Detail of GB 5009.284-2021

Introduction of GB 5009.284-2021

Codeofchina.com is in charge of this English translation. In case of any doubt about the English translation, the Chinese original shall be considered authoritative. National food safety standard - Determination of vanillin, methyl vanillin, ethyl vanillin and cumarin in food 1 Scope This standard specifies the method for the determination of vanillin, methyl vanillin, ethyl vanillin and cumarin in food. This standard is applicable to the determination of vanillin, methyl vanillin, ethyl vanillin and cumarin in formula foods/ complementary foods for infants and young children, pastry, confection, milk and dairy products, beverage and wheat flour. Method I Liquid chromatographic method 2 Principle Add water to the sample and mix them up; extract the mixture in an ultrasonic way after adding acetonitril; separate it by liquid chromatography; detect by UV detector or diode array detector; and quantify them by the external standard method. 3 Reagents and materials Unless otherwise specified, analytically-pure reagents and Class-I water specified in GB/T 6682 are used for the purpose of this method. 3.1 Reagents and materials 3.1.1 Acetonitrile (CH3CN): chromatographically pure. 3.1.2 Methanoic acid (HCOOH): chromatographically pure. 3.1.3 Methanol (CH3OH): chromatographically pure. 3.1.4 Sodium chloride (NaCl). 3.1.5 Concentrated hydrochloric acid (HCl): 12mol/L. 3.1.6 Organic-phase microporous membrane: 0.45μm.   3.2 Preparation of reagents 3.2.1 0.1% formic acid-methanol solution: dissolve 1mL of formic acid in methanol and dilute to 1,000mL; freshly prepare before use. 3.2.2 Hydrochloric acid solution (1mol/L): pipette 8.33mL of concentrated hydrochloric acid, dissolve it in water and dilute the solution to 100mL; freshly prepare before use. 3.2.3 Methanol-water solution (4+1): mix methanol and water at a ratio of 4:1 (volume ratio). 3.2.4 0.5% formic acid solution: dissolve 5mL of formic acid in water and dilute to 1,000mL; freshly prepare before use. 3.3 Standard substance 3.3.1 Vanillin standard sample (C8H8O3, CAS No.: 121-33-5): purity ≥98%, or its standard sample certified by the state and issued with the reference material certificate. 3.3.2 Methyl vanillin standard sample (C9H10O3, CAS No.: 120-14-9): purity ≥98%, or its standard sample certified by the state and issued with the reference material certificate. 3.3.3 Ethyl vanillin standard sample (C9H10O3, CAS No.: 121-32-4): purity ≥98%, or its standard sample certified by the state and issued with the reference material certificate. 3.3.4 Cumarin standard sample (C9H6O2, CAS No.: 91-64-5): purity ≥97%, or its standard sample certified by the state and issued with the reference material certificate. 3.4 Preparation of standard solution 3.4.1 Standard stock solutions (1,000mg/L): accurately weigh out 10mg (accurate to 0.1mg) of vanillin, methyl vanillin, ethyl vanillin and cumarin respectively, place them in 10mL volumetric flasks respectively, dissolve to the scale with 0.1% formic acid-methanol solution, and mix them up. Transfer the solutions to a brown glass container and store at -18℃ away from light; use them within the storage period of 8 months. 3.4.2 Mixed intermediate standard solutions (10mg/L): pipette 1.00mL of the standard stock solutions (1,000mg/L) of vanillin, methyl vanillin, ethyl vanillin and cumarin respectively, pour them into 100mL volumetric flasks respectively, add 0.1% formic acid-methanol solution to the scale, and mix them up. Transfer the solutions to a brown glass container and store at -18℃ away from light; use them within the storage period of 3 months.   3.4.3 Mixed standard series working solutions: pipette 0.2mL, 1.0mL and 5.0mL of mixed intermediate standard solution (10mg/L) respectively into a 10mL volumetric flask, and pipette 0.2mL, 0.5mL and 1mL of standard stock solution (1,000mg/L) of vanillin, methyl vanillin, ethyl vanillin and cumarin respectively into a 10mL volumetric flask, add methanol-water solution to the scale and mix them up. The mixed standard series working solutions shall have concentrations of 0.2mg/L, 1.0mg/L, 5.0mg/L, 20.0mg/L, 50.0mg/L and 100.0mg/L respectively; freshly prepare before use. 4 Apparatuses 4.1 Liquid chromatograph: equipped with diode array detector or ultraviolet detector. 4.2 Balances: with sensitivity of 0.1mg and 0.01g, respectively. 4.3 Vortex mixer. 4.4 Centrifuge. 4.5 Nitrogen evaporator. 4.6 Ultrasonic generator. 5 Analytical steps 5.1 Sample preconditioning 5.1.1 Sample preparation Shake the liquid samples well; directly use the homogeneous semi-solid or powdery solid sample to extract the specimen (5.1.2); homogenize or crush evenly samples of other states. 5.1.2 Specimen extraction 5.1.2.1 Formula milk powder and complementary foods for infants and young children Weigh 1.00g of the sample, add 10mL of water and 480μL of hydrochloric acid solution, shake it with a vortex mixer for 1min; add 20mL of acetonitrile, shake it with a vortex mixer for 1min, apply ultrasonic extraction for 30min, then add 5g of sodium chloride, shake it with a vortex mixer for 2min, centrifuge it at 8,000r/min for 5min; take the supernatant into a glass test tube, blow it with nitrogen at 40℃ until it is nearly dry; accurately add 1.0mL of methanol-water solution to dissolve the residue; filter it through a 0.45μm microporous membrane, and be ready to test. 5.1.2.2 Pastry, confection, milk and dairy products and beverage Weigh 1.00g of the specimen, add 5mL of water (if it is gum-based confectione, add 10mL of water and dissolve it in a 50℃ water bath), shake it with a vortex mixer for 1min; add 20mL of acetonitrile, shake it with a vortex mixer for 1min, and apply ultrasonic extraction for 30min; add 5g of sodium chloride, shake it with a vortex mixer for 2min, centrifuge it at 8,000r/min for 5min (if the sample is high-fat pastry, apply freezing centrifugation at 8,000r/min); take the supernatant into a glass test tube, blow it with nitrogen at 40℃ until it is nearly dry; accurately add 1.0mL of methanol-water solution to dissolve the residue; filter it through a 0.45μm microporous membrane, and be ready to test.

Contents of GB 5009.284-2021

1 Scope Method I Liquid chromatographic method 2 Principle 3 Reagents and materials 4 Apparatuses 5 Analytical steps 6 Calculation (expression) of analytic result 7 Precision 8 Miscellaneous Method II Liquid chromatography-mass spectrography (LC-MS)/ Mass spectrometry (MS) 9 Principle 10 Reagents and materials 11 Apparatuses 12 Analytical steps 13 Calculation (expression) of analytic result 14 Precision 15 Miscellaneous Method III Gas chromatography-mass spectrometry (GC-MC) 16 Principle 17 Reagents and materials 18 Apparatuses 19 Analytical steps 20 Calculation (expression) of analytic result 21 Precision 22 Miscellaneous Annex A Liquid chromatograms of vanillin, methyl vanillin, ethyl vanillin and cumarin Annex B GC-MS/MS characteristic ion chromatograms of the four compounds with corresponding deuteration isotope internal standard solutions Annex C GC-MS/MS characteristic ion scanning chromatograms and mass spectrograms of the four compounds with corresponding deuteration isotope internal standard solutions