IAC-SEP® Aflatoxin Total Immunoaffinity Column IAC103 Instruction Manual

IAC-SEP® Aflatoxin Total Immunoaffinity Column

(Product Code: IAC103)

user target audience

The IAC-SEP® Aflatoxin Total Immunoaffinity Column specifically purifies and concentrates aflatoxins B1, B2, G1, G2 in the sample. Aflatoxin immunoaffinity column is widely used in the extraction of food, food, feed, nuts, peanuts, soy sauce, vinegar, pepper, pepper, herbs and alcohol. The method is fast, easy to operate and highly accurate. It plays an important role in the accurate and sensitive determination of aflatoxins B1, B2, G1, G2 in the sample.

The IAC-SEP® Aflatoxin Total Immunoaffinity Column is suitable for the following criteria:

GB/T 18979-2003 Determination of aflatoxins in foods - Immunoaffinity chromatographic purification - High performance liquid chromatography

SN/T 1101-2002 Method for the detection of aflatoxins in oilseeds and grains

principle

The sample was extracted with methanol + water extract, filtered, diluted, and then passed through an immunoaffinity column to which aflatoxin specific antibody was bound. At this time, the aflatoxin is specifically adsorbed by the antibody in the affinity column, the impurities on the immunoaffinity column are removed with water or a buffer, and then the antibody is denatured with methanol to wash the aflatoxin from the affinity column. Take off. Finally, quantitative detection is performed using HPLC or a fluorometer.

Storage conditions

The affinity column is stored at 2-8 °C and must not be frozen. The shelf life is 18 months. It is recommended to use at room temperature (18-30 ° C).

Experimental solution preparation

Extract, methanol / water (8 + 2): Take 80 mL of methanol plus 20 mL of water, mix well.

Methanol / water (7 + 3): Take 70 mL of methanol and add 30 mL of water, mix well.

HPLC mobile phase, methanol/water (45+55): Take 450 mL of methanol plus 550 mL of water, mix well and filter through a 0.22 micron nylon screen.

Preparation of standard solution: Take aflatoxin mixed standard (G1010, Bug and mL of B1 and G1 is 0.3ug/mL), and the mobile phase is a diluent to prepare a standard solution. For example: take 100uL standard stock solution in a 10mL volumetric flask and dilute to 10mL with 10mL mobile phase, then aflatoxin B1 and G1 are 10ng/mL, aflatoxin B2 and G2 are 3ng/mL

Note: The prepared standard solution is stored at 2-8 ° C for 2 hours.

pH 7.0 PBS solution: Weigh 8.0 g of sodium chloride, 1.44 g of disodium hydrogen phosphate, 0.24 g of potassium dihydrogen phosphate, 0.2 g of potassium chloride, dissolve with 990 mL of pure water, and then adjust the pH to 7.0 with concentrated hydrochloric acid. Dilute to 1000mL with pure water

0.1% Tween/PBS: 1.0 mL Tween-20 was added to 1 L of pH 7.0 PBS solution and mixed.

Liquid chromatography conditions

Column: Cloversil-C18 4.6*150mm (5um) or 4.6*250mm (5um)

Mobile phase: methanol-water (45+55, v/v)

Flow rate: 0.8mL/min

Detector: fluorescence detector with excitation wavelength of 360 nm and emission wavelength of 440 nm

Injection volume: 20~100uL

Photochemical derivatization system: photochemical derivatizer (connected to the column and then to the fluorescence detector)

Analysis step

1. Sample extraction and dilution:

Rice, corn, wheat, nuts, seasonings, peanuts and their products: accurately weigh 25.0g of sample in a 250mL stoppered conical flask, add 5.0g of sodium chloride and accurately add 125.0mL of methanol-water (7+3) The mixture was extracted with a homogenizer at high speed for 2 min. The fluted filter paper was filtered, and 15.0 mL of the filtrate was accurately transferred and diluted with 30.0 mL of water, and filtered with a glass fiber filter paper until the filtrate was clarified and used.

Vegetable oil: 25.0 g of the sample was accurately weighed into a 250 mL stoppered conical flask, 5.0 g of sodium chloride and 125.0 mL of methanol-water (7+3) were added, and the mixture was extracted by a homogenizer at high speed for 2 min. The fluted filter paper was filtered, and 15.0 mL of the filtrate was accurately transferred and diluted with 30.0 mL of water, and filtered with a glass fiber filter paper until the filtrate was clarified and used.

Soy sauce: Weigh 50.0 g of the sample in a 250.0 mL stoppered Erlenmeyer flask, add 2.5 g of sodium chloride, and add methanol-water (8+2) to 100.0 mL, and extract with a homogenizer at high speed for 1 min. The fluted filter paper was filtered, and 10.0 mL of the filtrate was accurately transferred and diluted with 40.0 mL of water, and filtered with a glass fiber filter paper until the filtrate was clarified and used.

