Quantitative determination of total lipid hydroperoxides by a flow injection analysis system

A flow injection analysis (FIA) system coupled with a fluorescence detection system using diphenyl-1-pyrenylphosphine (DPPP) was developed as a highly sensitive and reproducible quantitative method of total lipid hydroperoxide analysis. Fluorescence analysis of DPPP oxide generated by the reaction of lipid hydroperoxides with DPPP enabled a quantitative determination of the total amount of lipid hydroperoxides. Use of 1-myristoyl-2-(12-((7-nitro-2-1,3-benzoxadiazol-4-yl)amino) dodecanoyl)-sn-glycero-3-phosphocholine as the internal standard improved the sensitivity and reproducibility of the analysis. Several commercially available edible oils, including soybean oil, rapeseed oil, olive oil, corn oil, canola oil, safflower oil, mixed vegetable oils, cod liver oil, and sardine oil were analyzed by the FIA system for the quantitative determination of total lipid hydroperoxides. The minimal amounts of sample oils required were 50 μg of soybean oil (PV=2.71 meq/kg) and 3 mg of sardine oil (PV=0.38 meq/kg) for a single injection. Thus, sensitivity was sufficient for the detection of a small amount and/or low concentration of hydroperoxides in common edible oils. The recovery of sample oils for the FIA system ranged between 87.2±2.6% and 102±5.1% when PV ranged between 0.38 and 58.8 meq/kg. The CV in the analyses of soybean oil (PV=3.25 meq/kg), cod liver oil (PV=6.71 meq/kg), rapeseed oil (PV=12.3 meq/kg), and sardine oil (PV=63.8 meq/kg) were 4.31, 5.66, 8.27, and 11.2%, respectively, demonstrating sufficient reproducibility of the FIA system for the determination of lipid hydroperoxides. The squared correlation (r ²) between the FIA system and the official AOCS iodometric titration method in a linear regression analysis was estimated at 0.9976 within the range of 0.35−77.8 meq/kg of PV (n=42). Thus, the FIA system provided satisfactory detection limits, recovery, and reproducibility. The FIA system was further applied to evaluate changes in the total amounts of lipid hydroperoxides in fish muscle stored on ice.     

Evaluation of MALDI-TOF/MS Technology in Olive Oil Adulteration

Adulteration of extra virgin olive oil (EVOO) by addition of other vegetable oils or lower-grade olive oils is a common problem of the oil market worldwide. Therefore, we developed a fast protocol for detection of EVOO adulteration by mass spectrometry fingerprinting of triacylglycerol (TAG) profiles based on MALDI-TOF/MS. For that purpose, EVOO TAG profiles were compared with those of edible sunflower oil and olive oil composed of refined olive oil and virgin olive oils. Adulteration of EVOO was simulated by addition of sunflower and mixture of refined olive oil and virgin olive oils at 1, 10 and 20% w/w. Results of mass spectrometry TAG profiling were compared with routinely assessed K values for identification of adulteration. MALDI-TOF/MS technology coupled with statistical analysis was proven as useful for detection of adulteration in EVOO at a rate down to 1%. In contrast, standard spectrophotometric methods failed to identify minor adulterations. In addition, the ability of MALDI-TOF/MS in detection of adulteration was tested on EVOO samples from different geographical regions. Results demonstrated that MALDI-TOF/MS technology coupled with statistical analysis is able to distinguish adulterated oils from other EVOO.     

Oil fractionation as a preliminary step in the characterization of vegetable oils by high-resolution 13C NMR spectroscopy

One hundred nine oil samples were separated chromatographically to obtain oil fractions with a decreased TAG content but with enhanced levels of the minor components that define oil genuineness and quality. The oils, which included virgin olive oils from different cultivars and regions of Europe and north Africa and refined olive, “lampante” olive, refined olive pomace, hazelnut, rapeseed, high-oleic sunflower, corn, grapeseed, soybean, and sunflower oils, were fractionated on a silica gel column with hexane/diethyl ether as the mobile phase eluent. The method was highly reproducible, and the fraction obtained contained about 15% unmodified TAG and 85% polar compounds, which included polymeric TAG, oxidized TAG, DAG, MAG, and FFA, in addition to other minor polar components of the oils. The presence of these compounds, in an enriched fraction, should provide information about the thermal, oxidative, and hydrolytic alterations of the oils, as well as many compounds of interest in determining oil genuineness. The results indicate that these fractions can provide more information than the original oils for NMR or other spectroscopic studies used in the determination of oil quality.     

