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1 Translated English of Chinese Standard: GB NATIONAL STANDARD OF GB THE PEOPLE S REPUBLIC OF CHINA National Food Safety Standard Determination of synthetic colorants in food stuffs How to BUY & immediately GET a full-copy of this standard? Search --> Add to Cart --> Checkout (3-steps); 3. No action is required - Full-copy of this standard will be automatically & immediately delivered to your address in 0~60 minutes. 4. Support: Sales@ChineseStandard.net. Wayne, Sales manager 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. Page 1 of 11

2 Table of Contents Foreword Scope Principle Reagents and materials Instruments and equipment Analysis steps Expression of analysis results Precision Other... 9 Annex A Colorant standard chromatogram Page 2 of 11

3 Foreword This Standard replaces GB/T Determination of synthetic colour in foods. Compared with GB/T , the main changes in this Standard are as follows: - modified the standard name to National Food Safety Standard - Determination of synthetic colorants in food stuffs ; - added the reagent level and formula; - added the standard sample; - modified the calculation equation; - modified the chromatogram; - deleted the second method of thin layer chromatography; - deleted the third method of oscillopolarography. Page 3 of 11

4 National Food Safety Standard Determination of synthetic colorants in food stuffs 1 Scope This Standard specifies the determination of synthetic colorants (excluding aluminum ingots) in beverages, formulated wines, hard candies, candied fruit, starch candy, chocolate beans and colored sugar-coated products. This Standard is applicable to the determination of synthetic colorants (excluding aluminum ingots) in beverages, formulated wines, hard candies, candied fruit, starch candy, chocolate beans and colored sugar-coated products. 2 Principle Synthetic colorants in food are extracted with polyamide or liquid-liquid distribution to make an aqueous solution. Fill it into a high performance liquid chromatograph. It is separated by reverse phase chromatography. Determine its nature according to retention time. Quantify it by comparison with peak area. 3 Reagents and materials Unless otherwise stated, the reagents used in this method are of analytical grade and water is grade one water specified in GB/T Reagents Methanol (CH3OH): chromatographic pure N-hexane (C6H14) Hydrochloric acid (HCl) Glacial acetic acid (CH3COOH) Formic acid (HCOOH) Ammonium acetate (CH3COONH4) Page 4 of 11

5 5.1.4 Chocolate beans and colored sugar products: weigh 5 g ~ 10 g (nearest to g), put into a 100 ml beaker, use water to repeatedly wash pigment till the chocolate beans have no pigment, then combine the pigment rinse solution as sample solution. 5.2 Pigment extraction Polyamide adsorption method: add sample solution plus citric acid solution, adjust ph to 6, heat to 60 C; add water into 1 g of polyamide powder to make it porridge, pour into sample solution, stir for a moment; use G3 vertical funnel to filter; use 60 C ph 4 water to rinse for 3 ~ 5 times; then use methanol-formic acid mixed solution to rinse for 3 ~ 5 times (use method for the sample containing erythrosine); and use water to rinse till neutral; use ethanol-ammonia-water mixed solution to desorb for 3 times ~ 5 times till the pigment is completely desorbed; collect desorption fluid; add acetic acid for neutralization; evaporate it to nearly dry; add water to dissolve; set volume to 5 ml. Filter it through a 0.45 μm microporous membrane. Analyze by high performance liquid chromatography Liquid-liquid distribution method (for samples containing erythrosine): place the well-prepared sample solution into a separating funnel; add 2 ml of hydrochloric acid, 10 ml ~ 20 ml of tri-n-octylamine-n-butanol solution; shake to extract; distribute the organic phase; repeat extraction till organic phase is colorless; combine the organic phase; use saturated sodium sulfate solution to wash 2 times, 10 ml per time; distribute the organic phase; put into evaporating dish; heat it in water bath and concentrate it to 10 ml. Transfer to the separating funnel. Add 10 ml of N-hexane. Evenly mix. Add ammonia solution to extract 2 ~ 3 times, 5 ml per time. Combine ammonia aqueous solution layer (containing water-soluble acid pigment). Use N-hexane to wash twice. Add acetic acid to ammonia layer and adjust it neutral. Heat the water bath and evaporate it to nearly dry. Add water and set volume to 5 ml. Filter through a 0.45 μm microporous membrane. Analyze it via high performance liquid chromatography. 5.3 Instrument reference conditions Chromatographic column: column C18, 4.6 mm 250 mm, 5 μm Sample injection volume: 10 μl Column temperature: 35 C Diode array detector wavelength range: 400 nm ~ 800 nm or UV detector detection wavelength: 254 nm See Table 1 for gradient elution. Page 7 of 11