GB 5009.33-2025National food safety standard - Determination of nitrite and nitrate in foods (English PDF)

3.1 Reagents

3.1.1 Acetic acid (CH3COOH). chromatographic pure.

3.1.2 Potassium hydroxide (KOH).

3.2 Preparation of reagents

3.2.1 Acetic acid solution (3%). Measure 3 mL of acetic acid into a 100 mL volumetric flask; use water to dilute to the mark; mix well.

3.2.2 Potassium hydroxide solution (1 mol/L). Weigh

5.6 g of potassium hydroxide; dissolve it in freshly boiled and cooled water; dilute to 100 mL; mix well.

3.2.3 Sodium hydroxide solution (1 mol/L). Weigh

4.0 g of sodium hydroxide; add freshly boiled and cooled water to dissolve it; dilute to 100 mL; mix well.

3.3 Standards

3.3.1 Sodium nitrite standard (NaNO2, CAS No.. 7632-00-0). purity >= 99%. Or nitrite standards that have been certified by the state and granted a standard substance certificate.

3.3.2 Sodium nitrate standard (NaNO3, CAS No.. 7631-99-4). purity >= 99%. Or nitrate standards that have been certified by the state and granted a standard substance certificate.

3.4 Preparation of standard solution

3.4.1 Sodium nitrite standard stock solution (100 mg/L). Accurately weigh 0.100 0 g of sodium nitrite dried to constant weight at 110 °C~120 °C; use water to dissolve and dilute to 1 000 mL; mix well; store in a refrigerator at 2 °C ~ 6 °C. The validity period is 3 months.

3.4.2 Sodium nitrate standard stock solution (100 mg/L). Accurately weigh 0.100 0 g of sodium nitrate dried to constant weight at 110 °C ~ 120 °C; use water to dissolve and dilute to 1 000 mL; mix well; store in a refrigerator at 2 °C ~ 6 °C. The validity period is 3 months.

3.4.3 Mixed standard intermediate solution of sodium nitrite and sodium nitrate (

2.00 mg/L). Accurately transfer

2.00 mL of each of the standard stock solutions of sodium nitrite and sodium nitrate into 100 mL volumetric flasks; use water to dilute to the mark. Prepare when necessary.

3.4.4 Mixed standard solution of sodium nitrite and sodium nitrate. Transfer the intermediate solution of the mixed standard of sodium nitrite and sodium nitrate; add water to dilute it stepwise to prepare a series of standard solutions with mass concentrations of 0 mg/L, 0.010 0 mg/L, 0.020 0 mg/L, 0.050 0 mg/L,

0.100 mg/L,

0.200 mg/L,

0.500 mg/L,

2.00 mg/L. Prepare when necessary.

Note. The range of the standard curve can be adjusted according to the content of the target analyte in the sample, and it shall include at least 5 concentration levels (excluding zero).

3.5 Materials

3.5.1 Membrane needle filter. water-based, 0.22 µm.

3.5.2 C18 purification column. 1 mL, or equivalent column. Before use, activate with 10 mL of methanol and 15 mL of water in sequence.

3.5.3 Ag purification column. column capacity

2.0 ion equivalent ~

2.2 ion equivalent, or equivalent column. Use 10 mL of water to activate before use.

3.5.4 Na purification column. column capacity

2.0 ion equivalent ~

2.2 ion equivalent, or equivalent column. Use 10 mL of water to activate before use.

5.1.1 Sample preparation

5.1.1.1 Liquid or semi-solid samples. Take an appropriate amount of sample and mix it thoroughly by stirring or shaking.

5.1.1.2 Solid powder samples. Take an appropriate amount of sample and mix it thoroughly by means of shaking or other methods.

5.1.1.3 Cheese. Take an appropriate amount of sample and grind it into a uniform slurry. To avoid moisture loss, excessive heat shall be avoided during the grinding process.

5.1.1.4 Other solid samples. Take an appropriate amount of sample and crush it into uniform powder or make it into slurry for later use.

