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GB 5009.86: Evolution and historical versions
| Standard ID | Contents [version] | USD | STEP2 | [PDF] delivery | Name of Chinese Standard | Status |
| GB 5009.86-2025 | English | 215 |
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National food safety standard - Determination of ascorbyl palmitate in food
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| GB 5009.86-2016 | English | 115 |
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National food safety standard - Determination of Ascorbic Acid in Foods
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| GB/T 5009.86-2003 | English | 239 |
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Determination of total ascorbic acid in fruits, vegetables and derived products -- Fluorometric method and colorimetric method
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GB 5009.86-2025: National food safety standard - Determination of ascorbyl palmitate in food
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GB
NATIONAL STANDARD OF THE
PEOPLE’S REPUBLIC OF CHINA
National Food Safety Standards - Determination of Ascorbyl
Palmitate in Food
Issued on: SEPTEMBER 2, 2025
Implemented on: MARCH 2, 2026
Issued by. National Health Commission of the People’s Republic of China;
State Administration for Market Regulation.
Table of Contents
Foreword... 3
1 Scope... 4
2 Principle... 4
3 Reagents and Materials... 4
4 Instruments and Equipment... 6
5 Analytical Procedures... 6
6 Expression of Analytical Results... 8
7 Precision... 9
8 Others... 9
9 Principle... 9
10 Reagents and Materials... 9
11 Instruments and Equipment... 11
12 Analytical Procedures... 11
13 Expression of Analytical Results... 14
14 Precision... 14
15 Others... 14
16 Principle... 15
17 Reagents and Materials... 15
18 Analytical Procedures... 16
19 Result Calculation... 17
20 Precision... 17
Appendix A Chromatogram of L-ascorbic Acid and D-isoascorbic Acid Standard
Solutions... 18
Appendix B Test Method for Iron Ions... 19
National Food Safety Standard - Determination of Ascorbyl
Palmitate in Food
1 Scope
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);
ρ---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.
......
Source: Above contents are excerpted from the full-copy PDF -- translated/reviewed by: www.ChineseStandard.net / Wayne Zheng et al.
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