GB 5009.308-2025National food safety standard - Determination of ascorbyl palmitate in food (English PDF)

3.1 Reagents

3.1.1 Methanol (CH4O). chromatographically pure.

3.1.2 Acetonitrile (C2H3N). chromatographically pure.

3.1.3 Phosphoric acid (H3PO4).

3.1.4 Citric acid (C6H8O7).

3.1.5 Isoascorbic acid (C6H8O6).

3.2.1 Citric acid-isoascorbic acid methanol solution. Weigh

1.0 g of citric acid and

1.0 g of isoascorbic acid, dissolve them in methanol and dilute to 1000 mL.

3.2.2 Phosphoric acid solution (0.1%). Measure

1.0 mL of phosphoric acid, dilute with water to 1000 mL, mix well and set aside.

3.2.3 Methanol-acetonitrile solution (50+50). Mix methanol and acetonitrile at a volume ratio of

50.50 and set aside.

3.3 Standard substances

3.3.1 Ascorbyl palmitate standard substance (C22H38O7, CAS No.. 137-66-6). with a purity of >=98%, or a standard substance that has obtained the national certification and been granted a standard substance certificate.

3.4.1 Ascorbyl palmitate standard solution (

1.00 mg/mL). Accurately weigh 25 mg (accurate to

0.1 mg) of the standard substance, dissolve it in citric acid-isoascorbic acid methanol solution and dilute to 25 mL, then mix well. Prepare fresh before use.

3.4.2 Ascorbyl palmitate standard intermediate solution (

0.100 mg/mL). Accurately pipette

1.00 mg/mL standard solution, dilute with citric acid-isoascorbic acid methanol solution and bring the volume to 10 mL, then mix well. Prepare fresh before use.

3.4.3 Ascorbyl palmitate standard working solution. Accurately pipette

0.10 mL,

0.20 mL,

0.50 mL,

5.0 mL of the intermediate standard solution, dilute with citric acid-isoascorbic acid methanol solution, and bring the volume to 10 mL. Mix well to obtain standard working solutions with mass concentrations of 1.00 µg/mL, 2.00 µg/mL, 5.00 µg/mL, 10.0 µg/mL, and 50.0 µg/mL, respectively. Prepare fresh before use. NOTE. The concentration points of the standard working solution can be adjusted appropriately within the linear range.

3.5 Materials Organic microporous filter membrane. 0.22 µm.

5.2.1 Sample extraction Weigh 5 g of the sample (accurate to

0.001

g) and place it in a 50 mL centrifuge tube. Add 30 mL of citric acid-isoascorbic acid methanol solution, tighten the cap, place the tube on a horizontal oscillator, and extract for 10 min by oscillating at a rate of not less than 250 times/min. Transfer the entire sample to a 50 mL volumetric flask, and dilute to the mark with citric acid-isoascorbic acid methanol solution. Mix well.

5.2.2 Purification The above sample extract is filtered through filter paper (discarding the initial filtrate) or centrifuged at 8000 r/min for 1 min to obtain the supernatant, and then filtered through a 0.22 µm organic microporous membrane into a brown sample vial for analysis by a high-performance liquid chromatograph. NOTE. If necessary, the sample solution can be diluted with citric acid-isoascorbic acid methanol solution to ensure that the concentration of ascorbyl palmitate in the sample solution is within the range of the standard working solution concentration; the sample solution shall be tested within 12 hours.

5.3 Blank test Except for the absence of a sample, all other steps are performed simultaneously with the sample processing as per 5.2.

5.4.1 Chromatographic column. C18 column (

4.6 mm×150 mm, 5 µm) or a column with equivalent performance.

5.4.2 Detection wavelength. 245 nm.