Some standard content:
ICS67.040
National Standard of the People's Republic of China
GB/T5009.24—2003
Replaces GB/T5009.24—1996
Determination of aflatoxins M, and B, in foods
Determination of aflatoxins M, and B, in foodsIssued on August 11, 2003
Ministry of Health of the People's Republic of China
Administrative Committee of Standardization of the People's Republic of China
Implementation on January 1, 2004
GB/T5009.24-2003
This standard replaces GB/T5009.24—1996 "Determination of aflatoxins M, and B, in foods". Compared with GB/T5009.24-1996, this standard has been modified as follows: the Chinese name of the standard has been modified, and the Chinese name of the standard has been changed to "Determination of aflatoxins M, 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. This standard is drafted by the Institute of Nutrition and Food Hygiene, Chinese Academy of Preventive Medicine. This standard was first issued in 1985 and revised for the first time in 1996. This is the second revision. 198
1 Scope
Determination of aflatoxins M, B, in foods GB/T5009.24-2003
This standard specifies the determination method of aflatoxins M, B in foods such as milk and its products, butter and fresh pig tissues (liver, kidney, blood and lean meat).
This standard applies to the determination of aflatoxin M, B in foods such as milk and its products, butter and fresh pig tissues (liver, kidney, blood and lean meat).
2 Normative references
The clauses in the following documents become the clauses of this standard through reference in this standard. For all dated references, all subsequent amendments (excluding errata) or revisions are not applicable to this standard. However, the parties who reach an agreement based on this standard are encouraged to study whether the latest versions of these documents can be used. For all undated references, the latest versions apply to this standard. GB/T5009.22—2003 Determination of aflatoxin B, in foods 3 Principle
After the sample is extracted, concentrated and separated by thin layer, aflatoxin M, B produces blue-violet fluorescence under ultraviolet light (wavelength 365nm), and the content is determined according to the minimum detection amount showing fluorescence on the thin layer. 4 Reagents
4.1 Same as Chapter 3 of GB/T5009.22~2003. 4.2 Isopropanol.
4.3 Silica gel G: For chromatography.
4.4 Sodium chloride and sodium chloride solution (40g/L). 4.5 Sulfuric acid (1+3).
4.6 Glass sand: Wash and dry after acid treatment, about equivalent to 20 mesh. 4.7 Aflatoxin M, standard solution: Prepare aflatoxin M, standard solution equivalent to 10μg per ml with chloroform. Use chloroform as blank reagent. The wavelength of the ultraviolet absorption peak of aflatoxin M. should be close to 357nm, and the molar extinction coefficient is 19950. Protect from light and store in a refrigerator at 4℃. 4.8 Aflatoxin M, and B mixed standard working solution: Use chloroform to prepare a solution with 0.04 of each of aflatoxin M and B per ml. Protect from light and store in a refrigerator at 4°C. 5 Instruments
Same as Chapter 4 of GB/T5009.22-2003. 6 Analysis steps
The entire operation shall be carried out in a dark room. 6.1 Sample extraction
6.1.1 Sample extraction preparation table, see Table 1. 199
GB/T5009.24-2003
Weighing sample weight/
Sample name
Milk powder
Pork lean meat
Amount of water added/
Amount of methanol added/
The amount of extract shall be calculated according to formula (1).
Table 1 Sample preparation
Amount of extract/
Where:
X-amount of extract, in milliliter (mL), amount of 40g/L sodium chloride solution added/
X(90+A+B)
Concentrated volume/bzxZ.net
Drop volume/
Method sensitivity/
pμg/kg
(1)
A—-Water content in the sample, in milliliter (mL) (the sampling volume of milk, condensed milk and pig tissue is 30g, and the sampling volume of milk powder and cheese is 15g),
Amount of water added, in milliliter (mL).
