Study On Detection Technology For Determination Of Aflatoxin B₁ With High Sensitivity

Aflatoxin B₁ (AFB₁) seriously threatened human and animal healthy or life safety. AFB₁ commonly contaminates agricultural commodities, food and animal feedstuffs, which is an important problem all through. Standard of AFB₁ limits and technical barriers to trade (TBT) were set by many countries. TBT led to impeded export of agricultural production and damaged the interests of farmers. Development of high sensitivity test method for AFB₁ determination is important and crucial to break out TBT. Detection technology with high sensitivity must be studied in order to guarantee agro-food quality control and management from farm to table, find out the contaminated production in time and prevent the production contained excess the standard AFB₁ into food chain.Based on the fluorescence characteristic of AFB₁, study on how to improve the sensitivity of detection technology was done. First, laser was introduced into the detection technology to induce fluorescence and laser induced fluorescence (LIF)-high performance capillary electrophoresis (HPCE) was developed with high sensitivity. Besides of laser lamp-house researchment, the new fluorescence enhancer of AFB₁ was researched and its scientific hypothesis of AFB₁ chelation fluorescence enhancement was established and deduced from experiments. The novel post-column derivatization method by high performance liquid chromatography (HPLC) was developed with high sensitivity by applied the new fluorescence enhancer. As fluorescence enhancer, HgCl₂ may be induced into IAC fast test method and improved sensitivity. The main conclusions were as follow.1. Laser diode double-pumped solid laser of 375nm wavelength was studied for a new excitation light source and coupled HPCE to detect AFB₁ in MECC separation mode. The optimized conditions were as follow. Buffer was composed of 50mmol/L NaDC, 6mmol/L Na₂B₄O₇, 10mmol/L Na₂HPO₄, 10% Acetonitrile. Excitation wavelength was at 375nm and detected emission wavelength was at 440nm. The applied voltage was 15kV and the current was 104μA. The capillary (75μm i.d., 57 cm total length, 50 cm length to detector) was chosen. The concentration of AFB₁ in samples was determined by LIF-HPCE. The results showed that repeatability expressed as RSD values was under 5% and intermediate precision expressed as RSD values was under 3%. Recovery was in the range of 84.1-96.1%. The data also fit the line y= 10⁶x + 319847 (R²=0.9988) in the linear range of AFB₁. For sample injection volume being little, the absolute limit of detection (0.17pg) and quantitative limit of AFB₁ (0.56pg) were far lower than other method. The absolute mass detection sensitivity of LIF-HPCE improved three magnitudes relatived to AFB₁ detection method2. Fluorescence spectrums and fluorescence signal changes of fluorescence system were researched and fluorescence enhancement technology chelated by metal ions was introduced into AFB₁ fluorescence detection. The result showed that HgCl₂ solvent could be a new enhancer to AFB₁ being enhanced the fluorescence intensity of AFB₁ rapidly, steadily, intensively in the room temperature. The enhanced condition influenced fluorescence system of AFB₁-Hg (II) was studied. The room temperature was accepted. The fluorescence enhancement reaction could balance and complete basically within the 2min when the molar ratio of Hg (II) and AFB₁ should exceed 1:1. In the same condition, enhanced fluorescence intensity by HgCl₂ was stronger than that of Br₂. The sensitivity of AFB₁ detection was improved by HgCl₂ enhancement. Fluorescence enhancement hypothesis of AFB₁ chelation with HgCl₂ was established and deduced. In the process of AFB₁ fluorescence enhancement, octahedral chelated resultant came into being and light redox reaction in LMCT occured and formed steady chelated products. Steady chelated products could extend rigidity plane or conjugate structure and fluorescence intensity was enhanced greatly by fluorescence efficiency being improved3. A novel post-column derivatization method for simultaneous enhancement of aflatoxin B₁, B₂, G₁, G₂, and M₁ by HPLC was developed. The aqueous solution of Mercuric Chloride was utilized as a new fluorescence enhancer for aflatoxin B₁, G₁, and M₁ in post-column. Full separation of five aflatoxins with fluorescence detector (λax=365nm,λam=440nm) could be achieved in the baseline by the mobile phase composed of methanol-acetonitrile-water (22:18:60 v:v:v) within 13 minutes. The flow rate of flow phase was 0.8mL/min and the flow rate of enhancer was 0.4mL/min. Sample injection was 10μL. The sensitivity was improved by the fluorescence intensity enhancement of HgCl₂. The limit of detection of AFB₁ was 0.05μg/kg and limit of quantitative of AFB₁ was 0.17μg/kg. The absolute mass detection sensitivity improved above two magnitudes relatived to AFB₁ detection method. Repeatability expressed as RSD values was 1.81% and intermediate precision expressed as RSD values was 4.28%. Recovery was in the range of 84.67-91.10%. The new method was proven to be an efficient method with high sensitivity, high accuracy and high repeatability for simultaneous determination of aflatoxins and successfully applied to analyze aflatoxins in contaminated peanut, rice, corn, peanut oil, and peanut butter.4. Fluorescence enhancement of HgCl₂ may be induced into IAC fast test method and sample pretreatment technology was developed for AFB₁ determination accurately, fast, conveniently in agricultural production and food. The limit of quantitative was 0.3μg/kg. The recovery of AFB₁ in spiked samples was above 90%. Repeatability expressed as RSD values was under 3% and R² was 0.99. The test time was shorter than 40min of single sample. The pretreatment process was simple and timesaving for whole process time of single sample being within 40min and the time was shorter when sample amount increased. The fast test method fit for test in spot and port etc with highlighted advantages such as high accuracy, low-cost, simple-manipulation, fast-run, high safety and so on.