“Hypermethylation” of anthranilic acid-labeled sugars confers the selectivity required for liquid chromatography-mass spectrometry

Analysis of the monosaccharides of complex carbohydrates is often performed by liquid chromatography with fluorescence detection. Unfortunately, methylated sugars, unusual amino- or deoxysugars and incomplete hydrolysis can lead to erroneous assignments of peaks. Here, we demonstrate that a volatile buffer system is suitable for the separation of anthranilic acid labeled sugars. It allows off-line examination of peaks by electrospray mass spectrometry. Approaches towards on-line mass spectrometric detection using reversed-phase or porous graphitic carbon columns fell short of achieving sufficient separation of the relevant isobaric sugars. Adequate chromatographic performance for isomeric sugars was achieved with reversed-phase chromatography of “hyper”-methylated anthranilic acid-labeled monosaccharides. Deuteromethyl iodide facilitates the discovery of naturally methylated sugars and identification of their parent monosaccharide as demonstrated with N-glycans of the snail Achatina fulica, where two thirds of the galactoses and a quarter of the mannoses were methylated.     

Determination of 1-phenyl-3-methyl-5-pyrazolone-labeled carbohydrates by liquid chromatography and micellar electrokinetic chromatography

In this paper, the method for the derivatization of carbohydrates with 1-phenyl-3-methyl-5-pyrazolone (PMP) was simplified. One-third of the derivatization time was saved. Five monosaccharide derivatives have been well separated by MEKC and HPLC under optimized conditions. Good reproducibility could be obtained with relative standard deviation (RSD) values of the migration times within 5.0 and 2.3%, respectively. Furthermore, the developed methods have been successfully applied to the analysis of carbohydrates in Aloe powder and food. These methods are quite useful for routine analysis of monosaccharides and oligosaccharides in real samples.     

A comparative study of monosaccharide composition analysis as a carbohydrate test for biopharmaceuticals

The various monosaccharide composition analysis methods were evaluated as monosaccharide test for glycoprotein-based pharmaceuticals. Neutral and amino sugars were released by hydrolysis with 4–7 N trifluoroacetic acid. The monosaccharides were N-acetylated if necessary, and analyzed by high-performance liquid chromatography (HPLC) with fluorometric or UV detection after derivatization with 2-aminopyridine, ethyl 4-aminobenzoate, 2-aminobenzoic acid or 1-phenyl-3-methyl-5-pyrazolone, or high pH anion exchange chromatography with pulsed amperometric detection (HPAEC-PAD). Sialic acids were released by mild acid hydrolysis or sialidase digestion, and analyzed by HPLC with fluorometric detection after derivatization with 1,2-diamino-4,5-methylenedioxybenzene, or HPAEC-PAD. These methods were verified for resolution, linearity, repeatability, and accuracy using a monosaccharide standard solution, a mixture of epoetin alfa and beta, and alteplase as models. It was confirmed that those methods were useful for ensuring the consistency of glycosylation. It is considered essential that the analytical conditions including desalting, selection of internal standards, release of monosaccharides, and gradient time course should be determined carefully to eliminate interference of sample matrix.Various HPLC-based monosaccharide analysis methods were evaluated as a carbohydrate test for glycoprotein pharmaceuticals by an inter-laboratory study.     

Analysis of the monosaccharide composition of purified polysaccharides in Ganoderma atrum by capillary gas chromatography

