Ifunnel 6550 qtofms

BRB and nectar samples were randomized for run order, and QC samples were positioned after every sixth injection. The use of QC samples allows for monitoring of instrument stability over the sample set. Untargeted full-scan data was acquired using a 1290 Infinity II series UHPLC (Agilent, Santa Clara, CA) coupled to an Agilent iFunnel 6550 QTOFMS. Samples were injected (3 μL) onto a 100 × 2.1 mm Agilent SB-Aq column (1.8 μm particle size) maintained at 50 °C. The mobile phase consisted of (A) 0.1% formic acid in water and (B) 0.1% formic acid in methanol at a flow rate of 0.6 mL/min. The mobile phase composition was as follows: 0–2 min, 0% B; 2–3 min increase to 10% B; 3–8 min, increase to 40% B; 8–14 min increase to 100% B; 14–16 min, hold at 100% B; 16–18 min, immediate switch to 0% B for a total run time of 18 min. The UHPLC was interfaced with the QTOF-MS with an ESI source operated in negative ion mode. Relevant MS parameters were as follows: gas temp 150 °C, drying gas 18 L/min, nebulizer 30 psig, sheath gas temp 350 °C, sheath gas flow 12 L/min, VCap 4000 V, nozzle voltage 2000 V, acquisition mode was 2 GHz extended dynamic range with a mass range of 50–1700 m/z.

Polar liver extracts were diluted 1:5 with 0.1% formic acid in water immediately prior to analysis on a 1290 Infinity ultra-high performance liquid chromatography (UHPLC) system coupled to an iFunnel 6550 QTOF-MS (Agilent, Santa Clara, CA, USA). Sample (1 μL) was separated at 0.4 mL min⁻¹ at 40 °C using a binary gradient on an Acquity UPLC HSS C₁₈ column (100 × 2.1 mm, 1.8 μm; Waters Corp, Milford, MA, USA). The mobile phases included A) 0.1% formic acid in water and B) 0.1% formic acid in acetonitrile with the following gradient profile: 0% B for 1 min, increase to 95% B over 7.5 min, hold for 2 min, restore initial conditions over 0.5 min. Injections were randomized and a QC sample was injected every eight samples. Compounds were ionized using an electrospray ionization source and resulting ions monitored from 50–1700 m/z at a scan rate of 3 spectra per second. Separate injections were performed for detection in positive and negative modes. MS nebulizer, gas, and voltage settings have been reported previously.^{[19 ]}