1 Scope
This standard specifies the method for determination of ethylenediaminetetraacetateedetate in food through liquid chromatograph.
This standard is applicable to the determination of ethylene diamine tetraacetic acid disodium in jam, preserved fruit, canned pickled vegetables, vegetable puree (sauce) except tomato sauce, canned nuts and seeds, canned mixed congee, compound seasonings and beverages and the determination of ethylene diamine tetraacetic acid disodium calcium in compound seasonings. This standard does not apply to the determination of ethylenediaminetetraacetic acid ferric sodium in iron fortified soy sauce.
2 Principle
Extract the specimen of to be determined ethylenediamine tetraacetic acid disodium by water. Add ferric chloride into the extracting solution for complexing. Purify the solution by mixed anion (PXA) solid phase extraction column, detect by liquid chromatograph, and quantify by external standard method. When determining compound seasoning, determine the total content of ferric chloride complex of ethylenediaminetetraacetateedetate firstly, and then the content of ethylenediamine tetraacetic acid disodium calcium. Identify the content of ethylenediamine tetraacetic acid disodium according to the results.
3 Reagents and materials
Unless otherwise stated, reagents used in this method are analytical reagents, and the water is Class-I water as specified in GB/T 6682.
3.1 Reagents
3.1.1 Methanol (CH3OH): chromatographically pure.
3.1.2 Hydrochloric acid (HCl).
3.1.3 Phosphoric acid (H3PO4)
3.1.4 Methanoic acid (CH2O2): chromatographically pure.
3.1.5 Tetrabutylammonium bromide (C16H36NBr).
3.1.6 Sodium acetate (CH3COONa).
3.1.7 Ferric chloride (FeCl3·6H2O).
3.2 Preparation of reagents
3.2.1 Ferric chloride solution: weigh 0.54 g of ferric chloride, dissolve it in 90 mL of water, add 0.10 mL of hydrochloric acid, transfer it to a 100-mL volumetric flask, dilute to scale with water, and mix it well.
3.2.2 5 % methanoic acid methanol aqueous solution: take 5 mL of methanoic acid and 20 mL of methanol and dilute to 100 mL with water.
3.2.3 Tetrabutylammonium bromide-sodium acetate mixed solution (pH 4.0): weigh 6.45 g of tetrabutylammonium bromide and 2.46 g of sodium acetate, add 1,000 mL of water for ultrasonic dissolution, and add phosphoric acid to adjust pH to 4.0±0.1.
3.2.4 Tetrabutylammonium bromide-sodium acetate mixed solution (pH 2.5): weigh 6.45g of tetrabutylammonium bromide and 2.46g of sodium acetate, add 1,000mL of water for ultrasonic dissolution, and add phosphoric acid to adjust pH to 2.5±0.1.
3.3 Standard
3.3.1 Ethylenediamine tetraacetic acid disodium standard (C10H14N2O8Na2•2H2O, CAS No.: 6381-92-6): purity ≥ 99 %
3.3.2 Ethylenediamine tetraacetic acid disodium (C10H14N2O8Na2Ca, CAS No.: 62-33-9): purity ≥ 99 %.
3.4 Preparation of standard solution
3.4.1 Ethylenediamine tetraacetic acid disodium standard solution: accurately weigh an appropriate amount of ethylenediamine tetraacetic acid disodium standard, dissolve it and dilute the solution in a 100-mL brown volumetric flask with water, so as to obtain a 10 mg/mL standard stock solution. Transfer the solution to liquid storage flask and store it at 4 ℃.
3.4.2 Ethylenediamine tetraacetic acid disodium standard series solution: pipet 1volume of ethylenediamine tetraacetic acid disodium standard solution and dilute it with 9 volumes of water into 1 mg/mL standard intermediate solution. Then dilute the standard intermediate solution with water to series standard working solutions with the concentrations of 0.5 μg/mL, 2.0 μg/mL, 5.0 μg/mL, 10.0 μg/mL and 50.0 μg/mL.
