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ASSESSMENT OF FATTY ACID AND VITAMIN E PROFILES IN EDIBLE OILS IN MALAYSIA

Cooking oil plays a pivotal role in our daily diet. The present investigation was designed to characterise the fatty acid composition and eight homologs of vitamin E in a total of 52 locally available vegetable oils [types: pure oils (n=10) and blended oils (n=6)] in Putrajaya, Malaysia. The analyses were performed by gas chromatography for fatty acid composition, the acetic acid-isooctane method for peroxide value (PV), and high-performance liquid chromatography (HPLC) for vitamin E profiling. Following multivariate discriminant analysis, the oils were categorised into two groups. Most studies and oil standards have reported only part of eight vitamin E homologs, therefore this current study is novel as it provides the complete vitamin E profiling of the tested oils. There was no major discrepancy found between the assayed values and packaging labels on the fatty acid composition, trans -fat, and vitamin E content. The PV and trans -fats were within the recommended range, indicating good quality compliance. Fatty acid and vitamin E content were within standard oil specifications. These observations implied that the oil manufacturers and retailers in Malaysia had good acquiescence to international standards and local regulations. This study provides consumers with technical insights in oil selection, based on the functionality needed.

Cooking oil is an essential part of our daily diet preparation. Fats and oils are made up of triglycerides which consist of a glycerol backbone esterified with three fatty acids ( Figure 1). Fats and oils play a pivotal role in maintaining good health and they provide more calories per gram (9 kcal g -1 ) than any other nutrients (Chowdhury et al., 2007). Fatty acids are grouped into three broad categories namely saturated fatty acids (SFA), monounsaturated fatty acids (MUFA) and polyunsaturated fatty acids (PUFA) depending on the number of double bonds (Chowdhury et al., 2007). The World Health Organization (WHO) recommends 20.0%-35.0% energy (E) total fat from total energy intake for adults (WHO, 2008). Intake of SFA and trans-fats are recommended to be <10.0% and <1.0% respectively of total energy intake. The acceptable range for total PUFA (n-6 and n-3 fatty acids) consumption is 6.0%-11.0% E from total energy intake of which n-3 fatty acid intake ranges between 0.5%-2.0% energy [>0.5% E alpha-linolenic acid (ALA) plus eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) (0.25-2.00 g/day)], whereas n-6 fatty acids (linoleic acid, LA) intake ranges between 2.5%-9.0% E (Elmadfa  and Kornsteiner, 2009;WHO, 2008). Wallingford et al. (2004) reported that the fatty acid profile of edible oils in China is often incompletely and incorrectly labelled. There are some literature on the characterisation of fatty acids in commercial oils in China (Haiyan et al., 2007;Wallingford et al., 2004), India (Dorni et al., 2018), the Spanish market (Rueda et al., 2014), Iran (Piravi-Vanak et al., 2009) and Bangladesh (Chowdhury et al., 2007). However, none were found in the Malaysian setting.
Vitamin E or tocols is a fat-soluble vitamin that is found abundantly in oilseeds and nuts (Aksoz et al., 2020). Vitamin E is a family of eight similar homologs that exists naturally as two families namely tocopherols and tocotrienols ( Figure 2). Each vitamin E family exists naturally in four isoforms namely alpha, beta, delta and gamma (Loganathan et al., 2021). Vitamin E is a potent antioxidant with lipoperoxyl radical scavenging activities which could protect the oils against oxidative deterioration (Loganathan et al., 2020a;2020b). Besides, the content of vitamin E in oil reflects the oil quality (Wen et al., 2020). Most studies (Aksoz et al., 2020;Gliszczynska-Swiglo et al., 2007;Matthaus et al., 2016;Petersen et al., 2012) and standards (Codex Alimentarius, 1999;2001;2019) have reported only part of eight vitamin E homologs, and more attention has been given to tocopherols as compared to tocotrienols. Therefore, there is a gap of knowledge in understanding the complete vitamin E profiling.

P R E S S
Fats and oils emanate a great deal of variation and uniqueness by nature in their fatty acid compositions, phytonutrient contents, and physiological properties. The aims of this work were to (1) characterise the fatty acid present and vitamin E content of locally available vegetable oils; (2) study the quality of oils on the shelf in terms of peroxide value (PV); (3) determine the accuracy of the packaging labels to the assayed values.

