An Agilent 7890B Gas Chromatograph (Hewlett-Packard, Atlanta, GA, USA) coupled with a Pegasus HT TOF mass spectrometer (Leco, St. Joseph, MI, USA) was used for identification and relative quantification of the metabolites. An aliquot of 1.0 μL of the derivatized sample was injected into the gas chromatograph in a splitless mode. An RTX-5Sil MS capillary column (30 m length, 0.25 mm inner diameter, and 0.25 μm film thickness; Restek, Bellefonte, PA, USA) with an additional 10-m-long integrated guard column was used for separation of the metabolites. Initially, the oven temperature was set at 50 °C for 1 min, and then ramped to 330 °C at a rate of 20 °C/min and held at 330 °C for 5 min. Mass spectra were recorded in a mass range of 85–500 m/z at an acquisition rate of 10 spectra/s. The temperatures of the ion source and transfer line were set to 250 °C and 280 °C, respectively. The ionization was performed on electron impact at 70 eV.
Pegasus ht tof mass spectrometer
Manufactured by Leco
Sourced in United States
The Pegasus HT TOF mass spectrometer is a high-throughput time-of-flight mass spectrometer designed for analytical applications. It features a high-speed data acquisition system and provides accurate mass measurements.
Automatically generated - may contain errors
Lab products found in correlation
Rtx 5sil ms capillary column, by Restek (2 mentions)
Chroma tof 4.3x software, by Leco (2 mentions)
Agilent 7890n, by Agilent Technologies (2 mentions)
7890 gas chromatograph system, by Agilent Technologies (1 mentions)
Rxi 5ms capillary column, by Restek (1 mentions)
7890a gas chromatograph, by Agilent Technologies (1 mentions)
Pulverisette 6, by Fritsch (1 mentions)
Db 5ms capillary column, by Agilent Technologies (1 mentions)
Agilent 7890, by Agilent Technologies (1 mentions)
N methyl n trimethylsilyl trifluoroacetamide mstfa, by Merck Group (1 mentions)
Integrated guard column, by Restek (1 mentions)
Fiehn rtx5 database, by Leco (1 mentions)
7890 gc system, by Agilent Technologies (1 mentions)
9 protocols using pegasus ht tof mass spectrometer
1
Metabolite Identification by GC-TOFMS
2
Fecal Metabolome Profiling by GC-TOF-MS
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Fecal samples were centrifuged, and supernatant (0.28 ml) was obtained and (28 (link)) analyzed by gas chromatography/time-of-flight mass spectrometry (GC-TOF-MS) using a 7890 Gas Chromatograph System (Agilent Technologies, Santa Clara, CA, USA) coupled with a Pegasus™ HT TOF Mass Spectrometer (LECO, Saint Joseph, MI, USA). Chroma TOF 4.3x software (LECO) and LECO-Fiehn Rtx5 database were used for extracting raw peaks, filtering, calibrating baselines, aligning peaks, performing deconvolution analysis, identifying peaks, and integrating peak area (29 (link)). Retention time index (RI) was used for peak identification, with an RI tolerance of 5,000. Metabolic features detected in <50% of quality control (QC) samples were removed (30 (link)). The identified differential metabolites were further validated by searching in the Kyoto Encyclopedia of Genes and Genomes (KEGG). Principal component analysis (PCA), enrichment analysis for the differential metabolites, and construction of random forest models were performed on the online platform MetaboAnalyst 4.0 (31 (link)).
3
Plasma Metabolite Analysis by GC-TOFMS
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Metabolites extracted from plasma samples were analyzed using an Agilent 7890N gas chromatograph coupled with a Pegasus HT TOF mass spectrometer (Leco Corporation). Briefly, a 1 μL aliquot of the derivatized solution was injected with the splitless mode. Rxi-5 ms capillary column (30 m × 250 μm I.D., 0.25-μm film thickness; Restek Corporation, Bellefonte, PA, USA) was used for metabolites separation, with helium as the carrier gas at a constant flow rate of 1.0 mL/min. The temperature settings for injection, transfer interface, and ion source were 260, 260, and 210 °C, respectively. The separation was achieved with the following GC temperature program: 80 °C for 2 min, 10 °C/min to 220 °C, 5 °C/min to 240 °C, and 25 °C/min to 290 °C, and kept at 290 °C for 8 min. The data was collected with full scan mode (m/z 40–600), and an acquisition rate of 20 spectra/s. Electron impact ionization (70 eV) was used.
4
Analysis of Vitamin E Compounds
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Genes that encoded for vitamin E-related proteins were screened by BLAST using 31,177 genes in the NCBI non-redundant database. A heat map was generated using DNAStar's ArrayStar 4 (http://www.dnastar.com ). Plant samples were lyophilized at -80°C for 4 days and pulverized into a very fine powder using a planetary mono mill (Pulverisette 6; Fritsch, Germany). Tocopherols and tocotrienols were identified by gas chromatography-time-of-flight mass spectrometry according to a previously described method [58 (link)]. Lipophilic compounds were extracted from 0.1 g of samples by adding 3 ml of ethanol containing 0.1% ascorbic acid (w/v); 0.05 ml of 5 α-cholestane (10 μg/mL) was used as an internal standard (IS). The extracts were lyophilized and then derivatized with 30 μl N-methyl-N-trimethylsilyltrifluoro-actamide (Sigma, USA) and 30 μl pyridine. The derivatized extracts were analyzed by a 7890A gas chromatograph (Agilent, USA) with a Pegasus HT TOF mass spectrometer (LECO, USA). Tocopherols and tocotrienols were quantified using calibration curves that plotted five concentrations of the commercial standards ranging from 0.01 to 10.0 μg and a fixed amount (0.5 μg each) of IS.
