development of hydrophilic interaction …...development of hydrophilic interaction chromatography...

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 withquadruple time-of-flight mass spectrometry for heparin and lowmolecular weight heparin disaccharide analysis

Yilan Ouyang1†, Chengling Wu1†, Xue Sun1, Jianfen Liu2**, Robert J. Linhardt3 andZhenqing Zhang1*1Jiangsu Key Laboratory of Translational Research and Therapy for Neuro-Psycho-Diseases and College of PharmaceuticalSciences, Soochow University, Suzhou, Jiangsu 215021, China2Xiehe Pharmaceutical Co. Ltd, Shijiazhuang, Hebei Province 050083, China3Departments 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. Thedetermination of their composition and structural heterogeneity still challenges analysts.METHODS: Disaccharide compositional analysis, utilizing heparinase-catalyzed depolymerization, is one of the mostimportant ways to evaluate the sequence, structural composition and quality of heparin and LMWH. Hydrophilicinteraction chromatography coupled with quadruple time-of-flight mass spectrometry (HILIC/QTOFMS) has beendeveloped to analyze the resulting digestion products.RESULTS: HILIC shows good resolution and excellent MS compatibility. Digestion products of heparin and LMWHsafforded up to 16 compounds that were separated using HILIC and analyzed semi-quantitatively. These included eightcommon disaccharides, two disaccharides derived from chain termini, three 3-O-sulfo-group-containingtetrasaccharides, along with three linkage region tetrasaccharides and their derivatives. Structures of these digestionproducts were confirmed by mass spectral analysis. The disaccharide compositions of a heparin, two batches of theLMWH, enoxaparin, and two batches of the LMWH, nadroparin, were compared. In addition to identifyingdisaccharides, 3-O-sulfo-group-containing tetrasaccharides, linkage region tetrasaccharides were observed havingslightly different compositions and contents in these heparin products suggesting that they had been prepared usingdifferent starting materials or production processes.CONCLUSIONS: Thus, compositional analysis using HILIC/QTOFMS offers a unique insight into different heparinproducts. Copyright © 2015 John Wiley & Sons, Ltd.

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

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

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

Most heparin products are isolated by extraction from animaltissues, such as porcine intestine.[6,7] The most commonrepeating 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 ofTranslational Research and Therapy for Neuro-Psycho-Diseases and College of Pharmaceutical Sciences,Soochow University, Suzhou, Jiangsu 215021, China.E-mail: [email protected]

** Correspondence to: J. Liu, Xiehe Pharmaceutical CO. LTD.,Shijiazhuang, Hebei province, 050083, ChinaEmail: [email protected]

† These two authors contributed equally to this work.

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

27

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

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

Copyright © 2015 John Wiley & Sons, Ltd.

7

Y. L. Ouyang et al.

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

However, each of these methods exhibits disadvantages.The instability observed in SAX chromatography makes itdifficult to assign peaks to individual disaccharides basedon relative retention times.[18] In addition, eluents used inSAX chromatography are often incompatible with massspectrometry (MS). In IPRP, ion-pairing reagents, even onesselected for compatibility with MS, often remain in the ionsource and even in the MS capillary suppressing itssensitivity and resolution.[19,20] Reversed-phase methodsrequire pre-column derivatization to improve the separationand detection sensitivity of heparin disaccharides, butderivatization often introduces impurities, resulting in theloss of structural information, and decrease in analyticalaccuracy.[20,21]

Hydrophilic interaction chromatography (HILIC), a rapidlydeveloping separation method, uses matrices with variousfunctional groups to separate polar compounds.[22] Severallaboratories have used HILIC to analyze heparan sulfate(HS)/heparin disaccharides and oligosaccharides.[23,24] Noderivatization is needed and HILIC buffer systems areusually MS friendly. In the current study, a new Xamidecolumn was applied to analyze the disaccharides derivedfrom heparin and two different low molecular weightheparins (LMWHs) without any additional derivatization.Amide functional groups, on the surface of resin in theXamide column, provide sufficient but not excessivelystrong interaction with highly sulfated heparin-deriveddisaccharides and oligosaccharides.[24,25] This allows the useof low concentrations of volatile salts in the eluent makingHILIC on Xamide columns well suited to MS analysis. It isalso important that the amide groups are attached to thechromatographic support with a hydrophilic linker,facilitating the use of an aqueous mobile phase required forthese highly polar analytes.[26] A new high-resolutionheparin disaccharide analysis method was developed using

