An accurate IDMS-based method for absolute quantification of phytohemagglutinin, a major antinutritional component in common bean

Phytohemagglutinin (PHA), a natural tetramer comprising PHA-E and PHA-L subunits that preferentially bind to red and white blood cells, respectively, constitutes a significant antinutritional and allergenic factor in common bean seeds. The accurate measurement of PHA content is a prerequisite for ensuring food safety inspections and facilitating genetic improvements in common bean cultivars with reduced PHA levels. Currently, mainstream methods for PHA quantification involve hemagglutination assays and immunodetection, but these methods often require fresh animal blood and lack specificity and accuracy. In this study, we present a novel liquid chromatography-mass spectrometry/MS-based method for PHA quantification, leveraging the advantages of isotope dilution mass spectrometry (IDMS). Two signature peptides each for PHA-E and PHA-L, along with a common signature peptide, were identified and employed for quantification, allowing differentiation between PHA-E and PHA-L subunits. The incorporation of amino acid analysis isotope IDMS enabled accurate determination of the synthetic signature peptides' purity during measurement, enhancing metrological accuracy. In addition, the trichloroacetic acid-acetone protocol was established as the optimized method for total protein extraction from dry bean seeds. Quantitative analysis of PHA-E and PHA-L subunits in six common bean varieties using the developed method demonstrated excellent linearity (R > 0.999), sensitivity (limit of detection and quantitation as low as 2.32 ng·mg ¹ and 7.73 ng·mg ¹, respectively), recovery (94.18%-104.47%), and precision (relative standard deviation