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Aflatoxin is a secondary toxic metabolite produced by parasitic Aspergillus and Aspergillus flavus in the genus Aspergillus, and belongs to the highly toxic small molecule substance category. Among them, aflatoxin B. The most toxic and harmful substance is Class 1A carcinogen, and studies have shown trace amounts of aflatoxin B. It can also cause carcinogenesis, teratogenicity, and damage to liver function in the human body, and has good thermal stability, only cracking at high temperatures above 268 \u00b0 C. When fungi are exposed to high temperatures (25-32 \u00b0 C), high humidity (90% -110%), or stress conditions that compete with other fungi, they produce mycotoxins, which are often detected in grains, oilseeds, and their products. At present, countries around the world have established corresponding limit standards for it. Therefore, accurate detection of aflatoxins in grain and oilseed crops is crucial.<\/p>\n
Sample pretreatment, as an important step in the analysis of aflatoxin B1 in edible oil, directly affects the accuracy and precision of the detection results. Currently, aflatoxin B1 is present in edible oil. The main pre-treatment purification methods include immunoaffinity column method, solid-phase extraction method, etc. Among them, immunoaffinity columns are widely used due to their low matrix interference, selective adsorption, strong specificity, and high sensitivity. There are already a large number of commercially available aflatoxin immunoaffinity columns at home and abroad. However, the pre-treatment process requires manual purification, which has the drawbacks of being cumbersome and time-consuming. Therefore, adopting an automatic purification pre-treatment method is of great significance.<\/p>\n
Immunomagnetic beads (IMBs) are a new type of magnetic nanoparticle material. In recent years, the immunomagnetic bead purification method has received widespread attention and research due to its advantages of high sensitivity, simple operation, short time consumption, batch processing of samples, and low personnel requirements. It has gradually been applied to the detection of fungal toxins. Chinese researchers used chemical co precipitation method to prepare superparamagnetic nano magnetic beads, which were mixed with aflatoxin B1. Multi antibody coupling to obtain immunomagnetic beads, combined with high-performance liquid chromatography for joint detection of vegetable oil<\/p>\n
The content of aflatoxin B1. This method has good linearity, a detection limit of 0.5 micrograms per liter, an average recovery rate of 96%, and a relative standard deviation of 12.5%. The R&D personnel used the immunomagnetic bead purification method to determine aflatoxin B1 in grains, with an average recovery rate of 88.58% to 107.61% and a coefficient of variation of less than 10%; Although the above immunomagnetic bead purification method can meet the separation and purification of aflatoxin B1, the steps are still relatively complex. Therefore, adopting automated and high-throughput pre-treatment methods is of great significance.<\/p>\n
The fully automatic purification instrument for fungal toxins is a specialized instrument used for fully automatic immunomagnetic bead purification in fungal toxin detection. This experiment uses a fungal toxin automatic purification instrument and an immunoaffinity column purification pre-treatment method, combined with ultra-high performance liquid chromatography to determine aflatoxin B1 in edible oil. The differences between the two pre-treatment methods are systematically analyzed, providing reference for the rapid and accurate detection of aflatoxin B1 in vegetable oil.<\/p>\n
Aflatoxin B1 standard; Quality control samples for analysis and detection of aflatoxin B1 in vegetable oil (naturally contaminated); VNYA aflatoxin B1 immunoaffinity column; Fungal toxin purification kit – VNYA aflatoxin purification kit for grain and oil; Methanol, acetonitrile; Glass fiber filter paper and double ring quantitative filter paper; Vegetable oil (low mustard pickled Chinese cabbage seed oil, fragrant peanut oil, goldfish soybean oil).<\/p>\n
VNYA mycotoxin fully automatic purification instrument; Ultra pure water system; Vortex oscillator; Nitrogen blowing instrument; Ultra high performance liquid chromatography with fluorescence detector.<\/p>\n
