ORIGINAL RESEARCH
published: 18 April 2019
doi: 10.3389/fmed.2019.00078
SAX-HPLC and HSQC NMR
Spectroscopy: Orthogonal Methods
for Characterizing Heparin Batches
Composition
Franco Spelta 1*, Lino Liverani 1 , Alessandra Peluso 1 , Maria Marinozzi 2 , Elena Urso 2 ,
Marco Guerrini 2 and Annamaria Naggi 2
1
Edited by:
Barbara Mulloy,
King’s College London,
United Kingdom
Reviewed by:
Marcelo Lima,
Federal University of São Paulo, Brazil
Paulo Antonio De Souza Mourão,
Federal University of Rio de Janeiro,
Brazil
*Correspondence:
Franco Spelta
f.spelta@opocrin.it
Specialty section:
This article was submitted to
Hematology,
a section of the journal
Frontiers in Medicine
Received: 07 November 2018
Accepted: 29 March 2019
Published: 18 April 2019
Citation:
Spelta F, Liverani L, Peluso A,
Marinozzi M, Urso E, Guerrini M and
Naggi A (2019) SAX-HPLC and HSQC
NMR Spectroscopy: Orthogonal
Methods for Characterizing Heparin
Batches Composition.
Front. Med. 6:78.
doi: 10.3389/fmed.2019.00078
Frontiers in Medicine | www.frontiersin.org
R&D Department, Opocrin S.p.A., Formigine, Italy, 2 Istituto di Ricerche Chimiche e Biochimiche “G. Ronzoni”, Milan, Italy
Heparin is a complex mixture of heterogeneous sulfated polysaccharidic chains. Its
physico-chemical characterization is based on the contribution of several methods, but
advantages of the use of complementary techniques have not been fully investigated yet.
Strong-Anion-Exchange HPLC after enzymatic digestion and quantitative bidimensional
1 H-13 C NMR (HSQC) are the most used methods for the determination of heparin
structure, providing the composition of its building blocks. The SAX-HPLC method is
based on a complete enzymatic digestion of the sample with a mixture of heparinases
I, II and III, followed by the separation of the resulting di- and oligo-saccharides by
liquid chromatography. The NMR-HSQC analysis is performed on the intact sample and
provides the percentage of mono- and di-saccharides by integration of diagnostic peaks.
Since, for both methods, accuracy cannot be proved with the standard procedures, it is
interesting to compare these techniques, highlighting their capabilities and drawbacks.
In the present work, more than 30 batches of porcine mucosa heparin, from 8
manufacturers, have been analyzed with the two methods, and the corresponding
results are discussed, based on similarities and differences of the outcomes. The critical
comparison of both common and complementary information from the two methods can
be used to identify which structural features are best evaluated by each method, and to
verify from the concordance of the results the accuracy of the two methods, providing
a powerful tool for the regular characterization of single, commercial preparations
of Heparin.
Keywords: heparin, characterization, composition, building blocks, SAX-HPLC, quantitative NMR, HSQC
INTRODUCTION
Heparin is the most important anticoagulant drug and has been used in clinical practice since
1939. Although heparin was discovered nearly a 100 years ago, its structure/function relationships
are still the subject of many studies (1). What makes investigations on the interaction of
heparin with biologic systems very difficult is that, unlike most biological substances, heparin
has an “intrinsically variant” structure. The structure of heparin chains is based on disaccharide
building blocks, all made up of a uronic acid and a glucosamine. Variations in the composition
1
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Heparin Characterization by SAX-HPLC and NMR-HSQC
of volumes of signals corresponding to the same monosaccharide
type (glucosamines or uronic acids) and the same carbonproton pair type (e.g., anomeric proton carbon pairs or C2
ring position pairs). Details of the method can be found in
published papers, where calculation formulas, the influence of
key experimental parameters and validation results are described
(19, 22). The main advantage of the HSQC method is that
no sample manipulation is required before the analysis and
that iduronic and glucuronic acid residues can be distinguished
and quantified. In contrast, information about which type of
glucosamine residue is sulfated or not in position 6 is not
available and only the overall amount of 6-O-sulfation can be
determined. A comparison of advantages and drawbacks of the
two methods is shown in Table 1 (23).
SAX-HPLC and HSQC methods are simple and can be
standardized, providing a high amount of complementary,
useful and quantitative information for the quality control of
commercial batches of heparin. Both methods provide molar
percentage distributions of heparin building blocks, but the two
methods resolve and identify different fragments (Tables 2, 3).
of heparin include distinct uronic acid content, i.e., different
glucuronic to iduronic ratios (GlcA/IdoA), different levels of Nacetylation/N-sulfation of the glucosamine and of 2-O- and 6O-sulfation (in iduronate and glucosamine, respectively), as well
as variable degrees of 3-O sulfation in glucosamine (2–4). The
industrial processes of extraction and purification of heparin can
cause further variability, affecting its structure in many points,
and at different levels (“process signatures”), causing partial
chemical modifications such as: depolymerization, N-desulfation
(5), O-desulfation with epoxidation and/or epimerization on C2C3 of sulfated iduronic acids (6), oxidation and/or disruption of
the linkage region (LR) (7, 8) and other minor defects (9–13).
Currently, the only source of heparin preparations in US, Europe
and Japan is porcine mucosa, but bovine and ovine heparin
preparations are available in other countries, and this might be
another cause of variability, as each source shows a typical set of
characteristic features (14–16). The most evident differences that
can be observed among heparin from different sources are related
to the sulfation pattern: e.g., porcine heparin is largely sulfated
in position 6 of glucosamine, in comparison to bovine mucosa
heparin, whereas the 2-O sulfation of iduronic acid is higher in
bovine mucosa heparin than in porcine (4).
A large number of analytical methods have been used to
address the high variability of heparin structure (17), but only
two of them are practical enough and, at the same time, able
to provide a sufficiently detailed and precise description of the
oligomeric composition of commercial batches: building block
analysis by Strong-Anion-Exchange HPLC after exhaustive
enzymatic digestion (SAX-HPLC) (18) and quantitative
NMR Heteronuclear-Single-Quantum-Coherence experiments
(HSQC) (19).
The SAX-HPLC method involves the enzymatic cleavage
of the heparin sample into its building blocks (mainly
disaccharides): a mixture of Heparinases I, II, and III cleaves the
linkage between the glucosamine and the uronic acid introducing
a double bond in position C4-C5 (“1”) of the uronic acid.
The mixture of di- and oligo-saccharides is resolved on a
chromatographic system, where the new double bond makes
it possible to detect all these saccharides by UV at 234 nm: a
simple calculation provides a molar ratio of each building block.
According to a general consensus, based on some papers (18, 20)
the same molar absorption coefficient for all 14-5 unsaturated
heparin di- and oligosaccharides has been used. As no clear
evidence of this sameness has been provided up to now, an
independent assessment of the response factor of the nine 145 unsaturated disaccharides, available on the market as reference
materials, was an additional aim of this work.
