Heparin sodium compliance to USP monograph: Structural elucidation of an atypical 2.18Â ppm NMR signal

► Unidentified NMR signal in the 2.10-3.20 ppm range is non compliant to the heparin US monograph. ► Some heparin sources containing an extra signal at 2.18 ppm were depolymerized by heparinase I. ► Heparin fractions were purified by LC to enrich moiety responsible of the NMR extra signal. ► From our investigation, a specific tetrasaccharide bearing an extra acetyl group was isolated. ► This chemical modification is characteristic of the heparin manufacturing process.