Accepted Manuscript Liquid chromatography-diode array detection-mass spectrometry for compositional analysis of low molecular weight heparins Zhangjie Wang, Daoyuan Li, Xiaojun Sun, Xue Bai, Lan Jin, Lianli Chi PII: DOI: Reference:
S0003-2697(14)00054-2 http://dx.doi.org/10.1016/j.ab.2014.02.005 YABIO 11644
To appear in:
Analytical Biochemistry
Received Date: Revised Date: Accepted Date:
22 November 2013 2 February 2014 4 February 2014
Please cite this article as: Z. Wang, D. Li, X. Sun, X. Bai, L. Jin, L. Chi, Liquid chromatography-diode array detection-mass spectrometry for compositional analysis of low molecular weight heparins, Analytical Biochemistry (2014), doi: http://dx.doi.org/10.1016/j.ab.2014.02.005
This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
Liquid chromatography-diode array detection-mass spectrometry for compositional analysis of low molecular weight heparins Short title: Compositional analysis of low molecular weight heparins Zhangjie Wanga,b, Daoyuan Lia,b, Xiaojun Suna, Xue Baia, Lan Jina, Lianli Chia,b,* a
National Glycoengineering Research Center, Shandong University, Jinan 250100, China
b
State Key Laboratory of Microbial Technology, Shandong University, Jinan 250100, China
*Corresponding author. Tel: +86 531 8836 3200, Fax: +86 531 8836 3002 E-mail address: [email protected] (L. Chi). Categories: carbohydrates; chromatographic techniques Abbreviations used: LMWH, low-molecular-weight heparin; RP, reversed phase; DAD, diode array detection; ESI, electrospray ionization; MS, mass spectrometry; AMAC, 2-aminoacridone; RE, reducing end; NRE, non-reducing end; HPLC, high performance liquid chromatography; SAX, strong anion exchange; UV, ultraviolet; HILIC, hydrophilic interaction liquid chromatography; RPIP, reversed phase ion pairing; CE, capillary electrophoresis; DMSO, dimethylsulfoxide; IT-TOF, ion trap-time of flight; TIC, total ion chromatogram.
1
ABSTRACT
Low molecular weight heparins (LMWHs) are important artificial preparations from heparin polysaccharide and
widely used as anticoagulant drugs. To analyze the structure and composition of LMWHs, identification and
quantitation of their natural and modified building blocks are indispensable. We established a novel reversed phase
(RP) high performance liquid chromatography (HPLC)-diode array detection (DAD)-electrospray ionization-mass
spectrometry (ESI-MS) approach for compositional analysis of LMWHs. After exhaustively digested and labeled
with 2-aminoacridone (AMAC), the structural motifs constructing LMWHs, including 17 components from
dalteparin and 15 components from enoxaparin, were well separated, identified, and quantified. Besides the 8 natural
heparin disaccharides, many characteristic structures from dalteparin and enoxaparin, such as modified structures
from the reducing end (RE) and non-reducing end (NRE), 3-O-sulfated tetrasaccharides, as well as trisaccharides,
have been unambiguously identified based on their retention time and mass spectra. Comparing to the traditional
heparin compositional analysis methods, the approach described herein is not only robust, but also comprehensive
because it is capable to identify and quantify nearly all components from lyases digests of LMWHs.
Keywords: LMWH; DAD; AMAC derivatization; ESI-MS; Compositional analysis
2
Heparin is a heterogeneous polysaccharide in terms of variable chain length, sulfo and N-acetyl substitutions, and epimerization. It consists of repeating disaccharide units of β-D-glucuronic acid (GlcA) or α-L-iduronic acid (IdoA) (1→4) linked to D-glucosamine. The hexuronic acid may be 2-O-sulfated and glucosamine may be 6-O-sulfated, N-sulfated, N-acetylated, or infrequently 3-O-sulfated. For a long time, heparin has been extensively used as clinical anticoagulant drugs for prevention and treatment of thrombotic diseases [1,2,3]. However, the negative effects of heparin, including bleeding and inducing thrombocytopenia and osteoporosis, limit its clinical application [4]. LMWHs are overtaking the market share of heparin as new anticoagulant and anti-thrombotic drugs in particular due to their improved bioavailability and reduced bleeding risk [5,6]. Several types of LMWHs have been produced by either controlled enzymatic or chemical depolymerization of heparin, including heparinase depolymerization, nitrous acid degradation, β-elimination, and hydrolytic cleavage with hydrogen peroxide [7]. The enzymatic and chemical reactions usually result in modified structures at the cleavage sites, i.e. the RE and NRE of LMWHs. For example, enoxaparin is manufactured by alkaline treatment of benzyl ester derivative of heparin. The NRE becomes an unsaturated hexuronic acid, and the RE is a 1,6-anhydro structure for 15-25% of the constitutes [8]. Dalteparin is produced by nitrous acid depolymerization. The NRE is a 2-O-sulfo-α-L-idopyranosuronic acid and the RE is a 6-O-sulfo-2,5-anhydro-D-mannitol structure [9]. Besides the natural disaccharides and modified structures at the RE and NRE, LMWHs also contain 3-O-sulfated pentasaccharide structures, which are responsible for their anticoagulant activity [10]. Peeling reaction is a common side reaction during preparation of heparin and LMWHs, which results in glucosamine structures at the RE [11]. Since heparin and LMWHs are heterogeneous macro biomolecules, they are usually degraded
3
completely into basic building blocks, and then analyzed using chromatographic or electrophoretic techniques. The cocktail of three types of heparinase (heparinase I, II and III) isolated from Flavobacterium heparinum are generally used to deconstruct heparin and LMWHs [12,13]. In the case of enoxaparin and dalteparin, the possible structures from exhaustive digestion include eight natural disaccharides, modified oligosaccharides from both termini, 3-O-sulfated tetrasaccharides, as well as trisaccharides derived from peeling reaction (Table 1). Compositional analysis is a fundamental approach to characterize heparin and LMWHs. It is required by the United States Food and Drug Administration to demonstrate the compositional sameness between generic and innovator’s LMWH products for an Abbreviated New Drug Application [14]. HPLC are conventional methods and have been extensively used to determine the composition of heparin and LMWHs. Strong anion exchange (SAX)-HPLC, regarded as a general analytic method, has been largely used to identify and characterize heparin and LMWHs-derived oligosaccharides based on their acidic properties and characteristic UV absorption at 232 nm [15]. Hydrophilic interaction liquid chromatography (HILIC) is capable to separate hydrophilic analytes, and has been reported to separate heparin and other glycosaminoglycan-derived disaccharides [16]. In recent years, reversed phase ion pairing (RPIP)-HPLC is drawing increasing attention. The ion pairing reagents modify the hydrophobicity of hydrophilic heparin-derived disaccharides and oligosaccharides, which can then be well resolved on traditional RP columns [17]. Capillary electrophoresis (CE) has been also employed to analyze LMWHs’ composition with relatively high resolution and low limit of detection (10-20 pg) [18,19]. However, most of the HPLC and CE methods mentioned above are only limited to detect eight natural heparin disaccharides. The exhaustive digestion of LMWHs results in more complicated disaccharide and oligosaccharide
4
composition. Unlike the natural heparin disaccharides, there are no commercial available reference standards for modified disaccharides and oligosaccharides. Moreover, some of these disaccharides and oligosaccharides lack unsaturated structures at NRE and cannot be monitored by UV detector in HPLC or CE analysis. Fluorescence derivatization techniques are frequently used to facilitate the separation and detection of heparin-derived oligosaccharides. A variety of fluorophores, such as 2-aminopyridine, 2-aminobenzamide, can be covalently attached to the RE of oligosaccharides through reductive amination reaction [20,21]. AMAC labeling is a newly developed method for heparin disaccharide analysis [22,23]. The labeled disaccharides can be directly separated on a C18 column, driven by the hydrophobicity of AMAC. The serial of retention times of eight disaccharides is reversed compared to the SAX and RPIP separations. The limitation of fluorescence derivatization methods is that they require aldehyde structure at the RE of oligosaccharides. Dalteparin chains contain 2,5-anhydro-D-mannitol structure while some of enoxaparin chains contain 1,6-anhydro structure at their REs. These non-reducible structures are not reactive to fluorescence reagents. MS techniques with soft ionization methods, such as ESI and matrix assisted laser induced ionization, have been remarkably successful in the structural characterization of heparin and LMWH oligosaccharides [24,25]. ESI-MS is more favored because it can be easily connected downstream to HPLC and CE separations. Typically, separation systems with no salts or low
concentration of volatile salts, including RPIP, HILIC, and GPC, are compatible with on-line ESI-MS analysis [26,27,28]. RPIP-ultra HPLC-MS methods have been applied to analyze the digests of heparin and LMWHs [29,30]. However, ion pairing reagents cause instrument contamination problem and are usually desired to avoid if possible. Hydrophobic fluorescence tags
5
can affect the retention properties of labeled heparin disaccharides on the regular RP columns. Several RP-HPLC-ESI-MS approaches have been attempted in the compositional analysis of different types of glycosaminoglycans [31,32]. The drawback of LC-MS methods is the relatively high instrument and maintenance costs. Moreover, quantitation analysis using MS data without isotopic standards is usually not optimal [33]. Generally it is preferred to use UV signals for quantitation analysis while using MS for peak identification. In this study, we established a RP-HPLC-DAD-MS approach for compositional analysis of two different LMWHs, dalteparin and enoxaparin. The DAD was used to detect disaccharides and oligosaccharides from the heparinase digests of LMWHs, by monitoring wavelengths at 232 nm for unsaturated uronic acids at NRE and 255 nm for the AMAC derivatives simultaneously. ESI-MS was used to identify the AMAC labeled or underivatized structures unambiguously. By using the synthetic heparin disaccharide ∆UA2S-GlcNCOEt6S (∆IP) as internal standard, eight heparin natural disaccharides, modified terminal structures, 3-O-sulfated tetrasaccharides, as well as trisaccharides were identified and quantified. Materials and methods Materials Dalteparin and enoxaparin reference standards were purchased from European Pharmacopoeia. Heparinase I, II and III were obtained from Aglyco (Beijing, China). Heparin disaccharide standards were purchased from Iduron Co. (Manchester, U.K.). AMAC, dimethylsulfoxide (DMSO) and sodium cyanoborohydride were from Sigma-Aldrich (St. Louis, MO, USA). All other reagents and chemicals were of the highest quality available. Enzymatic digestion of LMWHs
