Determination of plasma tocopherols by gas liquid chromatography

A method is described for the separation and determination of plasma tocopherols by gas liquid chromatography (GLC). Proteins in 0.1 g samples of plasma were precipitated with ethanol containing a known amount of 5,7-dimethyltocol which served as an internal standard. Tocopherols were extracted into petroleum ether, purified by thin layer chromatography and analyzed as trimethylsilyl ethers by gas liquid chromatography (GLC) on 0.5% Apiezon L. Recoveries of α- and γ-tocopherols averaged 100% and 93%, respectively. The mean total tocopherol content of eight human plasma samples was 8.5 μg/g by GLC and 9.9 μg/g by a ferric chloride-α,α′-dipyridyl method. The α- and γ-tocopherol contents of 16 human plasma samples ranged from 4.0 to 12.3 μg/g and 0.6 to 2.1 μg/g, respectively.     

A method for the quantitative estimation of cholesterol α-oxide in eggs

A method for the quantitation of cholesterol α-oxide in egg and egg products is described. Total lipids extracted from dry egg samples were fractionated on a silicic acid column to concentrate cholesterol oxides which were then quantitatively determined by gas liquid chromatography (GLC). Those samples which showed cholesterol oxides by GLC were further analyzed by high pressure liquid chromatography (HPLC) for the ratio of cholesterol α-oxide and cholesterol β-oxide. Cholesterol α-oxide content was calculated from the combined results of GLC and HPLC.     

Arachidonic acid autoxidation in an aqueous media effect of α-tocopherol,cysteine and nucleic acids

The autoxidation of arachidonic acid dispersed in aqueous media was evaluated simultaneously with and without different agents, e.g., α-tocopherol at different concentrations, cysteine, DNA and RNA. The autoxidation rate of arachidonic acid was evaluated by quantitative gas liquid chromatography (GLC) determination of the unoxidized acid and by spectrophotometric measurement of conjugated dienes. α-Tocopherol exhibited a prooxidant activity at concentrations of 1.25 × 10⁻⁴ M and 1.25 × 10⁻⁵ M and a weak antioxidant activity at a concentration of 1.25 × 10⁻⁶ M. Cysteine showed antioxidant activity and greatly reduced the prooxidant activity of α-tocopherol. DNA and RNA had no effect in either case. α-Tocopherol oxidation was followed by high pressure liquid chromatography (HPLC). The prooxidant effect was accompanied by a rapid oxidation of α-tocopherol, except in the presence of cysteine, which prevented the oxidation of α-tocopherol.     

Extraction and quantitation of total cholesterol,dolichol and dolichyl phosphate from mammalian liver

A procedure is described for the determination of total cholesterol, dolichol and dolichyl phosphate (Dol-P) in mammalian liver. It is based on extraction of these compounds into diethyl ether after alkaline saponification of the tissue. Extractability is affected by the length of saponification and concentration of potassium hydroxide (KOH) in the saponification mixture. After extraction, total cholesterol and dolichol are quantitated directly by reverse-phase high pressure liquid chromatography (HPLC) on C₁₈. Dol-P requires further purification before quantitation by HPLC, this is accomplished by chromatography on silicic acid. These methods gave recoveries of over 90% for cholesterol and dolichol and about 60% for Dol-P, using [4-¹⁴C]cholesterol, a polyprenol containing 15 isoprene units, and [1-¹⁴C]Dol-P as recovery standards. Concentrations of total cholesterol, dolichol and Dol-P in livers from one month-old-CBA mice were found to be 5.7±0.7 mg/g, 66.3±1.2 μg/g and 3.7±0.3 μg/g, respectively.     

Distribution of antithrombin III and glucosylceramide in human plasma lipoproteins and lipoprotein deficient plasma

We have investigated the distribution of antithrombin-III and glucosylceramide (Glc-Cer) in human plasma, plasma lipoproteins and lipoprotein-deficient plasma. Antithrom bin III activity was measured employing immunochemical and biological assays. Glc-Cer was quantified by gas liquid chromatography (GLC). Whole plasma contained 145 μg antithrombin III/ml plasma, all of which was associated with the lipoprotein-deficient plasma (d>1.25 g/ml). Whereas, most if not all the plasma GlcCer was associated with plasma low density lipoproteins (LDL) (d-1.022–1.055 g/ml) and high density lipoproteins (HDL) (d-1.063–1.25). GlcCer was not found in the lipoprotein-deficient plasma. We conclude that GlcCer on lipoproteins does not contribute to antithrombin III activity. Moreover, the absence of GlcCer in lipoprotein-deficient plasma does not impair antithrombin-III activity.     