Quantitative determination of phospholipids in sunflower oil

Phospholipids from sunflower oil samples were enriched by using solid-phase extraction (SPE) cartridges and subsequently separated and analyzed by high-performance liquid chromatography (HPLC) with an ultraviolet detector. The recovery of individual phospholipids at different total concentrations in model oils and the repeatability of the method were investigated. The results demonstrated the utility of SPE-HPLC for quantitative analysis of phospholipids in sunflower oil and the effectiveness for concentrating, separating, identifying, and quantitating phospholipids in samples with phosphatide contents as low as 0.1%. Samples of sunflower oil at different stages of processing were analyzed, and phospholipid profiles in hexane-extracted oil, hot-pressed oil, and water-degummed oils were compared.     

Triacylglycerol hydroperoxides not detected in pig small intestinal epithelial cells after a diet rich in oxidized triacylglycerols

The presence of TAG hydroperoxides in the epithelial cells of the small intestines in growing pigs was studied after they had consumed a diet rich in either nonoxidized or oxidized sunflower seed oil (PV in oils, 1 and 190 mequiv O₂/kg, respectively). To obtain molecular-level information on the oxidized TAG structures, a new approach based on TLC and HPLC-electrospray ionization-MS was used in the analysis of the samples. TAG hydroperoxides were not detected in the small intestinal mucosa or adipose tissue of either group, whereas TAG hydroxides, ketones, and epoxides were detected in all samples. The results suggest that dietary TAG hydroperoxides do not lead to the appearance of these molecules in the tissues.     

Thermoxidative stability of triacylglycerols from mutant sunflower seeds

Thermoxidative stability was evaluated in triaclyglycerols (TAG) from the oils of the mutant sunflower lines CAS-3, CAS-4, and CAS-8 (with a high percentage of stearic acid), CAS-5 (with a high percentage of palmitic acid), all from standard highlinoleic genetic backgrounds, and the mutant sunflower line CAS-12 (with a high percentage of palmitic acid), from a high-oleic genetic background. These oils contained unusually high contents of TAG molecular species with one or two saturated fatty acids at the sn-1,3 positions. Purified total TAG devoid of tocopherols were subjected to controlled thermoxidative treatment at 180°C. Polymerized TAG were determined at 2-h intervals for 10 h. After this time, total polar compounds, oxidized TAG monomers, TAG dimers, and TAG oligomers were determined. TAG from highly saturated sunflower oils with levels of linoleic acid similar to those found in conventional sunflower oils (40–50%) showed enhanced thermal stability. In these TAG, the amount of polar compounds formed during the thermoxidative treatment was similar to that formed in the high oleic acid line. Excellent results were obtained for the TAG of the CAS-12 oil, which had the highest thermal stability, producing half the amount of polar compounds as the conventional line and less than two-thirds that of the high-oleic line.     

Monitoring of 2,4‐decadienal in oils and fats used for frying in restaurants in Athens,Greece

