Fragment profiling of low molecular weight heparins using reversed phase ion pair liquid chromatography-electrospray mass spectrometry
Graphical abstract
Introduction
Heparin is a negatively charged, highly sulfated linear polysaccharide. It plays numerous significant roles in biological activities and physiologic processes through its interaction with multiple proteins.1 LMWHs are derived from the UFH by either controlled enzymatic digestion with heparinase or different chemical degradations.2 For example, enoxaparin is produced by alkaline degradation through chemical β-elimination reaction, while nadroparin is obtained by nitrous acid degradation.
Belonging to the family of glycosaminoglycans (GAG), heparin is composed of a repeating disaccharide building block of alternating 1,4-linked hexuronic acid (HexA) and glucosamine residue (GlcN). The HexA, either β-d-glucuronic acid (GlcA) or α-l-iduronic acid (IdoA), can be modified with 2-O-sulfo group. The GlcN may be N-acetylated (GlcNAc), N-sulfated (GlcNS), or unsubstituted, and its 3- and/or 6- positions can be substituted with O-sulfo group or unsubstituted.3 LMWHs have the same repeating disaccharide unit with heparin but different terminal structures altered by depolymerization processes. The non-reducing end (NRE) of enoxaparin is an unsaturated 4-enopyranose uronate structure, making ultraviolet (UV) absorption at 232 nm a versatile method to detect enoxaparin chains. Meanwhile, this kind of LMWH contains a specific 1,6-anhydro structure at the reducing end (RE) accounting for 15%–25% of the overall oligosaccharide chains.4 The characteristic terminal structures of nadroparin include a 2-O-sulfo-α-l-idopyranosuronic acid structure at the NRE and a 6-O-sulfo-2,5-anhydro-d-mannitol structure at the RE (Fig. 1).
Heparin and LMWHs have been widely utilized as anticoagulant drugs for prevention and treatment of thromboembolic diseases.5 Compared with UFH, LMWHs are currently more favorable drugs administrated to patients due to better bioavailability, longer biological half-lives and lower adverse effects.6 The occurrence of pharmaceutical heparin contamination with oversulfated chondroitin sulfate (OSCS) in 2008 aroused the introduction of sophisticated analytical methods to assure the quality and safety of pharmaceutical agent.7, 8 On the other hand, the U.S. Food and Drug Administration (FDA) developed five criteria to demonstrate the active ingredient sameness between generic and innovator LMWH products for an Abbreviated New Drug Application.9 Among these criteria, fragment mapping of LMWHs' partial digests from various enzymes provides global information on the sequences of oligosaccharides within the LMWHs' structures.
The fragment mapping analysis of LMWHs is in analogy to peptide mapping analysis of proteins. The enzymes including Hep I, Hep II and Hep III isolated from Flavobacterium heparinum are commonly used separately or as a cocktail to partially digest LMWHs.10, 11 Hep I shows a substrate specificity for α-l-IdoA2S (1→4) α-d-GlcNS. Hep II displays a broad selection for substrates comprised of either α-l-IdoA or β-d-GlcA. Hep III is selective for glycosidic bonds between α-d-GlcNS or α-d-GlcNAc and β-d-GlcA.12, 13 The fragments generated by various enzymatic reagents with diversely specific cleavage modes reflect global sequence of parent LMWHs and provide adequate evidence for sequence equivalence between generic and innovator LMWHs.
The mapping analysis can be achieved through various analytical techniques such as polyacrylamide gel electrophoresis (PAGE), capillary electrophoresis (CE), gel permeation chromatography (GPC), HPLC and MS. PAGE with relatively low resolution and MS incompatibility is a versatile method for the separation of heparin derived oligosaccharides in terms of size, conformation and charge.14 CE based on charge-to-size ratio provides high sensitivity and high separation resolution but still faces challenges on day-to-day reproducibility and CE/MS interface technique.15 GPC with relatively insufficient resolution is normally used as the first step to isolate a partially digested sample differing in size by disaccharide units.16 Strong anion exchange (SAX)-HPLC, a traditional approach for the separation and preparation of heparin components, usually possesses high resolution for low mass components ranging from disaccharides to decasaccharides.17, 18 Cetyltrimethylammonium (CTA) coated SAX-HPLC is powerful to separate highly sulfated heparin components with superior resolution.18 However, the identification of numerous peaks in SAX and CTA-SAX chromatograms is rather time-consuming and burdensome. Since both of them rely on high concentration nonvolatile salts in mobile phases, desalting of each peak is required prior to further structural analysis.