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.     

Determination of the total glucosinolate content in canola by reaction with thymol and sulfuric acid

Intact glucosinolates in seeds and meals of rapeseed and canola were isolated and purified on small DEA ion-exchange columns. After being eluted with potassium sulfate the glucosinolates were hydrolyzed with sulfuric acid to produce thioglucose which was determined as a complex with thymol. The method was compared to a gas liquid chromatography (GLC) procedure which determines aliphatic glucosinolates. The extra amount of glucosnilates found (ca. 14 ]gmmol/g oil-free) was equal to the sum of those not determined by GLC. The thymol method had a standard error of ±3 μmol/g compared with a standard error of ±1 μmol/g for the GLC procedure for the same set of 18 samples ranging from 10 μmol/g to 100 μmol/g (oil-free, 8.5% moisture basis) of aliphatic glucosinolates. This is paper No. 626 of the Canadian Grain Commission, Grain Research Laboratory, 1404-303 Main St., Winnipeg, Manitoba, Canada R3C 3G8     

Specific susceptibility of docosahexaenoic acid and eicosapentaenoic acid to peroxidation in aqueous solution

The peroxidation of different polyunsaturated fatty acids (PUFA) after photoirradiation in aqueous solution was evaluated by measuring fatty acid loss and malonaldehyde production in medium. The oxidation rates of eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA), two highly unsaturated fatty acids of the n−3 series, were surprisingly lower (14 and 22%, respectively) than the oxidation rates of linoleic, α-linolenic, γ-linolenic, dihomo γ-linolenic, and arachidonic acids (62–90%). The quantities of malonaldehyde (MA) produced were assayed simultaneously by gas chromatography (GC) and high performance liquid chromatography (HPLC). MA production was found to be related to both the degree of unsaturation and the metabolic series of the fatty acid. The maximum value was observed with arachidonic acid (MA production from 2 mM arachidonic acid in aqueous solution was estimated at 44.9±6.0 μM by GC and 46.8 ±4.0 μM by HPLC). Eicosapentaenoic acid and docosahexaenoic acid produced lower MA quantities compared to arachidonic acid (MA production from 2 mM EPA and 2 mM DHA was estimated at 17.9±1.5 μM and 37.9±0.7 μM, respectively, by GC, and 26.3±4.9 μM and 37.3±4.2 μM, respectively, by HPLC). The MA yield, defined as the amount of MA (nmols) produced per 100 nanomoles of oxidized fatty acid, was used to express the susceptibility of individual PUFA to peroxidation. The MA yield correlated well with the degree of unsaturation, but was independent of carbon chain length and metabolic series. The study suggests that adequate assessment of lipid peroxidation cannot be achieved by measuring MA formation alone, but it also requires knowledge of the fatty acid composition of the system studied.     

Fatty acid desaturase specificity in tetrahymena

The ciliate,Tetrahymena, was provided a supplement of the fatty acid [1-¹⁴C] 18∶2Δ6,9. After a period of growth the cells were claimed, the lipids extracted, the polar lipids recovered and the mild alkali-labile fatty acid methyl esters generated. The fatty acids were resolved by high pressure liquid chromatography (HPLC), the 18∶3Δ6,9,12 (γ-linolenic acid) was recovered and its identity verified by high pressure liquid chromatography (HPLC), gas liquid chromatography (GLC), hydrogenation and oxidation. Fifty-three percent of the cell-associated label was found in γ-linolenic acid; thus, a Δ12 fatty acid desaturase converts the 6,9 octadecadienoic acid to the 6,9,12 derivative. No carboxyl or methyl terminus restriction appears on Δ9 monoenoic or dienoic fatty acid desaturation in this cell as is found in higher plants and animals.     

Quantitative high-performance liquid chromatography analysis of plant phospholipids and glycolipids using light-scattering detection

Application of the evaporative light-scattering principle to quantitative high-performance liquid chromatography (HPLC) analyses of plant membrane lipids has received little study. Light-scattering detection response curves were generated for nine classes of plant membrane phospholipid and glycolipids. Quantitative results obtained by HPLC/light-scattering detection and conventional lipid analytical methods (thin-layer chromatography and lipid-P assay) were in close agreement, confirming the reliability of HPLC/evaporative light-scattering detection (ELSD) analyses. Only three of the nine plant lipid classes gave linear detector response functions above 10 μg injected lipid mass. This finding contradicts earlier precepts involving light-scattering detection of lipids. At a given mass, appreciable variation in ELSD signal intensity and detection limit was found to exist among the various plant membrane lipid classes. The variation in detector response among plant lipid classes is an important consideration in achieving accurate quantitative results in plant lipid analyses.     