6
The enzymatic digestion solution was a mixture of 10 µL of 20µg/ µL LMWH sample, 35 µL of 100 mM sodium acetate/2mM calcium acetate buffer (pH 7.0) containing 0.1 g/L bovine serum albumin, and 50 µL of enzyme cocktails containing 0.4 IU/mL each of heparinase I, II, and III. The mixture was
incubated in a water bath at 25 °C for 48 hr. The enzymatic reaction was terminated by boiling for 2 min. Derivatization of digested LMWHs with AMAC The derivatization was carried out as previously described by Jackson [34]. LMWH digest was lyophilized and reconstituted with 5 µL of a 0.1 M AMAC solution in glacial acetic acid/DMSO (3/17, v/v) at room temperature for 15 min. Then 5 µL of a 1 M sodium cyanoborohydride was added to the solution. The mixture was incubated at 45 °C for 4 hr. Finally, 40 µL of 50% DMSO was added to the sample solution. The aliquots were used for HPLC-DAD-MS analysis. HPLC-DAD-MS analysis The analysis of labeled dalteparin and enoxaparin digests were performed on an LC-20A system (Shimadzu Co., Kyoto, Japan) equipped with a DAD detector. The RP column was an ODS-2 HYPERSIL C18 (5 µm, 250 × 4.6 mm I.D.) from Thermo Scientific (Barrington, IL, USA). Mobile phase A was 40 mM ammonium acetate (pH 5.6) in water while mobile phase B was methanol. Step gradient of from 2 to 20% mobile phase B in 55 min, followed by isocratic 20% B for 45 min, and then from 20 to 50% B in 20 min was used at a flow rate of 0.6 mL/min. The wavelength ranged from 200 nm to 500 nm was scanned and recorded. On-line ESI-MS was carried out on an ion trap-time of flight (IT-TOF) hybrid mass spectrometer (Shimadzu Co., Kyoto, Japan). The MS analysis was performed in the negative ion mode with the following instrument parameters, interface voltage: -3.5 kV; nebulizing gas flow rate: 1.5 L/min; curved desolvation line temperature:
7
200 °C; heating block temperature: 200 °C; detector voltage: 1.8 kV; and scan range: 100-800. Quantitation analysis Disaccharide ∆IP was used as internal standard for quantitation analysis. The disaccharides and oligosaccharides derived from dalteparin and enoxaparin were quantified based on their UV absorbance either at 232 nm or at 255 nm, normalized using the peak area of ∆IP at these two wavelengths, respectively. Results and discussion Selection of chromatographic conditions The general LMWH analysis strategy depends on completely enzymatic degradation to produce building blocks and then analyzing these components by monitoring their UV absorbance at 232 nm for the unsaturated uronic acids at the NRE. However, the original NRE of dalteparin is a saturated structure lacking of UV absorption, which poses a significant challenge for comprehensive structure analysis. Fluorescent derivatization prior to analysis solved the problem to some extent. But the original REs of dalteparin and some of enoxaparin chains have no hemiacetal hydroxyl group and are not reactive to labeling reagents. As a result, single wavelength UV or fluorescence detection cannot provide detailed compositional analysis that covers both terminal structures. It was reported that AMAC labeled oligosaccharides processed a UV absorption peak at 254 nm or 255 nm [23,32,35]. Full wavelength scan of AMAC-labeled ∆IS using HPLC-DAD showed maximum UV absorption at 255 nm.
In this experiment, we used DAD to monitor the
AMAC-labeled LMWH digests and recorded the UV absorption at 232 nm and 255 nm simultaneously. Nearly all the dalteparin and enoxaparin building blocks were successfully detected. ESI-MS was used to identify and confirm the structures of these building blocks.
8
Analysis of AMAC-labeled dalteparin-derived components After exhaustively digested by heparinase cocktail and labeled with AMAC, a total of 17 dalteparin-derived structures were successfully characterized by RP-HPLC-DAD-ESI-MS analysis. The UV chromatograms and total ion chromatogram (TIC) in the negative ion mode are presented in Fig. 1. In addition to the 8 natural heparin disaccharides, structural motifs from both termini of original dalteparin chains were identified and quantified for the first time. Two 3-O-sulfated tetrasaccharides and two trisaccharides were also observed. The 3-O-sulfated tetrasaccharides were generated from the antithrombin III binding pentasaccharide sequence, since it is resistant to heparinases when 3-O-sulfo substitution is present [10,36]. Two disaccharides and one tetrasaccharide from the RE of dalteparin contain 2,5-anhydromannitol-6-sulfate structure and cannot be labeled with AMAC. Therefore, they eluted prior to other components with AMAC label since AMAC significantly improves the hydrophobicity of those components. The NRE of dalteparin contains saturated 2-O-sulpho-α-L-idopyranosuronic acid structure. Two disaccharides with different sulfo group substitutions from NRE were identified, which gave additional 18 Da mass compared to the corresponding unsaturated disaccharides in ESI-MS. MS results of all these components are present in Table 2 and representative MS spectra of selected components are shown in Fig. 2. In addition to the dalteparin components reported by Galeotti and Volpi [17], five components including two saturated disaccharides from the NRE, two trisaccharides and one 3-O-tetrasaccharide were revealed. Analysis of AMAC-labeled enoxaparin-derived components The labeled enoxaparin digests were analyzed using the same approach as dalteparin. A total of 16 enoxaparin-derived species were separated and identified (Fig. 3). Besides of 8 natural
9
disaccharides, two 3-O-sulfated tetrasaccharides, two trisaccharides, and characteristic structures of enoxparin, including two 1,6-anhydro disaccharides and one 1,6-anhydro tetrasaccharide, were revealed by RP-HPLC-DAD-MS analysis. However, the two 1,6-anhydro disaccharides possessed nearly the same retention times and could not be differentiated in the UV chromatogram. They were quantified as a group in the quantitative composition analysis. The original NRE of enoxaparin chains contains unsaturated structures and cannot be differentiated from other unsaturated disaccharides resulted from enzymatic digestion. A saturated trisulfated disaccharide was found from the enzymatic digests of enoxaparin, which was also reported by Galeotti and Volpi [31]. It is from saturated enoxaparin chains, which are minor structures that inherited from the NRE of unfractionated heparin [27]. The MS results of enoxaparin are summarized in Table 2 and representative MS spectra are shown in Fig. 4. Previous work from other groups reported a total of 18 enoxaparin-derived components [17,28,31]. We identified an additional monosulfated trisaccharide. But the two galacturonic acid disaccharides (∆IISgal and ∆IISgal) reported by Zhang et al., were not
detected possibly due to them possessing the identical MWs and retention times with their uronic acid isomers. Quantitative composition analysis of LMWHs Because most of the uncommon disaccharide and oligosaccharide standards are not commercial available, all components were normalized using disaccharide ∆IP. Instead of calculating the absolute
amount of each component, relative composition was obtained for LMWH characterization and comparison analysis. The artificial heparin disaccharide ∆IP has similar structure with natural heparin disaccharides but distinct molecular weight. Moreover, it can be well separated from any other components of LMWHs’ digests. ∆IP also possesses aldehyde structure at its RE and can be
10
labeled by AMAC. All these make ∆IP an ideal internal standard for quantitative compositional analysis of LMWHs. The peak areas corresponding to ∆IP at 232 nm and 255 nm were integrated separately and the ratio was calculated. The quantities of all components from LMWH digests were then calculated from the values of their peak areas relative to ∆IP at 232 nm, or additionally multiplied by the ratio of ∆IP’s peak areas at two different wavelengths, if using peak areas at 255nm. Some AMAC labeled components also showed up in the UV chromatogram at 232 nm because of the non-specific absorption of AMAC. They were not counted during quantitation analysis because their identities could be proved by ESI-MS. All the quantitative results were incorporated into Table 3. The relative amounts of natural disaccharides are similar to the results reported by B. Wang and coworkers [30]. And more importantly, the composition of characteristic structures from both termini, the anticoagulant structure motifs, as well as structures from side reactions were also determined for the first time. The quantitation analysis expressed excellent linearity when plotted as a function of amount of each component versus injected amount of the range from 10 µg to 200 µg (Fig. 5). The R2 values ranged from 0.9720 to 0.9996 for dalteparin components and 0.9711 to 0.9993 for enoxaparin components. Conclusions Many sophisticated analytic techniques, including different types of HPLC, CE, and LC-ESI-MS, have aided in compositional analysis of heparin and LMWHs. The enzymatic digests of LMWHs possess more complicated compositions than heparin. And chemical reference standards for most of these modified structures are not commercial available. In this research, we established a comprehensive AMAC derivatization RP-HPLC-DAD-ESI-MS method, which is capable to detect, identify and quantify nearly all possible building blocks of dalteparin and
11
enoxaparin. Once all components are identified unambiguously by on-line ESI-MS, the RP-HPLC-DAD method can be used solely as a robust and economic approach for routine analysis of LMWHs in pharmaceutical industry. Dual wavelength UV detectors can be used to replace the DAD detector since only two wavelengths are required for quantitation analysis. With the superior separation resolution, high sensitivity to detect minor components, and detailed structural information provided by ESI-MS, application of the method described herein may be extended for structural analysis of other glycosaminoglycans with significant biological, diagnostic or pharmaceutical values.
Acknowledgments The authors acknowledge support from the National Major Scientific and Technological Special Project for Significant New Drugs Development (2012ZX0950200-005), the National Basic Research Program of China (973 Program) (2012CB822102), the National Natural Science Foundation of China (31000367), and the Natural Science Foundation of Shandong Province, China (ZR2010CM038 and ZR2011CM041).
12
References
[1] W. Wei, M.R. Niñonuevo, A. Sharma, L.M. Danan-Leon, J.A. Leary, A comprehensive compositional analysis of
heparin/heparan sulfate-derived disaccharides from human serum, Anal. Chem. 83 (2011) 3703-3708.
[2] M. Petitou, B. Casu, U. Lindahl, 1976-1983, a critical period in the history of heparin: the discovery of the antithrombin
binding site, Biochimie 85 (2003) 83-89.
