Reply to Oversulfated chondroitin sulfate is not the sole contaminant in heparin

Determination of the degree of acetylation and the distribution of acetyl groups in chitosan by HPLC analysis of nitrous acid degraded and PMP labeled products

“…However, recently published studies demonstrate that 1 H NMR fails to detect up to 50% polysaccharides in chitosan and PEGylated chitosan copolymers [33] It has also been reported that even though the same M A N U S C R I P T A C C E P T E D ACCEPTED MANUSCRIPT 6 amount of oversulfated chondroitin sulfate (OSCS) and oversulfated dermatan sulfate (OSDS), two types of highly resembling chemically sulfated animal polysaccharides, possess the same numbers of acetyl groups, the 1 H NMR proton signal intensities reflecting the acetyl groups in OSDS are only half of that observed in OSCS. [34] Furthermore, 1 H NMR could detect 0.1% OSCS in heparin based on the proton signal intensity of the acetyl groups in OSCS but could not detect 30% oversulfated heparan sulfate (OSHS) in heparin because the proton signal of the acetyl groups in OSHS is totally disappeared when mixed with heparin, [35,36] which suggest that 1 H NMR invisible or non-quantitative phenomena might be associated with polysaccharide structural analysis in general. Although 1 H NMR-based chitin/chitosan analysis produces consistent spectra demonstrating great reproducibility towards polysaccharide analysis and has been chosen as a standard method by the American Standard Test Method organization to determining the DA for chitosan, [37] the invisible and un-quantitative phenomena observed in chitosan and other polysaccharides by 1 H NMR analysis raise questions about the reliability of using 1 H NMR analysis alone for chitin/chitosan DA measurement and for comprehensive structure analysis.…”

Complete Monosaccharide Analysis by High-Performance Anion-Exchange Chromatography with Pulsed Amperometric Detection

“…20,21 Various GAG functions result from their structural diversity including monosaccharide composition. 18,19 Analysis of only amino sugars is not sufficient to identify these GAGs, 22,23 and the uronic acids need to be confirmed. However, in the typical disaccharide analysis of GAGs, uronic acids are cleaved and converted to the same unsaturated uronic acid after digestion by GAG lyases so that the uronic acid composition information is lost.…”