LCMS Analysis of Alkaloid Compounds

The following extraction procedures were applied to aliquots of samples (80 mg): 1 mL of ethanol was added to the aliquot. The Eppendorf was vortexed and incubated for 60 min at 45 °C in a sonicator bath. A Shimadzu ultra-high-performance liquid chromatograph (Nexera XR LC 40) was paired with an MS/MS detector for qualitative analysis (LCMS 8060, Shimadzu Italy, Milan, Italy). The MS/MS used electrospray ionization and was controlled by Lab Solution software, which allowed for rapid change from a low-energy scan at 4 V (full scan MS) to a high-energy scan (10–60 V ramping) during a single LC run. The following source parameters were set: 2.9 L/min nebulizing gas flow, 10 L/min heating gas flow, 300 °C interface temperature, 250 °C DL temperature, 400 °C heat block temperature, and 10 L/min drying gas flow. The analysis was carried out by flow injection (i.e., no chromatographic separation), with acetonitrile: water + 0.01 percent formic acid (5:95, v/v) as the mobile phase. Instrument was set to positive mode for an SIM experiment [48 (link),49 (link)]. Samples were considered “positive” if the area under the curve was higher in magnitude than the blank. Pure standards used are listed in Table S1. Ergovaline, galanthamine, nor-galanthamine, anhydrolycorine, trisphaeridine, crinine, demethylmaritidine, N-formylgalanthamine and peramine were purchased from Sigma Aldrich.