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GB 5009.284-2021: National Food Safety Standard - Determination of vanillin, methyl vanillin, ethyl vanillin and coumarin in foods
GB 5009.284-2021
GB
NATIONAL STANDARD OF THE
PEOPLE’S REPUBLIC OF CHINA
National Food Safety Standard - Determination of
vanillin, methyl vanillin, ethyl vanillin and coumarin in
foods
ISSUED ON: SEPTEMBER 07, 2021
IMPLEMENTED ON: MARCH 07, 2022
Issued by: National Health Commission of the People's Republic of China;
State Administration for Market Regulation.
Table of Contents
1 Scope ... 3 
2 Principle ... 3 
3 Reagents and materials ... 3 
4 Instruments and apparatuses ... 5 
5 Analysis steps ... 5 
6 Expression of the analysis results ... 7 
7 Precision ... 8 
8 Others ... 8 
9 Principle ... 8 
10 Reagents and materials ... 8 
11 Instruments and apparatuses ... 10 
12 Analysis steps ... 11 
13 Expression of the analysis results ... 14 
14 Precision ... 15 
15 Others ... 15 
16 Principle ... 15 
17 Reagents and materials ... 15 
18 Instruments and apparatuses ... 16 
19 Analysis steps ... 16 
20 Expression of the analysis results ... 19 
21 Precision ... 19 
22 Others ... 19 
Appendix A Liquid chromatograms of vanillin, methyl vanillin, ethyl vanillin and
coumarin ... 20 
Appendix B Four compounds and corresponding deuterated isotope internal
standard liquid chromatography-mass spectrometry/mass spectrometry
characteristic ion chromatograms ... 21 
Appendix C Four compounds and their corresponding deuterated isotope
internal standard gas chromatography-mass spectrometry characteristic ion
scanning chromatograms and mass spectrograms ... 25 
National Food Safety Standard - Determination of
vanillin, methyl vanillin, ethyl vanillin and coumarin in
foods
1 Scope
This Standard specifies the determination methods of vanillin, methyl vanillin,
ethyl vanillin and coumarin in foods.
This Standard applies to the determination of vanillin, methyl vanillin, ethyl
vanillin and coumarin in infant formula, complementary foods for infants and
young children, cakes, candies, milk and dairy products, beverages, and wheat
flour.
Method I – Liquid chromatography
2 Principle
Add water to mix the sample; then, add acetonitrile for ultrasonic extraction; use
liquid chromatography for separation, ultraviolet detector or diode array
detector for detection, and external standard method for quantitation.
3 Reagents and materials
Unless otherwise specified, all the reagents in this method are analytical
reagents, the water is grade-1 water as specified in GB/T 6682.
3.1 Reagents and materials
3.1.1 Acetonitrile (CH3CH): chromatographic pure.
3.1.2 Formic acid (HCOOH): chromatographic pure.
3.1.3 Methanol (CH3OH): chromatographic pure.
3.1.4 Sodium chloride (NaCl)
3.1.5 Concentrated hydrochloric acid (HCl): 12 mol/L.
3.1.6 Microporous membrane: 0.45 μm, organic phase type.
3.4.3 Standard mixed series working solution: respectively draw 0.2 mL, 1.0 mL
and 5.0 mL of standard mixed intermediate solution (10 mg/L); put them in the
10 mL volumetric flasks; besides, respectively draw 0.2 mL, 0.5 mL and 1 mL
of standard stock solutions (1 000 mg/L) of vanillin, methyl vanillin, ethyl vanillin
and coumarin; put them in the 10 mL volumetric flasks; add methanol-aqueous
solution to fix volume to the mark; mix well. The concentrations of standard
mixed series working solutions are 0.2 mg/L, 1.0 mg/L, 5.0 mg/L, 20.0 mg/L,
50.0 mg/L, 100.0 mg/L, respectively; prepare them for immediate use.
4 Instruments and apparatuses
4.1 Liquid chromatograph: equipped with diode array or UV detector.
4.2 Balance: the sensitivity is 0.1 mg and 0.01 g.
4.3 Vortex mixer.
4.4 Centrifuge.
4.5 Nitrogen concentrator.
4.6 Ultrasonic generator.
5 Analysis steps
5.1 Sample pretreatment
5.1.1 Sample preparation
Shake liquid samples well; semi-solid samples and powder samples with
uniform base materials shall be directly used for the sample extraction in 5.1.2;
other samples need to be homogenized or pulverized uniformly.
