A simple and rapid preparation of alditol acetates for monosaccharide analysis

A simple and rapid method is described for the preparation of alditol acetates from monosaccharides. It can be performed in a single tube without transfers or evaporations. Monosaccharides are reduced with sodium borohydride in dimethyl sulphoxide and the resulting alditols acetylated using 1-methylimidazole as the catalyst. Removal of borate is unnecessary and acetylation is complete in 10 min at room temperature. Monosaccharides are quantitatively reduced and acetylated by this procedure. The alditol acetates are completely separated by glass-capillary, gas-liquid chromatography on Silar 10C. The method has been applied to the analysis of monosaccharides in acid hydrolysates of a plant cell-wall.     

Partially ethylated alditol acetates as derivatives for elucidation of the glycosyl linkage-composition of polysaccharides

The use of partially ethylated alditol acetates for the analysis by gas-liquid chromatography of the components of polysaccharides, and the glycosidic linkages of these components, is described. The derivatives are prepared by procedures analogous to those for the synthesis of partially methylated alditol acetates. Derivatization requires two successive ethylations and more-strenuous conditions of hydrolysis and reduction than for the methyl analogs. The partially ethylated alditol acetates are formed in nearly quantitative yield and give single, sharp peaks on gas chromatography. Retention-time data, relative to two internal standards, are given for 79 glycosidic linkage-isomers of mannose, galactose, glucose, arabinose, xylose, rhamnose, and fucose, on four g.l.c. columns. One of these columns is a newly developed, highly polar, capillary column. Direct comparisons of these retention times to retention times of partially methylated alditol acetates are made. The ethyl analogs are eluted sooner that the corresponding methyl derivatives, and the amount of this shift in elution time is dependent upon the number of alkyl groups in the derivative. This change in elution time allows separation of many polysaccharide components by g.l.c. that are not separable as their partially methylated alditol acetates. Others, separated as their O-methyl derivatives, are coeluted as their partially ethylated alditol acetates. The two derivatives thus provide excellent complementary procedures because of their differential chromatographic separation and because of the similarity of their preparation.     

A method of purification of partially methylated alditol acetates in the methylation analysis of glycoproteins and glycopeptides

A method for methylation analysis of intact glycoproteins is described. Starting with intact glycoprotein, the oligosaccharides are methylated, hydrolyzed, reduced, and acetylated. The partially methylated alditol acetates are then separated from noncarbohydrate contaminants on a silica gel G column. Partially methylated hexitol acetates are eluted from the column with petroleum ether:ethyl acetate (1:1, $\text{v}\text{v}$) and partially methylated N-acetylhexosaminitol acetates are subsequently eluted with methanol. Analysis by gas-liquid chromatography/mass spectrometry of the partially methylated alditol acetates shows no interfering contaminants. This method circumvents the need to make pronase glycopeptides and avoids the pitfalls of other methylation procedures.     

Evaluation of Poly S-179 as a stationary phase for the gas-liquid chromatography-mass-spectrometry of bile acid methyl ester acetates

The stationary phase Poly S-179 has been found to offer distinct advantages over the previously reported SP-525 for the gas-liquid chromatographic separation of bile acid methyl ester acetates. Relative retention times of these bile acid derivatives are compared on the two phases.     

Oligosaccharides derived from the xyloglucan isolated from the seeds of Hymenaea courbaril var. stilbocarpa

On aqueous extraction, Hymenaea courbaril var. stilbocarpa, known in Brazil as jatobá, furnishes a high yield of viscous xyloglucan (45%) from its seeds. The crude polysaccharide (B1) was hydrolysed and the products, analysed as alditol acetates, were glucose, xylose, galactose and arabinose in the ratio 50:35:13:2. After further fractionation on a DEAE-cellulose column (chloride form), the main fraction (70% yield, B2) was obtained. The basic structure of the xyloglucan was determined as a cellulose-type (1 → 4)-linked β-d-glucan backbone partially substituted with side chains at 06 of -d-xylopyranose, some of which were themselves substituted at 02 by the units of β-d-galactopyranose. Treatment of the xyloglucan (B2) with commercial cellulase from Trichoderma sp. yielded six oligosaccharides. These oligosaccharides were isolated by preparative paper chromatography, and their structures were determined by gas-liquid chromatography-mass spectroscopy of the derived partially O-methylated alditol acetates. These results confirm the structure proposed for jatobá seed xyloglucan.     

Structures of gangliosides from bovine adrenal medulla.