Introduction – Ganoderma, one of the best‐known traditional Chinese medicines, has attracted considerable attention owing to the fact that dozens of polysaccharides isolated from it have shown diverse and potentially significant pharmacological activities. However, no work has been reported on the analysis of monosaccharide composition of polysaccharide isolated from the aqueous extract of Ganoderma atrum yet. Objective – To develop a simple and sensitive GC‐based method for the analysis of monosaccharide composition of purified polysaccharides in Ganoderma atrum. Methodology – The polysaccharide was first hydrolysed to give the constituent monosaccharides, which were subsequently derived into acetylated aldononitriles and analysed by gas chromatography using a capillary column packed with a (5%phenyl) methylpolysiloxane stationary phase with the addition of acetyl inositol as the inner standard. High‐performance liquid chromatography was also used for comparison. Results – The stable derivatives of the most common monosaccharides could be separated and reproducibly determined with high sensitivity. The limits of detection and quantification were 0.013 and 0.043 mg/mL, respectively. The intermediary precision values (expressed as the RSD) were less than 10%. The mean recovery of the method was 100 ± 3%, with RSD values of less than 5%. The results obtained from GC and HPLC methods were found to be close to each other within acceptable error ranges. Conclusion – This study demonstrated that the developed method could be applied as an accurate method for the compositional analysis of monosaccharides in the field of biological and biochemical study. Copyright © 2009 John Wiley & Sons, Ltd.     

Determination of carbohydrates as their p-sulfophenylhydrazones by capillary zone electrophoresis.

p-Hydrazinobenzenesulfonic acid was explored as an ultraviolet labeling reagent for capillary electrophoresis of mono-, di- and trisaccharides. The labeling reaction that produces p-sulfophenylhydrazines took less than 8 min, and introduced both chromphore and charged groups into the carbohydrate molecules. The derivatives of nine mono- and disaccharides were completely separated in 9 min using a 100 mM borate buffer at pH 10.24. On-column UV detection at 200 nm allowed the detection of glucose with a mass detection limit of 17.6 fmol or a concentration limit of 3.6 microM. Reproducible quantification of carbohydrates was achieved in the concentration range of 0.1-9.1 mM in reaction solution. The method was applied successfully to determine the monosaccharide composition of laminaran.     

Gas-liquid chromatographic analysis of the monosaccharide composition of acid glycosaminoglycans (mucopolysaccharides) derived from animal tissues

Quantitative, gas-liquid chromatography was investigated for analysis of the monosaccharide composition of acid mucopolysaccharides from animal tissues. The method entailed the analysis of the trimethylsillyl (Me3Si) derivatives of methyl glycosides on two liquid phases. Good resolution of monosaccharides was achieved by use of columns of SE-30 and Apiezon-M. The procedure was tested with chondroitin 4-sulfate, and the results were slightly different from those of Mathews et al. When the analysis is performed according to this method, important points are: (1) absolutely anhydrous, methanolic hydrogen chloride is necessary, to ensure detection of hexosamines and sialic acid; and (2) high moisture in the air obstructs high recovery of methyl glycosides and their Me3Si derivatives, except in the case of neutral sugars.     

Application of high-performance capillary electrophoresis to analysis of carbohydrates, especially those in glycoproteins

Methods for the component monosaccharide analysis and oligosaccharide mapping for glycoprotein research, based on HPCE of reductively pyridylaminated (PA) derivatives, are described. the component monosaccharides released from glycoproteins by acid hydrolysis are converted to PA derivatives and analyzed by HPCE as borate complexes. They can be quantified in the picomole range (introduced amount) with high reproducibility. The oligosaccharides released by hydrazinolysis are similarly converted to PA derivatives. Two-dimensional mapping of the relative mobilities of these derivatives, obtained in an acidic phosphate buffer and an alkaline borate buffer, ensures reliable identification of the oligosaccharides.     

Sugar analysis of glycoproteins and glycolipids after methanolysis by high-performance liquid chromatography with pulsed amperometric detection

A procedure for the analysis of the monosaccharide composition of glycoproteins and glycolipids by methanolysis and high-performance liquid chromatography with pulsed amperometric detection is described. The advantage over previous methods is the analysis of underivatized methyl glycosides of all glycoconjugate monosaccharides including sialic acid and uronic acid in a single chromatographic step at the subnanomolar level.     

