Some standard content:
National Standard of the People's Republic of China
The method of determination ofaflatoxin B, in feedstuff
This standard adopts the international standard ISO6651-1983 (E) "Method of determination of aflatoxin B in feedstuff". 1 Scope of application
This standard is applicable to various single feeds and compound feeds. 2 Principle
UDC636.085
：543.062
GB 838187
After extraction, column chromatography, elution, concentration and thin layer separation, aflatoxin B in the sample produces blue-violet fluorescence under a wavelength of 365nm ultraviolet light. The content is determined based on the minimum detection amount of fluorescence displayed on the thin layer. 3 Reagents
3.1 Chloroform (GB682-78).
3.2 Hexane (HG31003—76).
3.3 Methanol (GB 683- 79).
3. 4 Benzene (GB 690—77). wwW.bzxz.Net
3. 5 Ethyl ether (HG B 3329--- 60). 3.6 Anhydrous ether or ether dehydrated with anhydrous sodium sulfate (HGB1002—76). 3.7 Acetone (GB 686—78)
The above reagents should be tested once for reagent blank before testing. If they do not interfere with the determination, they can be used. Otherwise, they need to be checked one by one and re-distilled. Benzene-acetonitrile mixture: Measure 98mL benzene and add 2mL acetonitrile to mix. 3.8
3.9 Trifluoromethane-methanol mixture: Take 97mL chloroform and 3mL methanol and mix. 3.10 Silica gel: 80~～200 mesh for column chromatography. 3.11
Silica gel G: For thin layer chromatography.
Trifluoroacetic acid.
Anhydrous sodium sulfate (HG3-123-76).
3.14 Diatomaceous earth.
3.15 Aflatoxin B standard solution
3.15.1 Instrument calibration: Determine the molar extinction coefficient of potassium dichromate solution to find the calibration factor for the instrument: Accurately weigh 25 mg of dried potassium dichromate (reference grade). Dissolve it in 0.009 mol/L sulfuric acid and accurately dilute to 200 mL (equivalent to 0.0004 mol/L solution). Pipette 25 mL of this dilution into a 50 mL volumetric flask and dilute to the mark with 0.009 mol/L sulfuric acid (equivalent to 0.0002 mol/L solution). Then take 25mL of this dilution into a 50mL volumetric flask, add 0.009mol/L sulfuric acid to dilute to the scale (equivalent to 0.0001mol/L solution). Use a 1cm quartz cup, use 0.009mol/L sulfuric acid as a blank at the wavelength of the maximum absorption peak (close to 350nm), and measure the absorbance of the above three different concentrations of molar solutions. And calculate the average of the molar extinction coefficients of the above three concentrations according to the following formula. Approved by the Ministry of Agriculture, Animal Husbandry and Fisheries of the People's Republic of China on November 20, 1987 and implemented on December 1, 1988
GB 8381-87
Where: E,—molar extinction coefficient of potassium dichromate solution; A—measured absorbance of potassium dichromate solution; Net-
—molar concentration of potassium dichromate solution.
Then compare this average with the molar extinction coefficient of potassium dichromate 3160, and calculate the correction factor of the instrument as follows. f=3160
Where:}…The correction factor of the instrument; —-The average value of the molar extinction coefficient of potassium dichromate is measured. M-
（2）
If it is greater than 0.95 or less than 1.05, the correction factor of the instrument can be ignored. 3.15.2 Preparation of 10ug/mL aflatoxin B standard solution: Accurately weigh 1mg~1.2mg of aflatoxin B standard, first add 2ml of acetonitrile to dissolve, then dilute to 100mL with benzene, and store in a refrigerator at 4℃. Use a UV spectrophotometer to measure the wavelength of the maximum absorption peak of this standard solution and the absorbance value at this wavelength, and calculate the concentration of the standard solution as follows.
X, = 4XMX1000Xf
Where: X. Concentration of aflatoxin B, standard solution, ug/mL; A-measured absorbance value;
M-molecular weight of aflatoxin B, 312;
E,-molar extinction coefficient of aflatoxin B, in benzene-acetonitrile mixture, 19800. According to calculation, the concentration of the standard solution is adjusted to 10μg/mL with benzene-acetonitrile mixture, and its concentration is checked with a spectrophotometer. · (3)
3.15.3 Determination of purity: 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, and observe the generation of fluorescence under ultraviolet light. The following conditions must be met:
After development, there is only a single fluorescent point, and no other impurity fluorescent points. a.
