| Patulin is a secondary metabolite produced by a wide range of fungal species and widely exist in food and animal feed. Patulin is toxic to animal, which including neurotoxicity, cytotoxicity, genotoxicity, etc. People have paid more and more attention to the toxicity of patulin in animal feed. The objectives of this study were to establish a high performance liquid chromatography that can determine patulin in animal feed and then survey the contamination of patulin in feed and ingredient in China, which provide technical and data support for establishing statute. The main results were as follows:The establishment of high performance liquid chromatography:Feed samples were crashed with 40-mesh sieve and misced bene. The samples were extracted with mixture of acetonitrile and water (4:1). After centrifugated,5mL of the supernatant was evaporated nearly dryness under the conditions of constant temperature water bath at 40℃. Then the residue was dissolved with 0.5mL mobile-phase and filted with 0.45μm membrane.20μL of the solution was injected into HPLC system with C18 and detected at 276nm, and mixture of acetonitrile and water (1:9) as mobile-phase with a flow rate of 1.0mL/min at 30℃. Retention time of patulin was approximately 10 min. In the present study, detection limit and quantitation limit of patulin in animal feed sample were 2.5μg/kg and 8.5μg/kg, respectively. Linearity of the detector response was verified with patulin standard solution in the range of 0.1～4μg/mL, the regression was y=127793x+854.16, R2=0.9999. The apple pomaces, barley malt, DDGS, corn, wheat middling, white distillers, formula feed wheat and rice bran were added with 50μg/kg,250μg/kg and 500μg/kg patulin. The recoveries of patulin from those samples were 66.2%～89.4%,76.1%～92.7%, 83.3%～97.7%, and the average levels were 73.8%,82.9%,90.0%, respectively. The coefficients of variations were in the range of 5.0%～10.1%. Precision experiments were carried out with samples that with three levels of patulin. The coefficients of variations in the parallelism test were in the range of 6.3%～12.2% and coefficient of variations in the repeatability test were in the range of 6.7%～10.4%. The coefficients of variations in reproducibility test were 9.5%,6.9%,5.5%, respectively in samples with three levels of patulin.The determination of patulin in animal feed:154 samples were determinated with the HPLC that established in this study. Positive rate of patulin was 32.4%, the average concentration of patulin was 152.3μg/kg. Patulin didn't detect in corn, corn gluten meal, corn bran, wheat seed, wheat flour and white distillers. The positive rate of patulin in the apple pomace was 90.9%, which was the highest one Followed by wheat bran, wheat germ and brewers dried grain with the positive rate of 80.0%,75.0% and 75.0%, respectively. The positive rate of patulin in the formula feed was 20.4%. The content of patulin in apple pomace that with the highest positive rate were in the range of 50.1～482.9μg/kg and the average content of patulin was 173.3μg/kg. The content of patulin in DDGS was in the range of 545.4～1636.2μg/kg, and the average was 514.9μg/kg, which was the highest one in all samples. Followed by rice bran, the conten of patulin in it was in the range of 19.3～1289.0μg/kg, and the average was 232.0μg/kg. Using TLC to check the positive samples had the same results compared with HPLC.The established HPLC in current study was accurate and stable, it can be use for detecting patulin in feed. The result of determination of patulin in feed suggested that feed and ingredient were contaminated by patulin. The positive rate of patulin in apple pomace was the highest one while the content of patulin in DDGS was highest one, we should pay attention to it and establish statute of patulin in feed.
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