Europine n oxide

Lasiocarpine (Las) and lasiocarpine N-oxide were ordered from Cfm Oskar Tropitzsch (Marktredwitz, Germany). Lycopsamine (Lyc), Lycopsamine N-oxide, europine (Eur), europine N-oxide, senecionine (Sen), senecionine N-oxide, echimidine (Ech), echimidine N-oxide, intermedine, intermedine N-oxide, jacobine, jacobine N-oxide, heliosupine, heliosupine N-oxide, retrorsine N-oxide, retronecine, and senkirkine were obtained from Phytolab (Vestenbergsgreuth, Germany). Retrorsine (Ret) was delivered by AppliChem (Darmstadt, Germany). Methanol (MeOH, LC–MS grade) and water (H₂O, LC–MS grade) were purchased from Merck KGA (Darmstadt, Germany). 7,9-Diglutathionyl-6,7-dihydro-1-hydroymethyl-5H-pyrrolizidine was obtained from ASCA (Berlin, Germany). Potassium cyanide (KCN), isotopically labeled potassium cyanide (KCX, K¹³C¹⁵N), glutathione and isotopically labeled glutathione (GSX, ¹³C₂¹⁵N-GSH), and all other chemicals and co-factors were purchased from Sigma-Aldrich (Steinheim, Germany). All chemicals were obtained in analytical grade, if available. Human liver microsomes (mixed gender from 50 donors; protein concentration 20 mg/mL; cytochrome P450 content 310 pmol/mg protein) and rat liver microsomes (male Sprague–Dawley rats; protein concentration 20 mg/mL; cytochrome P450 content 700 pmol/mg protein) were obtained from Corning (Corning, USA).

PA standards used for analysis were 7-acetylintermedine, 7-acetylintermedine-N-oxide, 7-acetyllycopsamine, 7-acetyllycopsamine-N-oxide, echinatine, echinatine-N-oxide, heliosupine, heliosupine-N-oxide, heliotrine, heliotrine-N-oxide, indicine hydrochloride, indicine-N-oxide, integerrimine, integerrimine-N-oxide, intermedine, lycopsamine, lycopsamine-N-oxide, retrorsine, riddelliine, riddelliine-N-oxide, rinderine, rinderine-N-oxide, senecionine, senecionine-N-oxide, seneciphylline, and senkirkine, from PhytoPlan (Heidelberg, Germany). Echimidine, Echimidine-N-oxide, erucifoline, erucifoline-N-oxide, europine hydrochloride, europine-N-oxide, intermedine-N-oxide, jacobine, jacobine-N-oxide, lasiocarpine, Monocrotaline-N-oxide, retrorsine-N-oxide, seneciphylline-N-oxide, senecivernine, and senecivernine-N-oxide were from PhytoLab (Vestenbergsgreuth, Germany). lasiocarpine-N-oxide was from Cfm Oskar Tropitzsch (Marktredwitz, Germany). Monocrotaline was from Carl Roth (Karlsruhe, Germany) and trichodesmine from Latoxan (Valence, France). For the palynological analysis and sample preparation, the following reagents were used: demineralized water, ultrapure water, dish soap, Kaiser’s glycerol gelatin, dry ice, 0.05 M sulfuric acid, ammonia solution, and methanol. For sample analysis, 5 mM ammonium formate and 0.1% (v/v) formic acid in methanol were used.

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).