This standard supersedes "Determination of Total Ascorbic Acid in Fruits, Vegetables and Derived Products—Fluorometric Method and 2,4-Dinitrophenylhydrazine Method" (GB/T 5009.86-2003), "Determination of Reductive-form Ascorbic Acid in Foods" (GB/T 5009.159-2003) and "Determination of Vitamin C in Vegetables and Fruits (2,6-Dichloro-Indophenol Titration Method)" (GB 6195-1986).
Compared with GB/T 5009.86-2003, this standard has the following main changes:
—the standard name was revised as "National Food Safety Standard—Determination of Ascorbic Acid in Foods";
—the applicable scope of the method was expanded;
—the limit of quantitation for high performance liquid chromatography and relevant methods was added;
—the 2,4-dinitrophenylhydrazine method was deleted;
—the 2,6-dichloro-indophenol titration method was added;
—the structure of the former standard was modified according to GB/T 20001.4-2015.
NATIONAL STANDARD
OF THE PEOPLE’S REPUBLIC OF CHINA
中华人民共和国国家标准
GB 5009.86-2016
National Food Safety Standard
Determination of Ascorbic Acid in Foods
食品安全国家标准
食品中抗坏血酸的测定
1 Scope
This standard specifies the determination of ascorbic acid in foods by high performance liquid chromatography, fluorometric method and 2,6-dichloro-indophenol titration method.
Method I of this standard is applicable to the determination of L(+)- ascorbic acid, D(+)- ascorbic acid and total content of L(+)- ascorbic acid in milk powder, cereal, vegetables, fruits and derived products, meat products, vitamin supplements, jelly, gum candy, eight ingredients porridge and wine. Method II is applicable to the determination of total content of L(+)- ascorbic acid in milk powder, vegetables, fruits and derived products. Method III is applicable to the determination of L(+)- ascorbic acid in fruits, vegetables and derived products.
2 Terms and Definitions
2.1 Ascorbic acid: an anti-oxidative organic compound, also known as "vitamin C", which is one of the essential nutrients for human body.
2.2 L(+)- ascorbic acid: levoform dextrorotary ascorbic acid, which is of strong reducing property and bioactivity for human body.
2.3 D(+)- ascorbic acid: also known as erythorbic acid, which is of strong reducing property, but no bioactivity basically for human body.
2.4 L(+)- dehydroascorbic acid: L(+)- ascorbic acid is extremely easy to be oxidized to L(+)- dehydroascorbic acid which may also be reduced to L(+)- ascorbic acid. It is generally referred to as dehydroascorbic acid.
2.5 Total content of L(+)- ascorbic acid: the total content of L(+)- ascorbic acid which is measured after the L(+)- dehydroascorbic acid in the sample is reduced to L(+)- ascorbic acid or the L(+)- ascorbic acid in the sample is oxidized to L(+)- dehydroascorbic acid.
Method I High Performance Liquid Chromatography
3 Principle
The ascorbic acid in the sample is dissolved by metaphosphoric acid, extracted by ultrasonic and then separated by reversed-phase chromatographic column, taking the ion pair reagent as mobile phase; L(+)- ascorbic acid and D(+)- ascorbic acid are determined directly by liquid chromatograph (of 245nm wavelength) equipped with ultraviolet detector. After L(+)- dehydroascorbic acid in the sample is reduced by L-cysteine solution, determine the total content of L(+)- ascorbic acid by ultraviolet detector (of 245nm wavelength), or obtain the content of L(+)- dehydroascorbic acid by subtracting the measured content of L(+)- ascorbic acid in the original sample. Carry out qualitative analysis by the retention time of chromatographic peak and quantitive analysis by external standard method.
4 Reagents and Materials
Unless otherwise specified, analytically-pure reagents and Grade I water (defined in GB/T 6682) are adopted for the purposes of this method.