[3] R.J. Linhardt, Heparin: an important drug enters its seventh decade, Chem. Ind. 2 (1991) 45-50.
[4] D.B. Brieger, K. Mak, K. Kottke-Marchant, E.J. Topol, Heparin-induced thrombocytopenia, J. Am. Coll. Cardiol. 31 (1998)
1449-1459.
[5] J. Fareed, W. Jeske, D. Hoppensteadt, R. Clarizio, J.M. Walenga, Low-molecular-weight heparins: pharmacologic profile
and product differentiation, Am. J. Cardiol. 82 (1998) 3L-10L.
[6] J. Hirsh, M.N. Levine, Low molecular weight heparin, Blood 79 (1992) 1-17.
[7] R.J. Linhardt, N.S. Gunay, Production and chemical processing of low molecular weight heparins, Semin. Thromb. Hemostasis
25 (1999) 5-16.
[8] J. Fareed, W.L. Leong, D.A. Hoppensteadt, W.P. Jeske, J. Walenga, R. Wahi, R.L. Bick, Generic low-molecular-weight
heparins: some practical considerations, Semin. Thromb. Hemostasis 30 (2004) 703-713.
[9] A. Bisio, D. Vecchietti, L. Citterio, M. Guerrini, R. Raman, S. Bertini, G. Eisele, A. Naggi, R. Sasisekharan, G. Torri, Structural
features of low-molecular-weight heparins affecting their affinity to antithrombin, Thromb. Haemostasis 102 (2009) 865-873.
[10] Z. Shriver, M. Sundaram, G. Venkataraman, J. Fareed, R. Linhardt, K. Biemann, R. Sasisekharan, Cleavage of the
antithrombin III binding site in heparin by heparinases and its implication in the generation of low molecular weight heparin,
PNAS 97 (2000) 10365-10370.
[11] L. Liverani, G. Mascellani, F. Spelta, Heparins: process-related physico-chemical and compositional characteristics,
fingerprints and impurities, Thromb. Haemostasis 102 (2009) 846-853.
13
[12] Z. Xiao, B.R. Tappen, M. Ly, W. Zhao, L.P. Canova, H. Guan, R.J. Linhardt, Heparin mapping using heparin lyases and the
generation of a novel low molecular weight heparin, J. Med. Chem. 54 (2011) 603-610.
[13] B. Yang, K. Solakyildirim, Y. Chang, R.J. Linhardt, Hyphenated techniques for the analysis of heparin and heparan sulfate,
Anal. Bioanal. Chem. 399 (2011) 541-557.
[14] S. Lee, A. Raw, L.Yu, R. Lionberger, N. Ya, D. Verthelyi, A. Rosenberg, S. Kozlowski, K. Webber, J. Woodcock, Scientific
considerations in the review and approval of generic enoxaparin in the United States, Nat. Biotechnol. 31 (2013) 220-226.
[15] A. Pervin, C. Gallo, K.A. Jandik, X. Han, R.J. Linhardt, Preparation and structural characterization of large heparin-derived
oligosaccharides, Glycobiology 5 (1995) 83-95.
[16] Y. Takegawa, K. Araki, N. Fujitani, J. Furukawa, H. Sugiyama, H. Sakai, Y. Shinohara, Simultaneous analysis of heparan
sulfate, chondroitin/dermatan sulfates, and hyaluronan disaccharides by glycoblotting-assisted sample preparation followed
by single-step zwitter-ionic-hydrophilic interaction chromatography, Anal. Chem. 83 (2011) 9443-9449.
[17] F. Galeotti, N. Volpi, Novel reverse-phase ion pair-high performance liquid chromatography separation of heparin, heparan
sulfate and low molecular weight-heparins disaccharides and oligosaccharides, J. Chromatogr. A 1284 (2013) 141-147.
[18] F. Lamari, M. Militsopoulou, X. Gioldassi, N. K. Karamanos, Capillary electrophoresis: a superior miniaturized tool for
analysis of the mono-, di-, and oligosaccharide constituents of glycan moieties in proteoglycans, Fresenius J. Anal. Chem. 371
(2001) 157-167.
[19] J.T. King, U.R. Desai, A capillary electrophoretic method for fingerprinting low molecular weight heparins, Anal. Biochem.
380 (2008) 229-234.
[20] A. H.K.Plaas, V. C.Hascall, R. J.Midura, Ion exchange HPLC microanalysis of chondroitin sulfate: quantitative derivatization
of chondroitin lyase digestion products with 2-aminopyridine, Glycobiology 6 (1996) 823-829.
[21] A. Kinoshita, K. Sugahara, Microanalysis of glycosaminoglycan-derived oligosaccharides labeled with a fluorophore
2-aminobenzamide by high-performance liquid chromatography: application to disaccharide composition analysis and
14
exosequencing of oligosaccharides, Anal. Biochem. 269 (1999) 367-378.
[22] M. Ambrosius, K. Kleesiek, C. Götting, Quantitative determination of the glycosaminoglycan ∆-disaccharide composition of
serum, platelets and granulocytes by reversed-phase high-performance liquid chromatography, J. Chromatogr. A 1201 (2008)
54-60.
[23] H. Kitagawa, A. Kinoshita, K. Sugahara, Microanalysis of glycosaminoglycan-derived disaccharides labeled with the
fluorophore 2-aminoacridone by capillary electrophoresis and high-performance liquid chromatography, Anal. Biochem. 232
(1995) 114-121.
[24] L. Chi, J. Amster, R.J. Linhardt, Mass spectrometry for the analysis highly charged sulfated carbohydrates, Curr. Anal. Chem.
1 (2005) 223-240.
[25] N. Volpi, R.J. Linhardt, High-performance liquid chromatography-mass spectrometry for mapping and sequencing
glycosaminoglycan-derived oligosaccharides, Nat. Protoc. 5 (2010) 993-1004.
[26] D. Li, L. Chi, L. Jin, X. Xu, X. Du, S. Ji, L. Chi, Mapping of low molecular weight heparins using reversed phase ion pair
liquid chromatography-mass spectrometry, Carbohydr. Polym. 99 (2014) 339-344.
[27] L. Li, F. Zhang, J. Zaia, R.J. Linhardt, Top-Down approach for the direct characterization of low molecular weight heparins
using LC-FT-MS, Anal. Chem. 84 (2012) 8822-8829.
[28] Q. Zhang, X. Chen, Z. Zhu, X. Zhan, Y. Wu, L. Song, J. Kang, Structural analysis of low molecular weight heparin by
ultraperformance size exclusion chromatography/time of flight mass spectrometry and capillary zone electrophoresis, Anal.
Chem. 85 (2013) 1819-1827.
[29] D.J. Langeslay, E. Urso, C. Gardini, A. Naggi, G. Torri, C.K. Larive, Reversed-phase ion-pair ultra-high-performance-liquid
chromatography-mass spectrometry for fingerprinting low-molecular-weight heparins, J. Chromatogr. A 1292 (2013)
201-210.
[30] B. Wang, L.F. Buhse, A. Al-Hakim, M.T. Boyne li, D.A. Keire, Characterization of currently marketed heparin products:
15
analysis of heparin digests by RPIP-UHPLC-QTOF-MS, J. Pharm. Biomed. Anal. 67-68 (2012) 42-50.
[31] F. Galeotti, N. Volpi, Online reverse phase-high-performance liquid chromatography-fluorescence detection-electrospray
ionization-mass spectrometry separation and characterization of heparan sulfate, heparin, and low-molecular weight-heparin
disaccharides derivatized with 2-aminoacridone, Anal. Chem. 83 (2011) 6770-6777.
[32] B. Yang, Y. Chang, A.M. Weyers, E. Sterner, R.J. Linhardt, Disaccharide analysis of glycosaminoglycan mixtures by
ultra-high-performance liquid chromatography-mass spectrometry, J. Chromatogr. A 1225 (2012) 91-98.
[33] Z. Zhang, J. Xie, H. Liu, J. Liu, R.J. Linhardt, Quantification of heparan sulfate disaccharides using ion-pairing
reversed-phase microflow high-performance liquid chromatography with electrospray ionization trap mass spectrometry,
Anal. Chem. 81 (2009) 4349-4355.
[34] P. Jackson, Polyacrylamide gel electrophoresis of reducing saccharides labeled with the fluorophore 2-aminoacridone:
subpicomolar detection using an imaging system based on a cooled charge-coupled device, Anal. Biochem. 196 (1991)
238-244.
[35] M. Militsopoulou, F.N. Lamari, A.H. Nikos, K. Karamanos, Determination of twelve heparin- and heparin sulfate-derived
disaccharides as 2-aminoacridone derivatives by capillary zone electrophoresis using ultraviolet and laser-induced
fluorescence detection, Electrophoresis 23 (2002) 1104-1109.
[36] M. Sundaram, Y. Qi, Z. Shriver, D. Liu, G. Zhao, G. Venkataraman, R. Langer, R. Sasisekharan, Rational design of
low-molecular weight heparins with improved in vivo activity, PNAS 100 (2003) 651-656.
