| As the fisheries continue to be over-exploited and depleted, the sea intensive farming has become an important way of seafood supply. Because the use of antibiotics not only can improve feed efficiency and the quality of seafood, also prevent disease, a variety of antibiotics have been widely used. Ant ibiotics bring economic benefits to marine fish farms, however, the excessive use of antibiotics has led to a serious problem of antibiotic residues. Antibiotic residues will constantly accumulate in the organisms and the natural environment, threaten to human health and cause environmental pollution. Therefore, exploring simple, rapid, efficient and environmentally friendly antibiotic residues detection method is of great significance for food safety and environmental monitoring.This paper choose the current widely used sulfa, quinolone and tetracycline antibiotics for the object under test, and established a method for simultaneous analysis and determination of these three classes of antibiotics with high performance liquid chromatography. Based on the high fat and high protein characteristics of seafood and amphoteric properties of antibiotics, a new method, QuEChERS-reversed-phase dispersive liquid-liquid microextraction, was developed for rapid detection of antibiotic multiresidue in seafood. QuEChERS(Quick, Easy, Cheap, Effective, Rugged and Safe) method is used to quickly and effectively extract the target from seafood, remove protein at the same time. Reversed-phase dispersive liquid-liquid microextraction method based on nonpolar system, with aqueous solution as micro extraction agent and extracting solution as dispersant, is used to extract the target for rapid separation, enrichment, and remove the fat.(1) This experiment, using high performance liquid chromatography(HPLC) method as a means of detection, examines the effects of chromatographic column types, mobile phase composition, mobile phase flow rate, composition of the sample solution and elution gradient on the chromatographic behavior of the target. Three classes of antibiotics optimal detection conditions were as follows: sample solution diluted with initial mobile phase; ZORBAX Eclipse XDB-C18(5 μm, 150 mm×4.6 mm) chromatographic column; 0.5% acetic acid aqueous solution as mobile phase A, acetonitrile as mobile phase B; the elution gradient: 0-18 min, 90%-82%A; 18-23 min, 82%-65%A; 23-26 min, 65%A; 26-27 min, 65%-90%A; 27-35 min, 90%A; a flow rate of 0.7 m L/min; the UV detection wavelength λ=280 nm. Eight kinds of antibiotic compounds all peak in 26 minutes, with symmetrical peak shape and good separating degree. The standard curves of 8 kinds of compounds was drawed with good linear relationship and the linear range was between 0.18-7.81 μg/m L. The experimental results show that the established HPLC method is applicable to rapid qualitative and quantitative analysis of sulfa, quinolone and tetracycline compounds.(2) QuEChERS-reversed-phase dispersive liquid-liquid microextraction with high performance liquid chromatography method was established for determination of sulfonamides and quinolones in seafood. This experiment examines the effects of the variety of extraction solvent and acid content, the volume of the dispersant, micro extraction solvent volume and p H, extraction time, etc., and the effects of extraction conditions on the target on the chromatographic behavior of the target. The optimum conditions were determined as follows: 1 m L of acetonitrile: water= 8:2(V:V) solution containing 1% acetic acid as extraction solvent, nitrogen blow extraction liquid to the volume of 200 μL as dispersant; 50 μL water containing 1% acetic acid as micro extraction agent; cyclohexane volume of 3 m L and a extraction time of 1 min.(3) The present method was applied to the analysis of five kinds of fish(including golden pompano, large yellow croaker, herring, silver carp and sea carp) samples and standard addition sample to verify applicability of the method. The calibration curves of six compounds was with good linear relationship and correlation coefficient was between 0.9951-0.9993. The detection limits of the method were 0.0218-0.0902 μg/g and the limits of quantitation were in the range of 0.0725-0.3007 μg/g. The average recoveries of 6 targets at two spiked concentration level ranged from 50% to 108%.(4) Reversed-phase dispersive liquid-liquid microextraction was established to direct extract antibiotics in high-fat sample(liquid). With similar properties, low price and easy access, butter was selected as a substitute for fish oil, and the application prospect of reversed-phase dispersive liquid-liquid microextraction in the extraction of antibiotics in fish oil was studied.The proposed method is suitable for the simple, rapid detection of antibiotics with simple operation, good degreasing effect, low organic solvent and short analysis time consumption. In addition, as to eliminate the influence of the fat, the method shows great application prospects in the analysis of polar compounds in high fat samples. Reversed-phase dispersive liquid-liquid microextraction coupled with different methods of extraction will further expand the method in detection of other compounds in different substrates. |