Differences in polyisoprenoid alcohols of mono-and dicotyledonous seeds

A lipid fraction enriched in polyisoprenoid alcohols was prepared from seeds of a number of crop plants, using Florisil chromatography. Analysis by HPLC of the fraction from soybeans showed a series of peaks corresponding to α-saturated homologues (dolichols) from 15 to 22 isoprene units in length. Similar results were obtained with seeds of other dicotyledonous species (rapeseed, peanuts, mung beans, navy beans and peas). In contrast, analysis of the seeds of monocotyledonous plants (wheat, rye, barley, rice and corn) by HPLC gave split peaks, indicating the presence of nearly equal amounts of 2 different homologous series of compounds. The polyisoprenoid material isolated from wheat germ was subsequently shown to consist of a mixture of dolichols and α-unsaturated homologues (polyprenols). Treatment with managanese dioxide selectively oxidized the polyprenols to the corresponding aldehydes, which were separated from the dolichols by TLC. The identity of the components was established by infrared-nuclear magnetic resonance (IR-NMR) spectroscopy and by comparison with authentic standards on high performance liquid chromatography (HPLC). The concentration of polyisoprenoid alcohols in seeds varied from 1–16 mg/100 g. Seeds of different species showed some differences in the pattern of homologues present.     

Rapid Preparation of Fluorescent 9-Anthrylmethyl Esters for Fatty Acid Analysis of Small Amount of Triacylglycerols

This paper proposes a one-step method for preparation of fluorescent 9-anthrylmethyl esters from triacylglycerols (TAG) ranging in amount from 0.1 to 5 μg. It involves base-catalyzed transesterification using potassium 9-anthracenemethoxide, prepared by proton exchange between 9-anthracenemethanol and potassium tert-butoxide. Transesterification for 10 min at room temperature gave the fatty acid 9-anthrylmethyl esters in nearly maximal yields (82–85%). The products could be analyzed by reversed-phase HPLC without purification. Excellent linear relationships were observed for standard curves of 10–250 pmol of TAG standards (16:0, 19:0, 18:2 and 22:6), and differences in the slopes were less than 5% among the standards. Almost consistent compositions of the esters were observed for the products formed from 0.5 to 5 μg or less of fish oils TAG, and they were similar to those obtained by HPLC of ordinary multi-step synthesis products and by GLC of methyl esters. The present method is a great improvement of derivatization time, and is powerful for fatty acid analysis of small amounts of natural TAG.     

Triacylglycerols of evening primrose oenothera biennis seed oil

The triacylglycerol (TG) composition of evening primrose(Oenothera biennis) seed oil (EPO) was studied using a combination of silver nitrate thin layer chromatography (AgNO```3``-TLC), reverse phase high performance liquid chromatography (HPLC) and capillary gas liquid chromatography (GLC). The important TGs in EPO are LLL (24.4%), LLO (23.9%), LLP (11.5%), LOO (7.2%), LOP (6.8%), LLS (4.8%), γLnLp (3.7%), LOS (3.3%), γLnLS (2.0%), γLnLL (2.0%), LPP (1.9%), OOO (1.7%), LSP (1.3%) and γLnLO (1.0%).     

Zur Analytik von Tocopherolen – Ein Methodenvergleich zwischen HPLC und GC

Analysis of Tocopherols by HPLC and GLC – A Method Comparison Study HPLC with straight phase columns as well as capillary GLC are suitable methods for the quantitative analysis of vitamin E isomers. In contrast to HPLC, it is necessary to saponify the oil for subsequent determination of tocopherols by means of GLC. Tocopherols are extracted quantitatively from the alkaline soap solution using cyclohexane. In order to test the accuracy of the GLC method, a BCR reference material was also included in the study and satisfying results were obtained. The random error, expressed as the standard error of the Y-estimate calculated by the method of least squares. indicated that 95% of the samples assayed by the two methods will agree within ± 16.7 μg/g.     

