GB 5009.284-2021National food safety standard - Determination of vanillin, methyl vanillin, ethyl vanillin and coumarin in foods (English PDF)

3.1 Reagents and materials

3.2 Preparation of reagents

3.2.1 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.

3.2.2 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.

3.3 Standard substances

3.3.1 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.

3.4 Preparation of standard solutions

3.4.1 Standard stock solution (1 000 mg/L). accurately weigh 10 mg (accurate

to 0.1 mg) of vanillin, methyl vanillin, ethyl vanillin and coumarin; place them in the 10 mL volumetric flasks respectively; use 0.1% formic acid methanol solution to dissolve and dilute to the mark; mix well.

3.4.2 Standard mixed intermediate solution (10 mg/L). respectively draw 1.00

mL of standard stock solutions (1 000 mg/L) of vanillin, methyl vanillin, ethyl vanillin and coumarin; place them in the 100 mL volumetric flasks; add 0.1% methanol formic acid to fix volume to the mark; mix well. Transfer the solution to a brown glass container; store at -18 °C in the dark for 3 months.

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.

4.6 Ultrasonic generator.

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 uL of hydrochloric acid solution; vortex for 1 min;

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;

5.2 Apparatus reference conditions

5.2.1 Chromatographic column. C18 column, 250 mm x 4.6 mm (inner diameter), 5 um (filler particle size) or equivalent.

5.2.2 Column temperature. 30 °C.

5.2.3 Injection volume. 10 uL;

5.2.4 Detection wavelength. 279 nm.

5.2.5 Flow velocity. 1.0 mL/min.

5.3 Preparation of the standard curve

Inject the standard mixed series working solutions into the liquid chromatograph respectively; measure the corresponding peak areas. Take the concentrations of vanillin, methyl vanillin, ethyl vanillin and coumarin in the standard series working solutions as the abscissa, and the peak area as the ordinate, to draw a standard curve. For the chromatograms of standard solutions of vanillin, methyl vanillin, ethyl vanillin and coumarin, see Figure A.1 in Appendix A.

10.1 Reagents and materials

10.1.4 Sodium chloride (NaCl).

10.1.5 Concentrated hydrochloric acid (HCl). 12 mol/L.

10.1.6 Polar group-containing reverse-phase polymer solid-phase extraction

column.

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.

10.3 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.

10.3.6 D3-methyl vanillin standard substance (C9H7D3O3, CAS number.

143318-06-3). purity >=98%.

10.4.4 Internal standard mixed intermediate solution (10 mg/L). draw 1.00 mL

of the standard stock solutions of D3-vanillin, D3-methyl vanillin, D5-ethyl vanillin and D4-coumarin (1 000 mg/L) respectively; place them in the 100 mL volumetric flasks; add 0.1% formic acid methanol solution to fix volume to the mark; mix well. Transfer the solution to a brown glass container; store at -18 °C in the dark for 3 months.

10.4.5 Standard and internal standard mixed series working solutions.

respectively draw 0.05 mL, 0.2 mL, 0.5 mL, 1 mL, 2 mL of the standard mixed intermediate solution (10 mg/L) and 0.1 mL of the internal standard mixed intermediate solution (10 mg/L); place them to in the 10 mL volumetric flasks;

add methanol-aqueous solution to the mark; mix well.

11.1 Liquid chromatography tandem quadrupole mass spectrometer. equipped

with electrospray ionization source.

11.2 Balance. the sensitivity is 0.1 mg and 0.01 g.

11.5 High-speed refrigerated centrifuge.

11.6 Nitrogen concentrator.

11.8 Solid-phase extraction device.

12.1 Sample pretreatment

12.1.1 Sample preparation

Shake liquid samples well; semi-solid samples and powder samples with uniform base materials shall be directly used for sample extraction in 12.1.2;

gum-based candies shall be crushed and stirred well; other samples need to be homogenized or crushed uniformly.

12.1.2 Sample extraction

12.2 Apparatus reference conditions

12.2.1 Liquid chromatography reference conditions

12.2.2 MS/MS reference conditions

12.2.2.1 Ionization mode. electrospray ionization, positive ion mode.

12.2.2.2 Scanning method. multiple reaction monitoring (MRM).