Conjugated linoleic acid ( CLA ) . Cis 9 , trans 11 and trans 10 , cis 12 isomer detection in crude and refined corn oils by capillary GC By

Conjugated linoleic acids (CLAs) exhibit protective effects against various types of cancer and heart diseases. With the newly developed capillary gas chromatographic method (GC), cis 9, trans 11 and trans 10, cis 12 octadecadienoic acid isomers of CLA (C18:2) were determined in crude and refined corn oils as qualitative and quantitative measurements. Cis 9, trans 11 C18:2 ( c 9, t 11 CLA) was the major CLA isomer in both oils. It was found that c9, t11 CLA was 0.62% of the total lipid in crude oil and 1.24% of the total lipid in refined oil. Using the refining process, the total CLA was 1.38% whereas that of crude corn oil was 0.62%. An approximate 2.2 fold increase in the total CLA was found in refined oil ( n = 9) ( p y = 2.782x + 0.046 (R 2 = 0.9999)] were performed ( p < 0.01).The proponed chromatographic procedure could be used for vegetable oil quality control


INTRODUCTION
Conjugated linoleic acid (CLA) is a term now used for a group of positional and geometrical isomers of linoleic acid and characterized by the presence of conjugated double bonds.It has been found that CLA exhibit a plethora of biological activities including its protective effects against various types of cancer and heart diseases (Pariza, 1999).It has also been shown that various CLA isomers inhibit platelet aggregation (Torres-Duerte and Vanderhoek, 2003).Conjugated linoleic acids (CLAs) are isomers of octadecadienoic acid containing conjugated double bonds at carbons 10 and 12 or 9 and 11, in all possible cis and trans combinations.Pariza et al. (2001) have recently reviewed the numerous positive biological effects of CLAs including anticarcinogenic, antidiabetic and antilipogenic.According to latest research, it is reported that CLA has anticancer effects on MCF-7 breast cancer cells (Guo et al., 2007), has positive effects on bone formation and rheumatoid arthritis problems (Hur and Park, 2007), has an important role in weight control, reducing body fat, obesity and psychiatric treatment (Katzman et al., 2007;  Watras et al., 2007).It is described that CLA increases skeletal muscle ceramide content and decreases insulin sensitivity in overweight, nondiabetic humans (Thrush et al., 2007).
It was demonstrated that cis9, trans11 C18:2 prevented the growth of human mammary cancer cells more effectively than trans10 ,cis12 C18:2.Ruminant meat and milk are the primary sources of CLA in the food chain.The quantitative amount of CLA ranges from 3.4 to 8.0 mg/g lipid in milk and dairy products and ranges from 2.7 to 5.6 mg/g lipid in ruminant meat products depending on the animal species, tissue and diet (Lin et al., 1995;Chin et al.,1992) while butter contains approximately 720 mg/100g food (wet weight) (Britton et al.,1992).It has been found that ruminant meats and milk contain ~80% of cis9, trans11 CLA and ~10% of trans10, cis12 CLA (Fogerty et al.,1988).
It has been reported that CLA content increases with manufacturing processes and hydrogen donor effects (Lin et al., 1995).The occurrence of CLA isomers in probiotic yogurt enhanced with fructooligosaccharide (FOS) and concentrated cream "kaymak" manufactured from cow's milk were identified (Akalin and Toku7oglu et al., 2005;2007).
The content of CLA in animal products is much higher than in plant oils.(Chin et al., 1992;Ackman et al., 1981;Fogerty et al.,1988).It has been shown that CLA ranges from 0.2-0.7 mg/g lipid in vegetable oils (Evans et al., 2002).Vegetable oils including safflower oil, sunflower oil, corn oil and peanut oil contain large amounts of linoleic (C18:2), oleic acid (C18:1); especially soybean oil contains high amounts of linolenic acid (C18:3).Not only their oils but also their meals have been used as animal feed for ruminant and nonruminant animals.
High oleic (cis9-C18:1) or high linoleic (C18:2n-6) feeding has affected the CLA formation in ruminant tissues.Cis9-C18:1 (oleic acid) and trans9-C18:1 (elaidic acid) are competitive inhibitors of linoleate isomerase activity (Kepler et  al., 1970).If present in large amounts, trans 9-C18:1 (derived from 18:2n-6 or cis9, trans11-C18:2 hydrogenation) and cis9-C18:1 could alter the profiles of intermediates produced in the rumen, thereby affecting the levels available for absorption in the small intestine.In this context, vegetable oils are important sources for CLA formation Due to the animal feed source and directly consumable food, vegetable oils are important for nutrition and it is necessary to know the quantitative CLA alterations in oil processing technology.The aim of this research is to develop a rapid GC method for CLA identification in corn oil and to monitor the conjugated linoleic acid (CLA) isomers in crude and refined corn oil by using this effective gas chromatographic method.

