Determination Of Mycotoxins In Black Rice By Compound Immunoaffinity-liquid Chromatography-tandem Mass Spectrometry

Mycotoxins are the most common type of fungal metabolites found in food and feed,which are carcinogenic,teratogenic and mutagenic.Crops are vulnerable to mycotoxin contamination during cultivation,production,processing,circulation,transportation and storage,and 25 percent of the world's food is contaminated annually by mycotoxins.In the field of food production and agricultural product safety,mycotoxins have been focused.The reports of mycotoxins in grains are mainly rice and wheat,and the reports of mycotoxins in black rice are less.Food and its products are contaminated by multiple toxins at the same time,the existing toxin detection methods are for a single toxin or a class of toxins,while the determination of multiple toxins is less.In this study,11 mycotoxins were used as the main targets,and a pretreatment method of composite immunoaffinity purification and a method of toxin detection by liquid chromatography-tandem mass spectrometry were established.Through methodological verification,the method was highly accurate and sensitive,and was applied to the detection and analysis of 11 toxins in 20 batches of black rice,which provided a reference for the development of a method for the simultaneous detection of multiple toxins in black rice,and was of practical significance for the study of combined contamination analysis of toxins.1.Establishment of pretreatment method for mycotoxin detection in black riceThe pretreatment methods were compared and analyzed from the aspects of extraction solution selection,extraction method,extraction time and purification method,and the use conditions of equipment and facilities,the methanol:acetonitrile:water solution with 0.1%formic acid(40:40:20 V/V)was used as the extraction solution.After shaking for 60 min,the method was purified by Myco6in1 immunoaffinity column.The recovery rates of the final 11toxins were above 75%,and effective extraction and purification were obtained.2.Establishment of HPLC-MS/MS method for mycotoxin detection in black riceThe detection method is established by optimising the mass spectrometry conditions such as voltage and collision energy to obtain the best response of parent ions,ions,select the appropriate chromatographic column,through the gradient elution program,sample size,column temperature,flow parameters optimization to obtain the best separation conditions.Finally,the Hypersil Gold liquid chromatography column was selected,and the water containing 0.2%formic acid and 2 mmol/L ammonium formate,and the methanol solution containing 0.2%formic acid and 2 mmol/L ammonium formate were used as the AB mobile phase for gradient elution.The column temperature was 35°C.The detection method of mycotoxins in black rice was established,and the methodology verification of the optimized method was carried out,mainly including linear relationship,linear range,detection limit,quantitative limit,precision,recovery rate and accuracy.The verification results showed this method is good accuracy,high sensitivity,can meet the needs of black rice in a variety of toxin test.3.Comparison of detection methods for mycotoxins in black riceSeven mycotoxins,AFB₁,AFB₂,AFG₁,AFG₂,DON,ZEN and OTA,were selected and compared with the HPLC method and the HPLC-MS/MS method to compare the linear range,detection limit,recovery,precision and accuracy of the two methods.The results showed that the two methods were highly accurate and precise,with correlation coefficients greater than0.99 in the linear range.The composite immunoaffinity-HPLC-MS/MS method achieved a single clean-up,one-shot injection,simple and rapid operation,and is more suitable for the screening and quantification of multiple toxins in batch samples.4.Detection of various toxins in black riceA total of 20 batches of samples were collected from the major black rice producing areas in China and detected by composite immunoaffinity-HPLC-MS/MS.The results showed that the black rice was polluted by fumonisin and deoxynivalenol.