Structural characterization of oligosaccharides by high-performance liquid chromatography, fast-atom bombardment-mass spectrometry, and exoglycosidase digestion

A method for structural characterization of oligosaccharides after preparing uv-absorbing derivatives is described. The derivatives can be rapidly analyzed and purified by high-performance liquid chromatography, with separation of various structures determined primarily by size and sugar composition. Derivatization requires as little as 0.5-1.0 nmol of oligosaccharide, and detection of down to 50 pmol of oligosaccharide is possible by monitoring absorbance at 229 nm. In addition, the carbohydrate portion of the derivative was found to retain its sensitivity to exoglycosidases, allowing sequential enzymatic digestions for determination of sugar sequence and anomerity to be performed. The derivatives also possessed a site of potential positive charge, making them amenable to analysis by fast-atom bombardment-mass spectrometry. Permethylation of the derivatives permitted their separation by capillary gas chromatography, thus allowing investigation of their structures by gas chromatography-mass spectrometry. The combination of these techniques will allow almost the complete structure of small amounts of oligosaccharides to be determined.     

Determination of plasma and urinary cortisol by high-performance liquid chromatography using fluorescence derivatization with dansyl hydrazine

A method is described for the determination of cortisol in human plasma and urine by high-performance liquid chromatography using fluorophotometric detection. After extraction with methylene chloride, cortisol is labelled with dansyl hydrazine, and then separated by high-performance chromatography. The eluate is monitored by a fluorophotometer at 350 nm (excitation) and 505 nm (emission). The optimum conditions for the determination, such as HCl and dansyl hydrazine concentrations, reaction time and reaction temperature, and for the eluent of high-performance liquid chromatography, are discussed. Linearity of the fluorescence intensity (peak height) with the amount of cortisol was obtained between 0.5 and 60 ng. The recoveries for 50 and 100 ng of added cortisol were 98.7 and 95.4% for plasma, and 96.4 and 90.6% for urine, respectively. Comparison with a radioimmunoassay gave a correlation coefficient of 0.978. The proposed method is suitable for the routine analysis of cortisol in plasma and urine.     

Separation of neutral oligosaccharides by high-performance liquid chromatography

We have developed a method for separating reduced, neutral oligosaccharides by high-performance liquid chromatography on columns of MicroPak AX-5 (Varian Associates) with a mobile phase consisting of acetonitrile:H₂O. Individual glucose oligomers containing from 1 to 20 glucose moieties can be separated in a single 1-h analysis with a solvent program of decreasing acetonitrile concentration. We have applied this method to both the analysis and preparative isolation of glycoprotein-derived oligosaccharides obtained by enzymatic release with endoglycosidases or chemical release by hydrazinolysis. Introduction of ³H by reduction with NaB³H₄ permits the detection of subnanomole quantities of oligosaccharides. This method offers previously unattainable rapidity and resolution for the analysis of oligosaccharides.     

Analysis of tissue glycoprotein sugar chains by two-dimensional high-performance liquid chromatographic mapping

Excellent separation of 45 pyridylamino derivatives of oligosaccharides were achieved by the two-dimensional combination of reversed-phase and size-fractionation high-performance liquid chromatography. The sugar chains of brain glycoproteins were derivatized into a mixture of pyridylamino-oligosaccharides from lyophilized brain tissue without any purification steps, and they were well separated by the system used. The pattern obtained was reproducible, and inter-individual variation was negligible. This finding demonstrated the possibility that this method could be applied to the detection of differences in the structure of glycoprotein sugar chains in crude preparations.     

Validation of a reversed-phase HPLC method for quantitative amino acid analysis.

A semi-automated method for amino acid derivatization and analysis has been validated for use in analysis of protein biopharmaceuticals. The method includes protein hydrolysis, o-phthalaldehyde derivatization, and reversed-phase high-performance liquid chromatography analysis in a general-purpose UV-visible high-performance liquid chromatography system. Amino-acid derivatization is performed automatically by the high-performance liquid chromatography autosampler right before injection. The required validation parameters, i.e., specificity, linearity, accuracy, precision, limit of detection, and limit of quantification, were studied for bovine serum albumin and for a recombinant human Fab fragment. The method can be employed as an absolute quantification method for determination of extinction coefficients of recombinant proteins.     

Precolumn labeling of reducing carbohydrates with 1-(p-methoxy)phenyl-3-methyl-5-pyrazolone: analysis of neutral and sialic acid-containing oligosaccharides found in glycoproteins.

