Food additive Vitamin B<Sub>6</Sub>detection

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6 Analysis Steps

6.1 Sample Preparation

6.1.1 Starch Containing Sample: Weigh approximately 5.0g of a uniformly mixed solid sample (accurate to 0.0001g), add approximately 25mL of water at 45 ℃ to 50 ℃, or weigh approximately 20.0g of a uniformly mixed liquid sample (accurate to 0.0001g) into a 150 mL conical flask. Add about 0.5 g of amylase (4.1), mix well, and then fill a conical flask with nitrogen. Cover the flask with a stopper and place it in a 50 ℃ to 60 ℃ incubator for about 30 minutes. Remove and cool to room temperature

6.1.2 Sample without starch: Weigh approximately 5.0g of a uniformly mixed solid sample (accurate to 0.0001g), add approximately 25mL of water at 45 ℃ to 50 ℃, or weigh approximately 20.0g of a uniformly mixed liquid sample (accurate to 0.0001g) into a 150 mL conical flask. Mix well and dissolve thoroughly, let stand for 5-10 minutes, and cool to room temperature

6.2 Preparation of the test solution

6.2.1 Adjust the pH value of the sample solution to 1.7 ± 0.1 with hydrochloric acid solution (4.6) and let it stand for about 1 minute. Adjust the pH of the sample solution to 4.5 ± 0.1 using sodium hydroxide solution (4.7)

Transfer the sample solution to a 50mL volumetric flask and rinse the conical flask with water. Combine the washing solution into a 50mL volumetric flask and dilute to 50mL with water

6.2.3 Place the above-mentioned volumetric flask into an ultrasonic oscillator and oscillate for about 10 minutes

6.2.4 Take another 50mL triangular flask, place a triangular funnel and filter paper on top, pour the sample solution shaken by ultrasound into it, and filter naturally. The filtrate is then pressurized and filtered through a 0.45um microporous filter membrane, and collected in a test tube to obtain the sample solution to be tested

6.3 Reference chromatographic conditions

Chromatographic column: C18 column (particle size 5um, 150mm) × 4.6mm) or equivalent performance chromatographic column

Mobile phase: methanol (4.4), 50mL, sodium octanesulfonate (4.2) 2.0g, triethylamine (4.5) 2.5mL, dissolved in water and brought to a constant volume of 1000mL. Adjust the pH to 3.0 ± 0.1 with glacial acetic acid (4.3), and then filter under pressure through a 0.45um microporous filter membrane

Flow rate: 1.0mL/min

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

Injection volume: 10uL

6.4 Quantitative Analysis

6.4.1 Standard Curve Drawing

Chromatographic determination of vitamin B6 mixed standard series determination solution (4.8.3) in sequence.

. Record the chromatographic peak area or peak height of each component, with the peak area or peak height as the vertical axis and the concentration of the standard measuring solution as the horizontal axis, and draw a standard curve

6.4.2 Sample Determination

Chromatographic determination of the sample solution (6.2.4).

. Record the peak area or height of each component in the chromatogram, and calculate the concentration Ci of each component of vitamin B6 in the sample solution according to the standard curve

7 Expression of analysis results

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 equation (1):

Where:

Xi - The content of the component in the sample, in micrograms per hundred grams (ug/100g)

Ci - The concentration of each component of vitamin B6 in the test solution of the sample, in micrograms per milliliter (ug/mL)

M - mass of the sample, in grams (g)

V - Volume of the sample solution, in milliliters (mL)

7.2 Calculation of vitamin B6 content in the sample

The content of vitamin B6 in the sample is calculated according to equation (2):

X=X alcohol+X aldehyde X1.012+X amine × 1.006 (2)

In the formula:

X - the content of vitamin B6 in the sample, in micrograms per hundred grams (ug/100g)

X alcohol - the content of pyridoxine in the sample, in micrograms per hundred grams (ug/100g)

X aldehyde - The content of pyridoxal in the sample, in micrograms per hundred grams (ug/100g)

X amine - The content of pyridoxamine in the sample, in micrograms per hundred grams (ug/100g)

1.012- coefficient for converting the content of pyridoxal to pyridoxol

1.006- The coefficient for converting the content of pyridoxamine to pyridoxine

Note: The content of vitamin B6 in the sample is calculated using pyridoxine

Represented as the arithmetic mean of two independent measurement results obtained under repeatability conditions, with the results retained to three significant digits

8 Precision

The absolute difference between two independent measurement results obtained under repeatability conditions shall not exceed 10% of the arithmetic mean.

9 Other

The detection limits of this standard are: pyridoxine 1.5ug/100g, pyridoxal 1.3ug/100g, and pyridoxamine 1.6ng/100g

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