Distribution Of Melamine Residues In Aquatic Products By Liquid Chromatography-Mass Spectrometry

This paper describes a method of HPLC-MS for determination of melamine in a widerange of food matrices including several aquatic products(Trachinotus blochii, Tilapia, andPenaeus vannamei), which were the main aquaculture varieties in the southern region ofChina. The detection methods of melamine in the aquatic and feed while the residueelimination of melamine in Penaeus vannamei were studied in this paper, using mathodsincluding the pre-treatment method, the optimization of chromatographic conditions andmass spectrometry conditions and so on. The main research contents and results are asfollows:1. Detectionation of Melamine in Aquatic Products by HPLC-MSA method for determination of melamine in aquatic products was established bydispersive solid phase extraction-high performance liquid chromatography-massspectrometry (DSPE-HPLC-MS). Samples were extracted with Methanol, pH4.5buffersolution of ammonium, and then cleaned with dispersive solid phase extraction. Afterextracted and separated on a Thermo Fisher Hypersil Gold C18(150mm×2.1mm，5μm),the analytes were detected by HPLC-MS with the selected reaction monitoring (SRM)anion mode. The results showed that: the calibration curves of elamine were linear in therange of5ng/mL~50ng/mL form, the correlation coefficient was0.9999, the limit ofquantization was0.01mg/kg, and the average recovery was in the range of82.8%~100.4%,RSD<5.0%. This method was suitable for the detection of melamine in aquatic products.2. Determination of Melamine in Feed by HPLC-MSA method for determination of melamine in feed stuff was established by highperformance liquid chromatography-mass spectrometry (HPLC-MS) with the optimizationof extraction, purification methods, choose a column. Determine the optimal extractionprograms for the corresponding extracts by orthogonal test. Samples were extracted inacetonitrile solution (V:V,70:30) by ultrasonic, the supernatant after centrifugation mixedwith an equal volume of acetonitrile for secondary protein precipitation, centrifugeddirectly after filtration membrane can be measured on the machine. After extracted andseparated on a Waters Atlantis HILIC Silica (150mm×2.1mm,5μm), The analytes weredetected by HPLC-MS with the selected reaction monitoring (SRM) mode. The resultsshowed that the calibration curves of elamine were linear in the range of 5.0ng/mL~50.0ng/mL form, the correlation coefficient was0.9996, the limit ofquantization was0.01mg/kg, and the average recovery was in the range of91.7%~98.5%,RSD<5.0%. The method has high accuracy and precision, the operation was easy andsuitable for the determination of melamine in feed of aquatic products.3. Determination of Cyanuric Acid in by HPLC-MSA method for determination of cyanuric acid in aquatic products was established byhigh performance liquid chromatography-mass spectrometry (HPLC-MS). PenaeusVannamei samples were extracted with0.1%formic acid in methanol solution afterdemulsificating with the diethylene glycol and centrifugation, the supernatant was filtermembrane direct determination on the machine. The results showed that: the calibrationcurves of elamine were linear in the range of5ng/mL~50ng/mL form, the correlationcoefficient was0.9998, the limit of quantization was0.01mg/kg, and the average recoverywas in the range of82.3%~101.4%, RSD<10.0%. This method was suitable for thedetection of cyanuric acid in Penaeus vannamei.4. Studying Residue and Elimination of Melamine in Penaeus VannameiSimulation the farming process of Penaeus Vannamei, At room temperature(23℃~29℃), Penaeus Vannamei were fed with containing different doses of melamine in feedfood for consecutive25days after domesticating with normal diet for7days, Studying onthe law about the residues and elimination of melamine in Penaeus vannamei blood,muscle and hepatopancreas organizations. Drug residues were detection by HPLC-MSafter extracting. The results showed that: adding melamine in the feed will affect theweight of Penaeus Vannamei have a significant effect (P <0.05), the feed conversion ratiohad not significantly effect (P>0.05). Melamine in blood, muscle and hepatopancreastissues samples could not be found respectively after2days,5days and7days. Thehighest residues was the hepatopancreas of Group E (800mg/kg), the residue was6.10mg/kg. Melamine in Penaeus vannamei can turn into cyanuric acid by metabolism.Cyanuric acid was only detected in the hepatopancreas tissue, not in blood and muscle, theresidue was disappeared after3days.