High performance liquid chromatography of the hydroperoxides produced by lipoxygenases.

Separation of 13-hydroperoxylinoleic acid or 13-hydroperoxylinolenic acid from linoleic acid or linolenic acid, respectively, was carried out easily and quickly by high performance liquid chromatography on porous polymer gel (TSK-Gel LS-140) using n-hexane/ethanol as an eluent. An eluent containing a large amount of n-hexane (96%) made possible the separation of 9- and 13-hydroperoxylinoleic acids. These methods were applicable for analyses of the products obtained by the incubation of soybean lipoxygenase-1 [linoleate: oxygen oxidoreductase, EC 1.13.11.12] with linoleic acid or 13-hydroperoxylinoleic acid.     

Reversed-phase high-pressure liquid chromatography of arachidonic acid metabolites formed by cyclooxygenase and lipoxygenases

High-pressure liquid chromatography is required to resolve the complex mixtures of arachidonic acid metabolites synthesized by many tissues. We have investigated some of the factors which affect the retention times of these substances in reversed-phase HPLC on columns of 5-micron octadecylsilyl silica. There are considerable differences in selectivity between mobile phases based on methanol and those based on acetonitrile, the latter being much better for cyclooxygenase products. The chromatographic behavior of peptidoleukotrienes (LTC4, LTD4, and LTE4) is quite different from that of other arachidonic acid metabolites which do not contain amino acids. Addition of phosphoric acid to the mobile phase results in very long retention times for peptidoleukotrienes. Very low concentrations of trifluoroacetic acid have effects similar to that of phosphoric acid, but as its concentration is raised, the retention times of peptidoleukotrienes decrease, whereas those of other arachidonic acid metabolites are unaffected. Changing the concentration of acetonitrile in the mobile phase also affects the retention times of peptidoleukotrienes differently from those of other metabolites. This information has been used to devise simple linear gradients which separate most of the major cyclooxygenase and lipoxygenase products of arachidonic acid metabolism.     

Determination of fatty acid content and composition in ultramicro lipid samples by gas-liquid chromatography

Simplified quantitative manipulations of very small amounts (30 micrograms) of lipids for determination of fatty acid content and composition by gas-liquid chromatography after (a) methanolysis (b) reduction and acetylation are described.     

Two-column gas-liquid chromatography of fatty acid methyl esters

Fatty acid methyl esters were separated into fractions according to chain length on a nonpolar gas-liquid chromatographic column. These fractions were collected and rechromatographed on a polar column. Temperature programming was used in both cases. Data are given for the accuracy of the double procedure applied to a synthetic mixture.     

Preparation of fatty acid methyl esters for gas-liquid chromatography

A convenient method using commercial aqueous concentrated HCl (conc. HCl; 35%, w/w) as an acid catalyst was developed for preparation of fatty acid methyl esters (FAMEs) from sterol esters, triacylglycerols, phospholipids, and FFAs for gas-liquid chromatography (GC). An 8% (w/v) solution of HCl in methanol/water (85:15, v/v) was prepared by diluting 9.7 ml of conc. HCl with 41.5 ml of methanol. Toluene (0.2 ml), methanol (1.5 ml), and the 8% HCl solution (0.3 ml) were added sequentially to the lipid sample. The final HCl concentration was 1.2% (w/v). This solution (2 ml) was incubated at 45°C overnight or heated at 100°C for 1–1.5 h. The amount of FFA formed in the presence of water derived from conc. HCl was estimated to be <1.4%. The yields of FAMEs were >96% for the above lipid classes and were the same as or better than those obtained by saponification/methylation or by acid-catalyzed methanolysis/methylation using commercial anhydrous HCl/methanol. The method developed here could be successfully applied to fatty acid analysis of various lipid samples, including fish oils, vegetable oils, and blood lipids by GC.     

Stable gas-liquid chromatography column for fatty acid analysis.

