GB 5009.259-2023 English PDF

GB 5009.259-2023: National food safety standard - Determination of biotin in foods
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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 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.
5.3 Reference Conditions of Instrument Determination
X---the content of biotin in the specimen, expressed in (g/100 g);
ρ---the mass concentration of biotin in the specimen calculated based on the standard curve,
expressed in (ng/mL);
V---the final constant volume of the specimen solution, expressed in (mL);
m---the mass of the specimen, expressed in (g);
100---the conversion factor for the specimen weight per 100 g;
1,000---the conversion factor for converting ng into g in the specimen.
The results shall retain 3 significant figures.
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.
Method II - Microbiological Method
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. In accordance with the standard curve of biotin content and absorbance,
calculate the biotin content in the specimen.
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.
10.2.2 Lactobacillus broth culture medium. see B.2 in Appendix B.
10.2.3 Medium for biotin determination. see B.3 in Appendix B.
NOTE. commercially available synthetic media can be used and operated in accordance with the
instructions.
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 (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.
10.4.3 Sulfuric acid solution B (1.0 mol/L). measure-take 54.4 mL of sulfuric acid, add it to
water and reach a constant volume of 1,000 mL.
10.4.4 Sulfuric acid solution C (0.5 mol/L). measure-take 27.2 mL of sulfuric acid, add it to
water and reach a constant volume of 1,000 mL.
10.4.5 Sodium hydroxide solution A (10 mol/L). weigh-take 400 g of sodium hydroxide, 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.
NOTE. when preparing sulfuric acid solution, prepare in a fume hood, wear gloves and pay
attention to safety. Slowly inject concentrated sulfuric acid into water and keep stirring it.
10.5 Reference Material
11.8 Refrigerator. 2 C ~ 8 C.
11.9 Sterile microplate.
11.10 Quantitative filter paper. with a diameter of 90 mm.
11.11 Test tube. 18 mm  180 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.
11.17 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.
NOTE. the cleaned glassware and metal appliances shall be dried at 200 C ~ 250 C for 1 h ~ 2 h.
12 Analytical Procedures
12.1 Preparation of Test Bacterial Suspension
12.1.1 Use an inoculating needle to pierce the strain of Lactiplantibacillus plantarum to the
lactobacillus agar culture medium, at 36 C  1 C, culture for 20 h ~ 24 h. After taking it out,
put it in the refrigerator and preserve it. It shall remain valid for 1 month. Propagate at least
once a month and store it as a reserve strain.
12.1.2 Inoculate the reserve strain to the lactobacillus agar culture medium, at 36 C  1 C,
culture for 20 h ~ 24 h to activate the strain and use it for the preparation of the inoculum
solutions. The reserve strain that has been stored for more than several weeks cannot be
immediately used to prepare the inoculum solutions. Before the test, continuously propagate
for 2 ~ 3 generations to ensure the viability of the strain.
12.1.3 Sub-cultivate the activated strain within 24 h to sterilized lactobacillus broth, at 36 C 
1 C, culture it for 16 h ~ 20 h. After taking it out, centrifuge the bacterial suspension and
discard the supernatant. Add 10 mL of physiological saline, use a vortex mixer to oscillate the
suspension. At 3,000 r/min ~ 5,000 r/min, centrifuge for 5 minutes, and discard the supernatant.
After repeating the above-mentioned operation for 2 ~ 3 times of cleaning, add 10 mL of
physiological saline and thoroughly mix it. Draw-take an appropriate amount of the bacterial
suspension into 10 mL of physiological saline and evenly mix it to prepare a test bacterial
suspension.
12.1.4 Use physiological saline as a blank, use a spectrophotometer to determine the
transmittance T (%) of the test bacterial suspension at a wavelength of 550 nm, and adjust the
amount of the above-mentioned bacterial solution added, so that the transmittance of the test
bacterial suspension is between 60% and 80%.
12.2 Specimen Extraction
NOTE. 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,...