4.1 Reagents
4.1.1 Metaphosphoric acid (HPO3)n: with content (expressed by HPO3) ≥38%.
4.1.2 Trisodium phosphate (Na3PO4·12H2O).
4.1.3 Potassium dihydrogen phosphate (KH2PO4).
4.1.4 Phosphoric acid (H3PO4): 85%.
4.1.5 L-cysteine (C3H7NO2S): guaranteed reagent.
4.1.6 Hexadecyl trimethyl ammonium bromide (C19H42BrN): chromatographically pure.
4.1.7 Methanol (CH3OH): chromatographically pure.
4.2 Preparation of reagents
4.2.1 Metaphosphoric acid solution (200g/L): weigh 200g (to the nearest of 0.1g) of metaphosphoric acid (4.1.1), dissolve it in water and dilute to 1L; such solution can be preserved for one month in the environment at 4℃.
4.2.2 Metaphosphoric acid solution (20g/L): take 50mL of 200g/L metaphosphoric acid solution and dilute it with water to 500mL.
4.2.3 Trisodium phosphate solution (100g/L): weigh 100g (to the nearest of 0.1g) of trisodium phosphate, dissolve it in water and dilute to 1L.
4.2.4 L-cysteine solution (40g/L): weigh 4g of L-cysteine, dissolve it in water and dilute to 100mL; prepare the solution at instant use.
4.3 Standard sample
4.3.1 Standard sample of L(+)- ascorbic acid (C6H8O6): purity ≥99%.
4.3.2 Standard sample of D(+)- ascorbic acid (erythorbic acid) (C6H8O6): purity ≥99%.
4.4 Preparation of standard solution
4.4.1 Standard stock solution of L(+)- ascorbic acid (1.000mg/mL): exactly weigh 0.01g (to the nearest of 0.01mg) of the standard sample of L(+)- ascorbic acid, scale the volume to 10mL with 20g/L metaphosphoric acid solution. Such stock solution can be preserved for a week at 2℃~8℃ in a dark place.
4.4.2 Standard stock solution of D(+)- ascorbic acid (1.000mg/mL): exactly weigh 0.01g (to the nearest of 0.01mg) of the standard sample of D(+)- ascorbic acid, scale the volume to 10mL with 20g/L metaphosphoric acid solution. Such stock solution can be preserved for a week at 2℃~8℃ in a dark place.
4.4.3 Mixed standard series working solution of ascorbic acid: pipet 0mL, 0.05mL, 0.50mL, 1.0mL, 2.5mL and 5.0mL of the standard stock solution of L(+)- ascorbic acid and D(+)- ascorbic acid respectively, scale the volume to 100mL with 20g/L metaphosphoric acid solution. The concentration of L(+)- ascorbic acid and D(+)- ascorbic acid in the standard series working solution is 0μg/mL, 0.5μg/mL, 5.0μg/mL, 10.0μg/mL, 25.0μg/mL and 50.0μg/mL respectively. Prepare the solution at instant use.
5 Apparatus
5.1 Liquid chromatograph: equipped with diode array detector or ultraviolet detector.
5.2 pH meter: with precision of 0.01.
5.3 Scale: with sensibility of 0.1g, 1mg and 0.01mg.
5.4 Ultrasonic cleaner.
5.5 Centrifuge: with rotation speed ≥4,000r/min.
5.6 Homogenizer.
5.7 Filter membrane: 0.45μm water phase membrane.
5.8 Oscillator.
Contents
Foreword i
1 Scope
2 Terms and Definitions
Method I High Performance Liquid Chromatography
3 Principle
4 Reagents and Materials
5 Apparatus
6 Analysis Procedure
7 Expression of Analysis Result
8 Precision
9 Miscellaneous
Method II Fluorometric Method
10 Principle
11 Reagents and Materials
12 Apparatus
13 Analysis Procedure
14 Calculation of Result
15 Precision
16 Miscellaneous
Method III 2,6-Dichloro-Indophenol Titration Method
17 Principle
18 Reagents and Materials
19 Determination
20 Calculation of Result
21 Precision
Appendix A
Standard Chromatograms of L(+)- Ascorbic Acid and D(+)- Ascorbic Acid
This standard supersedes "Determination of Total Ascorbic Acid in Fruits, Vegetables and Derived Products—Fluorometric Method and 2,4-Dinitrophenylhydrazine Method" (GB/T 5009.86-2003), "Determination of Reductive-form Ascorbic Acid in Foods" (GB/T 5009.159-2003) and "Determination of Vitamin C in Vegetables and Fruits (2,6-Dichloro-Indophenol Titration Method)" (GB 6195-1986).
