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Eur. J. Lipid Sci. Technol. 105 (2003) 683–687 DOI 10.1002/ejlt.200300837 683

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1 Introduction

Oils and fats of vegetable and animal origin are importantsources of energy in the human diet. In these oils fattyacids are bound to glycerol molecules called triacylglyc-erol or triglyceride. Although unsaturated fatty acids aregenerally present in nature with cis-double bond configu-ration, some fatty acids with trans-double bond configura-tion can also be formed. Trans isomers are formed by en-zymatic hydrogenation of dietary triacylglycerol fattyacids, containing more than one double bond in their hy-drocarbon chain. The process is catalyzed by an enzymeproduced by microorganisms in the rumen of ruminants[1]. For instance, trans-3-hexadecenoic acid is a substan-tial component of plant chloroplast lipids, and its isomertrans-11-octadecenoic acid (vaccenic) is found in the ru-men as a major by-product of biohydrogenation [2].Vaccenic acid is also found in all dairy products [3-5].

Furthermore, trans-9-octadecenoic acid (elaidic) exists inlow levels in human adipose tissues. It is acknowledgedthat most of the elaidic acid in human tissues is derivedfrom margarine consumptions [6, 7]. A large number ofgeometric and positional isomers of both cis- and trans-fatty acids are formed during the hydrogenation process –a major industry in many countries producing fixed-ediblefats with good organoleptic properties resistant to oxida-tion. Unsaturated fatty acids undergo two changes during

this process. One is geometric isomerization known as cisto trans transformation of double bonds. The second ispositional isomerization due to double bond migrationalong the fatty acid chain. As a result, trans-oleic (elaidicacid), cis-, trans-11-isooleic, and cis-, trans-7-isooleicacids are formed from oleic acid. In addition, more com-plex isomers are formed from linoleic and linolenic acidsknown as polyunsaturated fatty acids. Many factors canaffect the formation of these unusual products, the fore-most of which are temperature, alkalinity and the catalyst.Margarines are usually manufactured under high pres-sure and temperature in the presence of nickel (Ni) cata-lyst. The above mentioned reactions are observed underthese conditions. The effect of these conditions on transisomer formations and hydrogen concentration on thenickel catalyst surface, were investigated [8].

Melting points of trans fatty acid isomers are somewhathigher than that of cis isomers. For instance, meltingpoints of oleic acid and elaidic acid are 13.4 °C and 44 °C,respectively. It is reported that trans isomers of these fat-ty acids have negative effects on plasma lipoprotein lev-els and increase the risk of cardiovascular disease [9].Physiological effects of different isomers formed from var-ious natural fatty acids (oleic, linoleic and linolenic) arestill under investigation. Hence, determination of isomercontent of margarines is an important task. There aresome common methods reported in the literature for de-tecting trans isomers in margarines, such as capillary gaschromatography (GC), Fourier transform - infrared (FT-IR) spectroscopy, and high performance liquid chro-matography (HPLC) [10].

Mehmet Çetina

Ayhan Yıldırıma, A. Metin Sahinb

a Department of Chemistry,Faculty of Science andArts, Uludag University,Bursa, Turkey

b Helvacızade Food ProductInc., Konya, Turkey

Determination of fatty acids and someundesirable fatty acid isomers in selected TurkishmargarinesThe fatty acid composition and total trans fatty acid content in 10 margarines producedin Turkey were determined by capillary gas chromatography and Fourier transform - in-frared spectroscopy (FT-IR) spectroscopy. The fatty acid composition ranged as fol-lows: saturated fatty acids, C16:0 (palmitic) 11.3 to 31.8% and C18:0 (stearic) 5.7 to8.7%, monounsaturated fatty acids, C18:1 (oleic) 21.8 to 35.7% and C18:1 trans iso-mers 0.4 to 27.4%, polyunsaturated fatty acid, C18:2 linoleic acid 5.2 to 40.2%. Somepositional isomers of C18:1 as cis-11-octadecenoic acid varied from 0.7 to 4.6% andcis-13 trace to 2.4%. The total trans fatty acid contents were between 0.9 and 32.0%when measured with capillary gas chromatography and between 0 and 30.2% with FT-IR spectroscopy. Some of the margarines analyzed contained trace amount of transfatty acids which could not be detected by FT-IR spectroscopy.