Research Material and Standards
Corn oil was obtained from "Yonca Ege Oil Company, Manisa, Turkey" in April, 2003.Three bottle of raw corn oil (n = 9) and three bottle of refined corn oil (n = 9) were used for the analyses.Each was analyzed in duplicate .

Refining Process of Crude Oils
The conventional refining operation consisted of 4 steps .The first step was degumming to remove phospholipids.Secondly, FFAs were neutralized with sodium hydroxide (NaOH) to produce a soap which was then removed with remaining phospholipids by centrifugation.The pigments were then adsorbed by acid-activated bleaching clays.Finally, the oil was steam distilled under high vacuum to strip out trace amounts of FFAs, aldehydes, ketones and other volatile compounds (Toku7oglu and Ünal, 2003).

Proximate Composition of Crude and Refined Corn Oils
The moisture content of a 5.0g sample was determined at 110°C for 24 h in an oven by the procedure described by AOAC (1999).The total lipids in 5.0g of corn oil was determined by the procedure described by AOAC (1999).All proximate detections were done in triplicate.

Fatty Acid Methyl Esters of Corn Oil Samples
The fatty acid methyl esters (FAs) of corn oil samples were prepared from extracted lipids from each samples by esterification reaction with 14% boron trifluoride (BF 3 )-methanol complex according to the modified method described by Toku7oglu and Ünal (2003).
Extracted lipids from corn oil was refluxed with a NaOH solution containing 0.05 N methanol for 5 min and was esterified with 14% boron trifluoride (BF 3 )-methanol complex for 15 min and then equilibrated to room temperature (25 °C).All solutions were fractionated with hexane and saturated NaCl and then the methyl ester phase was separated.Anhydrous sodium sulfate (Na 2 SO 4 ) was then added to the methyl ester phase.Prior to GC injection, extract was sonicated for 2 min to remove oxygen and 1 µL of extract was injected into the GC.
The gas chromatograph was temperatureprogrammed to start at 70 °C for 1 min isoterm (initial temp.) and to increase at 10 °C/min to 150 °C and was held for 2 min isoterm (Ramp1) and then to increase at 2 °C/min to 265 °C and was held for 20 min isoterm (Ramp2).Injector and detector temperatures were set at 250 °C.Carrier gas was helium at a flow rate of 1.5 mL/min and split ratio was 50:1.The injection amount was 1 µL.FAs and CLA determinations were performed from 3 separate lipid extractions and esterifications.Each was injected in triplicate (n = 3).

Analytical Validation and Analytical Quality Control
Retention times (RT) were compared with known standards of FAMEs and CLA.Both FA standard mixture and CLA standard had linear calibration curves through the origin (R 2 = 0.9999) (p Ͻ 0.01).Analytical GC method was validated for FA determination of corn oil within the 95% confidence limits.Mean analytical recoveries determined from individual fatty acids in corn oil samples changed from 99.92% to 100%.

Statistical Analysis
Data were analyzed with Statistica (1998) by one-way analysis of variance (Kruskal-Wallis ANOVA) with corn oil individual fatty acids and conjugated linoleic acid isomers as the source of variance.