A mixed gas-liquid chromatography column of FFAP and Apolar-7CP has excelelnt thermal stability and shows excellent characteristics for the separation of the methylesters of fatty acids from Salmonella enteritidis call wall lipids.     

Stable gas-liquid chromatography column for fatty acid analysis.

A mixed gas-liquid chromatography column of FFAP and Apolar-7CP has excelelnt thermal stability and shows excellent characteristics for the separation of the methylesters of fatty acids from Salmonella enteritidis call wall lipids.     

Free fatty acid microdetermination by gas-liquid chromatography without transmethylating effects of methylation procedure

A fast and practicable gas-liquid chromatographic method for the quantitation of non-esterified fatty acids (C14:0-C18:2) from 100 microliter plasma is described. This technique includes extraction, purification using the solvents n-heptane and 0.5 M Na2CO3, and methylation by 0.1 M HCl-methanol. Extraction, quantification and optimal methylation conditions without transmethylation have been investigated. Reproducibility is good and results agree with values found in the literature.     

Determination of serum creatinine by gas-liquid chromatography

The gas-liquid chromatographic measurement of serum creatinine is described in the present study. A preliminary concentration on cation exchange resin is used. The different steps of the operating procedure are analysed. A statistical comparison is undertaken between the results obtained with gas-liquid chromatography and kinetic colorimetry method using Jaffé assay.     

Automated glass capillary gas-liquid chromatography of fatty acid methyl esters with reference to cis and trans isomers.

The availability of an excellent separation method for fatty acid methyl esters, including separation of cis and trans isomers and of isomers that differ only in the position of double bonds, has become more and more important. The present glass capillary chromatography system combines high separation power with high precision and easy handling. Moreover, the system is completely automated and therefore provides a time saving method. As compared to a conventional packed column, the glass capillary column provides about one hundred fold more theoretical plates (227,000), as well as narrower peaks, thus giving rise to less error when integrating with electronic integrators. The reproducibility for relative retention time is better with the capillary column (0.26%) and reproducibility of the weight percent values is at least similar to that of the packed column (1.53%). When handling only small sample amounts the capillary provides better values because of its low capacity. This powerful system should open up new possibilities in the field of fatty acid investigation.     

Determination of urinary hippuric acid by micellar electrokinetic capillary chromatography

We propose a method for the simultaneous determination of hippuric acid (HA) and creatinine based on capillary micellar electrokinetic chromatography. Experimental conditions were 20 mM sodium phosphate, pH 7.20, 25 mM sodium dodecyl sulfate, 5% (v/v) acetonitrile. Electropherograms evidenced HA and creatinine peaks in less than 12 min. The method showed good linearity for both analytes and satisfactory within-day precision. The present method, which is accurate, sensitive, rapid and simple, may be applied to single-spot urine samples.     

Determination of the specific radioactivity of fatty acids separated as their methyl esters by gas-liquid chromatography

Free or combined (3)H-labeled fatty acids are converted to their methyl-(14)C esters or, if labeled with (14)C, to their methyl-(3)H esters. For a given specific radioactivity of the methyl group, the nuclide ratio in the esters separated by GLC is a direct measure of the specific radioactivity of the fatty acids, and quantitative collection is unnecessary. Methods of methylation with minimum quantities of labeled methanol, and of deriving nuclide ratios from channel ratios in a scintillation spectrometer, are given.     

Determination of delta-aminolaevulinic acid in biological fluids by gas-liquid chromatography with electron-capture detection

A derivative of delta-aminolaevulinic acid (AmLev), 2-methyl-3-acetyl-4-(3-propionic acid pentafluorobenzyl ester)pyrrole, with favourable properties for g.l.c. with electron-capture detection, was synthesized. Less than 1 pg could be detected on the column. 6-Amino-5-oxohexanoic acid formed the analogous derivative under similar conditions and was used as the internal standard in the development of a highly sensitive and specific assay for AmLev. The method has been applied to peripheral-venous and umbilical-cord plasma and to cerebrospinal fluid of normal and porphyric subjects.