Determination of iduronic acid and glucuronic acid in glycosaminoglycans after stoichiometric reduction and depolymerization using high-performance liquid chromatography and ultraviolet detection

The reduction of uronic acids in glycosaminoglycans (GAGs) prior to depolymerization reactions is one way in which the uronic acid content of polysaccharides can be studied without major losses. The obtained monosaccharides can be recovered from the subsequent depolymerization with a yield better than 95%. Following reduction, depolymerization, and lyophilization, D-glucuronic acid is converted to D-Glc and L-iduronic acid to 1,6-anhydro-idose. Per-O-benzoyl derivatives of these monosaccharides can be separated and detected in nanogram amounts using reversed phase HPLC. A linear detector response was obtained for injections up to 22 nmol (4 micrograms) of Glc and 1,6-anhydro-idose and the detection limit was 5 and 7 pmol, respectively. Reduction, depolymerization, and derivatization with subsequent chromatography of various GAGs can be readily performed in the 1- to 30-micrograms range.     

Acidolysis-based component mapping of glycosaminoglycans by reversed-phase high-performance liquid chromatography with off-line electrospray ionization–tandem mass spectrometry: Evidence and tags to distinguish different glycosaminoglycans

Diverse monosaccharide analysis methods have been established for a long time, but few methods are available for a complete monosaccharide analysis of glycosaminoglycans (GAGs) and certain acidolysis-resistant components derived from GAGs. In this report, a reversed-phase high-performance liquid chromatography (RP–HPLC) method with pre-column 1-phenyl-3-methyl-5-pyrazolone (PMP) derivatization was established for a complete monosaccharide analysis of GAGs. Good separation of glucosamine/mannosamine (GlcN/ManN) and glucuronic acid/iduronic acid (GlcA/IdoA) was achieved. This method can also be applied to analyze the acidolysis-resistant disaccharides derived from GAGs, and the sequences of these disaccharides were confirmed by electrospray ionization–collision-induced dissociation–tandem mass spectrometry (ESI–CID–MS/MS). These unique disaccharides could be used as markers to distinguish heparin/heparan sulfate (HP/HS), chondroitin sulfate/dermatan sulfate (CS/DS), and hyaluronic acid (HA).     

Quantitative gas chromatography/mass spectrometry determination of C-mannosylation of tryptophan residues in glycoproteins

C-mannosylation of Trp residue is one of the most recently discovered types of glycosylation, but the identification of these mannosylated residues in proteins is rather tedious. In a previous paper, it was reported that the complete analysis of all constituents of glycoproteins (sialic acids, monosaccharides, and amino acids) could be determined on the same sample in three different steps of gas chromatography/mass spectrometry of heptafluorobutyrate derivatives. It was observed that during the acid-catalyzed methanolysis step used for liberation of monosaccharide from classical O- and N-glycans, Trp and His were quantitatively transformed by the addition of a methanol molecule on their indole and imidazole groups, respectively. These derivatives were stable to acid hydrolysis used for the liberation of amino acids. Since monosaccharide derivatives were also stabilized as heptafluorobutyrate derivatives of O-methyl-glycosides, it was suggested that C-mannosides of Trp residues could quantitatively be recovered. Based on the analyses of standard compounds, peptides and RNase 2 from human urine, we report that C((2))-mannosylated Trp could be quantitatively recovered and identified during the step of amino acid analysis. Analyses of different samples indicated that this type of glycosylation is absent in bacteria and yeasts.     

Quantitative analysis of sugar constituents of glycoproteins by capillary electrophoresis

