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Monosaccharide analysis is a critical way to profile the composition of complex carbohydrates. Methods to analyze neutral and amino sugars have been established for a long time, but methods for acidic sugars are rare. The acidic sugars, including uronic acids and sialic acids, are also important components in some complex carbohydrates. In this report, a high-performance anion-exchange chromatography method with pulsed amperometric detection was initially developed to analyze acidic sugars including different uronic acids and sialic acids. Subsequently, a method to profile complete monosaccharides, including most neutral, amino, and acidic sugars, was developed. This method has a limit of quantitation of ~12.5 × 10-3 nmol for each sugar as well as good linearity over a wide range. This is a convenient procedure because it avoids additional derivatization of monosaccharides and has a broad application to a wide range of complex carbohydrates. The monosaccharide compositions of a variety of complex carbohydrates such as different glycosaminoglycans, alginate, fucoidan, and glycans were profiled by this comprehensive method. In addition, the hydrolysis patterns of these complex carbohydrates are discussed. [ABSTRACT FROM AUTHOR]

Thin layer chromatography (TLC) is routinely used by synthetic organic chemists and natural products chemists working on a variety of types of molecules. Carbohydrate chemists and biochemists in the past largely relied on paper chromatography for rapid and inexpensive analysis. Carbohydrates are highly polar molecules and often require derivatization to be analyzed by TLC. Acidic carbohydrates pose an even more difficult challenge as they contain a formal negative charge and counterion, and can interact with commonly used stationary phases. This review describes TLC methods that have been developed for the analysis of acidic monosaccharides, disaccharides, and oligosaccharides derived from natural sources. [ABSTRACT FROM AUTHOR]

Abstract: Traditional chromatographic quantification methods for heparosan produced from the Escherichia coli K5 strain rely on extensive purification requiring laborious sample preparation. These methods are time-consuming, often resulting in sample loss during purification, and thus might not accurately reflect the amount of heparosan in the original mixture. A simple, sensitive 1H nuclear magnetic resonance (NMR) quantification method that directly quantifies heparosan K5 polysaccharide present in E. coli fermentation supernatant is described. [Copyright &y& Elsevier]

• SEC-MALS was firstly applied to measure the MW of ULC and SC. • The relationship between relative contents of ULC and PHR were investigated. • The relationship between sizes of ULC and PHR were investigated. • The relationship between heparin MW and the ULC size were investigated. Heparin-induced thrombocytopenia (HIT) is an immune complication of heparin therapy. Antibodies binding to complexes of platelet factor 4 (PF4) and heparin is the trigger of HIT. A method using size exclusion chromatography with multi-angle laser light scattering detector (SEC-MALS) was developed in this work. The soluble ultra-large complex (ULC) was separated from the small complex (SC) and their molecular weights (MWs) were firstly measured. The complexes of PF4 and three heparins with different MW, including unfractionated heparin (UFH), dalteparin (Daltep) and enoxaparin (Eno) were characterized using this method. The contents and the sizes of ULC increased gradually when heparins were added to PF4 to certain amounts. While, they reduced after more heparins were added. It is the first time to measure the MWs of the biggest ULC of PF4-heparins as millions of Dalton. at the proper ratios of PF4 to heparin (PHR). Meanwhile, those mixtures at those certain PHRs induced the higher expression of CD83 and CD14 markers on dendritic cells (DCs) suggesting that they had stronger immunogenicity and is critical for HIT. [ABSTRACT FROM AUTHOR]

Enoxaparin, one of the most important low-molecular-weight heparins (LMWHs), is widely used as a clinical anticoagulant. Different production processes and animal sources of its precursor (unfractionated heparin) can result in the structural diversity of enoxaparin. In this study, 38 lots of enoxaparin prepared at different times, from different providers and animal sources, were systematically analyzed. SEC and SAX were used to analyze the oligosaccharide dispersity and structural compositions (disaccharide domains) of enoxaparins by size and charge, respectively. The results provide clues as to whether the structural variations in enoxaparin, observed in oligosaccharide mapping and/or disaccharide analysis, are attributable to differences in the animal sources of its heparin precursor or enoxaparin production processes based on times or brands. The representative enoxaparins were fingerprinted with online multiple heart-cut two-dimensional liquid chromatography-mass spectrometry (MHC-2DLC-MS). The profiles in MHC-2DLC-MS showed the detailed structural information of enoxaparins. In addition, the binding capacities to antithrombin III (AT) of these 38 lots of enoxaparins were detected using surface plasmon resonance (SPR) with the competitive inhibition mode. The results showed that the glycan size distribution of an enoxaparin is more related to its production process. The disaccharide composition, sequence and the variety of glycans of an enoxaparin are more related to its AT binding-based anticoagulant activity. [ABSTRACT FROM AUTHOR]

