Pegasus ht tof ms

GC was performed on an HP 7890 GC (Agilent Technologies, Santa Clara, CA, USA), and the injector was used on a rail system (CTC, Gerstel, LEAP). The injection temperature was set at 250°C, and the injection type was set at split 10 : 1 mode. The other experimental parameters were set as follows: carrier gas He, flow rate 1.5 mL/min, column RTX-5 MS 30 m × 0.25 mm × 0.25 μm, and transfer line temperature (260°C).
Detection was performed on a LECO®PegasusHT® TOF-MS and controlled using a Chroma TOF (LECO, St. Joseph, MI, USA). The other experimental parameters were set as follows: acquisition delay 130 s, mass range (5–650 μ, acquisition rate 10 spectra/s, detector voltage 1650 V, electron energy 70 eV, and ion source temperature 250°C.

Gas chromatography time-of-flight mass spectrometry (GC-TOF-MS) analysis was performed as previously described by Jung et al. [50 (link)]. For analysis, all dried samples were oximated with 50 μL of methoxyamine hydrochloride (20 mg/mL in pyridine) for 90 min at 30 °C and silylated with 50 μL of N-methyl-N-(trimethylsilyl) trifluoroacetamide for 30 min at 37 °C. The derivatized samples were analyzed on an Agilent 7890A GC system (Santa Clara, CA, USA) coupled with an Agilent 7693 auto-sampler and Pegasus^® HT TOF MS (LECO Corp., St. Joseph, MI, USA). An Rtx-5MS column (30 m × 0.25 mm, 0.25-μm particle size, Restek Corp., St. Joseph, MI, USA) was used at a constant flow of 1.5 mL/min with helium used as the carrier gas. Next, 1 μL of the derivatized samples were injected into the GC in a splitless mode. The GC oven temperature was asset to 75 °C for 2 min, and then increased by 15 °C/min to 300 °C with a 3-min hold time as the final temperature. The mass data collection rate was set to 10 scans/s over a scan range of 50–1000 m/z followed by −70 eV of an electron ionization mode. The front inlet and transfer line temperatures were set to 250 °C and 240 °C, respectively.

As previously described [16 (link)] for GC/TOF MS analysis, the dried metabolites were derivatized with 5 μl of 40 mg/ml of methoxyamine hydrochloride in pyridine (Pierce, Rockford, IL) at 30°C for 90 min and 45 μl of N-methyl-N-(trimethylsilyl) trifluoroacetamide (Fluka, Buchs, Switzerland) at 37°C for 30 min. A mixture of fatty acid methyl esters (from C8 to C30) was added to the samples as internal retention index markers. GC/TOF MS analysis was conducted using an Agilent 7890B GC (Agilent Technologies, Wilmington, DE) equipped with a Pegasus HT TOF MS (Leco, St. Joseph, MI). For the separation of compounds in metabolite samples, an RTX-5Sil MS capillary column (30 m × 25 mm, 0.25 μm film thickness; Restek, Bellefonte, PA) with an additional 10-m long integrated guard column was used. Then, 1 μl of sample was injected into the GC in a splitless mode with oven temperature held at 50°C for 1 min, which was increased to 330°C at 20°C/min and held for 5 min. Mass spectra of metabolites were acquired in a mass range of 85 to 500 m/z at an acquisition rate of 17 spectra/s. The ionization mode was subjected to electron impact at 70 eV and the temperature for the ion source was set to 250°C. The transfer line was set to 250°C and 280°C, respectively.

An Agilent 7890A GC (Hewlett-Packard, Atlanta, GA) coupled to a Pegasus HT TOF MS (Leco, St. Joseph, MI) was used for the analysis of derivatized metabolite samples. The derivatized extract (1 µL) was injected into the GC in splitless mode. An RTX-5Sil MS capillary column (30 m length, 25 mm inner diameter, and 0.25 mm film thickness; Restek, Bellefonte, PA) and an additional 10-m long integrated guard column were used for GC separation. The sample was initially held at a constant temperature of 50°C for 1 min, after which it was ramped to 330°C at 20°C/min and then finally held for 5 min. The transfer line temperature was set at 280°C. Mass spectra were acquired in a scanning range of 85–500 m/z at an acquisition rate of 10 spectra/sec. The ionization mode was subjected to electron impact at 70 eV with an ion source temperature set at 250°C. GC/TOF MS data were preprocessed by Leco ChromaTOF software (version 3.34; Leco) by using automated peak detection and mass spectral deconvolution. Preprocessed MS data were processed using BinBase, an in-house programmed database for the identification of metabolites, as described previously [19] (link), [20] (link). The abundance of each identified metabolite was obtained by normalizing the peak intensity of each metabolite using the median of sums of peak intensities of all the identified metabolites in each sample [21] , [22] (link).

AH sample aliquots of 0.15 ml were transferred into cryovial tubes and stored at −80°C immediately. An Agilent 6890N gas chromatograph (Agilent Technologies) coupled to a Pegasus HT TOF MS (LECO Corp., St. Joseph, MI, USA) was used as the GC-TOF-MS platform. The sample preparation procedure and instrumental analysis were referred in the previously published methods (Qiu et al., 2014 (link)) with minor modifications, which was summarized in the Supplementary Materials.

1.5 µL of each derivatized sample was injected in (1:5) split mode into an Agilent 7890 B GC system (Agilent Technologies, Santa Clara, CA) coupled to a Pegasus HT TOF-MS (LECO Corporation, St. Joseph, MI). Separation was achieved on an Rtx-5 w/Integra-Guard capillary column (30 m × 0.25 mm ID, 0.25 µm film thickness; Restek Corporation, Bellefonte, PA) with helium as the carrier gas, at a constant flow rate of 1.0 mL/minutes. The temperatures of injection, transfer interface and ion source were 150 °C, 270 °C and 320 °C, respectively. GC temperature programming was set to 0.2 minutes of isothermal heating at 70 °C, followed by 6 °C /minutes oven temperature, ramping to 300 °C, a 4.0 minute isothermal heating of 270 °C, 20 °C/minute to 320 °C and a 2.0 minute isothermal heating of 320 °C. Electron impact ionization (70 eV) at full scan mode (40–600 m/z) was used, with an acquisition rate of 20 spectra per second in the TOF/MS setting.
Peak picking and alignments were performed using ChromaTof 4.7.2 (LECO Corporation). Mass spectra were compared to literature spectra available in the NIST database as well as the Fiehn library of compounds. A β-hydroxybutyric acid pure chemical standard was purchased (catalog #166898, Sigma-Aldrich, St. Louis, MO) for validation.

Extracts of the contents of the large intestine were prepared for metabolite profiling. The intestinal contents were extracted in 1 mL of 80% methanol by sonication for 10 min. After centrifugation (12,578 g, 4°C, 10 min), the supernatant was filtered using a 0.2-μm PTFE filter, and dried using a speed vacuum concentrator. The dried extracts were re-dissolved with 80% methanol (10mg/ml) for UPLC-Q-TOF-MS analysis under previously described analytical conditions [16 (link)]. For GC-TOF-MS analysis, dried samples were oximated with 50 μL of methoxyamine hydrochloride (20 mg/mL in pyridine) for 90 min at 30°C, and silylated with 50 μL of MSTFA for 30 min at 37°C. GC-TOF-MS analysis was performed using an Agilent 7890 gas chromatograph system (Agilent Technologies, Palo Alto, CA, USA), an Agilent 7693 auto-sampler (Agilent Technologies), and a Pegasus^® HT TOF MS (LECO, St. Joseph, MI, USA) system under previously described analytical conditions [13 ].