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• 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]

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]

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]