Some standard content:
ICS67.040
National Standard of the People's Republic of China
GB/T5009.22—2003
Replaces GB/T5009.22-1996
Determination of aflatoxin B in foods
Determination of aflatoxin B, in foodsIssued on August 11, 2003
Ministry of Health of the People's Republic of China
Standardization Administration of the People's Republic of China
Implementation on January 1, 2004
GB/T5009.22-2003
This standard replaces GB/T5009.22—1996 "Determination of aflatoxin BI in foods". Compared with GB/T5009.22-1996, the main changes of this standard are as follows: the Chinese name of the standard has been changed to "Determination of Aflatoxin B in Foods"; the structure of the original standard has been modified in accordance with GB/T20001.4-2001 "Standard Preparation Rules Part 4: Chemical Analysis Methods".
This standard is proposed and managed by the Ministry of Health of the People's Republic of China. The first method of this standard was drafted by the Institute of Nutrition and Food Hygiene, Chinese Academy of Preventive Medicine and Qingdao Import and Export Commodity Inspection Bureau of the People's Republic of China.
The second method of this standard was drafted by the Food Hygiene Supervision and Inspection Institute of the Ministry of Health and Beijing Institute of Nutrition Sources. The main drafters of the second method of this standard are: Ji Rong, Lu Ge, Luo Xueyun, Zhang Hao, Wang Jianwei. This standard was first issued in 1985 and revised for the first time in 1996. This is the second revision. 178
1 Scope
Determination of aflatoxin B in food
GB/T5009.22—2003
This standard specifies the determination method of aflatoxin B in various foods such as grains, peanuts and their products, potatoes, beans, fermented foods and alcoholic beverages.
This standard is applicable to the determination of aflatoxin B in various foods such as grains, peanuts and their products, potatoes, beans, fermented foods and alcoholic beverages.
In the first method, the minimum detection amount of aflatoxin B on the thin layer plate is 0.0004μg, and the detection limit is 5μg/kg. The detection limit of aflatoxin B in the second method is 0.01μg/kg. Method 1
2 Principle
After extraction, concentration and thin layer separation, the aflatoxin B in the sample will produce blue-violet fluorescence under ultraviolet light of wavelength 365nm. The content is determined according to the minimum detection amount of fluorescence on the thin layer. 3 Reagents
3.1 Chloroform.
3.2 Hexane or petroleum ether (boiling range 30℃～60℃ or 60℃～90℃). 3.3 Methanol.
3.4 Benzene.
3.5 Ethyl.
3.6 Anhydrous ether or ether dehydrated with anhydrous sodium sulfate. 3.7 Acetone.
When testing the above reagents, first conduct a reagent blank test. If they do not interfere with the determination, they can be used. Otherwise, they need to be re-distilled one by one. 3.8 Silica gel G: for thin layer chromatography.
3.9 Trifluoroacetic acid.
3.10 Anhydrous sodium sulfate.
3.11 Sodium nitride.
3.12 Benzene-acetonitrile mixture: Measure 98mL of benzene, add 2mL of acetonitrile, and mix. 3.13 Methanol aqueous solution: 55+45.
3.14 Aflatoxin B, standard solution
3.14.1 Instrument calibration: Determine the molar extinction coefficient of potassium dichromate solution to find the calibration factor of the instrument. Accurately weigh 25mg of dried potassium dichromate (reference grade), dissolve it in sulfuric acid (0.5+1000) and accurately dilute to 200mL, equivalent to [c(KzCrO,)=0.0004mol/LJ. Then draw 25mL of this dilution into a 50mL volumetric flask, add sulfuric acid (0.5+1000) to dilute to the scale, equivalent to 0.0002mol/L solution. Then take 25mL of this diluted solution into a 50mL volumetric flask and add sulfuric acid (0.5+1000) to dilute to the mark, which is equivalent to a 0.0001mol/L solution. Use a 1cm quartz cup and use sulfuric acid (0.5+1000) as a blank at the wavelength of the maximum absorption peak (near 350nm) to measure the absorbance of the above three molar solutions with different concentrations, and calculate the average value of the above three concentrations of the Myers extinction coefficient according to formula (1).
GB/T5009.22—2003
Wherein:
E, molar extinction coefficient of potassium dichromate solution: A
Measured absorbance of potassium dichromate solution:
Molar concentration of potassium dichromate solution
（1）
Then compare this average value with the extinction coefficient value of potassium dichromate 3160, that is, find the correction factor of the instrument, and calculate according to formula (2).
