| ObjectivesOchratoxinA(OTA) is the secondary metabolite product of some certain kinds of penicillium and aspergillus which is highly toxic, widely distributed. And OTA may cause serious pollution to farm products. The normal process of feed industry and stock raising will be seriously affected if the feed is contaminated with mycotoxin. So the determination of mycotoxin is closed linked to the health of animals and the safety of meat product. Dispersive liquid-liquid microextraction (DLLME) is a new technique to pretreat samples. This technique contains many advantages compared with conventional detection methods. This method is simple and fast to operate, uses less organic solvents, and the cost is low. What is more, the method has both enrichment and clean up effect. The main aim of this design is to establish a simple, sensitive and accurate pretreatment method to detect OTA in feed and pork. And the determination is performed by High Performance Liquid Chromatography-fluorescence detection(HPLC-FLD). We will investigate and analyte pollution level of OTA in feed produced by feed manufacturer and OTA accumulation in pork according to the established method.Methods1 Experiment method1.1 Extraction of OTAOTA in feed samples was extracted with the mixture of methanol and water by ultrasonic. OTA in pork samples was extracted with the mixture of methanol, phosphoric acid and sodium chloride solution by ultrasonic.1.2 Purification and enrichment methods of DLLMEAfter centrifugation and filtration,1.0 mL of the filtrate and extraction solvent was mixed and then rapidly injected into a centrifuge tube with a conical bottom filled with acetic acid solution. Then the mixture was centrifuged. After removing away the supernatant liquid, the sedimented phase was evaporated to nearly dryness under gentle nitrogen flow. Then the residue was redissolved with methanol for HPLC analysis.1.3 Chromatographic conditionsThe mobile phase was acetonitrile and 2% glacial acetic acid aqueous solution. The column temperature was 30℃. The flow rate of mobile phase was 1 mL/min. The excitation wavelength of fluorescence detector was 333 nm and the emission wavelength was 477 nm. Qualitative analysis was done by retention time and quantitative analysis by peak area.1.4 Method optimizationMany factors can affect the extraction efficiency of DLLME. In our experiment, factors such as kind and volume of extraction solvent, pH and volume of aqueous phase were optimized in our study. The ratio of mobile phase was also optimized in our experiment.1.5 Quality controlIn order to insure the analysis quality, some parameters of the method such as the linear, detection limit, recovery, within-day and inter-day precision were studied in our experiment.2 Survey of OTA content in feed and porkInvestigation of OTA content in feed from some provinces of China was performed in our experiment. The main categories of feed include bran, soya bean meal, cottonseed meal and Distillers Dried Grains with Solubles(DDGS). OTA accumulation in commercially available pork was also studied in the experiment.Results1. The optimum DLLME conditions:after centrifugation and filtration,1.0 mL of the filtrate and 200 μL chloroform was mixed and then rapidly injected into a centrifuge tube with a conical bottom filled with 5 mL water(adjusted to pH3.0 with glacial acetic acid). Then the mixture was centrifuged. After removing away the supernatant liquid, the sedimented phase was evaporated to nearly dryness under gentle nitrogen flow. Then the residue was redissolved with methanol for HPLC analysis. OTA in feed was determined with gradient elution of acetonitrile and acetic acid solution. And the ratio of acetonitrile and acetic acid solution was 50:50 when detecting OTA in pork.2. Under optimized conditions in pork and feed samples, the recoveries of OTA were both higher than 85%, relative standard deviations were less than 5%. The detection limit of OTA in pork was 0.21 μg/kg, and in feed was 0.25 μg/kg.3. In all feed samples, the total detection rate was 46.7%, and the average detection quantity of positive samples was 1.8μg/kg, the maximum level was 18 μg/kg. No sample exceeded the maximum level 100 μg/kg set by national standard. There are differences among the results of different feeds. The detction rate and detection quantity of soya bean meal and cottonseed meal were higher than the other two. The detction rate and detection quantity of OTA in the south is higher than the north and northwest.4. OTA can be detected in all the brand pork and 88.9% of off-brand pork. The highest detection quantity of OTA in brand pork was 2.2 μg/kg, and the average content was 0.37μg/kg. And in off-brand pork the highest detection quantity was 3.0 μg/kg, the average content was 0.36ug/kg. The distribution of OTA in brand pork was more concentrated than off-brand pork. The concentrations of OTA in all pork samples were lower than the national standard of OTA in cereals 5 μg/kg.Conclusions1. The method of DLLME-HPLC-FLD established in our experiment was easy and sensitive to detect OTA in feed and pork. The pretreatment procedure of DLLME reduced the use of chlorinated organic solvents, thus protected the environment and the health of operator; and the technique simplified the operation procedure, reduced the cost.2. The pollution level of OTA in the samples was lower than the national standard. Compared with bran and DDGS, soya bean meal and cottonseed meal were easier to be contaminated by OTA. The hazard of OTA in the southern area was more serious than in the northern and northwest area.3. OTA accumulation widely accurs in pork, but the detection level was lower than the national standard of OTA in cereals. The distribution of OTA in brand pork was more concentrated than off-brand pork. |
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