Analysis of trimethylsilyl O-methyloximes of carbohydrates by combined gas-liquid chromatography-mass spectrometry

We have concluded that the O-methyloxime derivative effectively “labels” the carbonyl portion of the surgar, and probably contributes to the stability of ions containing that part of the molecule. Electron impact-induced cleavage of the carbon-carbon bonds in the sugar, with charge retention on either fragment, provides a mass spectrum that may be used to analyze substituents on any carbon. We have examined the mass spectra of several classes of sugars, as their -O-methyloxime trimethylsilyl ethers, and as their -O-methyloxime peracetates and in most cases ions are found corresponding to the fragmentation pattern proposed here (unpublished results). In addition, the gas-liquid chromatographic retention behavior of these compounds suggests that they may be useful for quantitative determination of carbohydrates.     

Identification by gas-liquid chromatography-mass spectrometry of 4-O-acetyl-9-O-lactyl-N-acetylneuraminic acid,a new sialic acid from horse submandibular gland

The novel sialic acid 4-O-acetyl-9-O-lactyl-N-acetylneuraminic acid has been identified as a constituent of horse submandibular gland glycoproteins in addition to the already know equine sialic acids, N-acetylneuraminic acid, 4-O-acetyl-N-acetylneuraminic acid, 9-O-acetyl-N-acetylneuraminic acid, 4,9-di-O-acetyl-N-acetylneuraminic acid, N-glycolylneuraminic acid, 4-O-acetyl-N-glycolylneuraminic acidand 9-O-acetyl-N-glycolylneuraminic acid. The structure has been established by combined gas-liquid chromatography-mass spectrometry.     

Identification of benzophenanthridine alkaloids from Bocconia arborea by gas chromatography-mass spectrometry

A methanol extract of the bark of Bocconia arborea was fractionated on silica gel and the fractions analysed using gas chromatography coupled with mass spectrometry (GC-MS). Several benzophenanthridine alkaloids were identified including dihydrosanguinarine, oxysanguinarine, 11-acetonyldihydrochelerythrine, dihydrochelerythrine, chelerythrine, chelerythridimerine and angoline as the principal constituents. The results show that the direct GC-MS analysis of these alkaloids is possible with a clear distinction between the compounds. The technique is shown to be a valuable tool and an alternative technique to classical phytochemical procedures permitting the fast analysis of alkaloids mixtures.     

Identification of pyruvated monosaccharides in polysaccharides by gas-liquid chromatography mass spectrometry

Twelve bacterial polysaccharides of known structure containing a representative range of pyruvated monosaccharides, were methanolysed, trimethylsilylated, and analysed by g.l.c. and g.l.c.-m.s. Except for 3,4-O-(1-carboxyethylidene)-L-rhamnose, which was unusually labile, the pyruvic acid substituents were largely retained during methanolysis and the Me3Si derivatives of the resulting pyruvated methyl glycosides gave distinctive g.l.c. peaks with characteristic mass spectra. The pyranose rings of 4,6-O-(1-carboxyethylidene)-D-glucose, 4,6-O-(1-carboxyethylidene)-D-mannose, 4,6-O-(1-carboxyethylidene)-D-galactose, and 3,4-O-(1-carboxyethylidene)-D-galactose survived the methanolysis, but that of 2,3-O-(1-carboxyethylidene)-D-glucuronic acid was cleaved to give the methyl ester of 2,3-O-(1-carboxyethylidene)-aldehydo-D-glucuronic acid dimethyl acetal. In the case of 2,3-O-(1-carboxyethylidene)-D-galactose, cleavage of the pyranose ring was less complete; under the conditions used in these experiments two-thirds of the pyranose rings were intact while one-third were cleaved to give the methyl ester of 2,3-O-(1-carboxyethylidene)-aldehydo-D-galactose dimethyl acetal. A very small amount of 3,4-O-(1-carboxyethylidene)-L-rhamnose from one polysaccharide retained its pyruvic acid substituent after gentle methanolysis to give the methyl ester of 3,4-O-(1-carboxyethylidene)-aldehydo-L-rhamnose dimethyl acetal. Susceptibility to cleavage of the pyranose ring during methanolysis appears to be a property of pyruvated monosaccharides with trans-fused 1,3-dioxolane rings.     

