Insights into non-isoflavone flavonoid distribution and variation across soybean germplasm using HPLC-MS/MS

Flavonoids are key secondary metabolites in soybean, contributing to the plant defense mechanism and human health. The isoflavones in soybean have been well studied, and non-isoflavone flavonoids remain unexplored. This study optimized and validated a highly sensitive high-performance liquid chromatography coupled with mass spectrometry (HPLC-MS/MS) method to quantify 13 flavonoids from five subclasses (chalcone, flavanols, flavanones, flavones, and flavonols), representing 82 % of total flavonoid content (TFC) in soybean seeds. Profiling 711 diverse soybean accessions from China's three main ecoregions revealed significant variation, with TFC ranging from 667 to 24503 μg/100g, representing a 36-fold difference; four accessions exceeding 20000 μg/100g. The Huang Huai Hai Valley Region (HR) had the highest mean TFC (3341 μg/100g). Black seed coat accessions exhibited the highest flavonoid concentrations. The cumulative share of four major flavonoids, epicatechin, quercetin 3-galactoside, prunin, and apigenin, accounted for 77–81 % of TFC, particularly the dark-pigmented seedcoat enriched with epicatechin and quercetin derivatives. TFC correlated positively with most flavonoids, isoflavones, and protein, but negatively with oil content. Major flavonoid components were negatively associated with longitude, suggesting that geographical origin affects flavonoid accumulation. The findings provide actionable insights for breeding nutritionally enriched soybean varieties, with implications for functional food development. • Developed a robust HPLC-MS/MS method for quantifying 13 non-isoflavone flavonoids. • Profiling of 711 soybean accessions across three major ecoregions of China. • Flavonoid levels depend on accession type, ecoregion, seed coat color, and location. • Black and brown seed-coated accessions showed high flavonoid content. • The abundant flavonoids were epicatechin, quercetin 3-galactoside, prunin, and apigenin.