GB 5009.285-2022 PDF in English
GB 5009.285-2022 (GB5009.285-2022) PDF English
| Standard ID | Contents [version] | USD | STEP2 | [PDF] delivered in | Name of Chinese Standard | Status |
| GB 5009.285-2022 | English | 290 |
Add to Cart
|
0-9 seconds. Auto-delivery.
|
National food safety standard - Determination of Vitamin B12 in foods
| Valid |
Standards related to (historical): GB 5009.285-2022
PDF Preview
GB 5009.285-2022: PDF in English GB 5009.285-2022
NATIONAL STANDARD OF THE
PEOPLE’S REPUBLIC OF CHINA
National food safety standard - Determination of Vitamin
B12 in foods
ISSUED ON: JUNE 30, 2022
IMPLEMENTED ON: DECEMBER 30, 2022
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
2 Principle ... 4
3 Reagents and materials ... 4
4 Instruments and apparatuses ... 6
5 Analysis steps ... 6
6 Description of the analysis results ... 8
7 Precision ... 9
8 Others ... 9
9 Principle ... 9
10 Reagents and materials ... 9
11 Instruments and apparatuses ... 11
12 Analysis steps ... 12
13 Description of the analysis results ... 15
14 Precision ... 15
15 Others ... 15
16 Principle ... 16
17 Reagents and materials ... 16
18 Instruments and equipment ... 18
19 Test procedures ... 19
20 Description of the analysis results ... 21
21 Precision ... 22
22 Others ... 22
Appendix A Immunoaffinity column reference verification method ... 23
Appendix B Liquid chromatogram ... 24
Appendix C Liquid chromatography - mass spectrum ... 25
Appendix D Medium preparation method ... 26
National food safety standard - Determination of Vitamin
B12 in foods
1 Scope
This Standard specifies the determination method of vitamin B12 in foods.
Method I of liquid chromatography applies to the determination of vitamin B12 in foods
for infants and young children, milk and milk products, meat and meat products.
Method II of liquid chromatography - mass spectrometry applies to the determination
of vitamin B12 in foods for infants and young children, milk and milk products, meat
and meat products, ready-to-eat cereals, baked foods, jelly, and beverages.
Method III of microbiological method applies to the determination of vitamin B12 in
foods for infants and young children, milk and milk products.
Method I – Liquid chromatography
2 Principle
After the sample is enzymatically hydrolyzed, use potassium cyanide (or sodium
cyanide) solution to convert cobalamin isomers (hydroxocobalamin, methylcobalamin
and 5-deoxyadenosylcobalamin, etc.) to cyanocobalamin. After the sample solution is
purified and concentrated by the immunoaffinity column, use the reversed-phase liquid
chromatography column for separation, the ultraviolet detector for detection, and the
external standard method for quantification.
3 Reagents and materials
Unless otherwise specified, all the reagents are analytical reagents, and the water is
grade-1 water which is specified by GB/T 6682.
3.1 Reagents
3.1.1 Anhydrous sodium acetate (CH3COONa).
3.1.2 Acetic acid (CH3COOH).
3.1.3 Methanol (CH3OH): chromatographic grade.
3.1.4 Acetonitrile (CH3CN): chromatographic grade.
3.1.5 Trifluoroacetic acid (CF3COOH): chromatographic grade.
3.1.6 Potassium cyanide or sodium cyanide (KCN/NaCN).
3.1.7 Pepsin (CAS number: 9001-75-6, activity ≥ 400 U/mg).
3.1.8 Amylase (activity ≥ 50 U/mg).
3.1.9 Ethanol (C2H6O).
3.2 Preparation of reagents
3.2.1 Ethanol solution (25%): Weigh 250 mL of ethanol; add water to dilute to 1 000
mL; mix well.
3.2.2 Sodium acetate buffer (0.25 mol/L): Weigh 20.5 g of anhydrous sodium acetate;
dissolve in 950 mL of water; use acetic acid to adjust the pH to 4.0±0.1; use water to
dilute to 1 000 mL.