3.4.3 Ethylenediamine tetraacetic acid disodium calcium standard solution: accurately weigh an appropriate amount of ethylenediamine tetraacetic acid disodium calcium standard, dissolve it and dilute the solution in a 100-mL brown volumetric flask with water, so as to obtain 10 mg/mL standard stock solutions. Transfer the solution to liquid storage flask and store it at 4 ℃.
3.4.4 Ethylenediamine tetraacetic acid disodium calcium standard series solution: pipet 1volume of ethylenediamine tetraacetic acid disodium calcium standard solution and dilute it with 9 volumes of water into 1 mg/mL standard intermediate solution. Then dilute the ethylenediamine tetraacetic acid disodium calcium standard intermediate solution with water to series standard working solutions with the concentrations of 1.0 μg/mL, 10.0 μg/mL, 20.0 μg/mL, 50.0 μg/mL and 100.0 μg/mL.
4 Apparatus
4.1 Liquid chromatograph: equipped with ultraviolet detector.
4.2 Electronic balance: with a sensitivity of 0.1 mg and 0.01 g.
4.3 Centrifuge: rotating speed ≥ 7,500 r/min.
4.4 Vortex oscillator.
4.5 Ultrasonic cleaning machine.
4.6 pH meter: with an accuracy of 0.01.
4.7 24-hole solid phase extraction unit.
4.8 Millipore filter membrane: 0.45 μm.
4.9 Mixed anion (PXA) solid phase extraction column: 150 mg/6 mL or the equivalent. The main ingredient of PXA is quaternary group bonded to polystyrene/divinylbenzene copolymer with hydrophilic group.
5 Analysis procedures
5.1 Specimen preparation
5.1.1 Solid sample
Smash the sample by crusher, mix well. Then pack it in a clean container, seal and mark well. Samples with high sugar content need to be smashed after freezing. The specimen shall be preserved at 4 ℃.
5.1.2 Liquid sample
Samples with fruit particles need to be smashed by juicer and mixed well. Then pack it in a clean container, seal and mark well. Take 500 mL of carbonated beverage into a 1,000-mL beaker, heat it while stirring in a 70 ℃ water bath to remove part of carbon dioxide, after cooling to room temperature, pack it in a clean container, seal and mark well. The specimen shall be preserved at 4 ℃.
5.2 Determination
5.2.1 Determination of the total content of ethylenediaminetetraacetateedetate
5.2.1.1 Extraction
5.2.1.1.1 Samples immiscible with water
Weigh 5g of specimen (accurate to 0.01 g), put it in a 50-mL centrifuge tube, add 25 mL of water, mix well by subjecting it to vortex, subject it to ultrasound for 20 min, centrifuge it at 7,500 r/min for 5 min, and take the supernatant in a 50-mL glass colorimetric tube. Extract the remaining residue once again, and after centrifugation, combine the supernatants, and dilute to 50 mL with water. Pipet 5 mL of the diluted solution into a 15-mL centrifuge tube for complexation.
5.2.1.1.2 Samples miscible with water
Weigh 5 g of specimen (accurate to 0.01 g), put it in a 50-mL glass colorimetric tube, add 25 mL of water, mix well by subjecting it to vortex, after ultrasonic extraction for 20min, dilute the solution to 50 mL with water. Pipet 5 mL of supernatant into a 15-mL centrifuge tube for complexation.
5.2.1.2 Complexation
Add 0.5 mL of ferric chloride solution to the above extracted solution, mix for 1 min by subjecting it to vortex, subject it to ultrasound for 20 min, and centrifuge it for 5 min at 7,500 r/min for purification.
Foreword i
1 Scope
2 Principle
3 Reagents and materials
4 Apparatus
5 Analysis procedures
6 Expression of analysis results
7 Precision
8 Others
Annex A Liquid chromatograms of ethylenediamine tetraacetic acid disodium complex standard solution and ethylenediamine tetraacetic acid disodium calcium standard solution
1 Scope
This standard specifies the method for determination of ethylenediaminetetraacetateedetate in food through liquid chromatograph.
