High-performance liquid chromatography of selected phenolic compounds in olive oils

A reverse-phase high-performance liquid chromatographic technique with isocratic elution has been developed to separate and quantitate the major phenolic compounds of the hydroalcoholic extracts of olive oils. Hydroxytyrosol, tyrosol, caffeic acid,p-hydroxyphenylacetic acid and homovanillic acid were analyzed on a μBonapak C18 column with an acetonitrile/water/acetic acid (20:90:0.18, vol/vol/vol) mixture as a mobile phase. Electrochemical detection provided selectivity as well as sensitivity. The method was applied to the analysis of the most important phenolic compounds in olive oils.     

Synergistic effects of rosemary, sage, and citric acid on fatty acid retention of palm olein during deep-fat frying

A study to optimize the use of oleoresin rosemary extract, sage extract, and citric acid in refined, bleached, and deodorized (RBD) palm olein during deep-fat frying of potato chips was performed using response surface methodology. Results showed that the natural antioxidants used in this study retarded oil deterioration, as evidenced by retention of fatty acid profiles. The linoleic to palmitic (C18∶2/C16∶0) ratio was chosen as the parameter for optimizing the use of natural antioxidants in RBD palm olein during deep-fat frying. Linoleic (R ²=0.946) and palmitic (R ²=0.825) acids were found to be the most important dependent variables, giving highest R ² values to various antioxidant treatments after 25 h of frying. All three antioxidants had independent significant (P<0.05) effects on the C18∶2/C16∶0 ratio. In fact, significant effects on the C18∶2/C16∶0 ratio of RBD palm olein were also given by a second-order form. A combination of 0.076% oleoresin rosemary extract, 0.066% sage extract, and 0.037% citric acid produced the optimal retention of the essential fatty acid C18∶2. In addition, a synergistic effect among these antioxidants on the fatty acid ratio of RBD palm olein was found.     

Chia seeds as a source of natural lipid antioxidants

Chia (Salvia sp) seeds were investigated as a source of natural lipid antioxidants. Methanolic and aqueous extracts of defatted chia seeds possessed potent antioxidant activity. Analysis of 2 batches of chia-seed oils demonstrated marked difference in the fatty acid composition of the oils. In both batches, the oils had high concentrations of polyunsaturated fatty acids. The major antioxidant activity in the nonhydrolyzed extract was caused by flavonol glycosides, chlorogenic acid (7.1 × 10⁻⁴ mol/kg of seed) and caffeic acid (6.6 × 10⁻³ m/kg). Major antioxidants of the hydrolyzed extracts were flavonol aglycones/kaempferol (1.1 × 10⁻³ m/kg), quercetin (2.0 × 10⁻⁴ m/kg) and myricetin (3.1 × 10⁻³ m/kg); and caffeic acid (1.35 × 10⁻² m/kg). Two methods were used to measure antioxidant activities. Both were based on measuring bleaching ofβ-carotene in the coupled oxidation ofβ-carotene and linoleic acid in the presence of added antioxidants.     

Studies on the antioxidants: XI. oxidation products of concomitantly used butylated hydroxyanisole and butylated hydroxytoluene

The effects of antioxidants used concomitantly were studied for their contribution to the autoxidation process and for the function-al mechanism of the antioxidating process. In this study, an equal mixture of butylated hydroxyanisole (BHA) and butylated hydroxy-toluene (BHT) in benzene was irradiated with ultraviolet rays and the chemical structures and antioxidant activities of the resulting oxides were derived. The chemical changes of the substrates and oxides were followed quantitatively by gas chromatography. The resulting oxide was confirmed to be 3,3′,5′-tri-tert-butyl-5-methoxy-2,4′-dihydroxydiphenyl methane, which was a dehydrogenated dimer of BHA and BHT. The stability study on this compound by AOM showed its strong activity as an antioxidant on lard and soybean oil.     