16
Table 1 The structures of dalteparin, enoxaparin, and corresponding building blocks. -O3S
O O OH
O CO2OH
CH2OR1 O O
OR1
R=
OH
O NHR2 CO2O OH
O CO2OH
CH2OR1 O OR OR1
O
NHR2
OR1 O O
OR1
OH
O
O NHR2
OH O
OR1 n
O or H
OH
OSO3-
n
O CO2O OH OH OSO3-
CH2OR1 O
CO2-
NHSO3-
R1 = H or SO3Na, R2 = SO3Na or CO-CH3
R1= H or SO3Na, R2 = H or SO3Na or CO-CH3
Dalteparin natricum
Enoxaparin natricum
Name
Structure
∆IS
∆UA2S-GlcNS6S
∆IIS
∆UA-GlcNS6S
Common heparin disaccharides
∆IIIS
∆UA2S-GlcNS
∆IVS
∆UA-GlcNS
∆IA
∆UA2S-GlcNAc6S
∆IIA
∆UA-GlcNAc6S
∆IIIA
∆UA2S-GlcNAc
∆IVA
∆UA-GlcNAc
∆dp4(4OS,1NS) RE
∆UA2S-GlcNS6S-IdoA2S-Mnt6S-2,5-anhydro
∆dp2(2OS) RE
∆UA2S-Mnt6S-2,5-anhydro
Dalteparin characteristic structures
∆dp2(1OS) RE
∆UA-Mnt6S-2,5-anhydro
dp2(2OS,1NS) NRE
IdoA2S-GlcNS6S
dp2(1OS,1NS) NRE
IdoA2S-GlcNS
∆IIA-IVSglu
∆UA-GlcNAc6S-GlcA-GlcNS3S
∆IIA-IISglu
∆UA-GlcNAc6S-GlcA-GlcNS3S6S
∆dp3(3OS,1NS)
∆UA2S-GlcNS6S-IdoA2S
∆dp3(1OS)
∆UA-GlcNAc-IdoA (1OS)
1,6-anhydro ∆IS-ISepi
∆UA2S-GlcNS6S-IdoA2S-ManNS-1,6-anhydro
Enoxaparin characteristic structures
Synthetic heparin disaccharide
1,6-anhydro ∆IS
∆UA2S-GlcNS-1,6-anhydro
1,6-anhydro ∆IIS
∆UA-GlcNS-1,6-anhydro
dp2(2OS,1NS) NRE
IdoA2S-GlcNS6S
∆IIA-IVSglu
∆UA-GlcNAc6S-GlcA-GlcNS3S
∆IIA-IISglu
∆UA-GlcNAc6S-GlcA-GlcNS3S6S
∆dp3(3OS,1NS)
∆UA2S-GlcNS6S-IdoA2S
∆dp3(1OS)
∆UA-GlcNAc-IdoA (1OS)
∆IP
∆UA2S-GlcNCOEt6S
17
∆UA: unsaturated uronic acid. GlcA: glucuronic acid. IdoA: iduronic acid. GlcN: glucosamine. ManN: mannosamine. Ac: acetyl group. S: sulfo group. COEt: ethoxycarbonyl. Mnt: mannitol.
18
Table 2 Theoretical and experimental molecular mass of underivatized and AMAC-labeled LMWH building blocks. Number
Name
Theoretical MWmono
Theoretical MWmono
Major ions
(underivatized)
(AMAC-labeled)
(m/z)
Peak interpretation
Experimental MWmono
Dalterparin 1
∆dp4(4OS,1NS) RE
1058.9750
n.r.
528.4836
[M-2H]2-
1058.9829
2
∆dp2(2OS) RE
482.0036
n.r.
480.9989
[M-H]-
482.0067
401.0408
-
402.0486
3
∆dp2(1OS) RE
402.0468
n.r.
[M-H]
2-
1230.1313 1150.1689
4
∆IIA-IISglu
1036.0396
1230.1240
614.0578
[M-2H]
5
∆IIA-IVSglu
956.0828
1150.1672
574.0766
[M-2H]2-
384.5215
[M-2H]
22-
6
∆IS
576.9713
771.0557
771.0587
7
dp2(2OS,1NS) NRE
594.9819
789.0663
393.5270
[M-2H]
8
∆dp3 (3OS,1NS)
832.9602
1027.0446
512.5191
[M-2H]2-
1027.0539
9
dp2(1OS,1NS) NRE
515.0251
709.1095
353.5470
[M-H]2-
709.1097
-
789.0697
10
∆IIS
497.0145
691.0989
690.0963
[M-H]
691.1041
11
∆IIIS
497.0145
691.0989
690.0957
[M-H]-
691.1035
610.1392
-
611.1470
12
∆IVS
417.0577
611.1421
[M-H]
-
13
∆IA
539.0251
733.1095
732.1029
[M-H]
733.1107
14
∆dp3 (1OS)
635.1004
829.1848
413.5861
[M-H]2-
829.1879
15
∆IIA
459.0683
653.1527
652.1497
[M-H]-
653.1575
2-
16
∆IP
553.0407
747.1251
372.5553
[M-2H]
17
∆IIIA
459.0683
653.1527
652.1469
[M-H]-
653.1547
747.1263
18
∆IVA
379.1115
573.1959
572.1926
[M-H]-
573.2004
1,6-anhydro ∆IS-ISepi
1055.9753
n.r.
526.9811
[M-2H]2-
1055.9779
Enoxaparin 1 2
1,6-anhydro∆IS
479.0040
n.r.
477.9983
-
[M-H]
-
479.0061
1,6-anhydro∆IIS
399.0471
n.r.
398.0390
[M-H]
399.0468
3
∆IIA-IISglu
1036.0396
1230.1240
614.0560
[M-2H]2-
1230.1277
4
∆IIA-IVSglu
956.0828
1150.1672
574.0758
[M-2H]2-
1150.1673
2-
771.0515 789.0655
5
∆IS
576.9713
771.0557
384.5179
[M-2H]
6
dp2(2OS,1NS) NRE
594.9819
789.0663
393.5249
[M-2H]2-
512.5183
[M-2H]
2-
-
7
∆dp3 (3OS,1NS)
832.9602
1027.0446
1027.0523
8
∆IIS
497.0145
691.0989
690.0932
[M-H]
691.1010
9
∆IIIS
497.0145
691.0989
690.0936
[M-H]-
691.1014
610.1354
-
611.1432
10
∆IVS
417.0577
611.1421
[M-H]
-
11
∆IA
539.0251
733.1095
732.1049
[M-H]
733.1127
12
∆dp3 (1OS)
635.1004
829.1848
413.5863
[M-H]2-
829.1883
13
∆IIA
459.0683
653.1527
652.1462
[M-H]-
653.1540
2-
14
∆IP
553.0407
747.1251
372.5526
[M-2H]
15
∆IIIA
459.0683
653.1527
652.1467
[M-H]-
653.1545
16
∆IVA
379.1115
573.1959
572.1885
[M-H]-
573.1963
n.r.: components can not be reacted with AMAC. MWmono: monoisotopic molecular weight.
19
747.1209
Table 3 Quantitative composition analysis of dalteparin and enoxaparina. LMWHs Dalteparin
∆IS 63.42±1.10
∆ IIS 10.95±0.38
∆IIIS 5.32±0.36
Disaccharide (%) ∆IVS ∆IA 1.49±0.02 1.96±0.07
∆IIA 5.78±0.42
∆IIIA 1.42±0.05
∆IVA 2.08±0.23
Tetrasaccharide (%) ∆IIA-IVSglu ∆IIA-IISglu 0.03±0.00 0.24±0.05
Enoxaparin
63.13±0.79
11.91±0.63
6.63±0.40
3.52±0.10
4.33±0.15
1.69±0.07
3.70±0.12
0.03±0.00
1.61±0.06
LMWHs Dalteparin Enoxaparin
∆dp2(1OS) RE 0.77±0.10
Terminal structures (%) ∆dp4(4OS,1NS) RE 0.04±0.01
∆dp2(2OS) RE 0.26±0.03 dp2(2OS,1NS) NRE
1,6-anhydro ∆IS and 1,6-anhydro∆IS 0.18±0.01
1.61±0.39 a
In this experiment, the values were means of three measurements ± standard deviation (SD)
20
0.09±0.01
dp2(1OS,1NS) NRE 0.24±0.07 1,6-anhydro ∆IS-ISepi
0.15±0.00
Trisaccharide (%) ∆dp3(3OS,1NS) ∆dp3(1OS) 0.05±0.02 1.63±0.16 0.08±0.01
1.33±0.22
dp2(2OS,1NS) NRE 4.33±1.40
Figure legends
Figure 1. RP-HPLC-DAD-MS chromatograms of AMAC-labeled dalteparin-derived components (injected 50 µg). (A) TIC, (B)
chromatogram with UV detection at 255 nm, and (C) chromatogram with UV detection at 232 nm. Peak assignment: 1.
∆dp4(4OS,1NS) RE (underivatized), 2. ∆dp2(2OS) RE (underivatized), 3. ∆dp2(1OS) RE (underivatized); 4. ∆IIA-IISglu, 5.
∆IIA-IVSglu, 6. ∆IS, 7. dp2(2OS,1NS) NRE, 8. ∆dp3(3OS,1NS), 9. dp2(1OS,1NS) NRE, 10. ∆IIS, 11. ∆IIIS, 12. ∆IVS, 13. ∆IA, 14.
∆dp3(1OS), 15. ∆IIA, 16. ∆IP, 17. ∆IIIA, and 18. ∆IVA.
Figure 2. Representative ESI-MS spectra of characteristic dalteparin-derived components. (A) ∆dp4(4OS,1NS) RE
(underivatized), (B) ∆dp2(2OS) RE (underivatized), (C) ∆dp2(1OS) RE (underivatized), (D) dp2(2OS,1NS) NRE, and (E)
dp2(1OS,1NS) NRE.
Figure 3. RP-HPLC-DAD-MS chromatogram of AMAC-labeled enoxaparin-derived components (injected 30 µg). (A) TIC, (B)
chromatogram with UV detection at 255 nm, and (C) chromatogram with UV detection at 232 nm. Peak assignment: 1.
1,6-anhydro ∆IS-ISepi (underivatized), 2. 1,6-anhydro ∆IS (underivatized) and 1,6-anhydro ∆IIS (underivatized), 3. ∆IIA-IISglu, 4.
∆IIA-IVSglu, 5. ∆IS, 6. dp2(2OS,1NS) NRE, 7. ∆dp3(3OS,1NS), 8. ∆IIS, 9. ∆IIIS, 10. ∆IVS, 11. ∆IA, 12. ∆dp3(1OS), 13. ∆IIA, 14.
∆IP, 15. ∆IIIA, and 16. ∆IVA.
Figure 4. Representative ESI-MS spectra of characteristic enoxaparin-derived components. (A) 1,6-anhydro ∆IS-ISepi
(underivatized), (B) 1,6-anhydro ∆IS (underivatized), and (C) dp2(2OS,1NS) NRE.
Figure 5. Quantitative compositional analysis of LMWHs. The curves and linear equations of intensity as a function of
concentration for each building block are shown. The amounts of LMWHs used for RP-HPLC-DAD-MS analysis were 10, 25, 50,
100, and 200 µg, respectively. (A) the common disaccharides of dalteparin, (B) the characteristic building blocks of dalteparin, (C)
the common disaccharides of enoxaparin, and (D) the characteristic building blocks of enoxaparin.
