Natural heparin is a polysaccharide and consists of molecular chains of varying lengths. Low molecular weight heparin (LMWH) consists of short chains and is used as anticoagulant indiseases that feature thrombosis and for prophylaxis against it. Because LMWH has more predictable anticoagulant effect and pharmacokinetics as well as less side-effects, it is recommended over unfractionated heparin. Various methods of heparin depolymerization are used to manufacture LMWH.
LMWHs are defined as heparin salts having an average molecular weight of less than 8000 Da and for which atleast 60% of all chains have a molecular weight lessthan 8000 Da. GPC/SEC is the method of choice to measure the molecular weight (molar mass) and the molecular weight fractions above/below a molecular weight limit. Both, the Pharmaeuropa and the US Pharmacopeia, require GPC/SEC to characterize e.g. Enoxaparin Sodium or other low molar mass heparin. As recently again demonstrated, these methods allow to measure true molar masses with inexpensive standard lab equipment and lowest experimental effort.
Procedure, Results & Discussion
Although the calibration procedures described in the EP and USP are totally different, a dedicated GPC/SEC software can be used to analyze the heparin data in accordance with both standards precisely. The following figures show PSS WinGPC UniChrom raw data of the US calibration standards and the EP standard (Fig.1a and b) as well as the elugram data overlaid with the corresponding automatically constructed calibration curves (Fig.2a and b).
Once the calibration curve is constructed, the analysis of the data is comparable. All required molar mass information based on true heparin values are obtained.
Figure 3 shows the final results for the analysis of unknown low molecular weight heparin. PSS WinGPC UniChrom GPC/SEC software allows to set 2 additional markers at 2000 and 8000 Da, to display the measured percent below 2000 Da, above 8000 Da and in between these values. Of utmost importance is the y-axis of the result window. WinGPC UniChrom shows a true molar mass distribution with a w(log M) axis. Only this guarantees correct fractions above/below the molecular weight borders 2000 and 8000.5
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1 Application note by Wyatt Technology, Santa Barbara 2011
2 USP Enoxaparin sodium http://www.usp.org/pdf/EN/USPNF/enoxaparinSodium.pdf
3 EP Low-Molecular-Mass (LMM) heparins Monograph 0828
4 D. Held, Automated SEC of low molar mass heparins according to EP, LC/GC Europe Application book 12, page
5 D. Held, P. Kilz: "Qualification of GPC/SEC/SEC Data and results" chapter 13 in H.-J. Kuss/ S. Kromidas (eds.),
Quantification in LC and GC - A Practical Guide to Good Chromatographic Data, Wiley-VCH, Weinheim (2009)