5.1.2 Sample extraction
5.1.2.1 Infant formula, complementary foods for infants and young
children
Weigh 1.00 g of sample; add 10 mL of water and 480 μL of hydrochloric acid
solution; vortex for 1 min; add 20 mL of acetonitrile; vortex for 1 min; after 30
min of ultrasonic extraction, add 5 g of sodium chloride; vortex for 2 min;
centrifuge at 8 000 r/min for 5 min; take the supernatant to a glass test tube;
use nitrogen at 40°C to blow it to near dryness; accurately add 1.0 mL of
methanol-aqueous solution to dissolve the residue; pass it through a 0.45 μm
microporous membrane; wait for later test.
5.1.2.2 Cakes, candies, milk and dairy products, beverages
Weigh 1.00 g of sample; add 5 mL of water (for gum-based candy, add 10 mL
of water, and dissolve in a water bath at 50 °C); vortex for 1 min; add 20 mL of
acetonitrile; vortex for 1 min; after 30 min of ultrasonic extraction, add 5 g of
sodium chloride; vortex for 2 min; centrifuge at 8 000 r/min for 5 min (for cake
samples with high fat content, take frozen centrifugation at 8 000 r/min); take
the supernatant to a glass test tube; use nitrogen to blow at 40 °C to nearly dry;
accurately add 1.0 mL of methanol-aqueous solution to dissolve the residue;
pass it through a 0.45 μm microporous membrane for later test.
5.1.2.3 Wheat flour
Weigh 1.00 g of sample; add 10 mL of water and 240 μL of hydrochloric acid
solution; vortex for 1 min; add 20 mL of acetonitrile; vortex for 1 min; after 30
min of ultrasonic extraction, add 5 g of sodium chloride; vortex for 2 min;
centrifuge at 8 000 r/min for 5 min; take the supernatant to a glass test tube;
use nitrogen at 40°C to blow it to near dryness; accurately add 1.0 mL of
methanol-aqueous solution to dissolve the residue; pass it through a 0.45 μm
microporous membrane; wait for later test.
5.2 Apparatus reference conditions
5.2.1 Chromatographic column: C18 column, 250 mm × 4.6 mm (inner diameter),
5 μm (filler particle size) or equivalent.
5.2.2 Column temperature: 30 °C.
5.2.3 Injection volume: 10 μL;
5.2.4 Detection wavelength: 279 nm.
5.2.5 Flow velocity: 1.0 mL/min.
5.2.6 Mobile phase
Phase A: 0.5% formic acid solution;
Phase B: Acetonitrile.
Gradient elution is shown in Table 1.
Table 1 – Gradient elution procedure
polymer which is composed of two monomers, lipophilic divinylbenzene and
hydrophilic N-vinylpyrrolidone, in a certain proportion. Use 3 mL of methanol
and 3 mL of water to activate it before use.
10.1.7 Microporous membrane: 0.22 μm, organic phase type.
10.2 Preparation of reagents
10.2.1 0.1% formic acid methanol solution: draw 1 mL of formic acid and
dissolve it in methanol and dilute to 1 000 mL; prepare it for immediate use.
10.2.2 Hydrochloric acid solution (1 mol/L): draw 8.33 mL of concentrated
hydrochloric acid; dissolve it in water and dilute to 100 mL; prepare it for
immediate use.
10.2.3 Methanol-aqueous solution (4+1): mix methanol and water at 4:1
(volume ratio) uniformly.
10.2.4 0.5% formic acid solution: draw 5 mL of formic acid; dissolve it in water
and dilute to 1 000 mL; prepare it for immediate use.
10.3 Standard substances
10.3.1 Vanillin standard substance (C8H8O3, CAS number: 121-33-5): purity
≥98%, or a standard substance that is certified by the nation and granted a
standard substance certificate.
10.3.2 Methyl vanillin standard substance (C9H10O3, CAS number: 120-14-9):
purity ≥98%, or a standard substance that is certified by the nation and gra...