Rapid Characterization of Glycosaminoglycans Using a Combined Approach by Infrared and Raman Microspectroscopies

This study describes a complementary infrared (IR) and Raman spectroscopic investigations of a set of biomolecule representatives of glycosaminoglycans (GAGs) class. Both IR and Raman data exhibit characteristic spectral signatures that allow a direct molecular distinction of these compounds. Comparison of these molecular signatures clearly evidences the differences between heparan sulfate and heparin by computing the intensity ratio between the 1.248 cm(-1) and 1043 cm(-1) peaks, corresponding respectively to sulfate and C-O-C linkages. Identically, chondroitin-4-sulfate and chondroitin-6-sulfate are differentiated via the 730 cm(-1) and 853 cm(-1) bands (axial orientation) and the 822 cm(-1) and 1000 cm(-1) bands (equatorial orientation). These orientations concern the sulfate groups of these molecules. Secondly, multivariate statistical methods were employed to analyze the data set. Hierarchical cluster analysis (HCA) of both IR and Raman spectra showed a very good differentiation between the different structures. In addition, the sulfatation degree could be obtained from this classification. Principal component analysis using three principal components (PCs) confirmed the findings of HCA and, furthermore, comparisons of the PC loadings highlight the main molecular differences between the members of this class of biological molecules. It is expected that these microspectroscopic methods will be useful in identifying GAG spectral signatures in complex biological systems. (C) 2010 Wiley-Liss, Inc. and the American Pharmacists Association J Pharm Sci 100:441-450, 2011