Determination of aflatoxin B1 in tea by high performance liquid chromatography
Determination of aflatoxin B1 in tea by high performance liquid chromatography
1. Scope of application
The 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 high performance liquid chromatograph equipped with a fluorescence detector, and quantified by an external standard method.
3. Main reagents and instruments
3.1. Main reagents
Trichloromethane;
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 right amount of aflatoxin B1 standards, with benzene - acetonitrile in a brown flask, formulated at a concentration of 10 μ g / mL standard stock solution. If necessary, prepare a standard working solution at the appropriate concentration.
3.2. Instrument
High performance liquid chromatograph with fluorescence detector;
Tianjin Hengao silica gel column ;
Tianjin Hengao Oscillator: HMS Series ;
Tianjin Hengao Ultrasonic: HU , HS series;
Tianjin Hengao nitrogen blowing instrument ;
Tianjin Hengao filter membrane: organic system, 0.45 μ m ;
Tianjin Hengao microporous membrane filter
Rotary evaporator
Micro syringe
Centrifuge tube: 5mL with a stopper;
grinder.
4. Sample extraction and preparation
4.1. 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 out all the tea leaves on the plastic cloth separately, and take a representative sample of about 500g 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.
4.2. Sample preparation
The retrieved samples were all ground, passed through a 20 mesh sieve, mixed, and divided into two samples, placed in a clean container, sealed, and marked.
4.3. 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
A sample of 5.0 g (accurate to 0.1 g ) was weighed into a 100 mL stoppered Erlenmeyer flask, 15 mL of chloroform was added, and the mixture was 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
With a rotary evaporator above filtrate concentrated to approximately 1mL at 50 ℃ water bath, a 0.45 μ m filter membrane after filtration, injected into a small silica gel column. Wash the flask with 2 to 4 mL of n-hexane, rinse the cartridge, 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 eluate in a centrifuge tube. Slowly blow dry with a nitrogen meter for derivatization.
5.3. Derivative
5.3.1. Sample
Add 200 μ L of n-hexane and 50 μ L of trifluoroacetic acid to the centrifuge tube, a ground plug tightly, ultrasonic oscillation 1min, allowed to stand 10min, slowly with nitrogen blowing instrument to dryness. With acetonitrile - water (1 + 1) to 1.0 mL volume, ultrasonic 1min, with a 0.5 μ m membrane filter, the filtrate for liquid chromatography.
5.3.2. Standard working solution
Take 1.0 mL of the standard working solution and slowly dry it with a nitrogen blower. Follow the steps in 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 liquid, 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. Blank test
Except that no sample is added, the above measurement steps are carried out.
6. Calculation of results
Data processing with a chromatographic data processor
7. Low limit and recovery rate determination
7.1. Low limit
The lower limit of determination of this method is 0.001 mg/kg .
7.2. Recovery rate
Experimental data of recovery rate: The concentration of aflatoxin B1 was in the range of 0.001 to 0.5 mg/kg , and the recovery was 89.1% to 104.9% .
It can be seen from the above table that the expected results can be obtained by processing samples with Tianjin Hengao equipment .
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