Some frying by‐products of medium polarity, so‐called medium‐polarity materials (MPM), produced during domestic deep‐frying of French‐fried potatoes in edible vegetable oils, have recently been isolated and linearly correlated to % total polar materials and % polymerized triglycerides. The in vitro oxidation of low‐density lipoproteins in a dose‐dependent manner by MPM has also been reported. In the present study, the MPM constituents were identified after extraction of MPM from the oils, subsequent purification by RP‐HPLC, and GC‐MS analysis. The main constituent of MPM was trans,trans‐2,4‐decadienal, a compound that has previously been reported to be formed during peroxidation of linoleic and arachidonic acid. 2,4‐Decadienal was also quantified in oils and fats used for frying in restaurants in Athens, Greece, by direct injection of oil sample solutions in HPLC. For the most commonly used frying oils, 2,4‐decadienal concentration ranges were 0.3–119.7 mg/kg for sunflower oil, 13.3–92.7 mg/kg for cottonseed oil, 4.1–44.9 mg/kg for palm oil, and 2.0–11.3 mg/kg for vegetable cooking fats. Considering the common catering practices of frying, 2,4‐decadienal was more likely to be found in sunflower oil after deep‐frying of potatoes. Comparing the amounts of this aldehyde found in oils from restaurants to the amounts previously found for domestic frying (up to 30 mg/kg after the 8^th successive frying session in sunflower oil), the probability of consuming a level of 2,4‐decadienal in restaurant‐prepared food that is higher than the level in home‐fried food was determined to be approximately one third.     

Effects of Filtration on the Quality Properties of Extra Virgin Olive Oils during Storage

In this study, the effects of filtration on quality parameters, chemical characteristics, antioxidant activity, and oxidative stability (OS) of Turkish olive oils during the storage period of 12 months were investigated. The olive oil free acidity (% oleic acid per 100 g of olive oil) (free fatty acidity, FFA), peroxide values (PV) (meq O₂ kg⁻¹ oil), and UV spectrophotometric indices (K₂₃₂ and K₂₇₀ measurements) were used for evaluating the quality parameters of olive oils. α-tocopherol analysis, total phenolic content (TPC), total chlorophyll and carotenoid analyses, and 2,2-diphenyl-1-picrylhydrazyl (DPPH•) radical-scavenging activity (RSA) assays were carried out. Chromatographic methods were applied to determine the fatty-acid and triacylglycerol (TAG) composition, the content of methyl and ethyl esters (FAEE and FAME), and the content of fatty acids of olive oils. Univariate and multivariate statistical methods were performed to evaluate results. Univariate data analysis results showed that filtration of Ayvalık, Memecik, and Domat olive oils had no considerable influence on quality parameters, antioxidant compounds, FAEE and FAME, antioxidant activity, and OS, except TPC (P < 0.05). A significant difference between the samples was determined regarding storage times of the olive oils. Principal component analysis (PCA) analysis revealed that olive oils were grouped according to storage periods of the olive oils regarding fatty-acid and triacylglycerol (TAG) composition while there was no clear separation among the samples according to the filtration process. However, qualitative and quantitative changes took place on minor and major components of olive oils during the storage period.     

Triacylglycerol oxidation in pig lipoproteins after a diet rich in oxidized sunflower seed oil

The effects of two sunflower seed oil diets differing in oxidation levels (PV in oils 1 and 190 mequiv O₂/kg) on lipoprotein TAG and total lipid oxidation were investigated in growing pigs. For 2 wk, two groups of 10 pigs were fed either of the diets, after which blood samples were collected. A method based on RP-HPLC and electrospray ionization-MS was used for the analysis of oxidized TAG molecules in chylomicrons and VLDL. The baseline diene conjugation method was used for the estimation of in vivo levels of lipoprotein lipid oxidation. TAG molecules with a hydroxy, an epoxy, or a keto group attached to a FA, as well as TAG core aldehydes were detected in the samples. Typically, lipoprotein TAG and total lipids were more oxidized in the pigs fed on the oxidized oil compared with those fed on nonoxidized oil. Oxidation of dietary fat was thus reflected in the lipoprotein oxidation, which confirmed our earlier findings.     

