Research On Simultaneous Detection Of Multi-mycotoxins In Tradition Chinese Medicines And New Rapid Detection Techniques For Aflatoxin B₁

As an indispensable part of traditional Chinese medicine,traditional Chinese medicine is an important guarantee for the healthy and sustainable development of traditional Chinese medicine.In recent years,traditional Chinese medicine has been widely used due to its unique medicinal value,resulting in increasing demand.Various exogenous harmful substances that are contaminated during the cultivation,harvesting,processing,and storage of traditional Chinese medicines will affect the quality of Chinese medicine and cause potential harm to users.Mycotoxins are one of the most important exogenous pollutants affecting the safety of traditional Chinese medicines at the present stage.They are secondary metabolites produced by harmful fungi.They accumulate in the body and damage liver and kidney function,and even cause cancer,seriously harming human health.Therefore,the establishment of a sensitive,rapid,high-throughput detection technology to monitor and early-warn the contamination of mycotoxins in traditional Chinese medicine is of great significance for ensuring the quality,effectiveness,and safety of traditional Chinese medicines.Based on the main research content and conclusions of this paper are as follows:1.Established immunoaffinity column purification-high performance liquid chromatography-columnpost-photochemicalderivatization-fluorescence detection（IAC-HPLC-PCD-FLD）determination of aflatoxin B1,B2,G1 And G2in 8 kinds of traditional Chinese medicines such as Astragalus membranaceus and Polygala tenuifolia.Through the optimization of sample preparation and chromatographic conditions,an IAC-HPLC-PCD-FLD method was established for the simultaneous determination of aflatoxin B₁,B₂,G₁,and G₂ levels in 8 traditional Chinese medicines,including Radix Astragali and Polygala tenuifolia.Detection.The results showed that the linearity of the four aflatoxins was linear in their linear range.The correlation coefficient R was 0.9991-0.9996,the detection limit was 1.25μg·kg^-1,and the limit of quantitation was 2.5μg·kg^-1.The average recovery was 78.9%-111.8%.The sample test results showed that aflatoxin was detected in 1 batch of Polygala tenuifolia,and the total amount of aflatoxin did not exceed the limit standard of 10μg·kg^-1 for aflatoxin in some Chinese herbal medicines in the 2015 edition of the Chinese Pharmacopoeia;4 batches betel nuts was detected Aflatoxin,and the two indicators of the total amount of aflatoxin B₁ and aflatoxin in the two batches of betel nuts were exceeding the limit standard.The results of positive samples were further confirmed by liquid chromatography-tandem mass spectrometry.No false positives were found,indicating that the method was accurate and reliable.2.Investigate the transfer of aflatoxins in tradional Chinese Medicines during processing.The residue of mycotoxins in the processing of traditional Chinese medicines should be monitored and analyzed in real time to ensure that the quality of related products also meets the international limit level.Select Huangqi and Yuanzhi as the research subjects,through the detection of traditional Chinese medicines decoction pieces contaminated by Aspergillus flavus,and the content of aflatoxins in the formula granules and decocted filter residue prepared from the decoction pieces,to study the transfer rule of aflatoxin in the traditional Chinese medicines.The safety of traditional Chinese medicines provides a theoretical basis.The results of the study showed that Chinese Herbal Medicine Tablets contaminated with Aspergillus flavus contained a large of aflatoxins,of which mainly aflatoxins B₁ and B₂,and some contained aflatoxin G₂.At the same time,we found that the formula granules prepared from traditional Chinese medicines Tablets contaminated with Aspergillus flavus also contained the corresponding aflatoxins,and the transfer rate was 18.4%to 77.5%.In addition,the dregs remaining