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

Symbols Structure

ΔIVA ΔUA-GlcNAcΔIIIA ΔUA2S-GlcNAcΔIIA ΔUA-GlcNAc6SΔIA ΔUA2S-GlcNAc6SΔIVS ΔUA-GlcNSΔIIIS ΔUA2S-GlcNSΔIIS ΔUA-GlcNS6SΔIS ΔUA2S-GlcNS6SΔIVH ΔUA-GlcNΔIIIH ΔUA2S-GlcNΔIIH ΔUA-GlcN6SΔIH ΔUA2S-GlcN6SΔGlyser ΔUA-Gal-Gal-Xyl-serΔGlyserox1 ΔUA-Gal-Gal-Xyl-CH2-COOHΔGlyserox2 ΔUA-Gal-Gal-CH(CH2OH)-COOHΔdp2(0S)RE ΔUA-Mnt-2,5-anhydroΔdp2(1OS)RE ΔUA-Mnt6S-2,5-anhydroΔIVA-IVS3Sglu ΔUA-GlcNAc-GlcA-GlcNS3SΔIIA-IVS3Sglu ΔUA-GlcNAc6S-GlcA-GlcNS3SΔIIS-IIS3Sglu ΔUA-GlcNS6S-GlcA-GlcNS3S6S

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

an Xamide column. In addition to disaccharides, differenttypes of linkage region tetrasaccharides and 3-O-sulfo-group-containing tetrasaccharides could be separated andidentified using HILIC/LC/MS analysis. Small structuraldifferences in the composition of different heparins andLMWHs could be observed using this newly developedmethod.

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 wereobtained from Iduron Co. (Manchester, UK) (where ΔUA is4-deoxy-R-L-threo-hex-4-enopyranosyluronic acid, GlcN isglucosamine, Ac is an acetyl group, and S is a sulfo group).The structural information of these heparin disaccharides areprovided in Table 1. Heparin was obtained from the UnitedStates Pharmacopeia (USP, Rockville, MD, USA). Enoxaparinswere obtained from the USP and the market (a product ofSanofi Aventis). Nadroparins were obtained from theEuropean Pharmacopeia (EP) and the market (a product ofGlaxoSmithKline, GSK). Other chemical reagents were allHPLC or LC/MS grade.

Preparation of the disaccharides from heparin products

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

MWHs

Theoretical MW(Da)

Molecular ion[M–H]–/[M–2H]2–

379.1115 378.1030459.0683 458.0601459.0683 458.0592539.0251 538.0130417.0577 416.0480497.0145 496.0040497.0145 496.0037576.9713 575.9600337.1008 ——417.0577 ——417.0577 ——497.0145 ——719.2120 718.2024690.1854 689.1747588.1538 587.1408322.0900 321.0833402.0468 401.0387876.1260 437.0534956.0828 477.03091073.9859 535.9841

ley & Sons, Ltd. Rapid Commun. Mass Spectrom. 2016, 30, 277–284

Heparin disaccharide analysis using HILIC/MS

denatured enzyme was removed by centrifugation(15,000 rpm) for 10 min. The samples were diluted to1 mg/mL and 0.1 mg/mL before analysis.

Optimization of the HILIC method

The HILIC method was developed on an Agilent systemequipped with an HPLC system (1260) and an ESI quadrupletime-of-flight mass spectrometer (ESI-QTOFMS, model 6540,Agilent Technologies). Data was acquired with Mass Hunter6.0. (Agilent Technologies). Chromatograms were obtainedon a Xamide column (3.0 × 150 mm, 5 μm, Acchrom Corp.,Beijing, China) at a flow rate of 0.4 mL/min and withdetection at 232 nm and online MS. Mobile phase A wasaqueous solution (water), mobile phase B was aqueoussolution (water) containing 100 mM ammonium formate(pH was adjusted to pH 3.2 with formic acid), and mobilephase C was acetonitrile. The concentration of volatile salt(ammonium formate), mobile phase gradient, andtemperature were further considered as three importantfactors to optimize this method.

