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This standard replaces GB/T 5009.157-2003 Determination of Organic Acid in Foods.
The following main changes have been made with respect to GB/T 5009.157-2003 Determination of Organic Acid in Foods (the previous edition):
——This standard is renamed as National Food Safety Standard Determination of Organic Acid in Foods;
——Applicable scope is extended to jelly, solid beverage, fruit can and other foods;
——Such to-be-determined objects as lactic acid, fumaric acid and adipic acid are added.
National Food Safety Standard Determination of Organic Acid in Foods
1 Scope
This standard specifies determination method of tartaric acid, lactic acid, malic acid, citric acid, succinic acid, fumaric acid and adipic acid in foods.
This standard is applicable to seven organic acids in fruit juice and fruit juice beverage, carbonated beverage, gum candy, biscuit, pastry, jelly, fruit can, dough product and baked food filling.
2 Principle
Directly dilute or extract specimen with water, purify with strong anion exchanging solid phase extraction column, separate with reversed-phase chromatographic column and conduct qualitative determination by retention time and quantitative determination by external standard method.
3 Reagents and Materials
Unless otherwise specified, analytically-pure reagents and Class-I water (defined in GB/T 6682) are adopted for the purpose of this method.
3.1 Reagents
3.1.1 Methanol (CH3OH): chromatographically pure.
3.1.2 Absolute ethanol (CH3CH2OH): chromatographically pure.
3.1.3 Phosphoric acid (H3PO4).
3.2 Preparation of reagents
3.2.1 Phosphoric acid solution (0.1%): measure 2 mL phosphoric acid, add water to 100 mL and mix well.
3.2.2 Phosphoric acid-methanol solution (2%): measure 2 mL phosphoric acid, add water to 100 mL and mix well.
3.3 Standard products
3.3.1 Lactic acid (C3H6O3): purity≥99%.
3.3.2 Tartaric acid (C4H6O6): purity≥99%.
3.3.3 Malic acid (C4H6O5): purity≥99%.
3.3.4 Citric acid (C6H8N7): purity ≥98%.
3.3.5 Tartaric acid (C4H6N4): purity≥99%.
3.3.6 Fumaric acid (C4H4O4): purity≥99%.
3.3.7 Adipic acid (C6H10N4): purity≥99%.
3.4 Preparation of standard solutions
3.4.1 Mixed standard stock solution of tartaric acid, malic acid, lactic acid, citric acid, succinic acid and fumaric acid: respectively weigh 1.25 g tartaric acid, 2.5 g malic acid, 2.5 g lactic acid, 2.5 g citric acid, 6.25 g (accurate to 0.01 g) succinic acid and 2.5 mg (accurate to 0.01 mg) fumaric acid, put into a 50 mL small beaker, dissolve them with water, transfer them to a 50 mL volumetric flask with water, scale the volume, mix well and preserve at 4℃; in the solution, mass concentrations of tartaric acid, malic acid, lactic acid, citric acid, succinic acid and fumaric acid are 2,500 μg/mL, 5000 μg/mL, 5,000 μg/mL, 5,000 μg/mL, 12,500 μg/mL and 12.5 μg/mL respectively .
3.4.2 Mixed standard curve working solution of tartaric acid, malic acid, lactic acid, citric acid, succinic acid and fumaric acid: respectively pipet 0.50 mL, 1.00 mL, 2.00 mL, 5.00 mL and 10.00 mL mixed standard stock solution (3.4.1) into 25 mL volumetric flasks, scale the volume to the scale with phosphoric acid solution (3.2.1), mix well and preserve at 4℃.
3.4.3 Adipic acid Standard stock solution (500 μg/mL): accurately weigh 12.5 mg adipic acid converted to 100% mass according to its purity and put into a 25 mL volumetric flask, add water to the scale, mix well and preserve at 4℃.
3.4.4 Adipic acid standard curve working solution: respectively pipet 0.50 mL, 1.00 mL, 2.00 mL, 5.00 mL and 10.00 mL standard stock solution (3.4.3) into 25 mL volumetric flasks, scale the volume to the scale with phosphoric acid solution (3.2.1), mix well and preserve at 4℃.
3.5 Material
Strong anion solid phase extraction column (SAX): 1,000 mg and 6 mL. Activate it with 5 mL methanol and 5 mL water successively prior to use.