A convenient precolumn labeling method was developed for the analysis of neutral and sialic acid-containing oligosaccharides in glycoproteins using 1-(p-methoxy)phenyl-3-methyl-5-pyrazolone (PMPMP). PMPMP reacts with a reducing oligosaccharide under slightly alkaline conditions (pH 8.3) to form a 2:1 adduct (bis-PMPMP derivative). Sialic acid residues in the oligosaccharides remain intact during the reaction. Tryptic glycopeptides digested with glycopeptidase A for oligosaccharide liberation can be directly derivatized with PMPMP without prior treatment. Separation of the labeled oligosaccharides was performed by reverse-phase high-performance liquid chromatography on a C-18 column with aqueous acetonitrile, and positional isomers such as isomeric triantennary tetradecasaccharides from bovine fetuin were completely resolved. The bis-PMPMP derivatives were labile in alkaline media to form mono-PMPMP derivatives; however, the mono-PMPMP derivatives could be easily reconverted to the original bis-PMPMP derivatives. The proposed method is simpler than the reductive pyridylamination method, and detection sensitivity could reach subnanomole range with a uv detector. Oligosaccharides from ribonuclease B (bovine pancreas), ovalbumin, thyroglobulin (porcine thyroid), fetuin (bovine), and transferrin (human) have been successfully analyzed to demonstrate the usefulness of this method as an alternative to the existing methods.     

Determination of 3-methoxy-4-hydroxyphenylglycol in urine by high-performance liquid chromatography with amperometric detection

A reversed-phase high-performance liquid chromatographic method has been used for the quantitative determination of 3-methoxy-4-hydroxyphenylglycol (MHPG) in urine. After incubation with glusulase, free MHPG is extracted into ethyl acetate and further isolated by a combination of thin-layer chromatography (TLC) and high-performance liquid chromatography (HPLC). The addition of amperometric detection provides increased sensitivity to a highly specific assay.     

Simultaneous determination of guanine nucleotides in rat brain by high-performance liquid chromatography with dual-electrochemical detection

A new, sensitive, and specific assay method for guanine nucleotides using high-performance liquid chromatography with dual-electrochemical detection was developed. GTP, GDP, GMP, and cyclic GMP were separated with reversed-phase "ion-pair" chromatography and detected by a dual-electrochemical detector. Only guanine nucleotides among all purine and pyrimidine nucleotides responded to the electrochemical detector at 0.95 V. The peak heights for these guanine nucleotides were linear at concentrations between 0.5 pmol and 1 nmol. The regional distribution of these guanine nucleotides in the rat brain was studied by this new assay method.     

Simultaneous quantification of sialyloligosaccharides from human milk by capillary electrophoresis

The acidic oligosaccharides of human milk are predominantly sialyloligosaccharides. Pathogens that bind sialic acid-containing glycans on their host mucosal surfaces may be inhibited by human milk sialyloligosaccharides, but testing this hypothesis requires their reliable quantification in milk. Sialyloligosaccharides have been quantified by anion exchange high-performance liquid chromatography (HPLC), reverse- or normal-phase HPLC, and capillary electrophoresis (CE) of fluorescent derivatives; in milk, these oligosaccharides have been analyzed by high pH anion exchange chromatography with pulsed amperometric detection and, in our laboratory, by CE with detection at 205nm. The novel method described here uses a running buffer of aqueous 200mM NaH2PO4 (pH 7.05) containing 100mM sodium dodecyl sulfate (SDS) mixed with 45% (v/v) methanol to baseline resolve 5 oligosaccharides and separate all 12. This allows automated simultaneous quantification of the 12 major sialyloligosaccharides of human milk in a single 35-min run. This method revealed differences in sialyloligosaccharide concentrations between less and more mature milk from the same donors. Individual donors also varied in expression of sialyloligosaccharides in their milk. Thus, the facile quantification of sialyloligosaccharides by this method is suitable for measuring variation in expression of specific sialyloligosaccharides in milk and their relationship to decreased risk of specific diseases in infants.     

Applications of rapid-scanning multichannel detectors in chromatography : Plenary lecture

A review of progress in the field of multichannel detection in column and thin-layer chromatography is presented, together with some novel applications of a computer-based, linear photodiode array UV—visible spectrophotometer for detection in high-performance liquid chromatography (HPLC). Computer-aided methods for simultaneous monitoring of the elution profile at three wavelengths with automatic peak detection and capture of UV spectra are described. The continuous calculation of absorbance ratios during elution is discussed as an index of peak homogeneity. A novel technique for the enhancement of qualitative identification in HPLC, based on transformation of captured spectra to the second derivative or to the decadic logarithm, is proposed. These developments are exemplified by a model system of diacetylmorphine and its principal metabolites and degradation products, morphine and 6-acetylmorphine. The potential utility of three-dimensional projections of (A,ν,t) data is discussed in the context of pharmaceutical, bioanalytical and forensic applications.     

