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This standard replaces GB/T 5009.121-2003 Determination of Dehydroacetic Acid in Foods.
The following main changes have been made with respect to GB/T 5009.121-2003:
——This standard is renamed as National Food Safety Standard - Determination of Dehydroacetic Acid in Foods
——Liquid chromatography is added;
——Liquid chromatography given in GB/T 23377-2009 is adopted;
——Classification of specimens is added;
——Preparation and extraction methods of the sample in gas chromatography are improved;
——Chromatographic conditions of gas chromatography are modified.
National Food Safety Standard
Determination of Dehydroacetic Acid in Foods
1 Scope
This standard specifies determination methods of dehydroacetic acid content in fruit and vegetable juices, fruit and vegetable pulps, pickles, fermented bean products, butter, breads, pastries, baked food fillings, compound flavoring, preprocessed meat products and cooked meat products.
This standard is applicable to the determination of dehydroacetic acid content in fruit and vegetable juices, fruit and vegetable pulps, pickles, fermented bean products, butter, breads, pastries, baked food fillings, compound flavoring, preprocessed meat products and cooked meat products; other foods may also refer to this standard.
Method I Gas Chromatography
2 Principle
For solid (semi-solid) sample, pull down the protein and extract with ethyl acetate after degreasing and acidification; for samples of fruit and vegetable juices and pulps, extract with ethyl acetate after acidification; separate and determine with the gas chromatograph equipped with hydrogen flame ionization detector, conduct the qualitative determination according to the retention time of chromatographic peak and conduct the quantitative determination by external standard method.
3 Reagents and Materials
Unless otherwise specified, analytically-pure reagents and Class-II water (defined in GB/T 6682) are adopted for the purpose of this method.
3.1 Reagents
3.1.1 Ethyl acetate (C4H8O2): chromatographically pure.
3.1.2 n-hexane (C6H14): chromatographically pure.
3.1.3 Hydrochloric acid (HCl)
3.1.4 Zinc sulfate (ZnSO4?7H2O)
3.1.5 Sodium hydroxide (NaOH).
3.2 Preparation of reagents
3.2.1 Hydrochloric acid solution (1+1, volume ratio): measure 50 mL hydrochloric acid and add into 50 mL water.
3.2.2 Zinc sulphate solution (120 g/L): weigh 12 g zinc sulfate, dissolve it and dilute with water to 100 mL.
3.2.3 Sodium hydroxide solution (20 g/L): weigh 2 g sodium hydroxide, dissolve it and dilute with water to 100 mL.
3.3 Standard product
Dehydroacetic acid (C8H8O4; CAS: 520-45-6) standard product: purity ≥ 99.5%.
3.4 Preparation of standard solutions
3.4.1 Dehydroacetic acid standard stock solution (1.0 mg/mL): accurately weigh 0.1000 g (accurate to 0.0001g) dehydroacetic acid standard product into a 100 mL volumetric flask, dissolve with ethyl acetate and scale the volume, preserve the solution at 4℃, which will be valid for 3 months.
3.4.2 Dehydroacetic acid standard working solution: accurately pipet 0.01 mL, 0.1 mL, 0.5 mL, 1.0 mL and 2.0 mL into 10mL volumetric flasks respectively, dilute with ethyl acetate and scale the volume, prepare into the standard working solutions with the concentrations of 1.00 μg/mL, 10.0 μg/mL, 50.0 μg/mL, 100 μg/mL and 200 μg/mL, preserve at 4℃, which will be valid for 1 month.
4 Instruments and Apparatus
4.1 Gas chromatograph, equipped with hydrogen flame ionization detector.
4.2 Balance: with sensibility of 0.1 mg and 1 mg.
4.3 Centrifuge: rotation speed ≥ 4000 r/min.
4.4 Ultrasonic cleaner: with power of 35 kW.
4.5 Grinder.
4.6 Stainless steel high-speed homogenizer.
4.7 pH meter.
