Romanian Society of Pharmaceutical Sciences
 

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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

MOHD AMIR 1, MD SARAFROZ 2, WASIM AHMAD 3, MOHAMMAD KHALID 4, NAUSHAD ALI 5, ANZARUL HAQUE 6, AJAZ AHMAD 7, MOHD MUJEEB 8*

1 Department of Natural Products, College of Pharmacy, Imam Abdulrahman Bin Faisal University, Dammam, 1982, Saudi Arabia
2 Department of Pharmaceutical Chemistry, College of Pharmacy, Imam Abdulrahman Bin Faisal University, Dammam, 1982, Saudi Arabia
3 Department of Pharmacy, Mohammed Al-Mana College for Medical Sciences, Dammam-34222, Saudi Arabia
4 Department of Pharmacognosy, College of Pharmacy, Prince Sattam Bin Abdulaziz University, Alkharj, Saudi Arabia
5 Quality Unit, College of Pharmacy, King Saud University, 11451 Riyadh, Saudi Arabia
6 Central Laboratories Unit, Qatar University, 2713, Doha, Qatar
7 Department of Clinical Pharmacy, College of Pharmacy, King Saud University, 11451 Riyadh, Saudi Arabia
8 Department of Pharmacognosy and Phytochemistry, School of Pharmacy, Jamia Hamdard, New Delhi-62, India

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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.