1. Understanding plant-insect interactions is an active area of research in both ecology and evolution. Much attention has been focused 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 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 secondary metabolites in A. phalloides unlike the ‘Classic’ method that favors polar secondary metabolites.
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.

Amanita phalloides extracts were analyzed using Agilent 1260 Infinity II system, comprised of an Infinity II Binary Pump, Infinity Multisampler, Multicolumn Thermostat, Diode Array Detector (DAD, UV spectrometer), Agilent’s Instrument Control Framework, and Mass Selective Detector (MSD, mass spectrometer). The analysis was performed using Pursuit 5C18 150 x 4.6 mm column at 23 °C. The mobile phase comprised (A) water containing 0.2% formic acid and (B) acetonitrile containing 0.2 % formic acid. The following elution gradient was applied: 0-4 min 10% B; 4-20 min 10-55% B; 20-25 min 55% B; 25-30 min 55-100% B; 30-33 min 100% B; 33-35 min 100-10% B; 35-40 min 10% B. Five µL of the extract was injected onto the column and run at a 1.0 ml/min mobile phase flowrate. The eluate was monitored by DAD at 214 and 295 nm, and UV-VIS spectra within the range of 190-400 nm were stored for all the peaks. All the samples were analyzed on the MSD in electrospray positive ionization mode with 10 l/min drying gas flow rate, 50 psig nebulizer pressure, 350 °C drying gas temperature, and 4000 V capillary voltage. For fingerprinting, full scan acquisition mode was used with scan range: 100-1200.

Standard samples of α-amanitin, β-amanitin, phalloidin, and phallacidin obtained from SantaCruz Biotechnology (α-amanitin) and Cayman Chemical (all others). These four cyclopeptides are the only ones with commercially available standards to the best of the author’s knowledge. They were dissolved in double distilled water (α-amanitin, β-amanitin) or methanol (phalloidin, phallacidin) at a concentration of 1 µg/µL. These stock solutions were subsequently diluted ten-fold and used to confirm identification of selected cyclopeptides in the analyzed extracts. Other potential cyclopeptides were identified by their ionization pattern.