1. Instrument
High performance liquid chromatograph with fluorescence detector, oscillator (or high speed agitator), high speed centrifuge
2. Reagents and test solutions
Chromatography methanol, distilled water, acetonitrile + water (25 + 75), petroleum ether, 2.5 10% acetonitrile, sodium hydroxide solution (0.5 mol / L), glass fiber filter paper (PriboFast immunoaffinity column free of charge)
3. Determination method
This method uses high performance liquid chromatography (GB/T 18980-2003) to determine aflatoxin M1 in milk, milk powder and other products, unless otherwise specified, according to the following method.
3.1 Chromatographic conditions
Column: C18 column, 150 × 4.6mm, 5um
Detector: fluorescence detector; excitation wavelength 365 nm, emission wavelength 435 nm;
Mobile phase: acetonitrile-water (25:75)
Flow rate: 1.0ml/min
Injection volume: 20ul
Column temperature: room temperature
3.2 Preparation of standard solution
Aflatoxin M1 standard was taken and diluted to a standard stock solution of 0.5 ug/ml with acetonitrile. According to the needs of use, accurately absorb 10μl, 20μl, 50μl, 100μl, 200μl of aflatoxin standard stock solution, put it in a 5ml volumetric flask, dilute to the scale with mobile phase, and make it equivalent to 1ng/ml, 2g/ml, 5ng /ml, 10g/ml, 20ng/ml standard working solution, that is.
3.3 Sample preparation
3.3.1 Milk
Heating 100 mL milk water bath to 40 ° C;
Centrifuge at 4000r/min for 15min, collect 50mL of clear liquid for use;
3.3.2 Milk powder and powder infant formula
Weigh 10g milk powder sample, add 100mL 50 °C water several times, stir, so that the milk powder is fully dissolved;
Centrifuge at 6000r/min for 15min, collect 50mL of clear liquid for use;
3.3.3 fermented milk (including solid, semi-solid and fleshy)
Weigh 60 g of the mixed sample, and adjust the pH to 7.4 with a 0.5 mol/L sodium hydroxide solution on the acidity indicator;
High-speed homogenizer at 9500 rpm, high-speed homogenization for 5 min
Heating in a water bath to 40 ° C;
Centrifuge at 4000r/min for 15min, collect 50mL of clear liquid for use
3.3.4 cheese
Weigh 5 g of the sample which has been shredded and passed through a 10-hole circular sieve and place it in a 50 mL centrifuge tube, add 2 mL of water and 30 mL of methanol;
High-speed homogenizer at 9500r/min, high-speed homogenization for 5 min;
Ultrasonic extraction for 30 min;
After centrifugation at 6000 r/min for 15 min, the supernatant was collected and transferred to a 250 mL separatory funnel.
Add 30 mL of petroleum ether in a separatory funnel and shake for 2 min;
After stratification, the lower layer was transferred to a 50 mL beaker and the petroleum ether layer was discarded.
Repeated extraction with petroleum ether twice;
The lower layer solution was transferred to a 100 mL round bottom flask and concentrated to about 2 mL under reduced pressure;
The concentrate was poured into a centrifuge tube, and the flask was washed twice with 5 mL of acetonitrile-water solution (1+4), and the washing solution was poured into a 50 mL centrifuge tube;
Dilute to about 50 mL with water, centrifuge at 6000 rpm for 5 min, and clean the supernatant for purification.
3.3.5 Cream
Weigh 5g sample and place it in a 50 mL beaker;
It was dissolved in 20 mL of petroleum ether and transferred to a 250 mL stoppered Erlenmeyer flask.
Add 20 mL of water and 30 mL of methanol, shake for 30 min, then transfer all the liquid to the separatory funnel;
After stratification, the lower layer solution was all transferred to a 100 mL round bottom flask, concentrated to about 5 mL under reduced pressure in a rotary evaporator, and diluted with water to about 50 mL for purification.
3.4 Preparation of test solution
3.4.1 Operation of PriboFast ® aflatoxin M1 immunoaffinity column
The immunoaffinity column was attached to a 50.0 mL glass syringe. Accurately remove 50.0mL of purification solution into the glass syringe;
Connect the air pressure pump to the syringe, adjust the pressure so that the solution slowly passes through the immunoaffinity column at a flow rate of about 2-3 mL/min until 2 to 3 mL of air passes through the column;
A 10 ml syringe was replaced with the affinity column, 10 ml of water was added to the syringe, and the pressure was adjusted to wash the affinity column at a flow rate of about 6 mL/min.
Accurately add 4 mL of chromatographic grade acetonitrile to elute at a flow rate of 1 mL/min to 2 mL/min. Collect all eluates in glass tubes for testing. (It is recommended to add acetonitrile in 2-4 times. After each addition, let the methanol fully soak the gel in the column for 2 minutes. The eluted acetonitrile solution, preferably in a water bath at 30 ° C, is heated to dry. 10% acetonitrile to volume after liquid phase)
3.5 Determination
Precisely measure 20 ul of each standard working solution of 1 ng/ml, 2g/ml, 5ng/ml, 10g/ml, 20ng/ml, and inject into the liquid chromatograph to determine the peak area. The peak area is the ordinate and the concentration is the abscissa. , draw a standard curve. In addition, 20 ul of the test solution was accurately weighed, injected into a liquid chromatograph, and the peak area was measured. The amount corresponding to the aflatoxin M1 in the test sample was read from the standard curve, and the calculation was obtained.
1. This test should have corresponding safety and protective measures and must not pollute the environment.
2. The glassware containing the waste liquid or waste residue of aflatoxin M1 should be placed in a special storage container (containing 10% sodium chlorate solution), soaked for more than 24 hours, then rinse the glassware with clean water.
For technical inquiries, please contact: Beijing Tellabs Technology Co., Ltd.
Telephone 52453171 52453170 13311089404
Website: http://
E-mail :rapidbio @126.com
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