Biosynthesis-Informed Putative Annotation Reveals Alkaloid Profiles Across Tissues of Anisodus luridus

Background/Objectives: Tropane alkaloids (TAs) and phenylpropanoid polyamine alkaloids (PPAs) are major alkaloid constituents of solanaceous plants; however, the tissue-specific distribution of these compounds in Anisodus luridus, a medicinal plant native to southeastern Xizang, remains incompletely characterized. Methods: A biosynthesis-informed putative annotation workflow was developed using ultra-performance liquid chromatography–high-resolution mass spectrometry (UPLC-HRMS) to characterize alkaloid profiles across seven tissues (root, stem, leaf, calyx, corolla, ovary, and seed). Putative annotations were guided by substrate–product structural relationships, predicted enzyme-catalyzed modifications, and diagnostic tandem mass spectrometry (MS/MS) fragmentation patterns. Results: Eighteen alkaloid features were characterized, comprising 6 TAs and 12 PPAs. Hyoscyamine and scopolamine were identified using authentic standards, and the remaining 16 features were putatively annotated, including dihydroanisodamine, hexosylhyoscyamine, and several PPAs not previously reported in A. luridus. TAs were predominantly enriched in roots and ovaries, whereas PPAs accumulated in floral tissues and leaves. Orthogonal partial least squares discriminant analysis (OPLS-DA) identified scopolamine (variable importance in projection (VIP) = 1.30) as a principal variable associated with separation among tissue metabolic profiles. Conclusions: This workflow supports the putative annotation of alkaloids in non-model medicinal plants. The observed tissue-specific distribution patterns provide a basis for future comparative studies with A. belladonna and for evaluating cultivation, tissue-selective harvesting, and extraction strategies.