Analysis of triacylglycerols in leaves of citrus by HPLC

Lipids were extracted from leaves of cold-hardened and unhardened citrus plants and the crude triacylglycerol (TG) fraction of each sample was separated from other lipid components by silica gel column and thin layer chromatography (TLC). To determine TG levels and molecular species, the crude TG was subjected to High presure liquid chromatography (HPLC), 12 fractions collected and these fractions quantified by gas liquid chromatography (GLC). Levels of TG/g fresh leaf were 139 μg in unhardened sour orange, 2460 μg in cold-hardened sour orange and 672 μg in cold-hardened Valencia orange. Twenty-one molecular species were determined in the 36–46 equivalent carbon number range (ECN). Five of the TG species that contained linoleate accounted for over 60% of total TG in hardened sour orange and Valencia leaves. Hardened sour orange leaves contained 2–5 times more of these major TG species than hardened Valencia leaves. The increase in these TG species may relate to cold tolerance since sour orange seedlings are more cold hardy than Valencia budded on sour orange. Presented at the 73rd Annual Meeting of the AOCS, Toronto, May 1982. Southern Region, Agricultural Research Service, U.S. Department of Agriculture.     

Carotenoids and tocols of corn grain determined by HPLC

A high performance liquid chromatographic (HPLC) procedure has been developed that permits determinationof carotenoids and tocols in the same sample preparation of corn grain. For 15 inbreds, the total carotenoids ranged from 16 to 77 μg/g dry wt and the total tocols from 30 to 128 μg/g dry wt. For four inbreds, total carotenoids were concentrated in the horny endosperm (83±2%) and total tocols in the germ (77±6%). After six months storage at room temperature, the mean loss of total carotenoids for four inbreds was 42±4%, while the tocols had a mean loss of 5%. Presented at the AOCS meeting in Honolulu, HI in May 1986.     

Kinetic analysis of plasma VLDL-TG and VLDL-remnant-TG turnover in anesthetized rats

We have estimated the turnover and relative pool sizes of nascent-VLDL-TG and VLDL-remnants-TG in anesthetized rats. [1-¹⁴C]Palmitoyl- and [2-³H]glyceryl-labeled “VLDL”-TG (including nascent VLDL-TG and VLDL-remnants-TG) were prepared by injecting labeled palmitate and glycerol into donor rats. Labeled serum from these rats was then injected intravenously into nembutalized male rats and serial blood samples taken for 30 min. Special care was taken to define any early components in the labeled “VLDL”-TG disappearance curves. In other experiments, the donors were rendered functionally hepatectomized 30 min after injection of³H-glycerol and the endogenous labeled VLDL-TG was allowed to circulate 30–60 min before collection of the TG-labeled VLDL-remnants-containing serum. The latter was injected into 4 recipient nembutalized rats and the remnant-TG-turnover measured by serial sampling as above. In two cases,¹⁴C-“VLDL” and³H-VLDL-remnants were injected as a single bolus into ether-anesthetized rats. Despite its complex composition, “VLDL”-TG in most cases disappeared in a single exponential fashion for 30 min with an average half-life of 5.9 min in nembutalized and 2.8 in ether-anesthetized rats. VLDL-remnants-TG showed a more complex behavior, but contained a major rapid component with a mean t_1/2 of ca. 1.5 min in both groups. The data, analyzed by multicompartmental analysis, were fitted to a simple model in which turnover of a larger nascent VLDL-TG pool with formation of a more rapidly turning over smaller pool of VLDL-remnant-TG is the rate-limiting step in overall TG removal from the d<1.006 fraction of rat serum. The data are consistent with our theoretical prediction that under these conditions the kinetics of the VLDL-remnants cannot be resolved from analysis of the total composite “VLDL” (nascent plus remnant) pool.     

