Identification of Skin Lightening Agents in Cosmetics Using UHPLC with PDA, Mass Detection, and Empower 3 Software

Importance of the Topic

Skin lightening products often contain undeclared pharmaceutical actives that pose regulatory and health risks. Ensuring accurate identification of whitening agents in cosmetics is critical to protect consumers from adverse effects associated with prolonged use of steroids, hydroquinone, and retinoids. Advanced analytical methods can detect forbidden compounds at trace levels and help manufacturers and regulators enforce safety standards.

Objectives and Study Overview

This application note describes a workflow for the identification and quantitation of common skin-lightening agents in cosmetic creams and gels. The study focused on misbranded products purchased from online vendors in the United States and employed ultra-high performance liquid chromatography (UHPLC) with photodiode array (PDA) and mass detection. Empower 3 software was used for data acquisition, spectral matching, and reporting.

Methodology and Instrumentation

The analytical method combined UHPLC separation on a CORTECS® T3 column (2.7 μm, 3.0 × 100 mm) with gradient elution (0.1% formic acid in water and methanol) at 0.8 mL/min and column temperature of 30 °C. Detection was carried out by a 2998 PDA detector (210–400 nm) and an ACQUITY QDa mass detector in positive and negative electrospray modes. Data were processed in Empower 3 to match retention times, UV spectra, and m/z values to authentic standards.

Sample Preparation

Cream and gel matrices were spiked with known standards, equilibrated by vortexing and shaking in acetonitrile, centrifuged, and filtered prior to injection. Matrix-matched calibration curves (0.01–5% w/w for most analytes) were generated in blank cosmetic bases to achieve recoveries above 93% for key compounds.

Main Results and Discussion

Chromatographic separation of 12 target compounds (including nicotinamide, arbutin, hydroquinone, salicylic acid, four parabens, clobetasol propionate, betamethasone isomers, and tretinoin) was achieved in under 9 minutes. Several market samples contained prohibited actives:

• Clobetasol propionate detected in both gel and cream at 0.038–0.060% w/w.
• Hydroquinone found at 3.0–7.2% w/w, exceeding regulatory limits.
• Betamethasone valerates and tretinoin identified in multi-step skin treatments.

The combination of UV spectral matching and mass profiles, including chlorine isotope patterns, provided high confidence in compound identification. In many cases, labels did not declare the pharmaceutical ingredients.

Benefits and Practical Applications of the Method

This UHPLC–PDA–MS approach using a dual-flow path ACQUITY Arc system delivers:

• Rapid, high-resolution separations for polar and nonpolar analytes.
• Seamless method transfer between HPLC and UHPLC conditions.
• Enhanced detection confidence via combined UV and mass spectral data.
• Automated data processing and reporting with Empower 3.

This workflow supports routine quality control, regulatory compliance testing, and forensic screening of cosmetic products.

Future Trends and Applications

Emerging developments may include integration of high-resolution mass spectrometry for improved selectivity, miniaturized sample preparation techniques to increase throughput, and machine learning-driven spectral libraries for automated unknown identification. Expansion to other cosmetic matrices and incorporation of dermal absorption studies will further enhance consumer safety assessments.

Conclusion

The described UHPLC–PDA–MS method effectively identifies and quantifies prohibited skin-lightening agents in cosmetics. Its speed, sensitivity, and robust data processing make it a valuable tool for industry laboratories and regulatory authorities aiming to ensure product safety and compliance.

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