3.2.3 Potassium cyanide (or sodium cyanide) solution (10 mg/mL): Weigh 1.0 g of
potassium cyanide (or sodium cyanide) solid; add an appropriate amount of water to
dissolve; use water to dilute to 100 mL.
Note: Potassium cyanide (or sodium cyanide) is a highly toxic chemical; the operator
must wear protective equipment and prepare or use potassium cyanide (or
sodium cyanide) solution in a fume hood.
3.2.4 Trifluoroacetic acid solution (0.04%, volume ratio): Weigh 500 mL of water; add
200 μL of trifluoroacetic acid; mix well.
3.3 Standard
Vitamin B12 (cyanocobalamin) standard substance (C63H88CoN14O14P, CAS number:
68-19-9): purity ≥99%, or standard substance certified by the nation and granted a
certificate of reference material.
3.4 Preparation of standard solutions
3.4.1 Vitamin B12 standard stock solution (1 mg/mL): Weigh 10 mg (accurate to 0.01
mg) of standard vitamin B12 in a 50 mL beaker; use ethanol solution to dissolve it; then,
transfer it to a 10 mL volumetric flask; use ethanol solution to fix the volume to the
mark; shake well; transfer to a brown reagent bottle; store at -20 °C in the dark. This
solution is valid for 6 months.
3.4.2 Vitamin B12 standard working solution (10 μg/mL): Draw 1.00 mL of vitamin B12
standard stock solution; put it in a 100 mL volumetric flask; use ethanol solution to fix
mL of potassium cyanide (or sodium cyanide) solution in turn; mix well. Put the sample
solution into a water bath constant temperature oscillator; at 37 °C, carry out enzymatic
hydrolysis for 30 minutes (for meat samples, carry out enzymatic hydrolysis for 10 h ~
16 h). After enzymolysis, transfer it to a water bath at 100 °C; keep for 30 minutes; then,
take it out and cool to room temperature. Transfer the sample solution to a 100 mL
volumetric flask; use water to fix the volume to the scale; shake well. Pipette 40 mL of
the above solution into a 50 mL centrifuge tube; centrifuge at 10 000 r/min for 10 min;
take the supernatant and filter through a glass fiber filter paper for later use.
Note: For samples with cyanocobalamin as nutritional enhancer and no other forms of
cobalamin in the background, potassium cyanide (or sodium cyanide) may not
be used during sample extraction.
5.1.3 Purification
Connect the immunoaffinity column to the solid-phase extraction device. After
discarding the buffer in the immunoaffinity column, pipette an appropriate amount of
the above filtrate (containing 10 ng ~ 500 ng of vitamin B12) to pass through the column;
adjust the column passing speed to 2 mL/min ~ 3 mL/min. After the sample solution
has completely passed through the column, use 10 mL of water to rinse the
immunoaffinity column at a steady flow rate and drain. Place a 10 mL glass test tube
under the immunoaffinity column; use 3 mL of methanol to elute in three times; collect
all the eluates; use nitrogen flow to blow slowly below 60 °C until nearly dry; use 0.04%
trifluoroacetic acid solution to fix the volume to 1.0 mL; vortex for 30 s to dissolve the
residue; filter through a 0.22 μm filter; test.
Note: Strictly prohibit the mixture of potassium cyanide (or sodium cyanide) liquid
waste and acid. Add sodium hydroxide to adjust pH >10, and then potassium
permanganate powder (added as per 3% of the liquid waste mass) to decompose
the cyanide; discharge after 24 h.
5.2 Liquid chromatography reference conditions
5.2.1 Chromatographic column: C18 column (column length 150 mm, column inner
diameter 4.6 mm, packing particle size 2.5 μm), or equivalent.
5.2.2 Mobile phase: phase A, 0.04% trifluoroacetic acid solution; phase B, acetonitrile.
5.2.3 Gradient elution: 0 min ~ 6.0 min, 90% A; 6.0 min ~ 8.5 min, 90% ~ 0% A; 8.5
min ~ 14.0 min, 90% A.