Edible Oil Samples
Edible oil samples of 11 different varieties: palm oil (five samples), canola oil (five samples), sunflower oil (nine samples), corn oil (seven samples), soybean oil (four samples), olive oil (11 samples), rice bran oil (one sample), avocado oil (one sample), grapeseed oil (one sample), rapeseed oil (one sample), and blended oils (13 samples) were purchased from the local supermarket around Putrajaya, Malaysia area between October to November 2015. Oils were transferred into six dark amber bottles, blanketed with nitrogen, and stored at -20°C for subsequent analyses. All labels on oil samples were tabulated and analysed alongside.

Preparation of Fatty Acid Methyl Ester (FAME)
The fatty acid composition of the test fats was determined by converting fatty acids of triglyceride to FAME according to AOCS Official Method Ch 1-91 (The American Oil Chemists' Society, 2017c).

Fatty Acid Composition Analysis by Gas Chromatography (GC)
Analysis of fatty acid composition was carried out according to AOCS Official Method Ce 1a-13 (The American Oil Chemists' Society, 2017b) on GC Perkin Elmer Autosystem Model, USA. The GC was fitted with the SGE Capillary BPX70 column from SGE Analytical Science Pty. Ltd. (Milton Keynes, United Kingdom). The identification of fatty acids was based on AOCS oil reference mixture RM-6. The results were expressed as relative percentages of wet weight.

Vitamin E Analysis by HPLC
Vitamin E content was analysed by normal phase HPLC Agilent model 1100 series, the US with fluorescence detector. The mean recovery of internal standard, namely 2,2,5,7,8-pentamethyl-6-hydroxy-Chromane (PMC) was 93.07% ± 0.72%. The chromatographic analysis of the compounds was conducted on Phenomenex® Luna 5 μM Silica analytical column (250 mm x 4.6 mm i.d) and a mobile phase of hexane/dioxane/isopropyl alcohol (970:25:5) at a flow rate of 1.0 mL per min. Identification and quantification of the components were done by comparison of the peak areas with those pure standards. Values are presented as mg kg -1 of wet weight.

Peroxide Value (PV)
Analysis of PV was performed with reference to the AOCS Official Method Cd 8b-90 (The American Oil Chemists' Society, 2017a). Briefly, an oil sample weighing 5.0 g was placed in the conical flask and 50.0 mL of acetic acid/iso-octane solution (3:2) was added. The sample was stirred until it has been well dissolved before the addition of 0.5 mL of saturated potassium iodide. The solution was shaken thoroughly and thereafter, immediately, with 30.0 mL of distilled water and 0.5 mL of starch solution. The sample was titrated with 0.1 M sodium thiosulphate until the blue colour disappears. PV of the oil samples was measured in milliequivalent of peroxide per kg of oil sample (wet weight).

Data Presentation
All analytical determinations were performed at minimum in duplicates and presented as the mean values ± standard deviation. Oneway analysis of variance ANOVA followed by Tukey's post hoc test was applied to assess the differences between oils (p<0.05). Cluster analyses were performed by multivariate statistical classification methods. Following hierarchical cluster analysis, discriminant analysis was applied to analyse differences among the oil groups. Statistical analysis was conducted using GraphPad Prism version 9.3.1 and SPSS version 11.5.

