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GB 5009.35-2023: National food safety standard - Determination of synthetic colour in foods
GB 5009.35-2023
GB
NATIONAL STANDARD OF THE
PEOPLE'S REPUBLIC OF CHINA
National food safety standard - Determination of synthetic
colorants in food
ISSUED ON: SEPTEMBER 6, 2023
IMPLEMENTED ON: MARCH 6, 2024
Issued by: National Health Commission of the People's Republic of China;
State Administration for Market Regulation.
Table of Contents
Foreword ... 3
1 Scope ... 4
2 Principle ... 4
3 Reagents and materials ... 4
4 Instruments and equipment ... 6
5 Analysis steps ... 7
6 Presentation of analysis results ... 10
7 Precision ... 10
8 Others ... 10
Appendix A High-performance liquid chromatogram of standard solution ... 12
National food safety standard - Determination of synthetic
colorants in food
1 Scope
This standard specifies the liquid chromatography method for the determination of
synthetic colorants in food.
This standard is applicable to the determination of 11 synthetic colorants (tartrazine,
new red, amaranth, indigo, ponceau 4R, sunset yellow, allura red, brilliant blue, acid
red, quinoline yellow, and erythrosine) in food.
2 Principle
The synthetic colorant in the sample is extracted with ethanol ammonia solution,
purified by solid-phase extraction, then measured with a high-performance liquid
chromatograph equipped with a diode array detector, and quantified by the external
standard method.
3 Reagents and materials
Unless otherwise stated, the reagents used in this method are of analytical grade and the
water is first-grade water specified in GB/T 6682.
3.1 Reagents
3.1.1 Methanol (CH4O): chromatographically pure.
3.1.2 Methanol (CH4O).
3.1.3 Petroleum ether: The boiling range is 30 ℃~60 ℃.
3.1.4 Absolute ethanol (C2H6O).
3.1.5 Ammonia (NH3 • H2O): The content is 20%~25%.
3.1.6 Ammonium acetate (C2H7O2N): chromatographically pure.
3.1.7 Formic acid (CH2O2): The content is 98%.
3.2 Reagent preparation
3.2.1 Ethanol ammonia solution: Measure 700 mL of absolute ethanol, add 4 mL of
ammonia, dilute to 1 L with water, and mix well.
3.2.2 5% methanol aqueous solution: Pipette 5 mL of methanol (3.1.2), dilute with
water and make up to 100 mL, and mix well.
3.2.3 2% ammonia methanol solution: Pipette 2 mL of ammonia and dilute to 100 mL
with methanol (3.1.2).
3.2.4 Ammonium acetate solution (20 mmol/L): Weigh 1.54 g of ammonium acetate,
dissolve it in water, and dilute it to 1000 mL.
3.2.5 Ammonium acetate buffer solution, pH=9.0: Add ammonia water to the
ammonium acetate solution (3.2.4) and adjust the pH to 9.0.
3.2.6 2% formic acid aqueous solution: Pipette 2 mL of formic acid and dilute to 100
mL with water.
3.3 Standards
3.3.1 Tartrazine (CAS No.: 1934-21-0): with a purity of ≥95.0%, or a standard product
obtained national certification and awarded a reference material certificate.
3.3.2 New red (CAS No.: 220658-76-4): with a purity of ≥95.0%, or a standard product
obtained national certification and awarded a reference material certificate.
3.3.3 Amaranth (CAS No.: 915-67-3): with a purity of ≥95.0%, or a standard product
obtained national certification and awarded a reference material certificate.
3.3.4 Indigo (CAS No.: 860-22-0): with a purity of ≥90.0%, or a standard product
obtained national certification and awarded a reference material certificate.
3.3.5 Carmine (CAS No.: 2611-82-7): with a purity of ≥95.0%, or a standard product
obtained national certification and awarded a reference material certificate.
3.3.6 Sunset yellow (CAS No.: 2783-94-0): with a purity of ≥90.0%, or a standard
product obtained national certification and awarded a reference material certificate.
