GB 5009.285-2022 PDF English
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National food safety standard - Determination of Vitamin B12 in foods
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GB 5009.285-2022: National food safety standard - Determination of Vitamin B12 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/GB5009.285-2022
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
Foreword
This Standard replaces GB 5413.14-2010 National food safety standard -
Determination of vitamin B12 in foods for infants and young children, milk and milk
products.
Compared with GB 5413.14-2010, the major changes of this Standard are as follows.
-- Add Method I Liquid chromatography;
-- Add Method II Liquid chromatography - mass spectrometry;
-- Remove normative references;
-- Modify the original microbiological method to method III.
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
3.3 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
3.5 Materials
4 Instruments and apparatuses
4.1 Liquid chromatograph. equipped with a UV detector.
4.2 Balance. sensitivity 0.01 g, 0.001 g and 0.000 01 g.
4.3 pH meter. accuracy 0.01.
4.5 Ultrasonic cleaner.
4.6 Centrifuge. speed ≥ 10 000 r/min.
5 Analysis steps
Note. Avoid ultraviolet light during the operation, and operate as far away from light as possible.
5.1 Sample pretreatment
5.1.1 Sample preparation
Crush and grind the solid sample, or use a meat grinder to make it into chyme;
homogenize and mix. Shake and mix the liquid sample before testing.
5.1.2 Sample extraction
Weigh 5 g ~ 10 g of the sample (accurate to 0.01 g) into a 150 mL conical flask with
stopper; add 30 mL of sodium acetate buffer, 0.2 g of pepsin, 0.05 g of amylase and 2
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).
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.
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.
5.3 Preparation of the standard curve
Inject the standard series solutions into the liquid chromatograph from low
concentration to high concentration in turn; measure the corresponding
chromatographic peak areas.
5.4 Sample determination
Inject the to-be-tested sample solution into the liquid chromatograph, to obtain the peak
area of vitamin B12 in the to-be-tested solution; measure the concentration of vitamin
B12 in the test solution according to the standard curve. The response value of vitamin
B12 in the to-be-tested sample solution shall be within the linear range of the standard
curve. If it exceeds the linear range, the solution on the machine shall be diluted, or the
sampling amount shall be adjusted and the analysis shall be carried out again after
processing according to the sample analysis steps.
5.5 Blank test
Do not weigh the sample; operate according to the sample analysis steps. Substances
that interfere with the to-be-measured components shall not be contained.
6 Description of the analysis results
The content of vitamin B12 (calculated as cyanocobalamin) in the sample is calculated
according to Formula (1).
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.
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
10.3 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.
10.4.2 Vitamin B12 standard intermediate 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 dilute to the mark. Transfer it to a brown reagent bottle; store at 4 °C in the
dark. The validity period is 1 month.
10.5 Materials
10.5.1 Vitamin B12 immunoaffinity column, column capacity ≥800 ng, column recovery
≥85% (see Appendix A for the verification method).
10.5.2 Glass fiber filter paper.
10.5.3 Microporous membrane. water phase, 0.22 μm.
......
Source: Above contents are excerpted from the full-copy PDF -- translated/reviewed by: www.ChineseStandard.net / Wayne Zheng et al.
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