Wherein:
了——correction factor of the instrument;
f-3160
E—the average value of the molar extinction coefficient of potassium dichromate measured. If f is greater than 0.95 or less than 1.05, the correction factor of the instrument can be ignored. -（2）
3.14.2 Preparation of aflatoxin B standard solution: Accurately weigh 1mg~1.2mg of aflatoxin B standard, first add 2mL acetonitrile to dissolve, then dilute to 100mL with benzene, protect from light, and store in a refrigerator at 4℃. The standard solution is about 10μg/mL. Use an ultraviolet spectrophotometer to measure the wavelength of the maximum absorption peak of this standard solution and the absorbance value at this wavelength. Calculation of results: The concentration of aflatoxin B standard solution is calculated according to formula (3). X=AXMX1000Xf
Where:
Concentration of aflatoxin B, standard solution, in micrograms per milliliter (μg/mL); A—measured absorbance value;
at+→correction factor of the instrument used;
M—molecular weight of aflatoxin B, 312; E,—molar extinction coefficient of aflatoxin B, in benzene-acetonitrile mixture, 19800. .(3)
According to the calculation, the concentration of the standard solution is adjusted to 10.0μg/mL with benzene-acetonitrile mixture, and its concentration is checked with a spectrophotometer. 3.14.3 Purity determination: Take 5μL of 10μg/mL aflatoxin B standard solution and drop it on a silica gel G thin layer plate with a coating thickness of 0.25mm. Use methanol-chloroform (4+96) and acetone-chloroform (8+92) as developing agents to develop. Observe the generation of fluorescence under ultraviolet light. The following conditions should be met:
3.14.3.1 After development, there is only a single fluorescent point, and no other impurity fluorescent points. 3.14.3.2 There is no residual fluorescent substance at the origin. 3.15 Aflatoxin B, standard working solution: Accurately pipette 1mL of standard solution (10μg/mL) into a 10mL volumetric flask, add benzene-acetonitrile mixture to the scale, and mix well. Each milliliter of this solution is equivalent to 1.0μg of aflatoxin Bl. Take 1.0mL of this dilution, place it in a 5mL volumetric flask, add benzene-acetonitrile mixture to dilute to scale, this solution is equivalent to 0.2μg aflatoxin B1 per milliliter. Then take 1.0mL of aflatoxin B standard solution (0.2g/mL) and place it in a 5mL volumetric flask, add benzene-acetonitrile mixture to dilute to scale. This solution is equivalent to 0.04μg aflatoxin B per milliliter.
3.16 Sodium hypochlorite solution (for disinfection): Take 100g of bleaching powder, add 500mL of water, and stir evenly. Dissolve 80g of industrial sodium carbonate (NaCO·10HO) in 500mL of warm water, mix and stir the two liquids, clarify and filter. The filtrate contains hypochlorous acid at a concentration of about 25g/L. If prepared with bleaching powder, the amount of sodium carbonate can be doubled. The concentration of the resulting solution is about 50g/L. Contaminated glassware can be detoxified by soaking it in 10g/L sodium hypochlorite solution for half a day or in 50g/L sodium hypochlorite solution for a while. 4 Instruments
4.1 Small crusher.
4.2 Sample sieve.
4.3 Electric oscillator.
4.4 All-glass concentrator.
Glass plate: 5cm×20cm.
Thin layer plate applicator.
Development tank: inner length 25cm width 6cm, height 4cm. Ultraviolet lamp: 100W~125W, with a wavelength 365nm filter. 4.8
