REV. CHIM. (Bucharest) 63 No. 10 2012http://www.revistadechimie.ro984

Thermal Formation of trans Fatty Acids in Romanian VegetableOils Monitored by GC-MS and FT-IR Techniques

MIHAELA MIHALACHE1, AURELIA BRATU1, ANAMARIA HANGANU2*, NICOLETA-AURELIA CHIRA1, MARIA MAGANU2,MARIA-CRISTINA TODASC1, SORIN ROSCA11 University Politehnica of Bucharest, Faculty of Applied Chemistry and Materials Science, Costin D. Nenitzescu OrganicChemistry Department, 1-7 Polizu Str., 011061, Bucharest, Romania2 Romanian Academy, Organic Chemistry Center Costin D. Nenitzescu 202B Splaiul Independentei, 060023, Bucharest, Romania

Four different types of Romanian vegetable oils were heated in order to elucidate trans fatty acids accumulationduring thermal processing of oils in frequent domestic activities. The refined vegetable oils selected for thisstudy were sunflower, corn, soybean and linseed, heated at three different temperatures 180, 220 and 250oCfor 33 h. The amount of trans fatty acids formed during heating processes was determined using infraredspectroscopy (IR), based on trans specific bonds absorption at 966 cm-1 (while the absorption of the cisbonds appears at 724 cm-1). Based on the measurement of the absorption band at 966 cm-1, a calibrationcurve was obtained. The results were compared with those obtained by gas chromatography-massspectrometry technique (GC-MS) used as reference method to quantify the trans fats. The results obtaineddemonstrated that trans fatty acids formation in vegetable oils during heating, is closely related to processtemperature and time and also that trans fatty acids can only be formed under severe conditions.

Keywords: trans fatty acids, heated oils, Romanian vegetable oils, FT-IR, GC-MS

Trans fatty acids (TFAs) are unsaturated fatty acids thatcontain one or more isolated (non-conjugated) carbon-carbon double bonds in a trans configuration [1,2].

Naturally all unsaturated fatty acids in vegetable oils arein cis configuration, but during partial hydrogenation or as aresult of manufacturing processes (thermal refining,deodorization and bleaching) some cis-fatty acids may beisomerized to the trans configuration. Other process thatcan also lead to the formation of trans fatty acids arethermal processing of oils in frequent domestic activities(food-frying operations) [1-4].

Recent clinical and experimental studies reveal thatthere is a close correlation between high level of TFAsintake from the daily diet and the risk of cardiovasculardisease, by changing the plasma lipoprotein profile [1-4].TFAs produce an increase in low-density lipoproteins (LDL),so called bad cholesterol and a decrease of high-densitylipoproteins (HDL), so called good cholesterol [3,5].

It is suggested that fats for human daily consumption tocontain less than 2% (w/w) of the sum of all fatty acids(total fat) as trans [5]. Today it is widely recommended tobreak the trans fats group out of the total fat listing and theamount of trans fat to be added to the amount of saturatedfat [5,6].

Gas chromatography (GC) and infrared spectroscopy(IR) are the official methods used for the determination oftrans fatty acids in edible oils and fats [5,7].

American Oil Chemists Society developed using GCmethod a total of 21 fatty acids: 15 cis fatty acids, 1 transoleic acid, 3 trans linoleic acid and 2 trans linolenic acid[8].

Fourier transform (FT) infrared (IR) spectroscopy is asimple and rapid technique for determination of trans fattyacids with isolated trans-double bonds, applied directly onthe oil sample without any pretreatment, in comparisonwith gas chromatography-mass spectrometry (GC-MS)method that requires sample derivatisation of fatty acidsto fatty acid methyl esters (FAMEs) [3].

* email: ana.hanganu@cco.ro, tel : 0213167900

In FT-IR spectroscopy isolated trans fatty acids arequantified based on the measurement of trans peak areain the specific region from 990-945 cm-1 representing theC-H out of plane deformation absorption (in transconfiguration). Using GC method, lower trans fatty acidslevels can be measured and also offers both identificationand quantification of individual trans fatty acids [1, 9, 10].