Vinegar: Accurately weigh 5.0g sample, add 1.0g sodium chloride, dilute to 25.0mL with pH7.0 phosphate buffer solution, mix, filter by quantitative filter paper. 10.0 mL of the filtrate was added to 10.0 mL of a pH 7.0 phosphate buffer solution and mixed. Filter through glass fiber filter paper until the filtrate is clear and ready for use.

Feed: Accurately weigh 50.0 g of the sample into a 250 mL stoppered Erlenmeyer flask, add 5.0 g of sodium chloride and 100.0 mL of methanol-water (8+2), and extract with a homogenizer at high speed for 2 min. The fluted filter paper was filtered, and 10.0 mL of the filtrate was accurately transferred and diluted with 40.0 mL of pH 7.0 PBS, and filtered through a glass fiber filter paper until the filtrate was clarified and used.

2. Purification of samples

The column capacity of the total aflatoxin immunoaffinity column (maximum adsorption of aflatoxin) is 300 ng. When the aflatoxin in the sample exceeds the measurement range, please reduce the volume of the upper column appropriately, and make it within the detection range. The exact content is obtained.

Rice, corn, wheat, nuts, spices, peanuts and their products, vegetable oils and fats: (measurement range 0-300 μg/kg)

The immunoaffinity column was attached to a 20.0 mL glass syringe. Accurately transfer 15.0mL of sample extract into a glass syringe, connect the air pressure pump to the glass syringe, adjust the pressure to slowly pass the solution through the immunoaffinity column at a flow rate of about 2mL/min (1 drop/second) until 2~3mL of air Pass through the cylinder. The column was rinsed twice with 10.0 mL of water at a flow rate of 2-3 mL/min (1-2 drops/second), all of the effluent was discarded, and 2 mL to 3 mL of air was passed through the column. Accurately add 1.0 mL of chromatographic methanol elution at a flow rate of 1 mL/min to 2 mL/min (1 drop/sec) and collect all the eluate in a glass test tube for testing.

Soy sauce and vinegar: (measurement range 0-300 μg/kg)

The immunoaffinity column was attached to a 10.0 mL glass syringe. Accurately transfer 10 mL of the sample extract into a glass syringe, connect the air pressure pump to the glass syringe, and adjust the pressure to slowly pass the solution through the immunoaffinity column at a flow rate of about 2 mL/min (1 drop/second). Wash with 10.0 mL of 0.1% Tween/PBS at a flow rate of 2-3 mL/min (1-2 drops/sec), then wash the column twice with 10.0 mL of water, discard all effluent, and make 2 mL~3 mL of air. Pass through the cylinder. Accurately add 1.0 mL of chromatographic methanol elution at a flow rate of 1 mL/min to 2 mL/min (1 drop/sec). Collect all eluates in glass tubes for testing.

Feed: (measurement range 0-300 μg/kg)

The immunoaffinity column was attached to a 10.0 mL glass syringe. Accurately transfer 10 mL of the sample extract into a glass syringe, connect the air pressure pump to the glass syringe, and adjust the pressure to slowly pass the solution through the immunoaffinity column at a flow rate of about 2 mL/min (1 drop/second). The column was washed twice with 10.0 mL of water at a flow rate of 2-3 mL/min (1-2 drops/second), all of the effluent was discarded, and 2 mL to 3 mL of air was passed through the column. Accurately add 1.0 mL of chromatographic methanol elution at a flow rate of 1 mL/min to 2 mL/min (1 drop/sec). Collect all eluates in glass tubes for testing.

Standard chromatogram of aflatoxin B1, B2, G1, G2 detected by high performance liquid chromatography

Precautions

1. In the sample addition recovery test, the standard is added to the sample, and after standing for 2 hours or overnight, the sample is pretreated, otherwise the recovery rate is low.
2. Do not change the operating procedures in the operating instructions. If you need to change, please contact our technical department to confirm whether the changes are reasonable.
3. In the operation step, when the sample is passed through the column, rinsed, and eluted, the flow rate must be controlled, not too fast, otherwise the detection result will be low.
4. If the standard is directly passed through the column to verify the recovery rate, it must be ensured that the methanol concentration in the standard upper column liquid should not exceed 25%, otherwise it will affect the adsorption capacity of the column.
5. The glass instruments used in the test must be cleaned, especially those treated with hypochlorous acid, otherwise the test results will be low.

Equipment and reagents to be prepared (provided by Zhongkang Weikang)

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