Spectrophotometric vs. HPLC based Quantification of Vicine and Convicine in Faba Bean

Vicine and convicine (VC) are antinutritional pyrimidine glycosides of faba bean ( Vicia faba L.), commonly quantified either by spectrophotometric assays or by chromatographic methods. This study systematically evaluated the concordance between spectrophotometric extinction at 273 nm and high-performance liquid chromatography with diode array detection (HPLC-PDA) for VC determination across biologically and analytically diverse sample sets. A total of 301 samples from five experimental studies were analysed, covering multi-year and multi-location variety trials, drought stress conditions, tissue-specific samples, seed developmental stages, and different post-harvest drying procedures. Across the complete dataset, spectrophotometric extinction and HPLC-PDA-determined total VC showed a significant but moderate correlation (R = 0.77). However, stratified analysis revealed strong to very strong correlations within four of the five studies (R = 0.93–0.98), particularly in mature, homogeneous seed material. In contrast, the developmental series exhibited a lower correlation (R = 0.85) and marked shifts in regression parameters, reflecting matrix-dependent deviations. Differences among studies were driven by context-dependent shifts in slope and intercept rather than random analytical error, indicating that spectrophotometric and chromatographic methods are linked by context-specific relationships rather than a universal calibration. Untargeted LC-MS/MS analysis of aqueous spectrophotometric extracts identified a wide range of co-extracted polar metabolites, including nucleosides, nucleotides, sugars, and phosphorylated compounds, particularly enriched in immature seeds. Many of these compounds absorb in the same UV range as vicine and convicine and provide a mechanistic explanation for inflated extinction values in matrix-rich samples. The results demonstrate that spectrophotometric VC determination is reliable for high-throughput screening of mature, homogeneous seed material but becomes increasingly unreliable in developmentally dynamic or tissue-specific matrices. Chromatographic methods therefore remain essential for accurate VC quantification outside well-defined analytical contexts.