Determination of Residual Cyromazine and Its Metabolite Melamine in Pig Urine Samples Using Ultrahigh Performance Liquid Chromatography Coupled with Quadrupole£­Orbitrap Mass Spectrometry

KeyWord:pig urine  cyromazine  melamine  ultrahigh performance liquid chromatography coupled with quadrupole£­orbitrap mass spectrometry

Abstract:

      An ultrahigh performance liquid chromatography coupled with quadrupole£­orbitrap mass spectrometry£¨UHPLC£­Quadrupole£­Orbitrap MS£© was developed for the determination of cyromazine and its metabolite melamine in pig urine samples£®The pig urine sample£¨2.0 g£© was ultrasonically extracted with 1% trichloroacetic acid under the pH value of the extraction mixture lower than 3£¬followed by purification with a mixed cation solid phase extraction column£¨MCX£©£¬then the eluent from the MCX column was blow dried under nitrogen flow£¬and finally reconstituted with 95% acetonitrile aqueous solution£®The reconstituted solution 10 times diluted was injected into the UHPLC£­Quadrupole£­Orbitrap system using a Syncronis HILIC column£¨100 mm ¡Á 2.1 mm£¬1.7 ¦Ìm£© at 35 ¡æ£¬with 10 mmol/L ammonium acetate and acetonitrile as the mobile phases£®The mass spectrometer was operated in positive electrospray£¨ESI + £© and parallel reaction monitoring£¨PRM£© mode£®Qualitative analysis was carried out according to the accurate masses of each target compound£¬its fragment ions£¬and the abundance ratios of the fragment ions£®The external standard method and internal standard method were used for the quantification of cyromazine and melamine£¬respectively£®Method validation showed that there were good linear relationships for the two targets in the range of 0.2£­50.0 ng/mL£¬with their correlation coefficients£¨r2£© larger than 0.99£®At three spiked levels of 1.0£¬2.0 and 150.0 ¦Ìg/kg£¬the recoveries for cyromazine and melamine in pig urine sample ranged from 78.0% to 105%£¬with relative standard deviations£¨RSD£© no more than 11%£®The same spiked samples were used for comparison between PRM of orbitrap and multiple reaction monitoring£¨MRM£© mode of triple quadrupole£®The results obtained exhibited no significant difference£¬indicating that both methods have good quantitative ability for determination of cyromazine and melamine in pig urine sample£®With good recovery£¬dynamic range and quantification accuracy£¬the developed method could be applied to the simultaneous qualitative and quantitative analyses of cyromazine and melamine residues in pig urine samples£®