Application Of CCD Camera Imaging Based Solid Surface Fluorescence For Fast Screening Of Tetracyclines In Fish Muscle

Tetracyclines,(TCs)are broad-spectrum antibiotics showing activity against Gram-positive and Gram-negative bacteria including some anaerobes,by inhibiting the synthesis of bacterial proteins by binding to the 30 S bacterial ribosome and preventing the access of aminoacyl tRNA to the acceptor site on the mRNA-ribosome complex.TCs have been widely used in the prevention and treatment of infectious diseases,and as food additives for growth promotion as well, in the fish farming.The intensively use of these antibiotics have raised questions about the impact of veterinary medicines on organisms in the environment and on human health.Numerous studies suggest a link between antibacterial use in agriculture and antibacterial-resistant infections,and there is evidence that antibacterial resistance from agriculture can be transferred to humans.It is important to develop sensitive analytical methods to monitor the food supply to ensure that any antibiotic residues present are below the set tolerance level,thus promoting food safety and consumer confidence.A maximum residual limit of 100μg Kg^-1of TCs in aquiculture product is set by EU and China.This thesis is aimed at developing a new methodology for fast screening of TCs in fish muscle.Four chapters are included in this thesis,that is,introduction,method development and optimization,screening of TCs in fish muscles,and conclusion and expectation.In Chapter 1,a brief introduction of the use and effect of fishery drug in aquiculture and a review discussing the present stage of the art of the quantificational and qualitative methodology for TCs residues in aquatic product are given.A basic principle of solid-surface fluorescence(SSF)and its applications are introduced. Based on the update development in these areas,the research plan for the thesis was outlined. In Chapter 2,a previous developed quantificational method for the determination of TCs was optimized.This methodology is based on the fact that tetracyclines would transfer to highly fluorescent species on alkaline active solid silica gel G plates as a result of their alkaline degradation.By coupling the solid surface fluorescence with a CCD camera imaging,a CCD camera based SSF has been developed.In order to optimize the performance,the experiment parameters that would effect the result of determination were optimized.Being implemented with a multiple-spotting technique the sensitivity of this method was increased obviously,and the laborious time was also shorted.The method has demonstrated to bear some requiring advantages,such as its simplicity,rapidness,low-cost,less pollutants and reasonable sensitivity,on comparison with the official methods,HPLC.The calibration curves give linearity over a range of 0.20～1.0 ng spot^-1for all three most used tetracyclines,named tetracycline(TC),oxytetracycline(OTC)and chlorotetrecycline(CTC).The limits of detection(LOD),defined as 3σ,for TC,OTC and CTC are 0.14,0.15 and 0.16 ng spot^-1,respectively.The limit of quantification(LOQ),defined as 10σ,for TC,OTC and CTC are 0.47,0.50 and 0.53 ng spot^-1,respectively.In Chapter 3,the newly developed CCD camera based SSF was applied to the screening of the tetracyclines residues in fish muscle samples.The method was validated by evaluating the performance criteria:recovery,repeatability,calibration curve,limit of detection(LOD)and limit of quantification(LOQ),decision limit (CCα)and detection capability(CCβ),false positive and false negative rate, sensitivity and specificity rate,in accordance with European Commission Decision 657/2002.The average percentage recovery for fish muscle sample from 6 different fish matrix,named tilapia,weeever,crucian,silver carp,silver pomfret and besugo, fortified with TC at different level,was 63.1%with an average coefficient of variation of 8.2%.The coefficients of variation ranged from 2.5 to 5.8%and from 5.2 to 7.6% for intra-day and inter-day repeatability,respectively.The detection and quantification limit in fish muscle matrix was 16 and 53μtg Kg^-1of TCs,respectively.CCαand CCβ were found to be 107 and 114μg Kg^-1,respectively,determined on a basis of a MRL of 100μg Kg^-1.The calibration curve in different fish muscle matrix showed a good linearity in the whole range of tested concentrations(50～300μg Kg^-1)with a correlation coefficient(R)equal to 0.997.Setting a threshold as the(?)₁₀₀-3σ₁₀₀ allows for successful screening of TC in fish muscle fortified at 100μg Kg^-1, maximum tolerance level set by Ministry of Agriculture of the People's Republic of China.For 50 blank fish samples,each fortified at a randomly selected level in the range of 0～200μg Kg^-1,the sensitivity rate,specificity rate,false positive rate and false negative rate was 100%,88.9%,11.1%and 0%,respectively.This method can provide an easy,fast,and low-cost screening assay compared with HPLC.In Chapter 4,the research work was summarized and the prospect of the research field was given.