GB 5009.285-2022National food safety standard - Determination of Vitamin B12 in foods (English PDF)

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

4.1 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.

5.1 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 ~

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.

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.

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