The enzymatic cleavage causes the loss of information about
the epimerization of the uronic acids (iduronic or glucuronic),
which are no longer distinguishable. Moreover, some sequences
along the heparin chain (e.g., disaccharides made up of a
glucosamine with a sulfate in position 3, or the whole linkage
region) are not affected by the enzymatic activity resulting in the
formation of tetrasaccharides (21).
In the HSQC method the monosaccharides and disaccharides
composition (molar ratio) is calculated by normalizing volumes
of the signals of the 2-D NMR spectrum with reference to the sum
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TABLE 1 | Major advantages and drawbacks of the two methods.
SAX-HPLC
Advantages
Drawbacks
Di- and tetra-saccharide
composition: single building
blocks identified and quantified
Digestion with a mix of
Heparinases: the thorough yield
of the depolymerization reaction
should be confirmed (e.g., by
Size Exclusion Chromatography)
High Sensitivity: LOD 0.1%, LOQ Sequences with specific process
0.3% (for disaccharides that
signatures not cleaved by the
respond to the mixture
enzymes
of Heparinases)
Identification of specific
disaccharides containing
6-O-sulfated glucosamine
Iduronic/glucuronic structure not
distinguished. Information only
about uronic acid-glucosamine
sequences
Easily achievable in every
analytical lab;
standard equipment
Saturated residues at the
non-reducing end of heparin
chains not detectable
Quantification based on
consensus assumption that all
14-5 unsaturated di- and
oligo-saccharides have the same
molar absorption coefficient
HSQC-NMR No sample treatment necessary: Low sensitivity: specific for each
information about the
residue. LOD 0.5%, LOQ 2% on
overall structure
average
Mono- and
di-saccharide composition
Possible problems with signals
resolution
Iduronic and glucuronic acids
can be distinguished
Quantification possible only by
comparison of atoms with similar
magnetic properties
Information about both uronic
acid-glucosamine and
glucosamine-uronic
acid sequences
Only the overall 6-Osulfation of
glucosamine residues can be
determined*
*The possibility of differentiating 6-O-sulfated and non-sulfated glucosamine by 1D proton
NMR has been recently described (23). However, the resolution of the HSQC spectrum
does not make it possible to resolve these peaks, if not at a very high magnetic field.
2
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Heparin Characterization by SAX-HPLC and NMR-HSQC
TABLE 2 | Heparin building blocks identified by the SAX-HPLC method of the present study: relative retention time with respect to the main disaccharide 1Is, peak 14.
peak ID
Building block code
Building block structure
Relative retention time
Residue identified by:
1
L.R.
1GlcA-Gal-Gal-Xyl-Ser
0.190
Isolation through semi-prep. SAX-HPLC and LC/MS£
2
1IVa
1UA-GlcNAc
0.225
Comparison with standard disacch.
3
L.R., ox1
1GlcA-Gal-Gal-Xyl-CH2 COOH#
0.405
isolation through semi-prep. SAX-HPLC and LC/MS£
4
1IVsgal
1GalA-GlcNS$
0.465
Isolation through semi-prep. SAX-HPLC and LC/MS£
5
1IVs
1UA-GlcNS
0.475
Comparison with standard disacch.
6
1IIa
1UA-GlcNAc,6S
0.520
Comparison with standard disacch.
7
1IIIa
1UA,2S-GlcNAc
0.585
Comparison with standard disacch.
8
1Ih
1UA,2S-GlcN,6S
0.640
Comparison with standard disacch.
9
1IIsgal
1GalA-GlcNS,6S$
0.685
Isolation through semi-prep. SAX-HPLC and LC/MS£
10
1IIs
1UA-GlcNS,6S
0.700
Comparison with standard disacch.
11
1IIIs
1UA,2S-GlcNS
0.765
Comparison with standard disacch.
Comparison with standard disacch.
12
1Ia
1UA,2S-GlcNAc,6S
0.880
13
1IIa-IVsglu(3S)
1UA-GlcNAc,6S-GlcA-GlcNS,3S
0.975
Comparison with published data (18)
14
1Is
1UA,2S-GlcNS,6S
1.000
Comparison with standard disacch.
15
1IIa-IIsglu(3S)
1UA-GlcNAc,6S-GlcA-GlcNS,3S,6S
1.085
isolation through semi-prep. SAX-HPLC and LC/MS£
16
1IIs-IIsglu(3S)
1UA-GlcNS,6S-GlcA-GlcNS,3S,6S
1.175
Comparison with published data (18)
17
1Isglu(3S)
1UA,2S-GlcNS,3S,6S
1.265
Comparison with published data (18)
18
1Ia-IIsglu(3S)
1UA,2S-GlcNAc,6S-GlcA-GlcNS,3S,6S
1.300
Comparison with published data (18)
19
1Is-IIsglu(3S)
1UA,2S-GlcNS,6S-GlcA-GlcNS,3S,6S
1.360
Comparison with published data (18)
£ data not shown.
# “process signature”. Linkage region with an oxidized serine, due to heparin purification steps.
$ “process signature”. 2-O-desulfation of iduronic acid and its epimerization caused by strong alkaline processes and thermal stress.
Underlinings remark disaccharides with a glucuronic acid and a galactosamine with an additional sulfate in position 3.
The present work is therefore aimed at providing a solid
ground to the two methods, by comparing the results, looking for
similarities and possible discrepancies for identifying the more
reliable and accurate result in case of disagreement.
For this investigation, data from 33 Heparin Sodium batches,
USP and/or Ph. Eur. compliant, from 8 manufacturers, covering
a time span from 2011 to 2016, were collected and compared.
TABLE 3 | Heparin building blocks identified by the HSQC method.
Glucosamine residues
Uronic acid residues
GlcNH2 ,6x
GlcA-GlcNAc,6x
GlcNS,3S,6x
GlcA-GlcNS,6x
GlcNAc,6x-GlcA
GlcA-GlcNS,3S,6x
GlcNAc,6x-IdoA
GlcA,2S
GlcNS,6x-GlcA
IdoA-GlcNy
GlcNS,6x-IdoA
IdoA-GlcNy,6S
MATERIALS AND METHODS
GlcNS,6x-IdoA,2S
IdoA,2S-GlcNH2 ,6x
Samples
GlcNS,6x-GalA
IdoA,2S-GlcNy,3x,6x
GlcNS,6x-Epox
GalA
Thirty-three Heparin Sodium batches were selected from 87
samples of the updated “bona fide” library of the Ronzoni
Institute (24), aiming to cover the highest number of heparin
manufactures and the largest structural variability. All batches
were USP and/or Ph. Eur. compliant. A list of all batches, with
the relevant details, can be found in Supplementary Table 2.
Four additional, modified, heparin samples were prepared
in Opocrin for investigating specific process signatures that are
usually present at low levels in pharma grade heparin batches.
Two batches with a high level of N-desulfation on glucosamine,
and two batches with a high level of epoxide on the uronic acid
were produced. Details about the production of these 4 batches
are reported in section Production of heparin batches with higher
content of specific features.
