HPLC analysis of the molecular mass of low-molecular-mass heparins

Heparins are a class of mucopolysaccharide obtained from the porcine small intestine and is widely used in the pharmaceutical field as an anticlotting drug for the treatment and prevention of thromboembolism, treatment of disseminated intravascular coagulation syndrome, and as an anti-blood clotting agent when using extracorporeal blood circulating apparatus such as blood dialysis and artificial heart/lung. Low-molecular-mass heparin is produced by enzymatic treatment and chemical manipulation of heparin and is used as an anticlotting drug similarly to heparin due to its more predictable pharmacokinetics. Low-molecular-mass heparin is classified as parnaparin, dalteparin, enoxaparin etc., by decomposition method and molecular weight distribution.

In the European Pharmacopoeia 8th Edition (EP), the molecular weight measurement method is defined in the pharmaceutical products article of low-molecular-mass heparin, the average molecular weight and molecular weight distribution are also defined in the pharmaceutical article for lowmolecular-mass heparin (sodium) and low-molecular-mass heparin (calcium). Size exclusion chromatography (SEC) combined with a UV-Visible and Refractive Index detection is defined as the measurement method. In the analytical method, a molecular weight calibration curve using lowmolecular-mass heparin is created using low-molecular-mass heparin as the reference sample. In common molecular weight measurement methods using SEC, a calibration curve is created from the retention volume and molecular weight; this common method is used for oligomers and polymers where the molecular weight has already been determined and used as a standard sample. However, in this case the molecular weight has to be calculated by standardizing the coefficient and intensity ratio of RI and UV to define the calibration curve. This type of SEC analysis is considered so complicated that it can be difficult to use with commercial software.

In this application note, Jasco demonstrates the use of the optional low-molecular-mass heparin molecular weight calculation program for ChromNAV 2.0 to analyze the mass average molecular weight and molecular weight distribution of low-molecular-mass heparins using the method described in the European Pharmacopoeia.