Physicochemical characterization of galactosyldiglycerides and their quantitation in wheat flour lipids by high performance liquid chromatography

A high performance liquid chromatographic (HPLC) method was developed for analyzing digalactosyldiglycerides (DGDG) and monogalactosyldiglyceride (MGDG) in polar lipids fractionated from lipid extracts of wheat or flour. Wheat lipid samples were prepared by solvent extraction, then fractionated on a silica gel packed open column. A Spherisorb ODS (octadecyl silane) column with methanol/water elution system was used for separation of glycolipids in the polar lipid fractions. The detection limit of the refractive index detector with interferometric optics was 0.25μg for both DGDG and MGDG. Separating on nonpolar bonded phase columns permitted us to differentiate, based on fatty acid composition and position, among components within the specific glycolipid classes. Semipreparative HPLC on analytical columns was used to subfractionate the polar lipids. The glycolipids were collected for functional group characterization. Approximately 35% of each DGDG subfraction was accounted for as carbohydrate. The absence of phosphorus precluded phospholipids. Fatty acid analysis by gas chromatography showed the first DGDG to be linoleic acid, whereas the second DGDG peak was composed of linoleic, oleic and palmitic acids. Mass spectrometric analysis of the first DGDG peak showed linoleic acid in both the SN-1 and 2 positions. Mass spectrometric analysis revealed that palmitic or oleic acid in the second peak was preferentially located on the SN-1 position; linoleic acid was on the SN-2 position. Contribution no. 80-207J, Department of Grain Science and Industry, Kansas Agricultural Experiment Station, Kansas State University, Manhattan, KS. Part of a dissertation submitted by T.N. Tweeten in partial fulfillment of the PhD degree. Honored Student Award Presentation at AOCS annual meeting, San Francisco, April 1979.     

Cholesterol oxides in swedish foods and food ingredients: Butter and cheese

Fresh butter manufactured by batch or continuous technologies in Sweden contained only traces of 7-ketocholesterol at the detection limit (0.1 ppm in the lipids) and no detectable levels of other common cholesterol oxides. Storage at 4°C for up to four mo caused an increase in the levels of the isomeric 5,6-epoxycholestanols, the epimeric 7-hydroxycholesterols, 20α-hydroxycholesterol and 7-ketocholesterol as revealed by Lipidex chromatography, TEAP-Lipidex chromatography and GLC. Heating of butter for 10 min at the temperature interval 150–200°C under conditions similar to that in shallow-pan kitchen frying caused a gradual increase in the total amount of cholesterol oxides from 0 to 2.5 ppm, while at 200°C the total amount of cholesterol oxides was 1.6 ppm. Butter heated five min at 200°C also had less cholesterol oxides than that heated at 190°C for 10 min. Industrially manufactured butter oil and cream oil contained only low levels of some of the cholesterol oxides. Further, three types of processed cheese (soft-melted, hard-melted and grated cheese) were investigated. The soft-melted and the hard-melted cheese types contained only traces of some of the oxides, while the grated cheese samples had 0.5–2.2 ppm of the total cholesterol oxides in the lipids.     

Evaluation of the rapid micromethod for ultracentrifugal separation of labeled plasma lipoproteins

The fractionations of plasma lipoproteins by 2 methods were compared to evaluate the rapid separation (Airfuge^®) method for lipoprotein distribution studies. When [¹²⁵I] labeled very low density, low density, and high density lipoproteins (VLDL, LDL, HDL), were separately centrifuged in buffers at d=1.006, 1.06 or 1.2 g/ml by the conventional ultracentrifuge and the Airfuge^®, separations of the fractions in the Airfuge^® were incomplete at both 5 C and 24 C, especially at d=1.006. [³H] Benzo (a)pyrene, when added to plasma, associates with the plasma proteins and lipoproteins, especially LDL. Compared to the standard techniques, the Airfuge^® method greatly overestimated its distribution into VLDL. The distribution of [³H] vitamin D₃ into the VLDL plus LDL fraction was also overestimated by the Airfuge^® procedure. It is concluded that caution should be observed in quantitative studies of lipoproteins in the Airfuge^®. A careful comparison of the distribution into or fractionation of lipoproteins by the 2 methods should always precede any quantitative determinations involving the Airfuge^®.     

Automated AOM test for fat stability

A home-built version of the automated AOM test was used with Canola, corn, sunflower, olive and Crisco? oils, shortening and lard. The endpoint was found by measuring the conductivity of a solution of the exit gas from the reaction tube. Coefficients of variability of the samples ranged from 1.1% to 8.3%. The endpoint of the test was ca. 100 PV for Canola oil, ca. 200 PV for corn oil and 35 PV for lard. The aqueous solutions of the volatiles of three oils were used to determine the TBA value. Canola, sunflower and olive oil had TBA values ranging from 6–60 μg malonaldehyde/g at the end point. No apparent relationship was found between the TBA values of the volatiles’ solutions and the PV’s of the oils. Presented at the 73rd AOCS Annual Meeting, Toronto, 1982.     

