Micro-Scale Analysis of N-Acetylneuraminic Acid

Abstract

We have developed a micro-scale analytical method for the detection and quantification of Nacetylneuraminic acid (NANA)that does neither require any derivatization of NANAnoris it interfered by the presence of any monosaccharide component present on N-linked and O-linked carbohydrate chains of glycoproteins. The method involves acid hydrolysis of sub-nanomolar amounts of sialoglycoprotein (SGP) and subsequent neutralization, followed by high-pH anion-exchange chromatography (HPAE) with pulsed amperometric detection (PAD). The method wasvalidated, using a,-acid glycoprotein (AGP) (orosomucoid) as standard SGP. Six experimental series of decaplicates, each, were performed, varying i) the AGP concentration in PBS (i. e. 250, 500 and 750 ug/ml, respectively), ii) the sample amount used (i. e. 200, 100, 30, 15 and 10 ul, each, of defined AGP concentrations), iii) the mode of sample injection into the Dionex Bio-LC system (i. e. manual or automated injection by way of two different autoinjectors), iv) the sensitivity of the PAD detector(i. e. 1000 and 300 nAfull scale, respectively). Individual experimental series were repeated at different days and were occasionally performed by different persons. In each case, the NANA/AGP molar ratios were in the range of 15.0 +/- 0.4 mol/mol (corresponding with a standard deviation of <+/- 3 %), which is in excellent agreement with the NANA/AGP molar ratio described in theliterature.