Molar Ratio of Sulfate to Carboxylate: A Critical Discussion
Shreekant Deshpande, Michael Waldman and Raghvendra Sahai
Analytical Chemistry Division, Eutech Scientific Services, Inc., Highland Park, NJ 08904, USA
USP Method and its Limitations
Introduction
Results
Heparin belongs to a family of molecules known as glycosaminoglycans
(GAGs). These GAGs are differentiated into four different types depending
upon monosaccharide composition, configuration and position of their
glycoside linkages.
For evaluation of sulfate to carboxylate ratio, the GAG sample needs to be in
its acid form. Therefore ion chromatography was required to remove the
presence of free ions in it. The chromatogram obtained after eluting through
both type of resins are shown (Fig.2).
Method Optimization
LMWH
1.
2.
3.
4.
Discussion
Hyaluronic Acid
Chondroitin SO4/ Dermatan SO4
Heparin SO4
Keratin SO4
Inj. Volume Titrant Con. Dilution
SC
5mg/mL
1000 uL
0.01 N
60 mL
3.51
10mg/mL
1000 uL
0.01 N
60 mL
2.43
15mg/mL
1000 uL
0.01 N
60 mL
2.75
25mg/mL
1000 uL
0.1 N
60 mL
2.98
30mg/mL
1000 uL
0.1 N
60 mL
2.95
50 mg/mL
1000 uL
0.1 N
60 mL
2.55
The conductimetric curves obtained by titration of the acid form of GAGs
with 0.1N NaOH are more characteristic (Fig.3). The Initial conductance of
the solutions was relatively high due to the contribution of the mobile protons
of the sulfate groups, but decreases sharply as these protons were replaced by
Na+ ions. After all the SO3H groups have been neutralized, however, the
curves level out; in fact, the conductance barely changes during neutralization
of the carboxyl ions, giving the characteristic plateau regions in the titration
curves. In the plateau region, the small contribution due to neutralization of
the largely undissociated carboxyl protons is compensated by the positive
contribution from the increasing concentration of Na+ ions.
(Fig 4)
Validation of Method
No Injection volume is specified
Optimized method was validated for
precision, accuracy and specificity
for the GAGs. Both repeatability and
intermediate precision were carried
out with Enoxaparin sodium and the
RSD was about 5%. Water alone was
used to test the specificity.
Heparin
Heparin, a highly sulfated GAG, is a polymer of alternating residues of
uronic acid and glucosamine which are varyingly sulfated. This mixture of
chains of varying length and sulfation used widely as an injectable
anticoagulant, and has the highest negative charge density of any known
biological molecule. It can also be used to form an inner anticoagulant
surface on various experimental and medical devices such as test tubes and
renal dialysis machines[1].
Low Molecular Weight Heparins (LMWHs) are obtained by depolymerizing
or fragmenting unfractionated heparin (UFH) by different chemical or
enzymatic processes.
Method Modification
Optimized Procedure
Sample Solution: 50 mg/ mL Enoxaparin in CO2 free water.
Injection Volume: 1 mL of the 50 mg/ mL solution
Size of the Columns: Both anion (L64) and cation (L65) resin obtained from
Bio-Rad Laboratories are packed in 1.5-cm x 10-cm columns (MT20).
1. The eluted peak of interest from the ICS was collected for further
analysis.
2. The collected solution was diluted to 60 mL with CO2 free water.
3. This solution was then manually titrated against 100 µL of 0.1N NaOH.
4. The conductivity of the solution was monitored as a function of volume of
NaOH titrated. Test results are average of replicate runs.
(Fig 5)
Sulfate to Carboxylate ratio (SC)
3
4
5
6
Mean
Precision
1
2
Reapeatability
2.6
2.56
2.53
2.38
2.37
2.3
Intermediate
2.67
2.61
2.58
2.42
2.39
2.36
Specificity
1
2
Water
0
0
SD
rsd
2.46
0.122
5%
2.51
0.131
5%
0
0
As per USP method, a sample (LMWH) of 5mg/mL was used as starting
concentration, and the test was run with 25 µL injection volume, there were
no three linear sections, comprising initial downward slope, a middle slight
slope and a final rise. So we tried different concentrations of the sample
ranging from 10-100 mg/mL, higher loop volumes, and lower concentration
of titrant to decide what combination will be to produce consistent results. The
optimized method comes out to be 50 mg/mL LMWHs, 1000 µL injection
volume and titration with 0.1N NaOH.
