Ion max

The analysis was carried out using a Thermo Scientific HRAM LC-MS system (Thermo Scientific, MA, USA) consisting of an “Accela” liquid chromatograph with diode array detector and an “LTQ-Orbitrap XL” hybrid linear ion trap-orbitrap mass spectrometer. Chromatography was performed on a 150 × 3 mm, 3 μm Phenomenex Luna C18 column using a 400 μL/min flow rate. The separation was obtained using the following linear mobile phase gradient: 0 min: 90% A, 0% B, 10% C; 50 min: 0% A, 90% B, 10% C, 55 min: 0% A, 90% B, 10% C, 57 min: 90% A, 0% B, 10% C, 60 min 90% A, 0% B, 10% C (A: water, B: methanol, C: acetontirile containing 1% formic acid). The injection volume was 5 μL. Ammonia solution was added post column at a flow rate of 0.1 μL/min. The mass spectrometer was fitted with an electrospray source (Thermo “Ion Max”) operated in positive mode at a source voltage of 3.5 kV using sheath and auxiliary nitrogen flow rates of 60 and 20 units, respectively, and a capillary temperature of 300°C. High resolution MS1 scanning was performed in the orbitrap (m/z 250–2000; resolution 30,000). Low resolution data dependent MS2 scans were undertaken in the linear ion trap (isolation width 4 m/z units; collision energy 35%).

LC-MS analysis was performed using similar but not identical UHPLC systems coupled to Orbitrap-based mass analysers operated with the Thermo Scientific™ Ion Max™ source equipped with the HESI-II-probe (see Electronic Supplementary Material (ESM) Table S1). All data were acquired using Thermo Scientific™ Chromeleon™ Chromatography Data System (CDS) software 7.2.9 (Thermo Fisher Scientific).

A quality control sample (QC) taken from all samples was prepared in order to periodically assess the reproducibility of the measurements. The separation of the analytes contained in the saffron samples was achieved with a Fortis UPLC C₁₈ column (2.1 mm × 100 mm, 1.7 µm, Fortis Technologies Ltd., Cheshire, UK). The hyphenated LC-HRMS system comprised of an Accela UHPLC equipped with an autosampler, a vacuum degasser, a binary pump and a temperature-controlled column (Thermo Scientific, Germany) coupled to an Orbitrap Discovery XL, which was equipped with an IonMAX ion source (Thermo Scientific, Bremen, Germany). The mobile phase consisted of 0.1% aq. formic acid (v/v) (solvent A) and 0.1% formic acid in LC–MS grade ACN (v/v) (solvent B). The gradient program was for solvent B: 5% at 0 min, 5% at 3 min, 95% at 24 min, 95% at 26 min, 5% at 28 min, 5% at 30 min. The overall analysis time spanned for 30 min, whereas the injection volume was 5 µl keeping a flow rate of 400 µl min⁻¹. The positive ionization ESI mode was used using a mass range of 100–1000 amu. The “big three” approach, employing parallel scans, was used. The samples were centrifuged using a Mikro 200R centrifuge (Hettich Lab Technology, Tuttlingen, Germany), and for the solvent evaporation was performed on a GeneVac HT-4X EZ-2 series evaporator Lyospeed ENABLED (Genevac Ltd., Ipswich, UK).

Extracts of M. edulis (50 mg ml^-1, in methanol for 24 h) were analyzed by LC-ESIMS using an LTQ Orbitrap XL, linear ion trap/orbitrap hybrid mass spectrometer (Thermo Scientific, San Jose, CA, United States) with an electrospray ionization source (Ion Max, Thermo Scientific) coupled to an Acella 1250 uPLC system (Thermo Scientific). Samples were injected onto a Phenomenex Luna C₁₈(2) column (150 × 3 mm i.d., 3 μm particle size) and eluted at 400 μL min^-1 using a linear gradient as described previously (Green et al., 2017 (link)) and compounds eluted using MeOH (A), H₂O (B), and formic Acid (C) with A = 0%, B = 90% at T = 0 min; A = 90%, B = 0% at T = 20 min and held for 10 min with C at 10% throughout the analyses. Column temperature was 30°C with flow rate = 0.5 ml min^-1. Samples were scanned, using FTMS, from m/z 125–1250 in both positive and negative modes.