A multivariate optimization of triacylglycerol analysis by high-performance liquid chromatography

In the present study we have used statistical experimental design and multivariate optimization to formally optimize a reversed-phase high-performance liquid chromatography method for the analysis of triacylglycerol molecular species of natural oils. The optimal conditions found were, on an octadecylsilan-column, from acetonitrile/isooctane (90∶10, vol/vol) to acetonitrile/ethanol/isooctane (40∶35∶25, by vol), at a column temperature of 50°C and a flowrate of 1.5mL/min using a negative exponential gradient profile. Several examples of separations of natural seed and animal oils,i.e., soybean oil, rapeseed oil, palm oil, linseed oil, tallow and fish oil, are given. A version of the equivalent carbon number concept, utilizing the Snyder polarity index, was used to identity the molecular species.     

Identification of natural diacylglycerols as the 3,5-dinitrophenylurethanes by chiral phase liquid chromatography with mass spectrometry

This study reports a facile identification of the molecular species of enantiomeric diacylglycerols by combining chiral phase high-performance liquid chromatography with positive chemical ionization mass spectrometry. For this purpose the 3,5-dinitrophenylurethane (DNPU) derivatives ofsn-1,2(2,3)-diacylglycerols are separated on an (R+)-naphthylethylamine polymer column (25 cm × 0.46 cm ID) using an isocratic solvent system made up of hexane/dichloroethane/acetonitrile (85∶10∶5, by vol) or isooctane/tert-butyl methyl ether/acetonitrile/isopropanol (80∶10∶5∶5, by vol). About 1% of the column effluent (1 mL/min) was admitted to a quadrupole mass spectrometer (Hewlett-Packard, Palo Alto, CA)via direct liquid inlet interface, and positive chemical ionization spectra were recorded over the range of 200–900 mass units. The DNPU derivatives of diacylglycerols yield characteristic [M-DNPU]⁺ and [RCO+74]⁺ ions for each diacylglycerol species from which the molecular weight and exact pairing of fatty acids can be unequivocally obtained. The characteristic ions appear to be generated in nearly correct mass proportions as indicated by preliminary quantitative comparisons. The abbreviations 14∶0, 16∶1, 18∶2, etc. represent normal chain fatty acids of 14, 16, 18, etc. acyl carbons and 0, 1, 2, etc. double bonds, respectively; 16∶0–18∶1, etc. represent diacylglycerols containing 16∶0 and 18∶1 fatty acids of unspecified positional distribution;sn indicates stereospecific numbering of glycerol carbons;sn-1,2-diacylglycerols andsn-2,3-diacylglycerols are enantiomeric diacylglycerols of unspecified fatty acid composition;rac-1,2-diacylglycerols are racemic diacylglycerols representing equal amounts ofsn-1,2-andsn-2,3-enantiomers;sn-1,2(2,3)-diacylglycerols are a mixture ofsn-1,2-andsn-2,3-diacylglycerols of unspecified proportion of enantiomers and unspecified fatty acid compisition and positional distribution; X-1,3-diacylglycerols are diacylglycerols of unspecified fatty acid composition and reverse isomer content.     

Chromatographic separation of glucopyranosyl sinapate from canola meal

The compound 1-O-β-D glucopyranosyl sinapate (GPS), a phenolic glycoside, was separated from ethanolic extracts of defatted canola meal by a two-step chromatographic method. The first step involved Sephadex LH-20 chromatography with methanol as the eluting solvent. The solvent from the fraction containing GPS was evaporated, and glucopyranosyl sinapate was subsequently separated by a semi-preparative high-performance liquid chromatography method with an RP-18 column and a mobile phase consisting of water/acetonitrile/acetic acid (88∶10∶2, vol/vol/vol).     

Characterization and oxidative stability of enzymatically produced fish and canola oil-based structured lipids

