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

L-Canavanine is a non-protein amino acid analogous to L-arginine that occurs in several legumes and is associated with antimetabolic and toxic effects in animals and humans. The aim of this study was to develop and validate a simple, selective, and cost-effective high-performance thin-layer chromatography (HPTLC) method for the identification and quantitative determination of L-canavanine in plant-derived matrices. Chromatographic separation 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 at 500 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 resolution between L-canavanine and L-arginine was achieved. The method is suitable for routine analysis of L-canavanine in forage and food-related matrices, including edible legumes.