MS parameters

Nitrogen gas was used in the nebulizer at a pressure of30 psi. The spray voltage was 3.5 kV and a flow of nitrogengas of 8 L/min at 350 °C assisted in the drying process.Fragmentor voltage was set to 120 V. A full MS scan between100–2000m/z was performed. All data were acquired in thenegative mode.

27

RESULTS AND DISCUSSION

Optimization of chromatography

The HILIC separation was optimized using 12 heparindisaccharide standards. Three series of experiments wereperformed. The concentration of ammonium formate was firstinvestigated in a separation performed 25 °C. Mobile phase B(100 mM ammonium formate) was used at 5, 10 or 20% (thepercentage of mobile phase B is constant) with a lineargradient (0%/80% acetonitrile) of aqueous acetonitrile over a60 min period at a flow rate of 0.4 mL/min. Chromatogramscontaining analyte were monitored at 232 nm and a blankchromatogram monitored at the same wavelength wassubtracted to level the baseline (Supplementary Fig. S-1,Supporting Information). The starting condition of mobilephase in a method was usually used to equilibrate thecolumn for 30 min before the next injection. Most peakswere eluted after 25 min at a low salt concentration (5%mobile phase B, corresponding to 5 mM ammoniumformate). This suggested that 5 mM ammonium formatewas insufficient to properly separate all the disaccharides(Supplementary Fig. S-1(A)). High salt concentration (20%mobile phase B, corresponding to 20 mM ammoniumformate) eluted most peaks too quick resulting in poorresolution (Supplementary Fig. S-1(C)). Most peaks wereeluted within a reasonable time with good resolution when10% mobile phase B, corresponding to 10 mM ammoniumformate, was used (Supplementary Fig. S-1(B)) and theseconditions were selected going forward.

Rapid Commun. Mass Spectrom. 2016, 30, 277–284 Copyright © 2015 J

A multiple-step gradient (Supplementary Table S-1,Supporting Information) was selected based on several trialseparations at 25 °C of 12 heparin disaccharide standards.The chromatogram obtained for these disaccharide standardswas subtracted by a blank chromatogram (Fig. 1(A)).

One complication of this separation is the presence of peakdoubling, the result of the α- and β-configured isomers ofseveral disaccharides being separated. This was confirmed insubsequent MS analysis of the mixture of 12 heparindisaccharide standards (Supplementary Fig. S-2, SupportingInformation) and in chromatograms of each individualdisaccharide (data not shown). TheN-acetylated disaccharides,ΔIVA, ΔIIIA, ΔIIA and ΔIA, in particular showed separation ofthe α- and β-configured isomers. Some of these double peaksoverlapped with other disaccharides, adversely impacting thedisaccharide profiling of mixtures of 12 heparin disaccharidestandards (Fig. 1(A)). According to previous reports,[23,27]