GlcNAc,6x, αRed
Epox
GlcNS,6x, αRed
GlcA-Linkage Region
GlcNx,6S
The
precision
of
both
methods
is
assured
[Supplementary Table 1 for SAX-HPLC and (19) for HSQC]
but, because no official reference material (a heparin sample
with known composition) can be devised, accuracy of each
separate method cannot be evaluated. Only the similarity of the
results obtained on the same heparin sample by totally different
methods can support their accuracy. Since the two methods
do not provide information on the same residues, a direct
comparison of all results is not possible. This work combines
quantitative data of different residues in order to compare
broader and more general attributes, as in the case of the level of
sulfation on the different positions.
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Enzymatic Digestion
Heparin digestion was carried out by mixing 10 µL of a 10
mg/mL solution of sample in water with 20 µL of a calcium
buffer (bovine serum albumin 0.1 mg/mL, calcium acetate 2 mM,
3
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Heparin Characterization by SAX-HPLC and NMR-HSQC
sodium acetate 100 mM; pH 7.0) and 10 µL of a Heparinase I, II
and III mixture, 0.25 IU each one, in phosphate buffer (bovine
serum albumin 0.2 mg/mL, sodium phosphate 10 mM; pH 7.0).
The mixtures were incubated for 48 h at room temperature for a
complete digestion. Completeness of digestion was verified by a
double check:
a) The results of a Suitability sample, present in all analytical sets,
were compared with past, historical results on a control chart.
b) A selection of 15 digested samples were checked by Size
Exclusion Chromatography: the absence of hexasaccarides
or longer oligosaccharides, both by UV (234 nm) and
refractive index detection, was considered sufficient to
demonstrate the efficiency of the enzymatic digestion (details
in Supplementary Text 1).
FIGURE 1 | SAX-HPLC chromatogram of Heparin batch Man_D-2. Some
disaccharides show a double peak, identified by the extension “-i,” depending
on the elution of the two anomeric forms of the reducing end terminal. Early
peaks, eluting in the range between 0 and 5 min, are system peaks. Other very
small peaks could not be assigned, but no sample has shown an un-identified
peak area larger than 0.25%, or a total area of un-identified peaks larger
than 1.2%.
All reagents were of analytical grade and the three Heparinases
were from CPC Biotech (Monza, Italy) or Grampian Enzymes
(Aberdeen, UK). Water was from a Milli-Q purification
system (Millipore).
Strong-Anion-Exchange HPLC
Solutions of digested heparin (10 µL) were injected onto a
Spherisorb SAX column, 4.0 × 250 mm, 5 µm (Waters). The
mixture of saccharides was resolved according to the chain
length, the number of sulfates and their position by a linear
gradient elution with mobile phase A (sodium phosphate 2 mM,
pH 3.0) and B (sodium phosphate 2 mM, sodium perchlorate
1.0 M; pH 3.0): mobile phase B, t 0–0.5 min., 3%, t 20 min.,
35%; t 50 min., 100%. Flow rate was 0.8 mL/min., with a
column temperature of 40 ◦ C and UV detection at 234 nm.
Nineteen structures (12 di-, 5 tetra-, and 2 oligo-saccharides), all
carrying unsaturated uronic acid at the non-reducing end (20)
can be identified with this method: the 9 main, typical heparin
disaccharides were identified by comparison of their retention
time with that of pure disaccharides (Iduron; Alderley Edge, UK);
5 structures were isolated by semi-preparative SAX-HPLC and
identified by LC/MS (data not shown), and 5 were identified
by comparison with published data (18), in agreement with our
findings. The list of the 19 oligosaccharide structures is shown in
Table 2, in order of elution. A SAX-HPLC chromatogram, as an
example, is shown in Figure 1.
All identified peaks were integrated, a cumulative area
calculated, and a molar percentage distribution was obtained
applying the same molar absorption coefficient (see section
SAX-HPLC: building blocks and process signatures identified).
The method was validated, and its main characteristics are
summarized in Supplementary Table 1.
The 1 H-NMR spectra were recorded at 298 K on a 500
MHz Bruker AVANCE HD spectrometer equipped with a TCI
cryoprobe, using the following parameters: number of scans 16,
dummy scans 8, relaxation delay 25 s, spectral width 16 ppm,
transmitter offset 4.7 ppm. After exponential multiplication (line
broadening of 0.3 Hz), the spectra were Fourier-transformed,
phased and baseline corrected. First, the absolute concentration
of TSP (expressed in mol/g) was determined using a standard
of potassium phthalate monobasic (KHP) certified for NMR
(Sigma-Aldrich, product number 14659) as follows: about 20 mg
of KHP was dissolved in 2.0 mL of TSP, 0.6 mL of the solution was
transferred in a NMR tube and analyzed.
The following signals were integrated:
- The TSP signal at 0.00 ppm excluding the 1 H-13 C
satellite peaks
- The aromatic signals of KHP at 7.57 (H3) and 7.72 ppm (H2),
excluding the 1 H-13 C satellite peaks at 7.87 and 7.40 ppm; the
other satellite peaks are superimposed to the signals of KHP.
The concentration of TSP was determined as follows:
CTSP =
gKP and MWKHP are the amount and the molecular weight of
the potassium phthalate monobasic, respectively, ITSP is the
integration of the TSP signal, IKHP is the sum of the integrations
of the potassium phthalate monobasic signals, gsolvent is the
amount of the solvent, 0.995 is the correction coefficient due to
the overlap of 50 % of the 1 H-13 C satellite peaks of potassium
phthalate monobasic signals, 4 and 9 are the number of hydrogen
atoms of KHP and TSP, respectively.
To quantify the amount of disaccharides, two aliquots for each
sample solution (250 µL) were analyzed.
The integration of the following signals was measured:
Quantitative NMR Determination of the
Typical Heparin Disaccharides and of Their
SAX-HPLC Response Factor
The content of each vial of the 9 disaccharides (about 1 mg each,
from Iduron, UK) was dissolved with 1.0 mL of D2 O with TSP
0.002%. The molar concentration of these solutions was assessed
by proton NMR.
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gKP
1
ITSP
4
·
· ·
MWKHP 0.995 I KHP 9 gsolvent
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Heparin Characterization by SAX-HPLC and NMR-HSQC
- The TSP signal at 0.00 ppm excluding the
satellite peaks
- The 1U4 signal of the disaccharide
1 H-13 C
Uronic Acid, 2,3-epoxide
Two batches were produced from the same parent heparin of
the previous samples: two samples of ∼3 g of heparin were
dissolved separately with ∼27 mL of water. The solutions were
heated at ∼50◦ C, and 3.0 mL of NaOH 4M were added to each
one. The solutions were maintained at ∼50◦ C for 1 and 2.5 h,
respectively. Each solution was neutralized with HCl 4M, cooled
at room temperature, and the product precipitated with ∼78 mL
of methanol. The precipitate was recovered by centrifugation
and dissolved with ∼60 mL of water; the volume was reduced
to ∼30 mL on a rotary evaporator and the samples were freezedried, giving origin to batches epox_1 and epox_2, respectively.
The amount of disaccharides was calculated as follows:
mgdis = CTSP ·
Idis
· mgsolution · MWdis
ITSP /9
CTSP is the concentration of the TSP, Idis and ITSP are the
integral of H4 of the disaccharide and the TSP signal, respectively,
mgsolution is the amount of the solution, MWdis is the molecular
weight of the disaccharide, 9 the number of hydrogen atoms in
the TSP (Supplementary Figure 1).
