Thermo uhplc q exactive hf x system

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.

The LC–MS analysis of the samples was conducted on a Thermo UHPLC-Q Exactive HF-X system (Thermo Scientific, Massachusetts, USA) equipped with an ACQUITY HSS T3 column (100 × 2.1 mm i.d., 1.8 μm) at Majorbio Bio-Pharm Technology Co. Ltd. (Shanghai, China). Mobile phase A consisted of 95% water and 5% acetonitrile (containing 0.1% formic acid), whereas mobile phase B consisted of 47.5% acetonitrile, 47.5% isopropanol, and 5% water (containing 0.1% formic acid). The flow rate was 0.40 mL/min, column temperature was 40 °C, and injection volume was 3 μL. The samples were analyzed using electrospray ionization mass spectrometry in positive and negative ion scanning modes. The scan range was 70–1050 m/z; sheath gas flow rate was 50 psi; auxiliary gas flow rate was 13 psi; auxiliary gas heating temperature was 425 °C; ion transfer tube temperature was 325 °C; spray voltage was -3.5 kV and 3.5 kV in negative and positive ion modes, respectively; and the collision energy was set to 20, 40, and 60 eV. The first-stage mass resolution was 60,000, and the second-stage mass resolution was 7,500. Data acquisition was performed in the DDA mode.

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).