b. There is no residual fluorescent substance at the origin. 3.16 Aflatoxin B. Standard working solution: Accurately pipette 1mL of 10uμg/mL standard solution into a 10mL volumetric flask, add benzene-acetonitrile mixture to the scale, and mix well. This solution is equivalent to 1μg of aflatoxin B per milliliter. Pipet 1.0mL of this diluted solution into a 5mL volumetric flask, add benzene-acetonitrile mixture to dilute to the scale, and this solution is equivalent to 0.2ug of aflatoxin B per milliliter. Pipet another 1.0mL of this solution into a 5mL volumetric flask, and add benzene-acetonitrile mixture to dilute to the scale. Each milliliter of this solution is equivalent to 0.04μg of aflatoxin B. 3.17 Sodium hypochlorite solution (for disinfection): Take 100g of bleaching powder, add 500mL of water, and stir evenly. Separately, dissolve 80% of industrial sodium carbonate (Na,CO·10H,O) in 500mL of warm water, mix the two liquids, stir, clarify and filter. The filtrate contains about 2.5% sodium hypofluorite. If bleaching powder is used, the amount of sodium carbonate can be doubled. The concentration of the resulting solution is about 5%. The contaminated glassware can be detoxified by soaking it in 1% sodium hypochlorite solution for half a day or in 5% sodium hypofluorite solution for a while. 4 Instruments
4.1 Small pulverizer.
4.2 A set of sample sieves.
4.3 Electric oscillator.
4.4 The inner diameter of the chromatography tube is 22mm, the length is 300mm, with a piston at the bottom and a liquid reservoir at the top. 4.5 Glass plate: 5×20cm.
4.6 Thin layer plate applicator.
GB 8381- 87
4.7 Development tank: inner length 25cm, width 6cm, height 4cm. 4.8 Ultraviolet light: wavelength 365nm.
4.9 Balance.
4.10 Stoppered graduated test tube 10.0mL, 2.0mL. 4.11 Rotary evaporator or evaporating blood.
4.12 Micro syringe or hemoglobin pipette. 5 Operation method
5.1 Sampling
The high level of aflatoxin contamination in the sample can affect the determination result. Moreover, the proportion of toxic mold particles is small and the distribution is uneven. In order to avoid the error caused by sampling, a large amount of sampling must be taken and the large amount of crushed samples must be mixed evenly to obtain a relatively reliable result that can truly represent the batch sample. Therefore, sampling must be done with care. 5.1.1 Take representative samples according to regulations. 5.1.2 When testing samples that are partially moldy and deteriorated, separate samples should be taken for testing. 5.1.3 Each sample for analysis and determination should be reduced to 0.5-1kg by coarse crushing and continuous quartering multiple times, and all crushed. All samples should be passed through a 20-mesh sieve and mixed. Stir evenly when sampling. If necessary, three large samples can be taken from each batch of samples for sample preparation and analysis and determination. To observe whether the sample taken is representative to a certain extent. 5.2 Sample preparation
If the fat content of the sample exceeds 5%, it should be defatted before crushing. If defatted, the analysis result is based on the non-defatted sample. 5.3 Extraction
Take 20g of the prepared sample, place it in a ground-mouth conical flask, add 10g of diatomaceous earth (3.14), 10mL of water, 100mL of chloroform (3.1), add a stopper, shake on an oscillator for 30min, filter with filter paper, and the filtrate should be at least 50mL. 5.4 Column chromatography purification
5.4.1 Preparation of the column
Add about 2/3 of fluoroform (3.1) to the column, add 5g of anhydrous sodium sulfate (3.13), make the surface flat, add 10g of column chromatography silica gel (3.10) in a small amount, carefully eliminate bubbles, let it stand for 15min, then slowly add 10g of anhydrous sodium sulfate, open the piston, let the liquid flow down until the liquid reaches the upper surface of the sodium sulfate layer, and close the piston.
5.4.2 Purification
Take 50mL of the filtrate with a pipette, put it into a beaker, add 100mL of n-hexane (3.2), mix well, transfer the mixed solution quantitatively to the chromatography column, wash the beaker with n-hexane and pour it into the column. Open the piston, let the liquid flow down at 8-12mL/min until it reaches the upper surface of the sodium sulfate layer, then pour 100mL of ether (3.6) into the column, let the liquid flow to the upper surface of the sodium sulfate layer again, and discard the collected liquid. The whole process ensures that the column is not.
Elute the column with 150mL of chloroform-methanol solution (3.9), and collect all the eluent in a rotary evaporator flask. Distill under reduced pressure below 50℃, and quantitatively transfer the residue to the scale test butt with a benzene-acetonitrile mixture (3.8), and evaporate it in a water bath airflow below 50C until the liquid volume reaches 2.0mL. The eluent can also be evaporated to dryness in a water bath below 50°C in evaporation III, and then transferred to a stoppered graduated test tube with benzene-acetonitrile.