5 Analysis Steps
5.1 Preparation of specimens
5.1.1 Fruit and vegetable juices and pulps: weigh 2 g ~ 5 g sample (accurate to 0.001 g), put into a 50 mL centrifugal tube, add 10 mL water, shake for 1 min, add 1 mL hydrochloric acid solution (3.2.1) for acidification, accurately add 5.0 mL ethyl acetate, shake and extract for 2 min, keep still for layering, take the supernatant for the determination by gas chromatography.
5.1.2 Pickles and fermented bean products; homogenize the sample with stainless steel high-speed homogenizer. Weigh 2 g ~ 5 g (accurate to 0.001 g) samples, put into 50 mL centrifugal tubes, add about 15 mL water, 2.5 mL zinc sulphate solution (3.2.2), adjust pH to 7.5 with sodium hydroxide solution (3.2.3), conduct the ultrasonic extraction for 15 min, transfer to a 25 mL volumetric flask, and scale the volume with water. Transfer the sample solutions into the centrifugal tubes and centrifuge for 10 min at the rotation speed of 4000 r/min.
Take 10 mL supernatant, add 1 mL hydrochloric acid solution (3.2.1) for acidification, accurately add 5.0 mL ethyl acetate, shake for 2 min, keep still for layering and take the supernatant for the determination by gas chromatography.
5.1.3 Bread, pastries, baked food fillings, compound flavoring, preprocessed meat products and cooked meat products: grind the sample with a grinder or homogenize with stainless steel high-speed homogenizer. Weigh 2 g ~ 5 g (accurate to 0.001 g) sample, put into a 50 mL centrifugal tube, add about 15 mL water and 2.5 mL zinc sulphate solution (3.2.2), adjust the pH to 7.5 with sodium hydroxide solution (3.2.3), conduct the ultrasonic extraction for 15 min, transfer to a 25 mL volumetric flask and scale the volume with water. Transfer the sample solution into the separating funnel, add 5 mL n-hexane, shake for 1 min, keep still for layering, take lower-part water phase into the centrifugal tube, centrifuge for 10 min at the rotation speed of 4000 r/min. Take 10 mL supernatant, add 1 mL hydrochloric acid solution (3.2.1) for acidification, accurately add 5.0 mL ethyl acetate, shake for 2 min, keep still for layering, take the supernatant for the determination by gas chromatography.
5.1.4 Butter: Weigh 2 g ~ 5 g (accurate to 0.001 g) sample, put into a 50 mL centrifugal tube, add about 15 mL water and 2.5 mL zinc sulphate solution (3.2.2), adjust the pH to 7.5 with sodium hydroxide solution (3.2.3), conduct the ultrasonic extraction for 15 min, transfer to a 25 mL volumetric flask and scale the volume with water. Transfer the sample solution into the separating funnel, add 5 mL n-hexane, shake for 1min, keep still for layering, take lower-part water phase into the centrifugal tube, centrifuge for 10 min at the rotation speed of 4000 r/min. Take 10 mL supernatant, add 1 mL hydrochloric acid solution (3.2.1) for acidification, accurately add 5.0 mL ethyl acetate, shake for 2 min, keep still for layering, take the supernatant for the determination by gas chromatography.
5.2 Reference conditions of instruments
5.2.1 Capillary column: polar capillary column (chemical bonding and stationary phase of polyethylene glycol, 30 m × 0.32 mm × 0.25 μm) or equivalent.
5.2.2 Temperature rising procedure of column: with initial temperature of 150℃, rise to 210℃ at a rate of 10℃/min, rise to 240℃ at a rate of 20℃/min and maintain for 2 min.
5.2.3 Temperature of injection port: 240℃
5.2.4 Temperature of detector: 300℃.
5.2.5 Flow rate of carrier gas (N2): 1.0 mL/min.
5.2.6 Split injection: 5:1 for splitting ratio and 1.0 μL for injection volume.
Foreword i
1 Scope
2 Principle
3 Reagents and Materials
4 Instruments and Apparatus
5 Analysis Steps
6 Expression of Analysis Results
7 Accuracy
8 Other
9 Principle
10 Reagents and Materials
11 Instruments and Apparatus
12 Analysis Steps
13 Expression of Analysis Results
14 Accuracy
15 Other
Annex A Gas Chromatogram of Dehydroacetic Acid
Annex B Liquid Chromatogram of Dehydroacetic Acid
