GB 5009.89-2023National food safety standard - Determination of niacin and niacinamide in foods (English PDF)

3.1 Reagents

3.1.1 Hydrochloric acid (HCl). guaranteed reagent.

3.1.2 Sodium hydroxide (NaOH). guaranteed reagent.

3.2 Preparation of Reagents

3.2.1 Hydrochloric acid solution (5.0 mol/L). measure-take 415 mL of hydrochloric acid and

add water to reach a constant volume of 1,000 mL.

3.2.2 Hydrochloric acid solution (0.1 mol/L). draw-take 8.3 mL of hydrochloric acid and add

water to reach a constant volume of 1,000 mL.

3.3 Reference Materials

3.3.1 Niacin (C6H5NO2, CAS. 59-67-6). purity  98%, or a standard substance certified by the

state and awarded a reference material certificate.

3.4 Preparation of Standard Solutions

3.4.2 Niacin and niacinamide standard mixed intermediate solution (100.0 g/mL). respectively

and accurately draw-take 10.0 mL of niacin and niacinamide standard stock solution into a 100 mL volumetric flask, add water to reach a constant volume to the scale, and evenly mix it. It can be stored for 1 month when refrigerated at 2 C ~ 8 C.

3.4.3 Niacin and niacinamide standard mixed working solutions. respectively and accurately

draw-take 1.0 mL, 2.0 mL, 5.0 mL, 10.0 mL and 20.0 mL of the standard mixed intermediate solution into 100 mL volumetric flasks, add water to reach a constant volume to the scale and evenly mix them. Thus, standard mixed working solutions with a mass concentration of 1.0 g/mL, 2.0 g/mL, 5.0 g/mL, 10.0 g/mL and 20.0 g/mL are obtained. Prepare them right before use.

4.1 High performance liquid chromatograph. equipped with UV detector or diode array detector.

4.2 Balance. with a division value of 0.1 mg and 0.01 g, respectively.

4.4 Ultrasonic equipment.

4.5 pH meter. with an accuracy of 0.1.

4.6 Pulverizer.

5.1 Sample Pre-treatment

5.1.1 Specimen preparation

Sample pre-treatment. take at least 200 g of representative sample. For lumpy or granular samples, use a pulverizer to pulverize them; for powdery, pasty or liquid samples, thoroughly mix them and place them in a closed container.

5.1.2 Starches and starch-containing foods

5.1.3 Starch-free foods

5.2 Reference Conditions of Liquid Chromatography

5.3 Drawing of Standard Curve

In accordance with the chromatographic conditions that have been established, successively determine the niacin and niacinamide mixed standard series of determination solutions based on the above recommended chromatographic conditions (see Figure A.1 in Appendix A for the standard sample chromatogram). Record the chromatographic peak area of each component, take the mass concentration of the standard determination solutions as the x-coordinate, and the peak area as the y-coordinate to draw a standard curve.

5.4 Determination of Specimen Solution

Inject the specimen determination solution for determination in accordance with the above recommended chromatographic conditions.

6.1 Calculation of Niacin or Niacinamide Content in the Specimen

The content of niacin or niacinamide in the specimen is calculated in accordance with Formula (1).

6.2 Calculation of Total Niacin and Niacinamide Content in the Specimen

The total content (calculated by niacin) of niacin and niacinamide in the specimen is calculated in accordance with Formula (2).

10.1 Strain

Lactiplantibacillus plantarum (the former Lactobacillus plantarum) ATCC 8014, or equivalent strain.

10.2 Culture Media

10.2.1 Lactobacillus agar culture medium. see B.1 in Appendix B.

10.3 Reagents

10.3.1 Absolute ethanol (C2H5OH).

10.3.2 Sulfuric acid (H2SO4). 95% ~ 98%.

10.3.3 Sodium hydroxide (NaOH).

10.3.4 Sodium chloride (NaCl).

10.4 Preparation of Reagents

10.4.1 Ethanol solution (with a volume fraction of 25%). measure-take 250 mL of absolute

ethanol, add water to reach a constant volume of 1,000 mL.

10.4.2 Sulfuric acid solution A (10 mol/L). measure-take 560 mL of sulfuric acid, add it to water

and dilute it to 1,000 mL.

10.4.3 Sulfuric acid solution B (0.5 mol/L). measure-take 50 mL of sulfuric acid A (10 mol/L), add it to water and dilute it to 1,000 mL.

10.4.4 Sodium hydroxide solution A (10 mol/L). weigh-take 400 g of sodium hydroxide, add

water to dissolve it and dilute to 1,000 mL.

10.5 Reference Materials

10.5.1 Niacin (C6H5NO2, CAS. 59-67-6). purity  98%, or a standard substance certified by the

state and awarded a reference material certificate.

10.5.2 Niacinamide (C6H6N2O, CAS. 98-92-0). purity  98%, or a standard substance certified

by the state and awarded a reference material certificate.

10.6 Preparation of Standard Solutions

10.6.1 Niacin (or niacinamide) standard stock solution (50 g/mL). place niacin (or

niacinamide) reference material into a desiccator containing phosphorus pentoxide and dry it overnight. In accordance with purity, weigh it, so that the niacin (or niacinamide) content is

50.0 mg (accurate to 0.001 g), use ethanol solution (25%) to dissolve it and transfer it to a 1,000 mL volumetric flask, and reach a constant volume to the scale.

10.6.2 Niacin (or niacinamide) standard intermediate solution (500 ng/mL). draw-take 1.0 mL

of niacin (or niacinamide) standard stock solution (50 g/mL) into a 100 mL volumetric flask, use ethanol solution (25%) to reach a constant volume to the scale.

11.1 Analytical balance. with a division value of 0.1 mg.

11.2 Centrifuge.

11.3 Vortex mixer.

11.4 pH meter. with an accuracy of 0.01.

11.12 Volumetric flask. with a capacity of 100 mL, 200 mL, 250 mL and 500 mL.

11.13 One-mark pipette. 1 mL, 5 mL and 10 mL.

11.14 Graduated pipette. 5 mL (with a scale of 0.1 mL).

11.15 Glass funnel. with a diameter of 100 mm.

11.16 Conical flask. with a capacity of 250 mL.

11.17 Beaker. with a capacity of 100 mL.

11.18 Dispenser. 0 mL ~ 10 mL.

11.19 Micropipette. 1,000 L and 200 L.

11.20 Sterile centrifuge tube. 1.5 mL.

11.21 Syringe filter. with an aperture of 0.22 m.

12.1 Preparation of Test Bacterial Suspension

12.2 Specimen Extraction

Lumpy and granular specimens need to be crushed; powdered specimens, such as. milk powder and rice flour, need to be evenly mixed; fruits and vegetables, meat, eggs, fish and animal offal, etc., need to be made into chyme; semi-solid foods need to be homogenized and evenly mixed;

liquid specimens shall be shaken and mixed before use.

12.2.4 Dilution. in accordance with the niacin (or niacinamide) content in the specimen, use

water to appropriately dilute the extracting solution, so that the mass concentration of niacin (or niacinamide) in the specimen extracting solution after dilution is 5 ng/mL ~ 12 ng/mL.

12.3 Specimen Determination

12.3.1 Test tube culture method

12.3.1.2 Specimen series of tubes

In accordance with the sequence listed in Table 2, add water, specimen extracting solution and culture medium for niacin determination to the culture tube. Prepare 3 tubes for each number in the Table.