heparitin-sulfate and formic-acid

The analysis of heparan sulfate disaccharides poses a real challenge both from chromatographic and mass spectrometric point of view. This necessitates the constant improvement of their analytical methodology. In the present study, the chromatographic effects of solvent composition, salt concentration, and salt type were systematically investigated in isocratic HILIC-WAX separations of heparan sulfate disaccharides. The combined use of 75% acetonitrile with ammonium formate had overall benefits regarding intensity, detection limits, and peak shape for all salt concentrations investigated. Results obtained with the isocratic measurements suggested the potential use of a salt gradient method in order to maximize separation efficiency. A 3-step gradient from 14 mM to 65 mM ammonium formate concentration proved to be ideal for separation and quantitation. The LOD of the resulting method was 0.8-1.5 fmol for the individual disaccharides and the LOQ was between 2.5-5 fmol. Outstanding linearity could be observed up to 2 pmol. This novel combination provided sufficient sensitivity for disaccharide analysis, which was demonstrated by the analysis of heparan sulfate samples from porcine and bovine origin.

Topics: Animals; Cattle; Chromatography, Liquid; Disaccharides; Formates; Heparitin Sulfate; Hydrophobic and Hydrophilic Interactions; Limit of Detection; Linear Models; Sodium Chloride; Solvents; Swine

In the structural analysis of heparin and heparan sulfate, it is customary to combine or pool like-sized fractions obtained by size-exclusion chromatography (SEC) of enzymatically derived heparin oligosaccharides. In this study, we examine the heterogeneity of preparative-scale SEC fractions obtained from enzymatic digests of porcine intestinal mucosa heparin. Each fraction was profiled by capillary electrophoresis with UV detection (CE-UV) using a 60 mM formic acid running buffer at pH 3.43. Differences in the composition and relative concentration of components of the SEC fractions were observed for disaccharides and larger oligosaccharides. The heterogeneity of the fractions becomes more pronounced when heparin is digested using a heparin lyase cocktail. The heterogeneity of preparative SEC fractions was further investigated by reversed-phase ion-pairing ultraperformance liquid chromatography coupled with mass spectrometry (RPIP-UPLC-MS) using the ion-pairing reagent, tributylamine (Bu(3)N). Our results suggest that preliminary profiling of preparative SEC fractions prior to pooling may simplify efforts to identify and/or isolate rare structures.

Topics: Animals; Carbohydrate Conformation; Chromatography, Gel; Disaccharides; Electrophoresis, Capillary; Formates; Heparin; Heparitin Sulfate; Indicators and Reagents; Intestinal Mucosa; Kinetics; Models, Molecular; Oligosaccharides; Swine; Ultraviolet Rays