Different aliquots (2, 6, 10, and 14 µL) of the same solutions
were injected on the same SAX-HPLC system used for the present
study: the response factors of each disaccharide were calculated
as the mean of the 4 ratios “peak area/injected amount.” A
relative response factor, with reference to the disaccharide 1Is,
was calculated to make the comparison easier. Results are shown
in Supplementary Table 3.
RESULTS
SAX-HPLC: Building Blocks and Process
Signatures Identified
A SAX-HPLC chromatogram, as an example, is shown
in Figure 1, while a table with individual results can be
found in Supplementary Table 4: as previously reported, 19
oligosaccharide structures have been identified with the SAXHPLC method of the present study (Table 2).
Among these 19 structures, one is the “linkage region,” the
oligosaccharide that links the heparin chain to the protein core
of the parent proteoglycan (peak 1), 8 are the disaccharides
present in higher proportions, available as isolated reference
materials (peaks 2, 5, 6, 7, 10, 11, 12, and 14) and one is
the disaccharide 1UA,2S-GlcNS,3S,6S (peak 17), which is the
only disaccharide containing a sulfate in position 3 of the
glucosamine unit that can be obtained by enzymatic digestion.
This tetra-sulfated disaccharide is originated by the enzymatic
cleavage of the -IdoA2S-GlcNS,3S,6S- sequence, which can be
present in the heparin chain, or as an isolated disaccharide,
or in the pentasaccharide sequence containing an additional 3O-sulfated glucosamine (GlcNS,6S-GlcA-GlcNS,3S,6S-IdoA2SGlcNS,3S,6S) (25), or in antithrombin binding sequences recently
synthetized by chemoenzymatic methods, that might also be
present in natural sequences (GlcNS,6S-GlcA-GlcNS,6S-IdoA2SGlcNS,3S,6S-IdoA2S-GlcNS,6S-) (26).
Five residues are tetrasaccharides which were not cleaved
to disaccharides by the enzymes, due to the presence of
glucuronic acid followed by a 3-O-sulfated glucosamine: these
tetrasaccharides make up part of the pentasaccharide variants
responsible for the binding of heparin with antithrombin and, as
a consequence, for its anticoagulant properties (peaks 13, 15, 16,
18, 19) (27).
The remaining 4 structures are “process signatures” derived
from heparin extraction/purification processes, which make use
of strong oxidizing reagents, basic and acid pHs, and high
temperatures. One is related to an oxidized derivative of the
linkage region (peak 3) (6), two are related to the formation
of galacturonic acid (peaks 4 and 9) and the last one to the
disaccharide resulting from a N-desulfation of the glucosamine
(peak 8). The galacturonic acid is originated by alkaline and heat
treatments occurring during the heparin purification process:
alkalis cause the elimination of sulfate in position 2 of the
HSQC
The 1 H-13 C-HSQC spectra were measured on a Bruker AVANCE
III 600 MHz spectrometer equipped with a 5 mm TCI cryoprobe,
using the Bruker hsqcetgpsisp2.2 pulse sequence according to
the published method (19). Briefly, the spectra were recorded
at 298K using the following acquisition parameters: number of
scans 12, dummy scans 16, relaxation delay 2.5 s, spectral width
8 ppm (F2) and 80 ppm (F1), transmitter offset 4.7 ppm (F2)
and 80 ppm (F1), 1 JC−H = 150 Hz. 1024 points were recorded
for each of 240 increments (NUS of 75 % of 320 increments).
The FIDs were processed as follows: spectrum size 4096 (F2)
and 1024 (F1) (zero-filling in F2 and linear prediction in F1),
squared cosine window multiplication in both dimensions and
Fourier-transform. The diagnostic heparin building block signals
were integrated using Topspin software version 3.5 (Bruker
BioSpin, Rheinstetten, Germany) and the heparin composition
was computed from the integral values as previously described
(19). The list of the 23 heparin features identified and quantified
with this method is shown in Table 3.
Production of Heparin Batches With Higher
Content of Specific Features
Non N-Sulfated, Non N-Acetylated Glucosamine
Two batches were produced from the same parent heparin batch:
two samples of ∼3 g of heparin were dissolved separately with
∼26.6 mL of water. The solutions were heated at ∼50◦ C, and
3.4 mL of HCl 4M were added to each one. The solutions were
maintained at ∼50◦ C for 1 and 3 h, respectively. Each solution
was neutralized with NaOH 4M, cooled at room temperature,
and the product precipitated with ∼78 mL of methanol. The
precipitate was recovered by centrifugation and dissolved with
∼60 mL of water; the volume was reduced to ∼30 mL on a rotary
evaporator and the samples were freeze-dried, giving origin to
batches N-deS-1 and N-deS-2, respectively.
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considered small enough to justify the use of the same response
factors for all the peaks of the SAX-HPLC chromatograms.
HSQC
Figure 2 shows an example of HSQC spectrum of a heparin
sample: the signals used for quantitative analysis of individual
uronic acid and glucosamine residues are highlighted. The list
of the monomeric and disaccharidic structures—that could be
identified with the HSQC method—is shown in Table 3, while
individual results are shown in Supplementary Table 5. The
building block composition and the percentage of 6-O-sulfation
can be obtained directly by both HSQC and SAX-HPLC, but
information about the differently substituted uronic acid residues
(both glucuronic and iduronic) and the aminosugar-uronic acid
sequences are obtained only with HSQC. It is worth noting
that the results obtained by HSQC correspond to the molar
percentage of each residue compared to the total amount of
the corresponding monosaccharide type (i.e., glucosamine or
uronic acid).
Clustering of Data From SAX-HPLC
and HSQC-NMR
The specific heparin features identified by the two methods—
showed in Tables 2, 3—are not the same, as previously discussed.
However, data and outcomes obtained by each method can be
clustered according to homogeneous rules to provide comparable
information on more general characteristics of the sample. By
this process, data from the two methods can be evaluated by
looking for similarities or discrepancies between the outcomes.
The comparison of these data has been used to provide
information on the accuracy of the two methods and has allowed
more rational investigations on the possible discrepancies to be
carried out.
Eleven heparin attributes have been found suitable for this
clustering exercise: 7 related to the regular structure of heparin
and 3 to possible process signatures, all reported as percentages.
An additional attribute, the ratio sulfate to carboxylate ions (or
“degree of sulfation”), is reported as a pure number. The list of
these attributes, with a concise description of their meanings,
is shown in Table 4. The way the single data are combined
to originate information about each attribute can be found in
Supplementary Table 6.
FIGURE 2 | Low field region showing anomeric signals (A) and high field
region (B) of HSQC spectrum of Heparin batch Man_D-2 with relevant signal
assignments. Ovals identify the integrated signals. Glucosamine, iduronic acid
and glucuronic acid residues are indicated as A, I and G, respectively. X = H or
−
−
SO−
3 ; Y = H or Ac or SO3 ; Y’ = Ac or SO3 .
uronic acid residue, producing a 2–3 epoxide. This structure
can remain on the heparin chain, but the concurrent or
following heat treatments can cause it to open, with inversion
of the configuration of carbon 2 and 3, turning the L-iduronic
into L-galacturonic acid (6). The N-desulfated glucosamine
residue is mainly due to pH and thermal stress of heparin
during its manufacturing process, even if a small amount of
this disaccharide can be the natural marker of an incomplete
biosynthesis (5).
