GB 5009.259-2023National food safety standard - Determination of biotin in foods (English PDF)

3.1 Reagents and Materials

3.1.3 Ethanol (CH3CH2OH). chromatographically pure.

3.1.4 Ammonium formate (HCOONH4). chromatographically pure.

3.1.5 Perchloric acid (HClO4). 70% ~ 72%.

3.1.6 Sodium hydroxide (NaOH). purity  99.9%.

3.1.7 Amylase. Taka-amylase, CAS. 9001-19-8, enzyme activity  100 U/mg.

3.2 Preparation of Reagents

3.3 Reference Material

3.5 Preparation of Isotope Internal Standard Solutions

3.6 Preparation of Standard Series of Working Solutions

Respectively and accurately draw-take an appropriate amount of biotin standard working

solution into 10 mL volumetric flasks, accurately add 150 L of biotin-D4 isotope internal

standard working solution (1 g/mL), use mobile phase A to reach a constant volume to the

scale, so that the mass concentration of the standard series of biotin is respectively. 1.0 ng/mL,

2.0 ng/mL, 5.0 ng/mL, 10 ng/mL, 15 ng/mL, 20 ng/mL and 25 ng/mL. The mass concentration

of isotope in each series of solution is 15 ng/mL. Prepare them right before use.

4.1 Liquid chromatograph - tandem mass spectrometer.

4.2 Balance. with a division value of 0.001 g and 0.01 mg, respectively.

4.4 pH meter. with an accuracy of 0.01.

4.5 Ultrasonic oscillator.

4.6 Vortex mixer. 600 r/min ~ 3,200 r/min.

4.8 2 mm aperture test sieve (optional).

5.1 Sample Preparation

5.1.1 Solid samples

The sampling size needs to be greater than 0.5 kg. Use a high-speed pulverizer to pulverize

non-uniform samples, until all of them pass through a 2 mm aperture test sieve. After evenly

mixing, divide the samples to 100 g and store in a wide-mouth bottle. Seal it and reserve it for

testing. For uniform samples, directly evenly mix them and reserve them for testing.

5.1.2 Semi-solid samples

The sampling size needs to be greater than 0.5 kg. At least 3 packages (from the same batch)

need to be collected. After all samples are homogenized and evenly mixed in a container, store

any 100 g of them in a wide-mouth bottle. Seal it and reserve it for testing.

5.1.3 Liquid samples

The sampling size needs to be greater than 0.5 L. At least 3 packages (from the same batch)

need to be collected. After all samples are evenly mixed in a container, store any 100 mL of

them in a wide-mouth bottle. Seal it and reserve it for testing.

5.2 Pre-treatment of Samples

5.3 Reference Conditions of Instrument Determination

5.4 Qualitative Determination

The selected ions all appear, and in the same detection batch, compared with the relative

abundance of the standard solution of an equivalent concentration, the relative abundance of

two daughter ions of biotin in the sample shall have an allowable deviation of not exceeding

the range specified in Table 3.

5.5 Drawing of Standard Curve

In accordance with the concentration from low to high, successively inject the biotin standard

series of working solutions into the liquid chromatograph - tandem mass spectrometer, take the

ratio of biotin concentration to biotin internal standard concentration as the x-coordinate, and

the peak area ratio of biotin to biotin internal standard as the y-coordinate to draw a standard

curve of biotin.

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%.

10.4 Preparation of Reagents

10.4.1 Ethanol solution (50%). measure-take 500 mL of absolute ethanol, add it to water and

reach a constant volume of 1,000 mL.

10.4.2 0Sulfuric acid solution A (3.0 mol/L). measure-take 163.2 mL of sulfuric acid, add it to

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

10.4.6 Sodium hydroxide solution B (0.1 mol/L). draw-take 10 mL of sodium hydroxide

solution A (10 mol/L), add water to reach a constant volume of 1,000 mL.

10.4.7 Sterile physiological saline. weigh-take 8.5 g of sodium chloride and dissolve it in 1,000

mL of distilled water, divide it into stoppered test tubes, with 10 mL in each tube. At 121 C,

perform autoclaved sterilization for 15 min.

10.5 Reference Material

Biotin reference material (C10H16N2O3S). CAS. 58-85-5, 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 Biotin standard stock solution (100 g/mL). place the biotin reference material in a

desiccator containing phosphorus pentoxide and dry it overnight. In accordance with purity,

weigh-take it, so that the biotin content is 50 mg (accurate to 0.1 mg), use ethanol solution (50%)

to dissolve it and reach a constant volume of 500 mL.

11.1 Analytical balance. with a division value of 0.001 g and 0.1 mg, respectively.

11.2 Centrifuge. 3,000 r/min ~ 5,000 r/min.

11.3 Vortex mixer.

11.4 pH meter. with an accuracy of 0.01.

11.9 Sterile microplate.

11.10 Quantitative filter paper. with a diameter of 90 mm.

11.12 Volumetric flask. with a capacity of 100 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.

11.19 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.

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