A method for quantitative analysis of monosaccharides including N- acetylneuraminic acid derived from sialic acid-containing oligosaccharides and glycoproteins is presented. The analysis is based on the combination of chemical and enzymatic methods coupled with capillary electrophoretic (CE) separation and laser-induced fluorescence (LIF) detection. The present method utilizes a simplified acid hydrolysis procedure consisting of mild hydrolysis (0.1 M TFA) to release sialic acid and strong acid hydrolysis (2.0 N TFA) to produce amino and neutral sugars. Amino sugars released from strong acid hydrolysis of oligosaccharides and glycoproteins were reacetylated and derivatized with 8-aminopyrene-1,3,6-trisulfonate (APTS) along with neutral sugars in the presence of sodium cyanoborohydride to yield quantitatively the highly stable fluorescent APTS adducts. N- acetylneuraminic acid (Neu5Ac), a major component of most mammalian glycoproteins, was converted in a fast specific reaction by the action of neuraminic acid aldolase (N-acylneuraminate pyruvate-lyase EC 4.1.3.3) to N-acetylmannosamine (ManNAc) and pyruvate. ManNAc was then derivatized with APTS in the same manner as the other monosaccharides. This method was demonstrated for the quantitation of pure Neu5Ac and the species derived from mild acid hydrolysis of 6'-sialyl-N- acetyllactosamine and bovine fetuin glycan. Quantitative recovery of the N-acetylmannosamine was obtained from a known amount of Neu5Ac in a mixture of seven other monosaccharides or from the sialylated oligosaccharides occurring in glycoproteins. The sequence of procedures consists of acid hydrolysis, enzymatic conversion and APTS derivatization which produced quantitative recovery of APTS- monosaccharide adducts. The detection limits for sugars derivatized with APTS and detected by CE-LIF are 100 pmol for Neu5Ac and 50 pmol for the other sugars.      

Neutral, acidic, and basic derivatives of anthranilamide that confer different formal charge to reducing oligosaccharides

To facilitate the use of oligosaccharides as analytical tools in biological studies, we have designed, synthesized, and conjugated to maltosaccharides a novel series of homologous small fluorescent moieties that differ in formal charge. These moieties are amide derivatives of anthranilic acid: uncharged N-(2-aminobenzoyl)glycinamide (ABGlyAmide; 2), acidic N,N-dimethyl-N(')-(2-aminobenzoyl)ethylenediamine (ABGlyDIMED; 3), and basic N-(2-aminobenzoyl)glycine (ABGly; 1). Routes for synthesis and optimal reaction conditions for glycoconjugation by conventional reductive amination are presented, as is the compatibility of these adducts with common analytical and preparative chromatographic methods, including RP-HPLC and HPAEC-PAD. These novel anthranilic acid derivatives confer both fluorescence and defined charge to oligosaccharides, and so enhance the repertoire of chromatographic and analytical methods for which anthranilic acid can be used. Furthermore, because glucosaccharides have rigid solution structure, these small fluorescent adducts with different formal charge are ideal tools for molecular sizing studies of membrane pores.     

Quantification of glycosaminoglycans by reversed-phase HPLC separation of fluorescent isoindole derivatives

Glycosaminoglycans (GAGs) are linear polysaccharides made by all animal cells. GAGs bind to hundreds of proteins, such as growth factors, cytokines, chemokines, extracellular matrix components, protease inhibitors, proteases, and lipoprotein lipase, through carbohydrate and protein interactions. These interactions control many multicellular processes. The increased use of GAGs isolated from cells and small tissue samples in bioassays and binding experiments demands a sensitive and robust quantification method. We have developed such a method, which is based on a popular assay for amino acid analysis. We have refined it to enhance GAG quantification. It allows the quantification of glucosamine- and galactosamine-containing GAGs after the reversed-phase separation of their fluorescent isoindole derivatives. The derivatives are created by the reaction of o-phthaldialdehyde and 3-mercaptopropionic acid (3MPA) with the amino group of hexosaminitol monosaccharides generated from GAG acid hydrolysis and sodium borohydride reduction. The advantages of our method include automatic derivitization, a simple chromatograph with clean separation of glucosaminitol and galactosaminitol derivatives from contaminating amino acids, excellent sensitivity with 0.04 pmol detection, and linearity from 2.5 to 1280 pmol. A major advantage is that it can be readily implemented in any laboratory with typical reversed-phase high performance liquid chromatography (HPLC) equipment.     

