Abstract The experiment established an immunoaffinity chromatography-high performance liquid chromatography (HPLC) for the determination of aflatoxin B1 in feed, using methanol-water to extract aflatoxin B1 in the feed, purifying it with an immunoaffinity chromatography column, and using methanol With water as the mobile phase, reversed-phase high-performance liquid chromatography fluorescence detection method was used to determine the excitation wavelength of 365 nm, emission wavelength of 435 nm. The detection limit of the method was 0.01 μg / kg, the limit of quantification was 0.03 μg / kg, the average recovery was 77.5%, and the coefficient of variation was 0.03%.
Ding Ping Hou Yali Cheng Xiaowei Chongqing Feed Veterinary Medicine Supervision Office
Aflatoxin is a metabolite of strains produced by Aspergillus flavus and paramycin. It is a type of compound with similar chemical structure.According to the difference of its structure, it is called aflatoxin B1, B2, G1 and G2, Among them, aflatoxin B1 is the most common and one of the strongest chemical carcinogens known, which causes serious pollution to grain, oil and food. Aflatoxin B1 is often used as an indicator in feed testing. China's feed limit standard is usually 10 ~ 50μg / kg. The detection methods of aflatoxin B1 at home and abroad mainly include: thin layer chromatography, enzyme-linked immunoassay, high performance liquid chromatography and fluorescence photometry. The experiment used an immunoaffinity column to purify aflatoxin B1 in the feed, and studied the high-performance liquid chromatography fluorescence detection method, which provided a simple and feasible method for monitoring aflatoxin B1 in the feed.
1 Materials and methods
1. 1 instruments and reagent instruments Angilent 1100 high-performance liquid chromatograph, 2475 multi-wavelength fluorescence detector, METTLER AE 240 electronic balance, oscillator, vortex mixer, microporous membrane (0.45μm), glass fiber filter paper, Immunoaffinity chromatography column (provided by Beijing Anyi Century Trading Co., Ltd.).
Reagent Aflatoxin B1 standard solution (1 mg / L), provided by Sig2ma, trifluoroacetic acid, methanol (chromatographically pure), water is ultrapure water.
1. 2 Solution preparation
1. 2. 1 Take 550 mL of methanol in the sample extract, make up to 1 000 mL with water, and mix.
1. 2. 2 Take 450 mL of methanol in the mobile phase, dilute to 1000 mL with water, and mix.
1. 2. 3 Preparation of aflatoxin B1 derivatized standard samples Accurately absorb a certain amount of standard preparation solution, place it in a 5 mL glass test tube, blow dry under N2 flow in a 50 ℃ water bath, add 300 μL of derivatization reagent III Fluoroacetic acid was derivatized and placed at room temperature for 3 min. Blow dry under N2 flow in a 50 ℃ water bath, dissolve with a mixed solution of 1 mL methanol, acetonitrile and water, and configure it to have a concentration of 1.
2, 5, 10 and 20 μg / L standard solutions were tested by HPLC.
1. 3 Sample processing Weigh 5.0 g of sample accurately, place it in a 150 mL conical flask with a stopper, add 1.0 g of sodium chloride and 40.0 mL of sample extract, reciprocate and shake for 30 min. Fibre filter paper to filter. Pipette 10.0 mL of the filtrate accurately and add 30.0 mL of water to dilute it, inject it into an IAC cartridge that has been equilibrated to room temperature with water, rinse with 3 mL of water, and elute with 3 mL of methanol, collect the washing solution in a glass test tube, Blow dry under N2 flow in a 50 ° C water bath. HPLC detection after treatment according to standard derivative method.
1.4 Determination of recovery rate Weigh 5.0 g of blank feed and place it in a 150 mL conical flask with a stopper, add standard working solutions respectively, so that the drug concentration in the sample is 20.0, 10.0 and 2. 0μg / kg, mix and let stand for 10 min, set 4 parallels for each concentration level, and process and measure them separately according to the method of 1.3.
1.5 Chromatographic conditions Chromatographic column C18 column, 250 mm × 4.6 mm, particle size 5 μm; column temperature 25 ℃; mobile phase 450 mL methanol + 550 mL water; excitation wavelength 365 nm; emission wavelength 435 nm; injection volume 25μL.