Effect of roasting on oxidative and tocopherol stability of walnut oil during storage in the dark

The effect of roasting on the oxidative stability of oil from walnut (Juglans sinensis Dode) was investigated by observing changes in the characteristics of oils from unroasted and roasted kernels during storage in the dark at 60°C. Walnut kernels were roasted at 160°C for 15 min prior to oil extraction with the solvent, hexane. Roasting of kernels increased the peroxide value (POV) and conjugated dienoic acid (CDA) value of the oil. The rate of increase in the POV was significantly lower in roasted than in the unroasted walnut oil during storage at 60°C (1.90 vs. 1.06 and 4.45 vs. 3.55 meq/kg/day during induction period (IP) and post‐IP, respectively). Roasting of kernels significantly increased the IP of walnut oil from 0.89 to 3.39 days during storage. The total tocopherol content in roasted walnut oil was lower as compared to that in unroasted one (277.77 vs. 314.88 µg/g). However, the rate of degradation of total tocopherol during storage was lower in roasted walnut oil compared to unroasted one (1.18 vs. 2.17%/day), which showed that the tocopherol retention was higher in roasted walnut oil. These results indicate that roasting of kernels increased the oxidative and tocopherol stability of oil during storage in the dark.     

Oxidative stability of diacylglycerol oil and butter blends containing diacylglycerols

Diacylglycerol (DAG) oils produced from sunflower oil and traditional sunflower oil were stored for 20 wk at 38 °C, and their oxidative stability was measured. Moreover, two butter blends were produced containing 40 wt‐% DAG oil made from sunflower oil or rapeseed oil, respectively, as well as two control butter blends with sunflower oil or rapeseed oil. Their oxidative stability during storage at 5 °C for up to 12 wk was examined by similar means as for the pure oils. The storage study of the oils indicated that the DAG oil was oxidatively less stable as compared to sunflower oil, but that they had similar sensory quality. Storage of the butter blends revealed that blends with the two types of rapeseed oil (triacylglycerol (TAG) or DAG oil) were oxidatively more stable than the blends containing oils from sunflower. There was no unambiguous indication of DAG butter blends having a different stability than their respective control TAG blends. However, they had a significantly less salty and buttery flavour, which was ascribed to a much smaller water droplet size causing a delayed sensory perception in the mouth. The butter blend with DAG oil from rapeseed had a very neutral flavour. On the contrary, the butter blend with DAG oil from sunflower had a more rancid aroma and flavour than its control blend with sunflower oil.     

Monitoring PV in corn and soybean oils by NIR spectroscopy

NIR spectroscopy was used successfully in our laboratory to monitor oxidation levels in vegetable oils. Calibration models were developed to measure PV in both soy and corn oils, using partial least squares (PLS) regression and forward stepwise multiple linear regression, from NIR transmission spectra. PV can be measured successfully in both corn and soy oils using a single calibration. The most successful calibration was based on PLS regression of first derivative spectra. When this calibration was applied to validation sample sets containing equal numbers of corn and soy oil samples, with PV ranging from 0 to 20 meq/kg, a correlation coefficient of 0.99 between titration and NIR values was obtained, with a standard error of prediction equal to 0.72 meq/kg. For both types of oil, changes occurred in the 2068 nm region of the NIR spectra as oxidation levels increased. These changes appear to be associated with the formation of hydroperoxides during oxidation of the oils.     

Determination of volatile N-nitroso compounds in various samples of edible vegetable oils and margarine (commercially available products)

The examination of samples of various commer-cially available vegetable oils (olive oil, sunflower oil, thistle oil, linseed oil, plant germ oil, etc.) and of various samples of margarine for the presence of vola-tile N-nitroso-compounds yielded the following results. By means of the above mentioned procedure (gas liquid chromatography — AFID gas liquid chromatography — TEA), N-nitrosodimethylamine (NDMA) was found to be present in 21 of 61 dif-ferent samples of vegetable oil, in concentrations ranging from < 1 μg/kg to 23 μg/kg. 18 samples con-tained N-nitrosodiethylamine (NDEA) in concentra-tions varying between < 1μg/kg and 27.8 μg/kg. 37 out of 107 different samples of margarine were shown to contain N-nitroso compounds. N-nitroso-dimethylamine was found to be present in 15 samples. The range of concentrations determined was between < 1 μg/kg and 5.8 μg/kg. 33 samples con-tained N-nitrosodiethylamine in concentrations varying between < 1 μg/kg and 7.5 μg/kg.     