5.2.4 Flow velocity: 0.8 mL/min.
5.2.5 Column temperature: 40 °C.
5.2.6 Injection volume: 100 μL.
5.2.7 Detection wavelength: 361 nm.
1 000 – conversion coefficient.
Two significant figures shall be kept for the calculation results.
7 Precision
The absolute difference of two independent test results obtained under repeatability
cannot exceed 15% of the arithmetic mean value.
8 Others
When the sampling amount is 5.00 g, the detection limit of foods for infants and young
children, dairy products, meat and meat products is 0.2 μg/100 g, and the quantification
limit is 0.5 μg/100 g.
Method II – Liquid chromatography - mass spectrometry
9 Principle
After the sample is enzymatically hydrolyzed, use potassium cyanide (or sodium
cyanide) solution to convert cobalamin isomers (hydroxocobalamin, methylcobalamin
and 5-deoxyadenosylcobalamin, etc.) to cyanocobalamin. After the sample solution is
purified and concentrated by the immunoaffinity column, use the reversed-phase liquid
chromatography column for separation, the tandem mass spectrometry for detection,
and the isotope internal standard method for quantification.
10 Reagents and materials
Unless otherwise specified, all the reagents are analytical reagents, and the water is
grade-1 water which is specified by GB/T 6682.
10.1 Reagents
10.1.1 Anhydrous sodium acetate (CH3COONa).
10.1.2 Sodium hydroxide (NaOH).
10.1.3 Acetic acid (CH3COOH).
10.1.4 Acetonitrile (CH3CN): chromatographic pure.
10.1.5 Ammonium acetate (CH3COONH4): chromatographic pure.
10.1.6 Potassium cyanide or sodium cyanide (KCN/NaCN).
10.1.7 Pepsin (CAS number: 9001-75-6, activity ≥ 400 U/mg).
10.1.8 Amylase (activity ≥ 50 U/mg).
10.1.9 Ethanol (C2H6O).
10.2 Preparation of reagents
10.2.1 Ethanol solution (25%): Weigh 250 mL of ethanol; add water to dilute to 1 000
mL; mix well.
10.2.2 Sodium acetate buffer (0.25 mol/L): Weigh 20.5 g of anhydrous sodium acetate;
dissolve in 950 mL of water; use acetic acid to adjust the pH to 4.0±0.1; use water to
dilute to 1 000 mL.
10.2.3 Potassium cyanide (or sodium cyanide) solution (10 mg/mL): Weigh 1.0 g of
potassium cyanide (or sodium cyanide) solid; add an appropriate amount of water to
dissolve; use water to dilute to 100 mL.
Note: Potassium cyanide (or sodium cyanide) is a highly toxic chemical; the operator
must wear protective equipment and prepare or use potassium cyanide (or
sodium cyanide) solution in a fume hood.
10.2.4 Sodium hydroxide solution (1 mol/L): Weigh 4.0 g of sodium hydroxide; add an
appropriate amount of water to dissolve; use water to dilute to 100 mL.
10.2.5 Ammonium acetate solution (2.5 mmol/L): Weigh 0.19 g of ammonium acetate;
add an appropriate amount of water to dissolve; use water to dilute to 1 000 mL.
10.2.6 Acetonitrile solution (90%, volume ratio): Weigh 100 mL of water and add it to
900 mL of acetonitrile; mix well; ultrasonically degas.
10.3 Standard substance
10.3.1 Vitamin B12 (cyanocobalamin) standard substance (C63H88CoN14O14P, CAS
number: 68-19-9): purity ≥99%, or standard substance certified by the nation and
granted a certificate of reference material.
10.3.2 Vitamin B12 isotope internal standard solution (13C7-C63H88CoN14O14P): 1 μg/mL
methanol solution.
