
0.9377 | 0.9632 | 2.8 | 0.9366 | −0.1 | 0.9757 | 4.0 | 0.9609 | 1.0 | 0.9384 | 0.1 |
9 |
0.9313 | 0.9983 | 7.2 | 0.9375 | 0.7 | 0.9969 | 7.0 | 1.037 | 11.6 | 0.9756 | 4.8 |
9 |
0.9313 | 0.9400 | 1.0 | 0.9372 | 0.6 | 0.9459 | 1.6 | 0.9880 | 6.1 | 0.9390 | 0.8 |
6 |
0.9249 | 1.0419 | 12.7 | 0.9458 | 2.3 | 0.9576 | 3.5 | 0.9919 | 6.5 | 0.9302 | 0.6 |
9 |
0.9249 | 0.9259 | 0.1 | 0.9547 | 3.2 | 0.9410 | 1.7 | 0.9879 | 6.8 | 0.9405 | 0.2 |
19:0 | 0.9364 | 0.9895 | 5.7 | 0.9074 | −3.1 | 0.8798 | −6.0 | 0.9921 | 5.9 | 0.9079 | −3.0 |
7 |
0.9303 | 0.9338 | 0.4 | 0.9171 | −1.4 | 0.9449 | 1.6 | 1.0012 | 7.6 | 0.9390 | 0.8 |
20:0 | 0.9295 | 0.9712 | 4.5 | 0.9164 | −1.4 | 0.9186 | −1.2 | 0.9953 | 1.1 | 0.9400 | 0.6 |
5 |
0.9237 | 0.9777 | 5.9 | 0.9535 | 3.2 | 0.9350 | 1.2 | 1.0251 | 10.9 | 0.9312 | 0.2 |
8 |
0.9237 | 0.9960 | 7.9 | 0.8841 | −4.3 | 0.9019 | −2.3 | 1.0201 | 10.4 | 0.9258 | 0.8 |
11 |
0.9237 | 0.9724 | 5.3 | 0.9271 | 0.4 | 0.9154 | −0.9 | 1.0209 | 10.5 | 0.9273 | 1.7 |
11 |
0.9180 | 0.9612 | 4.7 | 0.9250 | 0.8 | 0.9268 | 1.0 | 1.0080 | 9.8 | 0.9241 | 0.4 |
8 |
– | 0.9278 | – | 0.9166 | – | 0.9409 | – | 1.0141 | – | 0.9272 | – |
11 |
– | 0.9466 | – | 0.9269 | 0.9247 | 1.0120 | 0.9336 | ||||
5 |
– | 0.8855 | – | 0.9467 | – | 0.8793 | – | 0.9754 | 0.9034 | – | |
5 |
– | 0.9371 | – | 0.9364 | – | 0.9478 | – | 1.0474 | – | 0.8891 | – |
22:0 | 0.9176 | 1.0016 | 9.8 | 0.8893 | −2.5 | 0.8941 | −0.2 | 0.9663 | 5.3 | 0.9096 | 1.6 |
13 |
0.9124 | 0.9459 | 4.3 | 0.9058 | −0.2 | 0.8995 | −0.9 | 1.0426 | 14.2 | 0.9022 | −0.3 |
13 |
0.9071 | 0.9624 | 6.1 | 0.8832 | −2.7 | 0.8980 | −1.0 | 1.0472 | 16.1 | 0.9238 | 2.8 |
13 |
0.9019 | 0.9503 | 5.4 | 0.9025 | 0.1 | 0.9213 | 2.0 | 1.0787 | 19.6 | 0.9472 | 6.1 |
7 |
0.8967 | 0.9112 | 1.7 | 0.9255 | 3.2 | 0.9270 | 3.3 | 1.0510 | 17.2 | 0.9211 | 0.5 |
7 |
– | 0.9369 | – | 0.9361 | – | 0.9613 | – | 1.1128 | – | 0.9682 | – |
4 |
– | 0.9448 | – | 0.9538 | – | 0.9855 | – | 1.1391 | – | 0.9954 | – |
24:0 | 0.9076 | 0.9759 | 8.7 | 0.8900 | −0.9 | 0.8850 | −1.5 | 1.0579 | 16.5 | 0.9525 | −1.3 |
15 |
0.9029 | 0.9073 | 1.6 | 0.8881 | −0.6 | 0.8930 | −0.1 | 1.0344 | 14.6 | 0.8977 | 5.0 |
Abbreviations: TCFr, Theoretical correction factor; ECF, Empirical correction factor; Δ, Difference between TCFr and ECF expressed as percentage. Values are the mean of triplicates.
