Intermedine

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).

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).

A mixture of 7-acetyl-intermedine/lycopsamine was from PlantaAnalytica (Danbury, US), retrorsine from Cfm O.Tropitzsch (Marktredwitz, Germany), and echimidine, intermedine, intermedine N-oxide, and senecionine were from PhytoLab (Vestenbergsgreuth, Germany). Echivulgarine was isolated in-house [38 (link)] and echimidine N-oxide and retrorsine N-oxide were N-oxidized in-house, starting from the free bases according to [39 (link)], except that dichloromethane was used instead of trichloromethane.
D3-atropine was from CDN isotopes (Quebec, Canada), methanol was from Honeywell (LC-MS grade, Grogg AG, Stettlen, Switzerland). Sulfuric acid, formic acid, and aqueous ammonia solution 25%, all of analytical grade, were from Merck (Grogg AG, Stettlen, Switzerland). Ultrapure water was used (Elga purelab ultra, Labtec, Villmergen, Switzerland).