GC and HPLC-RID method development and validation for the determination of twelve related substances, including several novel compounds in tetrabenazine

A series of GC-FID and HPLC-RID methods were developed and rigorously validated for the detection of various aldehyde and ketone impurities, including 5-methyl-2-hexanone and 5-methyl-3-hexen-2-one, as well as quaternary ammonium salt impurity that lack UV absorption, during the synthesis of tetrabenazine. Six novel compounds, three have not been previously reported, were synthesized and their structures confirmed. This was achieved through a comprehensive investigation into impurity transmission chains and their clearance throughout the synthesis process. The limits for all detected impurities, which included a total of twelve related substances, were established at approximately 0.1 %. The LOD for the developed analytical methods ranged from 0.3 μg mL −1 to 12.5 μg mL −1 .