Optimization of preparative HPLC method for the purification of thymol from the essential oil of Trachyspermum ammi (L.)

The escalating demand for high-purity bioactive compounds in pharmaceutical research and quality control underscores the critical role of preparative High-Performance Liquid Chromatography. This study introduces a novel and optimized preparative HPLC protocol for the large-scale purification of thymol from Trachyspermum ammi (L.) essential oil. Addressing a notable knowledge gap, this is the first reported application of Prep-HPLC for thymol isolation from this specific botanical source, establishing a new, scalable protocol. An initial analytical HPLC method, optimized with Methanol:Water (60:40, v/v), was successfully scaled to a semi-preparative level. The optimized preparative method, utilizing Methanol:Water (90:10, v/v) as the mobile phase, achieved thymol purification with an impressive 98.87 % purity. The structural identity of the purified compound was unequivocally confirmed through comprehensive spectroscopic analyses, including GC–MS, FTIR, XRD, and NMR. Crucially, the purified thymol exhibited potent and broad-spectrum antifungal activity against both susceptible and multidrug-resistant strains of Candida albicans and Candida auris. Minimum inhibitory concentrations were determined to be as low as 0.125 μg/mL against multidrug-resistant C. albicans and 0.25 μg/mL against multidrug-resistant C. auris. This robust efficacy, particularly against challenging drug-resistant isolates, strongly positions the purified thymol as a promising candidate for further therapeutic development against candidiasis. This research significantly advances separation science and highlights the industrial potential of Prep-HPLC for high-value natural product isolation.