If a small-diameter chromatography tube is used for chromatography, all reagents are reduced in proportion to the square of the inner diameter of the chromatography tube. 5.5 Determination by unidirectional development method
5.5.1 Preparation of thin layer plates: Weigh about 3g of silica gel G (3.11), add water equivalent to about two to three times the amount of silica gel, grind vigorously for 1 to 2 minutes until it becomes a paste, then immediately pour it into the applicator and push it into three thin layer plates of 5×20cm and a thickness of about 0.25mm. Dry in air for about 15 minutes, activate at 100°C for 2 hours, take out and dry, and store in a desiccator. It can generally be stored for two to three days. If it is stored for a long time, it can be reactivated before use.
5.5.2 Sample spotting: Scrape off the adsorbent attached to the edge of the thin layer plate, and drop the sample solution on the baseline 3cm away from the bottom of the thin layer plate with a micro syringe or a blood color 101
GB 8381 -- 87
pipet. Four points can be added to 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, and a hair dryer can be used to blow cold air while adding. The drop pattern is as follows: First point: 10uL0.04μg/mL aflatoxin B standard solution; second point: 16uL sample solution;
Third point: 16uL sample solution + 10μL0.04ug/mL aflatoxin B, standard solution; fourth point: 16L sample solution + 10uL0.2μL/mL aflatoxin B, standard solution. 5.5.3 Development and observation: Add 10mL of anhydrous ether to the development tank for pre-development of 12cm, take out and evaporate, then add 10mL of acetone-chloroform (8:92) to another development tank, develop 10-12cm, take out, and observe the results under ultraviolet light. The method is as follows: a. Due to the addition of a drop of aflatoxin B standard working solution on the sample 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 0.0004μ, 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 positioning role. The aflatoxin B in the fourth point on the thin layer plate: 0.002ug, mainly plays a positioning role. b. If there is no blue-purple fluorescent spot at the second point corresponding to the aflatoxin B standard point, it means that the aflatoxin B content in the sample is below 5ug/kg; if there is a blue-purple fluorescent spot at the corresponding position, a confirmation test is required. 5.5.4 Confirmation test: In order to confirm that the fluorescence of the sample solution on the thin layer plate is produced by aflatoxin B, add a drop of trifluoroacetic acid to produce a derivative of aflatoxin B. After development, the relative shift value of this derivative is about 0.1. Method;
Add two points in sequence on the left side of the thin layer plate. First point: 16μL sample solution;
Second point: 10μL 0.04μg/mL aflatoxin B standard solution; Add 1 small drop of trifluoroacetic acid (3.12) to each of the above two points and cover the sample point. 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℃. Then drop the following two points on the thin layer plate; the third point: 16μL sample solution;
the fourth point: 10μL0.04μg/mL aflatoxin B standard solution. Expand as before. Observe the sample solution under ultraviolet light to see if it produces the same derivative as the aflatoxin B standard point. The third and fourth points without trifluoroacetic acid can be used as blank controls for the sample solution and the standard derivatives. 5.5.5 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 aflatoxin B standard point (0.0004μg), the content of aflatoxin B in the sample is 5ug/kg. If the fluorescence intensity in the sample solution is stronger than the minimum detection amount, reduce the number of microliters to be added according to its intensity estimate or dilute the sample solution and then drop different microliters until the fluorescence intensity of the sample solution point is consistent with the fluorescence intensity of the minimum detection amount. The dripping pattern is as follows: First point: 10μL 0.04μg/mL aflatoxin B. standard solution; 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. 5.5.6 Calculation and expression of results
X,= 0. 0004 ×X×1000
Where: X,---the content of aflatoxin B in the sample, ug/kg; V,--the volume of the benzene-acetonitrile mixture added, mL; V,--the volume of the sample solution added when the minimum fluorescence appears, mL; D--the total dilution factor of the sample solution;
m-the mass of the sample equivalent to the dissolution of the benzene-acetonitrile mixture, g; 0.0004---the minimum detection amount of aflatoxin B, ug. 5.6 Determination by double-question development method
GB 8381-87
If the fluorescence intensity of aflatoxin B is obscured by the interference of impurities after development by the single-direction development method, the double-direction development method shall be adopted. The thin layer plate is first developed horizontally with anhydrous ether (3.6), and the interfering impurities are developed to one side of the sample point, aflatoxin B, and then acetone-chloroform (8:92) is used for vertical development. The impurity background color of the sample at the corresponding part of aflatoxin B is greatly reduced. Therefore, the sensitivity of the method is improved. If the two-point drop method is used in the double-direction development method, if there is still interference from impurities during development, the one-point drop method can be used instead. 5.6.1 Two-point drop method
5.6.1.1 Spot sampling: Take three thin layer plates and drop aflatoxin B standard solution and sample solution on the baseline 3 cm away from the lower end. That is, add 10μL of 0.04μg/mL aflatoxin B, standard solution at 0.8-1cm from the left edge of each of the three plates, add 16μL of sample solution at 2.8-3cm from the left edge, and then add 10uL of 0.04μg/mL aflatoxin B standard solution to the sample solution point of the second plate. Add 10μL of 0.2ug/mL aflatoxin B, standard solution to the sample solution point of the third plate. 5.6.1.2 Development
a. Horizontal development: Place a glass stand on the long side of the development tank and add 10mL of anhydrous ether. Place the long side of the above-pointed thin layer plate close to the standard point in the development tank for development. After the plate is developed to the end, take it out and evaporate it, or repeat the development 1-2 times as needed. Longitudinal development: The evaporated thin layer plate is developed with acetone: chloroform (8:92) to 10-12cm. The ratio of acetone to chloroform b.
