The metabolism and urinary excretion of N hydroxy 3,4 methylenedioxymethamphetamine (N OH MDMA), a newly banned narcotic in Japan, were explored to conˆrm biotransformation of N OH MDMA to 3,4 methylenedioxymethamphetamine (MDMA) and to discriminate between N OH MDMA and MDMA intake in forensic urine analysis. The in vitro and the in vivo experiences were performed with human liver S9 and rats, respectively, and the resultant products or metabolites were determined using liquid chromatography/mass spectrometry and liquid chromatography/tandem mass spectrometry.In both the in vitro and the in vivo experiences, MDMA and 3,4 methylenedioxyamphetamine (MDA) were detected, and the MDA levels exceeded the MDMA levels throughout the entire periods except for during 3 h after administration to the rats. This suggests the existence of the metabolic pathway(s) from N OH MDMA to MDA not via MDMA. In urine samples from the administered rats parent N OH MDMA and its demethylated metabolite N hydroxy 3,4 methylenedioxyamphetamine (N OH MDA) with very low levels during short period after administration (6 h) were detected. The ratios of the urinary MDA/ MDMA levels for N OH MDMA administered rats were higher than those for MDMA administered rats. In addition, the determination of urinary diastereomers of glucuronidated 4 hydroxy 3 methoxymethamphetamine (HMMA), MDMA metabolite, revealed that the relative peak intensity of l HMMA glucuronide to d