Determination of surfactant mixtures in shampoos and detergents by HPLC

A technique for separating 4 nonionic, 7 anionic and 4 amphoteric surfactants with n-dodecyl groups was studied by high performance liquid chromatography (HPLC) and applied to the determination of these surfactants in commercial shampoos and household detergents. Conditions used for the separation were: column packing and size, TSK-LS 410 (5μ) and 6 mm i.d. × 500 mm (2 connected, 250 mm columns); mobile phase, water/methanol (25/75, v/v) containing 0.25 M sodium perchlorate adjusted to pH 2.5 with phosphoric acid; column temp., 50 C; detector, RI. Surfactants in shampoos and detergents were clearly distinguished from each other and determined without column chromatographic pretreatment, e.g., ion-exchange chromatography.     

Analysis of milkfat by HPLC

A routine method for HPLC analysis of milkfat has been developed, which takes into account the specific phenomena of this complex and far-ranging system of triglycerides. The usual amount of injection of 1 mg has been reduced to 30 or 10 μg of fat. By this the composition of the mobile phase (acetone:acetonitrile, 35/65) and the temperature of the column (Nucleosil C18-5 μ, 15 cm + Microspher C18-3μ, 10 cm, in series, T=30–35 C) could be adjusted to a relatively high selectivity without inducing the high-melting fat components to crystallize on the column. A further advantage resulting from this consisted in permitting the eluent to be recycled over long periods of time. The extremely low amounts of samples necessitated a highly sensitive detection (Δn=5 × 10⁻⁷ RI units full scale deflection) which could be realized by an interferential refractometer in connection with a thermostat to keep up a stable temperature of the whole HPLC system (ΔT<0,005 K). By this it was possible to separate milkfat into 45 to 50 different types of triglycerides. By comparing soft and hard milkfats and fractions of milkfats, every fourth peak, starting with C 42, could be attributed to saturated triglycerides; apart from this, easily recognizable qualitative features appearing in the chromatograms permitted conclusions about the feeding regimen and energy supply of the cattle. Furthermore, due to the high stability of separating conditions achieved and due to computer software developed for this purpose it was possible to obtain and compare a large number of chromatograms under the same conditions.     

Fatty acid and sterol compositions of malagasy tamarind kernel oils

The oil contents of six samples of Malagasy tamarind (Tamarindus indica L.) kernel were determined by hexane extraction (6.0–6.4%) and chloroform/methanol extraction (7.4-9.0%). The protein contents were very low (trace-0.1%). Investigation by gas liquid chromatography revealed 15 fatty acids, mainly palmitic (14–20%), stearic (6–7%), oleic (15–27%), linoleic (36–49%), arachidic (2–4%), behenic (3–5%) and lignoceric (3–8%) acids. Testing for the sterol fraction enabled seven sterols to be separated and quantitatively analyzed by gas liquid chromatography. The main sterols were β-sitosterol (66–72%), campesterol (16–19%) and stigmasterol (11–14%).     

Determination of TBA number by high performance liquid chromatography

A high performance liquid chromatographic (HPLC) method for the quantitation of malonaldehyde in aqueous distillates was developed. Compared with the standard TBA test, the HPLC method was faster, and less affected by side feactions. A total of 5 min was necessary to assay each distillate and only malonaldehyde was detected. The standard curves were reproducible and standards were stable for up to 6 days. The HPLC method could detect malonaldehyde levels ranging from 1×10⁻¹¹ to 4×10⁻¹¹ mol/10μL and either peak height or peak area could be used to quantitate the malonaldehyde concentration. The coefficient of determination between absorbance values determined by the TBA test and peak heights determined by HPLC was 0.946. Twenty-one freeze-dried chicken samples with TBA numbers ranging from 3.93 to 16.6 were used for this correlation.     

Analysis of polymers from autoxidized marine oils by gel permeation HPLC using a light-scattering detector

A high-performance gel-permeation chromatographic method was developed for the isolation and quantification of polymers from autoxidized fish oils. The chromatographic system consists of a single pump, a 500 Å styrene/divinylbenzene column, and a light-scattering “mass” detector. Dichloromethane was used as the mobile phase, and quantifications were carried out using glycerol as internal standard. The polymer content of commercially available fish oils was found to be around 2%, but the value increased rapidly during oxidation at 35°C in air under light. The oxidized oils were also characterized by peroxide value, thiobarbituric acid value, ultraviolet (UV) measurements, thin-layer chromatography (TLC)/densitometry, and capillary gas chromatography. No correlation between polymer content and standard oxidation assessment parameters for the fish oils analyzed was found. However, a relationship between the polymer increase and the decrease of total fatty acids was observed, indicating that the polymers may be the main oxidation product in autoxidized marine oils.     

