Determination of 11 Synthetic Colorants in Red Wine (GB 5009.35-2023)

• 1. Scope of Application

This method is applicable for the determination of 11 synthetic colorants in food, including lemon yellow, new red, amaranth, indigo, carmine, sunset yellow, allura red, brilliant blue, acid red, quinoline yellow, and erythrosine.

Reference Standard: "GB 5009.35-2023 National Food Safety Standard - Determination of Synthetic Colorants in Food"

• 2. Reagents and Materials 

2.1 Reagents

(1) Reagents: Ammonium acetate (chromatographic grade), methanol (chromatographic grade), formic acid (suitable for HPLC), ammonia solution (NH3·H2O, 20%-25% concentration), ultrapure water

2.2 Preparation of Reagents

(1) 10% Aqueous Formic Acid Solution: Mix 10 mL of formic acid with 90 mL of water.

(2) 15% Ammonia Methanol Solution: Mix 15 mL of ammonia solution (25%-27%) with 85 mL of methanol.

(3) Ammonium Acetate Solution (20 mmol/L): Dissolve 1.54 g of ammonium acetate in water and dilute to 1000 mL.

2.3 Standards

(1) Quinoline Yellow (CAS No: 8004-92-0): Purity ≥ 95.0%, or a certified reference material with a standard substance certificate issued by a national certification body.

(2) Lemon Yellow (CAS No: 1934-21-0): Purity ≥ 95.0%, or a certified reference material with a standard substance certificate issued by a national certification body.

(3) New Red (CAS No: 220658-76-4): Purity ≥ 95.0%, or a certified reference material with a standard substance certificate issued by a national certification body.

(4) Amaranth (CAS No: 915-67-3): Purity ≥ 95.0%, or a certified reference material with a standard substance certificate issued by a national certification body.
(5) Carmine (CAS No: 2611-82-7): Purity ≥ 95.0%, or a certified reference material with a standard substance certificate issued by a national certification body.
(6) Sunset Yellow (CAS No: 2783-94-0): Purity ≥ 90.0%, or a certified reference material with a standard substance certificate issued by a national certification body.
(7) Allura Red (CAS No: 25956-17-6): Purity ≥ 95.0%, or a certified reference material with a standard substance certificate issued by a national certification body.
(8) Acid Red (CAS No: 3567-69-9): Purity ≥ 90.0%, or a certified reference material with a standard substance certificate issued by a national certification body.
(9) Erythrosine (CAS No: 16423-68-0): Purity ≥ 90.0%, or a certified reference material with a standard substance certificate issued by a national certification body.
(10) Brilliant Blue (CAS No: 3844-45-9): Purity ≥ 95.0%, or a certified reference material with a standard substance certificate issued by a national certification body.
(11) Indigo (CAS No: 860-22-0): Purity ≥ 90.0%, or a certified reference material with a standard substance certificate issued by a national certification body.

2.4 Preparation of Standard Solutions
(1) Standard Stock Solutions (1000 mg/L) for Lemon Yellow, Quinoline Yellow, New Red, Amaranth, Carmine, Sunset Yellow, Allura Red, Acid Red, Erythrosine, Brilliant Blue, and Indigo: Weigh 10 mg of each standard substance and place it into a 10 mL volumetric centrifuge tube. Dilute to 10 mL with ultrapure water. These standard stock solutions can be stored at 4°C in the dark for up to 6 months. Prepare the Indigo standard solution fresh before use.
(2) Mixed Standard Working Solution (50 mg/L): Pipette 0.5 mL of each of the above standard stock solutions (except Erythrosine) into a 15 mL centrifuge tube and dilute to 10 mL with water. Prepare the Erythrosine standard working solution (50 mg/L) separately.
(3) Mixed Standard Working Solutions: Pipette 0.12 mL, 0.3 mL, and 0.6 mL of the mixed standard working solution (excluding Erythrosine) into separate 15 mL centrifuge tubes, and dilute to 10 mL with water. The resulting concentrations will be 0.6 mg/L, 1.5 mg/L, and 3.0 mg/L, respectively.
(4) Mixed Standard Working Solutions for Erythrosine: Pipette 0.4 mL, 1.0 mL, and 2.0 mL of the Erythrosine standard working solution into separate 15 mL centrifuge tubes, and dilute to 10 mL with water. The resulting concentrations will be 2.0 mg/L, 5.0 mg/L, and 10.0 mg/L, respectively.

2.5 Instruments and Equipment

(1) High-Performance Liquid Chromatograph: Thermo Fisher U3000 with UV detector

(2) Balance: Minimum weighing capacity of 0.1 mg

(3) Ultrasonic Generator: Ultrasonic power not less than 700W, temperature control range of 20°C to 80°C

(4) Solid Phase Extraction (SPE) device

(5) Nitrogen Concentration Device

(6) Welchrom PAX Solid Phase Extraction Column: Specifications: 150 mg/6 mL

(7) Syringe Filter: Hydrophilic PTFE (polytetrafluoroethylene) membrane, pore size 0.22 μm

• 3. Pretreatment of Samples

Take 1 mL of red wine, add 0.5 mL of methanol and 0.3 mL of formic acid, mix well, and set aside for purification. Prepare blank, blank with standard, sample, sample with spike 1, and sample with spike 2.

• 4. Sample Purification
  Activation: Use 5 mL of methanol, followed by 5 mL of 10% aqueous formic acid, then discard.

Loading: Apply the entire sample preparation to the SPE column, then discard the eluate. Washing: Rinse with 5 mL of methanol (rinse the centrifuge tube), discard the eluate, and dry the column.

Elution: Elute with 8 mL of 15% ammonia methanol solution, collect the eluate in a 15 mL centrifuge tube, and dry the column.
Evaporate under nitrogen at 45°C to approximately 500 μL, then use the mobile phase to bring the volume to 5 mL (dissolve with a small amount of methanol), filter through a 0.22 μm PTFE filter, and proceed with HPLC detection.

• 5. Instrument Reference Conditions

Column: Welch Ultisil® XB-C18 (4.6×250 mm, 5 μm).

Injection Volume: 10 μL.

Column Temperature: 30°C.

UV Detector Wavelengths: 415 nm (Tartrazine, Quinoline Yellow), 520 nm (Ponceau 4R, Amaranth, Carmine, Sunset Yellow, Allura Red, Acid Red, Erythrosine), 610 nm (Indigo Carmine, Brilliant Blue).

The elution gradient is shown in Table 1, with mobile phase A being 20 mmol/L ammonium acetate solution and mobile phase B being methanol.

• 6. Preparation of the Standard Curve

Inject the standard series working solutions into the liquid chromatograph, measure the peak areas of the respective substances, and plot the standard curve using the concentration of the substance in the standard series working solutions as the x-axis and the response value of the peak area as the y-axis.

• 7. Sample Determination

(1) Blank Sample

(2) Blank Spiking

Welchrom PWAX Column:

(3) Sample

Under the same mobile phase conditions, no signal peaks corresponding to the coloring agents were observed in the sample as shown in the following figure.

(4) Sample Spiking

Welchrom PWAX Column

• 8. Summary Table of Spiking Recovery Rates

• 9. Relevant Product Information