Agilent 7890a gc system

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.

GC–TOF–MS analysis was performed using an Agilent 7890A GC system (Agilent Technologies, Palo Alto, CA, USA) equipped with an L-PAL3 autosampler and Pegasus^® HT TOF-MS system (LECO Corp., St. Joseph, MI, USA). Metabolites were separated using an RTX-5MS column (30 m length × 0.25 mm inner diameter × 0.25 μm particle size, Restek Corp., St. Joseph, MI, USA) with a constant flow of helium (1.5 mL) as the carrier gas. 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 (1 μL) were injected into the GC column in splitless mode. The analytical program for sample analysis was adopted from our previous study [22 (link)]. Moreover, metabolite analysis was performed in a random manner to reduce bias and systematic errors.

The derivatized samples were analyzed with an Agilent 7890A GC system (Agilent, Santa Clara, CA, USA) coupled to a quadrupole TOF mass spectrometer, G2-S QTOF (Waters Corporation, Manchester, UK), operating in APGC mode. The GC separation was performed using a fused silica DB5-MS capillary column (30m x 250μm I.D., 0.25μm film thickness; J&W Scientific, Folson, CA, USA). The initial GC oven temperature was 70°C. One min after injection, the GC oven temperature was increased with 15°C/min to 320°C and held for 4 min at 320°C. Splitless injections of 1 μL using a straight empty deactivated liner from Restek were carried out at 240°C. Helium was used as carrier gas at 2.0 mL/min. The interface temperature was set to 250°C using N₂ as auxiliary gas at 400 L/h, and cone gas at 150 L/h. The APCI corona pin was operated at 3.0 μA. The APGC source was operated in proton transfer mode by placing an uncapped vial with water (modifier) in a specially designed holder located in the source door (REF). The QTOF detection was operated in full-scan mode (m/z 50–650) with the resolution at ~20,000.

After separating the aliquot used for the DNA extraction, the tubes were centrifuged for 30 min at 4500 × g at 4°C. The supernatants were transferred into new tubes and centrifuged again at 4500 × g and 4°C for 15 min. Supernatants from faecal incubations were sterilised by filtration using a pore size of 0.22 μmØ.
Acetate, propionate, and butyrate were analysed using a gas chromatograph (Agilent 7890A GC system, Agilent Technologies, Santa Clara, CA, United States) equipped with a fuse-silica capillary column (DB-FFAP, 30 m × 0.32 mm × 0.5 μm) and a flame ionization detector (FID). The analysis was performed at Research Technical Services from UdG (Girona, Spain). Crotonic acid was used as internal standard. 0.5 μL of each sample was injected in split mode at 275°C. The analyses were performed using the following temperature programme: 3 min at 40°C, 5°C min^–1 to 70°C, 7°C min^–1 to 120°C, 10°C min^–1 to 180°C, and 35°C min^–1 to 250°C, hold for 5 min. All the analysed compounds were previously identified and calibrated by using a bench of standard solutions. The standard solutions were prepared by diluting a stock solution of the primary compounds.

The analysis was performed by GC/MS/MS: an Agilent 7890A GC system (Agilent Technologies, Palo alto, CA, USA) was equipped with an Agilent 7693 autosampler and was coupled to a triple quadrupole mass spectrometer 7000B (Agilent Technologies) and operated in electron ionization mode (EI −70 eV). Splitless injection of a 2-μL sample was separated by an HP-5 MS capillary column ((5%-phenyl)-methylpolysiloxane; 30 m × 0.25 mm ID and film thickness of 0.25 μm; Agilent Technologies). The oven temperature was programmed as follows: 70 °C (2 min hold) to 150 °C at a rate 25 °C/min⁻¹, increased to 200 °C at 3 °C/ min⁻¹, and finally to 280 °C at 8 °C/min⁻¹ and held for 10 min. Helium (99.9998% purity) was used as the carrier gas at a constant flow rate of 2.1 mL/min⁻¹. The total running time was 41.88 min. The temperatures of the transfer line, the ion source, first quadrupole, and second quadrupole were 280, 300, 180, and 180 °C, respectively. Helium (99.9998% purity) and nitrogen (99.9998% purity) were collision gases at a flow rate of 2.25 and 1.5 mL/min⁻¹, respectively. MassHunter quantitative analysis software (version B.06.00) (Agilent Technologies) was used for data processing. MRM transitions and other acquisition parameters can be found in Table S1.

Metabolites extracted from samples were analyzed as previously described with minor modifications (Tan et al. 2015 (link)). Briefly, the extracted metabolites were analyzed using an Agilent 7890A GC System coupled to an Agilent 7890A-5975C Inert XL EI/CI Mass Spectrometric Detector (MSD) System (Agilent Technologies, Santa Clara, CA, USA). Gas chromatography was performed on a DB-5 Capillary Column (30 m × 0.25 mm × 0.25 µm, Agilent J & W Scientific, Folsom, CA, USA) to separate the derivatives. The injection temperature was 280 °C, the interface was set to 150 °C, and the ion source was adjusted to 230 °C. The temperature gradient program was as follows: initial temperature of 60 °C for 1 min, +10 °C/min up to 300 °C, and a hold at 300 °C for 15 min. Mass spectrometry was determined by the full-scan method ranging from 33 to 600 (m/z). A series of n-alkanes (C7–C40) and blank control samples was analyzed along with samples to calculate retention indices (Schauer et al. 2005 (link)).