Simultaneous quantification of chondroitin sulfate and dermatan sulfate in crude heparins using 1H NMR and independent component analysis

Heparin, an essential anticoagulant in clinical practice, is susceptible to contamination with chondroitin sulfate (CS) and dermatan sulfate (DS). Ensuring the reliable quantification of these polysaccharides in crude heparins is critical for product safety and for monitoring the purification process, yet remains challenging due to their structural similarity and heterogeneous composition. ¹H NMR and Independent Component Analysis (ICA) were used to resolve the overlapping acetyl signals of CS and DS. Samples of crude heparin and synthetic mixtures were measured on a 500 MHz high-field NMR and an 80 MHz benchtop instrument. The ICA yielded models with root mean square errors (RMSEs) ≤ 1.7 % w/w (500 MHz) and ≤ 2.1 % w/w (80 MHz) for the synthetic mixtures (concentration range of 3-19 % w/w for CS and 3-40 % w/w for DS). The accuracy of the ICA method was confirmed by the conventional enzymatic digestion method for crude heparin samples. While the 500 MHz model completely conformed with the results of the enzymatic method, the 80 MHz benchtop system is best suited for screening applications where impurity levels are from moderate to high. The ICA-NMR workflow surpasses the traditional enzymatic assay in speed and simplicity, and opens the way towards the development of an automated and validated on‑site heparin quality control method.