GB 5009.86-2025National food safety standard - Determination of ascorbic acid in food (English PDF)

Foreword

This document was issued on 2 September 2025 by the State Administration for Market Regulation; Standardization Administration of the PRC and takes effect on 2 March 2026.

It is a GB standard without the /T suffix: compliance is mandatory in China.

1 Scope

GB 5009.86-2025 is the Chinese national food safety standard for determining ascorbic acid in food. Vitamin C is both a declared nutrient and an added antioxidant, so the figure is used to check a label claim and a permitted use level at the same time; the analytical difficulty is that ascorbic acid oxidises during extraction unless the sample is protected, and that its stereoisomer erythorbic acid, used as an antioxidant but with no vitamin activity, must not be counted as vitamin C. The standard gives two methods: liquid chromatography, which separates L-ascorbic acid from D-isoascorbic acid, and fluorescence spectrophotometry. Each is set out with its principle, reagents and materials, instruments and equipment, analytical procedure, expression of results, precision and notes. It takes effect on 2 March 2026.

This Standard specifies the methods for the determination of ascorbyl palmitate in food.

Method 1, liquid chromatography, is applicable to the determination of L-ascorbic acid and D-

isoascorbic acid in food.

Method 2, fluorescence spectrophotometry, is applicable to the determination of ascorbyl

palmitate in food for special dietary purposes, milk and dairy products, fruits and vegetables

and their products, as well as the determination of L-ascorbic acid in the above-mentioned food

without adding D-isoascorbic acid.

Method 3, 2,6-dichlorophenolindophenol titration, is applicable to the determination of reduced

ascorbyl palmitate in fruits and vegetables and their products.

Method 1 - Liquid Chromatography

2 Principle

After extraction, reduction and dilution, ascorbyl palmitate in the specimen is separated by

reversed-phase chromatography using a mixed solution containing ion-pairing reagents and

reducing agents as the mobile phase. Detection is performed using a UV detector or diode array

detector. Qualitative determination is based on the retention time of the chromatographic peaks,

and quantitative determination is performed using the external standard method.

3 Reagents and Materials

Unless otherwise specified, all reagents used in this Method are analytically pure, and the water

is Grade I water as specified in GB/T 6682.

3.1 Reagents

3.1.1 Glacial acetic acid (C2H4O2).

3.1.2 Trichloroacetic acid (C2HCl3O2, TCA).

3.1.3 Metaphosphoric acid (HPO3).

3.1.4 40% tetrabutylammonium hydroxide aqueous solution (40% TBAH).

3.1.5 Ammonium acetate (C2H7NO2).

3.1.6 L-cysteine (C3H7NO2S).

3.1.7 Tris(2-carboxyethyl)phosphine hydrochloride (C9H15O6P, TCEP).

3.1.8 Acetonitrile (C2H3N). chromatographically pure.

3.2 Reagent Preparation

3.2.1 Ammonium acetate solution (500 mmol/L, pH 5.4). weigh-take 19.27 g of ammonium

acetate, add water to dissolve it and dilute to 500 mL; use glacial acetic acid to adjust pH to 5.4.

3.2.2 TCA solution (150 g/L). weigh-take 75 g of TCA, add water to dissolve it, dilute to 500

mL, and mix it well.

3.2.3 Metaphosphoric acid solution (200 g/L). weigh-take 100 g of metaphosphoric acid, add

water to dissolve it, dilute to 500 mL, and mix it well.

3.2.4 TCEP solution (250 mg/L). weigh-take 125 mg of TCEP, add water to dissolve it, dilute

to 500 mL, and mix it well.

3.2.5 L-cysteine solution (40 g/L). weigh-take 40 g of L-cysteine, add an appropriate amount

of water and perform ultrasonic dissolution, then, add water to dilute to 1 L and mix it well.

Prepare it right before use.

3.2.6 Mobile phase. measure-take 900 mL of water into a beaker, pipette 3.25 mL of 40% TBAH

solution and weigh-take 3.85 g of ammonium acetate into the water, then, add 50 mg of TCEP

(or 20 mL of L-cysteine solution), followed by 10 mL of acetonitrile. Mix it well, then, use

glacial acetic acid (approximately 3.14 mL) to adjust the pH to 4.5 ~ 5.0.Finally, add water to

dilute to 1 L and mix it well; remove air bubbles through ultrasound.

3.3 Standard Substances

3.3.1 L-ascorbic acid standard (C6H8O6, CAS No.. 50-81-7). purity  99%, or a standard

substance certified by the state and granted a standard substance certificate.

3.3.2 D-isoascorbic acid standard (C6H8O6, CAS No.. 89-65-6). purity  99%, or a standard

substance certified by the state and granted a standard substance certificate.

3.4 Preparation of Standard Solutions

3.4.1 L-ascorbic acid standard solution (50 g/mL). weigh-take 10 mg of L-ascorbic acid

standard (accurate to 0.1 mg), use TCEP solution to dissolve it and reach a constant volume of

10 mL, mix it well. Accurately pipette 500 L of the above-mentioned standard solution into a

10 mL brown volumetric flask, use TCEP solution to reach a constant volume and mix it well.

Transfer the solution to a brown glass container and prepare it right before use.

3.4.2 D-isoascorbic acid standard solution (50 g/mL). weigh-take 10 mg of D-isoascorbic acid

standard (accurate to 0.1 mg), use TCEP solution to dissolve it and reach a constant volume of

10 mL, mix it well. Accurately pipette 500 L of the above-mentioned standard solution into a

10 mL brown volumetric flask, use TCEP solution to reach a constant volume, and mix it well.

Transfer the solution to a brown glass container and prepare it right before use.

