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  • Research Article

    Received: 31 August 2015 Revised: 22 October 2015 Accepted: 23 October 2015 Published online in Wiley Online Library

    Rapid Commun. Mass Spectrom. 2016, 30, 277–284

    Development of hydrophilic interaction chromatography with quadruple time-of-flight mass spectrometry for heparin and low molecular weight heparin disaccharide analysis

    Yilan Ouyang1†, Chengling Wu1†, Xue Sun1, Jianfen Liu2**, Robert J. Linhardt3 and Zhenqing Zhang1* 1Jiangsu Key Laboratory of Translational Research and Therapy for Neuro-Psycho-Diseases and College of Pharmaceutical Sciences, Soochow University, Suzhou, Jiangsu 215021, China 2Xiehe Pharmaceutical Co. Ltd, Shijiazhuang, Hebei Province 050083, China 3Departments of Chemistry and Chemical Biology, Chemical and Biological Engineering, Biomedical Engineering, Biology, Center for Biotechnology and Interdisciplinary Studies, Rensselaer Polytechnic Institute, 110 8th Street, Troy, NY 12180, USA

    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. Copyright © 2015 John Wiley & Sons, Ltd.

    (wileyonlinelibrary.com) DOI: 10.1002/rcm.7437

    Heparin, a highly sulfated glycosaminoglycan, has been widely used as a clinical anticoagulant since the 1930s.[1–3]

    As one of the most important biomacromolecules, heparin also participates in many other important biological processes, including viral and bacterial infection and entry, angiogenesis, inflammation, cancer, and development.[4,5]

    Most heparin products are isolated by extraction from animal tissues, such as porcine intestine.[6,7] The most common repeating disaccharide unit of heparin is 2-O-sulfo-α-L- iduronic acid (IdoA2S) 1→ 4-linked to 6-O-sulfo, N-sulfo-α- D-glucosamine (GlcNS6S), -IdoA2S (1→ 4)GlcNS6S-.[8,9]

    * Correspondence to: Z. Q. Zhang, Jiangsu Key Laboratory of Translational Research and Therapy for Neuro-Psycho- Diseases and College of Pharmaceutical Sciences, Soochow University, Suzhou, Jiangsu 215021, China. E-mail: z_zhang@suda.edu.cn

    ** Correspondence to: J. Liu, Xiehe Pharmaceutical CO. LTD., Shijiazhuang, Hebei province, 050083, China Email: liujianfen888@163.com

    † These two authors contributed equally to this work.

    Rapid Commun. Mass Spectrom. 2016, 30, 277–284

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    However, like all other natural polysaccharide products, heparin has heterogeneity in its molecular weight as well as its degree of sulfation, saccharide unit composition, and sequence, and this range of heterogeneity depends on tissue source and extraction process.[10,11] Different compositions, sequences and structures of heparin chains can lead to different activities and quality issues. In 2007–2008, a rapid onset, acute side effect associated with heparin was reported, which was believed to be caused by a contaminant, oversulfated chondroitin sulfate (OSCS), leading to hypotension and resulting in nearly 100 deaths.[12–14] This crisis initially went undetected as pharmacopeial methods to monitor heparin quality in addition to its activity and structure were lacking. A more detailed understanding of heparin structure and sequence was required to maintain its quality control for effective clinical application. Disaccharide analysis is one of the most important methods to describe the composition, sequence and structure of heparin.

    Many methods have been developed to analyze heparin disaccharides, such as strong anion-exchange (SAX) chromatography, ion-pairing reversed-phase (IPRP)

    Copyright © 2015 John Wiley & Sons, Ltd.

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    chromatography and ultra-performance reversed-phase chromatography with pre-column derivatization.[15–17]

    However, each of these methods exhibits disadvantages. The instability observed in SAX chromatography makes it difficult to assign peaks to individual disaccharides based on relative retention times.[18] In addition, eluents used in SAX chromatography are often incompatible with mass spectrometry (MS). In IPRP, ion-pairing reagents, even ones selected for compatibility with MS, often remain in the ion source and even in the MS capillary suppressing its sensitivity and resolution.[19,20] Reversed-phase methods require pre-column derivatization to improve the separation and detection sensitivity of heparin disaccharides, but derivatization often introduces impurities, resulting in the loss of structural information, and decrease in analytical accuracy.[20,21]