10.4 Preparation of standard solutions
10.4.1 Vitamin B12 standard stock solution (1 mg/mL): Weigh 10 mg (accurate to 0.01
mg) of standard vitamin B12 in a 50 mL beaker; use ethanol solution to dissolve it; then,
transfer it to a 10 mL volumetric flask; use ethanol solution to fix the volume to the
mark; shake well; transfer to a brown reagent bottle; store at -20 °C in the dark. This
solution is valid for 6 months.
12 Analysis steps
Note: Avoid ultraviolet light during the operation, and operate as far away from light
as possible.
12.1 Sample pretreatment
12.1.1 Sample preparation
Crush and grind the solid sample, or use a meat grinder to make it into chyme;
homogenize and mix. Shake and mix liquid samples before measurement.
12.1.2 Extraction
12.1.2.1 Foods for infants and young children, dairy products, meat and meat
products, baked foods, ready-to-eat cereals
Weigh 1 g ~ 5 g (accurate to 0.01 g) of the well-mixed sample into a 50 mL centrifuge
tube; add 100 μL of isotope internal standard working solution, 25 mL of sodium acetate
buffer, 0.04 g of pepsin, 0.01 g of amylase and 2 mL of potassium cyanide (or sodium
cyanide) in turn; mix well. Put the sample solution into a water bath constant
temperature oscillator; at 37 °C, shake and carry out enzymatic hydrolysis for 30
minutes (for meat samples, carry out enzymatic hydrolysis for 10 h ~ 16 h). After
enzymolysis, transfer it to a water bath at 100 °C; keep for 30 minutes; then, take it out
and cool to room temperature. Centrifuge at 10 000 r/min for 10 min; take the
supernatant and filter through a glass fiber filter paper for later use.
12.1.2.2 Beverages
Weigh 25 g (accurate to 0.01 g) of the well-mixed sample into a 50 mL centrifuge tube;
add 100 μL of the isotope internal standard working solution; use sodium hydroxide to
adjust the pH to 5 ~ 7 (carbonated beverages need to be degassed in an ultrasonic
oscillator for 10 minutes before pH adjustment). Centrifuge at 10 000 r/min for 10 min;
take the supernatant and filter it through a glass fiber filter paper for later use.
12.1.2.3 Jelly
Weigh 5 g (accurate to 0.01 g) of the mixed sample into a 50 mL centrifuge tube; add
100 μL of isotope internal standard working solution; add 40 mL of water; vortex and
mix; dissolve the sample in a 70 °C water bath; sonicate in a water bath at 50 °C for 20
min; use sodium hydroxide solution to adjust the pH to 5 ~ 7; centrifuge at 10 000 r/min
for 10 min; filter the supernatant through a glass fiber filter paper for later use.
Note: For samples with cyanocobalamin as nutritional enhancer and no other forms of
cobalamin in the background, potassium cyanide (or sodium cyanide) may not
be used during sample extraction.
12.1.3 Purification
Connect the immunoaffinity column to the solid-phase extraction device. After
discarding the buffer in the immunoaffinity column, transfer all the filtrate through the
column, and adjust the passing speed through the column to be 2 mL/min ~ 3 mL/min.
After the sample solution has completely passed through the column, use 10 mL of
water to rinse the immunoaffinity column at a steady flow rate and drain. Place a 10 mL
glass test tube under the immunoaffinity column; use 3 mL of methanol to elute in three
times; collect all the eluates; use nitrogen flow to blow slowly below 60 °C until nearly
dry; use ammonium acetate solution to fix the volume to 1.0 mL; vortex for 30 s to
dissolve the residue; filter through a 0.22 μm filter; test.
Note: Strictly prohibit the mixture of potassium cyanide (or sodium cyanide) liquid
waste and acid. Add sodium hydroxide to adjust pH > 10, and then potassium
permanganate powder (added as per 3% of the liquid waste mass) to decompose
the cyanide; discharge after 24 h.
12.2 Apparatus reference conditions
12.2.1 Liquid chromatography reference conditions
12.2.1.1 Chromatographic column: C18 column (column length 100 mm, column inner
diameter 2.1 mm, packing particle size 1.7 μm), or equivalent.
