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The seeds of Strophanthus kombé Oliv. are known to contain high levels of cardioactive compounds. However, the therapeutic use of Strophanthus in the treatment of cardiopathy requires more detailed knowledge of the compound profile to profit from the full potential of Strophanthus preparations. Therefore, the objective was to characterize the cardenolide profile and lipophilic constituents in S. kombé seeds using methods applicable in routine quality control. Freshly prepared S. kombé seed extracts were analyzed without previous sample clean-up using a novel HPLC-DAD-MSn method. In addition, seed oils were analyzed by GC-MS following derivatization of the lipids. More than 20 cardenolides were tentatively assigned in the seed extracts including strophanthidin, strophanthidol, periplogenin and strophanthidinic acid aglycones, carrying various saccharide moieties. The findings revealed the presence of eight novel cardenolides, which have not been described for S. kombé so far. The occurrence of strophanthidinic acid derivatives was verified by comparison with synthesized strophanthidinic acid-cymaropyranoside. GC-MS characterization of the oils mainly revealed the presence of fatty acids, especially oleic acid and linoleic acid, as well as phytosterols, the latter representing intermediates of cardenolide biosynthesis. In summary, these findings broaden our knowledge on the secondary metabolism of Strophanthus.