Identification of core aldehydes amongin vitro peroxidation products of cholesteryl esters

Synthetic cholesteryl 5-oxovalerate and 9-oxononanoate were used as reference standards for the isolation and identification of cholesteryl ester core aldehydes fromtert-butyl hydroperoxide/Fe⁺⁺ oxidation of synthetic and natural cholesteryl esters. The core aldehydes were recovered from the peroxidation products by thin-layer chromatography as the free aldehydes or the 2,4-dinitrophenylhydrazones and were identified, respectively, by gas-liquid chromatography (GLC) and by GLC combined with mass spectrometry (GC/MS) or by reverse-phase high-performance liquid chromatography (HPLC) and by HPLC with MS (LC/MS). The core aldehydes produced by peroxidation of cholesteryl linoleate were identified as mainly 9-oxononanoates of cholesterol and oxycholesterols, with smaller amounts of the 8-oxooctenoates, 10-oxodecenoates, 11-oxoundecenoates and 12-oxododecenoates. Peroxidation of cholesteryl arachidonate yielded 5-oxovalerates of cholesterol and the oxycholesterols as the main products with smaller amounts of the 4-oxobutyrates, 6-oxohexenoates, 7-oxoheptenoates, 8-oxooctenoates, 9-oxononenoates, 9-oxononadienoates and 10-oxodecadienotes. The oxycholesterols resulting from the peroxidation of the steroid ring were identified as mainly 7-keto-, 7α-hydroxy- and 7β-hydroxy-cholesterols and 5α, 6α-and 5β,6β-epoxy-cholestanols. Cholesteryl palmitate and oleate did not yield core aldehydes in the present peroxidation system. In these esters, the sterol and linoleic acid moieties appeared to be oxygenated at about the same rate, while the arachidonic acid moiety reacted more rapidly than did the sterol moiety.     

Lipid methodology — Chromatography and beyond. Part I. GC/MS and LC/MS of glycerolipids

This paper reviews recent examples of the application of combined high temperature gas-liquid chromatography (GLC) and reversed phase high pressure liquid chromatography (HPLC) with electron impact and chemical ionization mass spectrometry for structural studies of natural diacyl and triacylglycerols. It was concluded that the combination of reversed phase HPLC with direct liquid inlet chemical ionization mass spectrometry provides the most complete resolution and most reliable identification of natural acylglycerols, far exceeding the capabilities of either technique alone. The LC/MS method is suitable for quantitative analysis following appropriate calibration of the total or fragment ion response.     

Analysis of alpha tocopherol in blood plasma and platelets by gas liquid chromatography

A method is described for the analysis of α-tocopherol by gas liquid chromatography (GLC) with 0.3% Apiezon L as liquid phase. Impurities that interfere with GLC are removed by saponification. Cholesterol and other nonsaponifiables are separated from α-tocopherol by GLC.     

On the occurrence of α-tocopherolquinone in rat tissue

The amount of α-tocopherolquinone in rat liver has been reinvestigated comparing (a) a conventional procedure including saponification and thin layer chromatography followed by high peformance liquid chromatography (HPLC), with (b) the direct HPLC analysis of a total lipid extract. Recovery of added α-tocopherolquinone was quantitative with both procedures. In contrast to a recent report of 124 nmol/g in rat liver, we found no more than 1–4 nmol/g by procedure a and less than 1 nmol/g by procedure b.     

Comparison of analyses of surfactants in cosmetics using high-performance liquid chromatography and high-performance capillary electrophoresis

Separation of anionic, cationic, and amphoteric surfactants containing n-dodecyl groups and hydrophilic moieties was done by high-performance liquid chromatography (HPLC) and high-performance capillary electrophoresis (HPCE), and an ultraviolet-visible detector. Quantitation of surfactants in commercial cosmetic and toiletry products was also done using similar methods. Conditions used to separate mixtures of surfactants by HPLC are as follows: stationary phase: ODS 2; mobile phase: MeOH/H₂O (80∶20, vol/vol) containing 1.0M NH₄Cl, 0.003 M tetrabutylammonium hydrogen sulfate, and 0.005 M diammonium phosphate buffer solution at pH=6.0. Conditions for HPCE are as follows: an uncoated capillary (100 μm i.d., 110 cm in length, effective length of 75 cm) with 25 kV of applied voltage, and an aqueous buffer containing 80 mM sodium borate/20 mM NaOH at pH=9.2. Surfactants were eluted within 15 min. The accuracy of both techniques was evaluated by analyzing the recovery ratio of surfactants in the mixture.     

Diplocyclos palmatus L.: A new seed source of punicic acid

The seeds ofDiplocyclos palmatus L. (Cucurbitaceae) contained 23% oil and 15% protein. The UV, IR,¹H-NMR and¹³C-NMR spectrometry of the oil, and oxidation, reduction and gas liquid chromatography (GLC) of the methyl ester of conjugated fatty acid isolated by preparative thin layer chromatography (TLC) showed the presence of punicic (octadeca-cis-9,trans-11,cis-13-trienoic) acid. The fatty acid composition (wt %), as determined by GLC, is: punicic, 38.2; 18∶2, 43.9; 16∶0, 8.1; 18∶0, 4.9 and 18∶1, 4.9.