5.1.2 Sample storage

5.1.2.1 Perishable samples such as fruits, vegetables, meat, and aquatic products shall be stored at -18 °C or below.

5.1.2.2 Other samples shall be stored in accordance with the product's storage conditions.

5.2 Sample extraction

5.2.1 Milk and dairy products, infant formula, infant formula for special medical purposes, formula for special medical purposes, bird's nest and its products. Weigh 10 g of liquid sample and 1 g ~

2.5 g of other samples (accurate to

0.01 g); place them in a 150 mL stoppered conical flask; add 80 mL of water; shake well; sonicate for 30 min (heat if necessary to dissolve or fully swell); remove and place at room temperature; quantitatively transfer to a 100 mL volumetric flask; add 2 mL of 3% acetic acid solution (the amount can be adjusted appropriately according to the precipitation effect); add water to dilute to the mark; mix well. Transfer the solution into a 250 mL stoppered iodine flask and place it at 4 °C for 20 min; remove the flask and allow it to cool to room temperature; take a portion of the solution and filter it through filter paper; centrifuge the filtrate at 10 000 r/min for 15 min; keep the supernatant for later use.

5.2.2 High starch content samples that are easy to gelatinize. Weigh

2.5 g (accurate to

0.01

g) of the sample; place it in a 150 mL stoppered conical flask; add

0.2 g of alpha- amylase (the amount of enzyme can be adjusted according to the starch content); add 80 mL of water; shake well; enzymatically hydrolyze at 55 °C for 30 min. Follow the steps in section

5.2.1 starting from "Remove and let stand at room temperature".

5.2.3 Other samples. Weigh 2 g ~ 10 g (accurate to

0.01

g) of the sample; place it in a 150 mL stoppered conical flask; add 80 mL of water; extract by sonication for 30 min; shake once every 5 min to keep the sample completely dispersed. Place in a 75 °C water bath for 5 minutes; then follow the steps in section

5.2.1 starting from "Remove and let stand to room temperature".

5.3 Sample purification Take about 15 mL of the solution prepared in 5.2.1 ~

5.2.3 and pass it through a 0.22 µm aqueous filter needle filter and a C18 column; discard the first 3 mL (if the chloride ion concentration is greater than 100 mg/L or the target substance interferes, it is necessary to pass it through the needle filter, C18 column, Ag column and Na column in sequence; discard the first 7 mL); collect the eluent for analysis.

5.4 Blank test Except for not adding a sample, the other operations are the same as those for adding a sample.

5.5 Apparatus reference conditions

5.5.1 Column. a high-capacity anion exchange column with hydroxide selectivity, compatible with gradient elution, using a divinylstyrene-ethylstyrene copolymer matrix and alkanoyl quaternary ammonium salt functional groups, 4 mm × 250 mm (with a guard column of 4 mm × 50 mm), or an ion chromatography column with equivalent performance.

5.5.2 Eluent. Potassium hydroxide solution or sodium hydroxide solution, with a concentration of 6 mmol/L ~ 70 mmol/L. The elution gradients shall be 6 mmol/L for 30 min, 70 mmol/L for 5 min, and 6 mmol/L for 5 min; the flow rate shall be

1.0 mL/min. When chromatographic interference exists, the following elution methods can be selected. Potassium hydroxide solution or sodium hydroxide solution, with a concentration of 5 mmol/L ~ 50 mmol/L. The elution gradients shall be 5 mmol/L for 33 min, 50 mmol/L for 5 min, and 5 mmol/L for 5 min; the flow rate shall be

5.5.3 Detector. Conductivity detector, with a detection cell temperature of 35 °C; or ultraviolet detector, with a detection wavelength of 226 nm.

5.5.4 Injection volume. 50 µL (can be adjusted according to the content of the analyte ions in the sample).

5.6 Determination

5.6.1 Preparation of the standard curve Inject the standard series of working solutions into the ion chromatograph (see Appendix A, Figure A.1 for the standard chromatograms); plot the standard curve with the concentration of the standard working solution on the x-axis and the peak height or peak area on the y-axis.

5.6.2 Determination of sample solution Inject the blank and sample solutions into the ion chromatograph to obtain the peak height or peak area of the blank and sample solutions. Obtain the mass concentration of sodium nitrite or sodium nitrate in the test solution according to the standard curve.