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.     

High sensitivity quantitative lipidomics analysis of fatty acids in biological samples by gas chromatography-mass spectrometry

Historically considered to be simple membrane components serving as structural elements and energy storing entities, fatty acids are now increasingly recognized as potent signaling molecules involved in many metabolic processes. Quantitative determination of fatty acids and exploration of fatty acid profiles have become common place in lipid analysis. We present here a reliable and sensitive method for comprehensive analysis of free fatty acids and fatty acid composition of complex lipids in biological material. The separation and quantitation of fatty acids are achieved by capillary gas chromatography. The analytical method uses pentafluorobenzyl bromide derivatization and negative chemical ionization gas chromatography-mass spectrometry. The chromatographic procedure provides base line separation between saturated and unsaturated fatty acids of different chain lengths as well as between most positional isomers. Fatty acids are extracted in the presence of isotope-labeled internal standards for high quantitation accuracy. Mass spectrometer conditions are optimized for broad detection capacity and sensitivity capable of measuring trace amounts of fatty acids in complex biological samples. .     

Rapid separation of serum lipids for fatty acid analysis by a single aminopropyl column.

A rapid and simple method for separation of serum lipid classes for fatty acid analysis with a single aminopropyl solid phase glass column is described. The recoveries of cholesteryl esters, triglycerides, free fatty acids, and phospholipids were all at least 98%. Coefficients of variation less than 10% were obtained for absolute and relative amounts of most individual fatty acids analyzed after separation of serum lipid classes. This method provides an efficient and convenient tool to follow fatty acid patterns in serum lipid fractions.     

A simple and efficient frit preparation method for one‐end tapered‐fused silica‐packed capillary columns in nano‐LC‐ESI MS

A novel frit preparation method for one‐end tapered‐fused silica‐packed capillary columns in nano‐LC‐ESI MS was developed. A hollow‐fused silica capillary column with a tapered tip as nano‐spray emitter was filled with 5 μm C₁₈ beads, and then a sintered frit about 0.25 mm in length was prepared at the tip by butane flame. A stainless steel protection tube with 0.5 mm id was used to control the length of the frit and to protect the packed C₁₈ beads behind the sintered frit during the sintering. C₁₈ sintered frits were evaluated by BSA tryptic digests with nano‐LC‐LTQ. The sintered frits did not produce post‐column band broadening due to very small volume (about 0.2 nL) and did not produce adsorption to sample. The sintered frit columns had good separation reproducibility and separation performance compared with self‐assembled particles frit columns and commercial columns.     

Column-switching high-performance liquid chromatographic system with a laser-induced fluorimetric detector for direct,automated assay of salivary cortisol

In order to measure human stress, an easy and rapid, fully automated method for the determination of cortisol in saliva has been developed, using column-switching high-performance liquid chromatography with laser-induced fluorescence detection, which involves post-column labeling with sulfuric acid. The developed system requiers only 0.1 ml of saliva, and a simple pretreatment consisting of dilution and filtration is sufficient. The column-switching system consisted of a Polymer-Coated Mixed-Functional silica (PCMF) column for deproteinization, and a CN column for frontal concentration and separation. An ODS column in place of the CN provided a better separation, but required a post-column make-up of water for safe reaction. Detection limit of cortisol was 8 fmol (signal-to-noise ratio = 3), which is adequate for routine determination of normal levels of cortisol (1–20 pmol/ml). The analysis time was about 40 min and reproducibility was excellent with an R.S.D. of less than 5%.     

Analysis of thiohydantoins from amino acids by gas-liquid chromatography on short glass capillary columns

A method for separation of amino acid methyl or phenyl thiohydantoins by GLC on a short glass capillary column is described. Calculations of required parameters of the capillary column are presented. By the described methods, nineteen of twenty silylated methylthiohydantoins were separated in one run. The last one (histidine) can be identified on the same column by starting the analysis at a higher temperature. Cysteine and arginine were analysed as S-methylcysteine and ornithine, respectively.     

Routine amino acid capillary column chromatography of microquantities in the picomole range--procedures and modifications of a commercial analyzer

A commercial micro amino acid analyzer using capillary columns (internal diameter 0.7 mm) had to be modified greatly in order to get satisfactory separation and quantitative analysis on the microscale of free amino acids in biological fluids, tissue extracts, and hydrolysates. Furthermore, the usual analytical conditions were changed (i.e., lowering of the buffer pH) and a procedure and apparatus for sample deproteinization is described which allows simple handling of volumes in the range from 20 to 100 μl. The modifications described guarantee better and more reproducible analyses than reported so far. A design of a new “analytical unit” for capillary column chromatography is proposed.     

Transfomration of arachidonic acid into 12-hydroxy-5,8,10,14-eicosatetraenoic acid by mouse peritoneal macrophages.

Mouse peritoneal macrophages were incubated at 37 degrees C for 30 min with arachidonic acid (all-cis-5,8,11,14-eicosatetraenoic acid). Oxygenation of arachidonic acid in mouse peritoneal macrophages occurs by two major pathways: fatty acid cyclooxygenase and lipoxygenase. The major metabolite of the latter is 12-hydroxy-5,8,10,14-eicosatetraenoic acid which was identified by gas liquid chromatography on high resolution glass capillary column and mass spectrometry.     