Gas-liquid chromatography of the heptafluorobutyrate derivatives of the O-methyl-glycosides on capillary columns: a method for the quantitative determination of the monosaccharide composition of glycoproteins and glycolipids

We have developed a method involving the formation of hepta-fluorobutyrate derivatives of O-methyl-glycosides liberated from glycoproteins and glycolipids following methanolysis. The stable derivatives of the most common monosaccharides of these glycoconjugates (Ara, Rha, Xyl, Fuc, Gal, Man, Glc, GlcNAc, GalNAc, Neu5Ac, KDN) can be separated and quantitatively and reproducibly determined with a high degree of sensitivity level (down to 25 pmol) in the presence of lysine as an internal standard. The GlcNAc residue bound to Asn in N-glycans is quantitatively recovered as two peaks. The latter were easily distinguished from the other GlcNAc residues of N-glycans, thus allowing a considerable improvement of the data on structure of N-glycans obtained from a single carbohydrate analysis. The most common contaminants present in buffers commonly used for the isolation of soluble or membrane-bound glycoproteins (SDS, Triton X-100, DOC, TRIS, glycine, and polyacrylamide or salts, as well as monosaccharide constituents of proteoglycans or degradation products of nucleic acids) do not interfere with these determinations. A carbohydrate analysis of glycoproteins isolated from a SDS/PAGE gel or from PDVF membranes can be performed on microgram amounts without significant interferences. Since fatty acid methyl esters and sphingosine derivatives are separated from the monosaccharide peaks, the complete composition of gangliosides can be achieved in a single step starting from less than 1 microg of the initial compound purified by preparative Silicagel TLC. Using electron impact ionization mass spectrometry, reporter ions for the different classes of O-methyl-glycosides (pentoses, deoxy-hexoses, hexoses, hexosamines, uronic acids, sialic acid, and KDN) allow the identification of these compounds in very complex mixtures. The mass of each compound can be determined in the chemical ionization mode and detection of positive or negative ions. This method presents a considerable improvement compared to those using TMS derivatives. Indeed the heptafluorobutyrate derivatives are stable, and acylation of amino groups is complete. Moreover, there is no interference with contaminants and the separation between fatty acid methyl-esters and O-methyl glycosides is achieved.     

Electrochemically amplified detection for lipopolysaccharide using ferrocenylboronic acid

A novel electrochemical technique for lipopolysaccharide (LPS) detection has been developed using a combination of ferrocenylboronic acid derivatives and an enzyme-modified electrode. The enzyme-modified electrode was constructed from a gold electrode modified with a bovine serum albumin membrane containing diaphorase. Ferrocenylboronic acid derivatives are oxidized on the electrode, and then regenerated by a diaphorase-catalyzed reaction in the presence of NADH. The consumption/regeneration cycle for ferrocenylboronic acid derivatives resulted in a chemically amplified current response. The current response for ferrocenylboronic acid derivatives decreased in association with its complexation with glycosyl units of LPS, and this current decrease caused by LPS was also amplified by the recycling process. On the other hand, the addition of a monosaccharide such as D-mannose or D-galactose induced no response at the same LPS concentration. The enzyme membrane immobilized on the electrode plays an important role in selectivity as well as chemical amplification. In addition, the enzyme-modified electrode exhibited a rapid response of 5 min for LPS, which is much faster than the currently used method. The detection limit of LPS from Escherichia coli O127:B8 was as low as 50 ng ml-1.     

Sugar-mediated induction of Agrobacterium tumefaciens virulence genes: structural specificity and activities of monosaccharides.

The virulence genes of Agrobacterium tumefaciens are induced by specific plant phenolic metabolites and sugars (G. A. Cangelosi, R. G. Ankenbauer, and E. W. Nester, Proc. Natl. Acad. Sci. USA, in press). In this report, monosaccharides, derivatives, and analogs which induce the vir regulon have been identified and the structural requirements for monosaccharide-mediated induction have been determined. Pyranose sugars with equatorial hydroxyls at C-1, C-2, and C-3 displayed strong vir gene-inducing activity; the C-4 hydroxyl could be epimeric and a wide variety of substitutions at C-5 were permissible. The acidic monosaccharide derivatives D-galacturonic acid and D-glucuronic acid were the strongest inducers among the monosaccharides tested. Eight of the 11 inducing compounds are known plant metabolites, and 7 are monomers of major plant cell wall polysaccharides. A role for monosaccharides and plant phenolic compounds as wound-specific plant metabolites which signal the ChvE/VirA/VirG regulatory system is proposed.     