Two-kilogram quantities of structured lipids (SL) of menhaden fish and canola oils containing caprylic acids (8∶0) were produced in a laboratory-scale packed-bed bioreactor by acidolysis catalyzed by an immobilized lipase, Lipozyme IM, from Rhizomucor miehei. SL were characterized and their oxidative stabilities investigated. The SL contained 29.5% 8∶0 for fish oil and 40.15 for canola oil. Polyunsaturated fatty acids (PUFA) of fish oil remained unchanged after the modification while PUFA of canola oil were reduced from 29.6 to 21.2%. Monoenes, especially 18∶1n−9, were completely replaced by 8∶0 in fish oil and reduced from 61.9 to 34.7% in canola oil. Downstream processing of enzymatically produced SL led to loss in natural total tocopherol contents of the fish and canola oils. The effects of antioxidants such as α-tocopherol (TOC), tert-butylhydroxyquinone (TBHQ), and combinations thereof on the oxidative stability of SL were investigated. SL were analyzed for oxidative stability index, peroxide value, conjugated diene content, free fatty acid content, iodine value, saponification number, and thiobarbituric acid value. Iodine value of unmodified fish oil (154.71) was reduced to 144.10 and that of canola oil (114.49) to 97.27 after modification. The SN increased from 183.72 to 242.63 for fish oil and from 172.50 to 227.90 for canola oil. TBHQ exhibited better antioxidant effects than TOC. A combination of TBHQ/TOC also proved to be an effective antioxidant for SL. We suggest the addition of antioxidants to enzymatically produced and purified SL.     

Studies on the antioxidants XVII: Photooxidation products of concomitantly used butylated hydroxyanisole and propyl gallate

The effects of antioxidants were studied with a veiw to pursuing the fate of antioxidant molecules during the antioxidation processes. In this study, with the same view, an equal mixture of butylated hydroxyanisole (BHA) and propyl gallate (PG) in ethanol was irradiated with ultraviolet rays and the chemical structure and antioxidative activities of the resulting oxides were examined. The resulting oxides were identified as 2,3,7,8-tetrahydroxy [1] benzopyrano [5,4,3-cde] [1] benzopyran-5,10-dione (ellagic acid), which was formed from PG by coupling of dehydrogenated molecules of OH group and by dealcoholization, and propyl 3,4-dihydroxy-5-(2′-hydroxy-5′-methoxy-3′-tert-butylphenoxy)benzoate and propyl 3,5-dihydroxy-4-(2′-hydroxy-5′-methoxy-3′-tert-butylphenoxy) benzoate, both of which were dimers, one formed from a BHA/PG mixture by separation of hydrogen in aromatic hydrogen and the other from PG by dehydrogenation of OH group. All of the resulting oxides showed antioxidative activity on lard, soybean oil, and methyl oleate in the stability testing by the active oxygen method (AOM).     

Effect of natural antioxidants in virgin olive oil on oxidative stability of refined,bleached, and deodorized olive oil

The factors influencing the oxidative stability of different commercial olive oils were evaluated. Comparisons were made of (i) the oxidative stability of commercial olive oils with that of a refined, bleached, and deodorized (RBD) olive oil, and (ii) the antioxidant activity of a mixture of phenolic compounds extracted from virgin olive oil with that of pure compounds andα-tocopherol added to RBD olive oil. The progress of oxidation at 60°C was followed by measuring both the formation (peroxide value, PV) and the decomposition (hexanal and volatiles) of hydroperoxides. The trends in antioxidant activity were different according to whether PV or hexanal were measured. Although the virgin olive oils contained higher levels of phenolic compounds than did the refined and RBD oils, their oxidative stability was significantly decreased by their high initial PV. Phenolic compounds extracted from virgin olive oils increased the oxidative stability of RBD olive oil. On the basis of PV, the phenol extract had the best antioxidant activity at 50 ppm, as gallic acid equivalents, but on the basis of hexanal formation, better antioxidant activity was observed at 100 and 200 ppm.α-Tocopherol behaved as a prooxidant at high concentrations (>250 ppm) on the basis of PV, but was more effective than the other antioxidants in inhibiting hexanal formation in RBD olive oil.o-Diphenols (caffeic acid) and, to a lesser extent, substitutedo-diphenols (ferulic and vanillic acids), showed better antioxidant activity than monophenols (p- ando-coumaric), based on both PV and hexanal formation. This study emphasizes the need to measure at least two oxidation parameters to better evaluate antioxidants and the oxidative stability of olive oils. The antioxidant effectiveness of phenolic compounds in virgin olive oils can be significantly diminished in oils if their initial PV are too high.     