Determination of Fatty Acid Composition of Spore Lipids of the Moss Polytrichum commune by Glass Capillary Column Gas Chromatography

Fatty acid methyl esters of Polytrichum commune spore triglyceride and mono- and diglycosyl diglyceride fractions were analysed by glass capillary column gas chromatography provided with a precolumn system. The composition of the fatty acids in the lipid fractions differed only quantitatively: the diglycosyl diglyceride fraction was characterized by a high content of C 18: 3ω3 (67.7%), and the triglyceride and monoglycosyl diglyceride fractions by about 35%. The monoglycosyl diglyceride fraction contained a high proportion of C 14: 0 (18.4%). In all fractions the content of polyunsaturated C 20 acids was low, ranging from trace amounts to 4.9%.     

Analysis of bile acids in serum and bile by capillary gas-liquid chromatography

Various liquid phases for glass capillary columns have been evaluated for gas-liquid chromatographic analysis of methyl ester trimethylsilylether derivatives of bile acids from serum and bile. Bile acid analysis is rapid and exhibits high separation efficiency with a 20 X 0.3 mm glass capillary column whose internal surface is covered with a crystal layer of barium carbonate and coated with polyethyleneglycol 20000 as liquid phase according to Grob et al.     

Analysis of the stereochemistry of lipoxygenase-derived hydroxypolyenoic fatty acids by means of chiral phase high-pressure liquid chromatography

A chiral phase HPLC method was developed for the simultaneous determination of the positional and optical isomers of the lipoxygenase-derived hydroxypolyenoic fatty acids. With a Bakerbond chiral phase HPLC column (dinitrobenzoyl phenylglycine as chiral phase) the positional and optical isomers of the reduced dioxygenation products (by triphenylphosphine or borohydride) of linoleic acid and arachidonic acid were separated after methylation of the carboxylic groups. No cumbersome chemical derivatization such as conversion to a diastereomer was necessary. As compared with the methods used up till now chiral phase HPLC proved to be simpler and more sensitive. About 10 pmol of hydroxy fatty acids suffice for an analysis. The chiral phase HPLC can be used for the preparative separation of the optical antipodes of the lipoxygenase products. An optical purity of more than 90% can be reached in one preparative run. The method was applied to the determination of the stereochemistry of the dioxygenation products of polyenoic fatty acids formed by the lipoxygenases from soybeans, reticulocytes, pea seeds (isoenzyme I and II), tomato fruits, by the quasilipoxygenase activity of hemoglobin, and by the methylene blue-mediated photooxidation of arachidonic acid.     

Affinity chromatography and capillary electrophoresis for analysis of the yeast ribosomal proteins

We present a top down separation platform for yeast ribosomal proteins using affinity chromatography and capillary electrophoresis which is designed to allow deposition of proteins onto a substrate. FLAG tagged ribosomes were affinity purified, and rRNA acid precipitation was performed on the ribosomes followed by capillary electrophoresis to separate the ribosomal proteins. Over 26 peaks were detected with excellent reproducibility (<0.5% RSD migration time). This is the first reported separation of eukaryotic ribosomal proteins using capillary electrophoresis. The two stages in this workflow, affinity chromatography and capillary electrophoresis, share the advantages that they are fast, flexible and have small sample requirements in comparison to more commonly used techniques. This method is a remarkably quick route from cell to separation that has the potential to be coupled to high throughput readout platforms for studies of the ribosomal proteome.     

Differential chemical diagnosis of primary hyperoxaluria type II. Highly sensitive analysis of optical isomers of glyceric acid by GC/MS as diastereoisomeric derivatives

We established a separation method for the optical isomers of glyceric acid in urine by modifying the derivatization steps of the procedure used for the screening and diagnosis. The trimethylsilyl derivatization step in the mass screening procedure was replaced by O-acetyl-(+)-2-butylation, and the samples were analyzed under equivalent GC/MS conditions by capillary gas chromatography on a DB-5MS column. This method can be applied to cases that show a high urinary concentration of glyceric acid to obtain a differential diagnosis of primary hyperoxaluria type II and d-glyceric aciduria easily. l-Glyceric acid was also isolated from the urine of healthy controls as one of the main peaks.     

The Collection and Elution of Radioactive Fatty Acid Methyl Esters During Quantitative Gas-Liquid Chromatography

Abstract

An improved procedure is described for the collection and elution of low levels of radioactive fatty acid methyl esters separated by gas-liquid chromatography. A gas chromatographic effluent splitter was employed to partition fatty acid methyl ester samples in the column effluent, Condensation of a portion of the eluted fatty acids was accomplished in borosilicate glass tubing collectors maintained at ?70°C, Quantitation of nanomolar levels of fatty acid methyl esters was accomplished by calibrating the gas chromatographic flame ionization detectors with the splitters opened or closed. The elution of condensed radioactive fatty acid methyl esters from the glass collectors was complete when benzene followed by a toluene based scintillation fluid were employed as solvents. The method described may be applicable to the analysis of cis-trans isomers of unsaturated fatty acids.     

