HPTLC method for the identification and quantitative determination of the toxic antimetabolite L-canavanine in plant samples

Abstract

L-Canavanine is a non-protein amino acid analogous to L-arginine that occurs in several legumes and is associatedwith antimetabolic and toxic effects in animals and humans. The aim of this study was to develop andvalidate a simple, selective, and cost-effective high-performance thin-layer chromatography (HPTLC) method forthe identification and quantitative determination of L-canavanine in plant-derived matrices. Chromatographicseparation was achieved on silica gel plates using an isocratic ethanol-28% ammonium hydroxide (7:3, v/v)mobile phase, followed by post-chromatographic derivatization with ninhydrin and densitometric detection at500 nm. The method was validated according to ICH Q2(R2) guidelines, evaluating linearity, sensitivity, accuracy,precision, specificity, and robustness. Good linearity was obtained over the range of 50.0–400.0 ng/band,with limits of detection and quantification of 16.3 ng and 49.5 ng, respectively. Clear chromatographic resolutionbetween L-canavanine and L-arginine was achieved. The method is suitable for routine analysis of L-canavaninein forage and food-related matrices, including edible legumes.