4 Instruments and Apparatus
4.1 High-performance liquid chromatograph: with diode array detector or UV detector.
4.2 Balance: with sensibility of 0.01 mg and 0.01g.
4.3 High-speed homogenizer.
4.4 High-speed grinder.
4.5 Solid phase extraction device.
4.6 Water phase millipore filter: with pore diameter of 0.45 μm.
5 Analysis Procedures
Warning: experiment personnel shall wear glove and other protection tool when using liquid nitrogen to prevent accidental sprinkling and causing frozen injury.
5.1 Preparation and preservation of specimens
5.1.1 Liquid sample
As for samples of fruit juice and fruit juice beverage and fruity carbonated beverage, shake well, keep in portions, seal and preserve under normal temperature or by refrigeration.
5.1.2 Semi-solid sample
As for samples of jelly and fruit can, take their edible part and homogenize, stir uniformly, keep in portions, and seal and preserve them by refrigeration or freezing.
5.1.3 Solid sample
As for samples of biscuit, pastry and dough product with low moisture, grind them with high-speed grinder, keep in portions seal and preserve at a dark place under ambient temperature; as for solid beverage and other uniform powdery samples, directly keep them in portions and seal and preserve at a dark place under ambient temperature.
5.1.4 Special samples
As for gum candy and other special samples with larger viscosity, cut them into 2mm×2mm fragments with a pair of scissor and put into ceramic mortar, slowly pour liquid nitrogen, obtain uniform samples by grinding after being subjected to rapid freezing, keep in portions and seal and preserve by freezing.
5.2 Treatment of specimens
5.2.1 Fruit juice beverage, fruit juice and fruity carbonated beverage
Weigh 5 g (accurate to 0.01g) uniform specimen (remove carbon dioxide, if any, by heating), put into a 25 mL volumetric flask, add water to the scale, filter with 0.45 μm water phase filter and inject into high-performance liquid chromatograph for analysis.
5.2.2 Jelly and fruit can
Place 10 g (accurate to 0.01 g) uniform specimen, put into a 50 mL plastic centrifuge tube, add 20 mL water, homogenize and extract for 2 min at rotation speed of 15,000r/min, centrifuge for 5 min at rotation speed of 4,000 r/min, take extracting solution at upper layer into a 50 mL volumetric flask, re-extract the residues with 20 mL water, merge the extracting solution into the same volumetric flask, scale the volume to the scale with water, filter with 0.45 μm water phase filter and inject into high-performance liquid chromatograph for analysis.
5.2.3 Gum candy
Weigh 1 g (accurate to 0.01 g) uniform specimen, put into a 50 mL plastic centrifuge tube with stopper, add 20 mL water, conduct oscillating extraction for 2min in vortex mixer, centrifuge for 3min at rotation speed of 4,000 r/min, transfer supernatant to a 100 mL volumetric flask 100 mL volumetric flask, add 20 mL water to residues and re-extract once, merge extracting solution into the same volumetric flask, scale the volume with absolute ethanol (3.1.2) and shake well.
Accurately transfer 10 mL supernatant to a 100 mL obconic flask, add 10 mL absolute ethanol (3.1.2), conduct rotary concentration until nearly dry at 80℃±2℃, add 5 mL absolute ethanol (3.1.2), continue concentration until thoroughly dry and wash obconic flask twice with 1 mL ×1 mL water. Transfer all to-be-purified solution to pre-activated SAX solid phase extraction column with flow rate of 1 mL/min~2 mL /min and discard effluent. Wash purification column with 5 mL water, elute with 5mL phosphoric acid-methanol solution (3.2.2) at flow rate of 1 mL/min~2 mL/min, and collect eluent into a 50 mL obconic flask. Conduct rotary evaporation for eluent at 45℃ until nearly dry, add 5 mL absolute ethanol (3.1.2), continue concentration until thoroughly dry, conduct oscillating dissolution for residue with 1.0 mL phosphoric acid solution (3.2.1) and filter with 0.45 μm, and inject into high-performance liquid chromatograph for analysis.
Foreword i
1 Scope
2 Principle
3 Reagents and Materials
4 Instruments and Apparatus
5 Analysis Procedures
6 Expression of Analysis Results
7 Accuracy
8 Other
Annex A Standard Chromatogram of Acidity Regulator
