Abstract: Objective To identify the reference substance of mecobalamin and to explore the factors that affect its contents. Methods The reference substance was determined using HPLC, water titration, GC and high-resolution mass spectrometry. The dynamic vapor sorption (DVS) method was adopted to study hygroscopicity. Results The structure of the reference substance was confirmed while the adsorbed moisture content was analyzed as expected. In terms of hygroscopicity, the adsorbed moisture content was adjusted to 8%,which could ensure stable weighing when the ambient humidity ranged from RH 40% to RH 50%. Conclusion The changes in the hygroscopicity of the reference substance caused by alterations in the processing technology deserve attention. Studies on packaging, storage conditions and environments can help users better understand the characteristics of and considerations for mecobalamin.

Key words: Mecobalamin, Chemical Reference Substance(CRS), Hygroscopicity, Dynamic Vapor Sorption(DVS), Packaging, Storage, Quantitation