ABSTRACT: Individual plant parts from two to three specimens of one representative species of each subgeneric clade of Euphorbia were sampled (E. horrida Boiss., subg. Athymalus, three specimens; E. hirta L., subg. Chamaesyce, two specimens; E. lathyris L., subg. Esula, two specimens; E. milii Des Moul., subg. Euphorbia, two specimens) in the Botanical Garden in Copenhage, Denmark. Approximately 200 mg fresh plant material of each individual plant part was collected in 1.5 ml Eppendorf tubes and flash frozen under liquid nitrogen. The frozen plant material was disrupted in plastic tubes (Qiagen, RB 2 mL) in 1.3 ml 50/50 vol/vol methanol (Fisher Scientific, HPLC Grade)/acetonitrile (Fisher Scientific, Optima LC/MS) with stainless steel beads (VWR International, 5 mm) using a tissue lyser (Qiagen, TissueLyser II) at 25 Hz during 10 min. The samples were then extracted under sonication (Fisher Scientific) at 40 C during 15 min and centrifuged (Eppendorf Centrifuge 5418) for 10 min at 11,000 r.p.m. The extract supernatant was transferred to new plastic tubes and lyophilized to dryness (Labcono, Acid Resistant CentriVap Concentrator). Dried extracts were dissolved in 50/50 vol/vol methanol (Fisher Scientific, HPLC Grade)/acetonitrile (Fisher Scientific, Optima LC/MS) to a concentration of 1 mg/ml, 100 uL of each extract were transferred to a 96-well plate (Falcon, 96-well plates, 0.34 ml, polypropylene), sealed with Zone-Free Sealing Film (Excel Scientific) and centrifuged for 30 min. at 2000 r.p.m. at 4 C. 20 uL of each extract were injected into the LC-MS/MS equipment. For more details refer to the Material and Methods section and Supplementary Material in Ernst et al., (2019).
Pieter Dorrestein 
