Development of an HPTLC-MS Method for the Differentiation of Celosiae Semen: Celosia argentea Versus C. cristata

Celosiae Argentea Semen (CAS), derived from <i>Celosia argentea</i> L., is traditionally used in Korean and Chinese medicine to treat eye disorders and liver heat and is recognized in official Pharmacopeias. In contrast, Celosiae Cristatae Semen (CCS), despite its frequent presence in the market, is not officially listed. The morphological and chemical similarities between the two pose challenges for accurate identification. This study presents an integrative method combining digital image analysis and high-performance thin-layer chromatography coupled with mass spectrometry (HPTLC-MS) to differentiate CAS from CCS. Digital microscopy and ImageJ analysis showed that CCS has a projection area over twice that of CAS. Chemically, an optimized HPTLC method using ethyl acetate, methanol, water, and formic acid revealed distinct fingerprint patterns under UV 366 nm and white light. Notably, celosin F was exclusively detected in CAS, while celosin H, J, and K were characteristic of CCS. ESI-TOF-MS analysis confirmed these markers, resolving an overlap in <i>R</i>_F values. Repeatability tests showed total SDs of sucrose for intra-day, inter-day, and inter-analysis precision were 0.006, 0.004, and 0.005, respectively, confirming method reliability. This combined approach offers a rapid, reliable, and practical tool for distinguishing these two medicinal seeds, supporting enhanced quality control and regulatory standardization in pharmaceutical applications.