A total of 49 FAMEs were identified and quantified in the reference standard BCR-163; 26 FAMEs corresponded to the seven certified FAMEs including, as appropriate, their positional and geometrical isomers. The mean ± SD (n = 5) and recovery percentage for each certified FAME were calculated from the data presented by the five laboratories, reaching a final mean of 92.84 and 92.01 g·100g−1 total FAME using ECF and TCFr with recovery percentages of 97.4% and 96.5%, respectively (
Determined FAME content of the BCR 163 certified reference material and their respective recovery
FATTY ACIDS | Certified FAME content (g·100 g−1 FAME) | Determined FAME content by ECF (g·100 g−1 FAME) | Recovery (%) | Determined FAME content by TCFr (g·100 g−1 FAME) | Recovery (%) |
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14:0 |
2.29±0.04 | 2.16±0.11 | 94.3 | 2.16±0.07 | 94.3 |
16:0 |
25.96±0.30 | 25.28±0.65 | 97.4 | 25.42±0.36 | 97.9 |
16:1# | 2.58±0.16 | 2.37±0.22 | 91.9 | 2.26±0.22 | 87.6 |
18:0 |
18.29±0.17 | 17.80±0.66 | 97.3 | 18.04±0.40 | 98.6 |
18:1 |
38.30±0.40 | 37.50±0.69 | 97.9 | 36.52±2.53 | 95.4 |
18:2 |
7.05±0.17 | 6.95±0.13 | 98.6 | 6.85±0.16 | 97.2 |
18:3 | 0.86±0.14 | 0.78±0.07 | 90.7 | 0.76±0.07 | 88.4 |
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Mean value ± SD (n = 5). For abbreviations see
Includes, as appropriate, positional and geometrical (i.e. cis/trans) isomers.
The same modified temperature program indicated in 3.1 was applied in the GLC analysis of FAMEs from three commercial fat samples: a mixture of soybean and sunflower oil (80/20), fish oil and anhydrous butterfat. For the soybean/sunflower mixed oil, FAMEs were calculated by ECF and TCFr values, to compare both results according to the data discussed in 3.3. This procedure was not applied for fish oil or anhydrous butterfat (Mossoba and Kramer,
Eighteen FAs were identified and quantified in the soybean and sunflower oil mixture (80/20), seven SFAs, six MUFAs and five PUFAs, including
Fatty acid composition of refined mixture of soybean and sunflower oil
FATTY ACIDS | FAMEECF (g FAME·100 g−1 FAME) | FAECF (g FA·100g−1 FA) | TAGeECF (%) | FAMETCFr (g FAME·100 g−1 FAME) | FATCFr (g FA·100 g−1 FA) | TAGeTCFr (%) | *FACF | *TAGCF |
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14:0 | 0.09±0.02 | 0.09±0.02 | 0.09 | 0.09±0.02 | 0.09±0.02 | 0.09 | 0.9421 | 0.9945 |
16:0 | 10.50±0.17 | 9.96±0.15 | 10.45 | 10.51±0.19 | 9.96±0.18 | 10.46 | 0.9481 | 0.9950 |
17:0 | 0.10±0.02 | 0.09±0.01 | 0.10 | 0.10±0.01 | 0.09±0.01 | 0.10 | 0.9507 | 0.9953 |
18:0 | 4.43±0.19 | 4.23±0.18 | 4.41 | 4.49±0.19 | 4.28±0.16 | 4.47 | 0.9530 | 0.9955 |
20:0 | 0.15±0.05 | 0.14±0.04 | 0.15 | 0.15±0.05 | 0.14±0.05 | 0.15 | 0.9570 | 0.9959 |
