Researches Of Nitrofurazone And Semicarbazide In Marine Crustaceans

Semicarbazide (SEM), an amine small molecule, is considered as metabolite of the banned drug nitrofurazone (NFZ). SEM in animal-derived food usually originated from metabolism of NFZ used in animal breeding. At present, many countries detect SEM to judge illegal use of NFZ in the animal breeding process and to achieve the purpose of controlling NFZ. However, the occurrence of SEM in food is not solely attributable to the use of the parent compound. A number of sources have been identified in food where SEM has been detected. So this thesis focus on establishment of detection methods of SEM in marine crustaceans, exploration of sources of non-nitrofuran SEM and the experiments of establishing new test methods by another metabolite of NFZ---5-nitro-2-furaldehyde or its reactant with2,4-dinitrophenylhydrazine (DNPH-I)---5-nitro-2-furaldehyde-2,4-dinitrophenylhydrazone (NFDNPH-O). The main research areas in this thesis can be included as follows: 1. An ultra performance liquid chromatography-mass spectrometry with dispersive solid phase extraction (DSPE-UPLC-MS/MS) for the simultaneous determination of four metabolites of nitrofuran in crustacean aquatic products has been developed. Samples were washed with methanol-water mixtures (8:1) and then subjected to HCL for acidolysis, derivatived using2-nitrobenzaldehyde. The derivative was extracted by ethyl acetate, then concentrated and depurated by dispersive solid phase (PSA) extraction. The analysis was carried out in the multi-reaction monitoring (MRM) mode via positive electrospray ionization (ESI+) and internal standard isotope dilution method was used for quantification, internal standard was5.00μg/kg. Good linearity was obtained in the correlations between the peak areas and concentrations of the four metabolites of nitrofuran antibiotics over the concentration range from0.5to20.0ng/mL (the calibration coefficients were above0.997). The limits of detection were all0.1μg/kg. In the three added concentration0.5,1.0,2.5μg/kg, mean fortification recoveries were90.5%～104.5%and their relative standard deviation (RSD) were between1.56%and8.08%. The method could be used to identify and quantify the metabolites of nitrofuran antibiotics in aquatic fingerlings and shells with satisfactory sensitivity, efficience and repeatability. This method can quickly and accurately measuring the residual amount of nitrofurazone in complex marine crustaceans, providing methodological approach for SEM detecting.2. By using this DSPE-UPLC-MS/MS method,12different aquatic crustaceans were analyzed. Distribution of SEM in tissues was found from testing muscle, viscera, whole body and shell of each crustacean. SEM in different tissues showed the lowest SEM content distribution in muscle and highest SEM content in shells which proved the exsistence of endogenous semicarbazide. Shrimp Penaeus vannamei Boone, Parapenaeopsis hardwickii and crab Portunus trituberculatus were used in this assay to explore reasons for false positives in aquatic products. This study found that treatments of heating, hypochlorous acid (NaCIO) and adding azodicarbonamide (ADC) led SEM checkout:heat treatment had no effect on crab and shrimp muscles, but the values of SEM in shells changed a lot by heat treatment in different temperatures; muscles were sterilized by sodium hypochlorite (NaCIO) at different concentrations of active chlorine then compared with untreated muscles, SEM was detected in NaClO treated muscles and was proportional to concentrations of NaClO within6%; when adding ADC and baking, the SEM detectable quantity were0.587±0.124and0.42±0.121μg/Kg in coated shrimp and crab muscles. These confirmed the existence of non-NF SEM, and mechanisms of SEM production were briefly discussed.3. Currently the detection methods of NFZ were most depending on SEM by chromatography or spectral characteristics which can’t be distinguished SEM from different sources. In this study, another metabolite of NFZ---5-nitro-2-furfural (5-NF) was tried to use as biological marker to avoid confusion of non-NFZ originated SEM and NFZ-metabolic SEM. UPLC method was firstly attempted to establishing for5-NF:the liquid phase conditions was successfully selected, standard curve:y=2.65×104x-1.48×103, the concentration range was0.3-10.0μg/mL, the correlation coefficient R2=0.999745, and the limit of detection was0.1μg/mL. But because of the light sensitivity of5-NF, the recovery rates were always20%-40%, UPLC method was changed to be depended on its reactant with2,4-dinitrobenzene hydrazine (DNPH-I)---5-nitrate-2-furaldehyde-2,4-dinitrophenyl hydrazone (NFDNPH-O). UPLC conditions were purified, NFDNPH-O detection method was established:Standard curve y=2.29×104rx-3.37×103, the linear range was1.0-20μg/mL, R2=0.999282, the limit of detection was0.3μg/mL, the limit of quantification was1μg/mL. Advanced method was proposed to establish the internal standard method and liquid chromatography-mass spectrometry to improve recovery and reduce the limit of detection.