Thermo scientific q exactive orbitrap mass spectrometer

A 10-μL aliquot of each sample (prepared as described above) was auto-injected into a Thermo Scientific Ultimate 3000 UHPLC system (Thermo Fisher Scientific, Waltham, MA, USA). Chromatographic separation was achieved using an Agilent Zorbax C18 column (1.8 μm, 2.1 × 150 mm; Agilent). The flow phases used were (A) aqueous 0.1% (v/v) formic acid and (B) acetonitrile 0.1% (v/v) formic acid. The chromatographic conditions were as follows: 0–1 min, 2% B; 1–5 min, 2%–15% B; 5–13 min, 15%–70% B; 13–25 min, 70%–98% B; 25–30 min, 98% B; 30–30.2 min, 98%–2% B; 30.2–35 min, 2% B. High resolution accurate mass data was acquired both in positive and negative ion modes using a Thermo Scientific Q-Exactive Orbitrap mass spectrometer (Thermo Fisher Scientific) operated at a resolution of 70,000. The voltage of the electrospray source was set to 3.5 kV in the positive mode (ESI+) and 2.8 kV in the negative mode (ESI−). Mass spectra were recorded from m/z 100 to m/z 1000. The raw data was analyzed using the Component Extraction algorithm in SIEVE 2.0 software (Thermo Fisher Scientific) to detect the metabolites. The intensity threshold was set to 3,000,000, and four databases were chosen to identify the metabolites: the BioCyc Pathway, KEGG, Lipid Maps Databases, and NIST. The MZ tolerance was set to 5 ppm for the database search.

Ten microliters of each sample was injected into a Thermo Fisher Scientific Ultimate 3000 system (Thermo Fisher Scientific, Waltham, MA, USA) equipped with an Agilent Zorbax C18 column (3 µm, 2.1 × 150 mm, Agilent). The ionization source parameters were set as follows: positive mode; capillary temperature, 250 °C; and spray voltage, 2.3 kV. The flow phases used were A: 10 mM tributylamine plus 15 mM acetic acid in 97:3 water:methanol, and B: methanol. The gradient used was 99% A:1% B for 2.5 min; 80% A:20% B for 7.0 min; 35% A:65% B for 7.5 min; 5% A:95% B, for 9.01 min; and 99% A:1% B for 10 min. High-resolution accurate mass data were acquired in positive mode using a Thermo Scientific Q-Exactive Orbitrap mass spectrometer (Thermo Fisher Scientific) operated at a resolution of 70,000. The voltage of the electrospray source was set to 3.5 kV in the positive mode. Full scan MS spectra were acquired in a mass range from m/z 100 to 1000. The raw data were analyzed using the Component Extraction algorithm in SIEVE 2.0 software (Thermo Fisher Scientific) to detect the metabolites. The intensity threshold was set to 3,000,000, and three databases were chosen to identify the metabolites: Human Metabolome Database (HMDB), Metlin Metabolite Database, and Kyoto Encyclopedia of Genes and Genomes (KEGG). The m/z tolerance was set to 5 ppm for the database search.