SN/T 5276-2020Determination of multiple allergens in export foods Liquid chromatography-mass spectrometry/mass spectrometry (English PDF)

4.1 Reagents

4.1.1 Ammonium bicarbonate (NH4HCO3).

4.1.2 Dithiothreitol (C4H10O2S2, DTT).

4.1.3 Iodoacetamide (ICH2CONH2, IAA).

4.1.4 Calcium chloride (CaCl2).

4.1.5 Acetic acid (CH3COOH). chromatographically pure.

4.1.6 Formic acid (HCOOH). chromatographically pure.

4.1.7 Acetonitrile (CH3CN). chromatographically pure.

4.1.8 Alkaline trypsin. activity greater than 10 000 BAEE per mg protein.

4.1.9 Tris (C4H11NO3, Tris).

4.1.10 Hydrochloric acid (HCl).

4.1.11 n-hexane (C6H14). chromatographically pure.

4.2 Reagent preparation

4.2.1 Ammonium bicarbonate solution (500 mmol/L). Weigh 3.95 g of ammonium bicarbonate (4.1.1), dissolve in water and dilute to 100 mL.

4.2.2 Dithiothreitol solution (500 mmol/L). Weigh 0.771 g of dithiothreitol (4.1.2) and use ammonium bicarbonate solution (4.2.1) After dissolving, dilute to 10 mL. It can be stored at 4 °C for one month.

4.2.3 Iodoacetamide solution (500 mmol/L). Weigh 0.925 g of iodoacetamide (4.1.3) and use ammonium bicarbonate solution (4.2.1) After dissolving, dilute to 10 mL. Store at 4 °C in the dark and refrigerate for one month.

4.2.4 Calcium chloride solution (100 mmol/L). Weigh 0.111 g of calcium chloride (4.1.4), dissolve in water and dilute to 10 mL.

4.2.5 Acetic acid solution (1%, volume ratio). Pipette 0.1 mL of acetic acid (4.1.5), dilute with water and dilute to 10 mL.

4.2.6 Bovine trypsin solution (500 µg/mL). Weigh 5 mg alkaline bovine trypsin (4.1.8) and dissolve it with acetic acid solution (4.2.5) After solution, make the volume to 10 mL. The solution can be stored at -20 °C for 6 months.

4.2.7 Aqueous solution of formic acid (0.1%, volume ratio). accurately pipette 1.0 mL of formic acid (4.1.6), and dilute to 1 000 mL with water.

4.2.8 Acetonitrile solution of formic acid (0.1%, volume ratio). accurately pipette 1.0 mL of formic acid (4.1.6), and dilute to volume with acetonitrile (4.1.7) 1 000 mL.

4.2.9 Tris-HCl solution (40 mmol/L). Weigh 2.42 g of Tris (tris (trishydroxymethylaminomethane)), dissolve it in 800 mL of water, and use Adjust the pH to 8.0±0.10 with 10% hydrochloric acid, and dilute to 1000 mL with water.

4.3 Standard product

4.3.1 NIST SRM 8445.spray-dried whole egg for allergen detection (spray-dried whole egg for allergen detection).

4.3.2 NIST SRM 2387.peanut butter.

4.3.3 NIST SRM 1549a. whole milk powder.

4.3.4 NIST SRM 3234.soy flour.

(Or standard reference materials from other sources are also acceptable.)

4.4 Preparation of standard solution

4.4.1 NIST SRM 8445, SRM 2387, SRM 1549a, SRM 3234 protein extract standard stock solution. accurately weighed separately Take 1 g of NIST SRM 8445, SRM 2387, SRM 1549a, SRM 3234, add Tris-HCl (4.2.9) solution, and dilute to 20 mL. Shake uniformly to completely dissolve the sample, and place it in a water bath at 60 °C for heating and shaking for 3 hours. The extract is centrifuged at a high speed of 10 000 r/min After 5 minutes, the supernatant was too low protein adsorption 0.45 µm cellulose acetate filter membrane, the Bradford method was used to determine the concentration of total protein in the extract.

Temperature, the extract is stored in a refrigerator at -20 °C.

4.4.2 Because it is difficult to purchase standard materials, 1 g of the roasted hazelnuts, walnuts, and almonds were accurately weighed in the experiment, and Tris-HCl was added.

(4.2.9) Solution, dilute to 20 mL. Shake uniformly to completely dissolve the sample, and place it in a water bath at 60 °C for heating and shaking for 3 hours. extract the solution was centrifuged at a high speed of 10 000 r/min for 5 min. After the supernatant was too low protein adsorption on a 0.45 µm cellulose acetate filter, the Bradford formula was used.

Method to determine the concentration of total protein in the extract, the extract is stored in a refrigerator at -20 °C.

4.4.3 Series of standard working solutions. accurately pipette an appropriate amount of standard protein extraction stock solution, and prepare the final concentration with a blank matrix solution it contains whole milk powder, eggs. 5 mg/L, 10 mg/L, 20 mg/L, 100 mg/L, 500 mg/L, 1000 mg/L, peanuts, soybeans, Hazelnuts, almonds, walnuts. 10 mg/L, 20 mg/L, 40 mg/L,.200 mg/L, 800 mg/L, 1 600 mg/L matrix matching standard work Make a solution.

6.1 Sample preparation

6.1.1 Biscuits, pasta products, beverages, oatmeal

Take 500 g of a representative sample (accurate to 0.1 g), pulverize it with a pulverizer, then grind it into a fine powder, and put it in a clean Inside the sample container, seal and mark it.

6.1.2 Infant formula food (excluding formula milk powder)

Take a representative sample of 500 g (accurate to 0.1 g), put it into a clean sample container, seal and mark it.

6.2 Sample storage

During the operation of sample preparation, the sample should be prevented from being contaminated or from changing the content of residues.

7.1 Sample preparation

Weigh about 2.0 g of a well-mixed solid sample or about 10 g of a liquid sample (accurate to 0.001 g), and place it in a 50 mL plastic centrifuge tube with a stopper in. For samples with high fat content, add 10 mL of n-hexane (4.1.11), vortex for 3 minutes, and centrifuge at 4 500 r/min for 5 minutes.

Remove the upper layer solution. After repeating twice, dry with nitrogen. Add 20 mL Tris-HCl solution (4.2.9), shake evenly to completely dissolve the sample, Place it in a water bath at 60 °C for heating and shaking for 3 hours. The extract was centrifuged at a high speed of 10 000 r/min for 5 min, and the supernatant was too low for protein adsorption 0.22 µm cellulose acetate filter membrane.

Take 2 mL of the above crude protein extract and add it to an ultrafiltration centrifuge tube (5.5) with a molecular weight of 10 kDa cut-off membrane.

Centrifuge at high speed for 15 min, ultrafiltration to remove small molecule impurities; then add 500 µL of Tris-HCl solution (4.2.9) to the ultrafiltration centrifuge tube, and rinse the ultrafiltration membrane is centrifuged at a high speed of 10 000 r/min to about.200 µL of the remaining liquid on the membrane. The remaining volume can be read from the scale on the side wall of the ultrafiltration tube.

Collect the liquid on the membrane into a 1.5 mL low protein adsorption centrifuge tube (5.5).

7.2 Alkylation and enzymatic hydrolysis

Add 150 µL of ammonium bicarbonate solution (4.2.1) and 10 µL of dithiothreitol solution (4.2.2) to the above centrifuge tube, mix well and set Constant temperature water bath at 75 °C for 30 min; cool to room temperature, add 30 µL iodoacetamide solution (4.2.3), let stand in the dark for 30 min; add Into 10 µL calcium chloride solution (4...