4.9 Micro syringe or hemoglobin pipette.
5 Analysis steps
5.1 Sampling
GB/T5009.22—2003
One mold particle with high aflatoxin contamination in the sample can influence the test result, and the proportion of toxic mold particles is small and unevenly distributed. In order to avoid the error caused by sampling, a large number of samples should be taken, and the large number of samples should be crushed and mixed evenly, so that it is possible to obtain relatively reliable results that can truly represent a batch of samples. Therefore, the following points should be noted in sampling. 5.1.1 Take representative samples according to regulations. 5.1.2 When testing samples with local deterioration, separate samples should be taken. 5.1.3 Each sample for analysis and determination should be reduced from a large sample to 0.5kg~1kg by coarse crushing and continuous quartering method for multiple times, and then all crushed. All grain samples should be passed through a 20-mesh sieve and mixed. All peanut samples should be passed through a 10-mesh sieve and mixed. Or the good and bad samples can be measured separately, and then their content can be calculated. Peanut oil and peanut butter samples do not need to be prepared, but they should be mixed evenly when sampling. If necessary, three large samples can be taken from each batch of samples for sample preparation and analysis to observe whether the samples are representative. 5.2 Extraction
5.2.1 Corn, rice, wheat, flour, dried potatoes, beans, peanuts, peanut butter, etc. 5.2.1.1 Method A: Weigh 20.00g of crushed and sieved sample (flour and peanut butter do not need to be crushed), place in a 250mL stoppered conical flask, add 30mL of n-hexane or petroleum ether and 100mL of methanol aqueous solution, apply a layer of water on the stopper, and cover tightly to prevent leakage. Oscillate for 30 minutes, let it stand for a while, filter it into a separatory funnel with a folded rapid qualitative filter paper, and release the methanol aqueous solution into another stoppered conical flask after the lower layer of methanol aqueous solution is clear. Take 20.00mL methanol aqueous solution (equivalent to 4g sample) and place it in another 125mL separatory funnel, add 20mL chloroform, shake for 2min, let it stand and separate. If emulsification occurs, methanol can be added to promote separation. Release the chloroform layer, filter it into 50mL evaporating blood through a quantitative slow filter paper containing about 10g of anhydrous sodium sulfate moistened with chloroform in advance, add 5mL chloroform to the separatory funnel, shake and extract repeatedly, filter the chloroform layer into the evaporating dish, and finally wash the filter with a small amount of chloroform, and add the washing liquid into the evaporating dish. Place the evaporating III in a fume hood on a 65℃ water bath for ventilation and drying, then place it on an ice box to cool for 2min~3min, and accurately add 1mL of benzene-acetonitrile mixture (or evaporate the chloroform with a concentrated distiller under reduced pressure and blow to dry, and accurately add 1mL of benzene-acetonitrile mixture). Use the tip of a dropper with a rubber tip to mix the residue thoroughly. If benzene crystals are precipitated, remove the evaporating dish from the ice box, continue to dissolve and mix, and the crystals will disappear. Use the dropper to absorb the supernatant and transfer it to a 2mL stoppered test tube. 5.2.1.2 Method B (limited to corn, rice, wheat and their products): Weigh 20.00g of the crushed and sieved sample into a 250mL stoppered conical flask, add about 6mL of water with a dropper to moisten the sample, accurately add 60mL of trizoform, shake for 30min, add 12g of anhydrous sodium sulfate, shake, let stand for 30min, and filter with folded rapid qualitative filter paper into a 100mL stoppered conical flask. Take 12mL of filtrate (equivalent to 4g of sample) in an evaporating dish, evaporate it in a 65℃ water bath, add 1mL of benzene-acetonitrile mixture accurately, and operate according to 5.2.1.1 starting from "Use the tip of a dropper with a rubber tip to mix the residue thoroughly..." 5.2.2 Peanut oil, sesame oil, rapeseed oil, etc.
Weigh 4.00g of sample and place it in a small beaker. Use 20mL of n-hexane or petroleum ether to transfer the sample to a 125mL separatory funnel. Wash the beaker with 20mL methanol-water solution in batches, transfer the washing liquid to the separatory funnel, shake for 2 minutes, let stand and separate, transfer the lower methanol-water solution to the second separatory funnel, repeat the shaking extraction with 5mL methanol-water solution, transfer the extract to the second separatory funnel, add 20mL chloroform to the second separatory funnel, and shake for 2 minutes according to 5.2.1.1. “…” and operate according to 181
GB/T5009.22—2003
.