RESULTS AND DISCUSSION
All assayed samples had fatty acid profiles that fall within standard database values (Codex Alimentarius, 1999;2001;2019) and corresponded closely with the total fatty acid content of the packaging label. The fatty acid composition of the oils is shown in Table 1 and Figure 3. Heat map visualisation in Figure 4 provides a direct intuitive visualisation of the different contents of total SFA, total MUFA, and total PUFA in assayed edible oils by category.
Within the MUFA group, the oils were further classified into five sub-classes ( Figure 5). The first subclass had an equal composition of MUFA (45.11%-45.41%) and SFA (42.54%-43.42%). Palm olein is a member of the first subclass. Palm olein is derived from the mesocarp of the fruit of the oil palm, Elaeis guineensis (Kushairi et al., 2019). Olein fractions are widely used as cooking oils and suitable as a heavy-duty frying oil. There are two main grades of palm olein, namely singlefractionated palm olein and double-fractionated palm olein (Ahmad Tarmizi and Siew, 2008). All five tested palm olein were found to be of double fractionated grade. The tertiary blend of palm olein, peanut, and sesame oil (n=3) had an almost similar fatty acid composition to that of palm olein. The second subclass had MUFA concentrations ranging from 51.91%-64.06%. Low erucic acid canola and rapeseed oils classified in this group were derived from a variety of rapeseed cultivars of the plant family Brassicaceae (Codex Alimentarius, 1999;Matthaus et al., 2016). Similar to Matthaus et al. (2016), we too find these oils consisted mainly of oleic (C18:1) (51.80%-63.80%), linoleic (C18:2 N6C) (18.60%-25.30%), and n-3 fatty acid (C18:3N3, 8.00%-11.20%). The binary blend of high MUFA canola oil with high PUFA sunflower oil (n=4) as well as the blend of MUFA rich oils namely red palm olein and canola oil (n=1) also belongs to this group. These unique blends resulted in low SFA content (9.23%-18.18%) but high MUFA (51.91%-54.14%) and PUFA (27.68%-38.87%) profiles. High oleic sunflower oil is the only member of subclass three with distinctly high MUFA content. High oleic acid sunflower oil is produced from high oleic acid oilbearing seeds of the same plant (Helianthus annuus L.) (Codex Alimentarius, 1999). Following previous bibliographic data (Chowdhury et al., 2007;Petersen et al., 2012), the fractions corresponded almost entirely to C18:1N9C (84.53%). Olive and avocado oils were classified under sub-class four. Olive oil is the oil obtained from the fruit of the long-lived olive tree (Olea europaea L.) (Codex Alimentarius, 2001). We have evaluated three types of olive oils, namely extra virgin (n=3), extra light (n=2) and refined/ pure (n=5). All three types of olive oil had similar fatty acid compositions and this was in agreement with data from the literature (Piravi-Vanak et al., 2009) and standards (Codex Alimentarius, 2001;International Olive Council, 2006). Another member of this subclass namely avocado oil is an edible oil extracted from the fruit of Persea Americana (Haytowitz et al., 2011). Similar to the current study, Haiyan et al. (2007) also reported avocado oil had an almost similar fatty acid composition to that of canola/rapeseed oil, with predominantly MUFA (C18:1N9C). Avocado oil can be distinguished from other tested oil with a notable presence of C16:1 (7.80%), of which a similar observation was also reported in the literature (Rueda et al., 2014). A tertiary blend of soybean, palm olein, and canola oils was classified as subclass five. This blend had SFA (30.44%): MUFA (37.82%): PUFA (31.74%) content at a 1:1:1 ratio.
PUFA group, on the other hand, can be further divided into two big clusters. Sunflower, soybean, corn, binary blended oil (olive and sunflower), and tertiary blended oil (corn, soybean, and canola) were classified as one group (sub-class six). These oils have almost similar fatty acid composition (PUFA: 50.47%-62.49%, MUFA: 23.93%-37.06%, and SFA: 10.65%-15.71%). Sunflower (Helianthus annuus L.) is an annual crop that belongs to the family Asteraceae or Compositae (Codex Alimentarius, 1999). Sunflower oil is extracted from the sunflower seeds. Corn oil is a by-product from the wet or dry milling of maize germ (the embryos of Zea mays) (Jovanović et al., 2005). The primary product from this milling is starch (Jovanović et al., 2005). Soybean oil is derived from soybeans (Glycine max (L.) Merr.) that belong to the nitrogen-fixing leguminous plant (Codex Alimentarius, 1999). Soybean oil can be distinguished from the other members of the subclass as it had the highest levels of C18:3N3 (5.50%-7.10%) (Chowdhury et al., 2007;Haiyan et al., 2007). Grapeseed oil was classified as a separate subclass (no.7) under the PUFA group. Grapeseed oil is derived from the seeds of the grapes (Vitis vinifera L.) (Codex Alimentarius, 1999). Grapeseed oil is a winery industry by-product (Martin et al., 2020) and was found to have the highest PUFA content (72.00%) in the form of C18:2N6C as compared to other tested oils.
Most oil groups had no trans-fatty acid (C18:2N6T-linolelaidic acid) except for sunflower, corn, soybean, binary blends of canola and sunflower oil, and tertiary blends of soybean, palm olein, and canola oils ( Table 1). A slight variation was found between the assayed samples values as compared to packaging labels for canola oil (samples 6-9), sunflower oil (samples 11-17), and soybean oil (sample 27).
