Uhplc q exactive hf x mass spectrometer

Chromatographic separation of the metabolites was conducted via a Thermo UHPLC system with an ACQUITY UPLC HSS T3 column (100 mm × 2.1 mm i.d., 1.8 µm; Waters, Milford, CT, USA). The following chromatographic conditions were used: the injection volume was 2 μL, the column temperature was 40 °C, the mobile phase A composition was 95.5% water and 5% acetonitrile (including 0.1% formic acid), and the mobile phase B composition was 47.5% acetonitrile, 47.5% isopropanol, and 5% water (including 0.1% formic acid). The mass spectrometric data were collected using a Thermo UHPLC-Q Exactive HF-X mass spectrometer with an ESI source running in either positive or negative ion mode. The ideal conditions were as follows: normalized collision energy, 20–40–60 V rolling for MS/MS; heater temperature, 425 °C; capillary temperature, 325 °C; sheath gas flow rate, 50 arb; aux gas flow rate, 13 arb; and ion-spray voltage floating (ISVF), −3500 V in negative mode and 3500 V in positive mode. The resolution of the MS/MS scan was 7500, while the resolution of the entire MS scan was 60,000. The data-dependent acquisition mode was used to record the data. A mass range of 70–1050 m/z was employed for identification.

To compare the metabolites of the cell-free supernatant, a non-targeted metabonomic was performed. The sample preparation referred to our previous study [13 (link)]. LC-MS/MS determination was performed by Thermo UHPLC-Q Exactive HF-X Mass Spectrometer. After separation by an HSS T3 column, the samples were subjected to MS/MS detection for compound identification. Q Exactive HF-X mass spectrometer was operated as follows: −3500 V in negative mode and 3500 V in positive mode, capillary temperature at 325 °C, heater temperature at 425 °C, MS resolution at 60,000, MS/MS resolution at 75,000. Mass range was set to 70–1050. After determination, Metlin (https://metlin.scripps.edu/, accessed on 23 May 2023) and HMDB (http://www.hmdb.ca/, accessed on 3 June 2023) were used to identify the metabolites. The data were analyzed by Majorbio cloud platform (https://cloud.majorbio.com, accessed on 4 June 2023). Detailed information about the data filtered, data normalized, and data analyzed can be found in our previous study [13 (link)].

The mass spectrometric data were collected using a Thermo UHPLC -Q Exactive HF-X Mass Spectrometer, equipped with an electrospray ionization (ESI) source operating in either positive or negative ion modes. The optimal conditions were set as followed: heater temperature, 425°C; capillary temperature, 325°C; sheath gas flow rate, 50 arb; aux gas flow rate, 13 arb; ion-spray voltage floating (ISVF), −3,500 V in negative mode, and 3,500 V in positive mode, respectively. Normalized collision energy and 20-40-60V rolling were employed for MS/MS. Full MS resolution was 60,000, and MS/MS resolution was 7,500. Data acquisition was performed using the Data Dependent Acquisition (DDA) mode. The detection was carried out over a mass range of 70–1,050 m/z.