Keywords: Trans fatty acids, margarines, capillary gas chromatography (GC), FT-IRspectroscopy.

Correspondence: Mehmet Çetin, Department of Chemistry,Faculty of Science and Arts, Uludag University, 16059 Bursa,Turkey, Phone: + 90-224-442-9256 or -1305; Fax: + 90-224-4512767; e-mail: mcetin@uludag.edu.tr

In this study, capillary GC and FT-IR spectroscopic meth-ods were used to determine the amounts of some posi-tional and trans isomers of fatty acids, and the total transfatty acids content of 10 Turkish margarines.

2 Materials and methods

2.1 Samples

Ten margarines were purchased from local suppliers inKestel and Bursa, Turkey, between 2001 and 2002. Sam-ples were stored at –18 °C prior to analysis. Approximate-ly 1 g of each margarine sample was melted at 40-50 °Cin a 10 ml centrifuge tube. Samples were then centrifugedfor 4 min at 2000 rpm, and the supernatant removed witha Pasteur pipette. Fatty acid methyl esters were preparedby transesterification using 1% sulfuric acid in methanol[11].

2.2 Total trans fatty acids determination by FT-IR spectroscopy

An ATI Unicam Mattson 1000 FT-IR spectrometer with aRocket interchangeable glass syringe (2 ml capacity),and a Graseby Specac variable path length liquid cell withKBr windows (Serial No. 4785) were used. Methyl elai-date (99.9% GC grade, Sigma Chemical Co., St. Louis,MO, USA) was used as an external standard with CS2

(Merck, Darmstadt, Germany) as solvent. The analyticalprocedure according to Ratnayake [12] was adopted withminor modifications. A stock solution of methyl elaidatewas prepared by dissolving 400 mg in 20 ml CS2. To pre-pare a series of calibration standards, 0.5 ml, 1.0 ml, 1.5 ml, 2.0 ml, 2.5 ml, 3.0 ml, 4.0 ml and 5.0 ml of thisstock solution was diluted with CS2 to a final volume of 10 ml. The esterified 100 mg oil samples were diluted with10 ml CS2 before use. FT-IR spectra of standard andsample solutions were collected at 4 cm–1 resolution inthe 1110–910 cm–1 range. The regression equationY=5.3185X-0.0049, where Y = absorbance, X = g methylelaidate/10 ml CS2, and r2 = 0.9911.

2.3 Fatty acids composition determination bycapillary GC

We determined fatty acid composition by fatty acid methylester (FAME) analysis with a HP 6890 gas chromato-graph equipped with an auto sampler, flame ionization de-tector, and 6 m × 0.2 mm i.d. SP 2380 fused silica capil-lary column coated with 0.2 µm of stabilized poly-90% bis-cyanopropyl/ 10% cyanopropylphenyl siloxane (SupelcoInc., Bellefonte, PA, USA). Hydrogen was used as carriergas at a flow rate of 0.7 ml/min. Temperatures of injectorand detector were 250 °C and 260 °C respectively. Theoven was programmed at 50 °C initial temperature and2 min initial time. Thereafter the temperature increased

4 °C/min to 250 °C and held for 15 min at 250 °C. Totalruntime was 67 min. Samples were analyzed in splitmode (100:1), and FAME peaks were identified by com-parison with retention times of standards. Trans contentsare given in relative % of total GC areas of fatty acids.

It is known that high polarity of SP-2380 phase, allows theseparation of geometric (cis/trans) fatty acid methyl esterisomers as a group. Under the conditions described, cis-C18:1 and trans-C18:1 isomers separated into two dis-tinct peak groups in all samples except sample D. Somepositional isomers of cis-C18:1 were clearly separatedfrom each other (cis-9 C18:1, cis-11 C18:1) (Fig. 1). Chro-matogram (Fig. 1) of sample L was probably producedwithout hydrogenation. Consequently, trans fatty acid iso-mers of this margarine may originate from the refiningprocess. Due to high temperatures used in the deodoriza-tion step and lower activation energy, geometric isomersof this double bond may be present we did not however,investigate these. Cis-13 C18:1 oleic acid isomer was notdetected in sample L, but all other samples contained thisisomer.

