This standard supersedes GB/T 5009.29-2003 "Determination of Sorbic Acid and Benzioc Acid in Foods", GB/T 5009.28-2003 "Determination of Saccharin Sodium in Foods", GB/T 23495-2009 "Determination of Benzoic Acid, Sorbic Acid and Saccharin Sodium in Foods", GB 21703-2010 "National Food Safety Standard — Determination of Benzoic Acid and Sorbic Acid in Milk and Milk Products", SN/T 2012-2007 "Determination of Benzoic Acid and Sorbic Acid in Vinegar for Export and Import — Liquid Chromatography" and SB/T 10389-2004 "Determination of Sorbic Acid in Meat and Meat Products".
Compared with GB/T 5009.29-2003, the main changes in this standard are as follows:
— The standard name is modified to "National Food Safety Standard — Determination of Benzoic Acid, Sorbic Acid and Saccharin Sodium in Foods";
— "Multipoint calibration" method for plotting of standard curve is added;
— Pretreatment procedure of sample is modified;
— The provisions of separation of packed chromatographic column in gas chromatography are deleted;
— The provisions of separation of capillary chromatographic column in gas chromatography are deleted;
National Food Safety Standard — Determination of Benzoic Acid, Sorbic Acid and Saccharin Sodium in Foods
1 Scope
This standard specifies the determination method of benzoic acid, sorbic acid and saccharine sodium in foods.
Method I of this standard is applicable to the determination of benzoic acid, sorbic acid and saccharine sodium in foods; Method II is applicable to the determination of benzoic acid, sorbic acid and saccharine sodium in soy sauce, juice and jam.
Method I Liquid Chromatography
2 Principle
After the samples were extracted by water, the high fat samples were digested by normal hexane and the high protein samples were precipitated by protein precipitation agent, separated by liquid chromatography, tested by ultraviolet detector and quantified by external standard method.
3 Reagents and Materials
3.1 Reagents
Unless otherwise specified, reagents used in this method are all analytically pure, and water is the Grade 1 water as specified in GB/T 6682.
3.1.1 Ammonia Water (NH3·H2O).
3.1.2 Potassium ferrocyanide [K4Fe(CN)6·3H2O].
3.1.3 Zinc acetate [Zn(CH3COO)2·2H2O].
3.1.4 Absolute ethyl alcohol (CH3CH2OH).
3.1.5 Normal hexane (C6H14).
3.1.6 Methanol (CH3OH): chromatographically pure.
3.1.7 Ammonium acetate (CH3COONH4): chromatographically pure.
3.1.8 Formic acid (HCOOH): chromatographically pure.
3.2 Reagent preparation
3.2.1 Ammonia water solution (1+99): measure 1mL ammonia water and add to 99mL water, mix well.
3.2.2 Potassium ferricyanide solution (92g/L): weigh 106g potassium ferrocyanide and dissolve it with proper amount of water, scale the volume to 1000mL with water.
3.2.3 Zinc acetate solution (183g/L): weigh 220g zinc acetate and dissolve it with proper amount of water, add 30mL glacial acetic acid, scale the volume to 1000mL with water.
3.2.4 Ammonium acetate solution (20 mmol/L): weigh 1.54g ammonium acetate and dissolve it with proper amount of water, scale the volume to 1000mL with water; filter it with 0.22μmaqueous phase microporous filtering film, take the filtrate for use.
3.2.5 Formic acid-ammonium acetate solution (2 mmol/L formic acid + 20 mmol/L ammonium acetate): weigh 1.54g ammonium acetate and dissolve it with proper amount of water, add 75.2μL formic acid, scale the volume to 1000mL with water; filter it with 0.22μmaqueous phase microporous filtering film, take the filtrate for use.
3.3 Standards
3.3.1 Sodium benzoate (C6H5COONa, CAS No.: 532-32-1), purity ≥99.0%; or benzoic acid (C6H5COOH, CAS No.: 65-85-0), purity ≥99.0%; or other standard reference materials approved and awarded with reference material certificate by the nation.