Development of steryl ester analysis for the detection of admixtures of vegetable oils

The steryl ester content and composition of 28 samples from 10 vegetable oil types have been determined by isolation of the steryl esters by high-performance liquid chromatography and analysis by gas chromatography. The oils can be classified into oils with a high content (>4000 mg/kg) of steryl esters (corn and rapeseed); oils with a medium content (1400–2400 mg/kg) of steryl esters (sunflower oil and high-oleic sunflower oil); and oils with a low content (<1200 mg/kg) of steryl esters (safflower, soybean, cottonseed, groundnut, olive, and palm oils). The composition of the steryl ester fraction varies to a greater extent for different oil types than for different varieties of the same oilseed. The developed method is promising for authentication of some oils, and is particularly suitable for detecting admixtures of low levels of corn or rapeseed oils.     

Validation of a method for the analysis of phytosterols in sunflower seeds

Phytosterols are natural compounds that contribute to lower serum cholesterol in humans. Sunflower seeds and oils are rich sources of phytosterols. Breeding for phytosterol content in sunflower has been scarce thus far, mainly because of the lack of analytical methods suitable for use in plant breeding. The objective of this research was to validate a method for the analysis of phytosterols in small seed samples of sunflower. Samples consisting of six seeds were analyzed for phytosterol content in a set of 87 inbred lines using a method adapted to small samples. The accuracy of the method was evaluated through the standard error of the analysis of replicates of ground samples, which was 72.12 mg/kg compared to average values of 1665.3 and 1887.2 mg/kg seed in the samples. Sunflower inbred lines showed ranges of variation from 1426.0 to 4710.0 mg/kg seed and from 2855.2 to 9752.0 mg/kg oil. The method correlated strongly with the conventional method based on the analysis of extracted oils (r = 0.85). The results indicated that analysis of phytosterols on samples consisting of sunflower seeds is an accurate approach for breeding and genetic studies, in which extraction of the seed oil is not feasible. Practical applications : Phytosterols are usually analyzed in extracted oils. However, studies in plant breeding and plant sciences often require a direct analysis of phytosterols in seeds, without previous oil extraction (e.g. large‐scale screening of germplasm in breeding programs or genetic studies). Our results will be useful for plant scientists interested in the analysis of phytosterols in small samples of plant tissues.     

Characterization of volatile compounds and triacylglycerol profiles of nut oils using SPME‐GC‐MS and MALDI‐TOF‐MS

Several nut oil varieties mainly used as culinary and overall healthy food ingredients were subject of the present study. Headspace solid‐phase microextraction combined with gas chromatography‐mass spectrometry was employed in order to determine the qualitative composition of volatile compounds. Furthermore, matrix‐assisted laser desorption/ionization time‐of‐flight mass spectrometry was used in order to assess the profiles and relative composition of the prevalent triacylglycerols (TAG) within the oils. The headspace of the majority of oil samples was dominated by high contents of acetic acid (up to 42%) and hexanal (up to 32%). As nut oils are typically gained by cold‐pressing from previously roasted nuts, characteristic pyrazine derivatives as well as degradation products of long‐chain fatty acids were detected. TAG analysis of these oils revealed a quite homogeneous composition dominated by components of the C₅₂ and C₅₄ group composed mainly of oleic (18:1), linoleic (18:2), stearic (18:0) and palmitic (16:0) acid residues representing together between 65 and 95% of the investigated nut oils. The TAG profiles showed characteristic patterns which can be used as ‘fingerprints’ of the genuine oils. Nut oils exhibiting quite similar fatty acid composition (e.g. hazelnut, pistachio and beech oil) could be clearly discriminated based on TAG showing significant differences between the oils.     

