| The bacterial resistance and drug residual is major emerging issue in the livestock due to unusual use of broad spectrum antibiotics,which obviously affect either directly or indirectly on the animal's health.The Chinese Herbal Medicine(CHMs)is the best choice for the prevention and treatment of animal disease and also being used for the better production from livestock because of low toxicity,lower drug resistance and negligible drug residue.However,CHMs is easily contaminated by mycotoxins,from harvesting of plants up to the final step,under favorable conditions i.e.Humidity,temperature.The aim of this research is to develop fluorescence immunochromatographic assay and UPLC-MS/MS by using IAC for detection of AFB1 and ZEN in CHMs.This study provides technical support to monitor mycotoxins.In this research,the fluorescent backplane,the pH,the amount of antigen and antibody,incubation and reaction time were optimized.The background fluorescence immunochromatographic assay(BFIA)was successfully established for the determination of AFB1 and ZEN in CHMs.The IC50 of BFIA were 0.0192 ng/mL and 0.61 ng/mL for AFB1 and ZEN respectively.The cut-off of BFIA were 0.1 ng/mL and 2 ng/mL for AFB1 and ZEN in CHMs.The LOD of AFB1 was 0.38 ?g/kg and the recoveries of the spiked samples were 72.7%-88.0%with relative standard deviation less than 9.4%.The LOD of ZEN was 10 ?g/kg and the recoveries of the spiked samples were 74.9%-90.2%with relative standard deviation less than 8.9%.The results of stability shown that the strips could be stable at least 12 months at room temperature.Our research shows that,time-resolved fluorescent immunochromatographic assay(TRFIA)was also established for the detection of AFB1 and ZEN in CHMs.The Eu-nanospheres,the amount of antigen and antibody,reaction buffer system,detection mode,incubation and reaction time were optimized.Under the ideal conditions,the IC50 of TRFIA were observed 0.039 ng/mL and 0.018 ng/mL for AFB1 and ZEN.The cut-off of TRFIA were 0.1 ng/mL and 0.05 ng/mL for AFB1 and ZEN in CHMs.The LOD of AFB1 was 0.60 ?g/kg and the recoveries of the spiked samples were 73.0%-95.0%with relative standard deviation less than 9.5%.The LOD of ZEN was 0.40 ?g/kg and the recoveries of the spiked samples were 75.8%-90.0%with relative standard deviation less than 11.3%.The outcomes shown that the strips could be stable at least 12 months at room temperature.Immunoaffinity cleanup for the evaluation of AFB1 and ZEN in CHMs by ultra performance liquid chromatography tandem mass spectrometry(UPLC-MS/MS)was efficaciously developed.The immunoaffinity column of AFB1(AFB 1-IAC)was prepared by coupling NHS-hydroxysuccinimide-activated Sepharose 4B Fast Flow gel with the antibodies against AFB1 and ZEN.Then the ratio of methanol and the volume of elution were optimized.The IAC-UPLC-MS/MS method was developed and validated with three different matrices(Semen coicis,Rhizoma dioscoreae and Platycodon grandiflorus).The LOD of AFB1 was 0.03 ?g/kg and the recoveries of the spiked samples were 80.3%-98.1%with relative standard deviation less than 13.2%.The LOD of ZEN was 0.05 ?g/kg and the recoveries of the spiked samples were 73.2%-92.8%with relative standard deviation less than 12.8%.The method was proved as sensitive,accurate and suitable for the determination of real samples.Finally,it was used to detect 15 actual samples(Semen coicis,Rhizoma dioscoreae and Platycodon grandiflorus)and the result was in satisfactory with two fluorescent immunochromatographic assays.To sum up,the FICA and UPLC-MS/MS method based on immunoaffinity column purification have been established at the same time for simultaneous determination of AFB1 and ZEN.It has the characteristics of sensitive,accurate and simple,and meets the national related testing requirements.It can be used for screening and confirming the multiple types of mycotoxins in the actual samples. |
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