Structural analyses of asparagine-linked oligosaccharides of porcine pancreatic kallikrein

The structures of asparagine-linked oligosaccharides of porcine pancreatic beta-kallikrein are reported. Asparagine-linked neutral oligosaccharides were released by N-oligosaccharide glycopeptidase digestion, and the reducing ends of the oligosaccharides were derivatized with a fluorescent reagent, 2-aminopyridine. The mixture of pyridylamino oligosaccharides was separated by reverse-phase and amide-adsorption high-performance liquid chromatography. The pyridylamino oligosaccharides were separated into more than 50 kinds of oligosaccharides. The structures of 5 kinds of triantennary and 12 kinds of tetraantennary oligosaccharides were determined by the use of high-resolution proton nuclear magnetic resonance spectroscopy and methylation analysis. Furthermore, the structures of five kinds of oligomannose-type oligosaccharides were elucidated by a combination of exoglycosidase digestion and high-performance liquid chromatography. 1H NMR data for 14 out of the 17 kinds of N-acetyllactosamine-type oligosaccharides reported here have not previously been described in the literature. (1) It has been shown that fucose containing tri- and tetraantennary oligosaccharides is predominant in porcine pancreatic beta-kallikrein B. (2) It has also been shown that the heterogeneity of the structure in these types of oligosaccharides is derived from the variety of the positions of galactose residues linked to outer N-acetylglucosamine residues. (3) The distribution of oligosaccharides into two glycosylation sites, asparagine-95 and asparagine-239, of beta-kallikrein B was determined. It has been found that oligomannose-type oligosaccharides are exclusively present at asparagine-239, although N-acetyllactosamine-type oligosaccharides occur at both glycosylation sites.(ABSTRACT TRUNCATED AT 250 WORDS)     

Structural analysis of N-linked oligosaccharides by a combination of glycopeptidase, exoglycosidases, and high-performance liquid chromatography

A simple, sensitive, and rapid method for the analysis of structures of N-linked carbohydrates is reported. The method involves four steps: preparation of carbohydrate chains from glycopeptides by N-oligosaccharide glycopeptidase digestion; derivatization of the reducing ends of carbohydrate chains with a fluorescent reagent, 2-aminopyridine, by using sodium cyanoborohydride; separation of oligosaccharide derivatives by reverse-phase high-performance liquid chromatography; and structural analysis of oligosaccharides by sequential exoglycosidase digestion. The elution positions of 50 standard oligosaccharide derivatives were determined by HPLC. The structure of an unknown oligosaccharide can be characterized by comparison of its elution position with those of the standard compounds. The method was applied to elucidate the structures of oligosaccharides in the myeloma IgG protein, Yot.     

Analysis of the biogenic amines in the central nervous system of the tobacco hornworm by high-performance liquid chromatography with 16-sensor electrochemical detection.

A method was developed to analyze biogenic amines in extracts of the central nervous system of the tobacco hornworm, Manduca sexta by high-performance liquid chromatography with 16-sensor electrochemical detection (n-EC-HPLC). The amines, precursors, and metabolites were separated in two dimensions. The first dimension involves separation based upon retention time by reversed-phase HPLC, while the second dimension involves separation based upon the characteristic oxidation potentials achieved by n-EC. Biogenic amine identification was based upon maximum oxidation potential and peak height ratios in addition to retention time. The improved resolving power of this method allows for a simplified sample preparation procedure and simultaneous determination of a wide range of compounds, including phenylethylamine, catecholamines, indoleamines, and some of their precursors and metabolites.     

High-performance liquid chromatographic determination of urinary catecholamines by pre-column solid-phase dansylation on alumina

Sensitive and selective high-performance liquid chromatographic determination of catecholamines by pre-column solid-phase dansylation is described. After catecholamines are adsorbed on alumina, the amino groups not responsible for adsorption are dansylated by a solid-phase reaction. The excess reagent and fluorescent contaminants are washed out, and the dansylated catecholamines are eluted and separated by reversed-phase high-performance liquid chromatography. The four catecholamine derivatives can be separated within 10 min and no major interfering peak is observed on chromatograms. The response of each catecholamine is linear from 10 to 500 pmol per sample and the detection limit is 0.5 pmol. This method was applied to determination of catecholamines in human urine.     

Simultaneous determination of chlorpromazine and levomepromazine in human plasma and urine by high-performance liquid chromatography using electrochemical detection

A rapid, selective and sensitive method for the simultaneous determination of chlorpromazine and levomepromazine in human plasma and urine has been developed using high-performance liquid chromatography with electrochemical detection.The unchanged drugs and internal standard extracted from plasma and urine were separated by reversed-phase high-performance liquid chromatography. The influence of acetonitrile concentration and of the pH of the mobile phase were investigated. The detection limits were 100 pg for chlorpromazine and for levomepromazine. In comparison with three other detection systems this was found to be the most sensitive method.This method was successfully applied to the simultaneous determination of chlorpromazine and levomepromazine in human plasma and urine for pharmacokinetic studies.     