Chemical studies on corn embryos infected by various fungi

The occurrence of various fungi in corn kernels obtained from eight localities in Egypt in two successive years was studied. Values for refractive index, color, acid value, saponification value, iodine value, peroxide value and unsaponifiable matter content of oils extracted from corn embryos that were deliberately infected by various fungi were compared to those for oil extracted from healthy embryos. Spectrometric analyses (UV, visible and IR) were done to deduce differences in the functional groups of the oils. Corn oil extracted from embryos infected with various fungi contained the same lipid classes as the oil extracted from healthy embryos. Contents of mono- and diglycerides and free fatty acids were much smaller for the oil extracted from healthy embryos. The fatty acid and unsaponifiable compositions of oils were studied by gas liquid chromatography. The fatty acid composition of corn oil extracted from infected embryos showed that some new and short-chain fatty acids had appeared and that some of the 18:2 was converted to 18:0. Analysis of the hydrocarbon fraction of the unsaponifiables showed also that some new compounds had appeared and others disappeared. The sterols were greatly influenced by the fungi and the ratio between different sterols might be used to characterize the effect of fungi. Aflatoxin B₁ content of oil extracted from corn embryos infected byA. flavus was 300 μg/kg.     

Computation of conversion factors to determine the phospholipid content in peanut oils

Peanut oil was separated into the various lipid classes by column chromatography. The polar lipid fraction which contained phospho-lipids was separated into individual major components by 2-dimen-sional thin layer chromatography. Conversion factors for calculating the concentration of total phospholipid in peanut oil from percent elemental phosphorus were determined by estimation of molecular weights for the respective components. A conversion factor of 23.6, 24.8, 26.6, 22.2, and 24.4 was found for phosphatidylethanolamine, phosphatidylcholine, phosphatidylinositol, phosphatidic acid, and total phospholipid, respectively. These factors also were used to convert µg of phosphorus into µg of phospholipid.     

Confectionery fat analysis with high performance liquid chromatography

Natural triglyceride mixtures have been analyzed by a variety of techniques. Earlier methods, such as crystallization, were unsuitable because they were very time-consuming, required large amounts of material and were not reproducible. Later advances in chromatography gave successful separations of triglycerides but required a combination of techniques to give suitable separations. High performance liquid chromatography (HPLC) with very efficient columns has been successfully applied to triglyceride separations on a routine basis. In the present work, triglycerides of cocoa butter as well as several replacement fats were separated by HPLC. The system consisted of a highly efficient column packed with octadecylbonded spherical silica (5 μ) and a mobile phase composed of acetone and acetonitrile. Peaks corresponding to eluted components were collected and their composition determined by gas chromatography (GC) of the corresponding methyl esters to allow unambiguous assignment of triglyceride structure to the components. A profile of the triglycerides present in such fats can be obtained within 30 min.     

Gamma-irradiation of 7-ketocholesterol in aqueous dispersions and liposomes

7-Ketocholesterol in aqueous sodium stearate dispersions or incorporated into liposomes was exposed to gamma radiation at 0–4°C to a dose of 50 kGy. Substrate degradation was noted in all systems, but occurred to a much greater extent in dispersions (≈90%) than in liposomes (0–25%). The difference in degree of degradation is attributed to dissimilarities in the micro-environment of 7-ketocholesterol in the two systems. The principal decomposition product was identified as 7-ketocholestanol, a compound that can arise from hydrogenation of the double bond of the starting material. Also detected, but in lower concentration, were the epimeric 7-hydroxycholesterols resulting from the reduction of the original carbonyl function. Both 7-ketocholestanol and the epimeric 7-hydroxycholesterols are believed to be secondary species generated by the interaction of the starting material with the primary radiolysis products of water. In addition, many minor degradation products were noted but not identified.     