Compared with GB/T 5009.86-2003, this standard has the following main changes:
—the standard name was revised as "National Food Safety Standard—Determination of Ascorbic Acid in Foods";
—the applicable scope of the method was expanded;
—the limit of quantitation for high performance liquid chromatography and relevant methods was added;
—the 2,4-dinitrophenylhydrazine method was deleted;
—the 2,6-dichloro-indophenol titration method was added;
—the structure of the former standard was modified according to GB/T 20001.4-2015.
NATIONAL STANDARD
OF THE PEOPLE’S REPUBLIC OF CHINA
中华人民共和国国家标准
GB 5009.86-2016
National Food Safety Standard
Determination of Ascorbic Acid in Foods
食品安全国家标准
食品中抗坏血酸的测定
1 Scope
This standard specifies the determination of ascorbic acid in foods by high performance liquid chromatography, fluorometric method and 2,6-dichloro-indophenol titration method.
Method I of this standard is applicable to the determination of L(+)- ascorbic acid, D(+)- ascorbic acid and total content of L(+)- ascorbic acid in milk powder, cereal, vegetables, fruits and derived products, meat products, vitamin supplements, jelly, gum candy, eight ingredients porridge and wine. Method II is applicable to the determination of total content of L(+)- ascorbic acid in milk powder, vegetables, fruits and derived products. Method III is applicable to the determination of L(+)- ascorbic acid in fruits, vegetables and derived products.
2 Terms and Definitions
2.1 Ascorbic acid: an anti-oxidative organic compound, also known as "vitamin C", which is one of the essential nutrients for human body.
2.2 L(+)- ascorbic acid: levoform dextrorotary ascorbic acid, which is of strong reducing property and bioactivity for human body.
2.3 D(+)- ascorbic acid: also known as erythorbic acid, which is of strong reducing property, but no bioactivity basically for human body.
2.4 L(+)- dehydroascorbic acid: L(+)- ascorbic acid is extremely easy to be oxidized to L(+)- dehydroascorbic acid which may also be reduced to L(+)- ascorbic acid. It is generally referred to as dehydroascorbic acid.
2.5 Total content of L(+)- ascorbic acid: the total content of L(+)- ascorbic acid which is measured after the L(+)- dehydroascorbic acid in the sample is reduced to L(+)- ascorbic acid or the L(+)- ascorbic acid in the sample is oxidized to L(+)- dehydroascorbic acid.
Method I High Performance Liquid Chromatography
3 Principle
The ascorbic acid in the sample is dissolved by metaphosphoric acid, extracted by ultrasonic and then separated by reversed-phase chromatographic column, taking the ion pair reagent as mobile phase; L(+)- ascorbic acid and D(+)- ascorbic acid are determined directly by liquid chromatograph (of 245nm wavelength) equipped with ultraviolet detector. After L(+)- dehydroascorbic acid in the sample is reduced by L-cysteine solution, determine the total content of L(+)- ascorbic acid by ultraviolet detector (of 245nm wavelength), or obtain the content of L(+)- dehydroascorbic acid by subtracting the measured content of L(+)- ascorbic acid in the original sample. Carry out qualitative analysis by the retention time of chromatographic peak and quantitive analysis by external standard method.
4 Reagents and Materials
Unless otherwise specified, analytically-pure reagents and Grade I water (defined in GB/T 6682) are adopted for the purposes of this method.