|5.2.3 Soy sauce, vinegar
Weigh 10.00g of the sample in a small beaker, add 0.4g of sodium chloride to prevent emulsification during extraction, transfer to the separatory funnel, and use 15mL of chloroform. Wash the beaker several times and add the washing liquid into the separatory funnel. The following procedures are carried out according to 5.2.1.1 starting from "shake for 2 minutes, let stand and separate layers." Finally, add 2.5mL of benzene-acetonitrile mixture. Each milliliter of this solution is equivalent to 4g of sample. Or weigh 10.00g of sample, place it in a separatory funnel, add 12mL of methanol (replace water with the volume of soy sauce, so the volume ratio of methanol to water is still about 55+45), extract with 20mL of trichloromethane, and the following procedures are carried out according to 5.2.1.1 starting from "shake for 2 minutes, let stand and separate layers..." Finally, add 2.5mL of benzene-acetonitrile mixture. Each milliliter of this solution is equivalent to 4g of sample. 5.2.4 For dry sauces (including fermented black beans and fermented bean curd products), weigh 20.00g of evenly ground sample and place it at 25 0mL stoppered conical flask, add 20mL of n-hexane or petroleum ether and 50mL of methanol-water solution. Shake for 30min, let stand for a while, filter with folded rapid qualitative filter paper, let the filtrate stand for stratification, take 24mL of methanol-water layer (equivalent to 8g of sample, including the volume of 8g of dry sauce itself containing about 4mL of water) and put it in a separatory funnel, add 20mL of chloroform, and operate according to 5.2.1.1 from "shake for 2min, let stand for stratification...". Finally, add 2mL of benzene-acetonitrile mixture. Each milliliter of this solution is equivalent to 4g of sample. 5.2.5 Fermented wine
Same as 5.2.3 treatment method, but without adding sodium chloride. 5.3 Determination
5.3.1 One-way development method||tt| |5.3.1.1 Preparation of thin layer plates: Weigh about 3g of silica gel G, add water equivalent to about 2 to 3 times the amount of silica gel, grind vigorously for 1min to 2min until it becomes a paste, then pour it into the applicator and roll it into three thin layer plates of 5cm×20cm and a thickness of about 0.25mm. After drying in the air for about 15min, activate it at 100℃ for 2h, take it out and store it in a desiccator. It can generally be stored for 2 to 3 days. If it is stored for a long time, it can be used after activation.
5.3.1.2 Spotting: Scrape off the adsorbent attached to the edge of the thin layer plate, and use a microsyringe or hemoglobin pipette to drop the sample solution on the baseline 3cm away from the lower end of the thin layer plate. Four points can be dropped on one plate, with a distance of about 1cm from the edge and the distance between the points, and a diameter of about 3mm. The size of the drop points on the same plate should be consistent. When dropping, a hair dryer can be used to blow cold air while adding. The dripping pattern is as follows: First point: 10μL aflatoxin B standard solution (0.04μg/mL). Second point: 20μL sample solution.
Third point: 20μL sample solution + 10μL 0.04μg/mL aflatoxin B: standard solution. Fourth point: 20μL sample solution + 10μL 0.2μg/mL aflatoxin B standard solution. 5.3.1.3 Development and observation: Add 10mL of anhydrous ether to the development tank, pre-develop 12cm, take out and evaporate. Then add 10mL of acetone-trichloromethane (8+92) to another development tank, develop 10cm~12cm, and take out. Observe the results under ultraviolet light as follows. 5.3.1.3.1 Due to the addition of aflatoxin B standard solution on the sample solution point, the aflatoxin B standard point can overlap with the aflatoxin B fluorescent point in the sample solution. If the sample solution is negative, the aflatoxin B in the third point on the thin layer plate is 0.0004μg, which can be used to check whether the minimum detection amount of aflatoxin B in the sample solution appears normally; if it is positive, it plays a qualitative role. The aflatoxin B in the fourth point on the thin layer plate is 0.002μg, which mainly plays a positioning role. 5.3.1.3.2 If there is no blue-purple fluorescent spot at the second point corresponding to the aflatoxin purple B standard point, it means that the content of aflatoxin B in the sample is below 5μg/kg; if there is a blue-purple fluorescent spot at the corresponding position, a confirmation test is required. 5.3.1.4 Confirmation test: In order to confirm that the fluorescence of the sample solution on the thin layer plate is produced by aflatoxin B, add trifluoroacetic acid to produce a derivative of aflatoxin B. After development, the relative shift value of this derivative is about 0.1. Add two points in sequence on the left side of the thin layer plate. First point: 0.04μg/mL aflatoxin B, 10μL standard solution. Second point: 20μL sample solution.
Add a small drop of trifluoroacetic acid to each of the above two points and cover them. After reacting for 5 minutes, use a hair dryer to blow hot air for 2 minutes. Make sure that the temperature of the hot air on the thin layer plate is no higher than 40℃. Then add the following two points to the thin layer plate. 182
Third point: 0.04μg/mL aflatoxin B standard solution 10μL Fourth point: 20μL sample solution.