The SAX-HPLC results (Supplementary Table 4) and the
following considerations are based on the general consensus
assumption that response factors at 232-234 nm for all 14-5
unsaturated oligosaccharides are the same (18, 20). A specific,
concurrent study has been carried out to assess the actual
response factors of the 9 heparin disaccharides available as
reference materials (section Quantitative NMR determination
of the typical heparin disaccharides and of their SAX HPLC
response factor). This study demonstrates that the molar
absorption coefficients are very similar and only a few minor
disaccharides, present in heparin in small amounts, show small
differences (Supplementary Table 3). The differences noted were
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Comparison of Results
Results obtained by the two methods, for the 33 heparin batches
and for the 11 attributes, following the required calculations,
are shown in Supplementary Table 7 and are summarized and
compared in the box-plots of Figure 3. Data relevant to the
content of epoxide on uronic acid are not shown in this
table, as explained below. The comparison of the box-plots of
Figure 3A (SAX-HPLC vs. HSQC) shows that the two methods
detect similar contents of the main heparin attributes: Nsulfated glucosamine, N-acetylated glucosamine, 6-O-sulfated
glucosamine and iduronic acid with a sulfate in position 2. The
agreement of the two methods for these attributes is confirmed
by similar results of the molar ratio of sulfate to carboxylate ions
(Figure 3B), which summarizes the previous attributes.
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Heparin Characterization by SAX-HPLC and NMR-HSQC
TABLE 4 | List of the 11 heparin attributes that can be quantified by both the
SAX-HPLC and HSQC methods, combining information from the single, raw, data.
GlcNS
Regular structure
Content of N-sulfated glucosamine
GlcNAc
Regular structure
Content of N-acetylated glucosamine
GlcNx,6S
Regular structure
Content of glucosamine with a sulfate
in position 6
GlcNS,3S,6x
Regular structure
Content of a N-sulfated glucosamine
with an additional sulfate in position 3
GlcA- GlcNS,3S,6x
Regular structure
Content of a disaccharide made up of
a glucuronic acid and a glucosamine
with an additional sulfate in position 3
(GlcA-GlcNS,3S,6x, typical of the
“pentasaccharide” feature)
GlcNH2
Process signature
Content of glucosamine, non
N-sulfated, non N-acetylated;
N-desulfation due to pH and thermal
stresses. Possible natural marker of
an incomplete biosynthesis
IdoA,2S
Regular structure
Content of iduronic acid with a sulfate
in position 2
GalA
Process signature
Content of L-galacturonic acid, due
to the 2-O-desulfation of iduronic acid
and the following opening of the
epoxide, with inversion of
configuration
Epox
Process signature
Content of uronic acid with residual
epoxide in C2-C3 , not opened by
further steps of heparin processes
Linkage region
(LR)
Mixed information
The SAX-HPLC method identifies only
two major species: “native” LR and
one oxidized species; the HSQC
method detects all glucuronic acids
linked to a galactose, i.e., all “native”
and oxidized species
FIGURE 3 | Box-plots (median, first and third percentiles, range) of the main
heparin attributes of the 33 heparin batches : N-sulfated glucosamine (GlcNS),
6-O-sulfated glucosamine (GlcNx,6S) and 2-O sulfated iduronic acid (IdoA2S)
−
(A) molar ratio of sulfate to carboxylate ions (SO−
3 /COO ) (B) N-acetylated
glucosamine (GlcNAc), GlcA-A* or 3-O-sulfated-glucosamine (GlcNS,3S,6x),
glucuronic acid linked to 3-O-sulfated-glucosamine (GlcA-GlcNS,3S,6x),
N-desulfated glucosamine (GlcNH2 ), galacturonic acid (GalA) and linkage
region (LR) (C).
Despite a general agreement of results from the two methods,
a more accurate analysis of the data shows that some small
differences can be detected. Glucosamine residue with a sulfate
in position 6 (GlcNx,6S in Table 4 and Supplementary Table 6)
shows a first difference: figures from the SAX-HPLC method
are always higher, with a clear correlation between data from
the two methods (Figure 4, blue dots). The reason for this
systematic difference was investigated, and a longer spin-spin
relaxation time of protons (T2) of non-sulfated C6 compared
to sulfated C6 was considered responsible for unbalanced
outcomes for C6 of GlcNx,6S and GlcNx,6OH signal volumes.
The lower T2 of GlcNx,6S induces a higher loss of transverse
magnetization during the pulse sequence compared to that of
GlcNx,6OH, with a consequent underestimation of the residue
GlcNx,6S, according to Mauri et al. (19). This problem was
investigated on a subset of 6 heparin samples, expected to
cover the whole range of values from the HSQC method, with
additional 13 C-NMR spectra, where the proton T2 effect is
negligible. The results obtained from carbon spectra are, in this
case, very close to those obtained by the SAX-HPLC method
(Figure 4, red squares and Supplementary Table 8). According
to these data, more accurate information on the content of
glucosamine sulfated in position 6 can be obtained from the
SAX-HPLC method.
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Also figures of the content of 2-O-sulfated uronic acid are
similar, but values from the SAX-HPLC method are always a
bit lower, with a large scattering of the differences around a
value of −2.0%: a mix of random and systematic errors was
suspected. One point is that SAX-HPLC figures are obtained
by the sum of 7 peaks, while HSQC figures are obtained by
the sum of only two signals (three if the signal of 2-sulfated
glucuronic acid is higher than its LOD): this imbalance might
increase the scattering of the differences. On the other hand, a
systematic error can be due to a problem with the HSQC method:
the anomeric signal of galacturonic acid (GalA in Table 4) is
embedded in that of 2-sulfate iduronic acids. The HSQC method
tries to remove the GalA contribution by using the H5/C5 signal
of GalA instead of the anomeric signal. Unfortunately, as made
clear in a point below, this signal is always underestimated, so that
the final adjustment does not prove to be fully correct. Batches
with a higher content of GalA are more affected by this problem
and show slightly overestimated values of IdoA2S by the HSQC
method (Supplementary Table 7).
The molar ratio of sulfate to carboxylate ions resumes all the
main attributes of heparin, and the comparison of figures from
the two methods shows that they are very similar, with values
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Heparin Characterization by SAX-HPLC and NMR-HSQC
non-reducing end of the chains, recently described by some
authors (29, 30). This monosaccharide can be cleaved from the
non-reducing end by the enzymes in the SAX-HPLC method,
producing a saturated GlcNS,3S,6x residue, unnoticeable by UV
detection. Therefore, more accurate information on the content
of the monosaccharide GlcNS,3S,6x can be obtained from HSQC,
while more accurate information on the content of the different
sequences containing the disaccharide GlcA-GlcNS,3S,6x can be
obtained by SAX-HPLC.