The purpose of this research is to analyze theaccumulation of trans fatty acids in four types of Romanianvegetable oils during thermal processing. The four differenttypes of oils included sunflower, corn, soybean and linseedoils, heated continuously at three different temperatures180, 220 and 250oC for 33 h without any replenishment.

Experimental partThe commercially available vegetable oils used in the

thermal processes (sunflower, corn, soybean and linseedoils) were purchased from different local Romanianmarkets and stored at 4oC until use.

Heated oil samples were produced by heating the oilswithout frying any food at three different temperatures 180,220, 250oC for 33 h without any replenishment with freshoil.

Vegetable oils were heated in a commercially availableelectrical deep fat fryer. The temperature of the oil wasmonitored by a thermo recorder. Every day 500 mL of oilwas heated up for 1 h to reach the heating temperature(180, 220 or 250oC) and then other 6 h continuously (until33 h). Oil samples were collected at every 3 h. In total, 11samples were collected for each oil at one temperature.

The oil samples collected from the thermal experimentswere derivatised in order to obtain fatty acids methyl esters.

Fatty acid methyl esters (FAMEs) were prepared bytransesterification of oils with methanol, using BF3-MeOHcomplex as catalyst, according to the standard method[11].

The standard mixture of 37 fatty acids methyl esters(Supelco 37 Component FAME Mix) used for the gas-chromatographic analyses was purchased from Supelco.

REV. CHIM. (Bucharest) 63 No. 10 2012 http://www.revistadechimie.ro 985

Another standard mixture of fatty acids methyl esterscertified for its fatty acid profile used for the gas-chromatographic analyses was BCR-162R, soya-maize oilblend.

The gas-chromatograms of the fatty acid methyl estersmixtures were recorded on an Agilent Technologies model7890A instrument with mass detection AgilentTechnologies model 5975 C VL MSD with Triple AxisDetector and auto-sampler Agilent. Separation intocomponents was made on a capillary column especiallydesigned for the FAMEs analysis (Supelco SPTM 2560,characteristics: 100 m length, 0.25 mm inner diameter,0.2 m film thickness). The injection solutions wereprepared in CH2Cl2 of HPLC purity grade.

Fatty acids identification was made by comparing eachpeak retention time with those of two standard mixturesof fatty acid methyl esters (SupelcoTM 37 Component FAMEMix and BCR-162R). In the standard mixtures the exactconcentration of each component is known. Both standardmixtures and each of the fatty acid methyl esters of theanalyzed oils were chromatographically separated underthe same conditions, using the same temperature program(oven initial temperature 140oC to final temperature 240oC, heating rate 4oC/min.), injection volume 1L, split rate100:1, carrier gas He according to the Supelcospecifications. The calibration of the signals was made bytaking into account the concentration of each componentof the standard mixture, correlated with the detectorresponse.

FT-IR spectra were recorded on a Bruker Vertex 70spectrometer, with horizontal device for attenuated

reflectance and diamond crystal, on a spectral windowranging from 4000 to 400 cm-1, at a spectral resolution of2cm-1. Spectra were recorded without any samplepreparation and were processed with OPUS 5.5 software(Bruker). A series of trans calibration standard mixtures(used as primary standard) were prepared weighingaccurately to the nearest 0.0001 g (0.5-x) g of methyl oleateand x g of methyl elaidate into a vial, in order to prepare aseries of trans fat standards of known concentrations. Thesestandard solutions were (1:1 w/w) diluted with ciclo-hexane [7]. The FT-IR spectra of the trans fat standardsolutions were recorded.

Results and discussionsIR spectroscopy

The infrared spectroscopy and gas-chromatographyhave been widely used to determine the trans content inedible oils and fats. The simplest method is the directanalysis of oils by Fourier transform infrared (FT-IR)spectroscopy.

Typical FT-IR spectra of oils are presented in figure 1.By using specific trans absorption band at 966 cm-1,

several integration methods were performed using OPUS5.5 software for a series of trans calibration standardmixtures between fixed limits 990-945 cm-1. Severalcalibration equations were obtained depending on thespecific area integration procedure [12], as describedbelow. A series of linear calibration equations were obtainedwith variable correlation coefficients, as is shown intable 1.