after extraction and processing of traditional Chinese medicines still contain a large of aflatoxins,especially those containing high concentrations of aflatoxins.Since the solubility of aflatoxin is poor,the boiling residue is retained in the decoction residue.Most of the aflatoxins.Therefore,it is recommended that traditional Chinese medicines contaminated with Aspergillus flavus be put into the environment after proper treatment to avoid environmental pollution caused by aflatoxins.3.An ultra-high performance liquid chromatography-tandem mass spectrometry（UFLC-MS/MS）method was established for simultaneous detection of 21 mycotoxins in Astragalus membranaceus.Through the extraction solvent,extraction method and optimization of chromatography and mass spectrometry conditions of the sample,a simultaneous determination of aflatoxin B₁（AFB₁）,aflatoxin B₂（AFB₂）,aflatoxin G₁（AFG₁）,aflatoxin G₂（AFG₂）,ochratoxin A（OTA）,ochratoxin B（OTB）,ochratoxin（ST）,penicillic acid（PA）,HT-2 toxin（HT-2）,T-2 toxin（T-2）,Fumonisin B₁（FB₁）,Fumonisin B₂（FB₂）,Fumonisin B₃（FB₃）,cyclopiazonic acid（CPA）,Deoxynivalenol（DON）,Nivalenol（NIV）,Fusarenon X（Fus-X）,Alternariol（AOH）,Alternariol monomethyl ether（AME）,Tentoxin（TEN）,Zearalenone（ZEN）in Astragalus membranaceus was established.UFLC-MS/MS methods for contaminating levels of mycotoxins and used for actual sample detection.The results showed that the linearity of the 21 mycotoxins was linear in their respective linear ranges,the correlation coefficient R was 0.9995-1,the detection limit was 0.01 ng·m L^-10.1 ng·m L^-1,the limit of quantitation was 0.025 ng·m L^-10.25 ng·m L^-1,and the average recovery was Both are between 54.9-110.7%.Aflatoxins（AFB₁ and AFB₂）were detected in trans cultured samples of Astragalus membranaceus during the test of actual samples,and samples of naturally occurring Astragalus membranaceus were contaminated with ochratoxin（OTA and OTB）,but other mycotoxins were not detected.4.A rapid label-free fluorescent aptasensor strategy based on PicoGreen for Aflatoxin B1 analysis in Traditional Chinese MedicineA simple and rapid aptamer-based label-free strategy had been developed for highly selective and sensitive fluorescence detection of AFB₁ in complicated areca nut.In view of the high selectivity of aptamer to AFB₁ and ultra-sensitivity of PicoGreen for trace dsDNA,the proposed method realized an accurately quantified AFB₁ in nanomolar range.Because TCMs have complex matrix,the interference effect was overcome by a dissolution in TE buffer and background correction,largely simplifying the operation.The entire detection could be completed in 30 min.Due to its simple,label-free design,fast response and high selectivity,the proposed method may provide significant improvements in AFB₁ screening in TCMs safety monitoring.Importantly,it is promising as a universal method due to the exceptional selectivity of PicoGreen for dsDNA and the principle can be extended to the detection of other targets which aptamers have been selected.5.A fluorescently labeled nucleic acid aptamer sensor detects the aflatoxin B1 method.A new method for the detection of aflatoxin B₁（AFB₁）by aptamer recognition-fluorescence probe was established.When the conformation of the nucleic acid aptamer immobilized on the microtiter plate binds to the target substance AFB₁,the FAM-labeled short-stranded DNA hybridized with it will not be able to perform complementary base pairing with the aptamer,causing a change in the fluorescence signal.Quantitative detection of AFB₁ can be achieved.In the microplate,the avidin concentration was 50 mg·L^-1,the aptamer concentration was 50 nmol·L^-1,the FAM labeled sequence was 100 nmol L^-1,and the binding buffer was SSC buffer.Solution（750 mM NaCl,75 mM sodium citrate,pH 8.0）,reaction at 37°C for 30 min,the linear range of detection for AFB₁ was 0.1-20μg·L^-1;the detection limit was 0.5ng·m L;the relative standard deviation was 2.6%.The method has good selectivity and simple operation and can be used as a new method for aflatoxin B₁（AFB₁）.