Characterization of an Unknown Impurity in Glucosamine Sulfate Sodium Chloride by HPLC-Q-TOF MS and NMR

Abstract

Background: Glucosamine sulfate sodium chloride (glucosamine-SP) is mainly used for the treatment of osteoarthritis. During quality control of glucosamine-SP capsules, an unknown impurity was detected. Another unknown degradation product was generated together with above-mentioned impurity in heat condition. Objective: The study aimed to characterize an unknown impurity in glucosamine-SP capsules. Methods: A new volatile HPLC method compatible with mass spectrometry detection was set up. An amino column at 35 °C with a mobile phase consisting of water and acetonitrile (20: 80, v/v) was used at a flow rate of 1.5 ml/min at 297 nm. High-performance liquid chromatography quadrupole time-offlight mass spectrometry (HPLC-Q-TOF MS) was used to identify the impurity with the electrospray ionization (ESI) source in the positive ionization mode. Results: The results of HPLC-Q-TOF MS analysis indicated that the protonated molecule ions [M + H]+ of the unknown impurity and the novel degradation product were both at m/z 287. Preparative LC method was put into practice with a Prep-C18 column with a mobile phase consisting of water and acetonitrile (99: 1, v/v) at a flow rate of 20.0 ml/min at 297 nm. The assignment of the 1D and 2D NMR signals was performed for the unknown impurity. In addition, the formation of impurities was also studied. Conclusion: An unknown impurity and a degradation product in glucosamine-SP capsules were characterized. They were assigned as (1R, 2S, 3R)-1-(5-((S, E)-3, 4-dihydroxybut-1-en-1-yl) pyrazin-2-yl) butane-1, 2, 3, 4-tetraol and (1R, 2S, 3R)-1-(5-((S, Z)-3, 4-dihydroxybut-1-en-1-yl) pyrazin-2-yl) butane- 1, 2, 3, 4-tetraol.