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GB 5009.86-2016: National Food Safety Standard -- Determination of Ascorbic Acid in Foods
GB 5009.86-2016
GB
NATIONAL STANDARD OF THE
PEOPLE’S REPUBLIC OF CHINA
National Food Safety Standard -
Determination of Ascorbic Acid in Foods
ISSUED ON. AUGUST 31, 2016
IMPLEMENTED ON. MARCH 1, 2017
Issued by. National Health and Family Planning Commission of the
People's Republic of China
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Table of Contents
Foreword ... 3 
1 Scope .. 4 
2 Terms and definitions ... 4 
3 Principle.. 5 
4 Reagents and materials .. 5 
5 Instruments and equipment ... 6 
6 Analysis steps ... 7 
7 Expression of analysis results .. 8 
8 Precision... 9 
9 Other ... 9 
10 Principle... 10 
11 Reagents and materials .. 10 
12 Instruments and equipment .. 11 
13 Analysis steps .. 12 
14 Result calculation ... 13 
15 Precision .. 14 
16 Other .. 14 
17 Principle... 14 
18 Reagents and materials.. 14 
19 Determination ... 16 
20 Result calculation ... 16 
21 Precision .. 17 
Annex A L(+)- ascorbic acid, D(+)- ascorbic acid standard chromatogram ... 18 
Foreword
This Standard replaces GB/T 5009.86-2003 Determination of total ascorbic acid
in fruits, vegetables and derived products - Fluorometric method, GB/T
5009.159-2003 Determination of reductive-form ascorbic acid in foods and GB
6195-1986 Determination of vitamin C in vegetables and fruits (2,6-dechloro-
indophenol titration method).
Compared with GB/T 5009.86-2003, the main changes are as follows.
- modified the standard name to “National Food Safety Standard-
Determination of Ascorbic Acid in Foods”;
- expanded the application scope of the method;
- added the high-performance liquid chromatography and related
quantitation limitations;
- deleted 2,4-dinitrophenylhydrazine method;
- added 2,6-dichloro-indophenol titration;
- modified the structure of the previous edition according to GB/T 20001.4-
2015.
National Food Safety Standard -
Determination of Ascorbic Acid in Foods
1 Scope
This Standard specifies the high-performance liquid chromatography,
fluorescence method, 2,6-dichloro-indophenol titration method for the
determination of ascorbic acid in food.
Method One of this Standard applies to the determination of total amount of
L(+)- ascorbic acid, D(+)- ascorbic acid and L(+)- ascorbic acid in milk powder,
cereals, vegetables, fruits and their products, meat products, vitamin
supplements, jellies, gummies, rice pudding, wine. Method Two is applicable to
the determination of L(+)- total amount of ascorbic acid in milk powder,
vegetables, fruits and their products. Method Three applies to the determination
of L(+)- total amount of ascorbic acid in fruits, vegetables and their products.
2 Terms and definitions
2.1 Ascorbic acid. an organic compound with antioxidant properties. It is also
known as "Vitamin C," one of the essential nutrients in human body.
2.2 L(+)- ascorbic acid. left-right rotation ascorbic acid that has a strong
reduction. It has biological activity on the human body.
2.3 D(+)- ascorbic acid. also known as isoascorbic acid. It has a strong
reduction, but basically, it has no biological activity on the human body.
2.4 L(+)- dehydroascorbic acid. L(+)- ascorbic acid is easily oxidized to L(+)-
dehydroascorbic acid; L(+)- dehydroascorbic acid can also be reduced to L(+)-
ascorbic acid. It is often called as dehydroascorbic acid.
2.5 L(+)- total amount of ascorbic acid. total amount of ascorbic acid obtained
when L(+)- dehydroascorbate in the sample is reduced to L(+)- ascorbic acid
OR L(+)- dehydroascorbic acid is oxidized by L(+)- ascorbic acid in the sample.
