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Heparin is a linear sulfated polysaccharide with a complex structure. It is important to figure out the sequences at the terminals of the sugar chains, as it will help us understand the heparin structure deeper and control its quality properly. The tetrasaccharide linkage region (LR) could be a tag to help us find out heparin terminals after digestion by different combinations of heparinases. In this work, orthogonal chromatographic approaches including SAX, SEC-MS and 2D-LC-MS were applied to qualitatively and quantitatively analyze the heparinase released LR-terminals. The disaccharides next to LR are those ones with low or non-sulfation, UA-GlcNAc and UA-GlcNAc6S, and then they are extended with the highly sulfated disaccharides, IdoA2S-GlcNS and IdoA2S-GlcNS6S. It is suggested that the sulfo transferases did not work at the sugar residues next to LR terminal, especially the 2-O-sulfo and N-sulfo transferases, which could be affected by steric hindrance from LR, when heparin is biosynthesized. This conclusion will be theoretical fundamental to help us understand heparin's structure deeper. The methods provided in this work could be potential ways to control heparin's quality and monitor the production processes of heparin properly., Competing Interests: Declaration of Competing Interest None., (Copyright © 2022 Elsevier B.V. All rights reserved.)

Heparin is a linear sulfated polysaccharide. It is composed of a repeating disaccharide unit with different sulfo patterns. The compositional analysis after heparin was decomposed to disaccharides and enzyme resistant domains is an important way to delve into its structure. Strong anion exchange (SAX) chromatography is commonly used for the compositional analysis due to its high resolution, stability and capability of quantitation. However, nonvolatile salt in mobile phase is not compatible with MS, then the structural domains cannot be identified without standards. Here, a new two-dimensional liquid chromatography system, multiple heart cut (MHC), was developed and linked to mass spectrometry (MS) directly to provide a comprehensive analysis of enzyme digested heparin. SAX was applied as the first dimensional chromatography, in which 17 peaks were observed and integrated in the digested heparin. Size-exclusion chromatography (SEC) was used as the second dimensional chromatography to desalt efficiently. Structural information of each component was then obtained with MS, including eight common disaccharides, eight enzyme resistant tetrasaccharides and a heparin-core protein linkage domain. The comparison of enzyme digested heparins obtained from different vendors using this system suggested their similar major structure and activity, but slightly different production processes., Competing Interests: Declaration of Competing Interest There are no conflict of interest for authors., (Copyright © 2021 Elsevier B.V. All rights reserved.)

Heparin, a highly sulfated glycosaminoglycan, has been used as a clinical anticoagulant over 80 years. Low molecular weight heparins (LMWHs), heparins partially depolymerized using different processes, are widely used as clinical anticoagulants. Qualitative molecular weight (MW) and quantitative mass content analysis are two important factors that contribute to LMWH quality control. Size exclusion chromatography (SEC), relying on multiple angle laser scattering (MALS)/refractive index (RI) detectors, has been developed for accurate analysis of heparin MW in the absence of standards. However, the cations, which ion-pair with the anionic polysaccharide chains of heparin and LMWHs, had not been considered in previous reports. In this study, SEC with MALS/RI and inductively coupled plasma/mass spectrometry detectors were used in a comprehensive analytical approach taking both anionic polysaccharide and ion-paired cations heparin products. This approach was also applied to quantitative analysis of heparin and LMWHs. Full profiles of MWs and mass recoveries for three commercial heparin/LMWH products, heparin sodium, enoxaparin sodium and nadroparin calcium, were obtained and all showed higher MWs than previously reported. This important improvement more precisely characterized the MW properties of heparin/LMWHs and potentially many other anionic polysaccharides., (Copyright © 2017 Elsevier B.V. All rights reserved.)

Low molecular weight heparins (LMWHs), derived from unfractionated heparin (UFH) by chemical or enzymatic degradation, have lower side effects than that of heparin. Enoxaparin, nadroparin, and dalteparin are the most widely used LMWHs. Nadroparin and dalteparin are prepared through nitrous acid degradation followed by a subsequent reduction process, in which specific residue, 2,5-anhydro- d -mannitol (An-Man), is formed at the reducing terminals of generated sugar chains. However, few practicable analytical method was available to analyze An-Man qualitatively and quantitatively. In this work, a HPAEC-PAD-MS method was developed to analyze monosaccharides in heparin and LMWHs, especially An-Man. An ion suppressor is set up between HPAEC and MS to remove the nonvolatile salts from HPAEC and make the elute compatible to MS. Various monosaccharides were separated well with HPAEC. Online MS and MS/MS confirmed all sugar residues in the hydrolysates of heparin samples. The specific residue, An-Man, was only observed in the hydrolysates of LMWHs prepared with nitrous acid degradation and reduction. In addition, major glucosamine and minor arabinose/galactose were observed in all heparin samples. The amounts of these sugar residues were quantitated with PAD, simultaneously. The ratio of glucosamine to An-Man could be used to calculate the molecular weight of LMWHs. The calculated values are comparable to the value measured with size-exclusion chromatography-multiple angle laser scattering detection. Higher the ratio of glucosamine to An-Man higher the molecular weight. The HPAEC-PAD-MS platform is an accurate, precise and efficient way to identify LMWHs by determination of An-Man. It is also an alternative method to detect the MWs of LMWHs having An-Man for quality control purposes. [ABSTRACT FROM AUTHOR]