2.4. Indole diterpene alkaloid analysis

High performance liquid chromatography – high resolution mass spectrometry (HPLC-HRMS) analysis of plant material was based on published methods (Rasmussen et al. 2012; Lee et al., 2017). Samples were injected (10 μL) onto a Betasil C18 reversed phase column (5 μ; 100 × 2.1 mm i.d.) (Keystone Scientific, Inc. Bellefonte, PA, USA) protected by a guard column of the same phase. The samples were eluted from the column with a gradient flow consisting of 0.1% formic acid and acetonitrile at a flow rate of 0.300 mL/min. The mobile phase program was 0.1% formic acid-acetonitrile, 80:20, v:v for 1 min followed by a linear gradient to a composition of 100% acetonitrile at 40 min. The mobile phase was delivered and samples injected using an Ultimate 3000 HPLC (Thermo Scientific, San Jose, CA, USA) and the column eluent was connected to the heated electrospray source of an Exactive Plus Orbitrap high resolution mass spectrometer (Thermo Scientific) calibrated as per the manufacturer’s instructions and with a scan range 100 – 800 Da, resolution 70000, microscans 1, sheath gas flow 35, auxiliary gas flow 10, spray voltage 4 kV, capillary temperature 320 °C, S lens RF field 55, and auxiliary gas temperature 300 °C. Chromatographic peaks were identified by generating reconstructed HPLC-HRMS chromatograms with the calculated MH⁺ molecular weight of indole diterpene alkaloids to 5 decimals places and with a mass tolerance of 10 ppm.