3 Results and discussion

The fatty acid compositions of 10 brands of Turkish mar-garines are given in Tab. 1. The margarines were codedas A, B, C, D, E, F, G, H, K, and L, but not grouped as hardand soft species. C16:0 was the major saturated ingredi-ent in all samples, and ranged from 11.3 to 31.8% of peakareas in GC chromatograms (Tab. 1). All samples ana-lyzed had approximately the same amount of C18:0 fattyacid. Positional isomers of C18:1, trans-9, -10, and -11are the major trans fatty acids in hardened vegetable oils[13, 21].

Specific cis and trans isomers of C18:1 show co-elution.For instance, the trans-C18:1 isomer with double bonds inthe 12-, 13-, 14-, or 15-positions, co-elute with the cis iso-mers. Since we did not fractionate the fatty acids as satu-

684 Çetin et al. Eur. J. Lipid Sci. Technol. 105 (2003) 683–687

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Fig. 1. FT-IR spectroscopy analysis results.

rated, monounsaturated trans, monounsaturated cis, andpolyunsaturated with argentation thin-layer chromatogra-phy (Ag-TLC) prior to capillary GC analysis, it is possiblethat we were not able to obtain complete resolution for allcis and trans positional fatty acid isomers using capillarylong column coated with highly polar stationary phase.Fig. 2 shows chromatographic traces of FAMEs for sam-ple L. The C18:1 trans content of B, E and L margarines(produced by the same firm), were less than 2.5%, We didnot detect the deformation band of the =C–H bond nor-mally seen as absorbance at 965 cm-1 with FT-IR spec-

troscopy. Such margarines, containing nearly zero transfatty acids are desirable [14]. The amount of C18:1 cis-9fatty acid ranged from 21.8 to 35.7% (Tab. 1). The C18:1cis-11, formed during the hydrogenation process fromdouble bond migrations, was highest in sample C with avalue of 4.6%. Total possible amount of C18:2 trans iso-mers, was highest in sample D at 13.7% (Tab. 1). Fur-thermore, we found that C18:2 linoleic acids were entirelyconverted to various trans isomers in margarine D. We al-so encountered different trans isomers of C18:3 in allsamples.

Eur. J. Lipid Sci. Technol. 105 (2003) 683–687 Determination of fatty acids and some undesirable fatty … 685

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Tab. 1. Fatty acid composition (as percentage FAMEs) of various Turkish margarines.

Samples

Fatty acid A B C D E F G H K L

C16:0 (palmitic) 22.8 28.7 16.2 31.8 11.3 23.1 14.3 29.0 29.8 24.5C16:1 (trans)† – – – – – – – trace trace traceC16:1 (palmitoleic) 0.1 0.1 0.1 0.1 0.2 – 0.1 0.1 0.2 0.2C18:0 (stearic) 5.9 7.2 8.7 6.7 6.2 6.7 8.2 5.8 6.3 5.7C18:1 (trans) ‡ 19.0 0.6 24.0 0.2 0.9 16.7 27.4 15.9 8.7 0.4C18:1 (oleic) 24.8 22.8 34.4 31.6 35.6 25.8 22.6 35.7 34.3 21.8C18:1 (cis-vaccenic) 1.3 0.7 4.6 1.3 1.8 2.1 2.4 1.9 1.7 1.1C18:1 (cis-13-isooleic) 1.0 trace 0.6 1.5 0.4 0.7 1.6 2.4 1.4 –C18:2 (trans) § 2.7 0.4 1.33 13.7 0.3 1.0 4.4 2.3 1.5 0.3C18:2 (linoleic) 18.6 22.7 8.65 – 33.7 20.0 16.3 5.2 12.9 40.2C20:0 (arachidic) 0.4 0.4 0.4 0.5 0.4 0.4 0.4 0.4 0.4 0.4 C18:3 (trans) # 0.2 0.3 0.1 0.1 0.3 0.3 0.2 0.1 0.1 0.2C18:3 (α-linolenic) 1.2 1.7 0.2 0.7 3.6 2.6 1.7 0.3 0.9 2.9C20:1 (cis-11-eicosenoic) – 0.1 0.1 trace 0.4 – – 0.1 0.2 –C21:0 (henicosanoic) 0.3 0.2 0.4 0.6 0.5 0.2 – – – –C22:0 (behenic) trace – trace – 0.1 trace – – – –C22:1 (erucic) 0.3 0.1 0.2 trace – 0.1 – – – –

† Sum of C16:1 trans positional isomers.‡ Sum of C18:1 trans positional isomers.§ Sum of 9t,12c and 9c,12t isomers of C18:2.# Sum of unknown C18:3 trans isomers.