Mutagenic potential of ammonia-related aflatoxin reaction products in cottonseed meal

In a joint research effort, the Food and Drug Administration, the National Toxicology Program and the US Department of Agriculture studied the mutagenic potential of aflatoxin reaction products following ammoniation of contaminated cottonseed meal under conditions approximating those approved for commercial ammoniation of nonaflatoxin-contaminated meal. Uniformly ring-labeled¹⁴C-aflatoxin B₁ was added to cottonseed meal that contained ca. 4000 μg naturally incurred aflatoxin B₁/kg. Distribution of the radiolabeled compound was used to trace the modification of aflatoxin B₁ after treatment with ammonia. The radioactivity-to-weight ratio of the fractions isolated by solvent extractions and chemical and enzymic treatments was used to measure the relative concentration of an aflatoxin decontamination product. All extract fractions having a radioactivity-to-weight ratio ≥1 were tested for mutagenic activity using theSalmonella/microsome mutagenicity test (Ames test). Purified aflatoxin B₁ was mutagenic at a concentration of ca. 0.005 μg/plate. The methylene chloride extract of the ammoniated meal after Pronase digestion exhibited a similar response when 180 μg of this fraction was applied to each plate. This fraction represented 0.16% of the original added radioactivity. The other fractions produced no detectable mutagenic response at the concentrations tested (10–1000 μg/plate) onSalmonella tester strain TA100. Ammonia treatment of aflatoxin-contaminated cottonseed meal significantly decreased aflatoxin levels, and the aflatoxin decontamination products formed by the treatment had little or no mutagenic potential.     

Antineoplastic activity of the thioether lysophospholipid derivative BM 41.440 in vitro

Thioether lysophospholipid derivatives (TLP) inhibited the in vitro uptake of [³H]thymidine into blasts of eight leukemias and cells of 12 different solid tumors of human origin. This effect correlated with trypan blue dye exclusion, which was used to assess cell damage. Cytostatic and cytotoxic effects of TLP were dependent on dosage and incubation time. Destruction of leukemic blasts was completed with >5 μg/ml after an incubation of >48 hr, but 10 to 20 μg/ml were necessary in solid tumors. Ester-linked 2-lysophosphatidylcholine was ineffective in the same dose range, which points to the requirement of the alkyl moiety insn-1 and a stablesn-2 substitution of the molecule for the antineoplastic effect. To assess putative antileukemic selectivity, the cytotoxicity (trypan blue dye exclusion) of TLP was compared in human cell samples of 19 non-neoplastic bone marrows and 9 leukemias. Results revealed a significantly higher activity of the TLP BM 41.440 in leukemic blasts.     

Enzymatic acylation of ether and ester lysophospholipids in rat liver microsomes

The acylation of lysophospholipids by rat liver acyltransferases was studied. A comparison between ester and ether lysophospholipids as substrates revealed large differences in substrate properties. For instance, oleic acid from oleoyl-CoA and arachidonic acid from arachidonoyl-CoA were not incorporated into 1-O-octadecyl-sn-glycero-3-phosphocholine under experimental conditions that allowed an optimal transfer of oleic acid and arachidonic acid to 1-O-palmitoyl-sn-glycero-3-phosphocholine. However, we observed an acyl-CoA-independent transfer of arachidonic acid from 1-O-stearoyl-2-O-arachidonoyl-sn-glycero-3-phosphoinositol to 1-O-octadecyl-sn-glycero-3-phosphocholine.     

Method of analysis for deoxynivalenol and zearalenone from cereal grains

A method was developed to determine deoxynivalenol and zearalenone in corn, wheat, oats, rice and barley. The toxins are extracted with methanol/water (50:50, v/v) (2×) and partially purified by partitioning into ethyl acetate and then defatting with acetonitrile-petroleum ether. Toxins are isolated by silica gel column chromatography. Interfering materials are removed from the column with benzene; zearalenone is eluted with benzene/acetone (95:5, v/v), and after a column wash of chloroform/methanol (95:2, v/v), deoxynivalenol is eluted with chloroform/methanol (95:5, v/v). Zearalenone is quantitated by thin-layer chromatography and deoxynivalenol by gas-liquid chromatography of the trimethylsilyl derivative. The detection limit is about 0.02 μg/g for each toxin. Recoveries of added toxins varied with substrate and level of toxins. Recovery of deoxynivalenol ranged from 58% for 1 ppm in rice to 108% for 1 ppm in corn. Average recoveries for all levels (1, 2 and 5 ppm) ranged from 69% for barley to 89% for oats. Recovered zearalenone ranged from 40% for 5 ppm in wheat to 100% for 1 ppm in barley. Average recoveries for zearalenone at 1, 2 and 5 ppm varied from 53% for wheat to 87% for rice.