Europine

In total, 30 pyrrolizidine alkaloid standards were utilized in a high resolution accurate mass (HRAM) LC-MS/MS screen. Echimidine, erucifoline, europine, heliotrine, indicine, intermedine, jacobine, lasiocarpine lycopsamine, monocrotaline, retrorsine senecionine, seneciphylline, senecivernine, and their respective N-oxides, were purchased together with senkirkine and trichodesmine from Phytolab GmbH & Co. KG (Vestenbergsgreuth, Germany) and had a purity >89%. All other chemicals and solvents were of analytical reagent or HPLC grade purity. Water used for sample preparation and HPLC was Milli-Q purified (Merck Millipore, Darmstadt, Germany).

Analytical standards of all pyrrolizidine alkaloids and their N-oxide were purchased from Phytolab (Vestenbergsgreuth, Germany): Echimidine (Em), Echimidine-N-oxide (EmNO), Echinatine (En), Echinatine-N-oxide (EnNO), Erucifoline-N-oxide (ErNO), Europine (Eu), Europine-N-oxide (EuNO), Heliosupine (Hs), Heliosupine-N-oxide (HsNO), Heliotrine (Ht), Heliotrine-N-oxide (HtNO), Indicine (Id), Indicine-N-oxide (IdNO), Integerrimine (Ir), Integerrimine-N-oxide (IrNO), Intermedine (Im), Intermedine-N-oxide (ImNO), Lasiocarpine (Lc), Lycopsamine (Ly), Lycopsamine-N-oxide (LyNO), Retrorsine (Rt), Retrorsine-N-oxide (RtNO), Rinderine (Rn), Rinderine-N-oxide (RnNO), Senecionine (Sn), Senecionine-N-oxide (SnNO), Seneciphylline (Sp), Seneciphylline-N-oxide (SpNO), Senecivernine (Sv), Senecivernine-N-oxide (SvNO), Senkirkine (Sk), Spartioidine (St), Spartioidine-N-oxide (StNO), Usaramine (Us) and Usaramine-N-oxide (UsNO). Methanol (LC-MS grade) was from VWR Chemicals (Rosny-sous-Bois-cedex, France), sulphuric acid (96%) and acetonitrile (LC-MS grade) were from Carlo Erba Reagents (Val de Reuil Cedex, France), formic acid was from Carlo Erba Reagents (Milan, Italy) and ammonium formate (analytical grade) was from Sigma-Aldrich (St. Louis, MO, USA). Ultrapure water used throughout the experiments was produced by an EvoQua Water Technologies system (Diessechem, Milan, Italy).

Reference substances for 25 PA and PANO were purchased from Phytolab, Vestenbergsgreuth, Germany (echimidine, echimidine N-oxide, europine, europine N-oxide, erucifoline, erucifoline N-oxide, heliotrine, heliotrine N-oxide, intermedine, intermedine N-oxide, jacobine, jacobine N-oxide, lasiocarpine, lasiocarpine N-oxide, lycopsamine, lycopsamine N-oxide, monocrotaline, monocrotaline N-oxide, retrorsine, retrorsine N-oxide, senecionine, senecionine N-oxide, seneciphylline, seneciphylline N-oxide, and senkirkine). Stock solutions (c = 1.0 mg/mL) of each PA/PANO were prepared either with acetonitrile or acetonitrile/water (50/50, v/v), depending on the solubility, and stored at 4 °C in the dark. A PA/PANO mix solution (c = 1.0 µg/mL of each analyte) was used to prepare calibration standards and for the validation experiments. Water, acetonitrile and methanol were used in LC–MS grade (Th. Geyer, Renningen, Germany). Ammonia solution (32%) was purchased from Th. Geyer as well. Formic acid and ammonium formate used as eluent additive (LC–MS/MS grade) were from Sigma Aldrich (Deisenhofen, Germany).