21
22
23
24
25
26
S0003-2697(14)00054-2 http://dx.doi.org/10.1016/j.ab.2014.02.005 YABIO 11644
To appear in:
Analytical Biochemistry
Received Date: Revised Date: Accepted Date:
22 November 2013 2 February 2014 4 February 2014
Please cite this article as: Z. Wang, D. Li, X. Sun, X. Bai, L. Jin, L. Chi, Liquid chromatography-diode array detection-mass spectrometry for compositional analysis of low molecular weight heparins, Analytical Biochemistry (2014), doi: http://dx.doi.org/10.1016/j.ab.2014.02.005
This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
Liquid chromatography-diode array detection-mass spectrometry for compositional analysis of low molecular weight heparins Short title: Compositional analysis of low molecular weight heparins Zhangjie Wanga,b, Daoyuan Lia,b, Xiaojun Suna, Xue Baia, Lan Jina, Lianli Chia,b,* a
National Glycoengineering Research Center, Shandong University, Jinan 250100, China
b
State Key Laboratory of Microbial Technology, Shandong University, Jinan 250100, China
*Corresponding author. Tel: +86 531 8836 3200, Fax: +86 531 8836 3002 E-mail address: [email protected] (L. Chi). Categories: carbohydrates; chromatographic techniques Abbreviations used: LMWH, low-molecular-weight heparin; RP, reversed phase; DAD, diode array detection; ESI, electrospray ionization; MS, mass spectrometry; AMAC, 2-aminoacridone; RE, reducing end; NRE, non-reducing end; HPLC, high performance liquid chromatography; SAX, strong anion exchange; UV, ultraviolet; HILIC, hydrophilic interaction liquid chromatography; RPIP, reversed phase ion pairing; CE, capillary electrophoresis; DMSO, dimethylsulfoxide; IT-TOF, ion trap-time of flight; TIC, total ion chromatogram.
1
ABSTRACT
Low molecular weight heparins (LMWHs) are important artificial preparations from heparin polysaccharide and
widely used as anticoagulant drugs. To analyze the structure and composition of LMWHs, identification and
quantitation of their natural and modified building blocks are indispensable. We established a novel reversed phase
(RP) high performance liquid chromatography (HPLC)-diode array detection (DAD)-electrospray ionization-mass
spectrometry (ESI-MS) approach for compositional analysis of LMWHs. After exhaustively digested and labeled
with 2-aminoacridone (AMAC), the structural motifs constructing LMWHs, including 17 components from
dalteparin and 15 components from enoxaparin, were well separated, identified, and quantified. Besides the 8 natural
heparin disaccharides, many characteristic structures from dalteparin and enoxaparin, such as modified structures
from the reducing end (RE) and non-reducing end (NRE), 3-O-sulfated tetrasaccharides, as well as trisaccharides,
have been unambiguously identified based on their retention time and mass spectra. Comparing to the traditional
heparin compositional analysis methods, the approach described herein is not only robust, but also comprehensive
because it is capable to identify and quantify nearly all components from lyases digests of LMWHs.
Keywords: LMWH; DAD; AMAC derivatization; ESI-MS; Compositional analysis
2
Heparin is a heterogeneous polysaccharide in terms of variable chain length, sulfo and N-acetyl substitutions, and epimerization. It consists of repeating disaccharide units of β-D-glucuronic acid (GlcA) or α-L-iduronic acid (IdoA) (1→4) linked to D-glucosamine. The hexuronic acid may be 2-O-sulfated and glucosamine may be 6-O-sulfated, N-sulfated, N-acetylated, or infrequently 3-O-sulfated. For a long time, heparin has been extensively used as clinical anticoagulant drugs for prevention and treatment of thrombotic diseases [1,2,3]. However, the negative effects of heparin, including bleeding and inducing thrombocytopenia and osteoporosis, limit its clinical application [4]. LMWHs are overtaking the market share of heparin as new anticoagulant and anti-thrombotic drugs in particular due to their improved bioavailability and reduced bleeding risk [5,6]. Several types of LMWHs have been produced by either controlled enzymatic or chemical depolymerization of heparin, including heparinase depolymerization, nitrous acid degradation, β-elimination, and hydrolytic cleavage with hydrogen peroxide [7]. The enzymatic and chemical reactions usually result in modified structures at the cleavage sites, i.e. the RE and NRE of LMWHs. For example, enoxaparin is manufactured by alkaline treatment of benzyl ester derivative of heparin. The NRE becomes an unsaturated hexuronic acid, and the RE is a 1,6-anhydro structure for 15-25% of the constitutes [8]. Dalteparin is produced by nitrous acid depolymerization. The NRE is a 2-O-sulfo-α-L-idopyranosuronic acid and the RE is a 6-O-sulfo-2,5-anhydro-D-mannitol structure [9]. Besides the natural disaccharides and modified structures at the RE and NRE, LMWHs also contain 3-O-sulfated pentasaccharide structures, which are responsible for their anticoagulant activity [10]. Peeling reaction is a common side reaction during preparation of heparin and LMWHs, which results in glucosamine structures at the RE [11]. Since heparin and LMWHs are heterogeneous macro biomolecules, they are usually degraded
3
completely into basic building blocks, and then analyzed using chromatographic or electrophoretic techniques. The cocktail of three types of heparinase (heparinase I, II and III) isolated from Flavobacterium heparinum are generally used to deconstruct heparin and LMWHs [12,13]. In the case of enoxaparin and dalteparin, the possible structures from exhaustive digestion include eight natural disaccharides, modified oligosaccharides from both termini, 3-O-sulfated tetrasaccharides, as well as trisaccharides derived from peeling reaction (Table 1). Compositional analysis is a fundamental approach to characterize heparin and LMWHs. It is required by the United States Food and Drug Administration to demonstrate the compositional sameness between generic and innovator’s LMWH products for an Abbreviated New Drug Application [14]. HPLC are conventional methods and have been extensively used to determine the composition of heparin and LMWHs. Strong anion exchange (SAX)-HPLC, regarded as a general analytic method, has been largely used to identify and characterize heparin and LMWHs-derived oligosaccharides based on their acidic properties and characteristic UV absorption at 232 nm [15]. Hydrophilic interaction liquid chromatography (HILIC) is capable to separate hydrophilic analytes, and has been reported to separate heparin and other glycosaminoglycan-derived disaccharides [16]. In recent years, reversed phase ion pairing (RPIP)-HPLC is drawing increasing attention. The ion pairing reagents modify the hydrophobicity of hydrophilic heparin-derived disaccharides and oligosaccharides, which can then be well resolved on traditional RP columns [17]. Capillary electrophoresis (CE) has been also employed to analyze LMWHs’ composition with relatively high resolution and low limit of detection (10-20 pg) [18,19]. However, most of the HPLC and CE methods mentioned above are only limited to detect eight natural heparin disaccharides. The exhaustive digestion of LMWHs results in more complicated disaccharide and oligosaccharide
4
composition. Unlike the natural heparin disaccharides, there are no commercial available reference standards for modified disaccharides and oligosaccharides. Moreover, some of these disaccharides and oligosaccharides lack unsaturated structures at NRE and cannot be monitored by UV detector in HPLC or CE analysis. Fluorescence derivatization techniques are frequently used to facilitate the separation and detection of heparin-derived oligosaccharides. A variety of fluorophores, such as 2-aminopyridine, 2-aminobenzamide, can be covalently attached to the RE of oligosaccharides through reductive amination reaction [20,21]. AMAC labeling is a newly developed method for heparin disaccharide analysis [22,23]. The labeled disaccharides can be directly separated on a C18 column, driven by the hydrophobicity of AMAC. The serial of retention times of eight disaccharides is reversed compared to the SAX and RPIP separations. The limitation of fluorescence derivatization methods is that they require aldehyde structure at the RE of oligosaccharides. Dalteparin chains contain 2,5-anhydro-D-mannitol structure while some of enoxaparin chains contain 1,6-anhydro structure at their REs. These non-reducible structures are not reactive to fluorescence reagents. MS techniques with soft ionization methods, such as ESI and matrix assisted laser induced ionization, have been remarkably successful in the structural characterization of heparin and LMWH oligosaccharides [24,25]. ESI-MS is more favored because it can be easily connected downstream to HPLC and CE separations. Typically, separation systems with no salts or low
concentration of volatile salts, including RPIP, HILIC, and GPC, are compatible with on-line ESI-MS analysis [26,27,28]. RPIP-ultra HPLC-MS methods have been applied to analyze the digests of heparin and LMWHs [29,30]. However, ion pairing reagents cause instrument contamination problem and are usually desired to avoid if possible. Hydrophobic fluorescence tags
5
can affect the retention properties of labeled heparin disaccharides on the regular RP columns. Several RP-HPLC-ESI-MS approaches have been attempted in the compositional analysis of different types of glycosaminoglycans [31,32]. The drawback of LC-MS methods is the relatively high instrument and maintenance costs. Moreover, quantitation analysis using MS data without isotopic standards is usually not optimal [33]. Generally it is preferred to use UV signals for quantitation analysis while using MS for peak identification. In this study, we established a RP-HPLC-DAD-MS approach for compositional analysis of two different LMWHs, dalteparin and enoxaparin. The DAD was used to detect disaccharides and oligosaccharides from the heparinase digests of LMWHs, by monitoring wavelengths at 232 nm for unsaturated uronic acids at NRE and 255 nm for the AMAC derivatives simultaneously. ESI-MS was used to identify the AMAC labeled or underivatized structures unambiguously. By using the synthetic heparin disaccharide ∆UA2S-GlcNCOEt6S (∆IP) as internal standard, eight heparin natural disaccharides, modified terminal structures, 3-O-sulfated tetrasaccharides, as well as trisaccharides were identified and quantified. Materials and methods Materials Dalteparin and enoxaparin reference standards were purchased from European Pharmacopoeia. Heparinase I, II and III were obtained from Aglyco (Beijing, China). Heparin disaccharide standards were purchased from Iduron Co. (Manchester, U.K.). AMAC, dimethylsulfoxide (DMSO) and sodium cyanoborohydride were from Sigma-Aldrich (St. Louis, MO, USA). All other reagents and chemicals were of the highest quality available. Enzymatic digestion of LMWHs
6
