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GB 5009.259-2023English260 Add to Cart 0-9 seconds. Auto delivery. National food safety standard - Determination of biotin in foods GB 5009.259-2023 Valid GB 5009.259-2023
GB 5009.259-2016English75 Add to Cart 0-9 seconds. Auto delivery. National food safety standard -- Determination of free biotin in foods for infants and young children, milk and milk products GB 5009.259-2016 Obsolete GB 5009.259-2016



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GB 5009.259-2023: PDF in English
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, 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.
12.2.1 Solid specimens. accurately weigh-take the specimen (accurate to 0.001 g) and place it
in a 250 mL conical flask. Specifically speaking, 2 g ~ 5 g of fresh fruit and vegetable specimens;
0.2 g ~ 1 g of cereals, beans, nuts, offal, raw meat and dried specimens; 1 g ~ 3 g of special
dietary foods; 2 g ~ 3 g of milk powder and rice flour specimens; 0.2 g ~ 1 g of general nutrient
supplements or other foods.
12.2.2 Liquid beverages or liquid and semi-liquid specimens. weigh-take 5 g ~ 10 g of specimen
(or use a one-mark pipette to draw-take an appropriate volume) and place it in a 250 mL conical
flask. Special sports beverages do not need to be processed after sample weighing. After
weighing the sample, directly reach a constant volume of 100 mL (V); in accordance with 12.2.4,
dilute it.
12.2.3 In accordance with the specimen matrix, add 50 mL of sulfuric acid solution. for fortified
foods, such as. special dietary foods and prepared milk powder, add sulfuric acid solution C
(0.5 mol/L); for plant-derived foods, add sulfuric acid solution B (1.0 mol/L); for animal-
derived foods, add sulfuric acid solution A (3.0 mol/L).
Put the above-mentioned mixture into a pressure steam sterilizer, at 121 C, hydrolyze it for 30
min. Take it out and quickly cool it to room temperature in a water bath. Use sodium hydroxide
solution A and sodium hydroxide solution B to adjust pH to 4.5  0.2, transfer it to a 250 mL
volumetric flask, and use water to reach a constant volume to the scale (V1). Use quantitative
filter paper to filter it. The first 10 mL of filtrate shall be discarded. Draw-take 5 mL (V2) of the
filtrate into a 100 mL beaker and use sodium hydroxide solution B to adjust pH to 6.8  0.2,
transfer it to a 100 mL volumetric flask, and use water to dilute to the scale (V).
12.2.4 Dilution. in accordance with the biotin content in the specimen, use water to
appropriately dilute the extracting solution (f), so that the mass concentration of biotin in the
specimen extracting solution after dilution is 0.1 ng/mL ~ 0.2 ng/mL.
cooling, the sterilization test tubes shall not be too close to the inner wall of the sterilizer, and
the test tubes shall not be placed too densely, so as to avoid affecting air circulation.
12.3.1.4 Inoculation
Under sterile conditions, respectively add 1 drop (50 L ~ 100 L) of the test bacterial solution
to each of the above-mentioned tubes (except the sample blank tube and standard curve
uninoculated blank tube UN), cover them, and thoroughly oscillate to evenly mix all the test
tubes.
12.3.1.5 Culture
Put the test tubes into a constant-temperature incubator, at 36 C  1 C, culture for 18 h ~ 24
h.
12.3.1.6 Determination
After the culture is completed, visually inspect each test tube. The culture solution in the
uninoculated blank test tube (UN) shall be clear. There shall be a gradient difference in the
concentration of the culture solution in the standard curve series of tubes and the specimen
series of tubes. If the uninoculated blank test tube (UN) is turbid, then, the determination shall
be deemed as invalid.
12.3.1.6.1 Use the uninoculated blank test tube (UN) as a blank, adjust the spectrophotometer
absorbance (A) to 0%, and read the reading of the inoculated blank test tube (IN). Then, use the
inoculated blank test tube (IN) as the blank and adjust the absorbance to 0%.
12.3.1.6.2 Use a vortex mixer to thoroughly mix each test tube, then, immediately transfer the
culture solution into a cuvette, at a wavelength of 550 nm, determine the absorbance. After the
reading is stable, read the absorbance value. The stabilization time of each test tube shall be the
same. Successively read the absorbance of other test tubes. Culture tubes whose absorbance
exceeds the range of standard curve series of tubes S1 ~ S8 shall be discarded.
Take the concentration of the biotin reference material as the x-coordinate and the absorbance
as the y-coordinate to draw a standard curve.
12.3.1.6.3 The absorbance of the sample blank tube shall be read together. If the absorbance of
the sample blank tube is greater than 0.02, the absorbance value of a culture tube added with 1
mL of specimen extracting solution shall be subtracted by 1/5 of the absorbance value of the
sample blank tube; the absorbance value of a culture tube added with 2 mL of specimen
extracting solution shall be subtracted by 2/5 of the absorbance value of the sample blank tube,
and so on, which is used as the basis for calculation results.
12.3.2 Microplate culture method
12.3.2.1 Standard curve series of centrifuge tubes
Filter and sterilize the biotin standard curve working solution into a sterile centrifuge tube under
X---the content of biotin in the specimen, expressed in (g/100 g) or (g/100 mL);
ρ---the total average value of mass concentration of biotin in the specimen extracting solution
obtained through calculation, expressed in (ng/mL);
V---the constant volume of the specimen extracting solution, expressed in (mL);
m---the mass or volume of the specimen, expressed in (g) or (mL);
V1---the constant volume before filtration, expressed in (mL);
V2---the volume of the filtrate drawn after filtration, expressed in (mL);
f---the dilution factor of the specimen extracting solution;
100---conversion factor;
1,000---conversion factor.
The results shall retain 3 significant figures.
NOTE 1.for samples that do not require extraction, remove V1 and V2 from the Formula.
NOTE 2.if the biotin content in the sample is relatively high, it can be appropriately further diluted;
if the content is relatively low, the sampling size of the sample can be appropriately
increased, or the dilution factor can be reduced.
14 Precision
The absolute difference between the results of two independent determinations obtained under
repeatability conditions shall not exceed 20% of the arithmetic mean.
15 Others
15.1 For samples that need to be extracted. when the sampling size is 2 g, the quantitation limit
of this Standard is 2.5 g/100 g.
15.2 For samples that do not need to be extracted, for example, special-purpose beverages.
when the sampling size is 10 g, the quantitation limit is 0.1 g/100 g.
......

BASIC DATA
Standard ID GB 5009.259-2023 (GB5009.259-2023)
Description (Translated English) (National food safety standards Determination of sialic acid in food)
Sector / Industry National Standard
Classification of Chinese Standard X09
Word Count Estimation 15,118
Date of Issue 2023-09-06
Date of Implementation 2024-03-06
Issuing agency(ies) National Health Commission of the People's Republic of China, State Administration for Market Regulation
Summary This standard specifies the determination method of sialic acid in food. The first method of this standard, liquid chromatography-ultraviolet detection, is applicable to the determination of bound sialic acid in bird's nests and its products. The second method, liquid chromatography-fluorescence detection, and the third method, liquid chromatography-mass spectrometry/mass spectrometry, are applicable to Determination of sialic acid in liquid milk, milk powder, cakes and beverages.