A separation method for piperine and its isomers (chavicine, isochavicine and isopiperine) from a photoirradiated mixture of piperine, in solution and in suspension, by high performance liquid chromatography (HPLC), was investigated to clarify the relationship between the disappearance of the pungency in older, ground pepper and the photoisomerization of piperine. Piperine and its isomers were separated on a Sumichiral OA-2000 column with hexane/dichloromethane/ethanol (4811610.8) as an eluting solvent. Their structural elucidation was carried out using nuclear magnetic resonance spectroscopy and HPLC atmospheric pressure chemical ionization mass spectrometry (LC/APCIMS). Although the increment in the three isomers in commercial ground pepper after exposure to sunlight was observed by monitoring the MH+ ion on LC/APCIMS, the degree of increase varied very little. It is questionable whether the disappearance of the pungency in older, ground pepper is derived from the formation of tasteless isomers by photochemical changes in piperine.