This standard is applicable to the determination of ethylene diamine tetraacetic acid disodium in jam, preserved fruit, canned pickled vegetables, vegetable puree (sauce) except tomato sauce, canned nuts and seeds, canned mixed congee, compound seasonings and beverages and the determination of ethylene diamine tetraacetic acid disodium calcium in compound seasonings. This standard does not apply to the determination of ethylenediaminetetraacetic acid ferric sodium in iron fortified soy sauce.
2 Principle
Extract the specimen of to be determined ethylenediamine tetraacetic acid disodium by water. Add ferric chloride into the extracting solution for complexing. Purify the solution by mixed anion (PXA) solid phase extraction column, detect by liquid chromatograph, and quantify by external standard method. When determining compound seasoning, determine the total content of ferric chloride complex of ethylenediaminetetraacetateedetate firstly, and then the content of ethylenediamine tetraacetic acid disodium calcium. Identify the content of ethylenediamine tetraacetic acid disodium according to the results.
3 Reagents and materials
Unless otherwise stated, reagents used in this method are analytical reagents, and the water is Class-I water as specified in GB/T 6682.
3.1 Reagents
3.1.1 Methanol (CH3OH): chromatographically pure.
3.1.2 Hydrochloric acid (HCl).
3.1.3 Phosphoric acid (H3PO4)
3.1.4 Methanoic acid (CH2O2): chromatographically pure.
3.1.5 Tetrabutylammonium bromide (C16H36NBr).
3.1.6 Sodium acetate (CH3COONa).
3.1.7 Ferric chloride (FeCl3·6H2O).
3.2 Preparation of reagents
3.2.1 Ferric chloride solution: weigh 0.54 g of ferric chloride, dissolve it in 90 mL of water, add 0.10 mL of hydrochloric acid, transfer it to a 100-mL volumetric flask, dilute to scale with water, and mix it well.
3.2.2 5 % methanoic acid methanol aqueous solution: take 5 mL of methanoic acid and 20 mL of methanol and dilute to 100 mL with water.
3.2.3 Tetrabutylammonium bromide-sodium acetate mixed solution (pH 4.0): weigh 6.45 g of tetrabutylammonium bromide and 2.46 g of sodium acetate, add 1,000 mL of water for ultrasonic dissolution, and add phosphoric acid to adjust pH to 4.0±0.1.
3.2.4 Tetrabutylammonium bromide-sodium acetate mixed solution (pH 2.5): weigh 6.45g of tetrabutylammonium bromide and 2.46g of sodium acetate, add 1,000mL of water for ultrasonic dissolution, and add phosphoric acid to adjust pH to 2.5±0.1.
3.3 Standard
3.3.1 Ethylenediamine tetraacetic acid disodium standard (C10H14N2O8Na2•2H2O, CAS No.: 6381-92-6): purity ≥ 99 %
3.3.2 Ethylenediamine tetraacetic acid disodium (C10H14N2O8Na2Ca, CAS No.: 62-33-9): purity ≥ 99 %.
3.4 Preparation of standard solution
3.4.1 Ethylenediamine tetraacetic acid disodium standard solution: accurately weigh an appropriate amount of ethylenediamine tetraacetic acid disodium standard, dissolve it and dilute the solution in a 100-mL brown volumetric flask with water, so as to obtain a 10 mg/mL standard stock solution. Transfer the solution to liquid storage flask and store it at 4 ℃.
3.4.2 Ethylenediamine tetraacetic acid disodium standard series solution: pipet 1volume of ethylenediamine tetraacetic acid disodium standard solution and dilute it with 9 volumes of water into 1 mg/mL standard intermediate solution. Then dilute the standard intermediate solution with water to series standard working solutions with the concentrations of 0.5 μg/mL, 2.0 μg/mL, 5.0 μg/mL, 10.0 μg/mL and 50.0 μg/mL.