Data comparison shows a difference between the two methods
also for GlcNH2 residue, a “process signature” affecting the
glucosamine residue (1Ih, peak 8 in Table 2 and GlcNH2 in
Table 4) which is usually generated by uncontrolled pH and
thermal stresses during heparin manufacturing. This feature is
a major and important stability indicating attribute for heparin
formulations as well (31). Results in Supplementary Table 7
show that the SAX-HPLC method was unable to detect the
disaccharide 1Ih (1UA,2S-GlcNH,6S) in 29 out of the 33
heparin batches. Only 4 batches showed a content of 1Ih close
to the limit of detection (0.1%). On the contrary, the HSQC
method detected the GlcNH2 in almost all heparin batches, in
a range between 0.7 and 2.1 %, when the limit of detection is
close to 1%. The reasons for this difference were investigated with
specific experiments: a heparin sample was deliberately overstressed with a treatment with HCl 0.45 M, for 1 and 3 h at 50◦ C
(subsection Non N-sulfated, non N-acetylated glucosamine).
The two samples were recovered and analyzed by the two
methods, and compared with the parent heparin. Calculations
for the SAX-HPLC method required some adjustments to take
into consideration the unknown peaks (at least 6) originating
in these samples. A complete overview of results from these
experiments can be found in Supplementary Table 9a. These
results showed that the content of 1Ih from the SAX-HPLC
method increases, as was expected (from 0.1 to 2.4 and
6.9%, respectively), but the contents of the disaccharide 1Is
(peak 14 in Table 2) and of almost all tetrasaccharides with
glucosamines sulfated in position 3 are seriously affected by these
treatments. Other disaccharides with N-sulfated glucosamines
are affected too, even if to a lesser extent. Results from the
HSQC method (Supplementary Table 10a), on the other hand,
always show much higher contents of GlcNH2 compared to
the SAX method. These data suggest that the attribute GlcNH2 ,
from the disaccharide 1Ih, can be only partially quantified by
the SAX-HPLC method because the enzymatic depolymerization
originates many unidentified di- and tetra-saccharides that
spread on the chromatogram. Therefore, accurate information on
the content of N-desulfated glucosamine can be obtained from
the HSQC method only.
The content of galacturonic acid (peaks 4 and 9 in Table 2,
and GalA in Table 4), as a marker of alkaline treatments
during the heparin extraction/purification steps (6, 32), is an
important “process signature” able to identify batches produced
by treatments that are too strong. The SAX-HPLC method is very
sensitive to this feature: two disaccharides with a galacturonic
structure were identified in almost all the considered heparin
batches, with amounts of GalA ranging between the limit of
detection (0.1%) and 6.2% (Supplementary Table 7). However,
FIGURE 4 | Comparison of results for the content of glucosamine with a
sulfate in position 6 (GlcNx,6S): results from SAX-HPLC vs. HSQC (blue dots)
and 13 C-NMR (red squares). The distribution of blue dots shows a clear
correlation between the content of 6-sulfated glucosamine from the two
methods, where results from the SAX method are always higher than those
from the HSQC. Quantitative results from the carbon spectra of a sub-set of 6
heparin samples (red squares) show a much better agreement with SAX
results, confirming a greater accuracy of the enzymatic method than the
HSQC.
from HSQC just slightly lower than those from the SAX-HPLC
method (Figure 3B). This small difference is clearly in agreement
with the larger effect of the underestimation of the content
of glucosamine 6-sulfated by the HSQC method (Figure 3A).
According to these findings, slightly more accurate information
on sulfate to carboxylate ions ratio can be obtained from the
SAX-HPLC method.
The content of 3-O-sulfated glucosamine and of the
disaccharide glucuronic acid linked to 3-O-sulfated glucosamine
(GlcNS,3S,6x and GlcA-GlcNS,3S,6x, respectively; Table 4) are
important attributes of heparin, as these structures are somehow
related to the content of the antithrombin binding sequence
responsible for most of its anticoagulant properties (28). The
HSQC method can measure both the disaccharide GlcAGlcNS,3S,6x (by means of the signal of glucuronic acid linked
to the GlcNS,3S,6x residue) and the total amount of the
monosaccharide GlcNS,3S,6x, directly from its corresponding
C1 or C2 signals, respectively. On the contrary, the SAX-HPLC
method cannot provide the content of the monosaccharide
GlcNS,3S,6x, but only of the disaccharide 1IdoA2S-GlcNS,3S,6S
(peak 17 in Table 2) and of the tetrasaccharide variants
containing the disaccharide GlcA-GlcNS,3S,6x (peaks 13, 15,
16, 18, and 19 in Table 2). The combined information is used
to provide data on the content of the disaccharide GlcAGlcNS,3S,6S and to estimate the content of the monosaccharide
GlcNS,3S,6S. The content of the disaccharide GlcA-GlcNS,3S,6x
obtained by SAX-HPLC (Figure 3C) is significantly higher
compared to HSQC. A possible reason for the lower levels
detected by the HSQC method is that some sequence effects can
generate weak and in part undetectable signals in the HSQC
spectrum. On the other hand, the higher content of GlcNS,3S,6x
found by the HSQC method can be explained by the presence
in heparin preparations of 3-O-sulfated glucosamine at the
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Heparin Characterization by SAX-HPLC and NMR-HSQC
as discussed above, the HSQC method cannot quantify the
content of galacturonic acid from the anomeric signals, which
are embedded in the signal of 2-sulfate iduronic acids: the
content of GalA is obtained by integration of the corresponding
proton 5. This proton generates a broad and complex signal
due to sequence effects, making its integration difficult, with
a poor LOD/LOQ. The result of these considerations is that
the HSQC method identifies GalA only in batches containing a
high percentage of this residue and cannot provide accurate data
(Figure 3C and Supplementary Table 7).
The epoxide residue has not been detected in any of the 33
heparin batches of our survey, either by SAX or HSQC, making
the comparison of the two methods impossible. This residue is
another marker of alkaline treatments, as it is considered the
intermediate step of the process ending in galacturonic acid
(6). To address this problem, a couple of specific experiments
were designed: a heparin sample was deliberately over-stressed
with a treatment with NaOH 0.4 M, for 1.0 and 2.5 h at 50◦ C.
The two samples were recovered and analyzed by the two
methods, in comparison with the parent heparin (subsection
Uronic acid, 2,3-epoxide). The chromatograms of the SAXHPLC method showed the appearance of two major and, at
least, other 5 minor unknown, broad peaks, with λMax at
245 nm, when the typical λMax of all di- and oligosaccharides
from the enzymatic cleavage is about 234 nm. On the contrary,
the HSQC method could provide reliable data, through the
identification and quantification of the typical epoxide residue
signals (H2/C2 and H3/C3 at 3.71/54.5 and 3.78/53.5 ppm,
respectively), undetectable in the parent heparin and present
in amounts of 5.8 and 15.7% in the two samples, respectively.
A complete overview of results from these experiments can be
found in Supplementary Tables 9b, 10b. These results suggest
that the epoxide, as a minor marker of 2-O-desulfation of heparin
batches, can be quantified by the HSQC method only.