3.3.2 Potassium sorbate (C6H7KO2, CAS No.: 590-00-1), purity ≥99.0%; or sorbic acid (C6H8O2, CAS No.: 110-44-1), purity ≥99.0%; or other standard reference materials approved and awarded with reference material certificate by the nation.
3.3.3 Saccharine sodium (C6H4CONNaSO2, CAS No.: 128-44-9), purity ≥99.0%; or other standard reference materials approved and awarded with reference material certificate by the nation.
3.4 Preparation of standard solution
3.4.1 Benzoic acid, sorbic acid and saccharine sodium (Calculated in saccharin) standard stock solution (1000mg/L): accurately weigh (to the nearest 0.0001g) sodium benzoate, potassium sorbate and saccharine sodium 0.118g, 0.134g and 0.117g respectively, dissolve in water and scale the volume to 100mL respectively. Stored at 4℃, the storage period is 6 months. When benzoic acid and sorbic acid are used, them shall be dissolve in methanol and scale the volume.
Note: Saccharine sodium contain with crystal water, prior to use, it shall be baked 4h at 120℃, and then cooled in a desiccator to room temperature.
3.4.2 Benzoic acid, sorbic acid and saccharine sodium (Calculated in saccharin) mixed standard intermediate solution (200mg/L): accurately pipet benzoic acid, sorbic acid and saccharine sodium standard stock solution 10.0mL respectively and transfer into 50mL volumetric flask, scale the volume with water. Stored at 4℃, the storage period is 3 months.
3.4.3 Benzoic acid, sorbic acid and saccharine sodium (Calculated in saccharin) mixed standard series working solutions (200mg/L): accurately pipet benzoic acid, sorbic acid and saccharine sodium mixed standard intermediate solution 0mL, 0.05mL, 0.25mL, 0.50mL, 1.00mL, 2.50mL, 5.00mL and 10.0mL respectively, scale the volume to 10mL with water, to prepare mixed standard series working solutions with the mass concentration of 0 mg/L, 1.00 mg/L, 5.00 mg/L, 10.0 mg/L, 20.0 mg/L, 50.0mg/L, 100mg/L and 200mg/L respectively. Prepare immediately before use.
3.5 Materials
3.5.1 Aqueous phase microporous filtering film: 0.22μm.
3.5.2 Plastic centrifuge tube: 50mL.
4 Apparatus and Equipment
4.1 High performance liquid chromatography: equipped with ultraviolet detector.
4.2 Analytical balance: with sensitivity of 0.001g and 0.0001g.
4.3 Vortex oscillator.
4.4 Centrifuge: rotation speed ≥8000r/min.
4.5 Refiner.
4.6 Thermostat water bath.
4.7 Supersonic generator.
5 Analysis Procedure
5.1 Sample preparation
Direct mix several uniform samples of prepackaged beverage, liquid milk and other foods; non-uniform liquid, semisolid samples made into slurry by tissue refiner; solid samples crushed fully by grinder and stirred uniformly; cheese, grease, chocolate and other foods heated to molten at 50℃ to 60℃ and intensive mixing uniform while hot. Take 200g of such sample and put in a glass container, seal up; liquid sample preserved at 4℃, other samples preserved at -18℃.
5.2 Extraction of sample
5.2.1 General sample
Accurately weigh 2g (to the nearest 0.001g) sample in a centrifuge tube with stopper, add approximately 25mL water, mix well by vortex; ultrasonic processing 20min in 50℃ water bath, cool to room temperature; add 2mL potassium ferricyanide solution and 2mL zinc acetate solution, mix well, centrifuging 5min at 8000 r/min, and transfer the aqueous phase to a 50mL volumetric flask; add 20mL water to the residues, mix well by vortex; ultrasonic processing 5min, centrifuging 5min at 8000 r/min, and transfer the aqueous phase to the 50mL volumetric flask, dilute to the scale with water, mix well. Take right amount of supernatant and filter it with 0.22μm filter membrane, take the filtrate for liquid chromatographic determination.
Note: Protein precipitation reagent may not be required for determination of carbonated beverage, fruit wine, fruit juice and distilled liquor.