Note: The water content in the sample refers to the "Food Composition Table" compiled by the Institute of Nutrition and Food Hygiene, Chinese Academy of Preventive Medicine. Since each extract contains 48 mL of methanol, 39 mL of water is required to adjust the volume ratio of methanol to water to (55+45). Therefore, the amount of sodium chloride solution (40 g/L) added is equal to (87 mL) minus the amount of extract (mL). 6.1.3 Milk and condensed milk: Weigh 30.00 g of the mixed sample and place it in a small beaker. Then use 90 mL of methanol to transfer to a 300 mL stoppered conical flask and cover it tightly to prevent leakage. Oscillate for 30 minutes and filter with Folden fast filter paper in a 100 mL stoppered measuring cylinder. Collect 62 mL of milk and 52 mL of condensed milk (each equivalent to 16 g of sample) extract according to Table 1. 6.1.4 Milk powder: Take 15.00g of sample, place it in a stoppered conical flask, add 20mL of water, wet the sample, then add 90mL of methanol, and proceed according to 6.1.3, oscillate for 30min..., and collect 59mL of extract according to Table 1 (equivalent to 8g of sample). 6.1.5 Cheese: Weigh 15.00g of finely chopped and mixed sample that has been passed through a 10-mesh round sieve, place it in a stoppered conical flask, add 5mL of water and 90mL of methanol, and proceed according to 6.1.3, oscillate for 30min..., and collect 56mL of extract according to Table 1 (equivalent to 8g of sample). 6.1.6 Butter: Weigh 10.00g of sample, place it in a small beaker, dissolve the butter with 40mL of petroleum ether and transfer it to a stoppered conical flask. Add 45mL of water and 55mL of methanol, oscillate for 30min, and transfer all the liquid to a separatory funnel. Add 1.5g sodium nitride and shake to dissolve. After separation, collect 80mL of extract (equivalent to 8g sample) in a stoppered measuring cylinder according to Table 1. 6.1.7 Fresh pig tissue: Take fresh or frozen pig tissue samples (including liver, kidney, blood, lean meat), cut them into small pieces, mix them and weigh 30.00g, put them in a small mortar, add a little glass sand and grind them finely. Use a blender to mix fresh whole blood, or shake it with glass beads for anticoagulation. After mixing, weigh 30.00g, place each sample in a 300mL stoppered conical flask, add 90mL methanol, and operate according to 6.1.3 from "oscillate for 30min\". Collect 59mL of pig liver, 61mL of pig kidney, 58mL of lean pork and 61mL of pig blood extracts according to Table 1 (each equivalent to 16g of sample).
6.2 Purification
6.2.1 Purification by petroleum ether distribution: Transfer the above collected extracts to a 250mL separatory funnel, and then add a certain volume of sodium chloride solution (40g/L) according to various foods (see Table 1). Add 40mL of petroleum ether, shake for 2min, and after stratification, separate the lower layer of methanol-sodium nitride 200
GB/T 5009.24—2003
Transfer the water layer to the original measuring cylinder, pour out the upper petroleum ether solution from the top of the separatory funnel and discard. Then transfer the solution in the measuring cylinder to the original separatory funnel. Repeat the extraction with petroleum ether twice, 30mL each time, and finally transfer the solution in the measuring cylinder to the separatory funnel. The butter sample liquid is extracted twice with petroleum ether, 40mL each time.
6.2.2 Extract with chloroform: Add 20mL of chloroform to the original measuring cylinder, shake it, and then pour it into the original separatory funnel and shake for 2 minutes. After the layers are separated, transfer the lower chloroform to the original measuring cylinder, and repeat the extraction with chloroform twice, 10mL each time, and combine them in the original measuring cylinder. Discard the upper methanol-water solution. 6.2.3. Wash the chloroform layer with water and concentrate: Pour the combined chloroform layer back into the original separatory funnel, add 30mL of sodium chloride solution (40g/L), shake for 30s, and let stand. When the upper turbid liquid is partially clarified, the lower chloroform layer can be collected in the original measuring cylinder. Add 10g of anhydrous sodium sulfate, shake and place to clarify, and filter the liquid through a quantitative slow filter paper containing a small amount of anhydrous sodium sulfate into 100mL evaporation III. The sodium nitride water layer is extracted once with 10mL of chloroform, and filtered through the filter into evaporation III. Finally, pour the anhydrous sodium sulfate onto the filter paper, wash the measuring cylinder and anhydrous sodium sulfate with a small amount of trifluoromethane, and filter them into the evaporation blood together, ventilate and evaporate on a 65℃ water bath, and transfer the residue in the evaporation into the concentration tube with trifluoromethane. If there are too many residues in the evaporation blood, filter it into the concentration tube through the filter paper. At 65℃, the liquid was concentrated to less than 0.4mL by vacuum blowing method, and then the tube wall was washed with a small amount of chloroform, and then concentrated to 0.4mL for later use.
6.3 Determination
6.3.1 Preparation of silica gel G thin layer plate
The thickness of the thin layer plate is 0.3mm, activated at 105℃ for 2h, and can be stored in a desiccator for 1d~2d. 6.3.2 Point plate
Take two 5cm×20cm thin layer plates, and drop two points on the baseline 3cm from the bottom of the plate. 10μL of aflatoxin M and B mixed standard working solution was dropped at 0.8cm~1cm from the left edge of the first and second plates, and the same sample solution was dropped at 2.8cm~3cm from the left edge of each plate (the drop volume of various foods is shown in Table 1), and 10μL of aflatoxin M and B mixed standard working solution was dropped at the second point of the second plate. Generally, the thin layer plate can be placed in a chromatography tank filled with dry silica gel for dropwise addition, and the plate can be blown dry with cold air from a cold air blower while adding.