22:0 | 0.41±0.04 | 0.39±0.04 | 0.41 | 0.41±0.03 | 0.39±0.03 | 0.41 | 0.9604 | 0.9962 |
24:0 | 0.15±0.03 | 0.14±0.03 | 0.15 | 0.15±0.03 | 0.14±0.03 | 0.15 | 0.9633 | 0.9965 |
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9 |
0.09±0.01 | 0.08±0.02 | 0.09 | 0.08±0.02 | 0.08±0.01 | 0.08 | 0.9477 | 0.9950 |
9 |
21.63±0.41 | 20.61±0.42 | 21.53 | 21.54±0.44 | 20.52±0.45 | 21.44 | 0.9477 | 0.9950 |
11 |
1.27±0.04 | 1.21±0.03 | 1.26 | 1.25±0.05 | 1.19±0.05 | 1.24 | 0.9527 | 0.9955 |
5 |
0.83±0.12 | 0.79±0.13 | 0.83 | 0.77±0.05 | 0.74±0.05 | 0.77 | 0.9568 | 0.9959 |
8 |
0.68±0.06 | 0.65±0.05 | 0.68 | 0.66±0.06 | 0.63±0.06 | 0.66 | 0.9568 | 0.9959 |
11 |
0.15±0.02 | 0.14±0.03 | 0.15 | 0.14±0.01 | 0.14±0.01 | 0.14 | 0.9568 | 0.9959 |
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9 |
0.55±0.03 | 0.52±0.05 | 0.55 | 0.54±0.03 | 0.51±0.03 | 0.54 | 0.9524 | 0.9954 |
9 |
0.55±0.06 | 0.52±0.04 | 0.55 | 0.54±0.07 | 0.51±0.07 | 0.54 | 0.9524 | 0.9954 |
9 |
53.93±0.57 | 51.36±0.84 | 53.68 | 53.56±0.65 | 51.01±0.63 | 53.31 | 0.9524 | 0.9954 |
6 |
0.11±0.01 | 0.10±0.01 | 0.11 | 0.10±0.02 | 0.10±0.01 | 0.10 | 0.9520 | 0.9954 |
9 |
4.76±0.29 | 4.53±0.27 | 4.74 | 4.69±0.22 | 4.46±0.21 | 4.67 | 0.9520 | 0.9954 |
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Mean value ± SD (n = 5). TAGe, Triacylglycerol equivalents; CF, Conversion factor. For other abbreviations see
PUFA was the predominant group (60%), with linoleic acid being the main FA with a mean value of 54%. MUFAs were 24%, with oleic acid being the major constituent with 21%. SFAs represented 15% and the main one was palmitic acid, reaching 11%. The total g FAME·100 g−1 FAME using ECF or TCFr was up to 99% in both cases, and the conversion to g·100 g−1 FA and TAGe gave values of 95% and 99.9%, respectively, which were considered satisfactory. Thus, calculated ECF or tabulated TCFr can be used for quantitative purposes in this case. Chromatograms of the FAME separation and emerging time in two zones of the total FAMEs identified and quantified in this mixture of vegetable oils are presented in
A. Significant part of the chromatogram of mixed soybean oil/Sunflower oil (80/20) showing the zone of FAMEs 9
B. Significant part of the chromatogram of mixed soybean oil/sunflower oil (80/20) showing the zone of FAMEs 20:0 to 24:0 using the proposed temperature program.
C. Significant part of the chromatogram of fish oil capsules showing the zone of FAMEs 9
D. Significant part of the chromatogram of fish oil capsules showing the zone of FAMEs 5
E. Significant part of the chromatogram of fish oil capsules showing the zone of FAMEs 24:1 to 4
Ratnayake
In total, sixty three FAMEs were identified and quantified in the fish oil sample (
Fatty acid composition of fish oil
FATTY ACIDS | FAMEECF (g FAME·100 g−1FAME) | FAECF (g FA·100 g−1 FA) | TAGeECF (%) | *FACF | *TAGCF |
