Target audience

bisphenolA affinity column can specifically purify bisphenol A and bisphenol S in the sample. It uses column shaped agarose gel as the solid carrier, and agarose gel is coupled with bisphenol A antibody to form an immunoadsorbent, which is loaded into the column to make an immunoaffinity column. It can specifically purify bisphenol A in samples. Bisphenol A affinity columns are widely used in the extraction of food and beverage samples. This method is fast, easy to operate, and highly accurate, playing a very important role in improving the quality and safety of food.

This instruction manual applies the affinity column method to detect bisphenol in foodThe detection method of A.

GB 5009.305-2025 National Food Safety Standard - Determination of Bisphenol A, Bisphenol F, and Bisphenol S in Foods

Storage conditions

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

Preparation of experimental solution

lmethanol-Water solution (80+20):Measure methanol80mL, Add 20mL of water and mix well.

lmethanol-Water solution (40+60):Measure methanol40mL, Add 60mL of water and mix well.

l盐酸溶液(10+90):measureAdd 10mL of concentrated hydrochloric acid to 90mL of water and mix well.

lPBS solution:Translate into EnglishDissolve the PBS buffer reagent kit (item number: G3001) in 1000mL of pure water and mix well.

Analysis steps

GB 5009.305-2025 Method

1.0 Sample Extraction

1.1 Meat and meat products, aquatic products, solid and semi-solid dairy products (milk powder, fermented milk, etc.), infant formula food

Weigh out1g sample (accurate to 0.01g) is placed in a 50mL polypropylene centrifuge tube, and 50 μ L of isotope internal standard mixing solution is added. After shaking and mixing, it is left to stand for 30 minutes. Add 2mL of water, vortex for 30 seconds, and then add 5mL of acetonitrile for vortex mixing. Ultrasonic extraction of the mixed solution for 15 minutes, followed by centrifugation at 10000r/min for 10 minutes at 4 ℃. Take the supernatant and place it in a glass nitrogen blowing tube. Slowly blow it with nitrogen at 40 ℃ to about 2mL, add 8mL PBS and mix well. Wait for purification.

1.2 Eggs, milk, and liquid dairy products

Weigh out1g sample (accurate to 0.01g) is placed in a 50mL polypropylene centrifuge tube, and 50 μ L of isotope internal standard mixing solution is added. After shaking and mixing, it is left to stand for 30 minutes. Add 5mL of acetonitrile, vortex and mix, extract with ultrasound for 15 minutes, centrifuge at 10000r/min at 4 ℃ for 10 minutes. Take the supernatant and place it in a glass nitrogen blowing tube. Slowly blow it with nitrogen at 40 ℃ to about 2mL, add 8mL PBS and mix well. Wait for purification.

1.3 Grains and infant cereal supplements

Weigh out1g sample (accurate to 0.01g) is placed in a 50mL polypropylene centrifuge tube, and 50 μ L of isotope internal standard mixing solution is added. After shaking and mixing, it is left to stand for 30 minutes. Add 10 mL of methanol water solution (80+20), vortex for 30 seconds, extract with ultrasound for 15 minutes, and centrifuge at 10000 r/min for 10 minutes at 4 ℃. Take the supernatant and place it in a glass nitrogen blowing tube. Slowly blow it with nitrogen at 40 ℃ to about 2mL, add 8mL PBS and mix well. Wait for purification.

1.4 Vegetables and fruits

Weigh out1g sample (accurate to 0.01g) is placed in a 50mL polypropylene centrifuge tube, and 100 μ L hydrochloric acid solution (10+90) is added and mixed well. Then, 50 μ L of the same position is addedMixing solution of internal standardAfter oscillating and mixing, let it stand for 30 minutes. Add 5mL of acetonitrile, vortex for 30 seconds, extract with ultrasound for 15 minutes, centrifuge at 10000r/min at 4 ℃ for 10 minutes. Take the supernatant and place it in a glass nitrogen blowing tube. Slowly blow it with nitrogen at 40 ℃ to about 2mL, add 8mL PBS and mix well. Wait for purification.

1.5 Beverages

Weigh out1g sample (accurate to 0.01g) is placed in a 2mL polypropylene centrifuge tube, and 50 μ L of isotope internal standard mixing solution is added. After shaking and mixing, it is left to stand for 30 minutes. Centrifuge at 10000r/min for 10 minutes. Transfer the supernatant to a 50mL polypropylene centrifuge tube, add 10mL PBS solution and mix well, and wait for purification.

noteClear samples (such as purified water, mineral water, etc.) can be added directly to PBS without centrifugation and mixed well.

2.0 Sample Purification

2.1 Affinity Column Activation

Connect the immune affinity column toUnder a 10.0mL syringe. Adjust the pressure to slowly pass the column protection solution through the immunoaffinity column at a flow rate of approximately 2mL/min (1 drop/second) until a small amount of air passes through the column. Accurately measure 5mL of affinity column activation solution (standard for affinity column products) and rinse the affinity column at a flow rate of 1 drop/second until a small amount of air passes through the column. Rinse the column once with 5.0mL of pure water, discard all the effluent, and then rinse the column with 10.0mL of pH 7.4 PBS solution at a flow rate of 1 drop/second. When the last 2-3mL of solution fills the column, stop pressurizing and block the bottom outlet with a stopper for later use.

Note: In order to further improve bisphenol AThe purification and adsorption effects of the A immune affinity column require activation treatment before use. Please use the activated affinity column within 1 hour.

2.2 Affinity Column Purification

取All the purified liquid in step 1.0 is passed through the column at a flow rate of 1 drop/second. Discard all the effluent and rinse the immunoaffinity column with 10mL of water at a flow rate of 1-2 drops/second. After the water droplets are removed, use a vacuum pump to dry the immunoaffinity column. Add 1 mL of methanol for elution at a flow rate of 1-2 drops/second, collect the eluent in a glass nitrogen pipette, and slowly dry it with nitrogen gas at 40 ℃. Accurately add 0.40mL of methanol, vortex and mix for 10 seconds, then accurately add 0.60mL of water, vortex and mix, transfer to a 2mL polypropylene centrifuge tube, centrifuge at 10000r/min for 5 minutes, and take the supernatant for liquid chromatography tandem mass spectrometry measurement.

Precautions

1)Do not change the operating steps in the manual casually. If you need to make changes, please contact our technical department to confirm whether the changes are reasonable.

2)In the operation steps, the sample must be passed through the column, washed, and eluted. The flow rate must be controlled well and not too fast, otherwise it will result in lower detection results.

3） This experiment aims to minimize the use of plastic containers, especiallyPC material plastic products, experimental water should be distilled in glassware as much as possible.

Affinity Column Configuration List

Equipment and reagents that need to be prepared