is adjusted according to different conditions. 5.6.1.3 Observation and evaluation results
Observe the first and second plates under ultraviolet light. If the second point of the second plate shows the minimum detection amount at the corresponding position of the aflatoxin B standard point, and no fluorescent spot appears at the same position of the first plate as the second plate, the aflatoxin B content in the sample is less than 5ug/kg. If a fluorescent spot appears at the same position as the second plate on the first plate, compare the second plate with the third plate to see whether the fluorescent spot at the same position as the second point on the third plate overlaps with the aflatoxin B standard point. If it overlaps, then conduct a confirmation test. In the specific determination, the first, second and third plates can be made at the same time or in sequence. If they are made 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 made directly. 5.6.1.4 Confirmation test: Take two other thin layer plates. On the fourth and fifth plates, 10 μL of 0.04ug/mL aflatoxin B standard solution and a small drop of trifluoroacetic acid (3.12) were added at 0.8-1cm from the edge; 16 μL of sample solution and a small drop of trifluoroacetic acid were added to the fourth plate at 2.8-3cm from the left edge. On the fifth plate, 16 μL of sample solution, 10 μuL of 0.04ug/mL aflatoxin B, standard solution and a small drop of trifluoroacetic acid were added. The steps for producing derivatives were the same as those of the unidirectional development method. After development by the bidirectional development method, observe whether the sample solution produces derivatives related to aflatoxin B.Derivatives with overlapping standard points. During observation, the first plate can be used as a blank plate for the derivatives of the sample solution. For example, if the sample solution contains aflatoxin B, the content is high, dilute the sample solution and perform a confirmation test according to 5.5.4. 5.6.1.5 Dilution quantification: If the sample solution contains aflatoxin B, dilute the sample solution according to 5.5.5. If the aflatoxin B content is low and the dilution multiple is small, there is still impurity interference on the quantitative longitudinal development plate, which affects the judgment of the result. The sample solution can be subjected to a two-way development test to determine the content.
5.6.1.6 Calculation: Same as 5.5.6.
5.6.2 Drop-point method
5.6.2.1 Spot sampling: Take three thin layer plates and drop aflatoxin B, standard working solution and sample solution on the baseline 3 cm from the lower end. That is, add 16μL of sample solution at 0.8-1cm from the left edge of each of the three plates, add 10uL0.04μg/mL aflatoxin B standard solution to the point on the second plate, and add 10uL0.2ug/mL aflatoxin B standard solution to the point on the third plate. 5.6.2.2 Development: Same as the horizontal and vertical development in 5.6.1.2. 5.6.2.3 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 no fluorescent point appears at the same position on the first plate, the aflatoxin B in the sample is below 5ug/kg. If the first plate has a fluorescent point at the same position as the aflatoxin B standard point on the second plate, compare the first plate with the third plate to see whether the fluorescent point at the same position on the third plate as the first plate overlaps with the aflatoxin B standard point. If it overlaps, perform the following confirmation test. 5.6.2.4 Confirmation test: 16μE sample solution and 1 small drop of trifluoroacetic acid (3.12) are added to the fourth plate at 0.8-1cm from the left edge; 16μL sample solution, 10μL 0.04ug/mL aflatoxin B, standard solution and 1 small drop of trifluoroacetic acid are added to the fifth plate. The production of derivatives and the development method are the same as 5.6.1. Then observe the above two plates under ultraviolet light to determine whether the sample point produces derivatives that overlap with the standard point of aflatoxin B. During observation, the first plate can be used as a blank plate for the derivatives of the sample solution. After the above confirmation test is determined to be positive, dilution and quantification are performed. If it contains aflatoxin B, it does not need to be diluted or the dilution multiple is small, and the impurity fluorescence still has serious interference. According to the strength of the fluorescence of aflatoxin B in the sample solution, the two-way development method can be used for quantification directly, or combined with the one-way development method, the method is the same as above.
5.6.2.5 Calculation: Same as 5.5.6.
Additional Notes:
This standard was drafted by the China Veterinary Drug Administration. The main drafter of this standard is Shen Chichang.
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