3.3.7 Allura red (CAS No.: 25956-17-6): with a purity of ≥95.0%, or a standard product
obtained national certification and awarded a reference material certificate.
3.3.8 Brilliant blue (CAS No.: 3844-45-9): with a purity of ≥95.0%, or a standard
product obtained national certification and awarded a reference material certificate.
3.3.9 Acid red (CAS No.: 3567-69-9): with a purity of ≥90.0%, or a standard product
obtained national certification and awarded a reference material certificate.
3.3.10 Quinoline yellow (CAS No.: 8004-92-0): with a purity of ≥95.0%, or a standard
product obtained national certification and awarded a reference material certificate.
3.3.11 Erythrosine (CAS No.: 16423-68-0): with a purity of ≥90.0%, or a standard
product obtained national certification and awarded a reference material certificate.
3.4 Preparation of standard solution
3.4.1 Standard stock solution (1.0 mg/mL): Accurately weigh 100 mg (accurate to 0.1
mg) of each of tartrazine, new red, amaranth, ponceau 4R, sunset yellow, allura red,
brilliant blue, acid red, quinoline yellow and erythrosine, converted to 100 % by mass
according to their purity, dissolve in water and place in 100 mL volumetric flasks
respectively, dilute to volume, and shake well to obtain standard stock solutions with a
concentration of 1.0 mg/mL. The standard stock solution can be stored at 4 °C and away
from light for 6 months. Prepare the indigo standard solution fresh before use.
3.4.2 Mixed standard intermediate solution (50.0 μg/mL): Take 5.00 mL of each of the
above standard stock solution (3.4.1) and indigo standard solution (1.0 mg/mL) into a
100 mL volumetric flask, dilute to the mark with water, and shake well to obtain the
mixed standard intermediate solution (the concentration of each synthetic colorant is
50.0 μg/mL). Prepare the solution fresh before use.
3.4.3 Standard series working solutions: Take 0.2 mL, 0.5 ml, 1.0 mL, 2.0 mL, 5.0 mL,
and 10.0 mL of the mixed standard intermediate solution (3.4.2) in 50 mL volumetric
flasks, dilute to the mark with water, and shake well to obtain standard series working
solutions. The concentrations are 0.2 μg/mL, 0.5 μg/mL, 1.0 μg/mL, 2.0 μg/mL, 5.0
μg/mL and 10.0 μg/mL, respectively.
4 Instruments and equipment
4.1 High-performance liquid chromatograph: with a diode array detector.
4.2 Balance: The sensitivities are 1 mg and 0.1 mg, respectively.
4.3 pH meter: The accuracy is 0.01.
4.4 Electric stirrer: The speed range is 30 r/min~2000 r/min.
4.5 Vortex mixer.
4.6 Ultrasonic generator or constant temperature shaker: The ultrasonic power is not
less than 700 W; the temperature control range is 20 ℃~80 ℃; the shaker speed range
is 10 r/min~500 r/min.
4.7 High-speed centrifuge: The speed is not less than 15000 r/min.
(3.2.1), vortex for 1 min, extract for 20 min at 50 ℃ by ultrasonic or shaking (with a
speed of ≥250 r/min), and centrifuge at 8000 r/min for 5 min; take the supernatant and
place it in a 50 mL volumetric flask, add about 5 mL~10 mL of ethanol ammonia
solution (3.2.1) each time, repeat the extraction operation until the supernatant has no
obvious color, combine the supernatants after centrifugation, and make up to 50 mL
with ethanol ammonia solution (3.2.1) to obtain the extract. Accurately pipette 10 mL
of the extract, concentrate it to about 3 mL with nitrogen at 50 ℃, add a total of 10 mL
of 5% methanol aqueous solution 2~3 times to dissolve it, and use the solution as the
liquid to be purified.
5.1.2.3 Samples with high oil content (cocoa products, chocolate and chocolate
products, formulated milk powder, modulation cream powder, formulated
condensed milk, puffed food, processed nuts and seeds, cooked beans, pastries,
cooked meat products, compound seasonings, ice cream, popsicles, etc.)
Accurately weigh 2 g of the sample (accurate to 0.001 g)...