Study On Determination Methods Of Trichothecenes In Traditional Chinese Medicines

Trichothecenes are a group of toxic fungal metabolites produced mainly by fungi of the Fusarium genus,and found in a great variety of plants and agricultural products. Their presence is a risk for the human and animal health,since they may have adverse effects including immunotoxicity,hematotoxicity,dereased feed consumption,emesis and reduced growth.Moreover,trichothecenes have been implicated in two large outbreaks of gastrointestinal illness in humans.Fusarium species are the major pathogenic microbes associated with the root rot of some medicinal plants,such as Panax quinquefolium L.,Radix Notoginseng,etc.Moreover,natural contamination of Fusarium species in medicinal herbs is frequently found.Fusarium species can further produce trichothecene mycotoxins under some specific circumstances. Therefore,it is very important to develop some analytical methods for the determination of trichothecene mycotoxins in traditional Chinese medicine(TCM) in order to evaluate its potential hazard to human health.Due to the high toxicity and the great diffussion of trichothecenes,many different methods for the determination of trichothecenes have been published.At the same time,many countries have established legal regulations or recommendations for trichothecenes.To date,all analytical methods for the determination of trichothecenes have been concentrated mainly on agricultural products,foods and feeds.However, no one has been used for the determination of trichothecenes in TCM.Therefore, some methods had been developed for the determination of the most frequently natural contamination trichothecenes,such as NIV,DON and T-2 toxin in TCM in this thesis.The main contents and conclusions were as follows:1.The method was established to analyze deoxynivalenol in traditional Chinese medicine with GC-ECD and GC-MS.A new,sensitive and accurate method was developed and validated for the determination of deoxynivalenol(DON) in traditional Chinese medicine(TCM) by using gas chromatography with electron capture detection(GC-ECD).The sample was extracted with water,filtered twice and applied to an immunoaffinity column. After washing with water,DON was eluted from the column with methanol and determined as its heptafluorobuturyl esters,prepared by pre-column derivatization with N-(Heptafluoro-n-butyryl) imidazole(HFBI).DON then was analyzed on a fused-silica capillary column and positive results were further confirmed by GC-MS. Calibration curve was found to be linear with the range of 0.02-5μg/mL.Recoveries from different TCMs spiked with DON at levels ranging from 100 to 5000μg/kg were from 85.5 to 97.2%,with relative standard deviations(RSDs) of less than 6.3%.The limit of detection was 2 pg,based on a signal-to-noise ratio of 3:1.Out of a total 58 TCM samples,DON was only found at low concentration in three samples,ranging from 17.21μg/kg to 50.54μg/kg.The results indicated that the developed GC-ECD method can be readily utilized as a DON detecting method for TCM and its related products.This is the first report of DON contamination in TCM.2.The method was established to analyze T-2 toxin in traditional Chinese medicine with GC-ECD.Gas chromatography with electron capture detection(GC-ECD) has been employed for the determination of T-2 toxin in traditional Chinese medicines(TCM) for the first time.This method consists of extraction of the sample with MeOH:H₂O (80:20,v/v) followed by clean-up of the extract with immunoaffinity column.T-2 toxin was determined as its heptafluorobuturyl esters,prepared by pre-column derivatization with N-(Heptafluoro-n-butyryl) imidazole(HFBI).Effects of two important factors,the reaction temperature and time of derivatization,were investigated to acquire the optimum condition.Calibration curve was found to be linear with the range of 0.1-16ng of derivatized T-2.Recoveries from different TCMs spiked with T-2 at levels ranging from 50 to 1000μg/kg were from 82.2 to 98.6%, with relative standard deviations(RSD) of less than 7.5%.The limit of detection was 25 pg,based on a signal-to-noise ratio of 3:1.The method developed has been used on different origins in TCMs purchased from the markets(n = 32),none of the samples contained T-2 above the detection level.The proposed method has been successfully applied to determine T-2 toxin in TCM with satisfactory assay results. 3.The method was established to analyze nivalenol and deoxynivalenol simultaneously in traditional Chinese medicine with HPLC-UV.A simple and accurate method for simultaneous detremination of nivalenol(NIV) and deoxynivalenol(DON) in traitional Chinese medicine(TCM) is described.The method uses DON purification column for NIV and DON clean-up and liquid chromatography(LC) for toxins detection and quantification.After extracting the sample with acetonitrile - water(80:20,v/v),the extract was one-time clean-up by DON purification column.DON and NIV were then separated and determined by reversed-phase HPLC with RP C₁₈ column.A mixture of water:acetonitrile:methanol (90:5:5,v/v/v) was as mobile phase.The linear range for NIVwas 0.2-20μg/mL with a correlation coefficient of 0.9999 and that for DON was 0.2-20μg/mL with a correlation coefficient of 0.9999.The detection limits for NIV and DON were 2ng and 2.5ng(S/N=3).Recoveries from different TCMs spiked with NIV and DON at levels ranging from 500-10000μg/kg were 80.8-100.3%and 82.8-99.9%,respectively,and their relative standard deviations(RSD) range was 0.9-9.6%.Thirty TCM samples were analyed for NIV and DON with the developed method.NIV and DON were not detected in any of the samples above the detection level.4.The method was established to analyze trichothecenes qualitatively in traditional Chinese medicine and Fusarium cultures with LC-MS.The sample was extracted with acetonitrile - water(80:20,v/v),and the extract was one-time clean-up by Bond Elut Mycotoxin column.Then,the trichothecenes (NIV,DON and T-2) were analyzed with LC-MS.A linear gradient consisting of water-methanol(80:20,v/v) changing to water-methanol(80:20,v/v) in 10 min and holding 10 min was used.The MS experiments were performed in the ESI positive mode.The scan range was 15-650 m/z.Eight Fusarium cultures were analyed for NIV, DON and T-2 with the described method.NIV,DON and T-2 were not detected in any of the samples.