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12:0 | 0.11±0.02 | 0.10±0.01 | 0.11 | 0.9346 | 0.9937 |
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0.02±0.01 | 0.02±0.01 | 0.02 | 0.9386 | 0.9941 |
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0.05±0.01 | 0.05±0.01 | 0.05 | 0.9421 | 0.9945 |
14:0 | 7.35±0.43 | 6.92±0.45 | 7.31 | 0.9421 | 0.9945 |
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0.22±0.02 | 0.21±0.02 | 0.22 | 0.9453 | 0.9948 |
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0.06±0.01 | 0.06±0.01 | 0.06 | 0.9453 | 0.9948 |
15:0 | 0.52±0.02 | 0.49±0.03 | 0.52 | 0.9453 | 0.9948 |
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0.08±0.01 | 0.07±0.01 | 0.08 | 0.9481 | 0.9950 |
16:0 | 16.18±1.10 | 15.34±0.86 | 16.10 | 0.9481 | 0.9950 |
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0.20±0.03 | 0.19±0.03 | 0.20 | 0.9507 | 0.9953 |
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0.08±0.03 | 0.08±0.03 | 0.08 | 0.9507 | 0.9953 |
17:0 | 0.43±0.01 | 0.41±0.01 | 0.43 | 0.9507 | 0.9953 |
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0.14±0.03 | 0.13±0.02 | 0.14 | 0.9530 | 0.9955 |
18:0 | 3.21±0.27 | 3.06±0.19 | 3.20 | 0.9530 | 0.9955 |
20:0 | 0.19±0.03 | 0.18±0.03 | 0.19 | 0.9570 | 0.9959 |
22:0 | 0.16±0.05 | 0.15±0.04 | 0.16 | 0.9602 | 0.9962 |
24:0 | 0.03±0.02 | 0.03±0.02 | 0.03 | 0.9630 | 0.9965 |
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9 |
0.06±0.03 | 0.06±0.03 | 0.06 | 0.9417 | 0.9944 |
10 |
0.04±0.01 | 0.03±0.02 | 0.04 | 0.9449 | 0.9947 |
9 |
0.43±0.03 | 0.41±0.04 | 0.43 | 0.9477 | 0.9950 |
11 |
0.12±0.02 | 0.12±0.02 | 0.12 | 0.9704 | 0.9950 |
9 |
8.39±0.43 | 8.14±0.42 | 8.35 | 0.9704 | 0.9950 |
13 |
0.19±0.06 | 0.18±0.05 | 0.19 | 0.9704 | 0.9950 |
13 |
0.07±0.02 | 0.07±0.02 | 0.07 | 0.9704 | 0.9950 |
6 |
1.10±0.09 | 1.07±0.12 | 1.10 | 0.9704 | 0.9955 |
9 |
0.06±0.02 | 0.06±0.01 | 0.06 | 0.9704 | 0.9955 |
10 |
0.06±0.01 | 0.06±0.01 | 0.06 | 0.9704 | 0.9955 |
9 |
9.67±0.59 | 9.38±0.40 | 9.63 | 0.9704 | 0.9955 |
11 |
3.10±0.23 | 3.01±0.16 | 3.09 | 0.9704 | 0.9955 |
13 |
0.09±0.02 | 0.08±0.02 | 0.09 | 0.9704 | 0.9955 |
14 |
0.10±0.03 | 0.09±0.03 | 0.10 | 0.9704 | 0.9955 |
5 |
0.14±0.03 | 0.13±0.02 | 0.14 | 0.9520 | 0.9959 |
11 |
0.75±0.07 | 0.72±0.06 | 0.75 | 0.9568 | 0.9959 |
11 |
0.13±0.02 | 0.12±0.02 | 0.13 | 0.9600 | 0.9962 |
15 |
0.35±0.02 | 0.34±0.02 | 0.35 | 0.9628 | 0.9965 |
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9 |
0.29±0.06 | 0.27±0.06 | 0.29 | 0.9473 | 0.9950 |
6 |
0.19±0.03 | 0.18±0.03 | 0.19 | 0.9469 | 0,9950 |
7 |
0.08±0.01 | 0.08±0.01 | 0.08 | 0.9469 | 0.9950 |
3 |
0.08±0.02 | 0.08±0.02 | 0.08 | 0.9465 | 0.9950 |
4 |
0.13±0.03 | 0.12±0.03 | 0.13 | 0.9465 | 0.9950 |
6 |
1.04±0.13 | 0.98±0.14 | 1.03 | 0.9465 | 0.9950 |