12.2.1.2 Mobile phase: phase A, ammonium acetate solution (2.5 mmol/L); phase B,
acetonitrile solution (90%, volume ratio).
12.2.1.3 Gradient elution: 0 min ~ 0.5 min, 93% A; 0.5 min ~ 2.0 min, 93% ~ 85% A;
2.0 min ~ 2.5 min, 85% ~ 10% A; 2.5 min ~ 3.0 min, 10% A; 3.0 min ~ 3.5 min, 10%
~ 93% A; 3.5 min ~ 6.0 min, 93% A.
12.2.1.4 Flow rate: 0.3 mL/min.
12.2.1.5 Column temperature: 40 ℃.
12.2.1.6 Injection volume: 10 μL.
12.2.2 Mass spectrometry reference conditions
12.2.2.1 Ionization mode: ESI+.
12.2.2.2 Ion source temperature: 350 °C.
12.2.2.3 Cone hole backflushing gas flow: 50 L/h.
12.2.2.4 Desolvation gas temperature: 650 °C.
12.2.2.5 Desolvation gas flow rate: 900 L/h.
Method III – Microbiological method
16 Principle
Vitamin B12 is an essential nutrient for the growth of Lactobacillus leichmannii. Under
certain conditions, the growth of Lactobacillus leichmannii has a corresponding
relationship with the content of vitamin B12. According to the standard working curve
of vitamin B12 content and light transmittance (or absorbance value), calculate the
content of vitamin B12 in the sample.
17 Reagents and materials
Unless otherwise specified, all the reagents in this method are analytical reagents, and
the water used is grade-2 water specified by GB/T 6682.
17.1 Reagents
17.1.1 Sodium chloride (NaCl).
17.1.2 Anhydrous disodium hydrogen phosphate (Na2HPO4).
17.1.3 Anhydrous sodium metabisulfite (Na2S2O5).
17.1.4 Citric acid monohydrate (C6H8O7·H2O).
17.1.5 Sodium hydroxide (NaOH).
17.1.6 Hydrochloric acid (HCl).
17.1.7 Ethanol (C2H6O).
17.2 Strains
Lactobacillus leichmannii ATCC 7830 or an equivalent strain.
17.3 Standard
Vitamin B12 (cyanocobalamin) standard substance (C63H88CoN14O14P, CAS number:
68-19-9): purity ≥99%, or standard substance certified by the nation and granted a
certificate of reference material.
17.4 Preparation of reagents
17.4.1 Ethanol solution (25%): Weigh 250 mL of ethanol; add water to dilute to 1 000
mL; mix well.
19 Test procedures
19.1 Preparation of test bacteria solution
19.1.1 Strain recovery: After activating the freeze-dried strain of Lactobacillus
leichmannii (ACC 7830), inoculate it on the Lactobacillus agar medium, and culture at
36°C ± 1°C for 18 h ~ 24 h. Then, transfer 2 ~ 3 generations to enhance vitality. Store
in a refrigerator at 2 °C ~ 8 °C for later use. Transfer every 15 days; the number of
passages shall not exceed 15 times.
19.1.2 Bacterial suspension preparation: inoculate the activated strains into the
Lactobacillus broth medium; culture at 36 °C ± 1 °C for 18 h ~ 24 h; centrifuge at 2
000 r/min for 2 min ~ 5 min; discard the supernatant; add 10 mL of 9 g/L sodium
chloride solution; mix well; then, centrifuge for another 2 ~ 5 minutes, as before;
discard the supernatant; add 10 mL of 9 g/L sodium chloride solution; mix well.
19.1.3 Test bacteria solution: Absorb an appropriate amount of bacterial suspension into
10 mL of 9 g/L sodium chloride solution; mix well to prepare a test bacteria solution.
Use a spectrophotometer; use the 9 g/L sodium chloride solution as a blank; detect the
light transmittance of the test bacteria solution at a wavelength of 550 nm; make the
light transmittance of the test bacterial solution 60% ~ 80%.
