Development and validation of a size exclusion chromatography method for determination of molecular masses and molecular mass distribution in low molecular weight heparin.

A precise and rugged high performance size exclusion chromatography (HPSEC) method for determination of peak maximum molecular mass (Mp), weight average molecular mass (Mw), number average molecular mass (Mn), and molecular mass distribution (MMD) in Low Molecular Weight heparin (LMW heparin) has been developed and validated on two Ultrahydrogel 250 (ID 7, 8 mm, length 30 cm) columns in series at three laboratories using different equipment. The calibration is based on four local laboratory heparin standards with relatively narrow molecular mass distribution and Mp covering the range from 3,000 to 17,000 Da. The calibration curve describing the logarithm of the molecular mass versus retention time is linear in the range 3,000 to 17,000 Da. The precision (relative standard deviation) within-run and between-run is better than 2%. The between-laboratory variation is below 5%, 3% and 8% for Mp, Mw and Mn, respectively. This method is reproducible, rugged, and fast and is suited for the determination of average molecular masses and molecular mass distribution of LMW heparins on a routine basis.