A further quality attribute of heparin, investigated in this
study, is the “linkage region”, whose variants are useful tools
for providing information about the methods used for heparin
purification with regard to the oxidative stress. The presence
of high amounts of the “native” linkage region (LR, the
oligosaccharide -GlcA-Gal-Gal-Xyl-Serine) is a marker of a slight
stress, while the presence of different oxidized variants (e.g.,
the oligosaccharide -GlcA-Gal-Gal-Xyl-CH2 COOH) is a marker
of strong treatments (7). Also in this case, the two methods
do not provide the same kind of information: the SAX-HPLC
method identifies only two major species (“native” LR and
the oxidized species 1UA-Gal-Gal-Xyl-CH2 COOH), while the
HSQC method detects all glucuronic acids linked to a galactose,
including many other, more strongly oxidized, residues (-GlcAGal-remnant). The total amounts of linkage region detected by
SAX-HPLC are much lower than those obtained by HSQC. This
finding was expected, as the HSQC method detects a higher
number of oxidized variants, with the only limit of the presence
of glucuronic acid still linked to the first galactose. However,
the very high content of LR found in many heparin batches
(more than 5.0%, Supplementary Table 7), supports a possible
over-estimation of the content of LR, native or oxidized, by
the HSQC method. This over-estimation could be related to a
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higher “mobility” of the LR, which is not sulfated and located
at the reducing end terminal of heparin chains. This “extra”
mobility may induce a longer relaxation time compared to the
other parts of the chain, corresponding to a minor loss of
transverse relaxation time during the pulse sequence, with the
consequent slight over-estimation of the volume of the H1/C1
GlcA-Gal signal. According to these findings, neither SAX-HPLC
nor HSQC can provide accurate information on the LR content,
but the comparison of HSQC data and the content of 1UA-GalGal-Xyl-Serine by SAX-HPLC can be still considered suitable for
estimating the oxidative stress of heparin batches.
CONCLUSIONS
This study, comparing the outcomes of SAX-HPLC and HSQCNMR methods, proved to be very useful in investigating their
accuracy, and their strong points or shortcomings. Though both
SAX-HPLC and HSQC are unable to determine how the distinct
clusters (e.g., clusters of N-acetylated or N-sulfated glucosamine)
are distributed along the heparin chains, they provide a great
amount of high-quality and complementary information on
the building blocks composition of Heparin batches. Despite
some minor differences, both methods provide a consistent
comparison of heparin batches from the same or different
manufacturers, suitable for keeping under control the quality
of the drug and applicable to all the heparin types available on
the market.
Some major structural differences can be observed using
both techniques, i.e., sulfation level in position 2 of the uronic
acid residues and in position 2 and 6 of glucosamine. On the
other hand, the ratio between iduronic and glucuronic acid
is obtained only by HSQC-NMR, whereas the ratio between
the different antithrombin-binding pentasaccharide structures
can be determined exclusively by the SAX-HPLC method,
as tetrasaccharide sequences (15, 16). Many of the minor
discrepancies between the two methods, as far as the content of
some residues is concerned, can be related to the peculiarities
of each method. For instance, the content of 6-O-sulfated
glucosamine proved to be lower when calculated by HSQC due
to the different relaxation properties of the sulfated and nonsulfated C6 group. On the other hand, sequences containing
epoxide residues, induced by the alkaline treatment on the 2sulfated-iduronic acid, cannot be cleaved by enzymes and the
generated oligosaccharides are hardly detected by the SAXHPLC method. Moreover, it was experimentally demonstrated
that the molar absorption coefficients of the major 14-5
unsaturated heparin disaccharides are very similar and that
the small differences observed for few disaccharides present in
small amounts have no influence on the final results of the
SAX method.
In conclusion, the results described in the present study
demonstrated that both methods are sufficiently accurate
to determine the fingerprint of heparin, making them
suitable for monitoring the different steps of heparin
manufacturing process as well as for ensuring the quality
of the products on the market. Moreover, the combined
9
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Heparin Characterization by SAX-HPLC and NMR-HSQC
use of these methods increases the number of detectable
structural features useful also for differentiating between
heparins of different animal and organ sources more effectively.
Additionally, the application of chemometric analysis to
the large set of data achievable from these methods is a
promising tool for detecting structural anomalies and possible
cross-species contamination.
non-commercially available di- and oligosaccharides. All authors
contributed to revision of the manuscript and read and approved
the submitted version.
ACKNOWLEDGMENTS
The authors thank Dr. Lucio Mauri and Dr. Giovanni Boccardi
(Ronzoni Institute) for the useful discussion of results.
AUTHOR CONTRIBUTIONS
SUPPLEMENTARY MATERIAL
LL, FS, MG, and AN contributed to planning and writing the
paper. MM performed the NMR analyses. AP performed the
SAX-HPLC analysis. AP and EU gave support for the SAXHPLC data interpretation. EU performed the characterization of
The Supplementary Material for this article can be found
online at: https://www.frontiersin.org/articles/10.3389/fmed.
2019.00078/full#supplementary-material
REFERENCES
17.
1. Lever R, Mulloy B, Page CP. Heparin-a Century of Progress, Vol. 207. BerlinHeidelberg: Springer Science and Business Media (2012).
2. Lindahl U, Feingold DS, Rodén L. Biosynthesis of heparin. Trends Biochem
Sci. (1986) 11:221–5.
3. Lindahl U. ‘Heparin’–from anticoagulant drug into the new biology.
Glycoconjug J. (2000) 107:597–605. doi: 10.1023/A:1011030711317
4. Casu B, Guerrini M, Naggi A, Torri G, De-Ambrosi L, Boveri
G, et al. Characterization of sulfation patterns of beef and pig
mucosal heparins by nuclear magnetic resonance spectroscopy.
Arzneimittel-Forschung. (1996) 46:472–77.
5. Westling C, Lindahl U. Location of N-unsubstituted glucosamine
residues in heparan sulfate. J Biol Chem. (2002) 277:49247–
55. doi: 10.1074/jbc.M209139200
6. Jaseja M, Rej RN, Sauriol F, Perlin AS. Novel regio- and stereoselective
modifications of heparin in alkaline solution. Nuclear magnetic resonance
spectroscopic evidence. Can J Chem. (1989) 67:1449–56.
7. Viskov C, Mourier P. Process for Oxidizing Unfractionated Heparins and
Detecting Presence or Absence of Glycoserine in Heparin and Heparin Products.
U.S. Patent 2005/0215519A1, No. 10/808, 409 (2004).
8. Chen Y, Ange KS, Lin L, Liu X, Zhang X, Linhardt RJ. Quantitative analysis of
the major linkage region tetrasaccharides in heparin. Carbohydr Polym. (2017)
157:244–50. doi: 10.1016/j.carbpol.2016.09.081
9. Mourier PA, Guichard OY, Herman F, Viskov C. Heparin sodium
compliance to USP monograph: structural elucidation of an atypical
2.18 ppm NMR signal. J Pharmaceut Biomed Analy. (2012) 67:169–
74. doi: 10.1016/j.jpba.2012.04.015
10. Kellenbach E, Sanders K, Michiels PJA, Girard FC. 1 H NMR signal at 2.10
ppm in the spectrum of KMnO4 -bleached heparin sodium: identification of
the chemical origin using an NMR-only approach. Analyt Bioanalyt Chem.
