Mr. Parth Trivedi is an application specialist at Anchrom Enterprises Pvt. Ltd., Mumbai, India, working under the guidance of Mr. Akshay Charegaonkar, Managing Director at Anchrom Enterprises Pvt. Ltd., Mumbai, India. The company specializes in instrumental HPTLC and is dedicated to developing innovative, quantitative and regulatory-compliant analytical methods for a wide range of products, including pharmaceutical formulations, APIs, herbal products, food items, organic intermediates and dyes.
Mr. Trivedi utilizes the advantages of HPTLC due to its rapidity, cost-effectiveness, versatility, and ability to perform simultaneous analysis of multiple samples with minimal solvent use. His work focuses on the application of HPTLC for the detection of non-permitted substances, such as Rhodamine B, in food and cosmetic products, ensuring safety, quality control and regulatory compliance.
Introduction
Rhodamine B is a synthetic, water-soluble dye belonging to the xanthene family, characterized by its bright pink to reddish-purple appearance [1]. Widely used in textiles, paper, inks and biological research, it is recognized for its intense color and fluorescence. Its illegal use is often observed in brightly colored sweets, street foods and colored beverages. In cosmetics, it is frequently found in low-cost or unbranded products. However, Rhodamine B is not approved for use in any food or cosmetic products due to its toxic effect [2].
The Food Safety and Standards Authority of India (FSSAI) explicitly prohibits its presence in food, while the Drugs and Cosmetics Act 2006 bans its use in cosmetics. Rhodamine B is classified as a possible human carcinogen and mutagen, it has shown to cause damage to DNA, liver, kidney toxicity and even tumor formation in experimental studies [3]. HPTLC serves as a reliable analytical technique for the detection and identification of Rhodamine B in various food and cosmetic samples. Despite these restrictions, its unauthorized use persists, especially in low-regulated sectors, for coloring sweets, street foods and inexpensive cosmetic products.
This guidance aims to facilitate the detection of DEG and EG-contaminated drug components, helping to prevent further poisoning incidents. The HPTLC method, adapted from a TLC approach [2], has been optimized to enhance sensitivity and reproducibility.
Standard solution
10.0 mg Rhodamine B dissolved in 10.0 mL methanol – water 4:1 (V/V) (1.0 mg/mL stock).
Dilute 1.0 mL of this stock to 10.0 mL with the same solvent to obtain 0.1 mg/mL Rhodamine B standard solution.
Sample preparation
All food and cosmetic samples are prepared at a concentration of 500.0 mg/ml. Weigh 2500.0 mg of the sample and add 5.0 mL of methanol. Vortex thoroughly for at least 2-3 min. Sonicate the solution for 15 min, then centrifuge at 3000 rpm for 5 min. Collect the supernatant and use it for application.
Chromatogram layer
HPTLC plates silica gel 60 F254 (Merck), 20 × 10 cm are used.
Sample application
1.0 µL of Rhodamine B standard, 2.0 µL of each cosmetics sample and 5.0 µL of each food sample are applied with Automatic TLC Sampler 4 (ATS 4) as 8.0 mm bands, 15 tracks, track distance 11.4 mm, distance from left edge 20.0 mm, distance from the lower edge 8.0 mm.
Chromatography
Plates are developed in the ADC 2 with chamber saturation (with filter paper) for 20 min and after activation at 33 % relative humidity for 10 min using a saturated solution of magnesium chloride, development with tetrahydrofuran – toluene – formic acid – water 16:8:2:1 (V/V) to the migration distance of 70 mm (from the lower edge), followed by drying for 5 min.
Documentation
Images of the plate are captured with the TLC Visualizer 2 in UV 254 nm, UV 366 nm, and white light.
Results and discussion
In the analysis of food and cosmetic samples for Rhodamine B using HPTLC, several important observations were made. The Rhodamine B standard showed a distinct band at RF 0.50, which was visible under white light, UV 254 nm, and UV 366 nm, with a maximum (λmax) at 552 nm. This band served as a reference for comparison with the test samples. Among the tested samples, sugar-coated jelly candy and cotton candy clearly showed bands matching the Rhodamine B standard, indicating the presence of the dye. Similarly, sindoor (traditional red powder used in Hindu culture, liquid lipstick, blusher, eyeshadow and nail polish also exhibited bands at the same RF value, confirming contamination with Rhodamine B. In contrast, tutti frutti (candied fruit mix), jelly, halwa barfi (traditional Indian sweet), and lipstick did not show any corresponding bands, suggesting that these samples were free from the dye.
Spectral analysis further supported these results, as the spectra of positive samples closely overlapped with that of the Rhodamine B standard, confirming its identity. These observations highlight that HPTLC is a reliable and effective method for the detection of Rhodamine B in both food and cosmetic products. The study shows the importance of such testing for quality control and regulatory compliance to prevent harmful adulteration and ensure consumer safety.
HPTLC fingerprints: White light (A), UV 254 nm (B), UV 366 nm (C); Track 1 (left): shows Rhodamine B standard, track 1 (right): tutti frutti, track 2: sugar-coated jelly, track 3: jelly, track 4: cotton candy, track 5: ice barfi, track 6: cosmetic sample sindoor, track 7: lipstick, track 8: liquid lipstick, track 9: blusher, track 10: eye shadow, track 11: nail polish.
Literature
[1] Shabir, G. et al. Mini-Reviews in Organic Chemistry (2018) 166.
[2] Puttegowda, S. K. B. et al. Ind J Pharm Pract (2024) 8.
[3] Knudsen, K. B. et al. Journal of nanoparticle research (2014) 2221.