MOLECULAR-WEIGHT OF HEPARIN USING C-13 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 d etermination of this important drug has traditionally relied on gel pe rmeation chromatography, which requires the use of well-defined molecu lar weight standards that are not easily obtained. We have investigate d the use of C-13-NMR spectroscopy for measuring the number average mo lecular weight of heparin. The signal intensities of the reducing end and internal anomeric carbons, having distinctive chemical shifts in t he C-13-NMR spectrum, were used to determine the molecular Weight. Dis tortionless enhancement polarization transfer was found to provide a b etter quantitation of signal intensities of anomeric carbons than broa d band decoupling or selective decoupling of anomeric protons. Signal averaging over 300 000 transients, requiring approximately 48 h on a 3 60 MHz NMR spectrometer, resulted in the measurement of the number ave rage molecular weight (similar to 10 000 Da) of heparin. C-13- NMR spe ctroscopy does not require the use of difficult to obtain molecular we ight standards and thus is particularly well-suited for workers in the pharmaceutical industry.