7693 series autosampler

Samples and reference standards were analyzed using an Agilent 7890A GC System coupled to an Agilent 5975C inert XL MSD with triple-axis mass detector, an Agilent 7693 Series Autosampler, and a DB-5MS capillary column (30 m × 0.25 mm i.d. × 0.25 μm film thickness; Agilent Technologies, Santa Clara, CA). A volume of 1 μL of derivatized mixture was injected into the inlet heated at 280°C with 1:10 split ratio. Standard septum purge was performed after sample injection at 3 mL/min and helium carrier gas was at 1 mL/min constant flow rate. Transfer line, ion source, and quadrupole were heated at 250°C, 230°C, and 150°C, respectively. Oven was programmed initially at 70°C for 4 min, ramped up to 300ºC at 8°C/min, and then held at 300ºC for 5 min. Ionization was performed in an electron impact mode at 70 eV. Masses were scanned for full spectra from m/z 35 to 800 at 10,000 amu/s and 10.3 scans/s (m/z 0.2 step size). The solvent delay time was at 6 min.

Before analysis, VFA and NH₃ samples were thawed and filtered through four layers of cheesecloth. NH₃ concentration was determined using a portable meter (Orion meter model 290, Thermo Scientific Inc., Waltham, MA) coupled to an NH₃ ion selective electrode (Orion 9512 ammonia sensing electrode Thermo Scientific Inc., Waltham, MA) [19 (link)]. For VFA analysis, filtered samples were acidified with 25% metaphosphoric acid (w/v), centrifuged at 18,000 × g for 20 min, and the resulting supernatant was analyzed using a 7890A gas chromatography system equipped with a DB-FATWAX Ultra Inert column (30 m × 0.25 mm, 0.25 μm), a 5975C inert XL MSD with triple-axis mass detector, a 7693 series autosampler (Agilent Technologies, Santa Clara, CA, USA). Ionization was performed in an electron impact mode at 70 eV and a selected ion monitoring (SIM) mode was used to acquire ion abundance. Volatile fatty acids were quantified by an internal standard calibration with authentic volatile fatty acid standards. Isobutyric acid and isovaleric acid averaged 0.6 and 0.8 mol/100 mol across samples and were not included in further analyses.

Prior to analysis, samples for NH₃ and VFA were thawed. For NH₃ concentration, a portable meter (Orion meter model 290; Thermo Scientific Inc., Waltham, MA, USA) coupled to an NH₃ ion selective electrode (Orion 9512 ammonia sensing electrode; Thermo Scientific Inc., Waltham, MA, USA) was used. For VFA profile, 1 mL of filtered rumen fluid was mixed with 0.2 mL of an internal standard (2-mg/mL 2-ethyl butyric acid in 25% meta-phosphoric acid (w/v)). Subsequently, samples were centrifuged at 18,000×g for 20 min, and the resulting supernatant was analyzed using a 7890A gas chromatography system equipped with a DB-FATWAX Ultra Inert column (30 m × 0.25 mm, 0.25 µm), a 5975C inert XL MSD with triple-axis mass detector, and a 7693 series autosampler (Agilent Technologies, Santa Clara, CA, USA). Ionization was performed in an electron impact mode at 70 eV and a selected ion monitoring mode was used to acquire ion abundance. Volatile fatty acids were quantified by an internal standard calibration with authentic VFA standards.