9 |
0.08±0.01 | 0.08±0.02 | 0.08 | 0.9524 | 0.9954 |
8 |
0.15±0.04 | 0.14±0.04 | 0.15 | 0.9524 | 0.9954 |
9 |
2.04±0.12 | 1.94±0.14 | 2.03 | 0.9524 | 0.9954 |
11 |
0.36±0.05 | 0.34±0.05 | 0.36 | 0.9524 | 0.9954 |
9 |
0.28±0.03 | 0.27±0.03 | 0.28 | 0.9524 | 0.9954 |
7 |
0.24±0.03 | 0.23±0.03 | 0.24 | 0.9524 | 0.9954 |
6 |
0.20±0.02 | 0.19±0.03 | 0.20 | 0.9520 | 0.9954 |
9 |
0.71±0.08 | 0.68±0.07 | 0.71 | 0.9520 | 0.9954 |
6 |
2.34±0.34 | 2.23±0.36 | 2.33 | 0.9517 | 0.9954 |
11 |
0.17±0.03 | 0.16±0.03 | 0.17 | 0.9565 | 0.9958 |
8 |
0.11±0.03 | 0.11±0.03 | 0.11 | 0.9562 | 0.9958 |
11 |
0.32±0.01 | 0.31±0.02 | 0.32 | 0.9562 | 0.9958 |
5 |
0.84±0.06 | 0.80±0.07 | 0.84 | 0.9560 | 0.9958 |
8 |
0.05±0.01 | 0.04±0.01 | 0.05 | 0.9560 | 0.9958 |
5 |
16.85±0.99 | 16.10±0.87 | 16.78 | 0.9557 | 0.9958 |
4 |
0.62±0.07 | 0.59±0.08 | 0.62 | 0.9578 | 0.9961 |
13 |
0.80±0.12 | 0.77±0.12 | 0.80 | 0.9600 | 0.9962 |
13 |
0.04±0.01 | 0.04±0.02 | 0.04 | 0.9598 | 0.9961 |
7 |
0.09±0.01 | 0.09±0.02 | 0.09 | 0.9595 | 0.9961 |
4 |
0.31±0.06 | 0.30±0.06 | 0.31 | 0.9593 | 0.9961 |
7 |
2.19±0.14 | 2.10±0.14 | 2.18 | 0.9593 | 0.9961 |
4 |
10.93±0.79 | 10.48±0.75 | 10.89 | 0.9590 | 0.9961 |
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Mean value ± SD (n = 5). For abbreviations see
For the identification of FAMEs, different standards and literature data were consulted. The standard GLC 463 has 52 FAMEs, 34 of them being present in the fish oil analyzed (65%). Santercole
Chromatograms of the FAME in three zones of the total FAMEs identified and quantified in the fish oil sample analyzed are presented.
The PUFA group achieved the major percentage of the total FAs (40%), 20:5 n-3 and 22:6 n-3 being the most abundant with 16.9% and 10.9%, respectively. This FA composition agrees with data published for fish oils, where SFAs and MUFAs are in equivalent proportion and PUFAs are the prevalent group (SFAs: MUFAs: PUFAs/ 1.0: 0.9: 1.4) (Romero
Anhydrous butterfat is another complex animal fat to analyze FA composition by GLC due the large number of FAs with wide carbon chain numbers and different isomers, starting with butyric acid (4:0) as shown in
Significant parts of the chromatogram of anhydrous butterfat showing the zones of FAMEs: A. 4:0 to 14:0, B. 4
Each laboratory reported the mean value for each FAME identified, expressed as g FAME·100g−1 FAMEs at a final mean value ± SD (n = 5) for each FAME and the results are presented in
Fatty acid composition of anhydrous butterfat
FATTY ACIDS | FAMEECF (g FAME·100 g−1 FAME) | FAECF (g FA·100 g−1 FA) | TAGeECF (%) | FACF | TAGCF |
---|---|---|---|---|---|
4:0 | 2.33±0.73 | 1.99±0.62 | 2.30 | 0.8623 | 0.9868 |
5:0 | 0.06±0.05 | 0.05±0.05 | 0.06 | 0.8792 | 0.9884 |
6:0 | 1.89±0.31 | 1.69±0.28 | 1.87 | 0.8922 | 0.9897 |