5.2.2 Jelly, candy and other samples contain with gum base
Accurately weigh 2g (to the nearest 0.001g) sample in a centrifuge tube with stopper, add approximately 25mL water, dissolve the sample by heating in 70℃ water bath, ultrasonic processing 20min in 50℃ water bath, the rest procedure are the same with 5.2.1.
5.2.3 High fat samples of grease, chocolate, cream, fried foods
Accurately weigh 2g (to the nearest 0.001g) sample in a centrifuge tube with stopper, add 10mL normal hexane, heating in 60℃ water bath 5min, shaking slightly to dissolve the fat; add 25mL ammonia water solution (1+99) and 1mL ethyl alcohol, mix well by vortex; ultrasonic processing 20min in 50℃ water bath, cool to room temperature; add 2mL potassium ferricyanide solution and 2mL zinc acetate solution, mix well, centrifuging 5min at 8000 r/min, discard the organic phase and transfer the aqueous phase to a 50mL volumetric flask; re-extraction of the residues in accordance with 5.2.1, perform determination.
5.3 Reference conditions of apparatus
5.3.1 Chromatographic column: C18 column, 250mm in column length, 4.6mm in inner diameter and 5μm in particle size, or equivalent.
5.3.2 Mobile phase: methanol + ammonium acetate solution = 5+95.
5.3.3 Flow rate: 1mL/min.
5.3.4 Detection wavelength: 230nm.
5.3.5 Injection volume: 10μL.
Note: When there is interference peak or auxiliary qualitative is required, it can be determined by the mobile phase added with formic acid, for example, mobile phase: methanol + formic acid-ammonium acetate solution = 8 + 92. See Figure A.2 for the reference chromatogram.
5.4 Plotting of standard curve
Inject the mixed standard series working solution into liquid chromatography, determine the corresponding peak area, and plot the standard curve by taking the mass concentration of mixed standard working solution as horizontal coordinate and peak area as longitudinal coordinate.
5.5 Determination of sample solution
Inject the sample solution into liquid chromatography to obtain peak area, and obtain the mass concentration of benzoic acid, sorbic acid and saccharine sodium (calculated in saccharin) in the to-be-determined solution from standard curve.
This standard supersedes GB/T 5009.29-2003 "Determination of Sorbic Acid and Benzioc Acid in Foods", GB/T 5009.28-2003 "Determination of Saccharin Sodium in Foods", GB/T 23495-2009 "Determination of Benzoic Acid, Sorbic Acid and Saccharin Sodium in Foods", GB 21703-2010 "National Food Safety Standard — Determination of Benzoic Acid and Sorbic Acid in Milk and Milk Products", SN/T 2012-2007 "Determination of Benzoic Acid and Sorbic Acid in Vinegar for Export and Import — Liquid Chromatography" and SB/T 10389-2004 "Determination of Sorbic Acid in Meat and Meat Products".
Compared with GB/T 5009.29-2003, the main changes in this standard are as follows:
— The standard name is modified to "National Food Safety Standard — Determination of Benzoic Acid, Sorbic Acid and Saccharin Sodium in Foods";
— "Multipoint calibration" method for plotting of standard curve is added;
— Pretreatment procedure of sample is modified;
— The provisions of separation of packed chromatographic column in gas chromatography are deleted;
— The provisions of separation of capillary chromatographic column in gas chromatography are deleted;
National Food Safety Standard — Determination of Benzoic Acid, Sorbic Acid and Saccharin Sodium in Foods
1 Scope
This standard specifies the determination method of benzoic acid, sorbic acid and saccharine sodium in foods.
Method I of this standard is applicable to the determination of benzoic acid, sorbic acid and saccharine sodium in foods; Method II is applicable to the determination of benzoic acid, sorbic acid and saccharine sodium in soy sauce, juice and jam.
Method I Liquid Chromatography
2 Principle
After the samples were extracted by water, the high fat samples were digested by normal hexane and the high protein samples were precipitated by protein precipitation agent, separated by liquid chromatography, tested by ultraviolet detector and quantified by external standard method.
3 Reagents and Materials
3.1 Reagents
Unless otherwise specified, reagents used in this method are all analytically pure, and water is the Grade 1 water as specified in GB/T 6682.
