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GB 5009.259-2023 English PDF (GB5009.259-2023)

GB 5009.259-2023 English PDF (GB5009.259-2023)

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GB 5009.259-2023: National food safety standard - Determination of biotin in foods
GB 5009.259-2023
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
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
3.1.1 Formic acid (HCOOH). chromatographically pure.
3.1.2 Acetonitrile (CH3CN). chromatographically pure.
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
3.2.1 0.1% formic acid-10 mmol/L ammonium formate aqueous solution. weigh-take 0.63 g of
ammonium formate, use 100 mL of water to dissolve it, then, transfer it into a 1,000 mL reagent
bottle, add 1 mL of formic acid, use water to dilute to 1,000 mL, shake it well and reserve it for
later use.
3.2.2 Sodium hydroxide solution (2 mol/L). weigh-take 8.00 g of sodium hydroxide in a beaker,
add 100 mL of water to dissolve it, shake it well and reserve it for later use.
3.2.3 Ethanol solution (50%). accurately measure-take 500 mL of ethanol into a 1,000 mL
reagent bottle, use water to dilute it to 1,000 mL, shake it well and reserve it for later use.
3.3 Reference Material
3.3.1 Biotin reference material (C10H16N2O3S). CAS. 58-85-5, purity  98%, or a standard
substance certified by the state and awarded a reference material certificate.
3.3.2 Biotin-D4 (C10D4H12N2O3S). CAS. 1217850-77-5, purity  98%.
3.4 Preparation of Standard Solutions
3.4.1 Biotin standard stock solution (100 g/mL). in accordance with purity conversion,
accurately weigh-take 10.00 mg of biotin reference material (accurate to 0.01 mg), use ethanol
solution (50%) to dissolve it and reach a constant volume of 100 mL. Transfer the solution to a
brown glass bottle, seal and store it at 20 C. It shall remain valid for 3 months.
3.4.2 Biotin standard intermediate solution (10 g/mL). accurately draw-take 5.00 mL of biotin
standard stock solution (100 g/mL) into a 50 mL volumetric flask; use ethanol solution (50%)
to reach a constant volume of 50 mL. Transfer the solution to a brown glass bottle, seal and
store it at 20 C. It shall remain valid for 3 months.
3.4.3 Biotin standard intermediate solution (1 g/mL). accurately draw-take 1.00 mL of biotin
standard intermediate solution (10 g/mL) into a 10 mL volumetric flask; use ethanol solution
(50%) to reach a constant volume of 10 mL. Transfer the solution to a brown glass bottle, seal
and store it at 20 C. It shall remain valid for 3 months.
3.4.4 Biotin standard working solution (100 ng/mL). accurately draw-take 1.00 mL of the
standard intermediate solution (1 g/mL) and use mobile phase A to reach a constant volume
of 10 mL. Transfer the solution to a brown glass bottle, seal and store it at 4 C. It shall remain
valid for 1 month.
3.4.5 Biotin standard working solution (10 ng/mL). accurately draw-take 1.00 mL of biotin
standard working solution (100 ng/mL) and use mobile phase A to reach a constant volume of
10 mL. Prepare it right before use.
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
5.2.1 Starch-free specimens
Accurately weigh-take 2 g ~ 5 g (accurate to 0.001 g) of sample and place it in a 50 mL
centrifuge tube. Add 750 L of isotope internal standard working solution, then, add 30 mL of
warm water (35 C ~ 40 C); oscillate and evenly mix it, and conduct ultrasonic extraction for
15 min. After taking it out, quickly cool it to room temperature, and use perchloric acid to adjust
pH to about 1.6.At 4 C, at 8,500 r/min, centrifuge for 10 min. After filtering through glass
fiber, use sodium hydroxide solution to adjust pH to 4.6  0.1.Transfer the sample solution to
a 50 mL volumetric flask, use water to reach a constant volume to the scale and evenly mix it.
Then, transfer-take 1.5 mL of the extracting solution to a 2 mL centrifuge tube. At 10,000 r/min,
centrifuge for 10 min. Filter the sample solution through a 0.22 m water-based filter membrane
and analyze it on the machine.
5.2.2 Starch-containing specimens
Accurately weigh-take 2 g ~ 5 g (accurate to 0.001 g) of sample and place it in a 50 mL
centrifuge tube. Add 1% of the sample amount of amylase and 750 L of isotope internal
standard working solution, then, add 30 mL of warm water (35 C ~ 40 C), oscillate and evenly
mix it. Place it in a 50 C ~ 60 C incubator for about 30 min, take it out and perform ultrasonic
extraction for 15 minutes. After taking it out, quickly cool it to room temperature, and use
perchloric acid to adjust pH to about 1.6.At 4 C, at 8,500 r/min, centrifuge for 10 min. After
filtering through glass fiber, use sodium hyd...
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