Method One -- High performance liquid
chromatography
3 Principle
After ascorbic acid in the sample is dissolved with metaphosphoric acid and
extracted by sonication, use ion-pair reagent as mobile phase. Separate it
through reverse phase column. L(+)- ascorbic acid and D(+)- ascorbic acid are
measured directly with a liquid chromatograph (wavelength of 245 nm)
equipped with a UV detector. After L(+)- dehydroascorbate in the sample is
reduced by L-cysteine solution, the content of L(+)- dehydroascorbic acid is
determined by measuring the total amount of L(+)- ascorbic acid with a UV
detector (wavelength of 245 nm) or by subtracting the L(+)- ascorbic acid
content measured in the original sample. Determine the nature by the retention
time of chromatographic peak. Quantify in external standard method.
4 Reagents and materials
Unless otherwise specified, the reagents used in this method are of analytical
grade pure, water is of grade one specified in GB/T 6682.
4.1 Reagents
4.1.1 Metaphosphoric acid (HPO3)n. content (calculated as 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). excellent grade pure
4.1.6 Cetyltrimethylammonium bromide (C19H42BrN). chromatographically
pure
4.1.7 Methanol (CH3OH). chromatographically pure
4.2 Reagent preparation
4.2.1 Methanophosphoric acid solution (200 g/L). weigh 200 g (to the nearest
of 0.1 g) of metaphosphoric acid (4.1.1), dissolve in water and dilute to 1 L; this
solution can be stored in a 4°C environment for one month.
4.2.2 Methanophosphoric acid solution (20 g/L). weigh 50 mL of 200 g/L
partial phosphoric acid solution; dilute in water to 500 mL.
4.2.3 Trisodium phosphate solution (100 g/L). weigh 100 g (to the nearest of
0.1g) of trisodium phosphate; dissolve in water and dilute to 1 L.
6.4.1 Chromatographic column. C18 column, column length of 250 mm, inner
diameter of 4.6 mm, particle size of 5 μm, or chromatographic column of same
performance.
6.4.2 Detector. diode array detector or UV detector
6.4.3 Mobile phase. A. 6.8 g of potassium dihydrogen phosphate and 0.91 g
of cetyltrimethylammonium bromide; use water to dissolve and set volume to 1
L (adjust pH to 2.5 ~ 2.8 with phosphoric acid); B. 100% methanol. Mix
according to A.B=98.2; make it through 0.45 μm filter membrane; perform
ultrasonic degassing.
6.4.4 Flow rate. 0.7 mL/min
6.4.5 Detection wavelength. 245 nm
6.4.6 Column temperature. 25°C
6.4.7 Injection volume. 20 μL
6.5 Making of standard curve
Respectively determine the ascorbic acid mixed standard series of working
solutions. Take the mass concentration (μg/mL) of standard solution of L(+)-
ascorbic acid [or D(+)- ascorbic acid] as abscissa, L(+)- ascorbate [or D(+)-
ascorbate] peak height or peak area as ordinate to draw standard curve or
calculate regression equation. See Figure A.1 in Annex A for L(+)- ascorbic acid,
D(+)- ascorbic acid standard chromatogram.
6.6 Determination of sample solution
Determine the sample solution. Obtain the concentration (μg/mL) of L(+)-
ascorbate [or D(+)- ascorbate] according to the standard curve.
6.7 Blank test
The blank test refers that perform parallel operation by using identical analysis
steps, reagents and dosage, except not adding sample.
7 Expression of analysis results
The amount of L(+)- ascorbic acid [or D(+)- ascorbic acid] and the total amount
of L(+)- ascorbic acid in the sample are expressed in milligrams per hundred
grams, calculated according to equation (1).
11.2.3 Metaphosphoric acid - acetic acid - sulfuric acid solution. weigh 15 g of
metaphosphoric acid; add 40 mL of glacial acetic acid; drop 0.15 m...