Characterization of degradation products from alkaline wet oxidation of wheat straw

Alkaline wet oxidation pre-treatment (water, sodium carbonate, oxygen, high temperature and pressure) of wheat straw was performed as a 2(4-1) fractional factorial design with the process parameters: temperature, reaction time, sodium carbonate and oxygen. Alkaline wet oxidation was an efficient pre-treatment of wheat straw that resulted in solid fractions with high cellulose recovery (96%) and high enzymatic convertibility to glucose (67%). Carbonate and temperature were the most important factors for fractionation of wheat straw by wet oxidation. Optimal conditions were 10 min at 195 degrees C with addition of 12 bar oxygen and 6.5 g l(-1) Na2CO3. At these conditions the hemicellulose fraction from 100 g straw consisted of soluble hemicellulose (16 g), low molecular weight carboxylic acids (11 g), monomeric phenols (0.48 g) and 2-furoic acid (0.01 g). Formic acid and acetic acid constituted the majority of degradation products (8.5 g). The main phenol monomers were 4-hydroxybenzaldehyde, vanillin, syringaldehyde. acetosyringone (4-hydroxy-3,5-dimethoxy-acetophenone), vanillic acid and syringic acid, occurring in 0.04-0.12 g per 100 g straw concentrations. High lignin removal from the solid fraction (62%) did not provide a corresponding increase in the phenol monomer content but was correlated to high carboxylic acid concentrations. The degradation products in the hemicellulose fractions co-varied with the pre-treatment conditions in the principal component analysis according to their chemical structure, e.g. diacids (oxalic and succinic acids), furan aldehydes, phenol aldehydes, phenol ketones and phenol acids. Aromatic aldehyde formation was correlated to severe conditions with high temperatures and low pH. Apart from CO2 and water, carboxylic acids were the main degradation products from hemicellulose and lignin.     

Determination of endotoxin by the measurement of the acetylated methyl glycoside derivative of Kdo with gas-liquid chromatography-mass spectrometry

A gas-liquid chromatographic-mass spectrometric (GLC-MS) method was applied to the detection of 3-deoxy-d-manno-2-octulosonic acid (Kdo), a constituent of bacterial lipopolysaccharide (LPS, endotoxin). Samples containing LPS were dried, methanolyzed with 2 M HCl in methanol at 60 degrees C for 1 h and acetylated with acetic anhydride and pyridine (1:1, v/v) solution at 100 degrees C for 30 min, then the products were analyzed by GLC-MS or GLC-MSMS. Four acetylated methylglycoside methyl ester derivatives of Kdo are formed in these conditions, namely one with pyranose ring (Kdo1), two derivatives in the furanose form (Kdo2 and 3) and one derivative of anhydro Kdo (Kdo4), as results from their mass fragmentation patterns. Synthetic Kdo produced mainly Kdo4 derivative, whereas Kdo1 of pyranose ring shape was the predominating derivative formed from LPS. The ion fragment of m/z 375 was selected for the specific detection of this Kdo1 derivative, which might be applied for the endotoxin determination. That approach was used for the analysis of preparations of bacteria, bacteriophages and samples of animal sera. In order to ensure the removal of phosphate substitutions from Kdo, methanolyzed samples can be treated with alkaline phosphatase (2.6 U, pH 9.2, 37 degrees C, 15 min), what was elaborated on Vibrio LPS preparation.     

Characterization of enzymatic degradation products of carboxymethyl cellulose by gel chromatography

In order to investigate the molecular weight distribution of depolymerization products obtained by enzymatic degradation of carboxymethyl cellulose (CMC), a high resolution size exclusion chromatographic (SEC) system was developed.The SEC system using Fractogel® TSK-HW and an eluent containing sodium sulfate and sodium acetate enables an effective separation of the anionic cleavage products to be carried out. The experimental set-up was equipped with a sensitive detection system based on the post-column reaction of carbohydrates with orcinol.The elution patterns of enzymatic depolymerization products obtained from CMC with different degrees of substitution make it feasible to infer parts of the sequence and the distribution of carboxymethyl groups.     

Identification of glycation at the N-terminus of albumin by gas chromatography-mass spectrometry.

