NMR of heparin API: investigation of unidentified signals in the USP-specified range of 2.12-3.00 ppm

This article addresses the identification and quantification of the chemical species resulting in resonances at 2.17 and 2.25 ppm in the (1)H nuclear magnetic resonance (NMR) spectrum of pharmaceutical-grade heparin sodium. The NMR signals in question were first confirmed to arise from chemical moieties covalently attached to the heparin molecule through NMR diffusion experiments as well as chemical treatment of heparin active pharmaceutical ingredient (API) containing the resonances. The material responsible for the extra NMR signals was then demonstrated by NMR spiking studies to be something other than oversulfated chondroitin sulfate and was finally identified as an O-acetylation product of heparin through (13)C labeling experiments with subsequent NMR analysis. The extent of O-acetylation was quantified using three orthogonal techniques: (1)H NMR, ion chromatography, and headspace gas chromatography/mass spectrometry. The results of this work showed good agreement between the three quantitative methods developed to analyze the signals in the United States Pharmacopeia-specified region of 2.12-3.00 ppm for heparin API.