Gc 7890

The fatty acid composition of the LTL sample was determined based on the method described by Cherif et al [6 (link)], using a gas chromatograph (Agilent, GC7890, USA) equipped with a flame ionization detector and a 100% polymethylsiloxane capillary column (100 m×0.25 mm in diameter, 0.25-μm film thickness [HB-88]). The concentration of each fatty acid was expressed as milligrams of fresh muscle weight per gram quantified.

At the end of the experiment, approximately 3 h after morning feeding, rumen fluid was collected from five randomly selected lambs from each treatment group using an oral stomach tube (Wuhan Anscitech Farming Technology Co., Ltd, Wuhan, China). Rumen pH was measured using a pH meter (DDSJ-318; Shanghai INESA Scientific Instrument Co., Ltd, Shanghai, China). The rumen fluid samples were subsequently filtered through four layers of sterile cheesecloth. For NH₃-N analysis, 5 mL of filtered rumen fluid was placed in a centrifuge tube containing 1 mL of H₂SO₄ (20 g/L). To determine the volatile fatty acid (VFA) concentration, another 5 mL of filtered rumen fluid was placed in a centrifuge tube containing 1 mL of metaphosphoric acid (250 g/L). The samples for NH₃-N and VFA analysis were stored at −20°C until analysis.
The rumen fluid VFA concentration was measured using gas chromatography (Agilent, GC7890, Santa Clara, CA, USA) according to Jeon et al [18 (link)]. The rumen NH₃-N concentration was determined using a colorimetric spectrophotometer (UV-759; Shanghai Automation Instrument Co., Ltd, Shanghai, China) based on AOAC [16 ].

Nitrate and nitrite were analyzed on an NO_x analyzer (CLD 66s, Eco Physics) using the VCl₃ and NaI reduction assay, respectively (Braman and Hendrix, 1989 (link); Yang et al., 1997 (link)). Ammonium was analyzed with the salicylate method (Bower and Holm-Hansen, 1980 (link)). Isotopically labeled dinitrogen (¹⁵N-N₂) was analyzed in the headspace of exetainer samples on a gas chromatography-isotopic ratio mass spectrometer (GC-IRMS; Thermo Delta V Plus, Thermo Scientific) with the excess above natural abundance calculated according to Nielsen (1992) (link). Nitrous oxide was analyzed in the same exetainers on a gas chromatograph (GC 7890, Agilent Technologies). Both the prior sampling of the headspace for ¹⁵N-N₂ analysis and the amount of nitrous oxide dissolved in the seawater were accounted for in the calculation of the total amount of nitrous oxide in the sample. ¹⁵N-labeled NO₃^-, NO₂^-, and NH₄⁺ were analyzed with the cadmium/sulfamic acid, sulfamic acid, and hypobromite assay, respectively, followed by ¹⁵N-N₂ analysis on the GC-IRMS (Warembourg, 1993 ; McIlvin and Altabet, 2005 (link); Füssel et al., 2012 (link)).

Biomass was determined by gravimetric method. Lipid extraction method was same as Li et al. [5 (link)]. Fatty acid methyl esters (FAMEs) were prepared regarding to previous methods [33 (link)] with some modifications. First, 5 mL 0.5 M KOH–methanol was added to a tube containing total lipids. The tubes were heated in a water bath at 65 °C for 10 min and then 5 mL 30% BF₃-ether was added. The tubes were further heated in a water bath at 65 °C for 30 min and then 5 mL hexane was added when the tubes cooled down to room temperature. Then, 16 g/L methyl heptadecanoic acid (Sigma, San Francisco, USA) served as an internal standard and was added to mixtures. Then, the mixtures settled for separation of two phases after adding 1 mL saturated sodium chloride solution for preventing emulsification. FAMEs were applied to a gas chromatograph (Agilent GC 7890, Sacramento, USA) equipped with a 100 m × 0.25 mm capillary column (SP-2560, Sacramento, USA). The column was increased from 140 to 240 °C at 3 °C/min and then maintained at 240 °C for further 30 min. The temperature of the injector and detector were both set at 260 °C. Nitrogen was used as the carrier gas at 20 cm/s. Peaks were identified using authentic standards of supelco 37 component FAME mix (Sigma, San Francisco, USA). Fatty acids were quantified from the peak areas relative to the peak of the internal standard.

MCF derivatization of balsamic vinegars was carried out to determine the profile of amino and non-amino organic acids, and some primary amines and alcohols. The sample preparation protocol was adopted from that of Pinu et al. [30 (link)] and then optimized for the analysis of vinegars. An amount of 130 µL of vinegar was mixed with 20 µL internal standard l-alanine-2,3,3,3-d₄ (10 mM) and 50 µL of NaOH (2 M) in silanized reaction tubes. MCF derivatization was performed according to the method of Smart et al. [27 (link)] After derivatization, all the samples were injected into an Agilent GC 7890 coupled to an Agilent MSD 5975 with a quadrupole mass selective detector (Electron Ionization; positive mode) operated at 70 eV. The GC column used for all analyses was a Zebron ZB-1701 (Phenomenex, Torrance, CA, USA), 30 m × 250 µm (internal diameter) × 0.15 µm (film thickness), with a 5-m guard column. The MS was operated in scan mode (start after 6 min; mass range 38–650 a.m.u. at 1.47 scans/s). All the other analytical parameters are described in Smart et al. [27 (link)].

The homogenized sample (2.0 gm) was taken in a 50 mL polypropylene tube, dissolved in 10 mL MilliQ water, and mixed well. Chloroform (10 mL) was added to the sample and vortexed for 1 min followed by shaking for 30 min on a mechanical shaker. Sodium chloride (3 gm) was added and mixed with vigorous shaking. The sample was further centrifuged for 15 min at 4000 rpm. After centrifugation, the lower layer of chloroform was taken and filtered with a 0.2 μm microfilter and injected into GC-MS [32 (link)]. The GC-MS was performed using Agilent GC-7890 with an auto sampler. The column used was HP-5MS UI 15 m, 0.25 μm, 25 mm ID capillary column at 350 °C, with a run time of 12 min. The Inlet program was in split less mode, with 2.7324 mL/min of constant flow with helium gas type. MS conditions included the Agilent 6470 Triple Quad mass spectrometer with 2 filaments at 300 °C temperature and positive-mode electron ionization.