4.1 Reagents
4.1.1 Metaphosphoric acid (HPO3)n: with content (expressed by HPO3) ≥38%.
4.1.2 Trisodium phosphate (Na3PO4·12H2O).
4.1.3 Potassium dihydrogen phosphate (KH2PO4).
4.1.4 Phosphoric acid (H3PO4): 85%.
4.1.5 L-cysteine (C3H7NO2S): guaranteed reagent.
4.1.6 Hexadecyl trimethyl ammonium bromide (C19H42BrN): chromatographically pure.
4.1.7 Methanol (CH3OH): chromatographically pure.
4.2 Preparation of reagents
4.2.1 Metaphosphoric acid solution (200g/L): weigh 200g (to the nearest of 0.1g) of metaphosphoric acid (4.1.1), dissolve it in water and dilute to 1L; such solution can be preserved for one month in the environment at 4℃.
4.2.2 Metaphosphoric acid solution (20g/L): take 50mL of 200g/L metaphosphoric acid solution and dilute it with water to 500mL.
4.2.3 Trisodium phosphate solution (100g/L): weigh 100g (to the nearest of 0.1g) of trisodium phosphate, dissolve it in water and dilute to 1L.
4.2.4 L-cysteine solution (40g/L): weigh 4g of L-cysteine, dissolve it in water and dilute to 100mL; prepare the solution at instant use.
4.3 Standard sample
4.3.1 Standard sample of L(+)- ascorbic acid (C6H8O6): purity ≥99%.
4.3.2 Standard sample of D(+)- ascorbic acid (erythorbic acid) (C6H8O6): purity ≥99%.
4.4 Preparation of standard solution
4.4.1 Standard stock solution of L(+)- ascorbic acid (1.000mg/mL): exactly weigh 0.01g (to the nearest of 0.01mg) of the standard sample of L(+)- ascorbic acid, scale the volume to 10mL with 20g/L metaphosphoric acid solution. Such stock solution can be preserved for a week at 2℃~8℃ in a dark place.
4.4.2 Standard stock solution of D(+)- ascorbic acid (1.000mg/mL): exactly weigh 0.01g (to the nearest of 0.01mg) of the standard sample of D(+)- ascorbic acid, scale the volume to 10mL with 20g/L metaphosphoric acid solution. Such stock solution can be preserved for a week at 2℃~8℃ in a dark place.
4.4.3 Mixed standard series working solution of ascorbic acid: pipet 0mL, 0.05mL, 0.50mL, 1.0mL, 2.5mL and 5.0mL of the standard stock solution of L(+)- ascorbic acid and D(+)- ascorbic acid respectively, scale the volume to 100mL with 20g/L metaphosphoric acid solution. The concentration of L(+)- ascorbic acid and D(+)- ascorbic acid in the standard series working solution is 0μg/mL, 0.5μg/mL, 5.0μg/mL, 10.0μg/mL, 25.0μg/mL and 50.0μg/mL respectively. Prepare the solution at instant use.
5 Apparatus
5.1 Liquid chromatograph: equipped with diode array detector or ultraviolet detector.
5.2 pH meter: with precision of 0.01.
5.3 Scale: with sensibility of 0.1g, 1mg and 0.01mg.
5.4 Ultrasonic cleaner.
5.5 Centrifuge: with rotation speed ≥4,000r/min.
5.6 Homogenizer.
5.7 Filter membrane: 0.45μm water phase membrane.
5.8 Oscillator.
Contents of GB 5009.86-2016
Contents
Foreword i
1 Scope
2 Terms and Definitions
Method I High Performance Liquid Chromatography
3 Principle
4 Reagents and Materials
5 Apparatus
6 Analysis Procedure
7 Expression of Analysis Result
8 Precision
9 Miscellaneous
Method II Fluorometric Method
10 Principle
11 Reagents and Materials
12 Apparatus
13 Analysis Procedure
14 Calculation of Result
15 Precision
16 Miscellaneous
Method III 2,6-Dichloro-Indophenol Titration Method
17 Principle
18 Reagents and Materials
19 Determination
20 Calculation of Result
21 Precision
Appendix A
Standard Chromatograms of L(+)- Ascorbic Acid and D(+)- Ascorbic Acid