19.2 Sample pretreatment
19.2.1 Sample preparation
Crush and grind the solid sample; homogenize and mix. Shake and mix the liquid
sample before testing. Refrigerate all prepared samples and measure within one week.
19.2.2 Sample extraction
Weigh a certain amount of sample (accurate to 0.000 1 g, the vitamin B12 contained in
the sample is 50 ng ~ 100 ng) into a 250 mL autoclave bottle; mix it with 10 mL of the
extraction solution; then, add 150 mL of water; shake well; place in a pressure sterilizer
for hydrolysis at 121 °C for 10 min. After cooling, use hydrochloric acid solution (1
mol/L) to adjust the pH to 4.5±0.2; transfer it to a 250 mL volumetric flask; adjust the
volume to the mark. Mix well and filter. Pipette 5 mL of the filtrate; add 20 mL ~ 30
mL of water; use sodium hydroxide (1 mol/L) to adjust the pH to 6.8 ± 0.2; transfer it
to a 100 mL volumetric flask; adjust the volume to the mark; use it as the sample extract.
If necessary, further adjust the dilution ratio (represented by f), so that the mass
concentration of vitamin B12 in the sample extract is 0.01 ng/mL ~ 0.02 ng/mL, and the
mass concentration of sodium metabisulfite is less than 0.03 mg/mL.
19.3 Preparation of the standard curve
19.8.2 Use the uninoculated blank test tube (S1) as a blank; adjust the light
transmittance of the spectrophotometer to 100% (or the absorbance to 0); read the
reading of the inoculated blank test tube (S2). Then, take the inoculated blank test tube
(S2) as a blank; adjust the transmittance to 100% (or the absorbance to 0); read the
transmittance (or absorbance) of each other test tube in turn.
19.8.3 After fully mixing the culture in each test tube with a vortex shaker (or adding a
drop of antifoaming agent), immediately transfer the culture solution into a cuvette for
measurement, with a wavelength of 550 nm. After the reading is stable for 30 s, read
the transmittance; the stabilization time of each test tube shall be the same. The relative
standard deviation of the absorbance values of the 3 tubes at each standard curve
concentration point shall be less than 15%. If the relative standard deviation of the
absorbance of the 3 test tubes is greater than or equal to 15%, discard the standard curve
concentration point (the number of standard curve concentration points discarded shall
not exceed one). Take the vitamin B12 concentration in the standard series tubes as the
abscissa and the light transmittance as the ordinate to draw a standard curve.
19.8.4 Calculate the concentration of vitamin B12 in the to-be-tested solution from the
standard working curve according to the transmittance of the to-be-tested solution;
calculate the content of vitamin B12 in the sample according to the dilution factor and
the weighing sample. Discard the test value whose light transmittance exceeds the range
of the standard curve tubes S3 ~ S10.
19.8.5 Use the light transmittance of each test tube to calculate the concentration of
vitamin B12 per milliliter of each numbered test solution, and the average concentration
of vitamin B12 in the test solution of this number. The concentration measured in each
test tube shall not exceed this 15% of the average value, or shall be discarded. If the
number of tubes that meet this requirement is less than 2/3 of the total number of tubes
for the to-be-tested solution of all four numbers, the data used to calculate the content
of the sample is insufficient and needs to be re-examined. If the number of tubes that
meet the requirements exceeds 2/3 of the original number of tubes, recalculate the
average value of vitamin B12 content per milliliter of test solution in the valid numbered
sample tube, and calculate the total average value of all numbered sample tubes from
this average value as ρ. Use Formula (3) to calculate the content X of vitamin B12 in the
sample.
Note: Draw a standard curve to read both transmittance (T%) and absorbance (A).
20 Description of the analysis results
The content of vitamin B12 (calculated as cyanocobalamin) in the sample is calculated
according to Formula (3):
......
Source: Above contents are excerpted from the PDF -- translated/reviewed by: www.chinesestandard.net / Wayne Zheng et al.
|