Hematological and lipid changes in newborn piglets fed milk replacer diets containing vegetable oils with different levels of n−3 fatty acids

To test if linolenic acid (18∶3n−3) from vegetable oils would affect bleeding times and platelet counts in new-borns, piglets were used as a model fed milk replacer diets containing 25% (by wt) vegetable oils or oil mixtures for 28 d and compared to sow-reared piglets. The oils tested included soybean, canola, olive, high oleic sunflower (HOAS), a canola/coconut mixture and a mixture of oils mimicking canola in fatty acid composition. All piglets fed the milk replacer diets showed normal growth. Bleeding times increased after birth from 4–6 min to 7–10 min by week 4 (P<0.001), and were higher in pigs fed diets containing 18∶3n−3, as well as in sowreared piglets receiving n−3 polyunsaturated fatty acids (PUFA) in the milk, as compared to diets low in 18∶3n−3. Platelet numbers increased within the first week in newborn piglets from 300 to 550×10⁹/L, and remained high thereafter. Milk replacer diets, containing vegetable oils, generally showed a transient delay in the rise of platelet numbers, which was partially associated with an increased platelet volume. The oils showed differences in the length of delay, but by the third week of age, all platelet counts were >500×10⁹/L. The delay in rise in platelet counts appeared to be related to the fatty acid composition of the oil, as the effect was reproduced by a mixture of oils with a certain fatty acid profile, and disappeared upon the addition of saturated fatty acids to the vegetable oil. There were no alterations in the coagulation factors due to the dietary oils. Blood plasma, platelets and red blood cell membranes showed increased levels of 18∶3n−3 and long-chain n−3 PUFA in response to dietary 18∶3n−3. The level of saturated fatty acids in blood lipids was generally lower in canola and HOAS oil-fed piglets as compared to piglets fed soybean oil or reared with the sow. The results suggest that consumption of milk replacer diets containing vegetable oils rich in 18∶3n−3 does not represent a bleeding risk, and that the transient lower platelet count can be counterbalanced by the addition of saturated fatty acids to the vegetable oils.     

Optimization of physicochemical changes of palm olein with phytochemical antioxidants during deep-fat frying

Response surface methodology (RSM) was used to optimize the amounts of rosemary and sage extracts together with citric acid as synergist antioxidants in stabilizing refined, bleached, and deodorized palm olein during repeated deep-fat frying of potato chips. For all physicochemical properties studied, these phytochemical antioxidant treatments significantly (P<0.05) reduced the oxidation rate of the oil. During 5 d of frying, anisidine value, peroxide value, free fatty acid, polymer content, color units, viscosity, and absorbances at 232 and 268 nm gradually increased, whereas iodine value and ratio of 18∶2/16∶0 decreased. Further statistical analyses, including coefficient of determination (R ²) and probability of F values, indicated that mathematical models for each physicochemical parameter could be developed confidently in this study, with R ² for all parameters greater than 0.90. These results suggested that an optimal mixture of phytochemical antioxidants derived from rosemary and sage together with citric acid could be produced using RSM for stabilizing thermally processed oil. For many physicochemical parameters examined, the use of moderate levels of antioxidants could result in optimal responses.     

Antioxidant activity of extracts of defatted seeds of niger (<Emphasis Type="Italic">Guizotia abyssinica</Emphasis>)

Niger (Guizotia abyssinica) seed was ground and then defatted with hexane. The meal remaining after oil extraction was tested as a source of antioxidants. Three solvent systems, A [80∶20 (vol/vol) ethanol/water], B [80∶20 (vol/vol) acetone/water], and C (water) were evaluated as extraction media. Crude extracts were examined for their antioxidant activity in a β-carotene-linoleate and a meat model system. Extract A exhibited superior antioxidant activity, compared to extracts B and C, and its composition was studied further by using column chromatography and HPLC. Four fractions (I–IV) were obtained, of which fractions III and IV showed activity in the β-carotene-linoleate model system. Fraction IV was also highly effective in scavenging the 2,2-diphenyl-1-picrylhydrazyl radical but was less active when used in a bulk oil model system. Preparative TLC showed fraction IV as consisting of two components. UV spectroscopy suggested that the major active component pressent was a chlorogenic acid-related compound. Furthermore, HPLC analysis established that chlorogenic acid was dominant in the free phenolics fraction (2.6 mg/g). Upon hydrolysis, however, a substantial amount of caffeic acid (42.8 mg/g) was released, presumably from esterified and glycosylated chlorogenic acid. Thus, niger extracts derive their antioxidant activity, at least in part, from the chlorogenic acid-related compounds.     