It has previously been shown that neurotensin binds to high-affinity receptors in the adenocarcinoma HT29 cell line, and that receptor occupancy leads to inositol phosphate formation. The present study was designed to investigate further the effects of neurotensin on calcium mobilization and protein kinase C (PKC) activation in HT29 cells, and to assess the role of GTP-binding proteins (G-proteins) in the neurotensin response. Direct measurements of cytosolic Ca2+ variations using the fluorescent indicator quin 2 showed that neurotensin (0.1-1 microM) elicited Ca2+ transients in HT29 cells. These transients occurred after the neurotensin-stimulated formation of Ins(1,4,5)P3, as measured by means of a specific radioreceptor assay. In addition, the peptide induced a decrease in the 45Ca2+ content of cells previously equilibrated with this isotope. The peptide effect was rapid, long-lasting and concentration-dependent, with an EC50 of 2 nM. Phorbol 12-myristate 13-acetate (PMA) inhibited by 50% the neurotensin effects on both intracellular Ca2+ and inositol phosphate levels. The inhibition by PMA was abolished in PKC-depleted cells. Pertussis toxin had no effect on either the Ca2+ or inositol phosphate responses to neurotensin. Epidermal growth factor (EGF) receptors which are present in HT29 cells have been shown to be down-regulated through phosphorylation by PKC in a variety of systems. Here, PMA markedly (70-80%) inhibited EGF binding to HT29 cells. Scatchard analysis revealed that PMA abolished the high-affinity component of EGF binding, an effect that was totally reversed in PKC-depleted cells. In contrast, neurotensin slightly (10-20%) inhibited EGF binding to HT29 cells, and its effect was only partly reversed by PKC depletion. Neurotensin had no detectable effect on sn-1,2-diacylglycerol levels in HT29 cells, as measured by a specific and sensitive enzymic assay. In membranes prepared from HT29 cells, monoiodo[125I-Tyr3]neurotensin bound to a single population of receptors with a dissociation constant of 0.27 nM. Sodium and GTP inhibited neurotensin binding in a concentration-dependent manner. Maximal inhibition reached 80% with Na+ and 35% with GTP.IC50 values were 20 mM and 0.2 microM for Na+ and GTP respectively. Li+ and K+ were less effective than Na+ and the effects of GTP were shared by GDP and guanosine-5'-[beta gamma- imido]triphosphate but not by ATP. Scatchard analysis of binding data indicated that Na+ and GTP converted the high-affinity neurotensin-binding sites into lower affinity binding sites. The properties of the effects of Na+ and GTP on neurotensin-receptor interactions are characteristic of those receptors which interact with G-proteins.(ABSTRACT TRUNCATED AT 400 WORDS)     

Quantification of retinoic acid by gas-liquid chromatography-mass spectrometry: total versus all-trans-retinoic acid in human plasma

An assay based on negative ion chemical ionization mass spectrometry has been developed to quantify retinoic acid in plasma or serum. The lower limit of detection is 75 pg (240 fmol); normal values of retinoic acid can be determined on as little as 40 microliters of human plasma. The plasma concentrations of total retinoic acid in 12 healthy male volunteers taking no medication or vitamin supplementation ranged from 2.8 to 6.6 ng/ml; the mean was 4.9 ng/ml. The assay can be manipulated to measure all-trans-retinoic acid alone; about 75% of retinoic acid in human plasma or rat serum is all-trans-retinoic acid. Both retinol and retinoic acid can be quantified on the same 0.1-ml sample; the concentration of retinoic acid in human plasma or rat serum is at least 150-fold less than that of retinol.     

Hydrogels of N-acylchitosans and their cellulose composites generated from the aqueous alkaline solutions

Hydrogels of N-acetyl and N-propionylchitosans were prepared form aqueous solutions of sodium N-acylchitosan salts (alkaline N-acylchitosans) and sodium N-acylchitosan xanthate [O-(sodium thio)thiocarbonyl N-acylchitosan], respectively, by standing at room temperature and on heating. Novel hydrogels of N-acetylchitosan-cellulose and N-propionylchitosan-cellulose composites were also prepared from sodium cellulose xanthate [O-(sodiumthio)thiocarbonyl cellulose] solutions mixed with sodium N-acylchitosan salts and with sodium N-acylchitosan xanthates, respectively.     

Oxidized plant sterols in human serum and lipid infusions as measured by combined gas-liquid chromatography-mass spectrometry.

