Integrated LC/MS-MS approaches reveal toxicity differences across seed layers of <i>Cycas angulata</i>

Abstract Background and Aims Indigenous Australians have long recognised the toxicity of cycad seeds and have developed methods to process them for consumption. To date studies of cycad toxicity have focussed on individual toxins or groups of toxins rather than the broader chemical profile of seed layers. This has led to contradictory hypotheses about food preferences, herbivore defence and seed dispersal. To address this gap, we optimised protocols enabling concurrent or consecutive measurement of all major cycad toxins in the same tissues and applied them to assess the toxicity of each seed layer of the culturally important Cycas angulata. Methods Seeds were collected from the southwest Gulf of Carpentaria (N.T.). LC/MS-MS protocols were optimised to quantify the following key cycad toxins—the azoxyglycosides (AZGs), β-methylamino-L-alanine (BMAA), and β-sitosterol β-D-glucoside (BSSG) in the sarcotesta, sclerotesta, inner membrane and megagametophyte. Key Results Optimised extraction and analytical methods enabled quantification of five different key toxins from a single sample. AZGs were the most abundant across all seed layers, with cycasin predominating in the inner, and macrozamin in the outer layers. The sarcotesta and megagametophyte contained the highest concentration of toxins overall, while the inner membrane contained the lowest. Conclusions Contrary to previous work, the sarcotesta contained high macrozamin levels and may be as toxic as the megagametophyte. The high concentration of toxins in both tissues is consistent with a role in herbivore defence and that seed dispersal by animals would require toxin tolerance. These findings underscore the importance of measuring all putative toxins in the same tissues. The results support the detailed processing methods of Noongar people (southwest W.A.), highlighting the importance of considering socio-cultural factors when interpreting Indigenous cycad use. The optimised protocols provide a foundation for future comparative studies across cycad species, organs and tissues.