LC–MS/MS Quantification of Scopoletin in Trigonella foenum-graecum Seeds from Different Geographical Regions and Evaluation of Its In Vitro Antioxidant and Antimicrobial Activities

Abstract

This study reports the LC–MS/MS-based quantification of scopoletin in Trigonella foenum-graecum seeds collected from India, Egypt, and Pakistan, along with evaluation of their antioxidant and antimicrobial activities. Scopoletin, a hydroxycoumarin derivative structurally related to umbelliferone and possessing a methoxy substituent at the C-6 position, was identified using TLC and ¹H-NMR spectroscopy and quantified by a validated LC–MS/MS method. The method demonstrated good linearity over 10–1000 ng/mL (r² = 0.992), with LOD and LOQ values of 4.863 and 14.739 ng/mL, respectively. Recovery ranged from 98.56% to 101.22%, confirming method accuracy. Quantification revealed significant regional variation, with the highest scopoletin content observed in Indian samples (1.87 ± 0.08% w/w), followed by Pakistan (1.33 ± 0.09%) and Egypt (0.98 ± 0.06%). The extracts exhibited strong antibacterial activity against Staphylococcus aureus, while no activity was observed against Escherichia coli or Candida albicans. The findings demonstrate geographical influence on phytochemical composition and support the applicability of LC–MS/MS for quality control of herbal materials.