Accurate evaluation of sialic acids in biological matrices for a more reliable assessment of their role

An efficient procedure for the contemporaneous qualitative and quantitative determination of the most important sialic acids (SA), Neu5Ac and Neu5Gc, present in a large number of glycoproteins and glycolipids has been developed. The procedure represents the first protocol which uses isotope dilution liquid chromatography/tandem mass spectrometry without any sample derivatization, it involves the release of SA by acidic hydrolysis in presence of appropriate internal standards: the labeled analogues [1,2,3-13C3]-Neu5Ac and [1,2,3-13C3]-Neu5Gc. The use of isotopologous internal standards, considered the most appropriate in quantitative bioanalytical assays, compensates for variability in sample extraction, processing and analysis. The accomplishment of this method, which allows the chromatographic separation of Neu5Ac and Neu5Gc, has required the preparation of [1,2,3-13C3]-Neu5Gc and a home-made LC-column, conceived considering the interactions of alkylboronic acids with neutral and acidic carbohydrates. In order to confirm the reliability of this method, it was fully validated using fetuin as model glycoprotein and then applied to evaluate the presence of Neu5Ac and Neu5Gc in murine myoblasts (C2C12) and in human erythrocytes membranes of healthy subjects. Moreover, the levels of these SA were also determined in erythrocytes membranes of patients affected by diabetes or polycythemia vera. Because of the presence of the isotopologous internal standards starting from the hydrolysis of the biological substrates and the absence of any derivatization of SA, the method has permitted to evaluate some important biological events not observable by detecting SA by means of the previous techniques. The methodology appears to be of utility to support progresses in the field of the sialic acids biology.