elevated temperatures can eliminate the presence of separatepeaks corresponding to the α- and β-configured sugars. Thus,different temperatures were applied to further optimize thisseparation. Figures 1(B) and 1(C) show chromatograms of theheparin disaccharide standards obtained at 40 °C and 60 °C.Peak assignments were confirmed by MS and by the analysisof individual disaccharides (data not shown). Most peakdoublings were smaller at 40 °C and completely eliminated at60 °C (Figs. 1(B) and 1(C)). At 60 °C, a single peakcorresponding to each heparin disaccharide (ΔIVA, ΔIVH,ΔIIIA, ΔIIIH, ΔIIA/ΔIIH, ΔIVS, ΔIH, ΔIA, ΔIIIS, ΔIIS, andΔIS) was observed at 12.0, 15.5, 19.8, 21.0, 21.9, 29.2, 36.5,41.9, 43.8, 45.0, and 60.0 min, respectively. However, underthese conditions, the peak corresponding to ΔIIA stilloverlapped with the peak corresponding to ΔIIH.Disaccharides with a free amino group, such as ΔIIH, are rarelyobserved in heparin products.[20,28,29] The molecular ionscorresponding to those two overlapped disaccharides (m/z416 and 458) were individually extracted from the total ionchromatogram (TIC) of the heparin digestion product (Fig. 2).The m/z 416.0 extracted ion chromatogram (EIC) is shown inFig. 2(A). Four major peaks were present in the EIC. The peakof ΔIVS with molecular ion (m/z 416.0) was observed at~29.2 min; the peaks of ΔIIS and ΔIIIS (m/z 496.0) with loss ofa single sulfo group exhibited ions (m/z 416.0) at ~44.0 and45.0, respectively; and the peak of ΔIS (m/z 576.0) with two lostsulfo groups was observed at ~58.0 min. Only a small peak at~22.0 min corresponded to the rare disaccharide ΔIIH. Whenm/z 458.1 was extracted from the TIC of the heparin digestionproduct, the peak corresponding to ΔIIA, observed at~22.0 min, dominated the EIC, and ΔIIIA was present as asmall peak at ~21.0 min (Fig. 2(B)). This suggests that theoverlap of disaccharides ΔIIH and ΔIIA would not adverselyimpact the disaccharide analysis of heparin products, sinceeven minor amounts of ΔIIH in a heparin product canconveniently be confirmed by MS. In addition, the sameweight concentration of all standards was used in this work(Fig. 1), but the peak area refers to the quantity of eachdisaccharide (molar concentration). The disaccharidestandardswith highermass (more sulfated disaccharides) haverelatively lower peak areas.

Thus, a HILIC method was developed to analyze heparindisaccharides, in which at least ten disaccharide standardswere resolved effectively and the disaccharide compositioncould be calculated with peak integration.

ohn Wiley & Sons, Ltd. wileyonlinelibrary.com/journal/rcm

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Figure 1. Optimization of disaccharide analysis chromatography with differenttemperatures. Chromatograms obtained at (A) 25 °C; (B) 40 °C; and (C) 60 °C.

Figure 2. Extracted ion chromatograms (EICs) of heparindigested product: (A) m/z 416 EIC of heparin digestedproduct and (B) m/z 458 EIC of heparin product.

Figure 3. Linearity, equations and correlation coefficients ofeight common disaccharides obtained with QTOFMS.

Y. L. Ouyang et al.

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Quantitative disaccharide analysis of heparins and LMWHs

The composition of disaccharides from heparin products couldalso be analyzed and quantified by MS. Heparin disaccharideshave different ionization efficiencies. The peak areas of eachdisaccharide in the EIC, plotted as a function of itsconcentrations, gave good linearity over a proper range.(0.1–1.5 μg) The standard curve of each disaccharide is providedin Fig. 3 with the linear equation and correlation coefficient. A

wileyonlinelibrary.com/journal/rcm Copyright © 2015 John Wiley & Sons, Ltd. Rapid Commun. Mass Spectrom. 2016, 30, 277–284

Table

2.Com

position

sof

hepa

rin,

enox

aparinsan

dna

dropa

rins

Symbo

ls

UV

Com

position

(%)

MSCom

position

(%)

Com

position

(%)from

literature

Hp

Eno

(USP

)Eno

(San

ofi)

Nad

ro(EP)

Nad

ro(G

SK)

Hp

Eno

(USP

)Eno

(San

ofi)

Nad

ro(EP)

Nad

ro(G

SK)

Hp[

29]

Hp[

31]

Eno

[31]

LMWH

[31]