High-Resolution Performance of Polycaprolactone Functionalized with Guanidinium Ionic Liquid for Gas Chromatography

This work firstly reported a new polycaprolactone based material functionalized with guanidinium ionic liquid (PCL-GIL) as the stationary phase with high resolution performance for capillary gas chromatography (GC). It is composed of polycaprolactone (PCL) and guanidinium ionic liquid (GIL) with amphiphilic conformation. The PCL-GIL capillary column coated by static method exhibited high column efficiency of 3942 plates/m and moderate polarity. As a result, the PCL-GIL column exhibited high-resolution capability. For a mixture of 27 analytes with a wide ranging polarity and outperformed the PCL-2OH and HP-35 columns, showing its advantageous separation capability for analytes of diverse types. Moreover, the PCL-GIL column showed high resolving capability for various positional isomers and cis-/trans-isomers, including alkylbenzenes, chlorobenzenes, naphthalenes, bromonitrobenzenes, chloronitrobenzenes, benzaldehydes, phenols, alcohols, respectively. In a word, PCL derivatized by GIL units as a new type of stationary phase has a promising future in GC separations.     

Methods for the rapid separation and estimation of the major lipids of arteries and other tissues by thin-layer chromatography on small plates followed by microchemical assays

Methods are described for the rapid separation of the major individual phospholipids and neutral lipids of tissues by thin-layer chromatography on small glass plates (75 × 75 mm), and for the specific microchemical estimation of separated lipids and for determination of fatty acid composition and radioactivity. The overall method, involving tissues extraction, thin-layer chromatographic separation and assay has been evaluated using pure standards and biological samples and gives good reproducibility and almost complete recovery of lipids.     

A method for specific analysis of free fatty acids in biological samples by capillary gas chromatography.

The present report describes a simple method to selectively extract free fatty acids and analyze them by capillary gas-liquid chromatography. The procedure is based on the use of fumed silicon dioxide. In the presence of plasma, this material induces a rapid rise in the viscosity of the mixture and presents the ability to trap large particles such as emulsified lipids and lipoproteins. Albumin-bound fatty acids are thus left in the aqueous media. We present applications of our procedure for the analysis of free fatty acids in 0.2 ml of plasma from rat or human. By comparison with the method utilizing thin-layer chromatography for the separation of fatty acids and gas chromatography analysis, the present method has been found to be reliable and simple. The recovery of linoleic acid was 92.1 +/- 8.2%, a value which is about twice better than that obtained with the procedure using thin-layer chromatography. In particular, long-chain polyunsaturated fatty acids were better preserved. Our procedure does not require the use of organic solvents and its simplicity and reproducibility make it suitable for routine specific determination of the composition of free fatty acids in biological samples.     

Glass capillary or fused-silica gas chromatography—mass spectrometry of several monosaccharides and related sugars: Improved resolution

The gas chromatographic separation of several monosaccharides and related sugars derivatized by methoxylation and trimethylsilylation reactions was optimized with glass capillary (SP-2250) and fused silica (SP-2100) columns. Individual sugars included aldoses, ketoses, polyols, acidic forms and N-acetylated amino sugars. Peaks were detected by selected ion monitoring (SIM). The fused silica column gave complete resolution of all peaks (two per hexose and one per hexitol) arising from glucose, galactose, mannose, fructose, sorbitol, mannitol and dulcitol. The resolution of these sugars with the glass capillary column was not as good, but full differentiation was possible on the basis of SIM. Because the fused silica column gave a better resolution of 33 sugars tested and was more easily installed than the glass capillary column, it was utilized for quantitative analysis. A deuterated algal sugar mixture used for quantitation by isotope dilution was found to contain glucose, galactose, mannose, xylose, arabinose, ribose and rhamnose. Full recoveries were obtained of various amounts of glucose, galactose, mannose, fructose and xylose added to human serum.     

Use of large particles support for fast analysis of methadone and its primary metabolite in human plasma by liquid chromatography-mass spectrometry

A bioanalytical method was developed for the quantitation of methadone (MTD) and its primary metabolite, (EDDP) in plasma. The extraction step was performed within a capillary column packed with large particles (35x0.3 mm I.D.; d(p) 30 micrometer) at high flow-rate conditions (450 microliter/min). The separation was performed on a microbore analytical column (55x2 mm I.D.; d(p) 3 micrometer) coupled to a mass spectrometer (MS). This procedure was based on a column-switching unit. Analytes of interest were retained on the precolumn by hydrophobic interactions and backflushed from the precolumn to the analytical column. The detection was carried out with a MS single quadrupole equipped with an electrospray interface. The total analysis time was 6 min. The limits of quantification were evaluated at 10 and 25 ng/ml for MTD and EDDP, respectively. At this level, good accuracies were obtained for both analytes with repeatability values less than 18%.