Capillary column gas-liquid chromatographic resolution of oxidized cholesterol derivatives

Fused-silica capillary columns were evaluated for the resolution of oxidized cholesterol derivatives. Thermal instability of diol derivatives, epimeric 7 alpha- and 7 beta-hydroxy, 4 beta-hydroxy, and 25-hydroxycholesterol, was observed during gas chromatography. After derivatization as trimethylsilyl ethers the foregoing diols, alpha-epoxide, cholestane-triol, 7-ketocholesterol, and cholesta-3,5-dien-7-one were completely resolved on a DB-1 column. Each oxidized sterol revealed excellent response linearity as the trimethylsilylated sterol, enabling reliable quantification. The identity of each derivatized sterol was confirmed by mass spectrometry.     

Analysis of fatty acid composition of Candida species by gas-liquid chromatography using a polar column

The fatty acid composition of representative Candida species was examined by gas-liquid chromatography (GLC) using a polar column. The major fatty acids were C14:0, C16:0, C18:0 saturated, C16:1 and C18:1 monoenoic series, with or without C18 polyunsaturated acids (C18:2 and C18:3). In Torulopsis glabrata and Saccharomyces cerevisiae the C18:2 and C18:3 acids were not found, but the C10:0 and C12:0 acids were detected in S. cerevisiae. These results indicated that the Candida genus could be distinguished from Torulopsis and Saccharomyces genera by GLC analysis of fatty acids. Quantitative differences in the fatty acid composition between cells grown at high temperature (37 degrees C) and low temperature (25 degrees C) were found generally in Candida species, and the amounts of C18 polyunsaturated acids (C18:2 and C18:3) increased in the cells grown at 25 degrees C. Each Candida species showed a characteristic profile in fatty acid composition. Determination of the cellular fatty acid composition in Candida species is likely to be useful for the grouping or chemotaxonomy of newer isolates of Candida species.     

An improved gas-liquid chromatographic method for the determination of fecal neutral sterols

The analysis of fecal neutral sterols has been improved by use of a highly selective gas-liquid chromatography column packed with SP-2401. This chromatographic column allows separation of cholesterol and cholestanol and delta 5-5 alpha plant sterol homologs without employing silver nitrate thin-layer chromatography. Furthermore, there is no need to derivatize neutral sterols before injection. The main fecal neutral sterols are well resolved; retention times are reproducible; detector response is reproducible, linear, and sensitive to 0.2 micrograms. This method, successfully used for fecal samples, may be suggested as a routine method for the clinical study of cholesterol metabolism.     

Resolution of saturated and unsaturated 5 beta-cholanoic acids by gas-liquid and thin-layer chromatography.

The thin-layer and gas-liquid chromatographic properties of the methyl ester acetates were determined for a series of 20 monounsaturated 5 beta-cholanoic acids representing the simple chemical and enzymic dehydration products of the common bile acids. The unsaturated acids were generally indistinguishable from their saturated analogues by thin-layer chromatography on plain silica gel, but resolution was achieved on silica gel impregnated with silver nitrate for compounds having sterically exposed double bonds. The gas-liquid chromatographic behaviour of the unsaturated bile acids on the OV-225, SE-30, and Poly-S-179 liquid phases was closely similar to that observed for the saturated bile acids. The 5 beta-cholenoic acids obeyed the general rules of chromatographic mobility based on the overall shape of the molecule and the number and configuration of functional groups, with a constant retention factor attributable to the olefinic bond. The structural information provided by the chromatographic behaviour of the standard unsaturated bile acids allows a distinction to be made among most of the isomeric 5 beta-cholenoates. A complete identification of all isomeric olefins is possible when chromatographic and mass spectrometric data are combined.     

Highly simplified method for gas-liquid chromatographic quantitation of bile acids and sterols in human stool.

A simple method for the gas-liquid chromatographic quantitation of human fecal bile acids and sterols is described where bile acids are subjected to n-butyl ester derivatization, without prior isolation from the stool, followed by trimethylsilylation of the sterols and bile acids. Under these conditions, bile acid derivatives are well resolved from each other and from the trimethylsilyl ether derivatives of fecal sterols and no overlap occurs. The method was shown to be highly reproducible and recoveries were similar to those obtained with other methods used for fecal bile acid analysis. Application of the method for bile acid and sterol analysis in human stool is described.     

High resolution preparative column chromatographic system for gangliosides using DEAE-Sephadex and a new porus silica, Iatrobeads.

