Determination of Four Melamine and Its Derivatives in Urine by Ultra-Performance Liquid Chromatography-Tandem Mass Spectrometry

OBJECTIVE:To establish a method for simultaneous detection of melamine(MEL), ammeline(AMN), ammelide(AMD) and cyanuric acid(CYA) in urine by ultra-performance liquid chromatography-tandem mass spectrometry(UPLC-MS/MS).METHODS:After the urine sample was added to the internal standard, they were mixed by acetonitrile, ultrasonic extraction and centrifugation, enriched by MCX or MAX solid phase extraction column, eluted with 5% ammoniated methanol or 2% formic acid methanol, dried under nitrogen at 40 ℃, separated by Amide chromatography column(2.1 mm×100 mm, 1.7 μm), qualitatively and quantitatively analyzed by mass spectrometer in the simultaneous scanning mode of positive and negative ions.RESULTS:Melamine and its derivatives were completely separated with great chromatography peak. The limit of detection(LOD) for melamine, ammeline, ammelide and cyanuric acid were 0.03, 0.04, 0.04 and 0.05 ng/mL, and the limit of quantification(LOQ) were 0.11, 0.12, 0.14 and 0.15 ng/mL, respectively. The recovery rate of standard addition samples of 0.2, 5, 50 and 200 ng/mL ranged from 94.6% to 106.5%, the intra-day precision was between 0.73% and 8.34%, and the inter-day precision was less than 6.80%(n=6). At low concentrations, AMD and CYA mainly showed matrix enhancing effect, while AMN showed matrix inhibitory effect with increasing concentration. The overall detection rate of melamine and its derivatives was 75.0% in 60 parcels of random urine. The average concentration of urinary melamine and its derivatives were higher in men.CONCLUSION:UPLC-MS/MS can simultaneously determine four melamine and its derivatives in urine. The LOD and LOQ of the current method were low with great accuracy and precision.