Development Of The High-throughput And Rapid Detection Methods For Zearalenone And Its Metabolites In Coix Lacryma-Jobi L.

Coix lacryma-Jobi L.is a genus of Coix in the family Gramineae,and its seeds are used as medicine and food,with the function of diuresis,strengthening the spleen and stopping diarrhoea,detoxifying and dispersing knots.The straw and bran can be used as animal feed.Numerous studies have shown that Chinese herbal medicines are susceptible to mycotoxins contamination during planting,growing,harvesting,primary processing,transportation and storage.Zearalenone(ZEN)is detected in Coicis Semen at a high rate,and ZEN is hepatotoxic,immunotoxic and reproductive toxic,posing a serious threat to human and animal health.Therefore,it is important to detect the production of ZEN and its metabolites in different parts of Coix lacryma-Jobi L.and to understand the contamination of different parts of Coix lacryma-Jobi L.with Fusarium graminearum,in order to propose preventive and control measures for the growth and storage of Coix lacrymaJobi L..The detection of ZEN and its metabolites in Coicis Semen and its products is of great importance to improve the quality and safety of Coicis Semen,Coicis Semen and its products.mature seed kernels and its products,which is of great significance in safeguarding and improving the quality and safety of Chinese herbal medicines and food.It also provides a reference for the development of other rapid detection methods.In this study,a high performance liquid chromatography-tandem mass spectrometry(LC-MS/MS)method was developed for the determination of ZEN,α-zearalenol(α-ZEL),β-zearalenol(-ZEL),zearalenone(ZAN),zearalenol(α-ZAL)and β-zearalenol(β-ZAL)(ZENs)in various parts of Coix lacryma-Jobi L.at different periods to investigate whether endogenous plant hormones or exogenous fungi might be produced in the whole growth cycle of Coix Lacryma-Jobi L..ZEN production and its metabolites in Coix were detected by F.graminearum infecting different parts of Coix LacrymaJobi L..and cultured at 28℃ and 80%humidity for 7 days.After mouse immunization,cell fusion,monoclonal cell screening and antibody purification,a ZEN broad-spectrum antibody was prepared and used to develop an indirect enzyme-linked immunosorbent assay(ic-ELISA)and a colloidal gold immunochromatographic assay(GICA)for the detection of Coicis Semen and its products(Coicis Semen flour,Yimigao,Yishigao).ZEN was detected in seeds,roots,stems and leaves of Coix LacrymaJobi L.,ZEN content in seeds was 22.5 μg·kg-1,with lower levels in roots and leaves,only at the four-leaf stage,eight-leaf stage and jointing stage,and higher levels in stems compared to roots and leaves,detected in every period and reaching a maximum of 7.85 μg·kg-1 at the heading and flowering stage and contaminated ZEN seeds might not be transferred to their mature fruit during planting.Infection with F.graminearum resulted in higher ZEN content in stems and leaves and about five times more ZEN in Coicis Semen than in seeds.The IC50 values of the antibodies against ZEN,α-ZEL,β-ZEL,ZAN,α-ZAL and β-ZAL are1.13 ng·mL-1、1.69 ng·mL-1、2.06 ng·mL-1、0.66 ng·mL-1、1.20 ng-·L-1 and 0.94 ng·mL-1 with cross-reactivity(CR)of 100%,66.69%,54.85%,170.00%,94.17%and 120.21%,respectively.The recoveries of the established ic-ELISA methods ranged from 60.9%to 120.0%with relative standard deviations(RSDs)of 0.2%to 13.6%.The GICA method has a detection limit of 0.5 ng·mL-1 for ZEN,α-ZEL,β-ZEL,α-ZAL,β-ZAL and 0.25 ng·mL-1 for ZAN in 0.05%Tween-80 0.01 phosphate buffer salt(PBS)solution,and a detection limit of 10 to 20 μg·kg-1 for Coicis Semen and its products.Coix Lacryma-Jobi L.might produce the endogenous phytohormone ZEN during growth,be contaminated with exogenous fungi to produce ZEN or have ZEN in the seeds transferred to the above-ground parts.The stems and leaves of Coix Lacryma-Jobi L.were managed to enhance the prevention and control of the stems and leaves during storage,and the Coicis Semen shell provided protection against fungal contamination of Coicis Semen.The ZEN antibody was prepared with high sensitivity and broad-specificity for the simultaneous detection of six compounds.The detection results of two rapid test methods were in good agreement with the results of LC-MS/MS,and the results were accurate and reliable.The establishment of the method laid a foundation for the simultaneous detection of mycotoxin in Chinese medicinal materials.The ic-ELISA method allowed simultaneous testing of a large number of samples for high-throughput detection and the GICA method was rapid and allows for rapid detection in the field.