ΔIVA

2.9

3.3

2.6

2.8

2.0

3.6

3.9

3.7

3.6

3.2

2.3

6.6

5.2

4.5

ΔIIIA

1.0

1.7

1.2

1.3

0.8

2.1

2.3

2.1

2.2

2.0

1.7

0.8

0.9

0.9

ΔIIA

3.2

2.6

3.5

3.8

3.6

5.7

5.9

6.1

6.4

6.1

3.1

4.4

4.3

4.7

ΔIA

2.7

2.1

1.5

2.0

1.6

4.9

5.4

5.3

4.6

4.9

1.7

1.1

1.2

1.3

ΔIV

S1.1

1.4

1.1

1.3

1.5

1.4

1.6

1.4

1.6

1.7

2.5

4.8

4.9

4.0

ΔIIIS

6.0

6.7

7.0

6.2

4.9

6.5

7.1

7.3

6.9

5.9

8.9

7.9

8.4

7.2

ΔIIS

11.8

12.5

13.5

10.1

11.8

10.6

11.2

11.1

9.0

10.2

12.5

14.1

12.8

12.6

ΔIS

67.5

67.4

68.2

67.7

68.6

65.2

62.6

63.0

65.7

66.0

67.3

60.5

62.3

64.7

ΔGlyser

1.5

0.1

0.3

n.d.

n.d.

n.q.

n.q.

n.q.

n.d.

n.d.

n.d.

n.d.

n.d.

n.d.

ΔGlyser o

x10.2

1.1

0.3

0.5

0.3

n.q.

n.q.

n.q.

n.q.

n.q.

n.d

n.d

n.d

n.d

ΔGlyser o

x2n.d.

n.d.

n.d.

0.5

0.3

n.d.

n.d.

n.d.

n.q.

n.q.

n.d

n.d

n.d

n.d

Δdp2

(0S)RE

n.d.

n.d.

n.d.

0.9

1.1

n.d.

n.d.

n.d.

n.q.

n.q.

n.d

n.d

n.d

n.d

Δdp2

(1OS)RE

n.d.

n.d.

n.d.

1.7

1.7

n.d.

n.d.

n.d.

n.q.

n.q.

n.d

n.d

n.d

n.d

ΔIVA-IVS3

Sglu

0.9

0.6

0.3

0.7

1.2

n.q.

n.q.

n.q.

n.q.

n.q.

n.d

n.d

n.d

n.d

ΔIIA-IVS3

Sglu

0.7

0.1

0.1

0.2

0.2

n.q.

n.q.

n.q.

n.q.

n.q.

n.d

n.d

n.d

n.d

ΔIIS-IIS3

Sglu

0.8

0.4

0.4

0.5

0.7

n.q.

n.q.

n.q.

n.q.

n.q.

n.d

n.d

n.d

n.d

Hp,

hepa

rin;

Eno

,Eno

xapa

rin;

Nad

ro,N

adropa

rin;

n.d.,no

tdetected;n

.q.,no

tqu

antified

.



different response of each disaccharide in MS was observed inthis work. The response of each disaccharide in MS usuallyresults from the combination of polarity, quantity of charges,solubility, mass of the analyte, and even the setup of massspectrometry. In thiswork,ΔIVA has the lowest charge number,probably resulting in the relatively low efficiency of ionization.ΔIIIA has one sulfo group,which ismuch easier to be ionized inthe ion source. The specific position of the sulfo group, fairpolarity and relatively low mass could also be the reasons that

Figure 4. Disaccharide analysis chromatograms of heparin-related products obtained at 232 nm. Chromatogram of (A)eight common disaccharides; (B) digested heparin (USP);(C) digested enoxaparin (USP); (D) digested enoxaparin(Sanofi); (E) digested nadroparin (EP); and (F) digestednadroparin (GSK).


281

Y. L. Ouyang et al.

282

led to the high response of ΔIIIA inMS analysis. However, lackof some standards limits the quantitative application of this MSmethod. In particular, there are no standards available for the2,5-anhydromanitol disaccharides and enzyme-resistanttetrasaccharides (Table 1). Furthermore, the disaccharidepresent in the smallest amount (<1%) and that present in thelargest amount (~70%) could not be simultaneously quantified.Thus, a heparin disaccharide composition analysis requires twoexperiments using two different sample concentrations(0.1 mg/mL and 2 mg/mL), and normalized by intensity ofΔIIS, which presents ~10% in various heparin and LMWHs(data not shown). The disaccharide compositions of the heparinproducts were calculated using the quantitative MS methodand are provided in Table 2.

