GB/T 5413.13-1997National food safety standard -- Determination of vitamin B6 in foods for infants and young children, milk and milk products (English PDF)

6.1 Sample pretreatment

6.1.1 Starch-containing sample. Weigh about 5.0 g (accurate to 0.0001 g) of mixed homogeneous solid sample, add about 25 mL of 45 ° C ~ 50 ° C or about 20.0 g (exactly to 0.0001 g) in a mixed homogeneous liquid sample in a 150 mL Erlenmeyer flask. Add about 0.5g Amylase (4.1), mix well and then fill the conical flask with nitrogen, cover the stopper, set 50 °C ~ 60 °C incubator about 30min. Remove and cool to Room temperature.

6.1.2 Sample without starch. Weigh about 5.0 g (accurate to 0.0001 g) of mixed homogeneous solid sample, add about 25 mL of 45 ° C ~ 50 ° C or about 20.0 g (exactly to 0.0001 g) in a mixed homogeneous liquid sample in a 150 mL Erlenmeyer flask. Mix and full Dissolved, standing for 5min ~ 10min, cooling to room temperature.

6.2 Preparation of test solution

6.2.1 Adjust the pH of the sample solution to 1.7 ± 0.1 with hydrochloric acid solution (4.6) for about 1 min. And then with sodium hydroxide solution (4.7) Adjust the pH of the sample solution to 4.5 ± 0.1.

6.2.2 Transfer the sample solution to a 50 mL volumetric flask and rinse the conical flask with water. Wash the lotion in a 50 mL volumetric flask and set the volume with water to 50mL.

6.2.3 The above-mentioned volumetric flask into the ultrasonic oscillator, ultrasonic oscillation about 10min.

6.2.4 Another 50mL Erjiao bottle, the top into the triangle funnel and filter paper, the ultrasonic oscillation of the sample solution into which the natural filter.

The filtrate was further filtered through a 0.45 µm microporous membrane and collected with a test tube.

6.3 Reference chromatographic conditions

Column. C18 column (particle size 5µm, 150mm × 4.6mm) or equivalent column.

Mobile phase. methanol (4.4) 50mL, octane sulfonate (4.2) 2.0g, triethylamine (4.5) 2.5mL, dissolved in water and fixed to After 1000 mL, the pH was adjusted to 3.0 ± 0.1 with glacial acetic acid (4.3), and then filtered through a 0.45 µm microporous membrane.

Flow rate. 1.0 mL/min.

Detection wavelength. excitation wavelength 293nm, emission wavelength 395nm.

Injection volume. 10 µL.

6.4 Quantitative analysis

6.4.1 Standard curve drawing

The standard combination of standard solution (4.8.3) was used for the determination of the standard sample chromatogram (see Appendix A

Figure A. 1). Record the peak area or peak height of each component, the peak area or peak height as the ordinate, the standard test solution concentration for the sitting

Standard curve.

6.4.2 Specimen determination

The test solution (6.2.4) was subjected to chromatographic determination. Record the peak area or peak height of each component, according to the standard curve to calculate the sample to be

7.1 Calculation of the content of each component of vitamin B6 in the sample

The content of each component of vitamin B6 in the sample is calculated according to formula (1) (1) where.

V - The volume of the sample solution in milliliters (mL).

7.2 Calculation of the content of vitamin B6 in the sample

The content of vitamin B6 in the sample is calculated according to formula (2) X = X alcohol + X aldehyde x 1.012 + X amine x 1.006 (2) where.

X --- The content of vitamin B6 in the sample, in micrograms per gram (µg/100g);

X alcohol - the content of pyridoxine in the sample, in micrograms per gram (µg/100g);

X aldehyde - the content of pyridoxal in the sample, in micrograms per gram (µg/100g);

Xamine - the content of pyridoxamine in the sample, in micrograms per gram (µg/100 g);

1.012 --- pyridoxal content converted into pyridoxine coefficient;

1.006 --- The content of pyridoxamine is converted to the coefficient of pyridoxine.

Note. The content of vitamin B6 in the sample is based on pyridoxine.

The arithmetic mean of the two independent determinations obtained under repeatability is expressed as a result of the retention of three significant digits.