A new high-resolution preparative column chromatographic system was developed for efficient and rapid isolation of ganglioside molecular species. The system involved a combination of ion-exchange and adsorption chromatographies using DEAE-Sephadex A-25 and the newly developed, totally porous silica spheres, Iatrobeads. Using this system the brain gangliosides, GM1, GD1a, GD1b and GT1 were obtained in high purity and in milligram amounts, in a relatively short time, by simple procedures. The presence of a number of unidentified molecular species of gangliosides, which are present only in small amounts, was also demonstrated.     

Quantitative gas chromatographic analysis of amino acids on a short glass capillary column

A study of the quantitative gas chromatographic analysis of protein amino acids as their N-heptafluorobutyric amino acid n-propyl esters on a glass capillary column has been made. The analysis is completed within 35 min with good separation of the common protein amino acids in a single-column run.Hydrolyzed peptides have been analyzed. The analyses were performed with a precision varying between 1 and 6% (mean relative standard deviation) depending on the number of amino acid residues in the peptide. The amount taken for analysis was 20–300 μg. The results agree with the known sequences of the peptides and with the analyses by ion-exchange chromatography except for cysteine. This amino acid can be analyzed after modification as S-methylated cysteine.     

High-performance liquid chromatographic separation and on-line mass spectrometric detection of saturated and unsaturated oligogalacturonic acids

A method for the simultaneous determination of saturated and unsaturated oligogalacturonic acids up to degree of polymerization (dp) of 7 by high-performance liquid chromatography (HPLC) is presented. For this purpose, a Cyclobond I 2000 column and a volatile mobile phase consisting of ammonium formate and methanol were used, allowing direct coupling of HPLC to a mass spectrometer via an electrospray interface (ESI-MS) without additional desalting. The analytical system was used for the characterization of digests obtained by incubation of polygalacturonic acid with commercial enzyme preparations.     

Simultaneous quantitation of fatty acids,sterols and bile acids in human stool by capillary gas-liquid chromatography

A simple method for the simultaneous gas-liquid chromatographic quantitation of fatty acids, sterols and bile acids from human fecal samples is described. The various compounds are directly converted into the n-butyl ester-trimethylsilyl ether derivatives, without prior isolation from the stool. Under these conditions, fecal bile acid derivatives are well resolved from each other and from those of fecal fatty acids and sterols without overlaps. The method was found to be reproducible and recoveries were similar to those obtained after exhaustive solvent extraction of fecal sterols, fatty acids and bile acids. Optimum derivatization conditions that allowed maximum recovery of fecal components with minimal destruction and application of the method for simultaneous bile acid, fatty acid and sterol analysis in human stool are described.     

Determination of fatty acid compositions of Bacillus cereus and related bacteria: a rapid gas chromatographic method using a glass capillary column.

A rapid gas chromatographic method for the determination of fatty acid compositions of Bacillus cereus and related bacteria is presented. By the use of a free fatty acid phase-coated glass capillary column, the complete separation of fatty acids, including the branched ones, was achieved. The method enables a more distinct differentiation of Bacillus species than can be obtained with packed columns.     

Determination of fatty acid compositions of Bacillus cereus and related bacteria: a rapid gas chromatographic method using a glass capillary column.

A rapid gas chromatographic method for the determination of fatty acid compositions of Bacillus cereus and related bacteria is presented. By the use of a free fatty acid phase-coated glass capillary column, the complete separation of fatty acids, including the branched ones, was achieved. The method enables a more distinct differentiation of Bacillus species than can be obtained with packed columns.     

Simultaneous determination of cyclandelate and its metabolite in human plasma by capillary column gas-liquid chromatography

A method was developed for the simultaneous determination of cyclandelate and mandelic acid concentrations in plasma, involving extraction from plasma followed by trimethylsilylation and chromatography of the derivatives on a glass capillary column with hydrogen flame-ionization detection. Calibration graphs were linear down to at least 20 μg/ml for each substance. The precision was excellent with a pooled relative standard deviation of 6.3% and 6.4% for cyclandelate and mandelic acid serum samples, respectively. Concentrations below 500 ng/ml of each substance could be detected in human plasma. The method was developed for use in bioavailability and metabolism studies.     

Electron-capture, capillary column gas chromatographic determination of low-molecular-weight diols in serum

Research on alcoholism has revealed that concentrations of 1, 2-propanediol, d, l-2, 3-butanediol andmeso-2, 3-butanediol may be greater in the serum of chronic alcoholics than in the serum of social drinkers and nondrinkers. In connection with one of these studies, we developed methodology to determine these diols at the micromolar levels in 500 serum samples. The procedure consisted primarily of extraction of the serum with acetonitrile containing internal standard. The extract was then concentrated to dryness and reacted withp-bromophenylboric acid. The reaction mixture was injected into a gas chromatograph fitted with a capillary column and an electron-capture detector. The total coefficients of variation were best for 1, 2-propanediol, 6.82 and 10.00%, and worst ford, l-2, 3-butanediol, 13.64 and 19.22%. The observed means for the analytes were all within 10% of the spiked level.     