3.1.1 Ammonia Water (NH3·H2O).
3.1.2 Potassium ferrocyanide [K4Fe(CN)6·3H2O].
3.1.3 Zinc acetate [Zn(CH3COO)2·2H2O].
3.1.4 Absolute ethyl alcohol (CH3CH2OH).
3.1.5 Normal hexane (C6H14).
3.1.6 Methanol (CH3OH): chromatographically pure.
3.1.7 Ammonium acetate (CH3COONH4): chromatographically pure.
3.1.8 Formic acid (HCOOH): chromatographically pure.
3.2 Reagent preparation
3.2.1 Ammonia water solution (1+99): measure 1mL ammonia water and add to 99mL water, mix well.
3.2.2 Potassium ferricyanide solution (92g/L): weigh 106g potassium ferrocyanide and dissolve it with proper amount of water, scale the volume to 1000mL with water.
3.2.3 Zinc acetate solution (183g/L): weigh 220g zinc acetate and dissolve it with proper amount of water, add 30mL glacial acetic acid, scale the volume to 1000mL with water.
3.2.4 Ammonium acetate solution (20 mmol/L): weigh 1.54g ammonium acetate and dissolve it with proper amount of water, scale the volume to 1000mL with water; filter it with 0.22μmaqueous phase microporous filtering film, take the filtrate for use.
3.2.5 Formic acid-ammonium acetate solution (2 mmol/L formic acid + 20 mmol/L ammonium acetate): weigh 1.54g ammonium acetate and dissolve it with proper amount of water, add 75.2μL formic acid, scale the volume to 1000mL with water; filter it with 0.22μmaqueous phase microporous filtering film, take the filtrate for use.
3.3 Standards
3.3.1 Sodium benzoate (C6H5COONa, CAS No.: 532-32-1), purity ≥99.0%; or benzoic acid (C6H5COOH, CAS No.: 65-85-0), purity ≥99.0%; or other standard reference materials approved and awarded with reference material certificate by the nation.
3.3.2 Potassium sorbate (C6H7KO2, CAS No.: 590-00-1), purity ≥99.0%; or sorbic acid (C6H8O2, CAS No.: 110-44-1), purity ≥99.0%; or other standard reference materials approved and awarded with reference material certificate by the nation.
3.3.3 Saccharine sodium (C6H4CONNaSO2, CAS No.: 128-44-9), purity ≥99.0%; or other standard reference materials approved and awarded with reference material certificate by the nation.
3.4 Preparation of standard solution
3.4.1 Benzoic acid, sorbic acid and saccharine sodium (Calculated in saccharin) standard stock solution (1000mg/L): accurately weigh (to the nearest 0.0001g) sodium benzoate, potassium sorbate and saccharine sodium 0.118g, 0.134g and 0.117g respectively, dissolve in water and scale the volume to 100mL respectively. Stored at 4℃, the storage period is 6 months. When benzoic acid and sorbic acid are used, them shall be dissolve in methanol and scale the volume.
Note: Saccharine sodium contain with crystal water, prior to use, it shall be baked 4h at 120℃, and then cooled in a desiccator to room temperature.
3.4.2 Benzoic acid, sorbic acid and saccharine sodium (Calculated in saccharin) mixed standard intermediate solution (200mg/L): accurately pipet benzoic acid, sorbic acid and saccharine sodium standard stock solution 10.0mL respectively and transfer into 50mL volumetric flask, scale the volume with water. Stored at 4℃, the storage period is 3 months.
3.4.3 Benzoic acid, sorbic acid and saccharine sodium (Calculated in saccharin) mixed standard series working solutions (200mg/L): accurately pipet benzoic acid, sorbic acid and saccharine sodium mixed standard intermediate solution 0mL, 0.05mL, 0.25mL, 0.50mL, 1.00mL, 2.50mL, 5.00mL and 10.0mL respectively, scale the volume to 10mL with water, to prepare mixed standard series working solutions with the mass concentration of 0 mg/L, 1.00 mg/L, 5.00 mg/L, 10.0 mg/L, 20.0 mg/L, 50.0mg/L, 100mg/L and 200mg/L respectively. Prepare immediately before use.
