1. Scope of application This method is applicable to the inspection of the content of aflatoxin B1 in the exported tea.
2. Principle summary The sample is extracted with chloroform, the extract is purified by silica gel column, the purified extract is derivatized with trifluoroacetic acid, measured by a liquid chromatograph equipped with a fluorescence detector, and quantified by an external standard method.
3. Main reagents and instruments
3.1. The main reagent chloroform;
N-hexane
benzene;
Methanol: ultraviolet spectrum level;
Acetonitrile: ultraviolet spectrum level;
Trifluoroacetate;
Acetonitrile-water solution (1 + 1);
Chloroform-methanol solution (95 + 5);
Benzene-acetonitrile solution (98 + 2);
Aflatoxin B1 standard product: purity ≥99%;
Aflatoxin B1 standard solution: Accurately weigh an appropriate amount of aflatoxin B1 standard product, and use benzene-acetonitrile solution in a brown volumetric flask to prepare a standard stock solution with a concentration of 10 μg / mL. According to the need, formulate a standard working solution of appropriate concentration.
3.2. Instrument liquid chromatograph, equipped with fluorescence detector;
Silica cartridge: Waters Sep-pak Silica pretreatment cartridge or equivalent silica gel pretreatment cartridge;
Oscillator
Rotary evaporator, equipped with 100mL round bottom flask with tail tube;
Micro syringe
Centrifuge tube: 5mL with plug grinding port;
grinder;
Filter membrane: for organic system, 0.45μm;
Microporous membrane filter: for organic system, 0.5μm.
4. Sample extraction and preparation
4.1. The inspection lot shall not exceed 2000 pieces as an inspection lot.
Commodities of the same inspection lot should have the same characteristics, such as packaging, marking, place of origin, specifications, grade, etc.
4.2. Sampling quantity, batch, minimum sampling number, piece
1 ～ 5 1
6 ～ 50 2
51 ～ 500 11
501 ～ 1000 16
1001 ～ 1500 19
1501 ～ 2000 20
4.3. Sampling method Randomly select the number of pieces specified in 2.2 from the upper and lower parts of the entire batch of product stacks and open them one by one. Pour out all the tea leaves on the plastic cloth separately, and use a sampling shovel to remove about 500g of the representative sample from each piece. Mix the sample thoroughly, gradually divide it into 500g by the quartile method or the sampler, and put it into a clean and sealed sample cylinder. After sealing, mark it and send it to the laboratory in time.
4.4. Sample preparation Grind all the retrieved samples, pass through a 20-mesh sieve, mix well, divide into two samples, put them in a clean container, seal them, and mark them.
4.5. Sample storage Store the sample at room temperature.
Note: During the sampling and sample preparation operations, the sample must be protected from contamination or changes in residue content.
5. Brief description of the process
5.1. Extraction Weigh 5.0g of the sample (accurate to 0.1g) into a 100mL conical flask with a stopper, add 15mL of chloroform, extract on a shaker for 30min, and then filter through a glass fiber-filled funnel. The filtrate was collected in a round-bottomed flask with a rotary evaporator, and the filter residue was washed with chloroform, and the filtrate was collected to about 20 mL.
5.2. Purification The above filtrate was concentrated to about 1 mL in a 50 ° C water bath with a rotary evaporator, filtered through a 0.45 μm filter membrane, and poured into a small silica gel column. Wash the flask with 2 to 4 mL of n-hexane, then rinse the small column, and discard the effluent. Then elute with 3 to 4 mL of chloroform-methanol solution at a flow rate of 2 drops per second, and collect the eluent in a centrifuge tube. Slowly blow dry with nitrogen for derivation.
5.3. Derivation
5.3.1. Add 200 μL of n-hexane and 50 μL of trifluoroacetic acid to the centrifuge tube, cover the grinding plug tightly, oscillate ultrasonically for 1 min, let stand for 10 min, open the grinding plug, and slowly pass nitrogen gas to dryness. Dilute the volume to 1.0 mL with acetonitrile-water solution (1 + 1), sonicate for 1 min, filter with 0.5 μm filter membrane, and the filtrate is used for liquid chromatography.
5.3.2. Standard working solution Take 1.0 mL of standard working solution, slowly blow dry with nitrogen, and follow the steps in 5.3.1.
5.4. Determination
5.4.1. Chromatographic column: NOVA PAKc18, 300mm × 3.9mm (inner diameter);
Mobile phase: methanol-water solution (42 + 58);
Flow rate: 0.8mL / min;
Fluorescence detector: excitation wavelength 375 nm, emission wavelength 425 nm;
Column temperature: room temperature.
5.4.2. Determination According to the content of aflatoxin B1 in the sample solution, select a standard working solution with a similar peak height. The response values ​​of the aflatoxin B1 derivatives in the standard working solution and the sample solution should be within the linear range of the instrument detection. The standard working solution and the derivative solution of the sample solution are injected into the same volume for determination. Under the above chromatographic conditions, the retention time of aflatoxin B1 derivatives is about 8 min.
5.4.3. The blank test shall be carried out according to the above measurement steps except that no sample is added.
6. Calculate the result using a chromatographic data processor or the following formula to calculate the content of aflatoxin B1 in the sample
X = A · cs
As · c
In the formula: X —— the content of aflatoxin B1 in the sample, mg / kg;
A —— Peak area of ​​aflatoxin B1 derivative in the sample solution, mm2;
cs-the concentration of aflatoxin B1 in the standard working solution, μg / mL;
As —— Peak area of ​​aflatoxin B1 derivative in standard working solution, mm2;
c —— The concentration of the sample represented by the final sample solution, g / mL.
Note: The calculation results need to deduct the blank value.
7. Low limit and recovery rate determination
7.1. Low limit The determination of this method has a low limit of 0.001 mg / kg.
7.2. Recovery rate Experimental data of recovery rate: the added concentration of aflatoxin B1 is in the range of 0.001 to 0.5 mg / kg, and the recovery rate is 89.1% to 104.9%.
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