Five gangliosides, accounting for over 95% of the total ganglioside fraction, were isolated from bovine adrenal medulla by preparative thin-layer chromatography and the carbohydrate structures determined by a combination of periodate oxidation and permethylation techniques. Partially methylated alditol acetates were generated from the neutral sugars of the fully methylated glycolipids and identified by gas-liquid chromatography. Substitution on N-acetylgalactosamine was determined by methanolysis of the permethylated ganglioside, acetylation of the products, and identification of the resulting substituted methyl glycosides by GLC. Periodate oxidation followed by borohydride reduction confirmed some of the linkages and demonstrated the absence of (2-8) linkages between sialic acid units. Mass spectrometry of the permethylated gangliosides gave information on sugar sequence at the nonreducing end.     

A multiple-column approach to the methylation analysis of plant cell-walls

A procedure has been developed for the improved analysis of the neutral-sugar glycosidic linkages in plant cell-walls, utilising capillary g.l.c. and columns of three different phases for the separation of the products of methylation analysis. Retention coefficients are reported for a wide range of partially methylated alditol acetates on columns of CP-Sil88 and a bonded phase (BP-1) equivalent to OV-1. Using these phases and SP-1000, all cases of co-chromatography can be resolved. Computers were used to process the large amounts of data produced, to identify peaks and to assist in merging the results obtained using the three phases.     

Identification of l-iduronic acid as a constituent of the major extracellular polysaccharide produced by Butyrivibrio fibrisolvens strain X6C61

Abstract Butyrivibrio fibrisolvens strain X6C61 produces two extracellular polysaccharides (EPS-I and EPS- II) separable by anion-exchange chromatography. The neutral sugar constituents of EPS-I were identified by gas-liquid chromatography (GLC) as the alditol acetates of rhamnose, mannose, galactose, glucose, and an unidentified component. These results were confirmed using thin-layer chromatography (TLC). Neutral sugar analysis of EPS-II, which eluted from DEAE-Sephadex at 0.4 M NaCl, yielded the alditol acetates of rhamnose, galactose, glucose, and idose. However, idose was not found when hydrolysates of EPS-II were analysed by TLC. Further investigations showed that the iditol hexaacetate detected via GLC was an artifact of the commonly-used procedures for neutral sugar analysis. This compound was instead generated from l -iduronic acid, as shown by GLC-MS studies.     

Separation of mono-, di-, and trihydroxy stereoisomers of bile acids by capillary gas-liquid chromatography

Capillary gas-liquid chromatographic separation was studied for the complete set of the 26 theoretically possible isomers of mono-, di-, and trihydroxylated 5 beta-cholanic acids, which differ from one another in the number, position, and configuration of hydroxyl groups at C-3, C-7, and/or C-12 in the nucleus, as well as for some of their related acids. The bile acid samples were chromatographed as their methyl ester-trimethylsilyl (TMSi) ether derivatives and analyzed on three capillary columns coated with nonpolar OV-1, slightly polar OV-17, and polar SP-2340 as liquid phases. The retention times on capillary gas-liquid chromatography (GLC) responded dramatically to the minor structural differences, and almost complete separation of the positional and stereochemical isomers was achieved by the combined use of SP-2340 and OV-17 (or OV-1) capillary columns.     

Quantitative analysis by various g.l.c. response-factor theories for partially methylated and partially ethylated alditol acetates

Results are presented which demonstrate that the molar flame-responses of partially methylated partially ethylated alditol acetates should be calculated on an effective carbon response (e.c.r.) basis. The relative responses of 2,3,4,6-tetra-O-ethyl-D-glucitol 1,5-diacetate, 2,3,6-tri-O-ethyl-D-glucitol 1,4,5-triacetate, hexa-O-ethyl-D-glucitol, hexa-O-methyl-D-glucitol, α-D-galactopyranose pentaacetate were measured and compared to the predicted values from three theories: equal molar response, equal weight response, effective carbon response. The observed values agree very well (±0?6%) with the e.c.r.-calculated values. The other theories of relative response can result in as much as 100% error in quantitation. The e.c.r-calculated relative response-factors for all commonly found partially methylated partially ethylated alditol acetates are presented, and their use is suggested for accurate quantitation.     

