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<p>Niacinamide is widely incorporated into cosmetic formulations owing to its broad spectrum of topical benefits. However, its safety profile is often compromised by the unintentional or deliberate addition of hydroquinone, a potent skin‑lightening agent prohibited in several regions, including the European Union and the Gulf Standardization Organization (GSO). Conventional reversed-phase high-performance liquid chromatography methods frequently fail to achieve sufficient resolution between these two highly polar analytes, increasing the risk of co‑elution and false‑positive detection. This study aims to develop and validate an orthogonal, thin‑layer chromatography (TLC) method for the simultaneous quantification of niacinamide and hydroquinone in complex cosmetic matrices. Chromatographic separation was performed on precoated silica gel 60 F<sub>254</sub> plates using a mobile phase of chloroform–ethanol (9:1, <i>V/V</i>). Quantification was achieved by densitometric scanning at 280&#xa0;nm, complemented by postchromatographic derivatization with anisaldehyde–sulfuric acid (ASA) reagent. Method specificity was confirmed through superimposable reflectance spectra and three-dimensional spectral overlays of standards and commercial cream samples. Distinct separation was obtained with retardation factor (<i>R</i><sub>F</sub>) values of 0.22 for niacinamide and 0.27 for hydroquinone. The method exhibited excellent linearity across the tested ranges. Limits of detection (LOD) were determined as 0.45&#xa0;µg for niacinamide and 3.38&#xa0;µg for hydroquinone. Recovery values ranged from 98.57 to 102.5%, indicating negligible matrix interference from the emulsified cream base. The developed method also exhibited promising results when analyzed in other marketed cosmetic matrices, including body lotion, face wash, gel, etc. The developed TLC method offers a rapid, cost‑effective, and highly selective analytical tool for industrial quality control. By combining chemical derivatization with spectral fingerprinting, the method effectively eliminates the risk of false analyte identification, thereby strengthening product safety assurance and regulatory compliance within the cosmetic industry.</p>

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Thin-layer chromatography method to differentiate between hydroquinone and niacinamide in cosmetic formulation

  • 期刊论文

  • 发表日期：

    2026年4月1日

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  • Partha Ganguly,
  • Sourabh Saha,
  • Subhadeep Chowdhury,
  • Rahul Singh,
  • Punita Kalra

摘要

Niacinamide is widely incorporated into cosmetic formulations owing to its broad spectrum of topical benefits. However, its safety profile is often compromised by the unintentional or deliberate addition of hydroquinone, a potent skin‑lightening agent prohibited in several regions, including the European Union and the Gulf Standardization Organization (GSO). Conventional reversed-phase high-performance liquid chromatography methods frequently fail to achieve sufficient resolution between these two highly polar analytes, increasing the risk of co‑elution and false‑positive detection. This study aims to develop and validate an orthogonal, thin‑layer chromatography (TLC) method for the simultaneous quantification of niacinamide and hydroquinone in complex cosmetic matrices. Chromatographic separation was performed on precoated silica gel 60 F254 plates using a mobile phase of chloroform–ethanol (9:1, V/V). Quantification was achieved by densitometric scanning at 280 nm, complemented by postchromatographic derivatization with anisaldehyde–sulfuric acid (ASA) reagent. Method specificity was confirmed through superimposable reflectance spectra and three-dimensional spectral overlays of standards and commercial cream samples. Distinct separation was obtained with retardation factor (RF) values of 0.22 for niacinamide and 0.27 for hydroquinone. The method exhibited excellent linearity across the tested ranges. Limits of detection (LOD) were determined as 0.45 µg for niacinamide and 3.38 µg for hydroquinone. Recovery values ranged from 98.57 to 102.5%, indicating negligible matrix interference from the emulsified cream base. The developed method also exhibited promising results when analyzed in other marketed cosmetic matrices, including body lotion, face wash, gel, etc. The developed TLC method offers a rapid, cost‑effective, and highly selective analytical tool for industrial quality control. By combining chemical derivatization with spectral fingerprinting, the method effectively eliminates the risk of false analyte identification, thereby strengthening product safety assurance and regulatory compliance within the cosmetic industry.