Investigation of lipids profiles of Nigella, lupin and artichoke seed oils to be used as healthy oils

Nigella sativa, lupin and artichoke seed oils have been investigated. The oils were subjected to detailed studies using gas chromatographic analysis (GLC) for fatty acids (FA, as methyl esters) and whole sterols (as silyl derivatives). Whereas, high pressure liquid chromatography (HPLC) was employed for determination of molecular species of triacylglycerols (TAG), four sterol lipids (free and acylated sterols, FS and AS, and free and acylated sterylglycosides, FSG and ASG, as their anthroylnitrile derivatives) as well as tocopherol patterns (T). The results showed that the three seed oils are rich in oleic and linoleic acids whereas, lupin had high linolenic acid content. It was found that the TAGs of the three oils showed some similarity with sunflower oil. Lupin oil had higher sterol content and it was very rich in campe- and β-sitosterol. Nigella sativa oil had a high content of isofucosterol, whereas artichoke oil was unique in having a high content of 5-stigma-, 7-stigma-, and avena- sterol. Concerning the FS and AS, Nigella sativa oil had the highest content, whereas artichoke oil had the highest content of FSG and ASG. Nigella sativa and lupin oils contained over 90 % γ-T while, artichoke oil comprised about 100 % α-T. It is recommended to use the three oils as healthy oils and folk medicine.     

Diet and lipoprotein oxidation: Analysis of oxidized triacylglycerols in pig lipoproteins

Oxidized lipoproteins have a recognized role in atherogenesis, but molecular-level research on oxidized lipids in lipoproteins and the effect of diet on these molecules have been limited. In the present study, the effects of three sunflower seed oil diets differing in oxidation levels (PV in oils 1, 84, and 223 mequiv O₂/kg) on lipoprotein lipid oxidation in growing pigs were investigated. The emphasis was on the investigation of oxidized TAG molecules found in chylomicrons and VLDL. A method based on RP-HPLC and electrospray ionization-MS was used for the analysis of oxidized TAG molecules. The baseline diene conjugation method was used for the estimation of in vivo levels of lipoprotein lipid oxidation. Several oxidized TAG structures were found in the samples. These products consisted of TAG molecules with a hydroxy, an epoxy, or a keto group attached to a FA, and of TAG molecules containing an aldehyde structure derived from a FA. The lipoprotein lipids and TAG were more oxidized in the pigs fed on the most oxidized oil compared with those fed on nonoxidized oil. Oxidation of dietary fat was reflected in the lipoprotein oxidation. New, detailed information on oxidized TAG molecules of chylomicrons and VLDL was obtained.     

Chemiluminescence detection of mono-, bis-, and tris-hydroperoxy triacylglycerols present in vegetable oils

A reliable method was needed to analyze molecular species of oxidized vegetable oils. In order to accomplish this goal, mono-, bis-, and tris-hydroperoxides (Mono-OOH, Bis-OOH, and Tris-OOH, respectively) of triacylglycerols formed during autoxidation and photosensitized oxidation of oils were determined by reversed-phase high-performance liquid chromatography in combination with chemiluminescence detection (CL-HPLC). Mono-OOH was the major species (96% of total hydroperoxides) in trioleoylglycerol [peroxide value (PV) 0.16 meq/kg], and Bis-OOH and Tris-OOH showed prolonged accumulation with photooxidation. This profile was confirmed in photooxidation of trilinoleoylglycerol and trilinolenoylglycerol. Soybean oil (PV 6 meq/kg) contained Mono-OOH oleoyl-linoleoyl-linoleoylglycerol as the main peroxidic molecular species (50% of total hydroperoxides). Mono-OOH trilinoleoylglycerol was the principal species (61% of total hydroperoxides) in safflower oil (PV 5 meq/kg), and Mono-OOH oleoyl-oleoyl-linoleoylglycerol was the representative species (66% of total hydroperoxides) in olive oil (PV 3 meq/kg). The CL-HPLC method, which is specific for the detection of hydroperoxides, should prove useful in studies of triacylglycerol oxidation in foods and vegetable oils.