Referring to GB 5009.22-2016 “Determination of aflatoxin B and G groups in food”, the third method is high-performance liquid chromatography post column derivatization (without derivatizer method, direct detection in a large volume flow cell). Accurately weigh 5 g (accurate to 0.01 g) of oil sample and place it in a 50 mL centrifuge tube. Add 20 mL of methanol water (v\/v: 70\/30), vortex and mix well. Shake in a shaker for 20 minutes, centrifuge at 6000 r\/min for 5 minutes, and take the supernatant for later use.<\/p>\n
1.4.1 Immunoaffinity column purification<\/p>\n
Pass the supernatant obtained through 1.3 treatment through a quantitative filter paper, accurately transfer 10 mL of the filtrate, add 20 mL of water, mix well, and filter the paper. Restore the immunoaffinity column stored at low temperature to room temperature. After the liquid in the immunoaffinity column is drained, take 10mL of filtrate into a 10mL syringe barrel, adjust the dropping speed, and control the sample liquid to flow out at the speed of 1-2 drops per second. After the sample is dripped, add 20 mL of water to the syringe and rinse the immunoaffinity column at a flow rate of 2-3 drops per second. After the water drips, use a vacuum pump to dry the column. Wash with 1 mL of methanol and collect all eluent into a test tube. Slowly blow the eluent with nitrogen gas at 50 degrees Celsius until almost dry. Dilute with methanol water (v\/v: 50\/50) to a volume of 1.0mmL, vortex for 30 seconds to dissolve the residue, filter with a 0.22 micron filter membrane, and collect the filtrate in an injection bottle for injection.<\/p>\n
1.4.2 Fully automatic purification instrument for mycotoxin<\/p>\n
Accurately transfer 1 mL of 1.3 supernatant into the sample well of the reagent kit, place the reagent kit into the fully automatic purification instrument for human mycotoxins, start the instrument, and enter the program. Automatically complete the sample dilution, toxin enrichment, impurity cleaning, and target substance elution processes. After the program is completed, add 0.5mL of pure water to the 5 well position, mix and filter using a 0.22 micron filter membrane. Collect the filtrate in a sampling bottle for injection.<\/p>\n
The purification steps of the aflatoxin B1 fully automatic purifier include transferring magnetic beads to the sample well, mixing for 5 minutes, and magnetic suction for 7.5 minutes; Transfer the magnetic beads to the cleaning holes (2), clean each cleaning bag L for 1 minute, and magnetic suction for 1 minute; Transfer the magnetic beads to the elution hole, elute for 1 minute, magnetic suction for 1 minute, then transfer the magnetic beads to the magnetic bead recovery hole, and the purification is completed. The automatic purification time for aflatoxin B1 in a single reagent kit is 26 minutes.<\/p>\n
Prepare 10 micrograms per milliliter of standard intermediate stock solution for aflatoxin B1, and dilute gradient to prepare standard working solutions with concentrations of 0.625, 1.25, 2.5, 5.0, and 10.0 ng\/mL.<\/p>\n
Referring to GB 5009.22-2016, the derivatization free (direct detection in a large flow cell) method was used for sample injection determination. Mobile phase: A phase, water; B phase, methanol; Equal elution conditions: A, 50%; B\uff0c50%\uff1b Chromatographic column; Flow rate: 0.3mL\/min; Column temperature: 40 \u2103; Injection volume: 2 microliters; Excitation wavelength: 365nm; Emission wavelength: 436 nm.<\/p>\n
A series of standard solutions were prepared within the range of 0.625-10ng\/mL to establish standard curves. Fit a linear (first-order) calibration curve with peak area (Y) and standard solution mass concentration (X); A=-7.047 720e+004;b=13 699 236E+005\uff0cR2=0.9998\uff0c It has good linearity; The detection limit (LOD) of the method is 0.02 \u03bcg\/kg, and the quantification limit (LOQ) is 0.05 \u03bcg\/kg, which fully meets the determination requirements of aflatoxin B1 in oils and their products in China.<\/p>\n
2.2 Accuracy of test results<\/p>\n
To compare the accuracy of the two methods, a national certified standard substance containing corn aflatoxin B1 in peanut oil matrix was selected. The fully automatic immunomagnetic bead and immunoaffinity column purification method combined with ultra-high performance liquid chromatography were used to detect aflatoxin B1 in peanut oil. The detection results are shown in Table 1, and the measured values are within the standard value and its extended uncertainty range. The RSD is not more than 5%, indicating ideal detection results.<\/p>\n
The measured values of the mycotoxin automatic purification instrument and the immunoaffinity column purification method are 15.59 \u03bcg\/kg and 14.64 \u03bcg\/kg, respectively, which are within the standard value range of the reference substance. This indicates that both purification methods have high accuracy and can meet the detection requirements.<\/p>\n
