Source-induced fragmentation of heparin, heparan, and galactosaminoglycans and application

Sulfated glycosaminoglycans (GAGs) are difficult molecules for analysis by mass spectrometry due to their complexity in saccharide composition, polydispersity, and sequence heterogeneity. Structural information is typically derived from their enzymatic or chemical digests. Many analytical studies focused on the determination of disaccharide compositions. In this study a direct electrospray mass spectrometry method is described for the analysis of intact heparin, heparan sulfate, dermatan sulfate, and chondroitin sulfates. The GAGs were desalted by membrane filtration and analyzed in dilute formic acid (0.5%) in negative ion mode. At the dissociation cone voltage (defined as minimum cone voltage to dissociate all polymeric molecules), heparin, heparan sulfate, dermatan sulfate, and chondroitin sulfates produced simple mass spectra consisting primarily of monosaccharide and disaccharide ions derived from glycosidic bond cleavages. The type and abundance of the ions in the dissociation of each molecule were a good reflection of their saccharide compositions. The major ions of heparin were the hexuronic acid ion (m/z 175), glucosamine ion (m/z 240), and the disaccharide ion (m/z 416). Heparan sulfate produced the same set of fragments as heparin since they shared the same compositional saccharides. However, the formation of the m/z 175 ion dominated the source induced fragmentation process for heparan sulfate reflecting its high content of the nonsulfated disaccharide D-glucuronic acid-acetylated glucosamine (GlcA-GlcNAc). Chondroitin/dermatan sulfates contained only acetylated amino sugar (acetylated galactosamine (GalNAc). Consequently, the principle mono/disaccharide ions were all acetylated with m/z values of 282 and 458, respectively. The contrast among the dissociation features of the three types of molecules were sufficient to allow their ready differentiation. Additionally, sensitive detection of chondroitin/dermatan sulfate and heparan sulfate in heparin was made possible by the same differences in the dissociation chemistry of the three types of molecules. As low as 0.5% chondroitin/dermatan sulfate and 5% heparan sulfate in heparin can be reliably detected. This method was successfully used for the detection of oversulfated chondroitin sulfate in heparin as a contaminant following reports of increased adverse events associated with heparin injections from the end of 2007 to early 2008. Heparin is an important, widely prescribed anticoagulant. In light of this contamination event, quality assurance beyond standard activity assays proves to be important. This method provides a simple and fast way for the detection of low level chondroitin, dermatan, and heparan sulfates and their oversulfated derivatives in heparin raw material or formulation.