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Highlights • A method was developed to analyze the complex patterns of enoxaparin/PF4. • The analytical method was developed based on size-exclusion chromatography. • Stability, incubation time and ratios of enoxaparin/FP4 was investigated. • This method monitors the capacity of enoxaparin forming complexes with PF4. Abstract Heparin, a highly sulfated glycosaminoglycan, has been used as a clinical anticoagulant over 80 years. However, heparin-induced thrombocytopenia and thrombosis (HITT) is a serious side effect of heparin therapy, resulting in relatively high risk of amputation and even death. HITT is caused by forming of complexes between heparin and platelet factor 4 (PF4). Enoxaparin, one of the most commonly used low molecular weight heparin (LMWH), were developed in 1980's. The lower molecular weight of enoxaparin reduces the risk of HITT by binding to less PF4. To detect the binding capacity between enoxaparin and PF4 could be an effect way to control this risk before it goes to patients. In this work, a size exclusion chromatography (SEC) method was developed to analyze the patterns of complexes formed between PF4 and enoxaparin. The chromatographic condition was optimized to separate PF4, enoxaparin, ultra-large complexes and small complexes. The linearity and stability of this method were confirmed. The impacts of PF4/enoxaparin mixture ratios and incubation time on the forming complexes were investigated. Four enoxaparin samples were analyzed with this method to verify its practicability. It is a robust, accurate and practicable method, and provides an easy way to monitor the capacity of enoxaparin forming complexes with PF4, suggesting the HITT related quality of enoxaparin. [ABSTRACT FROM AUTHOR]

Highlights • 1H-NMR of heparins from different animals show distinct clusters in PCA and k means analysis. • Disaccharides components of heparins from different animals show distinct clusters in PCA and k means. • Tetrasaccharides components of heparins from different animals show distinct clusters in PCA and k means. • Chemometric analysis could help predict the heparin animal source and detect blending. Abstract Heparin is a polysaccharide anticoagulant drug isolated from animal tissues. There have been concerns on the safety and security of the heparin supply chain since 2007-8 when a contamination crisis led to its disruption. The current study applies a suite of modern analytical techniques to porcine, bovine and ovine intestinal mucosal heparins. These techniques include structural analysis by nuclear magnetic resonance spectrometry, disaccharide compositional analysis, bottom-up analysis of tetrasaccharides corresponding to heparin's antithrombin III binding site. Chemometric analysis was then applied to understand how these structural differences to predict the animal/tissue source of heparin and to help detect blending of heparins from various sources. [ABSTRACT FROM AUTHOR]

A simple and reliable high performance liquid chromatography coupled with electrospray ionization mass spectrometry (HPLC-ESI–MS) analysis method was established to simultaneously determine thirteen flavonoids of Xiaobuxing-Tang in intestine perfusate, namely onpordin, 3′- O -methylorobol, glycitein, patuletin, genistein, luteolin, quercetin, nepitrin, quercimeritrin, daidzin, patulitrin, quercetagitrin and 3-glucosylisorhamnetin. Detection was performed on a quadrupole mass spectrometer equipped with an electrospray ionization (ESI) source operating in negative ionization mode. Negative ion ESI was used to form deprotonated molecules at m / z 315 for onpordin, m / z 299 for 3′- O -methylorobol, m / z 283 for glycitein, m / z 331 for patuletin, m / z 269 for genistein, m / z 285 for luteolin, m / z 301 for quercetin, m / z 477 for nepitrin, m / z 463 for quercimeritrin, m / z 461 for daidzin, m / z 493 for patulitrin, m / z 479 for quercetagitrin, m / z 477 for 3-glucosylisorhamnetin and m / z 609.2 for rutin. The linearity, sensitivity, selectivity, repeatability, accuracy, precision, recovery and matrix effect of the assay were evaluated. The proposed method was successfully applied to simultaneous determination of these thirteen flavonoids, and using this method, the intestinal absorption profiles of thirteen flavonoids were preliminarily predicted. [ABSTRACT FROM AUTHOR]

Low molecular weight heparins (LMWHs), depolymerized from unfractionated heparin (UFH), are widely used as anticoagulant drugs in clinic. The variable degradation methods result the different types of LMWHs, such as enoxaparin prepared by alkaline degradation following benzylation and nadroparin degraded by nitrous acid and subsequent reduction. They have different anticoagulant activities, molecular weight and special oligosaccharide sequences. Oligosaccharide analysis of the heparinase-catalyzed digestion products of heparin and LMWHs is an important way to explore the fine structural composition. In this work, a MHC-2D-LC-MS system using SAX followed by SEC and tandem to MS was applied to analyze the heparinase-products of LMWHs. 15 components of enoxaparin and 20 components of nadroparin were separated and unambiguously characterized with mass spectrum, including eight common disaccharides, and the special structural domains resistant to enzyme digestion which have the 3- O sulfated residue and/or characteristic terminal residues and the linkage region tetrasaccharides. • The platform of MHC 2D-LC linked to MS was applied to analyze digested LMWHs. • SAX is the first dimensional, SEC is the second dimensional chromatography. • 15 and 20 structural domains in enoxaparin and nadroparin were identified. • Common structural framework and special structural domains were observed. [ABSTRACT FROM AUTHOR]