The enzymatic digestion solution was a mixture of 10 µL of 20µg/ µL LMWH sample, 35 µL of 100 mM sodium acetate/2mM calcium acetate buffer (pH 7.0) containing 0.1 g/L bovine serum albumin, and 50 µL of enzyme cocktails containing 0.4 IU/mL each of heparinase I, II, and III. The mixture was
incubated in a water bath at 25 °C for 48 hr. The enzymatic reaction was terminated by boiling for 2 min. Derivatization of digested LMWHs with AMAC The derivatization was carried out as previously described by Jackson [34]. LMWH digest was lyophilized and reconstituted with 5 µL of a 0.1 M AMAC solution in glacial acetic acid/DMSO (3/17, v/v) at room temperature for 15 min. Then 5 µL of a 1 M sodium cyanoborohydride was added to the solution. The mixture was incubated at 45 °C for 4 hr. Finally, 40 µL of 50% DMSO was added to the sample solution. The aliquots were used for HPLC-DAD-MS analysis. HPLC-DAD-MS analysis The analysis of labeled dalteparin and enoxaparin digests were performed on an LC-20A system (Shimadzu Co., Kyoto, Japan) equipped with a DAD detector. The RP column was an ODS-2 HYPERSIL C18 (5 µm, 250 × 4.6 mm I.D.) from Thermo Scientific (Barrington, IL, USA). Mobile phase A was 40 mM ammonium acetate (pH 5.6) in water while mobile phase B was methanol. Step gradient of from 2 to 20% mobile phase B in 55 min, followed by isocratic 20% B for 45 min, and then from 20 to 50% B in 20 min was used at a flow rate of 0.6 mL/min. The wavelength ranged from 200 nm to 500 nm was scanned and recorded. On-line ESI-MS was carried out on an ion trap-time of flight (IT-TOF) hybrid mass spectrometer (Shimadzu Co., Kyoto, Japan). The MS analysis was performed in the negative ion mode with the following instrument parameters, interface voltage: -3.5 kV; nebulizing gas flow rate: 1.5 L/min; curved desolvation line temperature:
7
200 °C; heating block temperature: 200 °C; detector voltage: 1.8 kV; and scan range: 100-800. Quantitation analysis Disaccharide ∆IP was used as internal standard for quantitation analysis. The disaccharides and oligosaccharides derived from dalteparin and enoxaparin were quantified based on their UV absorbance either at 232 nm or at 255 nm, normalized using the peak area of ∆IP at these two wavelengths, respectively. Results and discussion Selection of chromatographic conditions The general LMWH analysis strategy depends on completely enzymatic degradation to produce building blocks and then analyzing these components by monitoring their UV absorbance at 232 nm for the unsaturated uronic acids at the NRE. However, the original NRE of dalteparin is a saturated structure lacking of UV absorption, which poses a significant challenge for comprehensive structure analysis. Fluorescent derivatization prior to analysis solved the problem to some extent. But the original REs of dalteparin and some of enoxaparin chains have no hemiacetal hydroxyl group and are not reactive to labeling reagents. As a result, single wavelength UV or fluorescence detection cannot provide detailed compositional analysis that covers both terminal structures. It was reported that AMAC labeled oligosaccharides processed a UV absorption peak at 254 nm or 255 nm [23,32,35]. Full wavelength scan of AMAC-labeled ∆IS using HPLC-DAD showed maximum UV absorption at 255 nm.
In this experiment, we used DAD to monitor the
AMAC-labeled LMWH digests and recorded the UV absorption at 232 nm and 255 nm simultaneously. Nearly all the dalteparin and enoxaparin building blocks were successfully detected. ESI-MS was used to identify and confirm the structures of these building blocks.
8
Analysis of AMAC-labeled dalteparin-derived components After exhaustively digested by heparinase cocktail and labeled with AMAC, a total of 17 dalteparin-derived structures were successfully characterized by RP-HPLC-DAD-ESI-MS analysis. The UV chromatograms and total ion chromatogram (TIC) in the negative ion mode are presented in Fig. 1. In addition to the 8 natural heparin disaccharides, structural motifs from both termini of original dalteparin chains were identified and quantified for the first time. Two 3-O-sulfated tetrasaccharides and two trisaccharides were also observed. The 3-O-sulfated tetrasaccharides were generated from the antithrombin III binding pentasaccharide sequence, since it is resistant to heparinases when 3-O-sulfo substitution is present [10,36]. Two disaccharides and one tetrasaccharide from the RE of dalteparin contain 2,5-anhydromannitol-6-sulfate structure and cannot be labeled with AMAC. Therefore, they eluted prior to other components with AMAC label since AMAC significantly improves the hydrophobicity of those components. The NRE of dalteparin contains saturated 2-O-sulpho-α-L-idopyranosuronic acid structure. Two disaccharides with different sulfo group substitutions from NRE were identified, which gave additional 18 Da mass compared to the corresponding unsaturated disaccharides in ESI-MS. MS results of all these components are present in Table 2 and representative MS spectra of selected components are shown in Fig. 2. In addition to the dalteparin components reported by Galeotti and Volpi [17], five components including two saturated disaccharides from the NRE, two trisaccharides and one 3-O-tetrasaccharide were revealed. Analysis of AMAC-labeled enoxaparin-derived components The labeled enoxaparin digests were analyzed using the same approach as dalteparin. A total of 16 enoxaparin-derived species were separated and identified (Fig. 3). Besides of 8 natural
9
disaccharides, two 3-O-sulfated tetrasaccharides, two trisaccharides, and characteristic structures of enoxparin, including two 1,6-anhydro disaccharides and one 1,6-anhydro tetrasaccharide, were revealed by RP-HPLC-DAD-MS analysis. However, the two 1,6-anhydro disaccharides possessed nearly the same retention times and could not be differentiated in the UV chromatogram. They were quantified as a group in the quantitative composition analysis. The original NRE of enoxaparin chains contains unsaturated structures and cannot be differentiated from other unsaturated disaccharides resulted from enzymatic digestion. A saturated trisulfated disaccharide was found from the enzymatic digests of enoxaparin, which was also reported by Galeotti and Volpi [31]. It is from saturated enoxaparin chains, which are minor structures that inherited from the NRE of unfractionated heparin [27]. The MS results of enoxaparin are summarized in Table 2 and representative MS spectra are shown in Fig. 4. Previous work from other groups reported a total of 18 enoxaparin-derived components [17,28,31]. We identified an additional monosulfated trisaccharide. But the two galacturonic acid disaccharides (∆IISgal and ∆IISgal) reported by Zhang et al., were not
detected possibly due to them possessing the identical MWs and retention times with their uronic acid isomers. Quantitative composition analysis of LMWHs Because most of the uncommon disaccharide and oligosaccharide standards are not commercial available, all components were normalized using disaccharide ∆IP. Instead of calculating the absolute
amount of each component, relative composition was obtained for LMWH characterization and comparison analysis. The artificial heparin disaccharide ∆IP has similar structure with natural heparin disaccharides but distinct molecular weight. Moreover, it can be well separated from any other components of LMWHs’ digests. ∆IP also possesses aldehyde structure at its RE and can be
10
labeled by AMAC. All these make ∆IP an ideal internal standard for quantitative compositional analysis of LMWHs. The peak areas corresponding to ∆IP at 232 nm and 255 nm were integrated separately and the ratio was calculated. The quantities of all components from LMWH digests were then calculated from the values of their peak areas relative to ∆IP at 232 nm, or additionally multiplied by the ratio of ∆IP’s peak areas at two different wavelengths, if using peak areas at 255nm. Some AMAC labeled components also showed up in the UV chromatogram at 232 nm because of the non-specific absorption of AMAC. They were not counted during quantitation analysis because their identities could be proved by ESI-MS. All the quantitative results were incorporated into Table 3. The relative amounts of natural disaccharides are similar to the results reported by B. Wang and coworkers [30]. And more importantly, the composition of characteristic structures from both termini, the anticoagulant structure motifs, as well as structures from side reactions were also determined for the first time. The quantitation analysis expressed excellent linearity when plotted as a function of amount of each component versus injected amount of the range from 10 µg to 200 µg (Fig. 5). The R2 values ranged from 0.9720 to 0.9996 for dalteparin components and 0.9711 to 0.9993 for enoxaparin components. Conclusions Many sophisticated analytic techniques, including different types of HPLC, CE, and LC-ESI-MS, have aided in compositional analysis of heparin and LMWHs. The enzymatic digests of LMWHs possess more complicated compositions than heparin. And chemical reference standards for most of these modified structures are not commercial available. In this research, we established a comprehensive AMAC derivatization RP-HPLC-DAD-ESI-MS method, which is capable to detect, identify and quantify nearly all possible building blocks of dalteparin and
11
enoxaparin. Once all components are identified unambiguously by on-line ESI-MS, the RP-HPLC-DAD method can be used solely as a robust and economic approach for routine analysis of LMWHs in pharmaceutical industry. Dual wavelength UV detectors can be used to replace the DAD detector since only two wavelengths are required for quantitation analysis. With the superior separation resolution, high sensitivity to detect minor components, and detailed structural information provided by ESI-MS, application of the method described herein may be extended for structural analysis of other glycosaminoglycans with significant biological, diagnostic or pharmaceutical values.
Acknowledgments The authors acknowledge support from the National Major Scientific and Technological Special Project for Significant New Drugs Development (2012ZX0950200-005), the National Basic Research Program of China (973 Program) (2012CB822102), the National Natural Science Foundation of China (31000367), and the Natural Science Foundation of Shandong Province, China (ZR2010CM038 and ZR2011CM041).
12
References
[1] W. Wei, M.R. Niñonuevo, A. Sharma, L.M. Danan-Leon, J.A. Leary, A comprehensive compositional analysis of
heparin/heparan sulfate-derived disaccharides from human serum, Anal. Chem. 83 (2011) 3703-3708.
[2] M. Petitou, B. Casu, U. Lindahl, 1976-1983, a critical period in the history of heparin: the discovery of the antithrombin
binding site, Biochimie 85 (2003) 83-89.
[3] R.J. Linhardt, Heparin: an important drug enters its seventh decade, Chem. Ind. 2 (1991) 45-50.