Rapid separation of the major phospholipid classes on a single aminopropyl cartridge

A rapid method for the separation of the individual phospholipid classes phosphatidylcholine (PC), phosphatidylethanolamine (PE), phosphatidylserine (PS) and phosphatidylinositol (PI) by a single solid-phase extraction was developed. PC, PE, PS and PI were sequentially eluted from aminopropyl bonded silica with acetonitrile/n-propanol (2∶1, vol/vol), methanol, isopropanol/methanolic HCl (4∶1, vol/vol) and methanol/methanolic HCl (9∶1, vol/vol). Standard recoveries were over 95% for PC and PE and over 85% for PS and PI with undistorted fatty acid composition. The separation of complex lipid mixtures on aminopropyl minicolumns can be refined to the level of individual phospholipid classes.     

Concentration and stabilization of n-3 polyunsaturated fatty acids from sardine oil

A simple and relatively inexpensive procedure to obtain 90% eicosapentaenoic acid + docosahexaenoic acid concentrates from sardine oil involved a two-step winterization of the oil (10 and 4°C), followed by saponification and selective precipitation of saturated and less unsaturated free fatty acids by an ethanolic solution of urea. Antioxidant effects of butylated hydroxytoluene, dl-α-tocopherol, and two natural antioxidants, quercetin and boldine, added to the concentrate (0.5% wt/vol), were compared in the Rancimat at 60°C. dl-α-Tocopherol was unable to inhibit concentrate oxidation. Butylated hydroxyanisole and butylated hydroxytoluene had induction periods of 1.7–1.8 h compared to the control (1.0 h). A mixture of quercetin + boldine (2:1 w/w) significantly increased the induction period to 4.5 h.     

Analysis of free and esterified sterols in vegetable oils

In vegetable oils, phytosterols occur as free sterols or as steryl esters. Few analytical methods report the quantification of esterified and free sterols in vegetable oils. In this study, esterified and free sterols were separated by silica gel column chromatography upon elution with n-hexane/ethyl acetate (90∶10 vol/vol) followed by n-hexane/diethyl ether/ethanol (25∶25∶50 by vol). Both fractions were saponified separately and the phytosterol content was quantified by GC. The analytical method for the analysis of esterified and free sterols had a relative standard deviation of 1.16% and an accuracy of 93.6–94.1%, which was comparable to the reference method for the total sterol analysis. A large variation in the content and distribution of the sterol fraction between different vegetable oils can be observed. Corn and rapeseed oils were very rich in phytosterols, which mainly occurred as steryl esters (56–60%), whereas the majority of the other vegetable oils (soybean, sunflower, palm oil, etc.) contained a much lower esterified sterol content (25–40%). No difference in the relative proportion of the individual sterols among crude and refined vegetable oils was observed.     

Quenching mechanism and kinetics of ascorbyl palmitate for the reduction of the gamma irradiation-induced oxidation of oils

The effects of 0, 250, 500, and 1000 ppm (wt/vol) ascorbyl palmitate (AP) on the gamma irradiation-induced oxidation of soybean oil, cottonseed oil, corn oil, tallow, lard, or linoleic acid either in a solvent mixture (benzene/methanol, 4:1 vol/vol) or in methanol, was studied immediately after gamma irradiation with a dose of 1–5 kGy. Steady-state kinetic approximation was used to determine a quenching mechanism and quenching rate constant of AP on the gamma irradiation-induced oxidation of purified soybean oil in a solvent mixture (benzene/methanol, 4:1 vol/vol). Irradiation greatly increased oxidation of all oils, as was expected. AP was extremely effective at minimizing oxidation in all oils, and its effectiveness was concentration dependent. AP showed significantly greater antioxidative activity than α-tocopherol for the reduction of oxidation in all oils (P<0.05). The steady-state kinetic studies indicated that AP quenched oxygen only to minimize the oxidation of oils. The calculated total quenching rate of AP was 7.51×10⁷ M⁻¹s⁻¹. The present results clearly show the effective oxygen quenching ability of AP for the reduction of gamma irradiation-induced oxidation of oils.     

