High-performance thin-layer chromatography screening of selected steroidal compounds in herbal medicines: four corticosteroid adulterants and daucosterol

Adulteration of herbal medicines with corticosteroids remains a critical concern for product safety and quality control. Here we report a validated high-performance thin-layer chromatography (HPTLC) method for simultaneous detection of five steroid standards: daucosterol, prednisolone, betamethasone-17-valerate, dexamethasone, and triamcinolone acetonide. Twenty mobile phases were systematically evaluated, yielding five systems that provided reproducible resolution. Steroids were clearly separated and visualized under UV (254/366 nm) and visible detection with retention factors of 0.19–0.79. The method showed low limits of detection (333–603 ng/spot) and quantification (1009–1829 ng/spot), good linearity (0.2–1.0 mg/mL), and acceptable repeatability and intermediate precision. Densitometric evaluation supported both qualitative screening and quantitative determination. Applicability was confirmed in five spiked herbal medicine matrices, and slope-ratio analysis indicated limited matrix effects across the five mobile-phase systems. This protocol uses inexpensive solvents and readily available instrumentation, providing a robust and cost-effective tool for routine monitoring of steroid adulteration in herbal formulations.