AIM: Itopride, which is a dopamine D2 antagonist with acetylcholinesterase inhibitory actions, is often prescribed for patients with functional dyspepsia. The primary metabolite in humans is the N-oxide, generated by oxidation of the tertiary amine N-dimethyl group. The urinary excretions of itopride and its N-oxide were 3.7% and 75.4%, respectively, in healthy subjects after a single oral administration at a therapeutic dose. Studies support a predominant role of FMO3 in the formation of itopride N-oxide in human liver microsomes. In addition, itopride appears to be a suitable probe for human liver FMO3. Our aim was to establish a simple method adopting a one-step liquid-liquid extraction with dichloromethane followed by high-performance liquid chromatography (HPLC) with positive ion electrospray ionization tandem mass spectrometric (ESI-MS/MS) detection to simultaneously determine itopride and its N-oxide in human plasma and urine for FMO3 pharmacogenetics study, using sulpiride as an internal standard (IS). METHODS: Acquisition was performed in multiple reaction monitoring (MRM) mode, by monitoring the transitions: m/z 360.1>166.4 for itopride, m/z 376.1>165.5 for itopride N-oxide and m/z 342.9>112.2 for IS, respectively. Analytes were chromatographed on an Intersil ODS-3 reverse-phase chromatographic column (2.1 mm×150 mm, 5 μm) by isocratic elution with water (1000 mL water added 1 mL formic acid)-acetonitrile (60∶40, v/v), the flow rate was 0.2 mL/min with a total analysis time of 3 min per run. RESULTS: The linearity for itopride and its N-oxide were in the range of 0.25-1 000 ng/mL and 0.5-1 000 ng/mL, respectively, both in plasma and urine (r2=0.999). The recovery of itopride was 95.8% for plasma and 101.2% for urine and that of itopride N-oxide was 90.4% for plasma and 98.4% for urine. The intra-and inter-day RSD were <15% both in plasma and urine. CONCLUSION: The method described is successfully applied to the analysis of itopride and its N-oxide in plasma and urine samples generated after administration of 50 mg itopride hydrochloride in 18 healthy male volunteers who were genetyped according to FMO3 genetic polymorphism. AIM: Itopride, which is a dopamine D2 antagonist with acetylcholinesterase inhibitory actions, is often prescribed for patients with functional dyspepsia. The primary metabolite in humans is the N-oxide, generated by oxidation of the tertiary amine N-dimethyl group. The urinary excretions of itopride and its N-oxide were 3.7% and 75.4% respectively, in healthy subjects after a single oral administration at a therapeutic dose. Studies support a predominant role of FMO3 in the formation of itopride N-oxide in human liver microsomes. In addition, itopride appears to be a suitable probe for human liver FMO3. Our aim was to establish a simple method adopting a one-step liquid-liquid extraction with dichloromethane followed by high-performance liquid chromatography (HPLC) with positive ion electrospray ionization tandem mass spectrometric (ESI-MS / MS) detection to determine that itopride and its N-oxide in human plasma and urine for FMO3 pharmacogenetics study, using sulpiride as a METHODS: Acquisition was performed in multiple reaction monitoring (MRM) mode, by monitoring the transitions: m / z 360.1> 166.4 for itopride, m / z 376.1> 165.5 for itopride N-oxide and m / z 342.9> 112.2 for IS, respectively. Analytes were chromatographed on an Intersil ODS-3 reverse-phase chromatographic column (2.1 mm × 150 mm, 5 μm) by isocratic elution with water (1000 mL water added 1 mL formic acid) 60:40, v / v), the flow rate was 0.2 mL / min with a total analysis time of 3 min per run. RESULTS: The linearity for itopride and its N-oxide were in the range of 0.25-1000 ng / mL and 0.5-1 000 ng / mL, respectively, both in plasma and urine (r2 = 0.999). The recovery of itopride was 95.8% for plasma and 101.2% for urine and that of itopride N-oxide was 90.4% for plasma and 98.4% for urine. The intra-and inter-day RSD were <15% both in plasma and urine. CONCLUSION: The method described is successfully applied to the analysis of itopride and its N-oxide in p lasma and urine samples generated after administration of 50 mg itopride hydrochloride in 18 healthy male volunteers who were genetyped according to FMO3 genetic polymorphism.