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

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 G AGs, acid polysaccharide sequences of alternate monosaccharide units, amino sugar and uronic acid (galactose in keratan sulfate), has been measured in terms of their sugar components. To evaluate concentration of these circula ting sugars we considered blood samples obtained from healthy humans. Plasm a or serum was filtered through weak anion-exchange Ecteola-cellulose eithe r untreated or after mild alkaline treatment. GAGs adhering to resin were r ecovered by salt elution, and desalted on Bio-Gel P-2 resin. GAG fractionat ion by charge was carried out on a strong anion exchanger. GAG composition was evaluated in terms of galactose and aminosugars, measured in HPLC by th e proposed procedure using anion-exchange resin and pulsed amperometric det ection. The mobile phase consisted of 0.02 M NaOH and elution was carried o ut at flow-rate of 1.0 ml/min. The amperometric detector was set as follows : t(1) (0.5 s), E-1 (+0.1 V); t(2) (0.09 s), E-2 (+0.6 V); t(3) (0.05 s), E -3 (-0.6 V). The analysis required 14 min. Calibration standard curves for the six analytes were linear from 0.25 to 40 muM. RSD values for intra- and inter-day variabilities were less than or equal to5.3% at concentrations b etween 0.25 and 40 muM. Accuracy, expressed as percentage error, ranged fro m -16 to 14%. The method was specific and sensitive with quantitation limit s 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 result s of the assay showed higher GAG concentrations in serum than in plasma. (C ) 2001 Elsevier Science B.V. All rights reserved.