The original American Heart Association (AHA) Step I Diet fat recommended 30% E from dietary fat and fatty acid balance, SFA: MUFA: PUFA at 1:1:1 (Krauss et al., 2000), corresponding to soybean, palm olein, and canola blended oil. Currently, the AHA recommends slightly less SFA (<10% of calories), up to 10% from PUFA, and as much as 15% from MUFA (Krauss et al., 2000). Raising the dietary PUFA/SFA ratio has been recommended to reduce cardiovascular disease event risk and improve glycaemic control (Kang et al., 2005). WHO recommended PUFA/SFA ratio above 0.4 (Coskuntuna et al., 2015). Nevertheless, a high dietary PUFA/SFA ratio increases oxidative stress since PUFA are highly prone to lipid peroxidation (Kang et al., 2005). Only olive oil, avocado oil, and tertiary blended oil (soybean, palm olein, and canola oil) had a desirable PUFA/SFA ratio of 0.4-1.0. Palm olein had a lower than ideal PUFA/SFA ratio (>0.4). Whereas sunflower oil (5.90), grapeseed oil (8.83), and binary blended olive and sunflower oil (5.59) had a very high ratio of PUFA/SFA content. Edible oils are usually blended to contain a better proportion of SFAs, MUFAs, PUFAs, and essential fatty acids. The downside of blended oils was that the ratio of blending was not written on any of the labels. It was interesting to note that the same type of blended oil carried similar fatty acid composition, implying a similar ratio of blending, although the manufacturers were different.
The primary oxidation products are usually measured with a PV test. Peroxides are intermediate oxidation products of oil, that lead to the formation of a complex mixture of volatile compounds i.e. aldehydes, ketones, hydrocarbons, alcohols, and esters (Loganathan et al., 2020a;2020b). Peroxides form in oils, especially at undesirable storage conditions such as elevated temperatures as well as exposure to oxygen and/or light (Loganathan et al., 2020a;2020b). The acceptable range of PV in refined palm oil is less than 10 meq O 2 kg -1 . PVs of all the oil samples were within the recommended range (<10 meq O 2 kg -1 ) except for sample no. 6 ( Table 1).
All assayed samples had almost similar total vitamin E and its homologs content (Table 2 and Figure 6) as compared to standard database values (Codex Alimentarius, 1999;2001;2019). Additional to Codex Alimentarius standards' data, we also report beta-tocotrienol content in the assayed oils. Only 30 companies reported on vitamin E content in their packaging labels, whereby 50% of these labels (n=15) had vitamin E content within the assayed range (with ± 30% deviation), 14 were under- Note: Values are mean value ± SD. Analyses were done in replicates for fatty acid composition (%) and triplicates for PVs. Data was measured by one-way analysis of variance (ANOVA) with Tukey post hoc test. Statistical differences (p<0.05) were found between test oils except those indicated by the alphabet to represent the sample of each row.
The heat map in Figure 6 provides a clearer visual representation of the hierarchical concentrations of vitamin E homologs, total tocopherols, total tocotrienols, and total vitamin E in assayed edible oils by category. In accordance with previous findings, alpha-tocopherol was found to be the most abundant form of vitamin E in the studied vegetable oils and blends (Aksoz et al., 2020;Gliszczynska-Swiglo et al., 2007;Wen et al., 2020). The richest alpha-tocopherol source in the current pool of samples was sunflower oil (429 mg kg -1 ). Whereas, beta-tocopherol was found in low quantities in vegetable oils (Gliszczynska-Swiglo et al., 2007). Palm olein, grapeseed, and blended oils with palm olein (palm olein, peanut, and sesame blended oil; soybean, palm olein and canola blended oil; red palm olein and canola blended oil; and corn, soybean, and canola blended oil) were unique as it were the only oils bearing tocotrienols mainly in the forms of alpha, delta and gamma. Inferior to the current study, most studies have reported (deltaand gamma-) tocopherols together due to lack of separation (Aksoz et al., 2020;Gliszczynska-Swiglo et al., 2007). Soybean oil was found to be another rich source of vitamin E (549.2-862.3 mg/kg), mainly in the form of tocopherols: gamma (61.8%), delta (21.6%), and alpha (10.9%). Research conducted by Wen et al., (2020) reported higher concentrations of vitamin E in soybean oil 1053 mg kg -1 with almost similar concentrations of tocopherols: gamma 64.0%, delta 25.0%, and alpha 11.0% (Wen et al., 2020). Grapeseed oil had a balanced composition of tocopherols (45.2%) and tocotrienols (55.8%). Sunflower oil, olive oil, and their blend (sunflower and olive blended oil) resulted in more than 96.0% of tocopherols mainly in the form of alpha-tocopherol (>81.0%). Similar to Matthaus et al. (2016), we too report that corn oil, canola oil, and rapeseed oil can be distinguished from other analysed oils as they consist of only alpha-and gamma-tocopherols. Similar to the current study, Petersen et al. (2012) also observed that high oleic sunflower oil and sunflower oil possess comparable vitamin E content and composition. Corn, soybean, and canola blended oil display fairly high vitamin E content, mostly in the form of tocopherols: gamma (52.6%), alpha (17.7%), delta (10.2%), and trace amounts of gamma (7.7%) and alpha (6.8%) tocotrienols. Vitamin E content was lowest in avocado oil (88.8 mg kg -1 ) as compared to other assayed samples.

