Sequant zic philic 5 m 2.1 mm 100 mm column

Polar metabolites were analysed with an Accela 1250 quaternary UHPLC pump coupled to an Exactive Orbitrap mass spectrometry (Thermo Fisher Scientific, USA). Chromatographic separation was carried out at 25°C on a SeQuant^® ZIC^®-pHILIC 5 µm 2.1 mm × 100 mm column (Merck, Darmstadt, Germany) using the following solvent system: A = 10 mM ammonium formate in milliQ water, B = 0.1% formic acid in acetonitrile at a gradient program flow rate of 250 µL/min: 3–3% A (0.0–1.0 min), 3–30% A (1.0–12.0 min), 30–90% A (12.0– 14.5 min). 90% A was maintained for 3.5 min followed by re-equilibration with 3% A for 7 min. An injection volume of 2 µL was used. The electrospray probe was operated at room temperature (25°C) to avoid degradation of thermally labile compounds. External mass calibration of the Orbitrap prior to sample analysis was performed by the flow injection of the calibration mix solution according to manufacturer’s instruction. High resolution (25,000) data were acquired by full scan (m/z 55 to 1100) with a source voltage of 4000 V for both ESI + and ESI - ion modes. A capillary temperature of 325°C was set, and sheath, auxiliary, and sweep gas flow rates of 40, 10, and five arbitrary units were applied, respectively (42 ).

Polar metabolites were analysed with an Accela 1250 quaternary UHPLC pump coupled to an Exactive Orbitrap mass spectrometry (Thermo Fisher Scientific, USA). Chromatographic separation was carried out at 25 °C on a SeQuant® ZIC®-pHILIC 5 µm, 2.1 mm × 100 mm column (Merck, Darmstadt, Germany) with the following solvent system: A = 10 mM ammonium formate in water, B = 0.1% formic acid in acetonitrile. A gradient program was used at a flow rate of 250 µL/min: 3–3% A (0.0–1.0 min), 3–30% A (1.0–12.0 min), 30–90% A (12.0–14.5 min), 90% A was maintained for 3.5 min followed by re-equilibration with 3% A for 7 min. An injection volume of 2 µL was used. The electrospray probe was operated unheated at room temperature (20 °C) to avoid degradation of thermally labile compounds. External mass calibration of the Orbitrap prior to sample analysis was performed by flow injection of the calibration mix solution according to the manufacturer’s instruction. High resolution data (resolution 25,000) were acquired by full scan from m/z 55 to 1100 with source voltage of 4000 V for ESI + and − 4000 V for ESI − , capillary temperature of 325 °C, and sheath, auxiliary, and sweep gas flow rates of 40, 10, and five arbitrary units, respectively.