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Heparin NMR
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IntroductionHeparin is used as an injectable blood anticoagulant. The material is a naturally occurring sulfated carbohydrate derived from either porcine intestines or bovine lungs. Heparin is a mixture of {GlcN-GlcA/IdoA}n oligo/polymers (n varies widely), with GlcN and GlcA/IdoA each sulfated to variable extent. When GlcN is not sulfated (i.e., not GlcNS), it is acetylated (i.e., occurs as GlcNAc). In 2008, allergic reactions from contaminated heparin resulted in serious illnesses and deaths worldwide. The FDA traced the contamination to a related carbohydrate, oversulfated chondroitin sulfate (OSCS). See also Guerrini et al., Nature Biotechnology 26, 669-675 (2008) “Oversulfated chondroitin sulfate is a contaminant in heparin associated with adverse clinical events”. USP revised its Heparin Sodium monograph to include tests and specifications for this contaminant. A second revision of the monograph appeared in USP32; Pharmacopeial Forum 35(5) and became official in Sept-Oct 2009. The USP monograph requires 500-MHz 1H nuclear magnetic resonance (NMR) spectroscopy for identification of heparin and analysis for OSCS, as well as a number of other specifications. A signal diagnostic for OSCS appears in the 1H NMR spectrum at 2.16+0.03 ppm; this is the NAc signal of a GalN building block of OSCS. |
In 2009, Sanofi-Aventis reported a non-harmful contaminant present in some batches of their Lovenox. This impurity is believed to be a heparin analog with IdoA2S3Ac rather than IdoA2S(3S) as a building block. The 3Ac signal of this impurity appears in the 1H NMR spectrum at 2.18 ppm. While this is very close to the diagnostic OSCS NAc signal, in our experience it is easily resolved in most samples with our Bruker Avance III 500 MHz system. Other contaminants that have been detected in heparin samples usually come from process solvents such as ethanol, MIBK (methyl isobutyl ketone), etc., or are salts (such as acetate), all of which are allowed under the monograph. We typically verify the assignments of residual solvent signals in the 1H NMR spectra by subjecting the heparin sample to GC-MS analysis (see Residual Solvents).For more information on NMR ...
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Current USP SpecificationsThe specifications in the current monograph are presented below in an example report format: |
Exova (WCAS) provides NMR testing services for Heparin. |
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Reference: |
United States Pharmacopeia 32; Pharmacopeial Forum 35(5) Interim Revision Announcement |
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Monograph: |
Heparin Sodium |
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Sample ID: |
xxxxxxxx |
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Analysis |
Specification |
Result |
Pass/Fail |
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Identification A |
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1H NMR Spectrum |
Pass |
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The 500-MHz 1H NMR spectrum of the Sample solution exhibits: |
The 500-MHz 1H NMR spectrum of the Sample solution exhibited: |
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Signal 1 at 5.42 ± 0.03 ppm. |
Signal 1 at 5.420 ppm. |
Pass |
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Signal 2 at 5.21 ± 0.03 ppm. |
Signal 2 at 5.216 ppm. |
Pass |
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Signal 3 at 3.28 ± 0.03 ppm. |
Signal 3 at 3.284 ppm. |
Pass |
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Signal 4 at 2.05 ± 0.03 ppm. |
Signal 4 at 2.043 ppm. |
Pass |
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No unidentified signals greater than 4% of the mean of signal height of Signals 1 and 2 between 0.10 and 2.00 ppm. |
A triplet assigned to ethanol CH3 (residual solvent) at 1.186 ppm (signal height 95.6%); A singlet assigned to acetate at 1.913 ppm (signal height 20.6%) |
Pass |
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No unidentified signals greater than 4% of the mean of signal height of Signals 1 and 2 between 2.10 and 3.20 ppm. |
No signals greater than 4% of the mean of signal height of Signals 1 and 2 between 2.10 and 3.20 ppm. |
Pass |
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No unidentified signals greater than 4% of the mean of signal height of Signals 1 and 2 between 5.70 and 8.00 ppm. |
No signals greater than 4% of the mean of signal height of Signals 1 and 2 between 5.70 and 8.00 ppm. |
Pass |
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No signals greater then 200% signal height of the mean of the signal height of Signals 1 and 2 between 3.35 and 4.55 ppm. |
No signals greater then 200% signal height of the mean of the signal height of Signals 1 and 2 between 3.35 and 4.55 ppm. |
Pass |
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Analysis |
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Absence of Oversulfated Chondroitin Sulfate |
No features associated with oversulfated chondroitin sulfate between 2.12 and 3.00 ppm. |
No signals indicative of oversulfated chondroitin sulfate between 2.12 and 3.00 ppm. |
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Date Analyzed: |
12-11-09 |
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Quality Control Summary |
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Sample ID: |
System Suitability Solution |
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Analysis: |
1 H NMR Spectroscopy |
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Criterion |
Specification |
Result |
Pass/Fail |
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Heparin NAc Resonance |
2.05 ± 0.02 ppm |
2.045 ppm |
Pass |
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OSCS NAc Resonance |
2.16 ± 0.03 ppm |
2.151 ppm |
Pass |
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Sample ID: |
Standard Solution |
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Analysis: |
1 H NMR Spectroscopy |
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Criterion |
Specification |
Result |
Pass/Fail |
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Heparin NAc Resonance |
> 1000:1 |
1064:1 |
Pass |
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Date Analyzed: |
12-11-09 |
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1H NMR Spectra of Heparin
