Molecular weight of heparin using 13C nuclear magnetic resonance spectroscopy

Heparin is a polydisperse, heterogeneous polysaccharide that has been used as an anticoagulant for the past 50 years. The molecular weight determination of this important drug has traditionally relied on gel permeation chromatography, which requires the use of well-defined molecular weight standards that are not easily obtained. We have investigated the use of 13C-NMR spectroscopy for measuring the number average molecular weight of heparin. The signal intensities of the reducing end and internal anomeric carbons, having distinctive chemical shifts in the 13C-NMR spectrum, were used to determine the molecular weight. Distortionless enhancement polarization transfer was found to provide a better quantitation of signal intensities of anomeric carbons than broad band decoupling or selective decoupling of anomeric protons. Signal averaging over 300,000 transients, requiring approximately 48 h on a 360 MHz NMR spectrometer, resulted in the measurement of the number average molecular weight (approximately 10,000 Da) of heparin. 13C-NMR spectroscopy does not require the use of difficult to obtain molecular weight standards and thus is particularly well-suited for workers in the pharmaceutical industry.