High Resolution Analysis of Heparinand Heparin-like Impurities on
GlycomixTM – an Anion Exchange
ColumnAuthors
Xiaomi Xu
Yuna Li
Aiyan Cui
Haiying Chen

Glycan Separation

Abstract
Sepax Technologies, Inc.
5 Innovation Way
Newark, DE 19711
USAAnalysis of heparin is challenging due to its heterogeneity, both in its
size distribution and charge variance. Moreover, its impurities usually
have properties that resemble heparin which makes it difficult todistinguish heparin and its impurities using analytical methods. To
effectively separate heparin from its impurities, including naturally
occurring impurities during manufacturing process (e.g., dermatansulfate DS) and non-native contaminant from adulteration (e.g.,oversulfated chondroitin sulfate OSCS), U.S. Pharmacopeia (USP)
published a chromatographic method to identify heparin and heparin-like
impurities using ion-exchange liquid chromatography. However, current
commercially available ion-exchange column barely meets USP
separation resolution criteria; the situation calls for a new stationary
phase which can improve the resolution. Sepax has recently developed
an ion-exchange column – Glycomix TM SAX. It grafts charged and acertain level of hydrophilic functional groups to polymer-based resin.
Here we present the high efficiency separation of highly chargedglycans.

Flow rate: 0.22 mL/min
Column temperature: 25 °C
Detection: UV 202 nm
Injection volume: 10 μL
Pressure: 10 bar

Introduction
Heparin has been widely used as ananticoagulant or anti-thrombotic agent. It is acomplex mixture of sulfated glycosaminoglycans(GAGs), which are highly negatively charged.
During its extraction process from mammaliantissue (such as pig intestine), other polyanionic
GAGs can co-purify with heparin since theyshare heparin-like properties[1]. One
representative example of such impurities isdermatan sulfate (chondroitin sulfate B).
Oversulfated chondroitin sulfate (OSCS) was
identified as a non-native contaminant, which
can induce severe side effects even death. The
most recent heparin sodium monographpublished by U.S. Pharmacopeia[2] details theanalytical method that heparin manufacturers
must follow to ensure the quality of their heparinproducts. This application note reports theseparation of heparin, DS and OSCS using
GlycomixTM SAX with USP recommended
method. The performance of Glycomix SAX isalso discussed in comparison to competitor D’scolumn separation efficiency.

Results
Following the conditions listed on USP heparin
sodium monograph, Figure. 1 demonstrates theseparation of heparin from its impurities,
dermatan sulfate (DS) and oversulfatedchondroitin sulfate (OSCS). The three peaks,
according to elution order, are identified as DS,
heparin sodium salt and OSCS, the concentrationof which are 0.2 mg/mL, 20 mg/mL and 0.2mg/mL, respectively. The resolutions are 3.8
between DS and heparin and 5.8 between
heparin and OSCS; both are well beyond theUSP resolution requirements, which are 1.0 and1.5, respectively. GlycomixTM column shows theseparation resolution much higher than that ofCompetitor D column, which is 1.1 and 1.8[3]. It
was also stated in competitor D’s literature[3] thatdermatan sulfate may be unresolved fromheparin at >20 mg/mL concentrations as theheparin peak starts eluting from the column
within 2 min after the dermatan peak. Heparinloading is not a concern on GlycomixTM column.

Experimental
HPLC system
Agilent 1200 HPLC with binary pump

The high resolution between heparin anddermatan sulfate also enables identification of
any unknown impurities that elutes between
heparin and dermatan sulfate. As shown inFigure. 2, there is an impurity peak eluted after
dermatan sulfate, which is marked by an arrowon the graph. Comparing it with each single
standard, it is found that this unknown impurity
peak, which may be chondroitin sulfate, is alsopresent on heparin sodium salt (Sigma-Aldrich)sample. Figure 3 is another example of a real
heparin injection product analysis, which shows
the presence of OSCS.

Ion-exchange columns
GlycomixTM-SAX (4.6 x 250 mm, P/N 9016654625)
GlycomixTM-SAX guard column (4.6 x 50 mm,P/N 901665-4605)
Chemicals and ReagentsHeparin sodium salt and Dermatan sulfate (DSor chondroitin sulfate B) were purchased from
Sigma-Aldrich. Oversulfated chondroitin sulfate(part no. 1133580) was purchased from U.S.
Pharmacopeia. Monobasic sodium phosphateand sodium perchlorate were purchased fromEMD. All solutions were made with Milli-Qwater.

Glycomix™ can be used in the quantitative
analysis of heparin and its impurities. Figure 4
shows the calibration curve with different
heparin and impurity loadings over concentration
range from 0.5 mg/ml to 10 mg/ml for heparinand from 0.25 mg/ml to 4 mg/ml for OSCS. The
R2 values exceed 0.99 for both analytes(R2heparin=0.9997; R2OSCS=0.9972).

LC Method
Mobile phase:
A: 0.04% NaH2PO4, pH3.0;
B: 0.04% NaH2PO4+ 14% NaClO4,pH3.0
Gradient: 20% B to 90% B in 60 min

2Heparin

mAU
250200150

OSCS10050DS00510152025303540min

Figure 1. Elution profile of standard solution, containing 0.2 mg/mL dermatan sulfate (DS), 20 mg/mL
heparin sodium and 0.2 mg/mL oversulfated chondroitin sulfate (OSCS).

Heparin

mAU
400
350
300
250
200150OSCSDS1005000510152025303540min

Figure 2. Comparison of standard solution (1 mg/mL DS, 20 mg/mL heparin and 1 mg/mL OSCS) and one
heparin injection product provided by an undisclosed drug company.
HeparinOSCSHeparin injection
OSCSDS
Standard solution
0510152025303540min

Figure 3. Comparison of standard solution (0.2 mg/mL DS, 20 mg/mL heparin and 0.2 mg/mL OSCS) andanother heparin injection product provided by an undisclosed drug company, a different source than that onFigure 2.

3

Figure 4. Heparin calibration curve over the concentration range from 0.5 mg/ml to 10 mg/ml, OSCS from0.25 mg/ml to 4 mg/ml.

Conclusion
It has been demonstrated that with careful control of surface chemistry, high resolution separation ofheparin and its impurities is successfully achieved on Sepax Glycomix TM column. GlycomixTM column iswell suitable for quality control for heparin products, as well as quantitative analysis due to its highsensitivity.

Reference1.
2.
3.

Advances in the separation, sensitive detection, and characterization of heparin and heparinsulfate. Anal Bioanal Chem (2009) 393: 155-169
Heparin Sodium, Pharmacopeia Forum 2009, 35 (5), 1-4.
Determination of oversulfated chondroitin sulfate and dermatan sulfate in heparin sodium usinganion-exchange chromatography with UV detection. Dionex Application Note 235.

Ordering Information
ProductGlycomix SAX
Glycomix SAX guard column
Glycomix SAX Kit
Heparin Sodium
Chondroitin Sulfate B
Oversulfated Chondroitin Sulfate
Glycomix SAX

Description
4.6 x 250 mm
4.6 x 50 mm
Column + Guard
50 mg
5 mg
5 mg
Custom size

Part number
901665-4625
901665-4605
901665-KIT
HP-50
CS-5
OSCS-5Inquire

For more information on GlycomixTM products, please visit Sepax website: http://www.sepaxtech.com or contact us at 1-877-SEPAX-US4