RPIP-HPLC is an increasingly popular method for heparin derived oligosaccharides analysis according to the size, isomerization and the number of sulfo groups.19, 20 RPIP-HPLC is performed on a hydrophobic reversed phase column (typically C18) with volatile ion pairing reagents to improve the retention of heparin chains on the column. Recent efforts demonstrate the combination of on-line separation techniques with ESI-MS offers an obvious advantage in understanding heparin structures.21, 22 RPIP-ESI-MS technique has been established for GAG analysis by adding volatile ion pairing reagents for improved separation performance and sufficient MS response.23, 24 A series of ion pairing reagents ranging from propylamine (PPA) to hexylamine (HXA), and tributylamine (TrBA) were evaluated in previous studies.25, 26 With a post-column addition of acetonitrile/TrBA to improve the volatility of electrosprayed solution in the ion source, TrBA is applied for heparin derived disaccharides/oligosaccharides analysis at the cost of severe instrument contamination problem.27, 28 MS ion counts from mobile phases containing HXA are less than that from pentylamine (PTA) on the condition that both mobile phases are added 1,1,1,3,3,3-hexafluoro-2-propanol (HFIP) as an organic modifier to control solution pH and improve MS signal.26 However, similar to TrBA, the addition of HFIP also results in instrument contamination. In general, PTA is an optimal ion pairing reagent with efficient separation and ionization performance as well as MS friendly characteristic.
This paper described a RPIP-ESI-MS technique coupling a capillary HPLC with an IT-TOF mass spectrometer for analysis of partial enzymatic depolymerization products of enoxaparin and nadroparin. PTA avoiding the addition of HFIP was used in the mobile phases to achieve sufficient chromatographic separation as well as excellent and friendly MS performance. ESI-MS in positive ion mode was used to detect and identify LMWH derived oligosaccharides. This approach successfully provides fragment profiling of enoxaparin and nadroparin after treatment with each of Hep I, II and III. Both major oligosaccharides and some minor and characteristic components like terminal structures of enoxaparin and nadroparin were detected and identified.
Section snippets
Chemicals and samples
Two LMWHs reference standards, enoxaparin sodium and nadroparin calcium, were from European Pharmacopoeia Reference Standards. Nadroparin products include GlaxoSmithKline Fraxiparine injections (3 lots) and generic products (3 lots) from Hebei Changshan Biochemical Pharmaceutical Co. Ltd. Hep I, II and III were obtained from Aglyco (Beijing, China). PTA (purity >99%) was purchased from Sigma–Aldrich (St. Louis, MO, U.S.A.). Water (HPLC grade), acetonitrile (HPLC grade) and formic acid
PAGE analysis of LMWHs enzymatic digestion product
PAGE analysis with relatively low resolution and limited structural information was used to evaluate the degree of partial enzymatic digestion. GPC fractionated enoxaparin derived disaccharides and decasaccharides were used to determine the size ranges of LMWH oligosaccharides prepared through partial enzymatic digestion with each of heparinase (Fig. 2). The oligosaccharide mixtures under three kinds of heparinases displayed varying band patterns, indicating different digestion modes due to
Conclusions
In this work, a RPIP-ESI-MS approach was established to profile LMWHs' chain fragments prepared by controlled enzymatic depolymerization. The separation system adopted a C18 column and mobile phases containing PTA, which is more MS friendly than other ion pairing reagents such as TrBA. The use of HFIP was avoided to minimize the contamination to mass spectrometer without compromising LC resolution and MS ionization efficiency. Different types of LMWHs, enoxaparin and nadroparin, were readily
Acknowledgments
This work was supported by grants from the National Basic Research Program of China (973 Program) (2012CB822102), the National High Technology Research and Development Program of China (863 Program) (2012AA021504), the National Natural Science Foundation of China (21472115, 81102783), and the Natural Science Foundation of Shandong Province, China (ZR2010CM041, ZR2011HQ038).
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2018, Carbohydrate PolymersCitation Excerpt :The structural composition, including numbers of HexA residues, GlcN residues, terminal residues, sulfo group substitutions and N-acetyl group substitutions, are then calculated either manually or with the help of bioinformatics tools for individual oligosaccharides with size up to 30 saccharide units (Maxwell et al., 2012). In the bottom-up approaches, LMWHs are either exhaustively digested to disaccharides using a cocktail of heparinase I, II and III for basic building blocks analysis (Wang, Li, Sun, Bai, Jin, & Chi, 2014; Sun et al., 2016a,b) or partially degraded to oligosaccharide fragments by using only one kind of heparinase for fragment mapping analysis (Xu, Li, Chi, Du, Bai, & Chi, 2015; Li, Steppich et al., 2014). The top-down and bottom-up approaches provide in-depth structural elucidation for LMWHs, but both of these MS approaches, along with other technique, such as GC, LC, gel electrophoresis and NMR, pay more attention to the overall structural properties of LMWHs.
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