Thermo uhplc q exactive hf x mass spectrometer

A bone sample was ground with a grinding bead. A 400-μL extraction solution (methanol: water = 4:1 (v:v)) and an internal standard (L-2-chlorophenylalanine) were applied for metabolite extraction. Samples were ground by the Wonbio-96c frozen tissue grinder for 6 min (−10°C, 50 Hz), followed by low-temperature ultrasonic extraction for 30 min (5°C, 40 kHz). The bone samples were stored at −20°C for 30 min and then centrifuged for 15 min (4°C, 13,000 g). The supernatant was collected for LC-MS/MS analysis by a Thermo UHPLC Q Exactive HF-X system equipped with an ACQUITY HSS T3 column (100 mm × 2.1 mm i.d., 1.8 μm; Waters, USA). The mass spectrometric data were collected by a Thermo UHPLC Q Exactive HF-X Mass Spectrometer equipped with an electrospray ionization (ESI) source operating in positive mode and negative mode. The optimal conditions were set as follows: source temperature at 425°C; sheath gas flow rate at 50 arb; Aux gas flow rate at 13 arb; ion-spray voltage floating (ISVF) at −3,500 V in negative mode and 3,500 V in positive mode, respectively; normalized collision energy, 20–40–60 V rolling for MS/MS. Full MS resolution was 60,000, and MS/MS resolution was 7,500. Data acquisition was performed with the data-dependent acquisition (DDA) mode. The detection was carried out over a mass range of 70–1,050 m/z.

Metabolite of 0 h fermentation groups (CON-0, GLP-0, LEP-0, CMP-0, and PCP-0) and 8 h fermentation groups (CON-8, GLP-8, LEP-8, CMP-8, and PCP-8) were analyzed by non-targeted metabolomic analysis. The LC-MS/MS analysis of the sample was conducted on a Thermo UHPLC-Q Exactive HF-X system equipped with an ACQUITYHSS T3 column (100 mm × 2.1 mm i.d., 1.8 μm; Waters, Milford, MA, USA) at Majorbio Bio-Pharm Technology Co., Ltd. (Shanghai, China). 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 positive mode and negative mode. Data acquisition was performed using the Data Dependent Acquisition (DDA) mode. The detection was carried out over a mass range of 70–1050 m/z. Multivariate statistical analysis was performed using the ropls (Version 1.6.2) R package of Bioconductor on the Majorbio cloud platform (https://cloud.majorbio.com, accessed on 1 March 2023).

The LC/MS analyzing method was applied in our present study for the measurement of blood metabolic responses to Lactobacillus supplement under consecutive H₂S exposure treatment. To be simply stated, the internal standard was firstly prepared by dissolving 100 µL blood with 400 µL mixed liquor, which consisted of 80% methanol solution and 0.02 mg/mL L-2-chlorophenyl alanine. All samples were subsequently cleaned using ultrasound at 40 kHz and 5°C for 30 min, centrifuged with 13,000 g at 4°C for 15 min, and carefully transferred to sample vials for LC-MS/MS analysis. Chromatographic separation of the metabolites was conducted using the Thermo UHPLC system which was equipped with an ACQUITY UPLC HSS T3 (100 mm × 2.1 mm i.d., 1.8 µm; Waters, Milford, CT), followed by the mass spectrometric data collection by Thermo UHPLC-Q Exactive HF-X Mass Spectrometer equipped with electrospray ionization (ESI) source in either positive or negative ion mode.