Disaccharide composition analysis of heparins andLMWHs

Heparin (USP), enoxaparins (USP and Sanofi Aventis), andnadroparin (EP and GSK) were analyzed using our newlydeveloped method. As we observed previously, eight commondisaccharides were present as major components in all samples(Fig. 4). These showed baseline separation, although a risingbaseline was observed to the elution gradient.The UV chromatogram of heparin disaccharides is shown

in Fig. 4(B). In addition to the eight common disaccharides,five additional peaks were observed at ~40.5, ~41.5 ~ 55.0,

Figure 5. MS spectra of some disaccharidestetrasaccharide linkers and its derivatives, andsulfated disaccharide (Δdp2(0S)RE) with reducingdisaccharide (Δdp2(1OS)RE) with reducing termiΔIIA-IVS3Sglu; (E) ΔIIS-IIS3Sglu; (F) ΔGlyserox2; (


~61.5, and ~62.0 min, respectively, and their mass spectraare shown in Fig. 5. An ion at m/z 718.2 dominated thespectrum of the peak present at 40.5 min (Fig. 5(H)) and thiswas unambiguously assigned as the linkage regiontetrasaccharide located between the glycosaminoglycan andprotein core with serine (ΔGlyser); the structure is shown inTable 1. An ion at m/z 689.2 dominated the spectrum of thepeak present at ~41.5 min (Fig. 5(G)) and this was assignedas the linkage region tetrasaccharide located having anoxidized serine residue (ΔGlyserox1); the structure is shownin Table 1. All the glycosaminoglycan chains of proteoglycans,including heparin, are linked to a protein core through sucha linkage region tetrasaccharide.[24,30,31] This is the first timethat these structures have been detected using LC/MS.[16]

In heparin, the content of ΔGlyser is much greater than thatof ΔGlyserox1. The amount and the types of the linker reflectthe degree of oxidation (bleaching) in heparin production.[16]

The doubly charged ions at m/z 437.1, 477.0, and 536.0dominate the spectra as small peaks present at ~55.0, 61.5,and 62.0 min, respectively. These were assigned astetrasaccharides containing two, three, and five sulfo groups(Figs. 5(C), 5(D) and 5(E)). The heparinase-resistanttetrasaccharide typically contains a 3-O-sulfo group in theglucosamine residue at its reducing end.[24,30,31] Accordingto these reports, these heparinase-resistant tetrasaccharidescould be assigned as ΔIVA-IVS3Sglu, ΔIIA-IVS3Sglu, andΔIIS-IIS3Sglu, and their structures are shown in Table 1.

with reducing terminals from nadroparin,some 3-O-sulfated tetrasaccharides: (A) non-terminal from nadroparin; (B) mono-sulfatednal from nadroparin; (C) ΔIVA-IVS3Sglu; (D)G) ΔGlyserox1; and (H) ΔGlyser.



28

The EIC of three enzyme-resistant tetrasaccharides inheparin is shown in Supplementary Fig. S-3 (SupportingInformation).The UV chromatograms of enoxaparin disaccharides are

shown in Figs. 4(C) and 4(D). In addition to the eightcommon heparin disaccharides, the linkage regiontetrasaccharide and its derivatives (ΔGlyser and ΔGlyserox1)are also present in enoxaparin digestion products. Three3-O-sulfo-group-containing tetrasaccharides were alsoobserved at ~55.0, ~61.5, and ~62.0 min in both enoxaparinsamples. The contents of ΔGlyser and ΔGlyserox1 inenoxaparin (USP) were slightly different from those inenoxaparin (Sanofi) implying that different heparinprecursors, or slightly different processes, were applied toprepare these two batches of enoxaparin. The ΔGlyserox1,present as a major type of the linkage region tetrasaccharide,was different from those observed in heparin. Some of theΔGlyser present in heparin might also be degraded toΔGlyserox1 in production processes of enoxaparins.The UV chromatograms of nadroparin disaccharides are