植物组织中低聚糖乙酰化及毛细管气相色谱分析

建立一种用乙酰化衍生处理低聚糖并用毛细管气相色谱-FID进行分析的方法。以1-甲基咪唑为催化剂并以乙酸酐为乙酰化试剂,同时对植物样品中蔗糖、棉子糖和水苏糖等低聚糖乙酰化产物进行毛细管气相色谱分离和FID检测。确定了低聚糖乙酰化衍生物的毛细管气相色谱分析条件,并对低聚糖乙酰化反应条件及色谱分离条件进行了优化。结果表明,在80–1000ng·μL–1范围内线性关系良好,蔗糖、棉子糖和水苏糖的相关系数（R）分别为0.9952、0.9957和0.9877,并且精准度与回收率均较高。使用该方法对低聚糖进行乙酰化反应重现性好、所需样品材料及试剂量少且污染毒害小,能够得到理想的分离、检测和定量分析效果,适用于少量植物组织中低聚糖的定量分析。该方法在食品、医药检测和基础科学研究领域均具有广泛的适用性及参考价值。     

Improved method for simultaneous determination of alcohols, volatile fatty acids, lactic acid or 2,3-butanediol in biological samples

A rapid and sample procedure was developed to determine, by gas-liquid chromatography, the concentrations of C₂---C₄ alcohols, C₂---C₆ volatile fatty acids (VFA) and lactic acid or 2,3-butanediol in fermentation liquids. both lactic acid and 2,3-butanediol are oxidized to acetaldehyde by periodic acid and acetaldehyde was eluted before ethanol. A complete separation of the alcohols and acids was performed in <15 min on a column packed with 80/100 Chromosorb WAW, having GP 10% SP-1200/1% H₃PO₄ as the liquid phase. The method was suitable for the analysis of rumen fluid and fermentation products from microbial cultures. The detection limits for all compounds were <0.13 nmol · injection⁻¹.     

Klebsiella serotype 25 capsular polysaccharide: primary structure and depolymerization by a bacteriophage-borne glycanase.

By partial acid hydrolysis, methylation and gas-liquid chromatography-mass spectrometry of the methylated monomers (as the alditol acetates), mass spectrometry of trimethylsilylated disaccharide alditols, as well as proton magnetic resonance, the primary structure of the Klebsiella serotype 25 capsular polysaccharide was elucidated. A glycanase activity, associated with the particles of newly isolated Klebsiella bacteriophage no. 25, was shown to catalyze the hydrolysis of the glycan.     

Identification of L-altrose in the extracellular polysaccharide from Butyrivibrio fibrisolvens strain CF3

Abstract Butyrivibrio fibrisolvens strain CF3, a stricyly anaerobic bacterial isolate from the ovine cecum, produces extracellular polysaccharides (EPS) when grown on a defined medium containing glucose as the carbon source. EPS were purified from culture supernatants and their monosaccharide composition was determined by two different procedures. Analysis of EPS hydrolysates by thin-layer chromatography (TLC) yielded spots coincident with standard glucose, altrose, and 1,6-anhydroaltrose. These results were corroborated by both gas-liquid chromatography (GLC) and GLC-mass spectroscopy (GLC-MS) of alditol acetates prepared from EPS hydrolysates. Purification of the altrose from EPS hydrolysates was accomplished by preparative paper and column chromatography. Polarimetry demonstrated the isolated altrose to have the L -configuration. The occurrence of this hexose in nature has not yet been reported.     

A sensitive GLC-method for component sugars andO-glycosidic linkage monosaccharides of cartilage proteoglycans

A method is described for the measurement ofN-acetylgalactosamine,N-acetylglucosamine, galactose, mannose and xylose present in the different carbohydrate chains of cartilage proteoglycans (PG). Bovine articular cartilage PG samples corresponding to the minimum of 1 nmol of each monosaccharide were reproducibly quantified following hydrolysis with 2 M HCl and derivatization into alditol acetates. An on-column injection mode and an OV-1701 fused silica capillary column were used for chromatography.Alkaline borohydride treatment of the PG was exploited to reduce the acid labile xylose in the base of the chondroitin sulphate chain into more stable xylitol, allowing the assay of chondroitin sulfate chain length as anN-acetylgalactosamine/xylose ratio. A novel procedure is described for the measurement of the galactosaminitol evolving from the protein linkage of oligosaccharides and of keratan sulphate.     