5
GC-MS Analysis of Volatile Compounds
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The GC–MS analysis was performed using an Agilent 7890 gas chromatograph system (Agilent Technologies, Palo Alto, CA, USA) coupled with a Pegasus HT TOF mass spectrometer (LECO Corporation, St. Joseph, MI, USA). The system utilized a DB-5MS capillary column (30 μm × 250 μm inner diameter, 0.25 μm film thickness; J&W Scientific, Folsom, CA, USA) coated with 5% diphenyl cross-linked with 95% dimethylpolysiloxane. Helium was used as the carrier gas with a flow rate of 1 mL/min, and the injection volume was 1 μL without a split. The initial temperature was kept at 50 °C for 1 min, then raised to 300 °C at a rate of 10 °C/min, and maintained for 9 min at 300 °C. The injection, transfer line, and ion source temperatures were 280, 270, and 220 °C, respectively. The energy was − 70 eV in electron impact mode. The mass spectrometry data were acquired in a full-scan mode with the m/z range of 50–500 at a rate of 20 spectra per second after a solvent delay of 460 s.
6
Metabolite Derivatization and GC-TOF Analysis
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Before GC-time of flight (GC-TOF) analysis, metabolites were derivatized by adding 10 μL of methoxyamine hydrochloride in pyridine (40 mg/mL) to each dried sample followed by shaking at 30 °C for 90 min. A volume of 90 μL of N-methyl-N-(trimethylsilyl) trifluoroacetamide (MSTFA, Sigma-Aldrich, USA) was added for trimethylsilylation followed by the addition of C8–C30 fatty acid methyl esters (FAMEs, Sigma-Aldrich, USA) for retention time correction. Samples were shaken at 37 °C for 30 min. Derivatized samples were analyzed using Agilent 7890A GC coupled to a Leco Pegasus HT TOF mass spectrometer applying the same chromatographic and MS parameters previously described [25 ].
7
GC-TOF-MS Metabolome Analysis
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Metabolites extracted from plasma samples were analyzed using an Agilent 7890N gas chromatograph coupled with a Pegasus HT TOF mass spectrometer (Leco Corporation, St. Joseph, MI, USA). Electron impact ionization (70 eV) at full scan mode (m/z 40–600) was used, with an acquisition rate of 20 spectra/s in the TOF-MS setting. Data generated by GC-TOF-MS were semiquantitative data and are expressed in peak intensity. For details, see Supplementary Experimental Procedures .
8
Metabolomic Analysis Using GC-TOF-MS
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For metabolite analysis, an Agilent 7890B BC system (Agilent Technologies, Santa Clara, CA, USA) equipped with a Pegasus HT TOF mass spectrometer (LECO, St. Joseph, MI, USA) was used. An RTX-5Sil MS capillary column (30 m × 0.25 mm, 0.25 μm film thickness; Restek, Bellefonte, PA, USA), with an additional integrated guard column (10 m × 0.25 mm, 0.25 μm film thickness; Restek, Bellefonte, PA, USA) was injected with 1 µL of derivatives and subjected to an initial temperature of 50 °C with holding for 1 min, and then increased to 330 °C at 20 °C/min and held for 5 min. The injection temperature was 250 °C and the interface temperature was 280 °C. In the mass range of 85–500 m/z, mass spectra were collected by electron impact at 70 eV.
9
GC-TOF MS Metabolite Extraction Protocol
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The GC quadrupole TOF MS analysis and metabolites extraction were similar to a previous study (Zhang et al., 2019b) . Briefly, 450 μL of methanol/chloroform (volumetric ratio = 3:1) was added to 50 mg samples from PREP and POSP to extract metabolites. Equal aliquots of extract liquid from all experimental samples were pooled as quality control (QC) specimens. Adonitol was utilized as an internal standard. To perform the following GC TOF MS analysis of all samples, an Agilent 7890 GC system was used along with a Pegasus HT TOF mass spectrometer in splitless mode (LECO Corporation). For 1 min, the initial temperatures were maintained at 50°C, then incremented to 310°C at a rate of 10°C min -1 and kept at 310°C for 8 min. The ion source, injection, and transfer line temperatures were 250, 280, and 280°C, respectively. The MS data were obtained in a full-scan mode after a solvent delay of 6.33 min with the m/z range of 50-500 at a rate of 12.5 spectra per second. The Chroma TOF 4.3X software built-in with the LECO-Fiehn Rtx5 database (LECO Corporation) was used to preprocess and annotate the metabolomics data. The peaks detected less than 50% of QC specimens or relative standard metabolomics data deviation of more than 30% in QC specimens were eliminated (Dunn et al., 2011) .
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