[4] D.B. Brieger, K. Mak, K. Kottke-Marchant, E.J. Topol, Heparin-induced thrombocytopenia, J. Am. Coll. Cardiol. 31 (1998)
1449-1459.
[5] J. Fareed, W. Jeske, D. Hoppensteadt, R. Clarizio, J.M. Walenga, Low-molecular-weight heparins: pharmacologic profile
and product differentiation, Am. J. Cardiol. 82 (1998) 3L-10L.
[6] J. Hirsh, M.N. Levine, Low molecular weight heparin, Blood 79 (1992) 1-17.
[7] R.J. Linhardt, N.S. Gunay, Production and chemical processing of low molecular weight heparins, Semin. Thromb. Hemostasis
25 (1999) 5-16.
[8] J. Fareed, W.L. Leong, D.A. Hoppensteadt, W.P. Jeske, J. Walenga, R. Wahi, R.L. Bick, Generic low-molecular-weight
heparins: some practical considerations, Semin. Thromb. Hemostasis 30 (2004) 703-713.
[9] A. Bisio, D. Vecchietti, L. Citterio, M. Guerrini, R. Raman, S. Bertini, G. Eisele, A. Naggi, R. Sasisekharan, G. Torri, Structural
features of low-molecular-weight heparins affecting their affinity to antithrombin, Thromb. Haemostasis 102 (2009) 865-873.
[10] Z. Shriver, M. Sundaram, G. Venkataraman, J. Fareed, R. Linhardt, K. Biemann, R. Sasisekharan, Cleavage of the
antithrombin III binding site in heparin by heparinases and its implication in the generation of low molecular weight heparin,
PNAS 97 (2000) 10365-10370.
[11] L. Liverani, G. Mascellani, F. Spelta, Heparins: process-related physico-chemical and compositional characteristics,
fingerprints and impurities, Thromb. Haemostasis 102 (2009) 846-853.
13
[12] Z. Xiao, B.R. Tappen, M. Ly, W. Zhao, L.P. Canova, H. Guan, R.J. Linhardt, Heparin mapping using heparin lyases and the
generation of a novel low molecular weight heparin, J. Med. Chem. 54 (2011) 603-610.
[13] B. Yang, K. Solakyildirim, Y. Chang, R.J. Linhardt, Hyphenated techniques for the analysis of heparin and heparan sulfate,
Anal. Bioanal. Chem. 399 (2011) 541-557.
[14] S. Lee, A. Raw, L.Yu, R. Lionberger, N. Ya, D. Verthelyi, A. Rosenberg, S. Kozlowski, K. Webber, J. Woodcock, Scientific
considerations in the review and approval of generic enoxaparin in the United States, Nat. Biotechnol. 31 (2013) 220-226.
[15] A. Pervin, C. Gallo, K.A. Jandik, X. Han, R.J. Linhardt, Preparation and structural characterization of large heparin-derived
oligosaccharides, Glycobiology 5 (1995) 83-95.
[16] Y. Takegawa, K. Araki, N. Fujitani, J. Furukawa, H. Sugiyama, H. Sakai, Y. Shinohara, Simultaneous analysis of heparan
sulfate, chondroitin/dermatan sulfates, and hyaluronan disaccharides by glycoblotting-assisted sample preparation followed
by single-step zwitter-ionic-hydrophilic interaction chromatography, Anal. Chem. 83 (2011) 9443-9449.
[17] F. Galeotti, N. Volpi, Novel reverse-phase ion pair-high performance liquid chromatography separation of heparin, heparan
sulfate and low molecular weight-heparins disaccharides and oligosaccharides, J. Chromatogr. A 1284 (2013) 141-147.
[18] F. Lamari, M. Militsopoulou, X. Gioldassi, N. K. Karamanos, Capillary electrophoresis: a superior miniaturized tool for
analysis of the mono-, di-, and oligosaccharide constituents of glycan moieties in proteoglycans, Fresenius J. Anal. Chem. 371
(2001) 157-167.
[19] J.T. King, U.R. Desai, A capillary electrophoretic method for fingerprinting low molecular weight heparins, Anal. Biochem.
380 (2008) 229-234.
[20] A. H.K.Plaas, V. C.Hascall, R. J.Midura, Ion exchange HPLC microanalysis of chondroitin sulfate: quantitative derivatization
of chondroitin lyase digestion products with 2-aminopyridine, Glycobiology 6 (1996) 823-829.
[21] A. Kinoshita, K. Sugahara, Microanalysis of glycosaminoglycan-derived oligosaccharides labeled with a fluorophore
2-aminobenzamide by high-performance liquid chromatography: application to disaccharide composition analysis and
14
exosequencing of oligosaccharides, Anal. Biochem. 269 (1999) 367-378.
[22] M. Ambrosius, K. Kleesiek, C. Götting, Quantitative determination of the glycosaminoglycan ∆-disaccharide composition of
serum, platelets and granulocytes by reversed-phase high-performance liquid chromatography, J. Chromatogr. A 1201 (2008)
54-60.
[23] H. Kitagawa, A. Kinoshita, K. Sugahara, Microanalysis of glycosaminoglycan-derived disaccharides labeled with the
fluorophore 2-aminoacridone by capillary electrophoresis and high-performance liquid chromatography, Anal. Biochem. 232
(1995) 114-121.
[24] L. Chi, J. Amster, R.J. Linhardt, Mass spectrometry for the analysis highly charged sulfated carbohydrates, Curr. Anal. Chem.
1 (2005) 223-240.
[25] N. Volpi, R.J. Linhardt, High-performance liquid chromatography-mass spectrometry for mapping and sequencing
glycosaminoglycan-derived oligosaccharides, Nat. Protoc. 5 (2010) 993-1004.
[26] D. Li, L. Chi, L. Jin, X. Xu, X. Du, S. Ji, L. Chi, Mapping of low molecular weight heparins using reversed phase ion pair
liquid chromatography-mass spectrometry, Carbohydr. Polym. 99 (2014) 339-344.
[27] L. Li, F. Zhang, J. Zaia, R.J. Linhardt, Top-Down approach for the direct characterization of low molecular weight heparins
using LC-FT-MS, Anal. Chem. 84 (2012) 8822-8829.
[28] Q. Zhang, X. Chen, Z. Zhu, X. Zhan, Y. Wu, L. Song, J. Kang, Structural analysis of low molecular weight heparin by
ultraperformance size exclusion chromatography/time of flight mass spectrometry and capillary zone electrophoresis, Anal.
Chem. 85 (2013) 1819-1827.
[29] D.J. Langeslay, E. Urso, C. Gardini, A. Naggi, G. Torri, C.K. Larive, Reversed-phase ion-pair ultra-high-performance-liquid
chromatography-mass spectrometry for fingerprinting low-molecular-weight heparins, J. Chromatogr. A 1292 (2013)
201-210.
[30] B. Wang, L.F. Buhse, A. Al-Hakim, M.T. Boyne li, D.A. Keire, Characterization of currently marketed heparin products:
15
analysis of heparin digests by RPIP-UHPLC-QTOF-MS, J. Pharm. Biomed. Anal. 67-68 (2012) 42-50.
[31] F. Galeotti, N. Volpi, Online reverse phase-high-performance liquid chromatography-fluorescence detection-electrospray
ionization-mass spectrometry separation and characterization of heparan sulfate, heparin, and low-molecular weight-heparin
disaccharides derivatized with 2-aminoacridone, Anal. Chem. 83 (2011) 6770-6777.
[32] B. Yang, Y. Chang, A.M. Weyers, E. Sterner, R.J. Linhardt, Disaccharide analysis of glycosaminoglycan mixtures by
ultra-high-performance liquid chromatography-mass spectrometry, J. Chromatogr. A 1225 (2012) 91-98.
[33] Z. Zhang, J. Xie, H. Liu, J. Liu, R.J. Linhardt, Quantification of heparan sulfate disaccharides using ion-pairing
reversed-phase microflow high-performance liquid chromatography with electrospray ionization trap mass spectrometry,
Anal. Chem. 81 (2009) 4349-4355.
[34] P. Jackson, Polyacrylamide gel electrophoresis of reducing saccharides labeled with the fluorophore 2-aminoacridone:
subpicomolar detection using an imaging system based on a cooled charge-coupled device, Anal. Biochem. 196 (1991)
238-244.
[35] M. Militsopoulou, F.N. Lamari, A.H. Nikos, K. Karamanos, Determination of twelve heparin- and heparin sulfate-derived
disaccharides as 2-aminoacridone derivatives by capillary zone electrophoresis using ultraviolet and laser-induced
fluorescence detection, Electrophoresis 23 (2002) 1104-1109.
[36] M. Sundaram, Y. Qi, Z. Shriver, D. Liu, G. Zhao, G. Venkataraman, R. Langer, R. Sasisekharan, Rational design of
low-molecular weight heparins with improved in vivo activity, PNAS 100 (2003) 651-656.
