Polarisq mass spectrometer

The separation and identification of volatile organic compounds (VOCs), from the analyzed oil samples, were performed by using a Trace GC Ultra Gas Chromatograph from Thermo Scientific (Thermo Fisher Scientific Inc., Bremen, Germany) coupled with Thermo Electron Polaris Q Mass Spectrometer, equipped with an electron ionization source operating at 70 eV. For the chromatographic separation of the target compounds, a DB-5MS (30 m × 0.25 mm ID, 0.25 μm film thickness) capillary column (5% diphenyl, 95% dimethylpolysiloxane) was used. The injection volume of each diluted oil sample was 2 μL. The oven temperature program was 40 °C for 3 min, which was then increased from 40 to 300 °C with 10 °C min⁻¹, and kept at 300 °C for 10 min. The injector and transfer line were 250 °C and 300 °C, respectively. The carrier gas was helium with a flow rate of 1.5 mL min⁻¹. The constituents of the samples were identified and confirmed based on two different approaches: linear retention indices (LRIs) determined relative to a series of n-alkanes (C8–C20) and by comparison of the mass spectra with those available in the commercial libraries (NIST 2011) [59 ].

Dry matter content was determined according to the National Forage Testing Association method 2.1.4 [19 ]. Neutral detergent fiber (NDF) was determined as described by Van Soest et al. [20 (link)] using heat-stable α-amylase (FAA, Ankom Technology, Macedon, NY) and sodium sulfite. Acid detergent fiber (ADF) and lignin concentrations were determined according to AOAC method 973.18 [21 ]. Ash content was determined according to AOAC method 942.05 [21 ]. Hemicellulose was calculated as the difference between NDF and ADF. Cellulose was calculated by subtracting ash and lignin from ADF.
For measurement of ruminal VFA concentrations, rumen fluid samples were thawed and composited by day, animal, and period (n = 6). The samples were centrifuged for 30 min at 2500×g at room temperature to remove solid particles, and supernatant liquid was collected. An external tracer consisting of a mix of ¹³C-labelled acetate, propionate, and butyrate was added to each liquid sample, then rumen liquid samples were derivatized, and the derivatives were analyzed for isotopic ratio using a Thermo Electron Polaris Q mass spectrometer in tandem with a Thermo Electron Focus gas chromatography (GC-MS; Thermo Electron Corporation, Austin, TX) as described by Kristensen [22 (link)].

The SCFA analyzed were acetic (C2), propionic (C3), isobutyric (iC4), butyric (C4), isovaleric (iC5), valeric (C5) and hexanoic acids (C6). The SCFA content of the feces and chyme from the caecum was measured by SPME-GC-MS following a protocol previously described [31 (link)]. Between 20 and 25 mg of samples were introduced into a 20 mL glass vial. Forty µL of internal standard (2-methylvaleric acid) at a concentration of 0.2 mg/mL, 15 µL of 0.9 M sulfuric acid, and 920 µL of water were then added. The mixture was vortexed and placed on the autosampler of the SPME-GC-MS system until analysis. SCFA were extracted with a SPME fiber, separated on a Focus GC gas chromatograph (Thermo Fisher Scientific, Waltham, MA, USA) using a Supelcowax-10 column (30 m × 0.25 mm, 0.2 µm) (Supelco, Bellefonte, PA, USA) and analyzed with an ion trap PolarisQ mass spectrometer (Thermo Fisher Scientific, Waltham, MA, USA). The agitation temperature was set at 60 °C and the extraction time at 20 min.

Quantification of arachidonic acid release from cellular membranes was performed as previously described [31 (link), 32 (link)]. Briefly, freshly isolated PMNL (10⁷ cells/ml, in HBSS containing 1.6 mM CaCl₂ and 0.1% BSA) were incubated with adenosine deaminase (0.3 U/ml) and test compounds for 5 min at 37°C. Stimulation was initiated with the addition of 1 μM thapsigargin, followed by incubation at 37°C for 5 min. The reactions were stopped with the addition of two volumes of cold MeOH and 300 ng of arachidonic acid-d₈ (Cayman Chemicals) as internal standard. The samples were stored at −20°C overnight and then centrifuged the next day at 1000 g for 10 min. The supernatants were diluted with four volumes of acidified water (0.1%) then processed on an octadecyl (C18) column. Samples were eluted with the addition of 3 ml of MeOH and dried under nitrogen. Pentafluorobenzylesters were prepared with the addition 50 μl N,N-diisopropylethylamine (20% in acetonitrile) and 50 μl of 2,3,4,5,6-pentafluorobenzylesters (20% in acetonitrile). Samples were heated for 40 min at 40°C, then dried under nitrogen and resuspended in 100 μl of hexane. Samples were quantified by negative ion chemical ionization gas chromatography/mass spectrometry using TraceGC ultra column (Thermo) and a Polaris Q mass spectrometer (Thermo).

The FFAs analysis in the obtained samples was performed at Focus GC coupled with a PolarisQ mass spectrometer (Thermo Fisher, Waltham, MA, USA). The carrier gas was helium (1 mL/min) and the injected volume of the sample was 1 µL. The temperature program of the oven was as follows: the initial temperature 50 °C (1 min), then 25 °C/min to 200 °C and, immediately afterwards, 3 °C/min to 230 °C (held for 18 min). The injector was in split mode (50:1), while the temperatures of the injector, transfer line and ion source were 250 °C, 260 °C and 260 °C, respectively. The concentration of 18 fatty acids was investigate: FFAs abbreviations: C14:0—myristic; C15:0—pentadecylic; C16:0—palmitic; C16:1—palmitoleic; C17:0—margaric; C17:1—heptadecenoic; C18:0—stearic; C18:1c—oleic; C18:1t—elidic; C18:2c+t—linoleic/linolelaidic; C18:3n3c+t—linolenic; C20—arachidic; C20:1—gondoic; C20:2—eicosadienoic; C20:3n3—dihomo-α-linolenic; C20:3n4—dihomo-γ-linolenic; C20:3n6—eicosahexaenoic; C22:6—docosahexaenoic. Under applied chromatographic conditions cis- and trans- forms of C18:2 and C18:3n3 coelute at the same retention time, so their concentrations were calculated as the sum.

Analysis was performed on a GC–MS TRACE GC coupled to a Polaris Q mass spectrometer (Thermo Fisher Scientific) using the protocol of Plassmeier et al. (2007) (link). The instrument was equipped with a Rtx®-5MS column (30 m, iD 0.25, df 0.25 µm; Restek). A 1-µl sample was injected in splitless mode into the GC-MS column. The oven program consisted of 3 min at 80 °C, a ramp with 5 °C min^–1 up to 325 °C, and finally 325 °C for 2 min. The MS transfer line temperature to the quadruple was set to 250 °C and the electron impact (EI) ion source temperature was 220 °C. The spectra were recorded with a scanning range of 50–650 mz^–1 (Supplementary Table S1). Replicate samples were derivatized and measured separately with intervals of at least 3 d. A blank was run every four samples to check for carry-over of metabolites.