GB/T5009.22-2003
Expand again (same as 5.3.1.3) Observe the sample solution under ultraviolet light to see if it produces the same derivative as the standard point of aflatoxin B. Points 3 and 4 without trifluoroacetic acid can be used as blank controls for sample solution and standard derivatives. 5.3.1.5 Dilution quantification: If the fluorescence intensity of the aflatoxin B fluorescence point in the sample solution is consistent with the fluorescence intensity of the minimum detection amount of the aflatoxin B standard point (0.0004μg), the content of aflatoxin B in the sample is 5μg/kg. If the fluorescence intensity in the sample solution is stronger than the minimum detection amount, reduce the number of microliters added according to its intensity estimate or dilute the sample solution and then add different microliters until the fluorescence intensity of the sample solution point is consistent with the fluorescence intensity of the minimum detection amount. The drop pattern is as follows: First point: 10μL aflatoxin B, standard solution (0.04μg/mL). Second point: add 10μL sample solution according to the situation. Third point: add 15μL sample solution according to the situation. Fourth point: Add 20μL sample solution according to the situation. 5.3.1.6 Result calculation: The content of aflatoxin B in the sample is calculated according to formula (4). V.XD1000
X=0.000 4×
Wherein:
X—the content of aflatoxin B in the sample, in micrograms per kilogram (μg/kg); V,—the volume of the added benzene-acetonitrile mixture, in milliliters (mL); V,—the volume of the sample solution added when the minimum fluorescence appears, in milliliters (mL); the total dilution multiple of the same solution;
m-the mass of the equivalent sample when the benzene-acetonitrile mixture is dissolved, in grams (g); 0.0004-the minimum detection amount of aflatoxin B, in micrograms (μg). The result is expressed to the integer of the measured value.
(4)
5.3.2 Bidirectional development method
If the fluorescence intensity of aflatoxin B1 is obscured by the interference of impurities after the thin layer chromatogram is developed by the unidirectional development method, the bidirectional development method shall be adopted. The thin layer plate is first developed horizontally with anhydrous ether to spread the interfering impurities to one side of the sample solution spot while aflatoxin B1 remains unchanged. Then, acetone-chloroform (8+92) is used for longitudinal development. The impurity background color of the sample at the corresponding position of aflatoxin B1 is greatly reduced, thereby improving the sensitivity of the method. If there is still impurity interference when developing by the two-point drop method in the bidirectional development, the one-point drop method can be used instead. 5.3.2.1 Two-point drop method
5.3.2.1.1 Spot sample
Take three thin layer plates and drop aflatoxin B1 standard working solution and sample solution on the baseline 3 cm from the lower end. That is, add 10uL of aflatoxin B standard solution (0.04μg/mL) at 0.8cm~1cm from the left edge of each of the three plates, add 20uL of sample solution at 2.8cm~3cm from the left edge, then add 10μL of aflatoxin B standard solution (0.04μg/mL) to the sample solution point of the second plate, and add 10μL of 0.2μg/mL aflatoxin B standard solution to the sample solution point of the third plate. 5.3.2.1.2 Development
5.3.2.1.2.1 Horizontal development: Place a glass stand on the long side of the development tank, add 10mL of anhydrous ethanol, place the long side of the thin layer plate near the standard point in the development tank for development, and after developing to the end of the plate, take it out and evaporate it, or repeat the development 1~2 times as needed.
5.3.2.1.2.2 Longitudinal development: The evaporated thin layer plate is developed with acetone-chloroform (8+92) to 10cm~12cm. The ratio of acetone to chloroform is adjusted according to different conditions. 5.3.2.1.3 Observation and evaluation results
5.3.2.1.3.1 Observe the first and second plates under ultraviolet light. If the second point of the second plate has the lowest detection amount at the corresponding position of the standard point of aflatoxin B, and no fluorescent spot appears at the same position of the first plate as the second plate, the content of aflatoxin B in the sample is below 5pg/kg.
