Introduction

Robert F Kennedy Jr. (RFK) has spoken out against food dyes that is prominent in the American market. Food dyes exist in products like candy, beverages, and snacks. These food dyes go by names like Allura Red, Tartrazine, Sunset Yellow, Citrus Red 2, and many more. The primary focus in this experiment will be Red 40 (Allura Red), Yellow 5 (Tartrazine), and Blue 1 (Brilliant Blue FCF). All dyes were successfully detected in Green, Red, and Blue Gatorades. Tartrazine was the only analyte that needed more sufficient testing due to binding issues with the column.

Allura Red (Red 40)

Tartrazine (Yellow 5)

Brilliant Blue FCF (Blue 1)

Method

Sample Preparation

Powdered reagents of Brilliant Blue FCF (Supelco, 80717), Allura Red (Supelco, 38213), and Tartrazine (Supelco, 03322), were weighed and diluted, with water to create a 1000ppm solution of each individual target. Calibrant levels 10, 50, and 100ppm of all analytes together were created with the diluent being a 50:50 ratio of Ultrapure Water and HPLC Acetonitrile. As the target was gatorade which is majority water, the calibrant material was remade where the diluent was Ultrapure Water. 2mL of calibration solutions were filtered through 0.22uM Nylon Filters (Filtrous, SFN-AR-1013) to Amber HPLC Autosampler Vials (VAV-09-1020) before injection.

Procedure

Liquid based samples were poured into a clean beaker for easier use. 2mL of a liquid based sample was drawn with a syringe and filtered through a 0.22um Nylon Filter into an autosampler vial.

Soft and hard candy were weighed out to be measured at least 5mg. For every 1mg of candy used, 1mL of a 50:50 diluent of Water:Acetonitrile was used to immerse the sample in a conical tube. Samples were then sonicated for 60 minutes. After sonication, the samples were vortexed for 1 minute. 2mL of sample was drawn by a luer-lock syringe and then filtered through a 0.22um Nylon filter into an autosampler vial before injecting into the machine.

Results and Discussion

Separation and Detection

The detection of Tartrazine was linear, although it would elute within the dead volume of the injection of the sample, this made it difficult to detect in samples and is not good practice to detect the analyte. It would be ideal to change the aqueous conditions of the mobile phase or to change to a more polar column to detect Tartrazine with better retention. The linearity of all targets was consistent and detectable in the study.

Chromatograms below represent the separation of the dyes and its detectability at different wavelengths.

Quantitation of Dye Additives in Gatorade

Red Gatorade resulted in 41.63mg/mL of Allura Red dye. Yellow Gatorade resulted in 3.65mg/mL of Tartrazine dye. Blue Gatorade resulted in 5.39mg/mL of Brilliant Blue dye. These results were averaged across 3 repeats of sample preparation of the same source of beverage. Yellow Gatorade is the only quantity that is not the best practice to report due to the ability of Tartazine being able to bind to the column allowing for proper quantification and peak shape.

Conclusion

Utilizing the sample preparation methods and workflow highlighted above- the acceptable daily intake (ADI) as well as the median lethal dose (LD50)  of Allura Red (Red40) Tartrazine (Yellow 5), and Brilliant Blue (Blue 1) were calculated. The ADI determines the amount of a substance a human can consume every day throughout their lifetime without experiencing adverse health effects, while the LD50 is the dose of a substance that when administered to animals it was determined to be lethal to 50% of the population. These calculations were conducted with the synthetic food dyes as the analyte of interest and do not account for the ADI or LD50 of sugar, salt, water, or any other additives contained in gatorade. In Fruit Punch Gatorade, containing Red 40, the ADI was determined to be ~2.57 gallons, and the LD50 was ~1,839.5 gallons. Lemon-Lime gatorade, containing Yellow 5, had an ADI calculated to be ~31.45 gallons, with an LD50 of ~41,927 gallons. Cool Blue Gatorade, containing Blue1, had an ADI calculated to be ~17.07 gallons, with an LD50 of ~5,683.11 gallons.