Some oxidized forms of cholesterol (oxysterols) are thought to be atherogenic and cytotoxic. Because plant sterols are structurally related to cholesterol, we examined whether oxidized plant sterols (oxyphytosterols) could be identified in human serum and soy-based lipid emulsions. We first prepared both deuterated and nondeuterated reference compounds. We then analyzed by gas-liquid chromatography-mass spectrometry the oxyphytosterol concentrations in serum from patients with phytosterolemia or cerebrotendinous xanthomatosis, in a pool serum and in two lipid emulsions. 7-Ketositosterol, 7 beta-hydroxysitosterol, 5 alpha, 6 alpha-epoxysitosterol, 3 beta,5 alpha,6 beta-sitostanetriol, and probably also 7 alpha-hydroxysitosterol were present in markedly elevated concentrations in serum from phytosterolemic patients only. Also, campesterol oxidation products such as 7 alpha-hydroxycampesterol and 7 beta-hydroxycampesterol were found. Interestingly, sitosterol was oxidized for approximately 1.4% in phytosterolemic serum, which is rather high compared with the approximate 0.01% oxidatively modified cholesterol normally seen in human serum. The same oxyphytosterols were also found in two lipid emulsions in which the ratio of oxidized sitosterol to sitosterol varied between 0.038 and 0.041.In conclusion, we have shown that oxidized forms of plant sterols are present in serum from phytosterolemic patients and two frequently used soy-based lipid emulsions. Currently, it is unknown whether oxyphytosterols affect health, as has been suggested for oxysterols. However, 7 beta-hydroxycholesterol may be one of the more harmful oxysterols, and both sitosterol and campesterol were oxidized into 7 beta-hydroxysitosterol and 7 beta-hydroxycampesterol. The relevance of these findings therefore deserves further exploration.     

Identification of DNA degradation products by antitumor antibiotic, esperamicin

Esperamicin A1 is a DNA-damaging agent characterized by a unique ten-membered ene-diyne core. We studied the detailed reaction mechanism by using synthetic DNA oligomers. The cleavage site and activity depend on the sequence of the oligomers. d(GGATCC) and d(GGTACC) were cleaved by the drug while d(CCATGG) and d(CCTAGG) were not cleaved under the present conditions. d(GGTACC) gave two major 5'-fragments. The result of partial nuclease digestion experiments suggests that these products are trimer and pentamer with a modified 3'-end.     

Identification of 6-hydroxymelatonin in normal human urine by gas chromatography-mass spectrometry.

The major melatonin metabolite, 6-hydroxymelatonin, has been identified in normal human urine by gas chromatography-mass spectrometry (gc-ms) after rapid acid hydrolysis, extraction, and derivatization. An estimate of the amount detected (approximately 20 ng/ml) is consistent with calculated rates of melatonin synthesis and turnover.     

Method for quantitative analysis of PGA2 from plasma using deuterated carrier and gas-liquid chromatography-mass spectrometry

The preparation of [3,3,4,4,-²H₄, 17, 18-³H₂]prostaglandin A₂ (PGA₂) and [17, 18-³H₂]PGA₂ is described. These compounds have been prepared for use in quantitative determination of corresponding nondeuterated prostaglandin by gas-liquid chromatography-mass spectrometry. The method is based on addition of a known amount of carrier to the sample. After purification and derivatization, the ratio between the protium and deuterium form is measured in the mass spectrometer. Ions originating from deuterated and nondeuterated moleeules are focused intermittently on the electron multiplier using an accelerating voltage alternator. With this technique, 60 pg of PGA₂ can be determined with a precision of ±8.4%. The recoveries from plasma of added PGA₂ were about 97%. Basal levels of PGA₂ in human plasma were lower than 10 pg/ml.     

Characterization by gas-liquid chromatography-mass spectrometry and proton-magnetic-resonance spectroscopy of pertrimethylsilyl methyl glycosides obtained in the methanolysis of glycoproteins and glycopeptides.

The quantitative analysis by gas chromatography of monosaccharides present in glycoproteins and glycopeptides using methanolysis, followed by re-N-acetylation and trimethylsilylation, gives rise to several peaks for each monosaccharide. The identity of these peaks for xylose, fucose, mannose, galactose, glucose, N-acetylglucosamine, N-acetylgalactosamine and N-acetylneuraminic acid was established for alpha- and beta-methyl pyranosides and furanosides by combined g.l.c.-mass spectrometry and proton-magnetic-resonance spectroscopy. These data provide for the unambiguous interpretation of the gas chromatograms obtained in the application of this g.l.c. method, and supply basic information for the further application of mass spectrometry in this field.