Improved high-performance liquid chromatographic method to estimate aminosugars and its application to glycosaminoglycan determination in plasma and serum

Abstract

An improved isocratic high-performance liquid chromatography (HPLC) method for the analysis of l-(−)-fucose, d-(+)-galactosamine, d-(+)-glucosamine, d-(+)-galactose, obtained by hydrolysis of glycosaminoglycans (GAGs) and d-(+)-glucose and d-(+)-mannose is described. The presence in circulation of GAGs, acid polysaccharide sequences of alternate monosaccharide units, aminosugar and uronic acid (galactose in keratan sulfate), has been measured in terms of their sugar components. To evaluate concentration of these circulating sugars we considered blood samples obtained from healthy humans. Plasma or serum was filtered through weak anion-exchange Ecteola-cellulose either untreated or after mild alkaline treatment. GAGs adhering to resin were recovered by salt elution, and desalted on Bio-Gel P-2 resin. GAG fractionation by charge was carried out on a strong anion exchanger. GAG composition was evaluated in terms of galactose and aminosugars, measured in HPLC by the proposed procedure using anion-exchange resin and pulsed amperometric detection. The mobile phase consisted of 0.02 M NaOH and elution was carried out at flow-rate of 1.0 ml/min. The amperometric detector was set as follows: t₁ (0.5 s), E₁ (+0.1 V); t₂ (0.09 s), E₂ (+0.6 V); t₃ (0.05 s), E₃ (−0.6 V). The analysis required 14 min. Calibration standard curves for the six analytes were linear from 0.25 to 40 μM. RSD values for intra- and inter-day variabilities were ≤5.3% at concentrations between 0.25 and 40 μM. Accuracy, expressed as percentage error, ranged from −16 to 14%. The method was specific and sensitive with quantitation limits of 1 pmol for l-(−)-fucose, d-galactosamine and d-glucosamine, 3 pmol for d-(+)-galactose and d-(+)-glucose and 5 pmol for d-(+)-mannose. The results of the assay showed higher GAG concentrations in serum than in plasma.