Reprosil c18 column

An Ultimate 3000 Rapid Resolution HPLC system (LC Packings, DIONEX, Sunnyvale, USA) was used to perform metabolite separation. The system featured a binary pump and vacuum degasser, well-plate autosampler with a six-port micro-switching valve, a thermostated column compartment. Samples were loaded onto a Reprosil C18 column (2.0 mm × 150 mm, 2.5 µm - Dr Maisch, Germany) for metabolite separation. For lipids multi-step gradient program was used. It started with 8% solvent A (ddH20, 20 mmol L-1 ammonium formiate; pH 5) to 6% solvent A for 3 min than to 2% solvent A for 35 min and finally to 100% solvent B (methanol) in 30 min. At the end of gradient, the column was reconditioned with 8% solvent A for 5 min.

Flies were collected in screw cap tubes and weighed in groups of 10 using an ultramicro balance (with a high resolution of up to 0.0001 mg). For glucose measurements, samples were analyzed in agreement with previous reports [22 (link)]. Briefly, metabolite extraction was performed on fly samples by water/methanol/chloroform mixture (Bligh-Dyer method). Following mixing and centrifugation, fractions were transferred to Eppendorf tubes and then dried at 4 °C before resuspension in ultrapure water and analysis by LC-MS. Chromatographic separation was carried out on a Reprosil C18 column (2.0 mm × 150 mm, 2.5 μm—Dr. Maisch, Ammerbuch-Entringen, Germany) with a 0–100% linear gradient of solvent A (double-distilled 18 mΩ water, 10 mm ammonium acetate) to B (100% acetonitrile, 10 mm ammonium acetate) at a flow rate of 0.2 mL/min. Column flow was directed into the mass spectrometer (Q Exactive ThermoFisher Scientific, Monza Italy) operating in negative ion mode and scanning in full MS mode (2 μscans) at 70,000 resolution from 60 to 1000 m/z. Glucose concentrations were calculated via a six-point standard curve and related to the corresponding flyweights.

Twenty μL of extracted supernatant samples was injected into an ultra-high-performance liquid chromatography (UHPLC) system (Ultimate 3000, Thermo) and run on a positive mode: samples were loaded on to a Reprosil C18 column (2.0 mm× 150 mm, 2.5 μm—Dr Maisch, Germany) for metabolite separation. Chromatographic separations were made at a column temperature of 30 °C and a flow rate of 0.2 mL/min. For positive ion mode (+) MS analyses, a 0–100% linear gradient of solvent A (ddH2O, 0.1% formic acid) to B (Acetonitrile, 0.1%formic acid) was employed over 20 min, returning to 100% A in 2 min and holding solvent A for a 1-min post time hold. Acetonitrile, formic acid, and HPLC-grade water and standards (≥98% chemical purity) were purchased from Sigma Aldrich. The UHPLC system was coupled online with a Q Exactive mass spectrometer (Thermo) scanning in full MS mode (2 μscans) at resolution of 70,000 in the 67 to 1000 m/z range, a target of 1106 ions and a maximum ion injection time (IT) of 35ms with 3.8 kV spray voltage, 40 sheath gas, and 25 auxiliary gas. The system was operated in positive ion mode. Calibration was performed before each analysis against positive or negative ion mode calibration mixes (Pierce, Thermo Fisher, Rockford, IL) to ensure error of the intact mass within the sub ppm range.