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GB 5009.259-2023 PDF English

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GB 5009.259-2023: National food safety standard - Determination of biotin in foods
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GB 5009.259: Historical versions

Standard IDUSDBUY PDFDeliveryStandard Title (Description)Status
GB 5009.259-2023260 Add to Cart Auto, 9 seconds. National food safety standard - Determination of biotin in foods Valid
GB 5009.259-201675 Add to Cart Auto, 9 seconds. National food safety standard -- Determination of free biotin in foods for infants and young children, milk and milk products Obsolete

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GB 5009.26   GB 5009.260   GB 5009.258   GB 5009.268   

GB 5009.259-2023: National food safety standard - Determination of biotin in foods

---This is an excerpt. Full copy of true-PDF in English version (including equations, symbols, images, flow-chart, tables, and figures etc.), auto-downloaded/delivered in 9 seconds, can be purchased online: https://www.ChineseStandard.net/PDF.aspx/GB5009.259-2023
GB NATIONAL STANDARD OF THE PEOPLE’S REPUBLIC OF CHINA National Food Safety Standard - Determination of Biotin in Foods Issued on. SEPTEMBER 6, 2023 Implemented on. MARCH 6, 2024 Issued by. National Health Commission of the People’s Republic of China; State Administration for Market Regulation.

Table of Contents

Foreword... 3 1 Scope... 4 Method I - Liquid Chromatography - Tandem Mass Spectrometry... 4 2 Principle... 4 3 Reagents and Materials... 4 4 Instruments and Equipment... 6 5 Analytical Procedures... 6 6 Expression of Analysis Results... 9 7 Precision... 10 8 Others... 10 Method II - Microbiological Method... 10 9 Principle... 10 10 Reagents and Materials... 10 11 Instruments and Equipment... 12 12 Analytical Procedures... 13 13 Expression of Analysis Results... 18 14 Precision... 19 15 Others... 19 Appendix A Mass Spectrum Scan and MRM Chromatogram of Biotin Standard Solution... 20 Appendix B Preparation of Culture Medium... 21

Foreword

This Standard serves as a replacement of GB 5009.259-2016 National Food Safety Standard - Determination of Biotin in Foods. In comparison with GB 5009.259-2016, the main changes are as follows. ---Method 1 “Liquid Chromatography - Tandem Mass Spectrometry” is added, and the “Microbiological Method” is adjusted to Method 2; ---The microplate culture method is added to Method 2 - “Microbiological Method”; ---The scope of application, sample category and pre-treatment method of the Standard are modified; ---The expression of the drawing of standard curve and the preparation of sample extracting solution is modified; ---The detection limit, quantitation limit and linear range are modified. National Food Safety Standard - Determination of Biotin in Foods

1 Scope

This Standard specifies the methods for the determination of biotin in foods. Method 1 - liquid chromatography - tandem mass spectrometry is applicable to the determination of biotin in prepared milk powder and special dietary foods. Method 2 - microbiological method is applicable to the determination of biotin in foods. Method I - Liquid Chromatography - Tandem Mass Spectrometry

2 Principle

The specimen is dissolved and extracted, and the starch-containing specimen is subject to enzymatic hydrolysis by amylase, protein precipitation and centrifugal filtration, and separated on a C18 reversed-phase chromatography column. Adopt the liquid chromatography - tandem mass spectrometry multi-ion reaction monitoring mode for detection, and the isotope dilution internal standard method for quantitative determination.

3 Reagents and Materials

Unless it is otherwise specified, the reagents used in this Method are all analytically pure, and the water is Grade-1 water specified in GB/T 6682. 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 Instruments and Equipment

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 Analytical Procedures

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.

6 Expression of Analysis Results

The content of biotin in the specimen is calculated in accordance with Formula (1).

7 Precision

The absolute difference between the results of two independent determinations obtained under repeatability conditions shall not exceed 15% of the arithmetic mean.

8 Others

When the sampling size is 5.0 g and the constant volume is 50 mL, the detection limit of this Method is 0.300 g/100 g, and the quantitation limit is 1.00 g/100 g.

9 Principle

Biotin is an essential nutrient for the growth of Lactiplantibacillus plantarum. In the biotin determination culture medium, the growth of Lactiplantibacillus plantarum is correlated with the biotin content.

10 Reagents and Materials

Unless it is otherwise specified, the reagents used in this Method are all analytically pure, and the water is Grade-2 or Grade-1 water specified in GB/T 6682. 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 Instruments and Equipment

Except for the conventional sterilization and culture equipment of microbiology laboratory, other equipment and materials are as follows. 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. ......
Source: Above contents are excerpted from the full-copy PDF -- translated/reviewed by: www.ChineseStandard.net / Wayne Zheng et al.
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