Detection of Riboflavin 5 '- Sodium Phosphate as a Food Additive

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A. Determination of free riboflavin and riboflavin diphosphate

A.9.1 Reagents and materials

A.9.1.1 KH2PO4 solution: 0.054 mol/L

A.9.1.2; Methanol: Chromatically pure

A.9.1.3 Riboflavin standard: Riboflavin content shall not be less than 99.0% (mass fraction)

A.9.1.4 Riboflavin 5 '- sodium phosphate standard: Riboflavin content shall not be less than 74.0% (mass fraction)

A.9.2 Instruments and Equipment

High performance liquid chromatography with fluorescence detector (excitation wavelength 440nm; emission wavelength 470nm)

A.9.3 Reference chromatographic conditions

A.9.3.1 Chromatographic column: C18 column, 3.9mm × 300mm or other equivalent chromatographic column

A.9.3.2 Mobile phase: Prepare according to K2PO4 solution: methanol=85:15 (volume ratio), mix evenly, filter with 0.45um filter membrane, and use ultrasonic degassing for standby

A.9.3.3 Column temperature: room temperature

A.9.3.4 Mobile phase flow rate: 2.0mL/min

A.9.3.5 Injection volume: 100uL

A.9.4 Analysis Steps

A.9.4.1 Preparation of System Adaptable Solution

Dissolve 100mg of riboflavin 5 '- sodium phosphate standard in water to a mass concentration of 2.0mg/mL, add an equal volume of mobile phase, and mix well. Suck 8mL, dilute with mobile phase to 50mL, mix well and set aside

A.9.4.2 Preparation of Standard Solution

Accurately weigh 60.0mg of riboflavin standard into a 250mL volumetric flask, carefully add 1mL of hydrochloric acid to dissolve, dilute with water to the mark, and mix well.

. Pipette 4mL of riboflavin solution into a 100mL volumetric flask, dilute with mobile phase to the mark, and mix well for later use

A.9.4.3 Preparation of Sample Solution

Accurately weigh 100.0mg of the sample into a 100mL volumetric flask, dissolve in 50mL of water, dilute with mobile phase to the mark, and mix well.

. Pipette 8mL of solution into a 50mL volumetric flask, dilute with mobile phase to the mark, shake well, and set aside

A.9.4.4 System adaptability requirements

Take an appropriate amount of system adaptability solution and inject it into the chromatograph for chromatographic analysis.

. The separation degree between riboflavin 4 '- monophosphate and riboflavin 5' - monophosphate should not be less than 1.0; The relative standard deviation of two repeated injections of riboflavin 5 '- monophosphate is not more than 1.5%

The retention time of riboflavin 5 '- monophosphate is approximately 20 minutes to 25 minutes. The approximate relative retention time of each component is shown in Table A.1

Table A.1 Approximate Relative Retention Times of Each Component

A.9.4.5 Determination

Inject equal volumes (about 100uL) of standard solution, sample solution, and system adaptive solution into the chromatograph for chromatographic analysis, record the chromatogram, and measure the peak response values in the chromatograms of the standard solution and sample solution.

. Determine the peak to be measured in the sample solution chromatogram by comparing the retention time of the chromatographic peak in the system adaptive solution chromatogram

A.9.5 Result Calculation

The free riboflavin content is calculated as the mass fraction W3 of free riboflavin, and the value is expressed in%. Calculate according to formula (A.5):

In the formula:

625- the dilution ratio of the sample

Ws - The numerical value of riboflavin concentration in the standard solution, in milligrams per milliliter (mg/mL)

PF - peak response value of riboflavin in the chromatogram of the sample solution

PS - peak response value of riboflavin in the standard solution chromatogram

The content of riboflavin diphosphate is calculated as the mass fraction W4 of riboflavin diphosphate, and the value is expressed in%. Calculate according to formula (A.6):

Where:

625- dilution ratio of the sample

WS - The numerical value of riboflavin concentration in the standard solution, in milligrams per milliliter (mg/mL)

PS - peak response value of riboflavin in the standard solution chromatogram

PD - The total response values of three riboflavin diphosphate peaks in the chromatogram of the sample solution

The experimental results shall be based on the arithmetic mean of the parallel measurement results. The ratio of the absolute difference between two independent measurement results obtained under repeatability conditions and the arithmetic mean shall not exceed 1.5%

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