5.3.2.1.3.2 If a fluorescent spot appears on the first plate at the same position as the second plate, compare the first plate with the third plate to see whether the fluorescent spot on the second point on the third plate at the same position as the second point on the first plate coincides with the aflatoxin B and standard points. If so, conduct a confirmation test. In specific determinations, the first, second, and third plates can be done at the same time or in sequence. If done in sequence, when a negative result appears on the first plate, the third plate can be omitted. If the first plate is positive, the second plate can be omitted and the third plate can be done directly. 5.3.2.1.4 Confirmation test
Take two more thin layer plates, and add 10μuL of aflatoxin B, standard solution (0.04μg/mL) and a small drop of trifluoroacetic acid to the fourth and fifth plates at 0.8cm1cm from the left edge respectively; add 20μL of sample solution and a small drop of trifluoroacetic acid to the fourth plate at 2.8cm~3cm from the left edge; add 20μL of sample solution, 10μL of aflatoxin B, standard solution (0.04μg/mL) and a small drop of trifluoroacetic acid to the fifth plate. After reacting for 5 minutes, use a hair dryer to blow hot air for 2 minutes, so that the temperature of the hot air blowing to the thin layer plate is not higher than 40℃. After developing with the two-way development method, observe whether the sample solution produces derivatives that overlap with the standard point of aflatoxin B. When observing, the first plate can be used as a blank plate for the derivatives of the sample solution. If the sample solution has a high content of aflatoxin B, dilute the sample solution and perform a confirmation test according to 5.3.1.4. 5.3.2.1.5 Dilution Quantification
If the sample solution has a high content of aflatoxin B, perform the dilution quantification operation according to 5.3.1.5. If the content of aflatoxin B is low and the dilution multiple is small, there will still be impurities interfering on the quantitative longitudinal development plate, which will affect the judgment of the results. The sample solution can be further determined by the two-way development method to determine the content.
5.3.2.1.6 Result calculation
Quotient 5.3.1.6.
5.3.2.2 One-point drop method
5.3.2.2.1 Spot sampling: Take three thin layer plates and drop aflatoxin purple B standard working solution and sample solution on the baseline 3 cm from the lower end. That is, add 20μL of sample solution at 0.8cm to 1cm from the left edge of each of the three plates, add 10μL of aflatoxin B standard solution (0.04μg/mL) to the point on the second plate, and add 10μL of aflatoxin B standard solution (0.2μg/mL) to the point on the third plate. 5.3.2.2.2 Development: Same as the horizontal and vertical development in 5.3.2.1.2. 5.3.2.2.3 Observation and evaluation results: Observe the first and second plates under ultraviolet light. If the second plate has the lowest detection amount of aflatoxin B standard point, and the first plate has no fluorescent point at the same position, the aflatoxin B content in the sample is below 5μg/kg. If a fluorescent spot appears on the first plate at the same position as aflatoxin B on the second plate, compare the first plate with the third plate to see whether the fluorescent spot on the third plate at the same position as the first plate overlaps with the aflatoxin B standard spot. If it overlaps, perform the following confirmation test 5.3.2.2.4 confirmation test. Take two more plates and add 20μL sample solution and 1 drop of trifluoroacetic acid to the fourth plate at 0.8cm to 1cm from the left edge; add 20μL sample solution, 10μL 0.04μg/mL aflatoxin B standard solution and 1 drop of trifluoroacetic acid to the fifth plate. The method of generating derivatives and developing is the same as 5.3.2.1. Observe the above two plates under ultraviolet light to determine whether the sample solution spot produces derivatives that overlap with the aflatoxin B standard spot. The first plate can be used as a blank plate for the sample solution derivatives during observation. After the above confirmation test is determined to be positive, dilution and quantification are performed. If the aflatoxin B content is low, no dilution is required or the dilution multiple is small. If there is still serious interference from the impurity fluorescence, the two-way expansion method can be used directly for quantification according to the intensity of the aflatoxin B fluorescence in the sample solution. 5.3.2.2.5 Result calculation: Same as 5.3.1.6. Second method
6 Principle
The aflatoxin B in the sample is extracted, defatted, and concentrated, and then reacted with the quantitative specific antibody. The excess free antibody binds to the coated antigen in the ELISA plate, and the color is developed after adding the enzyme marker and substrate, and the content is determined by comparing with the standard. 7 Reagents
7.1 Anti-aflatoxin B, monoclonal antibody, quality control is carried out by the Food Hygiene Supervision and Inspection Institute of the Ministry of Health. 184
7.2 Artificial antigen: AFB-bovine serum albumin conjugate. GB/T5009.22--2003
7.3 Aflatoxin B: Standard solution: Prepare a 1 mg/mL solution of aflatoxin B with methanol, and then dilute it to about 10 μg/mL with methanol-PBS solution (20+80). Measure the optical density value of the maximum absorption peak of this solution with a UV spectrophotometer and substitute it into formula (5) to calculate: X=AxMX1000×f
Wherein:
-the concentration of aflatoxin B in the solution, in micrograms per milliliter (uμg/mL); A—measured optical density value;
-the molecular weight of aflatoxin B, 312; M-
-the molar extinction coefficient, 21800,
f is the correction factor of the instrument.