3.5 Materials
3.5.1 Aqueous phase microporous filtering film: 0.22μm.
3.5.2 Plastic centrifuge tube: 50mL.
4 Apparatus and Equipment
4.1 High performance liquid chromatography: equipped with ultraviolet detector.
4.2 Analytical balance: with sensitivity of 0.001g and 0.0001g.
4.3 Vortex oscillator.
4.4 Centrifuge: rotation speed ≥8000r/min.
4.5 Refiner.
4.6 Thermostat water bath.
4.7 Supersonic generator.
5 Analysis Procedure
5.1 Sample preparation
Direct mix several uniform samples of prepackaged beverage, liquid milk and other foods; non-uniform liquid, semisolid samples made into slurry by tissue refiner; solid samples crushed fully by grinder and stirred uniformly; cheese, grease, chocolate and other foods heated to molten at 50℃ to 60℃ and intensive mixing uniform while hot. Take 200g of such sample and put in a glass container, seal up; liquid sample preserved at 4℃, other samples preserved at -18℃.
5.2 Extraction of sample
5.2.1 General sample
Accurately weigh 2g (to the nearest 0.001g) sample in a centrifuge tube with stopper, add approximately 25mL water, mix well by vortex; ultrasonic processing 20min in 50℃ water bath, cool to room temperature; add 2mL potassium ferricyanide solution and 2mL zinc acetate solution, mix well, centrifuging 5min at 8000 r/min, and transfer the aqueous phase to a 50mL volumetric flask; add 20mL water to the residues, mix well by vortex; ultrasonic processing 5min, centrifuging 5min at 8000 r/min, and transfer the aqueous phase to the 50mL volumetric flask, dilute to the scale with water, mix well. Take right amount of supernatant and filter it with 0.22μm filter membrane, take the filtrate for liquid chromatographic determination.
Note: Protein precipitation reagent may not be required for determination of carbonated beverage, fruit wine, fruit juice and distilled liquor.
5.2.2 Jelly, candy and other samples contain with gum base
Accurately weigh 2g (to the nearest 0.001g) sample in a centrifuge tube with stopper, add approximately 25mL water, dissolve the sample by heating in 70℃ water bath, ultrasonic processing 20min in 50℃ water bath, the rest procedure are the same with 5.2.1.
5.2.3 High fat samples of grease, chocolate, cream, fried foods
Accurately weigh 2g (to the nearest 0.001g) sample in a centrifuge tube with stopper, add 10mL normal hexane, heating in 60℃ water bath 5min, shaking slightly to dissolve the fat; add 25mL ammonia water solution (1+99) and 1mL ethyl alcohol, mix well by vortex; ultrasonic processing 20min in 50℃ water bath, cool to room temperature; add 2mL potassium ferricyanide solution and 2mL zinc acetate solution, mix well, centrifuging 5min at 8000 r/min, discard the organic phase and transfer the aqueous phase to a 50mL volumetric flask; re-extraction of the residues in accordance with 5.2.1, perform determination.
5.3 Reference conditions of apparatus
5.3.1 Chromatographic column: C18 column, 250mm in column length, 4.6mm in inner diameter and 5μm in particle size, or equivalent.
5.3.2 Mobile phase: methanol + ammonium acetate solution = 5+95.
5.3.3 Flow rate: 1mL/min.
5.3.4 Detection wavelength: 230nm.
5.3.5 Injection volume: 10μL.
Note: When there is interference peak or auxiliary qualitative is required, it can be determined by the mobile phase added with formic acid, for example, mobile phase: methanol + formic acid-ammonium acetate solution = 8 + 92. See Figure A.2 for the reference chromatogram.
5.4 Plotting of standard curve
Inject the mixed standard series working solution into liquid chromatography, determine the corresponding peak area, and plot the standard curve by taking the mass concentration of mixed standard working solution as horizontal coordinate and peak area as longitudinal coordinate.
5.5 Determination of sample solution
Inject the sample solution into liquid chromatography to obtain peak area, and obtain the mass concentration of benzoic acid, sorbic acid and saccharine sodium (calculated in saccharin) in the to-be-determined solution from standard curve.