Rapid separation of oligomers of polysialic acid by high-performance liquid chromatography

A rapid procedure utilizing high-performance liquid chromatography was developed for the separation of homooligomers of sialic acid (N-acetylneuraminic acid). The method utilizes the anion exchanger Mono-Q HR 5/5 and can resolve sialyl oligomers with degrees of polymerization (DP) from 2 to 20 in 25 min. Previous methods required 1 to 9 days. Recoveries are quantitative and the method can be used either analytically to analyze the enzymatic digestion products of polysialic acid or semipreparatively to prepare sialyl oligomers of defined length. The method is potentially useful for analyzing other anionic oligosaccharides.     

Preparation and structure of heparin lyase-derived heparan sulfate oligosaccharides

Porcine intestinal mucosal heparan sulfate was exhaustivelydepolymerized on a large scale using beparin lyase II (heparinaseII) or heparin lyase III (heparitinase, EC 4.2.2.8 [EC] ). The oligosaccharidemixtures formed with each enzyme were fractionated by low pressuregel permeation chromatography. Size-uniform mixtures of disaccharides,tetrasaccharides, and hexasaccharides were obtained. Each size-fractionatedmixture was then purified on the basis of charge by repetitivesemipreparative strong-anion-exchange high-performance liquidchromatography. This approach has led to the isolation of 13homogenous oligosaccharides. The purity of each oligosaccharidewas demonstrated by the presence of a single peak on analyticalstrong-anion-exchange high-performance liquid chromatographyand reversed polarity capillary electrophoresis. The structuresof these oligosaccharides were established using 500 MHz one-and two-dimensional nuclear magnetic resonance spectroscopy.Three of the thirteen structures that were solved were novelwhile the remaining 10 have been previously described. All ofthe structures obtained using heparin lyase III contained a     

Size fractionation of anionic oligosaccharides and glycopeptides by high-performance liquid chromatography

A rapid method for the fractionation of anionic oligosaccharide and glycopeptide species on the basis of net carbohydrate content utilizing high-performance liquid chromatography has been developed. Amine-bearing bonded-phase columns are eluted with a mobile phase consisting of a water:acetonitrile gradient containing 3% acetic acid titrated to pH 5.5 with triethylamine. Phosphorylated and sialylated oligosaccharides within various charge classes differing in their hexose or hexosamine contents but bearing the same number of anionic species can be resolved without prior removal of the anionic moieties. Glycopeptides containing at least as many as six amino acids are also well fractionated on the basis of carbohydrate content. A variety of detection methods may be used and sensitivity in the subnanomole range is possible with fluorescent or radiolabeling techniques. This method offers a significant improvement in the rapidity and resolution attainable for the size fractionation of anionic complex carbohydrates.     

Analysis of N-glycosylation of phospholipase A2 from venom of individual bees by microbore high-performance liquid chromatography-electrospray mass spectrometry using an ion trap mass spectrometer

The N-linked oligosaccharides were released from the phospholipase A2 (PLA) with glycopeptidases and reductively aminated with the chromophore, p-aminobenzoic acid ethyl ester (ABEE). The ABEE-labeled oligosaccharides were separated by microbore high-performance liquid chromatography (micro-HPLC) using a reversed-phase column and analyzed by electrospray mass spectrometry. Differentiation between alpha-1,3 and alpha-1,6 core-fucosylated glycans was achieved by comparison the glycans released by glycopeptidases peptide-N-glycanase A (PNase A) and peptide-N-glycanase F (PNase F). All N-linked oligosaccharides except 3B and 3C could be identified in this approach. The analysis of PLA oligosaccharides from the venom of individual bees indicated that glycosylation patterns between the younger and the older bees were similar.     

Analysis of asparagine-linked oligosaccharides from plasma membranes of rat normal liver and ascites hepatoma cells

Isolated plasma membranes from rat liver and ascites hepatoma cells were shown by SDS-polyacrylamide gel electrophoresis and concanavalin A reactivity to contain a variety of glycoproteins having asparagine-linked oligosaccharides. Membrane oligosaccharides were released by almond glycopeptidase digestion, and the pyridylamino derivatives were analyzed by high-performance liquid chromatography. Forty-four percent of the total carbohydrates in the original membranes were released and suggested to be of the complex type. Hepatoma membranes showed different oligosaccharide patterns from normal.