With Different LMWHs
Materials and Instruments used
Thermo Dionex ICS-2100 with manual injection
Thermo Scientific Orion Star A215 pH/Conductivity Meter
Orion 013005MD Conductivity Cell
Fluka 0.1N NaOH, Fluka 319511, Lot#SHBC3764V
Water: Grade I.
Enoxaparin
I
2.78
SC
II
2.85
Average
2.55
Deltaparin
3.01
2.95
2.98
Nadroparin
2.52
2.68
2.60
Parnaparin
2.12
2.27
2.20
Tinzaparin
2.26
2.23
2.25
LMWHs
Validation parameters performed on this optimized method yielded RSD of
repeatability and intermediate precisions was about 5%. Stability studies was
also carried out with different time period of titration and the output clearly
details the environmental CO2 neutralizes the acidity of both analyte and
water. The initial conductivity get reduces with the time period and consumes
little high amount of titrant to dissociate carboxyl groups.
Column dimensions are least important as long as they contain sufficient
amount of resins to convert loaded LMWHs into acidic form and their
repetitive regeneration in between the runs. 35-50 mg of LMWHs are required
to give correct SC ratio when titrating with 0.1N NaOH and 10-15 mg is for
0.01N NaOH titration.
(Fig 6)
(Fig 1)
Different LMWHs were characterized for this optimized SC method. They all
behaved same and produced characteristic response. Enoxaparin curve was
relatively shallow compare to other LMWHs (Fig 6).
Differences in the specificity of interaction between GAGs and their binding
proteins result from the structural diversity of GAGs.
The degree of sulfation in a UFH/ LMWH sample is related to its biological
function and hence to its quality.
Thus USP has made mandatory determining the molar ratio of sulfate to
carboxylate for appropriate quality control of each GAGs/ LMWHs.
Acknowledgement
Stability Analysis
It includes type, size, saccharide composition, charge density, sequence and
molecular weight.
Charge density is often referred to as degree of sulfation or molar ratio of
sulfate to carboxylate, and has a great impact on the proper functioning of
GAG, compared to other structural properties.
After neutralization of all the carboxyl groups, the conductance increases
sharply again because of the added contribution of the mobile OH¯ ions. The
curves are rounded off near the equivalence points, because of hydrolysis
effects, the impact of which is different for different samples. Extrapolation of
the three branches of the conductimetric curve gives two intersections, the
first corresponding to the equivalence point of the sulfates, and the second to
that of the carboxyl ions. The conductimetric titration of GAGs in the acid
form, therefore appears to provide a simple method for determining both the
sulfate and the carboxyl groups.[3]
Miss. Keyaara Robinson and Dr. Shailendra K Saxena for their technical assistance
(Fig 2)
(Fig 3)
Eluant peak was collected from the cation- exchange column in a beaker at the
outlet until the ion detector reading returns to the baseline value and used it for
conductivity titration. Although there is not much difference in SC ratio while
considering the collection of full run.
Column dimensions are least important as long as they contain sufficient
amount of resins to convert loaded LMWHs into acidic form and their repetitive
regeneration in between the runs.
(Fig 7)
SC
0h
6h
24h
48h
1
2.56
3
2.53
2.45
2
2.54
2.69
2.41
2.12
Avg
2.55
2.85
2.47
2.29
Enoxaparin sodium was titrated in
different time periods and determined
the SC ratio. The environmental
bicarbonate affects the ratio by
shifting the curve to the higher side
of the graph by consuming more
amount of titrant.
References
[1] Goodman and Gilman's The Pharmacological Basis of Therapeutics, Twelfth Edition.
[2] Linhardt R.J. & Gunay, M.S. (1999) Semin Thromb Hemost. 25 (3), 5-16.
[3] Casu, B. & Gennaro U. (1975) Carb Res. 39, 168-176.
[4] USP37 NF32 Enoxaparin Sodium (Revised bulletin 2011)
[5] Calero, V.R., Puignou L., Diezi, M & Galcern M.T. (2001) Analyst 126,169-174.
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