Conifer seeds: Oil content and fatty acid composition

The seed oils from twenty-five Conifer species (from four families—Pinaceae, Cupressaceae, Taxodiaceae, and Taxaceae) have been analyzed, and their fatty acid compositions were established by capillary gas-liquid chromatography on two columns with different polarities. The oil content of the seeds varied from less than 1% up to 50%. Conifer seed oils were characterized by the presence of several Δ5-unsaturated polymethylene-interrupted polyunsaturated fatty acids (Δ5-acids) with either 18 (cis-5,cis-9, 18∶2,cis-5,cis-9,cis-12 18∶3, andcis-5,cis-9,cis-12,cis-15 18∶4 acids) or 20 carbon atoms (cis-5,cis-11 20∶2,cis-5,cis-11,cis-14, 20∶3, andcis-5,cis-11,cis-14,cis-17 20∶4 acids). Pinaceae seed oils contained 17–31% of Δ5-acids, mainly with 18 carbon atoms. The 20-carbon acids present were structurally derived from 20∶1n-9 and 20∶2n-6 acids. Pinaceae seed oils were practically devoid of 18∶3n-3 acid and did not contain either Δ5-18∶4 or Δ5-20∶4 acids. Several Pinaceae seeds had a Δ5-acid content higher than 50 mg/g of seed. The only Taxaceae seed oil studied (Taxus baccata) had a fatty acid composition related to those of Pinaceae seed oils. Cupressaceae seed oils differed from Pinaceae seed oils by the absence of Δ5-acids with 18 carbon atoms and high concentrations in 18∶3n-3 acid and in Δ5-acids with 20 carbon atoms (Δ5-20∶3 and Δ5-20∶4 acids). Δ5-18∶4 Acid was present in minute amounts. The highest level of Δ5-20∶4 acid was found inJuniperus communis seed oil, but the best source of Δ5-acids among Cupressaceae wasThuja occidentalis. Taxodiaceae seed oils had more heterogeneous fatty acid compositions, but the distribution of Δ5-acids resembled that found in Cupressaceae seed oils. Except forSciadopytis verticillata, other Taxodiaceae species are not interesting sources of Δ5-acids. The distribution profile of Δ5-acids among different Conifer families appeared to be linked to the occurrence of 18∶3n-3 acid in the seed oils.     

Studies on the antioxidants: X. oxidation products of concomitantly used butylated hydroxyanisole and ethyl protocatechuate

The effects of antioxidants were studied from the viewpoints of (a) the products generated during the autoxidation process and (b) the functional mechanism of antioxidation. The chemical structure and antioxidant activity of the oxide obtained by irradiating an ethanol solution of an equal mixture of butylated hydroxyanisole (BHA) and ethyl protocatechuate (EP) with the ultraviolet (UV) ray and its secondary decomposition were studied. The oxide was identified as 2,2′-dihydroxy-3-tert-butyl-5-methoxy-5′-carboethoxy-diphenyl ether which was a dimer of free radicals generated by BHA and EP. The resulting oxide showed a strong antioxidant activity on lard and methyl oleate in the stability testing by active oxygen method (AOM).     

Simultaneous determination of ascorbyl palmitate and nine phenolic antioxidants in vegetable oils and edible fats by HPLC

This study describes an HPLC method for the simultaneous determination of ascorbyl palmitate (AP) and synthetic phenolic antioxidants (SPA) in vegetable oils and edible fats in a single run. To achieve this, citric acid was used in combination with isoascorbic acid for stabilization of AP in standard and sample solutions and for deactivation of oxidizing agents in the HPLC system. SPA and AP were directly extracted from samples with methanol containing 1 mg/mL each of citric acid and isoascorbic acid. HPLC analytical and guard columns were pretreated with 90% methanol/acetonitrile 1∶1 (vol/vol), containing 4 mg/mL each of citric acid and isoascorbic acid, and 10% water at pH 3, for 30 min. Under these conditions, AP was stable for about 7 h at room temperature. The relative SD of repeatability for AP (0.5–3.6%) was comparable to that for SPA (0.3–2.8%). Average recovery from spiked samples was 100% for AP, 98–103% for SPA, and 85% for BHT (up to 90% using double extraction with methanol).