(2011) 399:621–8. doi: 10.1007/s00216-010-4177-7
11. Beccati D, Roy S, Yu F, Gunay NS, Capila I, Lech M, et al. Identification of a
novel structure in heparin generated by potassium permanganate oxidation.
Carbohyd Polym. (2010) 82:699–705. doi: 10.1016/j.carbpol.2010.05.038
12. Lee SE, Chess EK, Rabinow B, Ray GJ, Szabo CM, Melnick B, et al.
NMR of heparin API: investigation of unidentified signals in the USPspecified range of 2.12–3.00 ppm. Anal Bioanal Chem. (2011) 399:651–
62. doi: 10.1007/s00216-010-4262-y
13. Beccati D, Roy S, Lech M, Ozug J, Schaeck J, Gunay NS, et al. Identification
of a novel structure in heparin generated by sequential oxidative–reductive
treatment. Anal Chem. (2012) 84:5091–5096. doi: 10.1021/ac3007494
14. Watt DK, Yorke SC, Slim GC. Comparison of ovine, bovine and porcine
mucosal heparins and low molecular weight heparins by disaccharide analyses
and 13 C NMR. Carbohyd Polym. (1997) 33:5–11.
15. Fu L, Li G, Yang B, Onishi A, Li L, Sun P, et al. Structural characterization
of pharmaceutical heparins prepared from different animal tissues. J Pharmac
Sci. (2013) 102:1447–57. doi: 10.1002/jps.23501
16. Naggi A, Gardini C, Pedrinola G, Mauri L, Urso E, Alekseeva A, et al.
Structural peculiarity and antithrombin binding region profile of mucosal
Frontiers in Medicine | www.frontiersin.org
18.
19.
20.
21.
22.
23.
24.
25.
26.
27.
28.
29.
10
bovine and porcine heparins. J pharmacy Biomed Anal. (2016) 118:52–
63. doi: 10.1016/j.jpba.2015.10.001
Capila I, Gunay NS, Shriver Z, Venkataraman, G. Methods for structural
analysis of heparin and heparan sulfate. In: Garg HG, Linhardt RJ, Hales CA,
editors. Chemistry and Biology of Heparin and Heparan Sulfate. Oxford, UK:
Elsevier (2005).
Mourier P, Anger P, Martinez C, Herman F, Viskov C. Quantitative
compositional analysis of heparin using exhaustive heparinase digestion
and strong anion exchange chromatography. Anal Chem Res. (2015) 3:46–
53. doi: 10.1016/j.ancr.2014.12.001
Mauri L, Boccardi G, Torri G, Karfunkle M, Macchi E, Muzi L, et al.
Qualification of HSQC methods for quantitative composition of heparin
and low molecular weight heparins. J Pharm Biomed Anal. (2017) 136:92–
105. doi: 10.1016/j.jpba.2016.12.031
Yoshida K, Miyauchi S,Kikuchi H, Tawada A, Tokuyasu K. Analysis
of unsaturated disaccharides from glycosaminoglycuronan by highperformance liquid chromatography. Anal Biochem. (1989) 177:
327–32.
Zhao W, Garron ML, Yang B, Xiao Z, Esko JD, Cygler M, et al.
Asparagine 405 of heparin lyase II prevents the cleavage of glycosidic linkages
proximate to a 3-O-sulfoglucosamine residue. FEBS Lett. (2011) 585:2461–
6. doi: 10.1016/j.febslet.2011.06.023
Keire DA, Buhse LF, Al-Hakim A. Characterization of currently marketed
heparin products: composition analysis by 2D-NMR. Anal Methods. (2013)
5:2984–94. doi: 10.1039/C3AY40226F
Vilanova E, Vairo BC, Oliveira SMCG, Glauser BF, Capillé NV, Santos
GRC, et al. (2019) Heparins sourced from bovine and porcine mucosa
gain exclusive monographs in the brazilian pharmacopeia. Front Med.
6:16. doi: 10.3389/fmed.2019.00016
Rudd TR, Gaudesi D, Skidmore MA, Ferro M, Guerrini M, Mulloy
B, et al. Construction and use of a library of bona fide heparins
employing 1 H NMR and multivariate analysis. Analyst. (2011) 136:1380–
9. doi: 10.1039/c0an00834f
Guerrini M, Elli S, Mourier P, Rudd TR, Gaudesi D, Casu B, et al.
An unusual antithrombin-binding heparin octasaccharide with an
additional 3-O-sulfated glucosamine in the active pentasaccharide
sequence. Biochem J. (2013) 449:343–51. doi: 10.1042/BJ201
21309
Wang Z, Hsieh, P.H.., Xu Y, Thieker D, Chai EJ, Xie S, et al. Synthesis
of 3-O-sulfated oligosaccharides to understand the relationship between
structures and functions of heparan sulfate. J Am Chem Soc. (2017) 139:5249–
56. doi: 10.1021/jacs.7b01923
Lindhal U, Thunberg L, Bäckström G, Riesenfeld J, Nordling K, Bjork I.
Extension and structural variability of the antithrombin-binding sequence in
heparin. J Biol Chem. (1984) 259:12368–12376.
Lindahl U, Bäckström G, Thunberg L, Leder IG. Evidence for a 3-O-sulfated
D-glucosamine residue in the antithrombin-binding sequence of heparin.
Proc Natl Acad Sci USA. (1980) 77:6551–5.
Alekseeva A, Casu B, Torri G, Pierro S, Naggi A. Profiling glycol-split heparins
by high-performance liquid chromatography/mass spectrometry analysis of
April 2019 | Volume 6 | Article 78
Spelta et al.
Heparin Characterization by SAX-HPLC and NMR-HSQC
their heparinase-generated oligosaccharides. Anal Chem. (2013) 434:112–
22. doi: 10.1016/j.ab.2012.11.011
30. Rudd TR, Macchi E, Muzi L, Ferro M, Gaudesi D, Torri G, et al.
Unravelling structural information from complex mixtures utilizing
correlation spectroscopy applied to HSQC spectra. Anal Chem. (2013)
85:7487–93. doi: 10.1021/ac4014379
31. Beaudet JM, Weyers A, Solakyildirim K, Yang B, Takieddin M, Mousa S, et al.
Impact of autoclave sterilization on the activity and structure of formulated
heparin. J Pharm Sci. (2011) 100:3396–404. doi: 10.1002/jps.22527
32. Liverani L, Mascellani G, Spelta F. Heparins: process-related
physico-chemical and compositional characteristics, fingerprints and
impurities. Thromb Haemost. (2009) 102:846–853. doi: 10.1160/TH09-0
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Conflict of Interest Statement: FS, LL, and AP were employed by Opocrin S.p.A.
The remaining authors declare that the research was conducted in the absence of
any commercial or financial relationships that could be construed as a potential
conflict of interest.
Copyright © 2019 Spelta, Liverani, Peluso, Marinozzi, Urso, Guerrini and Naggi.
This is an open-access article distributed under the terms of the Creative Commons
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April 2019 | Volume 6 | Article 78