    Hydrophilic interaction chromatography (HILIC), a rapidly developing separation method, uses matrices with various functional groups to separate polar compounds.[22] Several laboratories have used HILIC to analyze heparan sulfate (HS)/heparin disaccharides and oligosaccharides.[23,24] No derivatization is needed and HILIC buffer systems are usually MS friendly. In the current study, a new Xamide column was applied to analyze the disaccharides derived from heparin and two different low molecular weight heparins (LMWHs) without any additional derivatization. Amide functional groups, on the surface of resin in the Xamide column, provide sufficient but not excessively strong interaction with highly sulfated heparin-derived disaccharides and oligosaccharides.[24,25] This allows the use of low concentrations of volatile salts in the eluent making HILIC on Xamide columns well suited to MS analysis. It is also important that the amide groups are attached to the chromatographic support with a hydrophilic linker, facilitating the use of an aqueous mobile phase required for these highly polar analytes.[26] A new high-resolution heparin disaccharide analysis method was developed using

    Table 1. Products of the heparinase digestion of heparin and L

    Symbols Structure

    ΔIVA ΔUA-GlcNAc ΔIIIA ΔUA2S-GlcNAc ΔIIA ΔUA-GlcNAc6S ΔIA ΔUA2S-GlcNAc6S ΔIVS ΔUA-GlcNS ΔIIIS ΔUA2S-GlcNS ΔIIS ΔUA-GlcNS6S ΔIS ΔUA2S-GlcNS6S ΔIVH ΔUA-GlcN ΔIIIH ΔUA2S-GlcN ΔIIH ΔUA-GlcN6S ΔIH ΔUA2S-GlcN6S ΔGlyser ΔUA-Gal-Gal-Xyl-ser ΔGlyserox1 ΔUA-Gal-Gal-Xyl-CH2-COOH ΔGlyserox2 ΔUA-Gal-Gal-CH(CH2OH)-COOH Δdp2(0S)RE ΔUA-Mnt-2,5-anhydro Δdp2(1OS)RE ΔUA-Mnt6S-2,5-anhydro ΔIVA-IVS3Sglu ΔUA-GlcNAc-GlcA-GlcNS3S ΔIIA-IVS3Sglu ΔUA-GlcNAc6S-GlcA-GlcNS3S ΔIIS-IIS3Sglu ΔUA-GlcNS6S-GlcA-GlcNS3S6S

    wileyonlinelibrary.com/journal/rcm Copyright © 2015 John Wi

    an Xamide column. In addition to disaccharides, different types of linkage region tetrasaccharides and 3-O-sulfo- group-containing tetrasaccharides could be separated and identified using HILIC/LC/MS analysis. Small structural differences in the composition of different heparins and LMWHs could be observed using this newly developed method.

    EXPERIMENTAL

    Materials

    Unsaturated disaccharide standards of heparin, ΔUA-GlcNAc (ΔIVA), ΔUA2S-GlcNAc (ΔIIIA), ΔUA-GlcNAc6S (ΔIIA), ΔUA2S-GlcNAc6S (ΔIA), ΔUA-GlcNS (ΔIVS), ΔUA2S-GlcNS (ΔIIIS), ΔUA-GlcNS6S (ΔIIS), ΔUA2S-GlcNS6S (ΔIS), ΔUA- GlcN (ΔIVH), ΔUA2S-GlcN (ΔIIIH), ΔUA-GlcN6S (ΔIIH) and ΔUA2S-GlcN6S (ΔIH), and heparinase I, II and III were obtained from Iduron Co. (Manchester, UK) (where ΔUA is 4-deoxy-R-L-threo-hex-4-enopyranosyluronic acid, GlcN is glucosamine, Ac is an acetyl group, and S is a sulfo group). The structural information of these heparin disaccharides are provided in Table 1. Heparin was obtained from the United States Pharmacopeia (USP, Rockville, MD, USA). Enoxaparins were obtained from the USP and the market (a product of Sanofi Aventis). Nadroparins were obtained from the European Pharmacopeia (EP) and the market (a product of GlaxoSmithKline, GSK). Other chemical reagents were all HPLC or LC/MS grade.

    Preparation of the disaccharides from heparin products

    Each heparin product (2 mg) was dissolved in 0.4 mL water and incubated with lyase mixture (heparinase I, II and III, 0.02 U for each) at 37 °C overnight. The digestions were complete and the solutions were boiled for 20 min. The

    MWHs

    Theoretical MW (Da)

    Molecular ion [M–H]–/[M–2H]2–

    379.1115 378.1030 459.0683 458.0601 459.0683 458.0592 539.0251 538.0130 417.0577 416.0480 497.0145 496.0040 497.0145 496.0037 576.9713 575.9600 337.1008 —— 417.0577 —— 417.0577 ——

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