(5)
According to the calculation, the solution is prepared into a 10 μg/mL standard solution. When testing, dilute the standard solution to the required concentration with methanol-PBS solution.
7.4Chloroform.
7.5Methanol.
7.6Petroleum ether.
7.7Bovine serum albumin (BSA).
O-phenylenediamine (OPD).
Horseradish peroxidase (HRP) labeled goat anti-mouse IgG. 7.9
Sodium carbonate.
Sodium bicarbonate.
Potassium dihydrogen phosphate.
Sodium hydrogen phosphate.
Sodium chloride.
Potassium chloride.
Hydrogen peroxide (H,O).
Sulfuric acid.
ELISA buffer is as follows:
Preparation of coating buffer (pH 9.6 carbonate buffer): Na.cOsWww.bzxZ.net
Add distilled water to 1000mL.
7.18.2 Preparation of phosphate buffer (pH 7.4 PBS): KH,PO.
Na,HPO,·12H,O
Add distilled water to 1000mL.
7.18.3 Preparation of washing solution (PBS-T): PBS plus 0.05% Tween-20. Preparation of antibody diluent: BSA 1.0g plus PBS-T to 1000mL. 7.18.4
7.18.5 Preparation of substrate buffer is as follows: 7.18.5.1A solution (0.1mol/L citric acid aqueous solution): citric acid (CHO,.H,O) 21.01g, add distilled water to 1000mL. 7.18.5.2 Solution B (0.2 mol/L sodium dihydrogen phosphate aqueous solution): Na2HPO4·12Hz071.6g, add distilled water to 1000mL. 185
GB/T5009.22—2003
7.18.5.3 Prepare before use according to the ratio of solution A + solution B + distilled water of 24.3 + 25.7 + 50 (volume ratio). 7.18.6 Preparation of blocking solution: same as antibody diluent, 8 Instruments
8.1 Small pulverizer.
8.2 Electric oscillator.
8.3 Microplate reader with built-in 490nm filter. 8.4 Constant temperature water bath.
8.5 Constant temperature incubator.
8.6 Microplate for enzyme labeling.
8.7 Micropipette and matching pipette tip.
9 Analysis steps
9.1 Sampling
Same as 5.1.
9.2 Extraction
9.2.1 Extraction of rice and millet (fat content <3.0%)) After the sample is crushed and passed through a 20-mesh sieve, weigh 20.0g and add it to a 250mL stoppered conical flask. Accurately add 60mL of trifluoromethane, cover the stopper and seal it tightly with water. Oscillate at 150r/min for 30min. After standing, filter it into a 50mL beaker with a rapid qualitative filter paper. Immediately take 12mL of the filtrate (equivalent to 4.0g of sample) in a 75mL evaporator and evaporate it to dryness in a 65℃ water bath. Dissolve and thoroughly rinse the coagulated material in the evaporated blood with 2.0mL 20% methanol-PBS three times (0.8mL, 0.7mL, 0.5mL), transfer it to a small test tube, cover it, shake it, and let it stand for testing. Each milliliter of this solution is equivalent to 2.0g of sample.
9.2.2 Extraction of corn (fat content 3.0%~5.0%) After the sample is crushed, pass it through a 20-mesh sieve, weigh 20.0g, add it to a 250mL stoppered conical flask, accurately add 50.0mL methanol-water (80+20) solution and 15.0mL petroleum ether, cover the stopper and seal it tightly with water. Oscillate at 150r/min for 30min. Filter it into a 125mL separatory funnel with a quick qualitative filter paper. After stratification, release the lower layer of methanol-water solution into a 50mL beaker, and take 10.0mL (equivalent to 4.0g sample) from it into 75mL evaporating blood. The following operations are carried out according to 9.2.1 starting from "65℃ water bath ventilation evaporate to *". 9.2.3 Extraction of peanuts (fat content 15.0% to 45.0%) After removing the shell and skin of the sample and crushing it, weigh 20.0g and add it to a 250mL stoppered conical flask. Accurately add 100.0mL methanol-water (55+45) solution and 30mL petroleum ether, cover the stopper and seal it tightly with water. Oscillate at 150I/min for 30min. After standing for 15min, filter it with a fast qualitative filter paper into a 125mL separatory funnel. After stratification, release the lower layer of methanol-water solution into a 100mL beaker, take 20.0mL (equivalent to 4.0g sample) from it and place it in another 125mL separatory funnel, add 20.0mL trichloromethane, shake for 2min, let it stand for stratification (if emulsification occurs, add methanol to promote stratification), and release trichloromethane into a 75mL evaporating dish. Add 5.0mL of chloroform to the separatory funnel and shake repeatedly to extract, release the chloroform and put it in evaporation III, and operate according to 9.2.1 starting from "65℃ water bath ventilation and dry...".
