Üveges et al-suppl data_ontogeny of chem def.xlsx (57.89 kB)

Data: Age- and environment-dependent changes in chemical defences of larval and post-metamorphic toads

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We used high-performance liquid chromatography with diode-array detection and mass spectrometry (HPLC-DAD-MS) to identify and quantify bufadienolide compounds. We homogenized specimens of the common toad (Bufo bufo) using a VWR VDI 12 homogenizer with an IKA S12N-7S dispersing tool. After drying samples in vacuo at 45 °C using a Büchi Rotavapor R-134 rotary evaporator, we measured dry weight of samples using an Ohaus Pioneer PA-114 analytical balance to the nearest 0.1 mg and subsequently re-dissolved samples in 1 ml HPLC-grade absolute methanol, which was further aided by exposing the samples briefly to ultrasound in a Tesla UC005AJ1 bath sonicator. We filtered the samples using FilterBio nylon syringe filters (pore size = 0.22 μm). We identified compounds as bufadienolides by inspecting the UV spectrum of peaks (Hagman et al. 2009, Hayes et al. 2009, Bókony et al. 2016) and by using commercially acquired bufalin, bufotalin, resibufogenin, gamabufotalin, areno- and telocinobufagin (Biopurify Phytochemicals, Chengdu, China), cinobufagin (Chembest, Shanghai, China), cinobufotalin (Quality Phytochemicals, New Jersey, USA) and digitoxigenin (Santa Cruz Biotechnology, Dallas, TX, USA) as standards. Identification of compounds present in low quantities was further aided by the analysis of a sample obtained from an adult male common toad by gently massaging the parotoid glands.
A single-quadrupole HPLC-MS system (Model: LC-MS-2020, Shimadzu, Kyoto, Japan) equipped with a binary gradient solvent pump, a vacuum degasser, a thermostated autosampler, a column oven, a diode array detector and a mass analyser with electrospray ionization (ESI-MS) was used. Chromatographic separations were carried out at 35 °C on a Kinetex C18 2.6 µm column (100 mm x 3 mm i.d.) in series with a C18 guard column (4 mm × 3 mm i.d.) using 10 µL injections. The mobile phase consisted of water containing 0.05% formic acid (solvent A) and acetonitrile containing 0.05% formic acid (solvent B). The flow rate was 0.8 mL/min and the gradient was as follows: 0-2 min, 15-25% B; 2-15 min, 25-35% B; 15-24 min, 30-50% B; 24-25 min, 50-90% B; 25-30 min 90% B; 30-35 min 15% B. ESI worked under the following conditions: desolvation line (DL) temperature, 250 °C; heat block temperature, 400 °C; drying N2 gas flow, 15 L/min; nebulizer N2 gas flow, 1.5 L/min; positive ionization mode. Data was acquired and processed using the programme LabSolutions 5.42v (Shimadzu, Kyoto, Japan).