7:0 | 0.03±0.00 | 0.03±0.00 | 0.03 | 0.9027 | 0.9907 |
8:0 | 1.24±0.11 | 1.13±0.13 | 1.23 | 0.9114 | 0.9915 |
9:0 | 0.03±0.01 | 0.03±0.01 | 0.03 | 0.9186 | 0.9922 |
10:0 | 2.81±0.27 | 2.60±0.26 | 2.79 | 0.9247 | 0.9928 |
11:0 | 0.06±0.02 | 0.06±0.01 | 0.06 | 0.9296 | 0.9933 |
12:0 | 3.34±0.43 | 3.12±0.42 | 3.32 | 0.9346 | 0.9937 |
|
0.05±0.01 | 0.05±0.01 | 0.05 | 0.9386 | 0.9941 |
|
0.07±0.01 | 0.07±0.01 | 0.07 | 0.9386 | 0.9941 |
14:0 | 10.74±0.58 | 10.12±0.68 | 10.68 | 0.9421 | 0.9945 |
|
0.59±0.07 | 0.56±0.07 | 0.59 | 0.9453 | 0.9948 |
15:0 | 1.18±0.14 | 1.12±0.14 | 1.17 | 0.9453 | 0.9948 |
|
0.26±0.03 | 0.25±0.03 | 0.26 | 0.9481 | 0.9950 |
16:0 | 26.74±1.01 | 25.35±1.05 | 26.61 | 0.9481 | 0.9950 |
Iso-17:0 | 0.09±0.01 | 0.08±0.02 | 0.09 | 0.9507 | 0.9953 |
17:0 | 0.62±0.06 | 0.60±0.06 | 0.62 | 0.9507 | 0.9953 |
18:0 | 11.27±0.83 | 10.74±0.73 | 11.22 | 0.9530 | 0.9955 |
19:0 | 0.08±0.03 | 0.08±0.02 | 0.08 | 0.9551 | 0.9957 |
20:0 | 0.15±0.03 | 0.14±0.03 | 0.15 | 0.9570 | 0.9959 |
22:0 | 0.07±0.02 | 0.07±0.02 | 0.07 | 0.9602 | 0.9962 |
24:0 | 0.05±0.01 | 0.04±0.01 | 0.05 | 0.9630 | 0.9965 |
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9 |
0.24±0.05 | 0.22±0.04 | 0.24 | 0.9239 | 0.9927 |
9 |
0.08±0.02 | 0.08±0.02 | 0.08 | 0.9339 | 0.9937 |
12 |
0.12±0.02 | 0.11±0.02 | 0.12 | 0.938 | 0.9941 |
9 |
0.36±0.16 | 0.34±0.15 | 0.36 | 0.9416 | 0.9944 |
9 |
0.88±0.10 | 0.83±0.09 | 0.88 | 0.9416 | 0.9944 |
9 |
0.13±0.03 | 0.12±0.02 | 0.13 | 0.9477 | 0.9950 |
11 |
0.38±0.17 | 0.36±0.16 | 0.38 | 0.9477 | 0.9950 |
9 |
1.26±0.24 | 1.19±0.23 | 1.25 | 0.9477 | 0.9950 |
10 |
0.50±0.06 | 0.47±0.22 | 0.50 | 0.9477 | 0.9950 |
11 |
0.16±0.04 | 0.15±0.04 | 0.16 | 0.9477 | 0.9950 |
9 |
0.23±0.04 | 0.22±0.04 | 0.23 | 0.9503 | 0.9952 |
4 |
0.04±0.01 | 0.04±0.01 | 0.04 | 0.9527 | 0.9955 |
6 |
0.33±0.06 | 0.31±0.06 | 0.33 | 0.9527 | 0.9955 |
9 |
0.26±0.02 | 0.25±0.01 | 0.26 | 0.9527 | 0.9955 |
10 |
0.57±0.03 | 0.54±0.04 | 0.57 | 0.9527 | 0.9955 |
11 |
2.39±0.12 | 2.28±0.11 | 2.38 | 0.9527 | 0.9955 |
13 |
0.22±0.06 | 0.21±0.06 | 0.22 | 0.9527 | 0.9955 |
9 |
21.80±0.98 | 20.77±1.02 | 21.70 | 0.9527 | 0.9955 |
11 |
0.90±0.05 | 0.86±0.04 | 0.90 | 0.9527 | 0.9955 |
12 |
0.27±0.02 | 0.26±0.02 | 0.27 | 0.9527 | 0.9955 |
13 |
0.08±0.02 | 0.08±0.02 | 0.08 | 0.9527 | 0.9955 |
14 |
0.06±0.00 | 0.06±0.01 | 0.06 | 0.9527 | 0.9955 |
16 |
0.49±0.07 | 0.47±0.07 | 0.49 | 0.9527 | 0.9955 |
15 |
0.18±0.07 | 0.17±0.07 | 0.18 | 0.9527 | 0.9955 |
8 |
0.05±0.01 | 0.05±0.01 | 0.05 | 0.9568 | 0.9959 |
11 |
0.01±0.01 | 0.01±0.00 | 0.01 | 0.9568 | 0.9959 |
15 |
0.04±0.01 | 0.03±0.01 | 0.04 | 0.9628 | 0.9965 |
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9 |
0.11±0.01 | 0.11±0.02 | 0.11 | 0.9524 | 0.9954 |
9 |
0.15±0.01 | 0.14±0.01 | 0.15 | 0.9524 | 0.9954 |
