| In recent years, with the large extent of traditional Chinese medicines (TCMs) being used in the word, safety of TCMs has been paid more and more attention. Trace mycotoxin residues in TCMs as toxic and harmful exogenous substances generally do not exhibit acute toxicity, but their accumulative, carcinogenic, teratogenic and mutagenic effects seriously affect the safety of TCMs. The dissertation systematically developed the detection methods for mycotoxins in TCMs, investigated the effects of the storage conditions on the mycotoxins production, and studied the pharmacokinetics of the typical mycotoxins in TCMs. The mainly purpose was to provide the theoretical and experimental basis for improving the quanlity control standards, enhancing the harvesting and storage methods, preventing mycotoxins contamination and ensuring the safety use of TCMs. The contents and results of the dissertation were described as follows:1. The ultra-high-performance liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS) was utilized for the establishment of the multi-component mycotoxin detection system for the first time, including two aspects:One was to establish the exclusive methods which could be used for the determination of the aflatoxins, ochratoxins, fumonisins, type B-trichothecenes and zearalenone and its derivatives, respectively;the other one was to establish a general method for simultaneous determination of 35 mycotoxins in different TCMs. Because of the variety of the TCMs, along with their complex composition, the matrix effects usually affects the accuracy of the LC-MS/MS methods. After optimization of extraction solvent and extraction methods, development of the home-made clean-up cartridges and combined using of the isotope internal standard, the matrix effects were successfully eliminated and accurate quantitation was achieved. The established method was further validated by determining the linearity, sensitivity, average recovery, precision and the quality control samples. Such accurate determination methods improved upon existing technologies for the detection of mycotoxins in terms of high sensitivity, rapidity of analysis and specificity for the toxin for high throughput testing.2. The established UHPLC-MS/MS methods were applied to the determination of the 38 mycotoxins in a total of 201 TCMs widely used in China. The results showed that among the 201 samples,157 were contaminated with the mycotoxins, ranging from 0.1 to 166.0μg kg-1. Then, each type of mycotoxins were analyzed. It could be obviously seen that aflatoxins could easily contaminate seeds and fruits (incidence 62.2%, range 0.2-23.8μg kg-1), rhizomes and roots (incidence 31.4%, range 0.1-27.5μg kg-1), leaves and grasses (incidence 37.1%, range 0.1-62.6μg kg-1), but only a few in flowers (incidence 12.8%, range 0.5-2.4μg kg-1). The rhizomes and roots contained highest contents of ochratoxins (incidence 27.9%, range 0.1-70.0μg kg-1). The leaves and grasses could also be easily contaminated by ochratoxins (incidence 25.7%, range 0.2-62.3μg kg-1). The incidence (contamination levels) of ochratoxins in seeds and fruit was 18.9%(0.3-10.1μg kg-1). The flowers contained fewest ochratoxins (incidence 5.1%, range 0.2-1.5μg kg-1). The fumonisins frequently existed in rhizomes and roots (incidence 18.6%, range 0.4-60.2μg kg-1) and leaves and grasses (incidence 22.9%, range 1.1-83.2μg kg-1), there were a few fumonisins in seeds and fruit, as well as in flowers. The incidences were 13.5% and 7.7%, and the content ranges were 0.6-8.2μg kg-1 and 4.8-19.2μg kg-1, respectively. Type B-trichothecenes were also detected in TCMs, with the contents no more than 88.4μg kg-1, within the acceptable limits set by European Union or China. Only very few ZEN and its derivatives were found in rhizomes and roots, seeds and fruit, leaves and grasses (no more than 33.7μg kg-1), no flower samples were contaminated with these mycotoxins. It could be summarized that the TCMs could be easily contaminated with mycotoxins, even with the contents higher than the relative regulation. It emphasized