P R E S S
Antioxidants are intentionally added at times to improve the oxidative stability of oils (Loganathan et al., 2020b). All the oils used in the current study were in their natural form and none were fortified. The recommended dietary allowance (RDA) for Vitamin E by the Institute of Medicine National Academies (Russell et al., 2001) is 15 mg day -1 . All the oils except for olive and avocado oils could fulfil the required daily intake of vitamin E. In general, vitamin E deficiency is very rare in healthy people as vitamin E is found in a variety of foods (Aksoz et al., 2020).
Limitations of the current study were erucic acid (C22:1n9 cis), elaidic acid (C18:1n9t) and stereospecific analysis of fatty acids were not analysed. There could be a little variation in fatty acid composition and vitamin E content within the same type/cultivar of oil. This could be due to variations in oilseed/plant, the degree of fruit ripeness/plant maturity, the geographical location of plantations, climatic conditions, oil extraction and processing method, storage conditions, and analytical method (Aksoz et al., 2020;Loganathan et al., 2017;Matthaus et al., 2016;Wen et al., 2020).
It's important to have a healthy balance of the fatty acids in our diets. Each oil has a different fatty acid profile and unique uses in meal preparations. This can be attained by choosing the right commercially available blended oil. Another option is using a combination of edible oils. For example, in a meal, sesame oil can be used to season rice porridge, sunflower oil to sauté green leaves, palm olein to deep fry chicken fillets, and olive oil to prepare the salad dressing. Regardless of how healthy or how balanced it is, whatever is consumed in excess is deleterious to health. Hence, appropriate calorie intake and a balanced diet are important.

CONCLUSION
By analysing the pattern of fatty acid and vitamin E profiles, the current study provides novel evidence that edible oils in the Malaysian market are of good quality and appropriately labelled. We did not find any discrepancy in the fatty acid composition labelling in any of the samples assayed. Although vitamin E content was underreported on a few labels of the assayed samples, however, all assayed samples had almost similar fatty acid and vitamin E profiles as compared to standard oil specifications. The PVs were also within the recommended range, indicating good quality compliance. These observations implied that the oil manufacturers had good acquiescence to international standards and local regulations. In Note: Yellow ( ) indicates fatty acids distributed at high concentration and blue ( ) indicates fatty acids distributed at a low concentration).
SFA (saturated fatty acids), MUFA (monounsaturated fatty acids), and PUFA (polyunsaturated fatty acids).  Figure 6: Heatmap explains the different contents of vitamin E homologs, total tocopherols, total tocotrienols and total vitamin E (tocols) wet weight basis in assayed edible oils by category. Note: Yellow (n) indicates fatty acids distributed at high concentration and blue (n) indicates fatty acids distributed at a low concentration). SFA (saturated fatty acids), MUFA (monounsaturated fatty acids), and PUFA (polyunsaturated fatty acids).