Method for using zearalenone immunoaffinity column

IAC-SEP® Zearalenone Immunoaffinity Column

(Product Code: IAC10 5)

user target audience

The IAC-SEP® Zearalenone Immunoaffinity Column specifically purifies and concentrates zearalenone in the sample. Zearalenone 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 accurate. High, plays an important role in the accurate and sensitive determination of zearalenone in the sample.

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

GB/T 23504-2009 Determination of Zearalenone in Foods - Immunoaffinity Chromatography Purification High Performance Liquid Chromatography

Determination of Zearalenone in Grains GB/T 5009.209-2008

SN/T 1172-2006 Determination of Zearalenone in Import and Export Grains - Immunoaffinity Column - Liquid Chromatography

Determination of Zearalenone in Import and Export Soybean Rapeseed and Edible Vegetable Oils by SN/T 1175-2006

principle

The sample was extracted with methanol + water extract, filtered, diluted, and then passed through an immunoaffinity column to which a specific antibody to zearalenone was bound. At this time, zearalenone 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, thereby removing zearalenone from the pro And elute on the column. 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, acetonitrile / water ( 8+2 ): Take 80 mL of acetonitrile plus 20 mL of water and mix well .

Acetonitrile / water (9+1): Take 90 mL of methanol plus 10 mL of water and mix well .

HPLC mobile phase, acetonitrile / water / methanol ( 4 6 + 46 + 8): 4 60 mL of acetonitrile and 80 mL of methanol were taken in a 1 L volumetric flask , made up to 1 L with purified water and filtered over a 0.22 micron nylon screen.

Preparation of the standard solution: Take the standard stock solution of zearalenone, the mobile phase is the diluent, and prepare the standard solution. For example: take 100uL standard stock solution 10ug/mL in a 10mL volumetric flask and dilute to 10mL with mobile phase . Then, the concentration of zearalenone standard is 100ng/mL.

Note: The prepared standard solution is valid for 7 days .

pH 7.0 PBS solution: Weigh 8.0 g of sodium chloride, 1. 44 g of disodium hydrogen phosphate, 0.2 4 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 . Finally, dilute to 1000 mL with pure water .

Liquid chromatography conditions

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

Mobile phase: acetonitrile / water / methanol ( 4 6 + 46 + 8)

Flow rate: 0.8mL/min

Detector: fluorescence detector, excitation wavelength 274nm, emission wavelength 440nm; UV detector, 265nm

Injection volume: 20~100uL

Analysis step

The column capacity of the zearalenone immunoaffinity column ( maximum adsorption zearalenone ) is 1500 ng . When the zearalenone in the sample exceeds the measurement range, please reduce the volume of the upper column appropriately to make it in the detection range. Within, calculate the exact content.

1) Sample extraction and dilution:

Samples of grains, grain, peanuts and their products, soybeans, vegetable oils, spices, etc.: (measurement range 0-1875 μg/kg) Weigh 40g of ground sample, 5g of sodium chloride; placed in a 250mL stoppered conical flask, Or in a homogenized cup. Add 100 mL of acetonitrile/water (9+1) solution. The homogenizer was used for high-speed homogenization for 2 min, or the shaker was shaken for 60 min. Remove the lid, place the extract on the fluted filter paper, and collect the filtrate in a clean container. Pipette 10 mL of the filtrate from the previous step and place in a clean container. Dilute with 40 mL of pure water and mix. The previous dilution was passed through a glass microfiber filter, and the filtrate was collected in a glass syringe barrel, and 10 mL of the sample was prepared for purification on the column.

Feed samples: (assay range 0-1875 μg/kg) Weigh 40g of ground sample, 5g of sodium chloride; place in a 250mL conical flask, or homogenized cup. Add 100 mL of acetonitrile/water (8+2) solution. The homogenizer was used for high-speed homogenization for 2 min, or the shaker was shaken for 60 min. Remove the lid, place the extract on the fluted filter paper, and collect the filtrate in a clean container. Pipette 10 mL of the filtrate from the previous step and place in a clean container. Dilute with 40 mL of pH 7.0 PBS and mix. The previous dilution was passed through a glass microfiber filter, and the filtrate was collected in a glass syringe barrel, and 10 mL of the sample was prepared for purification on the column.

Alcohol: (measuring range 0-3750 μg/kg) Weigh 20g sample (containing carbon dioxide wine before storage in a refrigerator at 30 °C for 30min, then filter or ultrasonic sample), placed in a 50mL volumetric flask, add acetonitrile Make up to 50mL and shake well. Pipette 10 mL of the previous step and place in a clean container. Dilute with 40 mL of pure water and mix. The previous dilution was passed through a glass microfiber filter, and the filtrate was collected in a glass syringe barrel, and 10 mL of the sample was prepared for purification on the column.

Soy sauce, vinegar, sauce and sauce products: (measurement range 0-3000 μg/kg) Weigh 25g sample, place it in a 100mL volumetric flask, add acetonitrile to a volume of 100mL, and ultrasonically extract for 2min. The extract was placed on a fluted filter paper and the filtrate was collected in a clean container. Pipette 10 mL of the filtrate from the previous step and place in a clean container. Dilute with 40 mL of pure water and mix. The previous dilution was passed through a glass microfiber filter, and the filtrate was collected in a glass syringe barrel, and 10 mL of the sample was prepared for purification on the column.

2) Purification of samples

The above 10 mL of the filtrate was passed through the zearalenone immunoaffinity column at a flow rate of 1 drop/second until the air entered the affinity column. The affinity column was rinsed twice with 10 mL of pure water at a flow rate of 1-2 drops/second. The affinity column was rinsed with 1.5 mL of HPLC grade methanol at a flow rate of 1 drop/second, and all sample eluent (1.5 mL) was collected in a glass test tube. 1.5 mL of eluent was diluted with 1.5 mL of pure water, mixed, and injected into HPLC; or 1.5 mL of eluent was blown dry under nitrogen at 50 ° C, and mixed with 1 mL of mobile phase, mixed, and injected into HPLC.

Standard chromatogram of zearalenone detected by high performance liquid chromatography

Precautions

1) When the sample is added and recovered, the standard is added to the sample, and after standing for 2 hours or overnight, the sample is pretreated, otherwise the recovery rate will be low.

2) Do not change the operation steps 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 passes through the column, rinse, and elute, 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 concentration of acetonitrile in the standard column liquid should not exceed 20%, 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.

6) When the eluent is concentrated with nitrogen, the nitrogen flow should not be too large, otherwise some zearalenone will be lost.

Equipment and reagents to be prepared

Contributed by Xue Lili

Phone 13366849880