the need for further and regular studies of TCMs for the various mycotoxins for the assessment and prevention of risk to consumers.3. The TCM matrix, temperature and humidity conditions for the mycotoxins production were investigated. A total of 8 dried TCMs were selected:(1) Seeds and fruit:Semen Persicae, Semen Armeniacae Amarum;(2) Rhizomes and roots:Radix Polygoni Multiflori, Radix Scutellariae; (3) Grasses and leaves:Folium Sennae, Herb of Spanishneedles;(4) Flowers:Flos Carthami, Flos Magnoliae Officinalis. After the selected TCMs had been stored under different temperatures (40℃,25℃and 10℃) and humidities (31.6-33.5%,53.2-60.7% and 82.3-86.8%) for one month, the mycotoxins were determined. The results showed that the contents of most of the mycotoxins were improved and the maximal increment was 56.7μg kg-1. The TCM matrix, temperature and humidity conditions all could effect the production of the mycotoxins, but the extents were different. Among them, the matrix of the TCM had a significant effect on the production of the mycotoxins. The easily contaminated TCMs usually contained high contents of mycotoxins under different storage conditions though the species and quantities of mycotoxins were various. The temperature and humidity conditions also played a vital role. For example, the aflatoxins could be generated under all conditions, but the ochratoxins needed high humidity, and so on. Further study was needed since the simulated conditions were different from the actual conditions, along with the small number of samples analyzed.4. In order to study the toxicity of the typical mycotoxins in TCMs, a fast UHPLC-MS/MS method has been developed for simultaneous determination of aflatoxin B1, ochratoxin A and T-2 toxin in rat plasma, heart, liver, kidney, spleen, lung and brain. Compared to the previous methods, the established quantitative method, which could be applied to the determination of aflatoxin B1, ochratoxin A and T-2 toxin in all matrices, had many advantages including simple pretreatment, rapid determination, high sensitivity and selectivity. Radix Paeoniae Alba was selected as the typical TCM, which could be easily contaminated by mycotoxins. The pharmacokinetics were studied after oral administration of the aflatoxin B1, ochratoxin A and T-2 toxin spiked extracts of Radix Paeoniae Alba in rat. The results showed that the times to peak plasma concentration of aflatoxin B1, ochratoxin A and T-2 toxin were 0.78 h,2.01 h and 1 h, respectively, and the half-lives were 20.4 h,48.95 h and 32.02 h, respectively. The clearance of AFB1 and T-2 were faster than OTA in the tissues. After investigation of the tissues accumulation, all of the three mycotoxins exhibited accumulative effects. The highest concentration of aflatoxin B1 was observed in liver, followed by kidney. The highest tissue concentration of T-2 toxin was observed in spleen. Ochratoxin A usually accumulated in kidney.5. An accurate mass quadrupole time of flight (Q-TOF) mass spectrometry was used for the analysis of the metabolites of aflatoxin B1 and ochratoxin A after oral administration of the aflatoxin B1 and ochratoxin A spiked extracts of Radix Paeoniae Alba in rat. The results showed that aflatoxin B1 could be oxidized by cytochrome P450s (CYP1A2, CYP3A4, CYP2A6, et al.) to aflatoxin M1 and aflatoxin B1 exo-8,9-epoxide (unstable). The highly active aflatoxin B1 exo-8,9-epoxide could react with serum albumin to generate lysine adducts. After hydrolysis, the highly active aflatoxin B1 exo-8,9-epoxide could also be converted to dihydrodiol, monoalcohol and dialcohol. Ochratoxin A could be converted to ochratoxin B, ochratoxin a, ochratoxinβ, hydroxy-ochratoxin A and hydroxy-ochratoxin B by the detoxification reactions. Meanwhile, a lactone-opened ochratoxin A was also identified as a toxic metabolite ofˉochratoxin A in tissues and urine. After analysis of the main metabolites, the possible biological transformation roadmaps of aflatoxin B1 and ochratoxin A spiked in Radix Paeoniae Alba were developed.
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