Optimised extraction and LC-MS/MS analysis of flavonoids reveal large field variation in exudation into Lupinus Angustifolius L. rhizosphere soil

Knowledge about agricultural crops and their mechanisms is crucial in a time focused on climate. Flavonoids are plant secondary metabolites present in many plant species, including narrow-leafed lupin (Lupinus angustifolius L.). To gain a better understanding of the biological effect of flavonoids in the soil, the link between roots and soil must be elucidated. Plants were grown in a long-term crop rotation field experiment, where roots and rhizosphere soil were sampled under L. angustifolius and reference spring barley (Hordeum vulgare L.). Three high-performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS) methods were developed and validated for the identification and quantification of 27 flavonoids in roots and rhizosphere soil, with flavonoids present in the soil representing exudation. Soil extraction using acetonitrile (ACN) and methanol (MeOH) was compared to 70% MeOH. In addition, to elucidate the bioavailable flavonoids, water-based extractions were investigated. The optimal soil extraction method was using 70% MeOH, achieving acceptable recoveries for several compounds. L. angustifolius roots contained 14 of 27 flavonoids at concentrations of 0.05–736 μg/g DW; genistein, genistin, and luteone being the most abundant. Four flavonoids were detected in L. angustifolius soil, but not in H. vulgare soil. None of the water-based extractions resulted in flavonoid quantities similar to those obtained with 70% MeOH extraction. L. angustifolius roots contained larger quantities of flavonoids than H. vulgare roots. The presence of four flavonoids in L. angustifolius soil, and not in H. vulgare soil, suggests that the flavonoids were exuded from the L. angustifolius roots. Sonication using 70% MeOH was an efficient soil extraction method, and enabled quantification of the flavonoid pool present in soil at the sampling time point.