Sequential GC/MS analysis of sialic acids,monosaccharides, and amino acids of glycoproteins on a single sample as heptafluorobutyrate derivatives

A GC/MS procedure was developed for the analysis of all major constituents of glycoproteins. The rationale for this approach is that by using GC/MS analysis of the constituents as heptafluorobutyrate derivatives, it was possible to quantitatively determine the sialic acid, monosaccharide, fatty acids (when present), and the amino acid composition with the sample remaining in the same reaction vessel during the entire procedure. A mild acid hydrolysis was used to liberate sialic acids and was followed by formation of methyl-esters of heptafluorobutyrate (HFB) derivatives. After GC/MS analysis of sialic acids, the remaining material was submitted to acid-catalyzed methanolysis followed by the formation of HFB derivatives. After GC/MS analysis of the monosaccharides, the sample was supplemented with norleucine (as internal standard) and hydrolyzed with 6 M HCl followed by the formation of isoamyl-esters of HFB derivatives and GC/MS analysis. His and Trp residues were modified during the step of acid-catalyzed methanolysis, but the resulting derivatives were stable during acid hydrolysis and quantitatively recovered by GC/MS analysis. As a result, all constituents of glycoproteins (sialic acids, monosaccharides (or di- and trisaccharides) and amino acids) are identified in the electron impact mode of ionization and quantified using three GC/MS analysis in the same chromatographic conditions and using a limited number of reagents, a considerable advantage over previous techniques. This method is very sensitive, all data (qualitative and quantitative) being obtained at the sub-nanomolar level of initial material.     

CarboPac PA20: a new monosaccharide separator column with electrochemical detection with disposable gold electrodes

This report documents the development of a new monosaccharide separator column (CarboPac PA20, 3×150 mm) that allows fast, efficient monosaccharide separations with good spacing. It is based on a new chemistry with a reduced resin particle size (from 10 to 6.5 μm). Faster, more efficient separations of glycoprotein monosaccharides with better spacing were achieved across a range of isocratic NaOH concentrations at lower flow rates. Detection sensitivity was improved, enabling routine low to sub pmol monosaccharide determinations. Glycoprotein monosaccharides eluted in less than 10 min at a flow rate of 0.5 ml/min. Furthermore, when used with an AminoTrap guard column, the protein matrix consisting of amino acids and peptides (released by acid hydrolysis of glycoprotein) did not interfere with monosaccharide analysis. Compared to previous CarboPac columns (CarboPac PA1 and CarboPac PA10), the CarboPac PA20 has improved selectivity with respect to glycoprotein monosaccharides. The improved selectivity results in better separation of glucosamine and galactose, enabling the accurate determination of monosaccharide ratios for undergalactosylated glycoproteins. Finally, disposable gold working electrodes that eliminate the possibility of working electrode recession affecting peak area response were used.     

高效液相色谱法测定金钱菇多糖的单糖组成

采用高效液相色谱法,测定金钱菇多糖的单糖组成.用超声辅助提取金钱菇多糖,通过1-苯基-3-甲基-5-吡唑酮(PMP)衍生水解后的单糖,高效液相色谱法检测衍生物.结果表明：金钱菇多糖由甘露糖(Man)、核糖(Rib)、鼠李糖(Rha)、葡萄糖(Glc)、半乳糖(Gal)、木糖(Xyl)组成,其摩尔为1．00∶0．90∶0．91∶28．03∶1．58∶0．11.该方法快速、简便、重现性好,可用于测定金钱菇多糖的单糖组成.