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To evaluate the accuracy and precision of the two methods, three different levels of aflatoxin B1 were added to negative samples of peanut oil, corn oil, and soybean oil (samples without detected aflatoxin B1) for spiked recovery rate determination. The results are shown in Table 2. The recovery rates of the fully automatic immunomagnetic bead purification method in three different oils ranged from 96.65% to 109.06%; The recovery rate of immunoaffinity column purification method was 88.48% to 105.76%. The relative standard deviation (RSD) of both methods is within 5%, which meets the needs of daily testing.<\/p>\n
17 different types of plant oil positive samples (samples capable of detecting aflatoxin B1) were detected using fully automated immunomagnetic beads and immunoaffinity column purification methods. Use paired t-test to evaluate the consistency of the results obtained by the two purification methods. As shown in Table 3, it can be concluded through t-test that the t-value (0.75) is less than the theoretical t0.05<\/p>\n
(17) The value (2.11), P>0.05, indicates that there is no significant difference in the measurement results between the two purification methods.<\/p>\n
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The Bland Altman method was used to analyze the differences between the fully automatic immunomagnetic bead and immunoaffinity column purification methods. The results are shown in Figure 1, and the two purification methods have the same effect. Only 5.9% (1\/17) of the points in both methods are outside the 95% consistency threshold. Among them, within the consistency limit, the maximum absolute value of the difference between the measured values of the two purification methods is 2.34 \u03bcg\/kg, and the average value of the difference is -0.57 \u03bcg\/kg. The difference between these two purification methods is within an acceptable range, so it can be considered that the purification effects of these two methods have good consistency. When purifying and enriching aflatoxin B1 in vegetable oil, they can be used interchangeably.<\/p>\n
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The fungal toxin fully automatic purifier used in conjunction with the immunomagnetic bead purification method can process 10 samples at once, with an average purification time of about 2-3 minutes per sample, achieving high-throughput automatic purification of aflatoxin B1 in grains. Immune affinity column manual purification, one person can process up to 10 samples simultaneously, and the entire process takes nearly 1 hour. The average purification time for each sample is about 5-6 minutes; From this, it can be seen that processing a sample using magnetic bead purification method can save 2-3 minutes of time, and compared to manual purification using immunoaffinity columns, magnetic bead purification has an absolute advantage in purification time. When processing large quantities of samples, it can greatly improve work efficiency.<\/p>\n
This experiment compared two pre-treatment methods, fully automatic immunomagnetic bead method and immunoaffinity column purification method, for the determination of aflatoxin B1 in edible oil. The accuracy of the data obtained by the two pre-treatment methods was verified by adding different gradients of aflatoxin to different varieties of edible oil to determine their spiked recovery rates, and using nationally certified standard substances.<\/p>\n
The experimental results show that both pre-treatment methods can achieve experimental results and meet the experimental requirements when detecting aflatoxin B1 in edible oil. The cost of fully automatic immunomagnetic bead purification of aflatoxin B1 in vegetable oil is lower, and the detection cost of each sample is about half of that of immunoaffinity column method. Compared with the widely used immunoaffinity column purification method, the fully automatic purification method using immunomagnetic beads has the characteristics of simple operation, short detection time, high detection efficiency, and low cost. In the experiment, the instrument is automatically processed, and the reagent kits used are disposable consumables with less contact with harmful reagents. It can be used for the detection of aflatoxin B1 in edible oil.<\/p>","protected":false},"excerpt":{"rendered":"
Aflatoxin is a secondary toxic metabolite produced by parasitic Aspergillus and Aspergillus flavus in the …<\/p>\n