Fig.1 FT-IR spectra of oilscontaining trans fatty acids

obtained in the spectralregion between 1000 and

930 cm-1, centered at 966 cm-1

Table 1CORRELATIONS COEFFICIENTS OBTAINED FOR

DIFFERENT INTEGRATION METHODS

REV. CHIM. (Bucharest) 63 No. 10 2012http://www.revistadechimie.ro986

Gas-chromatographyAnother method used for identification and

quantification of trans fatty acids was gas-chromatography,technique that allows to identify individual fatty acids. Usingthis method we could notice when trans isomers begin toform in thermally processed samples.

During the heating processes carried out, the followingtrans isomers are identified:

- 9-cis 18:1 (oleic acid) generates 9-trans 18:1 (elaidicacid);

- 9-cis, 12-cis 18:2 (linoleic acid) generates 9-trans, 12-trans 18:2 (linolelaidic acid), 9-cis, 12-trans 18:2 and 9-trans 12-cis,18:2.

Table 3 provides an overview on the individual trans fattyacids composition detected in heated oils samples at threedifferent temperatures studied.

Elaidic acid (9-t C18:1) appeared after 18 h of severethermal degradation of oils (250oC) increasing with theheating time. Considerable amounts of trans isomersderived from linoleic acid are also formed in all edible oils.

Table 2CALCULATED PERCENTS OF TOTAL TRANSISOMERS FOR THE CONTROLLED HEATED

OIL SAMPLES USING IR SPECTRA

Fig.2. Calibration curve and correlationcoefficient obtained with the bases of peak

areas between 990-945 cm-1

It can be remarked from table 1 that procedure B ofintegration in the IR spectrum has the best linear correlationcoefficient, therefore the calibration curve obtained wasused to determinate the trans concentration in heated oilsamples by linear regression.

The calibration curve obtained correlates the areas ofthe absorbance peak with corresponding methyl elaidateconcentrations (fig. 2).

Exploiting the specific trans absorption band from theIR spectra of heated oils by linear regression, the calibrationequation y=0.0975x + 0.014 was used to calculate thepercent of trans isomers from the samples, using theappropriate software.

The results are reported in table 2.As it can be observed from table 2 there is an increase

in total trans isomers in all thermal experiments as theheating time increases. The highest amount of trans fattyacids was accumulated in the first thermal experiment(250oC), followed by 220oC experiment, while 180oCexperiment has the smallest quantity of trans fats farmed.

REV. CHIM. (Bucharest) 63 No. 10 2012 http://www.revistadechimie.ro 987

As it can be noticed drastic temperatures conditionsproduced trans fatty acids.

In order to verify the similarity of the information providedfrom the two methods used for TFAs quantification werealized a series of calibration curves with good linearcorrelation coefficients. An example of this type ofcalibration curves for sunflower oil and corn oil, are givenin figure 3.

Figure 3 reveals that FT-IR and GC techniques givecomparable results. By plotting the results obtained fromGC and FT-IR a satisfactory calibration was achieved. It isvery clear that FT-IR results are in agreement with GCresults for sunflower and corn oil in which the major fattyacids are linoleic acid and oleic acid, responsible for thetrans fatty acids formed during heating. In case of soybeanand linseed oils responsible for trans fatty acids formationis linolenic acid also.

Table 3 TRANS FATTY ACID COMPOSITION OF HEATED OILS AT 250OC

Fig.3. Plot of trans values obtained from GC and FT-IR forsunflower oil and corn oil

According to literature specifications, during heatingprocesses, linolenic acid (9-cis, 12-cis, 15-cis C18:3)generates trans isomers like [13]:

- 9-trans, 12-cis, 15-cis C18:3;- 9-cis, 12-trans, 15-cis C18:3;- 9-cis, 12-cis, 15-trans C18:3;- 9-trans, 12-trans, 15-cis C18:3;- 9-trans, 12-cis, 15-trans C18:3;- 9-cis, 12-trans, 15-trans C18:3;- 9-trans, 12-trans, 15-trans C18:3.The standard mixtures previously presented used for

trans fatty acids identification did not allowed us to identifytrans isomers derived from linolenic acid during heating,so it is very clear that the results obtained using FT-IRmethod (total amount of TFAs) differ from the resultsobtained using GC techniques. The difference between theresults of the two methods represents linolenic acid transisomers not identified by GC.