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]

RATIONALE: Heparin and low molecular weight heparin (LMWH) are widely used as clinical anticoagulants. The determination of their composition and structural heterogeneity still challenges analysts. METHODS: Disaccharide compositional analysis, utilizing heparinase-catalyzed depolymerization, is one of the most important ways to evaluate the sequence, structural composition and quality of heparin and LMWH. Hydrophilic interaction chromatography coupled with quadruple time-of-flight mass spectrometry (HILIC/QTOFMS) has been developed to analyze the resulting digestion products. RESULTS: HILIC shows good resolution and excellent MS compatibility. Digestion products of heparin and LMWHs afforded up to 16 compounds that were separated using HILIC and analyzed semi-quantitatively. These included eight common disaccharides, two disaccharides derived from chain termini, three 3-O-sulfo-group-containing tetrasaccharides, along with three linkage region tetrasaccharides and their derivatives. Structures of these digestion products were confirmed by mass spectral analysis. The disaccharide compositions of a heparin, two batches of the LMWH, enoxaparin, and two batches of the LMWH, nadroparin, were compared. In addition to identifying disaccharides, 3-O-sulfo-group-containing tetrasaccharides, linkage region tetrasaccharides were observed having slightly different compositions and contents in these heparin products suggesting that they had been prepared using different starting materials or production processes. CONCLUSIONS: Thus, compositional analysis using HILIC/QTOFMS offers a unique insight into different heparin products. [ABSTRACT FROM AUTHOR]

Alginate is a linear and acidic polysaccharide, composed of (1 → 4) linked β- d -mannuronic acid (ManA) and α- l -guluronic acid (GulA). The ratio of ManA to GulA ( M / G ) is one of the most important factors for the application of alginate and its derivatives in various areas. In this work, a robust and accurate method was developed to analyze M / G using high-performance anion-exchange chromatography with pulsed amperometric detection (HPAEC-PAD). The impact of hydrolysis conditions on the release patterns of ManA and GulA from alginate and its derivatives was investigated. The release patterns of ManA and GulA need to be considered separately to obtain an accurate M / G . Several hydrolysis conditions were established that released ManA and GulA completely and maintained these saccharide residues intact. The proper M / G of alginates from different sources and its derivatives could then be calculated by integration of the corresponding ManA and GulA peaks. [ABSTRACT FROM AUTHOR]

Abstract: Peramivir is a novel influenza neuraminidase inhibitor. In this article, hydrophilic interaction chromatography coupled with tandem mass spectrometry was developed to determine peramivir in human plasma. The positive ion MRM mode was performed and the precursor to the product ion transitions of m/z 329→100 and 285→138 were used to measure peramivir and Ro 64-0802 (I.S.). Chromatographic separation was performed on an Amide-80 column with acetonitrile–water–formic acid (70:30:0.1, v/v/v, 0.5mL/min). The method was linear over a concentration range of 10–10,000ng/mL. The average inter-day/intra-day precision values were 3.7±1.8% and 4.3±1.8%, respectively, while the accuracy values were 97.0±4.8%. This method has been successfully applied to Phase I of clinical research of peramivir. [Copyright &y& Elsevier]

Abstract: A sensitive and specific high-performance liquid chromatography–tandem mass spectrometry method (LC/ESI/MS) was developed and validated for the identification and quantification of the novel lead compound of anticholinergic drug thiencynonate in rat plasma. The analytes were determined using positive electrospray ionization mass spectrometry in the selected reaction ion monitoring (SRM). The chromatography separation was on BetaBasic-18 column (150mm×2.1mm i.d., 3μm). The mobile phase was composed of methanol–water (70:30, v/v), containing 0.5‰ formic acid, which was pumped at a flow rate of 0.2ml/min. Phencynonate was selected as the internal standard (IS). Simultaneous MS detection of thiencynonate and IS was performed at m/z 364.4 (thiencynonate), m/z 358 (phencynonate), and the SRM of the two compounds were both at 156. Thiencynonate eluted at approximately 2.8min, phencynonate eluted at approximately 2.9min and no endogenous materials interfered with their measurement. Linearity was obtained over the concentration range of 1–100ng/ml in rat plasma. The lower limit of quantification (LLOQ) was reproducible at 1ng/ml in rat plasma. The precision measured was obtained from 2.47 to 9.28% in rat plasma. Extraction recoveries were in the range of 67.63–76.76% in plasma. This method was successfully applied to the identification and quantification of thiencynonate in pharmacokinetic studies. [Copyright &y& Elsevier]