A single column packing for the gas-liquid chromatographic separation of various biologically important compounds.

The use of a single, commercially available column packing, TabsorbR, is described for the g.l.c. separation of a large number of different compounds. The resolution of the homologous members of the following series of compounds was achieved: (1) saturated fatty acids (C1-C18), (2) normal aliphatic saturated dicarboxylic acids (C2-C14), (3) normal aliphatic saturated alcohols (C1-C24), (4) normal aliphatic saturated amines (C1-C12), (5) the common amino acids except arginine, histidine and cysteine, (6) aliphatic hydrocarbons (C10-C20) and (7) monosaccharides. It should be noted that twenty-two monosaccharides including three hexosamines and two anhydrohexoses, could be resolved as alditol acetates in a single run. In addition, galacturonic, glucuronic and iduronic acids could be separated from one another as their 1,4-lactones. The resolution achieved in these series of compounds was found to be consistent and highly reproducible. It is of further interest that certain isomers of the higher fatty acids and hydrocarbons with one double bond could also be separated from the normal and saturated compounds, respectively. The applicability of "Tabsorb" for the g.l.c. separation, although noted above to be considerably broad, is by far not yet exhausted. These procedures which form the basis for the quantitative determinations of the various compounds studied as demonstrated by analysis of glycopeptides for neutral hexoses and proteins for the amino acids, can readily be adapted to preparative methods. From the biochemical point of view "Tabsorb" is an extremely versatile column packing in that it can be used for the identification of many of the common building blocks of natural products.     

Determination by capillary gas-liquid chromatography of the absolute configurations of unsaturated fatty acid hydroperoxides formed by lipoxygenases

The absolute configurations of a number of unsaturated hydroperoxy fatty acids obtained by lipoxygenase catalysis were investigated by capillary gas-liquid chromatography after proper derivatization. To this end the hydroperoxy groups were reduced and the resulting hydroxyl groups acetylated. Oxidative ozonolysis of the acetylated methyl esters yielded acetylated 2-hydroxycarboxylic acids, which were converted into R-(--)-2-butyl esters and then reacetylated. The ratio of the resulting diastereomers, which reflects the optical purity of the chiral centers in the parent hydroperoxy fatty acids, was determined by capillary gas-liquid chromatography. Application of this simple method to a number of mono- and dihydroperoxy fatty acids obtained by incubation with soybean lipoxygenase-1 or -2, or by corn-germ lipoxygenase yields enantiometric compositions which are in good agreement with results obtained by other methods.     

Determination of molecular species of enantiomeric diacylglycerols by chiral phase high performance liquid chromatography and polar capillary gas-liquid chromatography

A simple method is described for the determination of molecular species of enantiomeric sn-1,2- and sn-2,3-diacylglycerols derived from natural triacylglycerols by Grignard degradation. The method is based on a preparative separation of the enantiomeric diacylglycerols as 3,5-dinitrophenylurethane (DNPU) derivatives by high performance liquid chromatography (HPLC) on a chiral column (25 cm x 4.6 mm ID) containing R-(+)-1-(1-naphthyl)ethylamine as a stationary phase. This is followed by polar capillary gas-liquid chromatography (GLC) of the trimethylsilyl (TMS) ether derivatives of the enantiomeric diacylglycerols derived from the DNPU derivatives using trichlorosilane, which does not cause acyl migration and racemization during the reaction. The cleavage is better than 94% complete. The method was standardized with synthetic sn-1,2- and sn-2,3-dipalmitoyl- and rac-1,2-dioleoylglycerols and was applied to the identification and quantitation of individual molecular species of enantiomeric diacylglycerols generated by Grignard degradation of the triacylglycerols from corn oil, cocoa butter, and lard.     

Preparative chromatographic resolution of racemates using HPLC and SFC in a pharmaceutical discovery environment

The preparative chromatographic resolution of racemates has become a standard approach for the generation of enantiomers in pharmaceutical discovery laboratories. This paper will discuss the use of preparative HPLC and SFC to generate individual enantiomers for discovery activities. Analytical HPLC and SFC method development to rapidly screen chiral stationary phases and solvent combinations will be presented. The usefulness of preparative chromatographic resolution of racemates will be demonstrated through the presentation of numerous non-routine case studies from the laboratories at Amgen.