9.2.4 Extraction of vegetable oil
Use a small beaker to weigh 4.0g of the sample, use 20.0mL of petroleum ether, transfer the sample to a 125mL separatory funnel, wash the beaker with 20.0mL of methanol-water (55+45) solution several times, and transfer the solution to the separatory funnel (4.0g of refined oil sample is 4.525mL, directly add it to the separatory funnel with a pipette, and then add the solvent and shake), and shake for 2min. After standing for stratification, release the lower layer of methanol-water solution and put it into 75mL evaporator III, and then use 5.0mL methanol-water solution to shake and extract again. Add the extract to the evaporated blood, and follow the steps in 9.2.1 from "evaporate to dryness in a 65℃ water bath".
1) For fat content, refer to "Food Composition Table", compiled by the Institute of Nutrition and Food Hygiene, Chinese Academy of Preventive Medicine, August 1991, first edition. 186
9.2.5 Extraction of other foods
GB/T 5009.22—2003
You can follow the steps in 5.2.1.1, 5.2.3, and 5.2.4 of the first method until "evaporate to dryness in a 65℃ water bath in a ventilated cabinet", and follow the steps in 9.2.1 from "use 2.0mL 20% methanol-PBS three times..." and follow the steps in 9.2.1. 9.3 Indirect competitive enzyme-linked immunosorbent assay (ELISA) 9.3.1 Coating microplate: Coat the ELISA plate with AFB,-BSA artificial antigen, 150μL/well, overnight at 4℃. 9.3.2 Antibody-antigen reaction: Dilute the purified monoclonal antibody of aflatoxin B and a) mix and shake with the same amount of aflatoxin B standard solution of different concentrations in a 2mL test tube, and then let it stand at 4℃. This solution is used to prepare the standard inhibition curve of aflatoxin B. b) Mix and shake with the same amount of sample extract in a 2mL test tube, and then let it stand at 4℃. This solution is used to determine the content of aflatoxin B in the sample.
9.3.3 Blocking: Wash the coated ELISA plate with washing solution 3 times, 3 minutes each time, and then add blocking solution, 250μL/well, and place at 37℃ for 1h.
9.3.4 Determination: After washing the ELISA plate for 3×3min, add antibody-antigen reaction solution (add antibody diluent or Sp2/0 culture supernatant as negative control to the appropriate wells of the ELISA plate) 130μL/well, 37℃, 2h. Wash the ELISA plate for 3×3min, add enzyme-labeled secondary antibody [1:200 (volume fraction) J100μL/well, 1h. Wash the ELISA plate with washing solution for 5×3min. Add substrate solution (10mgOPD) plus 25mL substrate buffer plus 37μL30%H, O2, 100μL/well, 37℃, 15min, then add 2mol/LHzSO, 40μL/well to terminate the color reaction, and measure the OD value at 490nm with an ELISA reader.
9.4 Calculation of results
The concentration of aflatoxin B: is calculated according to formula (6). x
Concentration of aflatoxin B (ng/g) = c×
Wherein:
-aflatoxin B, content, in nanograms (ng), the corresponding standard curve is calculated by numerical interpolation; V——the volume of the sample extract, in milliliters (mL); V,—the volume of the added sample solution, in milliliters (mL); a dilution factor,
the mass of the sample, in grams (g).
（6）
Since the unit of aflatoxin B concentration (ct) directly obtained according to the standard curve is nanograms per milliliter, and the volume of sample extraction added to the measuring well is 0.065 mL, so in formula (6), c=0.065mLXcl
and V=2mL, Vz=0.065mL, D=2, m=4g Substituting into formula (6), then 0.065×2x
ci(ng/g）
Aflatoxin B, concentration (ng/g) = 0.065×c1× Therefore, when the sample extraction is carried out completely according to this method, the value c1 directly obtained from the standard curve is the concentration of aflatoxin B in the test sample (ng/g).
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