Exhaustive extraction of cyclopeptides from Amanita phalloides: Guidelines for working with complex mixtures of secondary metabolites

1. Understanding plant‐insect interactions is an active area of research in both ecology and evolution. Much attention has been focused on the impact of secondary metabolites in the host plant or fungi on these interactions. Plants and fungi contain a variety of biologically active compounds, and the secondary metabolite profile can vary significantly between individual samples. However, many experiments characterize the biological effects of only a single secondary metabolite or a subset of these compounds. 2. Here, we develop an exhaustive extraction protocol using an accelerated solvent extraction protocol to recover the complete suite of cyclopeptides and other secondary metabolites found in Amanita phalloides (death cap mushrooms) and compare its efficacy to the “Classic” extraction method used in earlier works. 3. We demonstrate that our extraction protocol recovers the full suite of cyclopeptides and other secondary metabolites in A. phalloides unlike the “Classic” method that favors polar cyclopeptides. 4. Based on these findings, we provide recommendations for how to optimize protocols to ensure exhaustive extracts and also the best practices when using natural extracts in ecological experiments.