Structural Diversity of Steroidal Glycoalkaloids in Potato (Solanum tuberosum) Leaves Revealed by Untargeted UHPLC–QTOF Metabolomics

Steroidal glycoalkaloids (SGAs) are nitrogenous defence metabolites characteristic of Solanum species. Although the major potato SGAs α-solanine and α-chaconine are well known, the full structural scope of glycoalkaloids in vegetative tissues remains largely uncharacterized. Here, we applied untargeted ultra-high-performance liquid chromatography–quadrupole time-of-flight mass spectrometry (UHPLC-QTOF-MS) to profile SGA diversity in the leaves of three cultivars (Lady Rosetta, Charlotte, and Bintje). High-resolution MS/MS data enabled annotation of over 30 tri-glycosylated alkaloids differing in both the steroidal backbone and glycosylation topology. Three compounds, α-solanine, α-chaconine, and α-solamargine, were verified with authentic standards (Metabolomics Standards Initiative (MSI) level 1). Besides classical solanidine-based SGAs, multiple metabolites were consistent with solasodine- and solaverol-type aglycones (MSI level 2–3), revealing unrecognized biosynthetic branches. SGA composition varied strongly among cultivars: Lady Rosetta exhibited the broadest chemical repertoire and the highest abundance of oxygenated SGAs, whereas Bintje displayed a restricted solanidine-dominant profile. Several low-abundance SGAs bearing partially unsaturated or over-oxidized aglycones ( m/z 396, 412 and 428) were reproducibly detected and likely represent genuine metabolites, hinting at additional, low-flux biosynthetic routes. This comprehensive profile extends the structural landscape of potato SGAs and underscores the capacity of untargeted UHPLC–QTOF metabolomics to resolve complex steroidal metabolite networks. The observed cultivar-specific signatures suggest differential regulation of late-stage oxidation and glycosylation steps, potentially contributing to varietal variation in chemical defence.