CHROMATOGRAPHIC SEPARATION AND PHOTODIODE ARRAY IDENTIFICATION OF SYNTHETIC INDUSTRIAL DYES IN FOODS, BEVERAGES, OVER THE COUNTER (OTC) DRUGS, AND COSMETICS

Significance of the Topic

Synthetic food dyes are widely used in beverages, candies, over-the-counter drugs and cosmetics to enhance visual appeal. Recent studies and regulatory actions have raised concerns about neurobehavioral effects in children, carcinogenic risks and misbranding violations. A robust analytical method for rapid screening and confirmation of multiple dyes across diverse consumer matrices is essential for quality control, regulatory compliance and public health monitoring.

Objectives and Overview

This study demonstrates a single high-performance liquid chromatography method with photodiode array detection for simultaneous separation, identification and quantification of ten common synthetic dyes. Key aims include achieving baseline resolution of dye standards, establishing spectral purity checks, creating a library of reference spectra and evaluating dye levels in various food, beverage, drug and cosmetic products.

Methodology and Used Instrumentation

Sample Preparation

• Solid samples were dissolved in water (10–200 mL per serving), sonicated or stirred, then centrifuged at 15 000 RPM for 10 minutes.
• Sports drinks were diluted 1 in 10 in water to reduce turbidity.
• Dye standards (Yellow 5, Yellow 6, Blue 1, Blue 2, Red 3, Red 40, Green S, Green 3, Carmoisine, Patent Blue V) were prepared in water at 0.1 mg/mL; serial dilutions yielded 0.003–0.375 µg on column.

Chromatographic Conditions

• LC System: Alliance iS HPLC with PDA Detector, Empower 3 software v 3.8.0.
• Column: XBridge Premier BEH C18 2.5 µm, 4.6 × 150 mm at 40 °C; sample temperature 20 °C.
• Mobile phases: A = 10 mM ammonium acetate, pH 7; B = methanol; C = acetonitrile. Gradient from 97 % A/2 % B/1 % C to 50 % A/25 % B/25 % C over 15 min; flow rate 1.6 mL/min; injection 30 µL.
• Detection: PDA scanned 200–800 nm at 10 Hz, resolution 1 nm. Extracted channels: 455 nm for yellow dyes, 520 nm for red dyes, 628 nm for blue and green dyes.
• PDA processing: Purity angle/threshold analysis and library matching using Empower 3 processing methods.

Main Results and Discussion

The method achieved baseline separation of all ten dye standards within a single 15 minute run. Linearity was confirmed from 0.003 to 0.750 µg on column with correlation coefficients above 0.995 (data not shown). Peak purity analysis verified spectral homogeneity and absence of co-elutions in complex matrices. A PDA spectral library enabled unambiguous identification based on match angle and threshold criteria. Quantitative analysis of consumer samples revealed the highest total dye content in spicy snack sticks (predominantly Yellow 6 and Red 40), while candies ranged from 4.8 mg to 18.6 mg per serving. Sports drinks showed intermediate levels due to larger serving sizes. OTC drugs and cosmetics exhibited minimal dye content (< 0.5 mg per dose), consistent with non-ingestible formulations.

Benefits and Practical Applications

• Single-run analysis reduces instrument time and solvent usage.
• PDA spectral purity checks enhance confidence in quantification by excluding co-eluting interferents.
• Library matching accelerates identification without additional confirmatory techniques.
• Applicable to a wide range of matrices for quality control in food, pharmaceutical and cosmetic industries.
• Supports regulatory monitoring for color additive compliance and import/export screening.

Future Trends and Applications

Future developments may integrate the HPLC-PDA approach with mass spectrometry for structural confirmation of unknown dyes and degradation products. Automated high-throughput sample preparation and data analysis pipelines could further improve laboratory productivity. Expanded spectral libraries and chemometric tools, including machine learning algorithms, promise enhanced discrimination of closely related colorants. Portable or miniaturized PDA detectors could facilitate on-site screening for rapid field testing.

Conclusion

A versatile HPLC-PDA method was established for the separation, identification and quantification of ten synthetic food dyes across diverse consumer product matrices. Baseline chromatographic resolution, robust spectral purity validation and reliable library matching deliver accurate, high-throughput analysis suitable for routine quality control and regulatory surveillance.

References

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4. West Virginia State Legislation, Food Dye Law, accessed April 4 2025.
5. U.S. FDA, FDC Red No. 3 Phase-Out, accessed April 4 2025.
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7. Federal Register, Food Labeling Revision of Nutrition and Supplement Facts Labels, accessed April 8 2025.
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9. PubChem, Dye Reference Data, accessed April 7 2025.