RESULTS AND DISCUSSION
Figure 1 shows standard GC chromatogram of conjugated linoleic acid cis-9, trans-11 and trans-10, cis-12 isomers and Figure 2 shows good separation of the above-mentioned compounds with other fatty acids simultaneously in refined corn oil at the same chromatographic conditions (Figure 1 and Figure 2).Conjugated linoleic acid cis-9, trans-11 and trans-10, cis-12 isomers were perfectly separated with the proposed chromatographic conditions.This GC procedure provided the good baseline separation of t10, c12 CLA (CLA isomer 1) (t retention time = 20.37 min) and c9, t11 CLA (CLA isomer 2) (t retention time = 20.58min) as shown in standard chromatogram (Figure 1).CLA isomer 1 (t retention time = 20.39 min) and CLA isomer 2 peaks (t retention time = 20.58min) were confirmed in the GC chromatogram of refined corn oil (Figure 2) .
The analytical parameters of crude and refined corn oils are shown in Table 1.Interday precision, relative standard deviation (RSD), recovery, and detection limit data of cis-  trans-10, cis-12 CLA were supported to good resolution (Table 1).
With the proposed chromatographic method, it was found that c9, t11 CLA was 0.62% of the total lipid content (as percent of total lipid) in crude oil whereas 1.24% of total lipid in refined oil (n = 9) (p Ͻ 0.01) (Table 2).With the refining process of oil, it was found that this CLA isomer increased 1.99 fold.t-10, c-12 CLA isomer was 0.14 % in refined corn oils whereas this isomer was not detected in crude corn oils.It is shown that t-10, c-12 CLA increased with the refining process (Table 2).
Some quality parameters of crude and refined corn oils including total lipid, volatile matter data and fatty acid contents were also determined.In particular, the total lipid level is most important in order to evaluate CLA content in oils (Table 3).It has been shown that crude oil contains 99.35 Ϯ 0.03g oil /100g sample whereas refined oil contains 99.62 ± 0.02g oil /100g sample (Table 3).According to some proximate data table (Table 3), volatile matter was 0.38% in refined corn oils and was 0.65% in crude corn oils (n = 9) (p Ͻ 0.01).With the refining process, volatile matter has been decreased (Table 3).
Due to the refining process, the total CLA content of refined corn oil was 1.38% whereas that of crude corn oil was 0.62% (Table 2).An approximate 2.2 fold alteration for lipid constituent CLA was found in refined oil.In this context, the alteration in the CLA amount could be used an indicator for refining process quality due to CLA contribution to polyunsaturated fatty acids (PUFAs) (n = 9) (p Ͻ 0.01) (Table 2).As shown in Table 2, fatty acids (FAs) in crude and refined oils were determined in the same chromatographic conditions.With the refining process used, total polyunsaturated acid (Σ PUFA) amounts were 57.02%.It is shown that Σ PUFA content had increased ∼1.0 fold with refining (n = 9) (p Ͻ 0.01) (Table 2).Total saturated FA levels (Σ SAT) were 13.24% and had reduced ∼1.05 fold (n = 9) (p Ͻ 0.01).Linoleic acid (omega-6) (C18:2n-6) and linolenic acid (omega-3) (C18:3n-3) increased in trace amounts with refining whereas palmitic acid (C16:0) decreased 1.07 fold (Table 2).
Current studies and clinical investigations have made evident that polyunsaturated fatty acids (PUFAs) exert beneficial effects on human health and play an important role in the prevention and treatment of coronary artery disease, hypertension, inflammatory, autoimmune disorders and cancer (Ricardo Uauy and Valenzula, 2000).
Crude vegetable oils are obtained by oilseed crushing, followed by solvent extraction.Crude vegetable oils contains 95% triacylglycerols and remaining compounds such as phospholipids, free fatty acids (FFAs), pigments, sterols, carbohydrates, proteins and degradation products (Toku7oglu, 2003).
Preexpelled and solvent extracted crude oil is not appropriate for dietary use.Crude vegetable oils undergo a complex refining process to achieve the desired quality and to produce a more stable product because the above-mentioned substances may impart undesirable flavor and color and shorten the shelf life of the oil shelf-life (Toku7oglu, 2003).
Refining is the final process for high quality vegetable oil.In our study we determined that CLA levels increased after the refining process.This may occur due to the manufacturing process of oil.
According to Ha et al. (1987) in the presence of oxygen and catalysts such as metals, the autooxidation of linoleic acid produces hydroperoxy acids that have the specific characteristics of a cisor trans-conjugated dienic system (Ha et al., 1987).Jung et al. (2002) reported that the CLA formation in oils during the hydrogenation process as affected by catalyst types, catalyst contents, hydrogen pressure, and oil species.
The lipids in food undergo a variety of chemical changes as a result of heat treatment through manufacturing processes, cooking, roasting, frying, pasteurization, etc. or the extrusion process.Pakdeechanuan et al. (2007) indicated that the effect of extrusion parameters on the CLA of corn extrudates.It is reported that CLA content increased from 1.2 mg/g of oil in feed to 7.8 mg/g of oil in corn extrudates at 150 °C.
In this research, with the developed GC method, we effectively separated CLA isomers in both crude and refined corn oil.This above-mentioned procedure provided a reproducible and sensitive quantitative fatty acid and CLA detection and can be used in the quality control area of the oil manufacturing industry.The study is in progress under different refining conditions and CLA separation.As it is known, recently, the commercial membrane separation processes and the use of a combination of membrane and supercritical fluid (SCFE) technologies in oilseed processing and edible oil refining are possible in the areas of minor ingredient purification such as tocopherols, phosphatidyl-choline and lecithin except for protein purification and waste treatment applications.CLA purification from vegetable oils in the refining process may be possible using these technologies.