Putative identification of Taspine and Magnoflorine precursors by liquid chromatography-high resolution mass spectrometry (LC-HRMS)

Leaf, stem, root, and flower samples (n = 3 per tissue) were lyophilized in a freeze dryer (Labconco FreeZone®). The sample corresponding to inflorescence (inflorescence with floral stem) was not included in the phytochemical analysis as removal of the flowers from the floral stem results in a leafless flowering stem that we considered likely to be similar to stems or flowers, both of which were analysed separately. Fruits were not included because, like flowers, they are seasonal organs, the abundance of which depends on pollination frequencies. Plant extracts were prepared using an accelerated solvent extraction (ASE) system (ASE 350, Dionex, Thermo Scientific). Briefly, 3 g of dry material were mixed with 1 g of diatomaceous earth and placed in a 34 mL Dionex stainless-steel cell (2.9 cm diameter). The cell was filled with methanol to a pressure of 10.342 MPa and heated to 60 °C for 5 min. Then the cells were washed with methanol (HPLC grade, Sigma-Aldrich) up to 30% of the cell volume and, to avoid bias, a single sample from each organ (1 mL) was prepared by pooling the methanolic extracts of the three replicate samples. A Waters Class I UPLC coupled to a Synapt G2-Si HDMI mass spectrometer was used as analytical platform. 2 μL of each sample extract pool obtained by ASE system was injected in a Waters Acquity BEH (1.7 μm, 2.1 × 50 mm) column. The sample and column temperatures were 15 and 40 °C, respectively. Water (A) and acetonitrile (B), both with 0.1% formic acid (MS grade, Sigma-Aldrich), were used as mobile phase. The solvent gradient was 0–13 min, 1–80% B; 13–14 min, 80% of B; 14–15 min 80–1% of B with a constant flow rate of 0.3 mL/min. It was used an electrospray ionization source in positive mode with a capillary, sampling cone and source offset voltages of 3000, 40 and 80 V, respectively. The source and desolvation temperatures were 100 and 20 °C, respectively. The desolvation gas flow was 600 L/h, and the nebulizer pressure was 0.65 MPa. As lock mass a mixture of leucine-enkephalin (556.2771, [M + H]⁺) it was used and settings were: Mass range 50–1200 Da, function 1 CE, 6 V, function 2 CER 10–30 V, with a scan time of 0.5 s. MassLynx software (v4.1, Waters™) was used to analyse the generated spectrometric information. The tentative identification of taspine and magnoflorine and their precursors such as (S)-reticuline, (S)-corytuberine and magnoflorine methine was performed based on the analysis of accurate mass spectra and fragmentation patterns (maximum error allowed was 3 ppm) with those reported in METLIN-Scripps databases (https://metlin.scripps.edu/).