Scope of Application This method is applicable to the test of aflatoxin B1 content in tea leaves.
2. Principle Summary The sample was extracted with chloroform, and the extract was purified by a silica gel column. The purified extract was derivatized with trifluoroacetic acid, and determined 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 spectral level;
Trifluoroacetate;
Acetonitrile-water solution (1+1);
Trichloromethane-methanol solution (95+5);
Benzene-acetonitrile solution (98+2);
Aflatoxin B1 standard: purity ≥99%;
Aflatoxin B1 standard solution: Accurately weigh the appropriate amount of aflatoxin B1 standard, and prepare a standard stock solution with a concentration of 10 μg/mL in a brown volumetric flask with a benzene-acetonitrile solution. If necessary, prepare a standard working solution at the appropriate concentration.
3.2. Instrument liquid chromatograph equipped with a fluorescence detector;
Silica gel cartridge: Waters Sep-pak Silica pretreatment cartridge or equivalent silica pretreatment cartridge;
Oscillator
Rotary evaporator equipped with a 100 mL round bottom flask with a tailpipe;
Micro syringe
Centrifuge tube: 5mL with stopper grinding;
grinder;
Filter membrane: for organic use, 0.45 μm;
Microporous membrane filter: for organic use, 0.5 μm.
4. Sample extraction and preparation
4.1. The inspection lot shall be no more than 2000 pieces for one inspection lot.
Goods in the same inspection lot should have the same characteristics, such as packaging, marking, origin, specifications, grades, etc.
4.2. Sampling quantity batch, piece zui low sampling number, piece
1~5 1
6 to 50 2
51～500 11
501~1000 16
1001～1500 19
1501～2000 20
4.3. Sampling method Randomly extract the number of pieces specified in 2.2 from different parts of the whole batch of products, and open them one by one. Pour all the tea leaves on the plastic cloth separately, and take a representative sample of about 500 g from each piece with a sampling shovel. The sample taken is thoroughly mixed, and 500g is gradually reduced by a quarter method or a sampler, and placed in a clean sealed sample cylinder. After sealing, the mark is marked and sent to the laboratory in time.
4.4. Sample Preparation All the samples taken were ground, passed through a 20 mesh sieve, mixed, and divided into two samples, placed in a clean container, sealed, and marked.
4.5. Sample Storage The sample was stored at room temperature.
Note: Samples must be protected from contamination or changes in residue levels during sampling and sample preparation operations.
5. Process brief
5.1. Extraction Weighing 5.0 g (accurate to 0.1 g) was placed in a 100 mL stoppered Erlenmeyer flask, 15 mL of chloroform was added, extracted on a shaker for 30 min, and then filtered through a funnel padded with glass fibers. The filtrate was collected in a round bottom flask with a rotary evaporator, and the 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 using a rotary evaporator, filtered through a 0.45 μm filter, and poured into a silica gel cartridge. The flask was washed with 2 to 4 mL of n-hexane, and the column was rinsed, and the effluent was discarded. Then, it was eluted with 3 to 4 mL of a chloroform-methanol solution at a flow rate of 2 drops per second, and the eluate was collected in a centrifuge tube. Blow dry slowly with nitrogen for derivatization.
5.3. Derivative
5.3.1. Add 200 μL of n-hexane and 50 μL of trifluoroacetic acid to the above centrifuge tube, cover the ground plug, shake it for 1 min, let stand for 10 min, open the grinding plug, and slowly pass nitrogen to dry. The volume was adjusted to 1.0 mL with an acetonitrile-water solution (1+1), sonicated for 1 min, filtered through a 0.5 μm filter, and the filtrate was used for liquid chromatography.
5.3.2. Standard working solution Take 1.0mL standard working solution, slowly dry with nitrogen, and operate according to step 5.3.1.
5.4. Determination
5.4.1. Chromatographic conditions Column: NOVA PAKc18, 300mm × 3.9mm (inside diameter);
Mobile phase: methanol-water solution (42+58);
Flow rate: 0.8 mL/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, a standard working solution with similar peak heights is selected. The response of the aflatoxin B1 derivative 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 were measured by equal volume insertion. Under the above chromatographic conditions, the aflatoxin B1 derivative retention time was about 8 min.
5.4.3. The blank test was carried out in accordance with the above measurement procedure except that no sample was added.
6. Calculation of results The content of aflatoxin B1 in the sample is calculated by a chromatographic data processor or as follows:
X= A·cs
As·c
Where: X —— the content of aflatoxin B1 in the sample, mg/kg;
A —— the peak area of ​​the 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 zui final sample, g/mL.
Note: The calculation result needs to be deducted from the blank value.
7. Low limit and recovery rate determination
7.1. Low limit The lower limit of the method is 0.001 mg/kg.
7.2. Experimental data of recovery rate recovery: The concentration of aflatoxin B1 is in the range of 0.001 to 0.5 mg/kg, and the recovery is 89.1% to 104.9%.
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