Fig. 2. GC chromatogram of sample L.

The total trans content of the margarines determined byGC was higher than that detected with FT-IR spec-troscopy, except for samples F and D. In addition, transfatty acid percentages were similar to each other with thetwo techniques in all samples, but GC showed consider-ably high value for sample F. Low and irregular resonanceenergy of FT-IR spectrometer seen during analysis of thismargarine, may have caused false absorbance measure-ment. Conjugated trans/trans- (about 990 cm-1) orcis/trans- (between 990 and 950 cm-1) double bonds ex-hibited absorption band interference with the isolatedtrans-double bond absorption. Broad bands of thesebonds overlap with trans absorptions at 966 cm-1.

The GC peaks of some cis and trans C18:1 isomers canoverlap and do not resolve well. Consequently, accuratedetermination is difficult due to poor resolution [15].Peaks produced by mono-cis and mono-trans C18:1 iso-mers using GC, did not completely separate from eachother for sample D. Consequently, higher trans contentfound by GC can be attributed to low resolution.

Margarines B, E and L were probably manufactured fromnon hydrogenated vegetable oils using an alternativemodification process known as interesterification. (Mar-garines produced by interesterification contain zero transfatty acids [14]). However, we did not investigate this hy-pothesis further. Trace amounts of trans isomers mightform during the deodorization step of the refining process.Usually at this stage monotrans isomers of polyunsaturat-ed fatty acids are formed [16].

The trans fatty acid percentages determined by FT-IRspectroscopy and GC are shown as bar graphics in Fig. 1and Fig. 3. Our results are in agreement with earlier re-ports [17, 18] on fatty acid composition of selected Turk-ish margarines. Tab. 2 shows a summary of earlier re-ports on the trans fatty acid content of margarines fromvarious countries. The highest trans fatty acid contentsreported are from Turkish and Argentinean margarines, incontrast to margarines manufactured in countries such asSweden, Finland, Hungary and Austria which can containvery little trans fatty acids.

4 Conclusions

FT-IR spectroscopy only provides measurement of the to-tal trans isomers whereas capillary GC provides veryclear separation and good quantifications for the various

686 Çetin et al. Eur. J. Lipid Sci. Technol. 105 (2003) 683–687

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Tab. 2. Reported trans fatty acid content of margarines in various countries.

Country Method Trans content Reference

New Zealand Ag-TLC/GC 14.70% [19]Australia FT-IR/GC 16.3%by FT-IR 14.5% by GC [20]Portuguese GC 9.0% [21]Argentina GC 31.9% [22]Scotland Ag-TLC/GC 15.3% [23]Germany Ag-TLC/GC 6.2% [24]Bulgaria Ag-TLC 11.2% [25]Belgium Ag-TLC/GC 3.1% [26] Czech Republic Ag-TLC/GC 9.6% [26]Finland Ag-TLC/GC 1.4% [26]Hungary Ag-TLC/GC 1.9% [26]Japan Ag-TLC/GC 18.5% [26]Norway Ag-TLC/GC 16.3% [26]Poland Ag-TLC/GC 16.7% [26]Sweden Ag-TLC/GC 1.1% [26]United States Ag-TLC/GC 14.7% [26]Austria Ag-TLC/GC 2.4% [26]Denmark Ag-TLC/GC 4.8% [26]Turkey FT-IR/GC 0-30.2% by FT-IR 0.9-32.0% by GC [ *]

[*] Our results.

Fig. 3. GC analysis results.

cis and trans fatty acid positional isomers. Fused silicacapillary columns are widely used to separate these iso-mers. We have determined the trans fatty acid contents ofsome Turkish margarines. The data obtained in this studyindicate that there are different margarines with variouslevels of saturated, trans and polyunsaturated fatty acidsavailable to the Turkish consumer. Results by FT-IR spec-troscopy and GC indicate that many domestic margarineshave higher trans contents than those from various otherEuropean countries.

Acknowledgements

This project was fully supported by Uludag University Re-search Foundation (Project No. 2001/69). Valuable dis-cussions with Dr. Demir C. are gratefully acknowledged.

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[Received: May 9, 2003; accepted: August 7, 2003]

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