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Automated USP and EP GPC/SEC analysis of low molecular weight heparin (LMWH) Application Note Pharmaceutical Analysis

AuthorsDaniela Held and Friedhelm Gorescontact: [email protected] and [email protected]

IntroductionNatural heparin is a polysaccharide and consists of molecular chains of varying lengths. Lowmolecular weight heparin (LMWH) consists of short chains and is used as anticoagulant indiseases that feature thrombosis and for prophylaxis against it. Because LMWH has morepredictable anticoagulant effect and pharmacokinetics as well as less side-effects, it isrecommended over unfractionated heparin. Various methods of heparin depolymerization areused to manufacture LMWH.

LMWHs are defined as heparin salts having an averagemolecular weight of less than 8000 Da and for which atleast 60% of all chains have a molecular weight less than 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.

System RequirementsUSP1 EP2

SECcurity GPC1200isocratic pump

• flow rate [mL/min]: 0.6• mobile phase: 0.5M LiNO3

• flow rate [mL/min]: 0.5• mobile phase: Na2PO4 28.4 g/L

PSS SECcurity GPC1200 Autosampler

Columns column packing L59• PSS PFG precolumn 6*40mm • PSS PFG 10µ 100Å (7.8*300mm)• PSS PFG 10µ 300Å (7.8*300mm)

silica based, 5µ • PSS PROTEEMA 5µ 100Å

(7.5*300mm)

Detectors Refractive index • SECcurity 1200 RI

Refractive index • SECcurity 1200 RIadditional for calibration UV: • SECurity 1200 UV @ 234 nm

Calibration • USP Enoxaparin Sodium Calibrant ARS and B RS (narrow standard)

• Verification: USP test solution

• Heparin Low-Molecular-Mass forcalibration CRS batch 2 Mn = 3800

Software GPC software• PSS WinGPC UniChromoptional: Compliance Pack

• PSS WinGPC UniChrom withheparin module3

optional: Compliance Pack

PSS Polymer Standards Service GmbH In der Dalheimer Wiese 5 55120 Mainz | Germany

Polymer Standards Service-USA, Inc.160 Old Farm Rd, Suite A Amherst | MA 01002 | USA



Procedure, Results & Discussion

Although the calibration procedures described in the EP and USP are totally different, adedicated GPC/SEC software can be used to analyze the heparin data in accordance with bothstandards precisely. The following figures show PSS WinGPC UniChrom raw data of the UScalibration standards and the EP standard (Fig.1a and b) as well as the elugram data overlaidwith the corresponding automatically constructed calibration curves (Fig.2a and b).

Once the calibration curve is constructed, the analysis of the data is comparable. All requiredmolar mass information based on true heparin values are obtained.

Figure 3 shows the final results for the analysis ofunknown low molecular weight heparin. PSSWinGPC UniChrom GPC/SEC software allows toset 2 additional markers at 2000 and 8000 Da, todisplay the measured percent below 2000 Da,above 8000 Da and in between these values.

Of utmost importance is the y-axis of the resultwindow. WinGPC UniChrom shows a true molarmass distribution with a w(log M) axis. Only thisguarantees correct fractions above/below themolecular weight borders 2000 and 8000.4

Literature1 USP Enoxaparin sodium http://www.usp.org/pdf/EN/USPNF/enoxaparinSodium.pdf2 EP Low-Molecular-Mass (LMM) heparins Monograph 08283 D. Held, Automated SEC of low molar mass heparins according to EP, LC/GC Europe Application book 12, page35 (2006) 4 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)

Fig. 1a: Raw data Calibrant A and B, USP Fig. 1b: Raw data CRS 2, UV and RI signal

Fig.2b: Elugram CRS2 overlaid with the calibrationcurve constructed from RI and UV signal

Fig.2a: Elugram USP test standard overlaid withheparin calibration curve

Fig.3: Result comparison Enoxaparin sodium,sample A passes, sample B fails

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