3.4.3 Ethylenediamine tetraacetic acid disodium calcium standard solution: accurately weigh an appropriate amount of ethylenediamine tetraacetic acid disodium calcium standard, dissolve it and dilute the solution in a 100-mL brown volumetric flask with water, so as to obtain 10 mg/mL standard stock solutions. Transfer the solution to liquid storage flask and store it at 4 ℃.
3.4.4 Ethylenediamine tetraacetic acid disodium calcium standard series solution: pipet 1volume of ethylenediamine tetraacetic acid disodium calcium standard solution and dilute it with 9 volumes of water into 1 mg/mL standard intermediate solution. Then dilute the ethylenediamine tetraacetic acid disodium calcium standard intermediate solution with water to series standard working solutions with the concentrations of 1.0 μg/mL, 10.0 μg/mL, 20.0 μg/mL, 50.0 μg/mL and 100.0 μg/mL.
4 Apparatus
4.1 Liquid chromatograph: equipped with ultraviolet detector.
4.2 Electronic balance: with a sensitivity of 0.1 mg and 0.01 g.
4.3 Centrifuge: rotating speed ≥ 7,500 r/min.
4.4 Vortex oscillator.
4.5 Ultrasonic cleaning machine.
4.6 pH meter: with an accuracy of 0.01.
4.7 24-hole solid phase extraction unit.
4.8 Millipore filter membrane: 0.45 μm.
4.9 Mixed anion (PXA) solid phase extraction column: 150 mg/6 mL or the equivalent. The main ingredient of PXA is quaternary group bonded to polystyrene/divinylbenzene copolymer with hydrophilic group.
5 Analysis procedures
5.1 Specimen preparation
5.1.1 Solid sample
Smash the sample by crusher, mix well. Then pack it in a clean container, seal and mark well. Samples with high sugar content need to be smashed after freezing. The specimen shall be preserved at 4 ℃.
5.1.2 Liquid sample
Samples with fruit particles need to be smashed by juicer and mixed well. Then pack it in a clean container, seal and mark well. Take 500 mL of carbonated beverage into a 1,000-mL beaker, heat it while stirring in a 70 ℃ water bath to remove part of carbon dioxide, after cooling to room temperature, pack it in a clean container, seal and mark well. The specimen shall be preserved at 4 ℃.
5.2 Determination
5.2.1 Determination of the total content of ethylenediaminetetraacetateedetate
5.2.1.1 Extraction
5.2.1.1.1 Samples immiscible with water
Weigh 5g of specimen (accurate to 0.01 g), put it in a 50-mL centrifuge tube, add 25 mL of water, mix well by subjecting it to vortex, subject it to ultrasound for 20 min, centrifuge it at 7,500 r/min for 5 min, and take the supernatant in a 50-mL glass colorimetric tube. Extract the remaining residue once again, and after centrifugation, combine the supernatants, and dilute to 50 mL with water. Pipet 5 mL of the diluted solution into a 15-mL centrifuge tube for complexation.
5.2.1.1.2 Samples miscible with water
Weigh 5 g of specimen (accurate to 0.01 g), put it in a 50-mL glass colorimetric tube, add 25 mL of water, mix well by subjecting it to vortex, after ultrasonic extraction for 20min, dilute the solution to 50 mL with water. Pipet 5 mL of supernatant into a 15-mL centrifuge tube for complexation.
5.2.1.2 Complexation
Add 0.5 mL of ferric chloride solution to the above extracted solution, mix for 1 min by subjecting it to vortex, subject it to ultrasound for 20 min, and centrifuge it for 5 min at 7,500 r/min for purification.
Contents of GB 5009.278-2016
Foreword i
1 Scope
2 Principle
3 Reagents and materials
4 Apparatus
5 Analysis procedures
6 Expression of analysis results
7 Precision
8 Others
Annex A Liquid chromatograms of ethylenediamine tetraacetic acid disodium complex standard solution and ethylenediamine tetraacetic acid disodium calcium standard solution