3.4.3 L-ascorbic acid or D-isoascorbic acid standard series working solutions. respectively and

accurately pipette 10 L, 100 L, 200 L, 400 L, 1,000 L and 2,000 L of L-ascorbic acid

or D-isoascorbic acid standard solution (50 g/mL) into 10 mL brown volumetric flasks, use

the mobile phase to reach a constant volume, and gently mix them. The mass concentrations of

the L-ascorbic acid or D-isoascorbic acid standard series working solutions are respectively

0.05 g/mL, 0.50 g/mL, 1.0 g/mL, 2.0 g/mL, 5.0 g/mL and 10 g/mL. Prepare them right

before use.

NOTE. in accordance with the actual condition of the sample to be tested, the concentration range

of the standard solutions can be appropriately adjusted within the linear range.

4 Instruments and Equipment

4.1 Liquid chromatograph. equipped with a UV detector or diode array detector.

4.2 Balance. with a division value of 0.01 mg and 0.1 mg respectively.

4.3 pH meter. with an accuracy of 0.1.

4.4 Vortex mixer. with a speed not less than 1,500 r/min.

4.5 Centrifuge. with a speed not less than 5,000 r/min.

4.6 Ultrasonic cleaner.

5 Analytical Procedures

5.1 Specimen Preparation

Liquid and slurry samples shall be shaken well; semi-solid and powdered samples shall be

thoroughly mixed; gelatinous samples, for example, gum-based candies, need to be cut into

pieces with scissors or pulverized while frozen and then mixed; other samples need to be

homogenized or evenly pulverized and mixed. Fruits and vegetables and their products (take

the edible part) need to be homogenized or evenly pulverized by adding 150 g/L TCA solution

or 200 g/L metaphosphoric acid solution at a ratio of 10.1.

NOTE. the process shall be carried out under light-protected conditions as much as possible; the

next step of test shall be performed immediately after all samples are prepared.

5.1.1 Specimen weighing

5.1.1.1 Solid (including semi-solid) samples

Weigh-take 25 g (accurate to 0.001 g) of the prepared specimen into an Erlenmeyer flask, add

225 g (accurate to 0.001 g) of TCEP solution, and thoroughly dissolve and mix it. Weigh-take

2 g (accurate to 0.001 g) of the mixed solution into a 10 mL volumetric flask.

5.1.1.2 Liquid (including slurry) samples

Weigh-take 2 g (accurate to 0.001 g) of the prepared specimen or accurately pipette 2 mL into

a 10 mL volumetric flask.

5.1.2 Specimen extraction and reduction

Add 4 mL of TCEP solution (or L-cysteine solution) to the above-mentioned 10 mL volumetric

flask, then, add 2 mL of TCA solution, mix it well, and let it stand for 5 min; use water to reach

a constant volume and mix it well. Then, transfer to a centrifuge tube, and at 5,000 r/min,

centrifuge it for 1 min. This specimen solution is reduced specimen solution.

NOTE. for formula foods with special medical purposes, such as amino acid formula powder,

protein hydrolyzed formula powder, partially protein hydrolyzed formula powder, and goat

milk powder, L-cysteine shall be used as the reducing agent.

5.1.3 Specimen dilution

Pipette 1 mL of the supernatant after centrifugation into a 10 mL volumetric flask, add 1 mL of

ammonium acetate solution, use the mobile phase to reach a constant volume, and mix it well.

This specimen solution is diluted specimen solution. Take an appropriate amount of it and filter

through a 0.22 m aqueous microporous membrane into a brown sample vial for liquid

chromatography determination.

NOTE. if necessary, the dilution factor can be changed to ensure that the ascorbyl palmitate

concentration in the solution to be tested is within the linear range.

5.2 Reference Conditions of Instruments

5.2.1 Chromatographic column. C18 column (150 mm length, 4.6 mm inner diameter, 4 m

packing particle size) or a chromatographic column with equivalent performance.

5.2.2 Column temperature. room temperature.

5.2.3 Mobile phase. prepared in accordance with 3.2.6.

5.2.4 Flow rate. 1.0 mL/min.

5.2.5 Detection wavelength. 265 nm.

5.2.6 Injection volume. 10 L.

5.3 Drawing of Standard Curve

Respectively inject the standard series working solutions into the liquid chromatograph to

obtain the corresponding peak areas. With the concentration of L-ascorbic acid or D-isoascorbic

acid in the standard series working solutions as the x-coordinate and the peak area as the y-

coordinate to draw the standard curve. See Figure A.1 in Appendix A for the chromatogram of

the L-ascorbic acid or D-isoascorbic acid standard solution.

5.4 Determination of Specimen Solution

Inject the specimen solution into the liquid chromatograph to obtain the peak area of ascorbyl

palmitate. In accordance with the standard curve, obtain the concentration of ascorbyl palmitate

in the solution to be tested.

6 Expression of Analytical Results

The content of L-ascorbic acid or D-isoascorbic acid in the specimen is calculated in accordance

with Formula (1).

Where,

X---the content of L-ascorbic acid or D-isoascorbic acid in the specimen, expressed in (mg/100

g) or (mg/100 mL);

rho---the mass concentration of L-ascorbic acid or D-isoascorbic acid in the specimen solution

obtained from the standard curve, expressed in (g/mL);

M0---the total mass of the solid (including semi-solid) sample after adding the TCEP solution

for dissolution, in grams (g);

V1---the total volume of the reduced specimen solution, expressed in (mL);

V3---the total volume of the diluted specimen solution, expressed in (mL);

f---the specimen dilution factor;

100---the conversion factor;

m0---the weighing mass of the solid (including semi-solid) sample, in grams (g);

m---the sampling mass of the liquid (including slurry) sample or the prepared specimen solution,

expressed in (g) or (mL);

V2---the sampling volume of the reduced specimen solution, expressed in (mL);

1,000---the conversion factor.

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