9 |
0.05±0.03 | 0.05±0.03 | 0.05 | 0.9524 | 0.9954 |
9 |
0.52±0.31 | 0.50±0.35 | 0.52 | 0.9524 | 0.9954 |
11 |
0.08±0.02 | 0.08±0.02 | 0.08 | 0.9524 | 0.9954 |
9 |
2.11±0.20 | 2.01±0.21 | 2.10 | 0.9524 | 0.9954 |
9 |
0.08±0.02 | 0.08±0.03 | 0.08 | 0.9524 | 0.9954 |
6 |
0.03±0.01 | 0.03±0.01 | 0.03 | 0.9520 | 0.9954 |
9 |
0.75±0.22 | 0.71±0.21 | 0.75 | 0.9520 | 0.9954 |
9 |
1.19±0.43 | 1.13±0.42 | 1.18 | 0.9524 | 0.9954 |
9 |
0.02±0.00 | 0.02±0.00 | 0.02 | 0.9524 | 0.9954 |
10 |
0.05±0.03 | 0.05±0.03 | 0.05 | 0.9524 | 0.9954 |
9 |
0.03±0.01 | 0.03±0.01 | 0.03 | 0.9524 | 0.9954 |
11 |
0.03±0.01 | 0.03±0.01 | 0.03 | 0.9524 | 0.9954 |
8 |
0.08±0.01 | 0.08±0.01 | 0.08 | 0.9524 | 0.9954 |
7 |
0.03±0.02 | 0.03±0.02 | 0.03 | 0.9524 | 0.9954 |
11 |
0.10±0.02 | 0.09±0.02 | 0.10 | 0.9565 | 0.9958 |
8 |
0.07±0.02 | 0.06±0.02 | 0.07 | 0.9562 | 0.9958 |
11 |
0.06±0.05 | 0.06±0.05 | 0.06 | 0.9560 | 0.9958 |
13 |
0.04±0.02 | 0.04±0.02 | 0.04 | 0.9600 | 0.9962 |
5 |
0.04±0.02 | 0.04±0.02 | 0.04 | 0.9560 | 0.9958 |
5 |
0.06±0.03 | 0.06±0.03 | 0.06 | 0.9557 | 0.9958 |
7 |
0.08±0.01 | 0.08±0.02 | 0.08 | 0.9593 | 0.9961 |
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Mean value ± SD (n = 5). For abbreviations see
Seventy-three FAMEs were identified and quantified. They were organized by groups, SFAs, MUFAs and PUFAs, including their respective positional and geometric isomers, as shown in
The SFAs represented more than 60%, MUFAs about 32% and PUFAs the lowest percentage at 5.7%, which is characteristic of ruminant fats. Seppänen-Laakso
Due to the complexity of CLA separation, combined techniques have been used in dairy and beef fats (Kramer
Rozema
Rozema
Kramer
This work shows the advantages of the improved temperature program developed and applied among the five laboratories for FA -analysis in some commercial fat samples compared with the one indicated in the AOCS Method Ce-1j 07 (AOCS,
This work was financed by Universidad Nacional del Litoral-Cursos de Acción para la Investigación y Desarrollo (CAI + D 2009) - Secretaría de Ciencia y Técnica- UNL- Argentina and Agencia Nacional de Promoción Científica, Tecnológica y de Innovación (ANPCyT - FITS # 001/2010) Argentina. This research was also supported with funds from CAPES, Brasil; Instituto de Nutrição Universidade Federal do Rio de Janeiro, RJ, Brasil; Faculdade de Ciências Farmacêuticas Universidade de San Paulo, SP, Brasil; INCIENSA, San José, Costa Rica and Facultad de Ciencias Químicas y Farmacéuticas, Universidad de Chile, Santiago, Chile. Finally, the authors wish to thank CYTED (Programa de Ciencia y Tecnología para el Desarrollo – Ministerio Español de Ciencia y Tecnología–BFI2002-00218) for the financial support of the Red Temática 208RT0343, where this research was integrated.