6.3.3 Development
6.3.3.1 Horizontal development: Add 15 mL of anhydrous acetaldehyde that has been dehydrated with anhydrous sodium sulfate in advance into the tank (add 20 g of anhydrous sodium sulfate to 500 mL of anhydrous ether). Place the long side of the thin layer plate close to the standard point into the tank, develop to the end of the plate, take it out and evaporate it, and then continue to develop it once as above. 6.3.3.2 Vertical development: After being developed horizontally twice and evaporated, the thin layer plate is then developed vertically with a mixed developing agent of isopropanol-acetone-benzene-n-hexane-petroleum ether (boiling range 60℃～90℃)-trichloromethane (5+10+10+10+10+55) until the front edge is 10cm~12cm away from the origin, and then take it out and evaporate it. 6.3.3.3 Horizontal development: After the vertical development has been evaporated, the plate shall be horizontally developed once or twice with ether, and the development method is the same as that in 6.3.3.1. 6.3.4 Observation and evaluation results
6.3.4.1 Compare and 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 M and B standard points (the ratio of M and B is 0.25 and 0.43 respectively), and no fluorescent point appears at the same position of the first plate, then the content of aflatoxin M and B in the sample is below the sensitivity of the method (see Table 1). 6.3.4.2 If the fluorescent points of aflatoxin M and BI appear at the same position of the first plate, check whether the sample solution points at the second point of the second plate overlap with the added standard points. If they overlap, perform the following quantitative and confirmation tests. 6.3.5 Dilution Quantification
If the fluorescence intensity of the aflatoxin M and B fluorescence point in the sample solution is consistent with the fluorescence intensity of the minimum detection amount of aflatoxin M and B (0.0004μg), the content of aflatoxin M and B in milk, condensed milk, milk powder, cheese and butter samples is 0.1, 0.2, 0.5, 0.5 and 0.5μg/kg respectively; the fresh pig tissue (liver, kidney, blood, lean meat) samples are all 0.2μg/kg (see Table 1). If the fluorescence intensity of aflatoxin M and BI in the sample solution is stronger than the minimum detection amount, then measure them one by one according to their intensity, estimate the number of microliters to be added or add different microliters after dilution until the fluorescence intensity of the sample solution point is consistent with the fluorescence intensity of the minimum detection amount point. 6.3.6 Confirmation test
On the thin layer plate after the qualitative or quantitative test, circle the points of aflatoxin M and B to be confirmed with a pin. Spray with sulfuric acid solution (1+3), let it stand for 5 minutes, and observe under ultraviolet light. If the aflatoxin M and B points in the sample solution turn into yellow fluorescence like the standard point, it is further confirmed that the detected fluorescent point is aflatoxin M, and the content of aflatoxin M or B is calculated according to formula (2). 6.3.7 Result calculation
The content of aflatoxin M or B is calculated according to formula (2). xDx1000
Wherein:
X-aflatoxin M, or B content, in micrograms per kilogram (μg/kg); V,--the volume of the sample solution after concentration, in milliliters (mL); V,--the drop volume of the sample solution with the lowest fluorescence, in milliliters (mL); D--the total dilution multiple of the concentrated sample solution;
--the equivalent sample mass in the concentrated sample solution, in grams (g); 0.0004--the minimum detection amount of aflatoxin M, or B, in micrograms (μg). The results are expressed to the integer of the measured value.7 Calculation of results
The content of aflatoxin M, or B, is calculated according to formula (2). xDx1000
Wherein:
X-aflatoxin M, or B content, in micrograms per kilogram (μg/kg); V,--the volume of the sample solution after concentration, in milliliters (mL); V,--the drop volume of the sample solution with the lowest fluorescence, in milliliters (mL); D--the total dilution multiple of the concentrated sample solution;
--the equivalent sample mass in the concentrated sample solution, in grams (g); 0.0004--the minimum detection amount of aflatoxin M, or B, in micrograms (μg). The results are expressed to the integer of the measured value.7 Calculation of results
The content of aflatoxin M, or B, is calculated according to formula (2). xDx1000
Wherein:
X-aflatoxin M, or B content, in micrograms per kilogram (μg/kg); V,--the volume of the sample solution after concentration, in milliliters (mL); V,--the drop volume of the sample solution with the lowest fluorescence, in milliliters (mL); D--the total dilution multiple of the concentrated sample solution;
--the equivalent sample mass in the concentrated sample solution, in grams (g); 0.0004--the minimum detection amount of aflatoxin M, or B, in micrograms (μg). The results are expressed to the integer of the measured value.
Tip: This standard content only shows part of the intercepted content of the complete standard. If you need the complete standard, please go to the top to download the complete standard document for free.