REV. CHIM. (Bucharest) 63 No. 10 2012http://www.revistadechimie.ro988

ConclusionsDrastic temperature conditions applied to edible oils

induce changes in fatty acids composition generating transisomers. No trans fatty acids were formed in sunflower,corn and linseed oils during heating at 180oC. Trans fattyacids accumulation in heated oils is closely related toprocess temperature and heating time.

In conclusion, using edible oils in frequent domesticactivities (food-frying operations) at lower temperatures,major degradation processes do not occur.

Both modern physical methods used, give similar results,but both have advantages and disadvantages:

- FT-IR is a simple and rapid method applied directly onoil samples, but the results obtained are reported as totaltrans fats;

- GC is a more laborious method (oil samples requirederivatisation), but provides information about individualtrans fatty acids.

Further research is necessary to study the formation oftrans fatty acids in vegetable oils during heating byexploiting the specific absorption band in the FT-IR spectra.

In a previous paper it was studied the authentication ofvegetable oils by [1] H-NMR [14].

Acknowledgments: The work has been funded by the SectoralOperational Programme Human Resources Development 2007-2013of the Romanian Ministry of Labour, Family and Social Protectionthrough the Financial Agreement POSDRU/88/1.5/S/60203.

References1. SHERAZI, S., KANDHRO, A., ARAIN, S., Food Chem., 114, 2009,p. 323.2. TSUZUKI, W., MATSUOKA, A., USHIDA, K., Food Chem., 123, 2010,p. 976.32.CHO, I.K., KIM, S., KHURANA, H.K., LI, Q.X., JUN, S., Food Chem.,125, 2011, p. 1121.4. BANSAL, G., ZHOU, W., TAN, T.W., NEO, F.L., LO, H.L., Food Chem.,116, 2009, p. 535.5. PRIEGO-CAPOTE, F., RUIZ-JIMNEZ, J., LUQUE DE CASTRO, M.D.,Food Chem., 100, 2007, p. 859.6. NAZ, S., SIDDIQI, R., SHEIKH, H., SAYEED, S.A., Food Res. Int., 38,2005, p. 127.7. PRIEGO-CAPOTE, F., RUIZ-JIMNEZ, J., GARCA-OLMO, J., Luquede Castro, M.D., Anal. Chim. Acta, 517, 2004, p. 13-20.8. LIU, W.H., INBARAJ, B.S., CHEN, B.H., Food Chem., 104, 2007,p. 1740.9. MAHESAR, S.A., KANDHRO, A.A., CERRETANI, L., BENDINI, A.,SHERAZI, S.T.H., BHANGER, M.I., Food Chem., 123, 2010, p. 1289.10. KANDHRO, A.A., SHERAZI, S.T.H., MAHESAR, S.A., BHANGER, M.I.,TALPUR, M.Y., RAUF, A., Food Chem., 109, 2008, p. 207.11. LI, Y., WATKINS, B. A., Current Protocols in Food AnalyticalChemistry, Ed. John Wiley and Sons Inc., New York, 2001, p. D1.2.1-D1.2.1512.TODASC, M.C., CHIRA, N, DELEANU, C, ROSCA, S., UPB Sci. Bull.,Series B, 69(4), 2007, p.3.13. DIJKSTRA, A. J., HAMILTON, R. J., HAMM, W., Trans fatty acids,Ed. Blackwell, 2008, p. 3114. MIHALACHE, M., BRATU, A., HANGANU, A., CHIRA, N.-A., TODASCA,M.C., ROSCA, S., Rev. Chim. (Bucharest), 63, no. 9, 2012, p. 877

Manuscript received: 2.05.2012