STATISTICAL DESIGN SUPPORTED METHOD DEVELOPMENT AND SIMULTANEOUS QUANTITATIVE ANALYSIS OF HARMALINE, HARMINE, AND PEGANINE BY HPTLC FROM IN VITRO AND NATURALLY GROWN SEEDS OF PEGANUM HARMALA

This study developed and validated a high-performance thin-layer chromatography (HPTLC) method for the simultaneous quantification of these alkaloids in methanolic extracts of P. harmala seeds and in vitro–derived callus. Chromatographic separation was achieved on aluminum-backed silica gel plates using toluene:ethyl acetate:diethylamine (7:2.5:0.5, v/v/v), yielding compact spots with Rf values of 0.77 ± 0.04 (harmaline), 0.46 ± 0.09 (harmine), and 0.58 ± 0.06 (peganine). The method demonstrated high accuracy (recoveries 98.03–102.74%) and precision with low intra-and inter-day %RSD. Robustness, assessed via a Central Composite design evaluating wavelength, chamber saturation time, and toluene volume, indicated minimal influence of small parameter variations on peak response. Seeds were cultured on Murashige and Skoog media supplemented with varying concentrations of 2,4-D, 6BA, and IAA for germination and callus induction. The validated method is specific, reliable, rapid, and cost-effective for routine alkaloid analysis in P. harmala.