2 Results and analysis
2.1 The linear response of the measurement method Take the configured standard solution for measurement under the selected chromatographic conditions and draw a standard curve (see Figure 1). The tailing peak is at a concentration of 1 to 50 μg / L. The former is rare. > The peak area of ​​the chromatogram is linearly related to the concentration. The regression equation is y = 2.524 1 x + 0.272 2 and the correlation coefficient r2 = 0.9999 8. Its linear response range fully meets the measurement requirements, indicating that the method is reliable for quantitative determination.
2. 2 The sensitivity of the measurement method is calculated under the test conditions, and the signal-to-noise ratio (S / N) is 3, and the detection limit of aflatoxin B1 in the feed is 0.01 μg / kg, and the S / N is 10 After calculation, the quantitative limit of aflatoxin B1 in the feed was 0.03 μg / kg, which met the requirements of quantitative analysis.
2. 3 Addition recovery test The addition recovery rate and the coefficient of variation of the results are shown in Table 1. The chromatograms of standard samples, blank feed samples and standard-added feed samples are shown in Figure 2. Under this method, the retention time of aflatoxin B1 is about 8.3 min.
It can be seen from Table 1 that at the three feed addition levels, the average recovery rate of aflatoxin B1 is 74.9% ~ 81.6%, and the coefficient of variation is 0.01% ~ 0.04%.
3 Discussion This method uses methanol-water to extract feed samples, purify and concentrate them through an IAC column, and determine the content of aflatoxin B1 by HPLC fluorescence detection. From the chromatogram of the blank feed sample and the chromatogram of the added feed sample, it can be seen that there is no impurity peak near the chromatographic peak of aflatoxin B1, indicating that the impurities in the feed do not interfere with this determination method.
It can be seen from Table 1 that the HPLC method has a higher recovery rate (average 77.5%) and good reproducibility (average coefficient of variation 0.03%). Due to the high toxicity of aflatoxin, the limit requirement for aflatoxin is now 2 to 4μg / kg or even lower. The lower the limit of aflatoxin content, the higher the requirements for detection instruments and methods. Complex operations, a large number of organic reagents, strict protection conditions and damage to operators are not suitable for the development of the situation. In the experiment, immunoaffinity chromatography-HPLC method was used to replace the commonly used thin layer method, reducing the operation steps and the loss of organic solvents. The determination of aflatoxin in the national standard food uses post-column derivatization HPLC-fluorescence spectrometry. This method is adopted in the test, and the requirements for the instrument are reduced.
The experiment investigated the effect of injection volume on the peak shape of aflatoxin B1. When the injection volume was 100 μL, the peak shape of the sample peak was very asymmetric, with a prolonged peak. When the injection volume was changed to 50μL later, the peak shape was greatly changed and also symmetrical. Since 25μL injection can also meet the quantitative requirements, the test finally determined the injection volume is 25μL. When the sample is extracted, the shaking time should be sufficient. When the test is started, the extraction time is 15 min, and the recovery rate is very low, which cannot meet the quantitative requirements. When the shaking time is extended to 30 min, the recovery rate is greatly improved, which can meet the requirements of feed detection.
The test results show that the immunoaffinity chromatography-HPLC method is simple and easy to operate and can meet the analysis requirements. It is a sensitive and reliable method for determining aflatoxin B1 in feed.
references
1 Joerg S, Elke A. Development of a simplified densit2ometer for the determination of aflatoxins by thin -layer chromatography. Journal of Chromatography A, 2002 (904): 263 ～ 268
2 Niu Chengyu, Li Zhixiang, Wang Yong. Liquid Chromatographic Determination of Aflatoxin in Agricultural Products. Agricultural Environmental Protection, 1994, 14 (1): 43 ～ 45
3 Anders K, Barbro A, Kurt A. Immunoaffinity columnclean-up for the high performance liquid chromato2graphic determination of aflatoxinsB1, B2, G1, G2, M1 and Q1 in urine. Journal of Chromatography B, 1995 (627): 253 ～ 259
4 Joerg S, Elke A. Development of a simplified densito2meter for the determination of aflatoxins by thinlayerchromatography. Journal of Chromatography A, 2003 (1011): 49 ～ 54
5 Liu Zhaohui, Yang Jizhou, Yang Xiangying. Application of immunoaffinity column fluorescence photometry in the detection of aflatoxin. Quarantine Inspection, 2000, 11 (21): 32
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