Development Of Detection Critical Technology And Application For Multi-mycotoxins In Animal Biological Fluids And Tissues

Mycotoxins are secondary metabolites produced by different fungal species. They can contaminate many agricultural commodities during harvest or in storage. Currently, more than400mycotoxins have been identified in the world, although only a few of them present a significant source of food-borne illnesses and are of major concern worldwide. They can be categorized into Aflatoxins (AFs) family (produced by Aspergi//us spp.), Ochratoxin A (OTA), produced by Aspergillus and Penicillium spp., Zearalenones (ZENs), Fumonisins (FBs) and thricothecenes (TCT), produced by Fusarium spp. A group of carcinogenic mycotoxins is produced by some fungi under certain conditions. Therefore, monitoring carcinogenic mycotoxins in human and animal plasma and urine is important. Fusariums pose a significant potential threat to human and animal, so detecting fusariums in animal plasma and urine would be very useful. In addition, developing and validating methods to determine Enniatins(ENA), Enniatins A1(NA1), Enniatins B(ENB), Enniatins B1(ENB1), Moniliformin (MON), Beauvericin(BEA) in biological matrices would be very useful because they could be tools during toxicokinetic and toxicology studies.The aim of the work was to develop a high-throughput method based on LC-MS/MS for the detennination of mycotoxins and metabolites in animal biological matrices and tissues. Contents include the following:Section one:Quantitative determination of carcinogenic mycotoxins in animal biological matrices and tissues using HPLC-MS/MS methods.The analytes were extracted using PLE first, then,’dilute, and shoot’approach,’dilute, evaporate and shoot’approach and ’QuEChERS’procedure have been developed. These procedures should be exhaustive with respect to the target compounds, as well as fast, simple and environmentally friendly for routine analysis. High-throughput was achieved by using PLE pre-treatment and without the need for any cleanup. The optimum extraction conditions were a static time of5min, the extraction pressure and the temperature was1500psi and140(?), respectively, the flush volume was60%. For the tissues samples, the extraction solvent was acetonitrile/water/hexane/acetic acid (60/14/25/1, v/v/v/v), for the plasma and urine samples, the extraction solvent was acetonitrile/acetic acid (99/1, v/v).The limits of detection were defined as CCa varied from0.028ug/kg (ug/L) to0.402ug/kg (ug/L). The recoveries of spiked samples from0.25ug/kg (ug/L) to3.2ug/kg (ug/L) ranged from64.6%to101.6%with the relative standard deviations of less than15.3%. To our knowledge, this is the first method for large-scale testing of carcinogenic mycotoxins in all kinds animal of biological fluids and tissues using HPLC-MS/MS.Section two:Quantitative determination of enniatins and beauvericin in animal biological matrices and tissues using HPLC-MS/MS Methods.The method is to develop a simultaneous method for the determination of including Enniatins(ENA), Enniatins A1(ENA1), Enniatins B(ENB), Enniatins B1(ENB1), Moniliformin (MON) and Beauvericin(BEA) in swine, chicken, rat and rabbit plasma, urine and tissues. The samples extracted with PLE and’dilute, evaporate and shoot’approach and ’QuEChERS’ procedure.CCa were0.110to0.220u.g/kg (ug/L) and CCB were0.210to0.512ug/kg (ug/L), respectively. The recoveries of enniatins and beauvericin, within the spiked level of quantitative limits, were from72.0%-99.2%. All the relative standard deviations of less than15.7%.Section three:Quantitative determination of22fusariums in animal biological matrices and tissues using HPLC-MS/MS Methods.The analytes were extracted using PLE first, then,’dilute, and shoot’approach,’dilute, evaporate and shoot’approach and’QuEChERS’procedure have been developed. The optimum PLE extraction conditions were a static time of7min, the extraction pressure and the temperature was1500psi and100(?), respectively, the flush volume was60%. For the urine samples, the extraction solvent was methanol/acetic acid (99.5/0.5, v/v/v), for the tissues samples, the extraction solvent was methanol/water/hexane/acetic acid (70/10/19/1, v/v/v), for the plasma samples, the extraction solvent was methanol/acetic acid (80/19/1, v/v/v). The limits of detection were defined as CCa varied from0.040ug/kg (ug/L) to0.680ug/kg (ug/L). The recoveries of spiked samples from0.210ug/kg (ug/L) to1.353ug/kg (ug/L) ranged from69.8%-98.2%with the relative standard deviations of less than15.5%.Section four:Quantitative determination of gliotoxin in human and animal biological matrices and tissues using HPLC-MS/MS Methods.The analytes were extracted using PLE first, then,’dilute, and shoot’approach,’dilute, evaporate and shoot’approach and "QuEChERS" procedure have been developed. The optimum PLE extraction conditions were a static time of6min, the extraction pressure and the temperature was2000psi and100(?), respectively, the flush volume was60%. For the urine samples, the extraction solvent was methanol/acetic acid (99/1, v/v), for the tissues samples, the extraction solvent was methanol/water/hexane (70/10/20, v/v/v). for the plasma samples, the extraction solvent was methanol.The limits of detection were defined as CCa varied from0.090to0.150ug/kg (ug/L). The recoveries of spiked samples from0.30ug/kg (ug/L) to2.4ug/kg (ug/L) ranged from76.9%~99.6%with the relative standard deviations of less than14.9%.Section five:Application of carcinogenic mycotoxins method in rat plasma and tissues.The method was applied in real samples obtained from rats. It could be used to check the efficacy of detoxification strategies (adsorbents, detoxification microorganisms, and enzymes) in vivo, and reduce mycotoxin toxicity in farm animals. In the future, this method can be widely applied in mycotoxins exposure assessment of human and animals.