16
Table 1 The structures of dalteparin, enoxaparin, and corresponding building blocks. -O3S
O O OH
O CO2OH
CH2OR1 O O
OR1
R=
OH
O NHR2 CO2O OH
O CO2OH
CH2OR1 O OR OR1
O
NHR2
OR1 O O
OR1
OH
O
O NHR2
OH O
OR1 n
O or H
OH
OSO3-
n
O CO2O OH OH OSO3-
CH2OR1 O
CO2-
NHSO3-
R1 = H or SO3Na, R2 = SO3Na or CO-CH3
R1= H or SO3Na, R2 = H or SO3Na or CO-CH3
Dalteparin natricum
Enoxaparin natricum
Name
Structure
∆IS
∆UA2S-GlcNS6S
∆IIS
∆UA-GlcNS6S
Common heparin disaccharides
∆IIIS
∆UA2S-GlcNS
∆IVS
∆UA-GlcNS
∆IA
∆UA2S-GlcNAc6S
∆IIA
∆UA-GlcNAc6S
∆IIIA
∆UA2S-GlcNAc
∆IVA
∆UA-GlcNAc
∆dp4(4OS,1NS) RE
∆UA2S-GlcNS6S-IdoA2S-Mnt6S-2,5-anhydro
∆dp2(2OS) RE
∆UA2S-Mnt6S-2,5-anhydro
Dalteparin characteristic structures
∆dp2(1OS) RE
∆UA-Mnt6S-2,5-anhydro
dp2(2OS,1NS) NRE
IdoA2S-GlcNS6S
dp2(1OS,1NS) NRE
IdoA2S-GlcNS
∆IIA-IVSglu
∆UA-GlcNAc6S-GlcA-GlcNS3S
∆IIA-IISglu
∆UA-GlcNAc6S-GlcA-GlcNS3S6S
∆dp3(3OS,1NS)
∆UA2S-GlcNS6S-IdoA2S
∆dp3(1OS)
∆UA-GlcNAc-IdoA (1OS)
1,6-anhydro ∆IS-ISepi
∆UA2S-GlcNS6S-IdoA2S-ManNS-1,6-anhydro
Enoxaparin characteristic structures
Synthetic heparin disaccharide
1,6-anhydro ∆IS
∆UA2S-GlcNS-1,6-anhydro
1,6-anhydro ∆IIS
∆UA-GlcNS-1,6-anhydro
dp2(2OS,1NS) NRE
IdoA2S-GlcNS6S
∆IIA-IVSglu
∆UA-GlcNAc6S-GlcA-GlcNS3S
∆IIA-IISglu
∆UA-GlcNAc6S-GlcA-GlcNS3S6S
∆dp3(3OS,1NS)
∆UA2S-GlcNS6S-IdoA2S
∆dp3(1OS)
∆UA-GlcNAc-IdoA (1OS)
∆IP
∆UA2S-GlcNCOEt6S
17
∆UA: unsaturated uronic acid. GlcA: glucuronic acid. IdoA: iduronic acid. GlcN: glucosamine. ManN: mannosamine. Ac: acetyl group. S: sulfo group. COEt: ethoxycarbonyl. Mnt: mannitol.
18
Table 2 Theoretical and experimental molecular mass of underivatized and AMAC-labeled LMWH building blocks. Number
Name
Theoretical MWmono
Theoretical MWmono
Major ions
(underivatized)
(AMAC-labeled)
(m/z)
Peak interpretation
Experimental MWmono
Dalterparin 1
∆dp4(4OS,1NS) RE
1058.9750
n.r.
528.4836
[M-2H]2-
1058.9829
2
∆dp2(2OS) RE
482.0036
n.r.
480.9989
[M-H]-
482.0067
401.0408
-
402.0486
3
∆dp2(1OS) RE
402.0468
n.r.
[M-H]
2-
1230.1313 1150.1689
4
∆IIA-IISglu
1036.0396
1230.1240
614.0578
[M-2H]
5
∆IIA-IVSglu
956.0828
1150.1672
574.0766
[M-2H]2-
384.5215
[M-2H]
22-
6
∆IS
576.9713
771.0557
771.0587
7
dp2(2OS,1NS) NRE
594.9819
789.0663
393.5270
[M-2H]
8
∆dp3 (3OS,1NS)
832.9602
1027.0446
512.5191
[M-2H]2-
1027.0539
9
dp2(1OS,1NS) NRE
515.0251
709.1095
353.5470
[M-H]2-
709.1097
-
789.0697
10
∆IIS
497.0145
691.0989
690.0963
[M-H]
691.1041
11
∆IIIS
497.0145
691.0989
690.0957
[M-H]-
691.1035
610.1392
-
611.1470
12
∆IVS
417.0577
611.1421
[M-H]
-
13
∆IA
539.0251
733.1095
732.1029
[M-H]
733.1107
14
∆dp3 (1OS)
635.1004
829.1848
413.5861
[M-H]2-
829.1879
15
∆IIA
459.0683
653.1527
652.1497
[M-H]-
653.1575
2-
16
∆IP
553.0407
747.1251
372.5553
[M-2H]
17
∆IIIA
459.0683
653.1527
652.1469
[M-H]-
653.1547
747.1263
18
∆IVA
379.1115
573.1959
572.1926
[M-H]-
573.2004
1,6-anhydro ∆IS-ISepi
1055.9753
n.r.
526.9811
[M-2H]2-
1055.9779
Enoxaparin 1 2
1,6-anhydro∆IS
479.0040
n.r.
477.9983
-
[M-H]
-
479.0061
1,6-anhydro∆IIS
399.0471
n.r.
398.0390
[M-H]
399.0468
3
∆IIA-IISglu
1036.0396
1230.1240
614.0560
[M-2H]2-
1230.1277
4
∆IIA-IVSglu
956.0828
1150.1672
574.0758
[M-2H]2-
1150.1673
2-
771.0515 789.0655
5
∆IS
576.9713
771.0557
384.5179
[M-2H]
6
dp2(2OS,1NS) NRE
594.9819
789.0663
393.5249
[M-2H]2-
512.5183
[M-2H]
2-
-
7
∆dp3 (3OS,1NS)
832.9602
1027.0446
1027.0523
8
∆IIS
497.0145
691.0989
690.0932
[M-H]
691.1010
9
∆IIIS
497.0145
691.0989
690.0936
[M-H]-
691.1014
610.1354
-
611.1432
10
∆IVS
417.0577
611.1421
[M-H]
-
11
∆IA
539.0251
733.1095
732.1049
[M-H]
733.1127
12
∆dp3 (1OS)
635.1004
829.1848
413.5863
[M-H]2-
829.1883
13
∆IIA
459.0683
653.1527
652.1462
[M-H]-
653.1540
2-
14
∆IP
553.0407
747.1251
372.5526
[M-2H]
15
∆IIIA
459.0683
653.1527
652.1467
[M-H]-
653.1545
16
∆IVA
379.1115
573.1959
572.1885
[M-H]-
573.1963
n.r.: components can not be reacted with AMAC. MWmono: monoisotopic molecular weight.
19
747.1209
Table 3 Quantitative composition analysis of dalteparin and enoxaparina. LMWHs Dalteparin
∆IS 63.42±1.10
∆ IIS 10.95±0.38
∆IIIS 5.32±0.36
Disaccharide (%) ∆IVS ∆IA 1.49±0.02 1.96±0.07
∆IIA 5.78±0.42
∆IIIA 1.42±0.05
∆IVA 2.08±0.23
Tetrasaccharide (%) ∆IIA-IVSglu ∆IIA-IISglu 0.03±0.00 0.24±0.05
Enoxaparin
63.13±0.79
11.91±0.63
6.63±0.40
3.52±0.10
4.33±0.15
1.69±0.07
3.70±0.12
0.03±0.00
1.61±0.06
LMWHs Dalteparin Enoxaparin
∆dp2(1OS) RE 0.77±0.10
Terminal structures (%) ∆dp4(4OS,1NS) RE 0.04±0.01
∆dp2(2OS) RE 0.26±0.03 dp2(2OS,1NS) NRE
1,6-anhydro ∆IS and 1,6-anhydro∆IS 0.18±0.01
1.61±0.39 a
In this experiment, the values were means of three measurements ± standard deviation (SD)
20
0.09±0.01
dp2(1OS,1NS) NRE 0.24±0.07 1,6-anhydro ∆IS-ISepi
0.15±0.00
Trisaccharide (%) ∆dp3(3OS,1NS) ∆dp3(1OS) 0.05±0.02 1.63±0.16 0.08±0.01
1.33±0.22
dp2(2OS,1NS) NRE 4.33±1.40
Figure legends
Figure 1. RP-HPLC-DAD-MS chromatograms of AMAC-labeled dalteparin-derived components (injected 50 µg). (A) TIC, (B)
chromatogram with UV detection at 255 nm, and (C) chromatogram with UV detection at 232 nm. Peak assignment: 1.
∆dp4(4OS,1NS) RE (underivatized), 2. ∆dp2(2OS) RE (underivatized), 3. ∆dp2(1OS) RE (underivatized); 4. ∆IIA-IISglu, 5.
∆IIA-IVSglu, 6. ∆IS, 7. dp2(2OS,1NS) NRE, 8. ∆dp3(3OS,1NS), 9. dp2(1OS,1NS) NRE, 10. ∆IIS, 11. ∆IIIS, 12. ∆IVS, 13. ∆IA, 14.
∆dp3(1OS), 15. ∆IIA, 16. ∆IP, 17. ∆IIIA, and 18. ∆IVA.
Figure 2. Representative ESI-MS spectra of characteristic dalteparin-derived components. (A) ∆dp4(4OS,1NS) RE
(underivatized), (B) ∆dp2(2OS) RE (underivatized), (C) ∆dp2(1OS) RE (underivatized), (D) dp2(2OS,1NS) NRE, and (E)
dp2(1OS,1NS) NRE.
Figure 3. RP-HPLC-DAD-MS chromatogram of AMAC-labeled enoxaparin-derived components (injected 30 µg). (A) TIC, (B)
chromatogram with UV detection at 255 nm, and (C) chromatogram with UV detection at 232 nm. Peak assignment: 1.
1,6-anhydro ∆IS-ISepi (underivatized), 2. 1,6-anhydro ∆IS (underivatized) and 1,6-anhydro ∆IIS (underivatized), 3. ∆IIA-IISglu, 4.
∆IIA-IVSglu, 5. ∆IS, 6. dp2(2OS,1NS) NRE, 7. ∆dp3(3OS,1NS), 8. ∆IIS, 9. ∆IIIS, 10. ∆IVS, 11. ∆IA, 12. ∆dp3(1OS), 13. ∆IIA, 14.
∆IP, 15. ∆IIIA, and 16. ∆IVA.
Figure 4. Representative ESI-MS spectra of characteristic enoxaparin-derived components. (A) 1,6-anhydro ∆IS-ISepi
(underivatized), (B) 1,6-anhydro ∆IS (underivatized), and (C) dp2(2OS,1NS) NRE.
Figure 5. Quantitative compositional analysis of LMWHs. The curves and linear equations of intensity as a function of
concentration for each building block are shown. The amounts of LMWHs used for RP-HPLC-DAD-MS analysis were 10, 25, 50,
100, and 200 µg, respectively. (A) the common disaccharides of dalteparin, (B) the characteristic building blocks of dalteparin, (C)
the common disaccharides of enoxaparin, and (D) the characteristic building blocks of enoxaparin.
21
22
23
24
25
26