shown in Figs. 4(E) and 4(F). In addition to the eight commondisaccharides, seven peaks were present in the chromatogramat 9.5, 18.0, 40.0, 41.5, 55.0, 61.5 and 62.0 min. Ions at m/z321.1 and 401.1 dominated the MS spectra of the peaks at 9.5and 18.0 min, respectively. Because the production process ofnadroparin relies on nitrous acid, 2,5-anhydromannitolresidues are formed at most reducing ends of the resultingproducts. These two peaks were assigned as non-sulfated andmono-sulfated disaccharide from nadroparin reducing ends(Δdp2RE, Δdp2RE1S) (Figs. 5(A) and 5(B)) and their structuresare shown in Table 1. An ion at m/z 587.1 dominated the MSspectrum of the peak at 40.0 min, assigned as a linkage regiontetrasaccharide with an oxidized xylose (ΔGlyserox2) (Fig. 5(F))and its structure is shown in Table 1. The peak present at 41.5 isΔGlyserox1 and is the same as that present in the heparin andthe enoxaparin digestion products. Since nadroparin isproduced from heparin by nitrous acid degradation, allΔGlyser present in heparin is likely oxidized to ΔGlyserox1and ΔGlyserox2. The linkage region tetrasaccharide, ΔGlyserox2,is exclusively present in nadroparin and ΔGlyser is notobserved. In addition, the contents of the derivatives of thelinkage region tetrasaccharide in nadroparin (EP) are slightlyhigher than those present in nadroparin (GSK) suggestingdifferent heparin precursors or slightly different productionprocesses have been used. Finally, three 3-O-sulfo-group-containing tetrasaccharides are also present in the digestionproducts of the two nadroparins.In heparin production processes, proteases, base and

oxidants are all used to remove proteins.[14] The linkageregion tetrasaccharide with serine (ΔGlyser) and serinederivative (ΔGlyserox1) could result from the use of thesereagents. Different processes undoubtedly result in differentcontents or types of these linkage region tetrasaccharides. Inthe heparin product analyzed in this work, ΔGlyser is themajor type of linkage region present in heparin, whileΔGlyserox1 is the major type of the linkage region present inenoxaparin. The reaction applied to produce enoxaparin fromheparin might convert some ΔGlyser into ΔGlyserox1.Similarly, all ΔGlyser could be oxidized to ΔGlyserox1 andΔGlyserox2 when heparin is converted into nadroparin usingnitrous acid, which also produces the 2,5-anhydromannitolresidue Δdp2RE and Δdp2RE1S, exclusive to nadroparin.


CONCLUSIONS

In this study, a HILIC/QTOFMS method was used to analyzethe heparinase-catalyzed digestion products of heparin andLMWHs. The disaccharide compositions of different heparinsand LMWHs obtained with this method are comparable tothose obtained with other methods (Table 2),[29,31] suggestingthe feasibility and reliability of this method. In addition, 16compounds in the final digested heparin and LMWHproducts were separated and quantitatively analyzed withthis method, in which rare disaccharides, 3-O-sulfo-group-containing tetrasaccharides, linkage region tetrasaccharidesand their derivatives (Table 1) could only be comparedbetween the samples as peak areas normalized to the one ofthe disaccharides. The MS compatibility of HILIC provideda reliable way to identify rare domains in heparin andLMWHs, which contains important structural information.The disaccharide compositions of a heparin, two enoxaparinsand two nadroparins were compared. The types and contentsof linkage region tetrasaccharides reflect the startingmaterials and production processes used for differentLMWHs. Slight differences in content between two batchesof enoxaparin and two batches of nadroparin could beobserved suggesting that this method might be useful forassessing the processes used to prepare LMWHs.

AcknowledgementsThe authors are grateful to the National Natural ScienceFoundation of China (81473179), Jiangsu Specially-AppointedProfessor Research Funding (SR13200113), Priority AcademicProgram Development of Jiangsu Higher EducationInstitutions (PAPD, YX13200111), and the funding for JiangsuKey Laboratory of Translational Research and Therapy forNeuro-Psycho-Diseases (BM2013003).

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