The rapid,quantitative determination of neutral sugars (as aldononitrile acetates) and amino sugars (as O-methyloxime acetates) in glycoproteins by gas-liquid chromatography

O-Methyloximes have been prepared from 2-amino-2-deoxy-d-glucose, -d-mannose, and -d-galactose. The acetates of these derivatives yield stable compounds which are readily separated quantitatively by gas-liquid chromatography on a number of polar phases. The above compounds along with the aldononitrile acetates of neutral sugars can be easily separated from one another on a single column in one chromatographic run. The procedures developed were tested on a number of glycoproteins of known composition as reported by other workers utilizing more classical methodologies, resulting in excellent agreement in terms of sugar composition. An improved method is also described for converting neutral sugars to oximes which can be either converted to the trimethylsilyl derivatives or, upon acetylation, derivatized to aldononitrile acetates.     

A comprehensive procedure for preparation of partially methylated alditol acetates from glycoprotein carbohydrates.

Various steps involved in the preparation of partially methylated alditol acetates (PMAAs) from glycoprotein-derived carbohydrates were improved to obtain the derivatives in a rapid manner with excellent yields. Carbohydrates were permethylated in dimethyl sulfoxide (DMSO), using a fine suspension of sodium hydroxide and methyl iodide (CH3I). The fine suspension of NaOH was prepared conveniently from commercially available 50% aqueous NaOH in DMSO by sonication and washing the precipitate with DMSO. Methylation of ovalbumin and fetuin glycopeptides using the fine suspension of NaOH and CH3I was complete within 5 min, and the methylation reaction did not generate any nonsugar artifacts. Methylated carbohydrates without any purification were hydrolyzed in a mixture of volatile organic acids, which permitted rapid removal of the acids from samples by evaporation. Acetylation of partially methylated alditols with acetic anhydride for 2-4 h at ambient temperature using 4-N,N'-dimethylaminopyridine as a catalyst and the reaction was free from generating nonsugar reaction artifacts. The reaction time course for methylation, hydrolysis, and acetylation was determined to obtain optimum reaction conditions for preparation of the PMAAs. The procedure facilitated rapid identification and quantitation of PMAAs due to diminished reaction artifacts and the quality of the chromatogram depended only on the purity of starting material and the reagents used for the methylation analysis. Utility of these simple methods for rapid methylation analysis was demonstrated in the characterization of oligosaccharides isolated in small amounts using a carbohydrate analyzer.     

Hydrolysis of plant polysaccharides and GLC analysis of their constituent neutral sugars

The release and degradation of sugars from onion cell walls and potato cell wall polysaccharides were followed during hydrolysis with trifluoroacetic acid so that the optimum hydrolysis conditions could be determined. After 6 hr hydrolysis in 2 M acid at 100°, calculated recovery factors of different monosaccharides from potato pectic fractions ranged from 61 to 96%. Lower yields of monosaccharides were obtained from intact onion cell walls, while the yield from cellulose was less than 0.2%. A new GLC column for the separation of alditol acetates derived from cell wall sugars is described.     

High-resolution proton nuclear magnetic resonance characterization of seminolipid from bovine spermatozoa

The high-resolution one- and two-dimensional proton nuclear magnetic resonance (1H-NMR) characterization of seminolipid from bovine spermatozoa is presented. The 1H-NMR data was confirmed by gas-liquid chromatography-mass spectrometric analysis of the partially methylated alditol acetates of the sugar unit, mild alkaline methanolysis of the glyceryl ester, mobility on normal phase and diphasic thin-layer chromatography (HPTLC), and fast atom bombardment mass spectrometry (FAB-MS). The structure of the molecule corresponds to 1-O-hexadecyl-2-O-hexadecanoyl-3-O-beta-D-(3'-sulfo)-galactopyranosyl- sn-glycerol.     

Differential gas-liquid chromatography method for determination of uronic acids in carbohydrate mixtures

An integrated gas-liquid chromatography method is described for the quantitation of mixtures containing simple monosaccharides in addition to mannuronic, glucuronic, and/or galacturonic acids. A hydrolyzed sample is divided into two portions. One portion is analyzed by the standard aldononitrile method. Glucuronic, galacturonic, and mannuronic acids are converted into compounds that do not chromatograph in the region of the standard aldononitrile acetates. Thus, this analysis gives an accurate estimation of the neutral monosaccharide content. The second portion is analyzed by a modified alditol acetate procedure. The reduction step is repeated three times to convert mannuronic, galacturonic, and glucuronic acids to their corresponding alditols via their intermediate lactones. The results of this gas-liquid chromatography analysis reflect the sum of the monosaccharides present plus their corresponding uronic acids. The difference between the values obtained by the aldononitrile acetate method and the modified alditol acetate method, therefore, is a measure of the uronic acid(s) present.