| Mycotoxins are secondary metabolites mostly produced by filamentous fungi Aspergillus, Penicillium and Fusarium, which could cause toxic response when they were ingested by human and animals. So far, more than 400 different kinds of mycotoxins were identified in the world, and most of mycotoxins were widely found in agricultural products, even several mycotoxins were simultaneously found in them. Knowing that toxicity mechanisms are structure dependent, numerous acute toxic and chronic carcinogenic, mutagenic, teratogenic, or estrogenic effects have been link to mycotoxins exposure in human and animals. Therefore, maximum residue levels and detection methods in agricultural products have been seted up by many countries and regions for mycotoxins. However, these single mycotoxin detection methods are time-consuming, intensive laboratory procedures and high cost. Besides, some blind spots in detecting single mycotoxin made the high exposure risk of mycotoxin to the consumers. So, development and validation of quick, effective, low-cost and high throughput analysis methods can not only increase the detecting efficiency, but also can reduce the mycotoxin exporsue risk and protect agricultural porducts’safety effectively.In this paper, the familiar important mycotoxins, including Aflatoxin B1(AFB1), Citrinin(CIT), Ochratoxin A(OTA) and Zeralenone(ZEN) were.focused The Quick, Easy, Cheap, Effective, Rugged, and Safe(QuEChERS) extraction method and the Reverse Phase High Performance Liquid Chromatography, Multi-Channel Fluorescence Detector (HPLC-FLD) detection method for simultaneous determination of AFB1, CIT, OTA and ZEN were established. The results showed that the established extraction and detection methods could be used for determining trace AFB1, CIT, OTA and ZEN simultanously. On this basis, 70 species of cereal samples were detected by the established methods for AFB1, CIT, OTA and ZEN.1 The establishment of HPLC-FLD simultaneous detection methodIn order to establish the HPLC-FLD simultaneous detection method, the separating effect of AFB1, CIT, OTA and ZEN with different organic solvents (methanol, acetonitrile),7 types of Cig columns and three different column temperatures were compared. The influences of phosphoric acid on chromatographic peaks, retention times and resolution were analyzed, and the chromatographic simulation software DryLab was used to optimized the mobile phase conditions. In addition, the spectrophotometer was used to research the fluorescence property of AFB1, CIT, OTA and ZEN, especially the differences of fluorescence property of CIT in different pH values. Finally, the established HPLC-FLD conditions for AFB1, CIT, OTA and ZEN were followed:stationary phase:Intersil ODS-3 (250 mm x 4.6 mm,5μm), mobile phase:pH=2.7with phosphoric acid, water:acetonitrile= 50:50 (v/v), flow rate:0.8 mL/min, column temperature:30℃, and detector:FLD.By this method, the retention times of AFB1, CIT, OTA and ZEN were 7.2,15.5,17.9 and 19 min, respectively. The linearity rang of them were 0.5～6 ng,1～15 ng,0.5～6ng and 10～100 ng, respectively. The correlation coefficient (R2) were 0.9918,0.9918,0.9975 and 0.9984, respectively, and the linear equations of them were y= 842680x + 224255, y= 2101765x-70546, y= 2948376x-634601 andy= 79111x-285656, respectively.2 The establishment of simultaneous extraction and purification methods for mycotoxinsBecause AFB1, OTA and ZEN are water-insoluble, CIT are certain water-soluble and weak acidic, a single solvent could not simultaneously extract all of them. In this study, the extraction effect of different mixed solvents on AFB1, CIT, OTA and ZEN were analysised by using Liquid Liquid Extraction (LLE) and QuEChERS technique. The results showed that the extract containing 24% ethyl acetate-42% acetonitrile-14% methanol-5% acetic acid in the LLE method gave the best extraction effect, and its recovery of AFB 1, CIT, OTA, ZEN were 80.08%,60.44%,91.53% and 102.03% respectively. Then, in the QuEChERS method, the extraction effect of 60% acetonitrile - 5% acetic acid was the best, and the recovery of AFB1, CIT, OTA and ZEN reached 80.65%,90.56%,96.63% and 69.95% respectively. Compared with the LLE method, the QuEChERS methods was easy, quick, safe, using easier mixed extraction solvents (60% acetonitrile-5% acetic acid-35% water), and had a better extraction effect on the simultaneous extraction of AFB1, CIT, OTA and ZEN. So the QuEChERS method was selected as the pretreatment method. 3 The evaluation of HPLC-FLD detection method and determination of specimenIn the QuEChERS-HPLC-FLD method, its detection limits of AFB1, CIT, OTA, ZEN were 2.12μg/kg,5.46μg/kg,2.39μg/kg,53.10μg/kg respectively, and the quantitaion limits were 8.48μg/kg,21.82μg/kg,9.57μg/kg,212.38μg/kg. Under the national limited standards of AFB1, OTA and ZEN and the industrial standard of CIT level, the recovery of the four kinds of mycotoxins in feed and rice were ranged from 75% to 120%, and the standard deviation of them were between 1.22 and 5.81. Finally, the 70 species of cereal samples were analyzed with this method, the results showed that OTA was detected in 2 samples, AFB1 was found in 6 samples and ZEN was obtained in 4 samples, however, among them 2 samples contained AFB1 and ZEN were reveaed. On the other hand, all samples were not detected containing CIT. |
PDF Full Text Request