Gas chromatography mass spectrometry

The composition of the marjoram oil was determined by Gas Chromatography Mass Spectrometry (Agilent Technologies, Santa Clara, CA, USA).

The measurement of SCFAs in mice feces was carried out by gas chromatography-mass spectrometry (Agilent Technologies, Palo Alto, CA, USA) as described by Han et al. [17 (link)] with modifications. In brief, 0.1 g of fecal samples were mixed with 600 μL ultrapure water oscillated for 1 min. The suspension was acidified by 50% concentrated sulfuric acid (Sinopharm, Beijing, China), kept at room temperature for 5 min and vortexed, and then centrifuged at 5000× g for 10 min. The supernatants were mixed with anhydrous ether (Sinopharm, Beijing, China) at 1: 1 (v/v), vortexed for 30 s and centrifuged at 5000× g for 10 min. The upper ether layer was taken for further analysis. Chromatographic analysis was performed on HP–FFAP column (30 m × 250 μm × 0.25 μm; Agilent Technologies, Palo Alto, CA, USA). The procedure was set at an initial temperature of 90 °C and held for 2 min, increased to 150 °C at a rate of 12 °C/min and then increased to 220 °C at a rate of 20 °C/min and kept for 4.5 min. The running time was 15 min. Acetic, propionic and butyric acids (Macklin Biochemical, Shanghai, China) were used as standard solutions.

An amount of 5 g of homogenized sample was placed into a 40 mL vial (Supelco, Bellefonte, PA, USA). The extraction of volatile compounds was performed using solid phase microextraction (SPME) with carboxen/polydimethylsiloxane fiber (CAR/PDMS, 75 µm, Supelco, Bellefonte, PA, USA). The sample was kept at 30 °C for 1 h in a thermal block (Supelco, Bellefonte, PA, USA) to collect the volatile compounds. After equilibration, the SPME fiber was exposed to the sample headspace at 30 °C for 2 h. Gas chromatography/mass spectrometry (Agilent, Santa Clara, CA, USA) was used to identify volatile compounds. A DB-624 (J&W Scientific, 30 m × 0.25 mm × 1.4 μm film) was used as the column and the carrier gas was helium. The oven temperature was first set to 40 °C for 6 min, then gradually increased to 210 °C and then held at 210 °C for 12 min. The injector port was in splitless mode. The GC/MS interface was maintained at 280 °C. Mass spectra were obtained by electron impact at 70 eV, and the quadrupole mass spectrometer scan range was 40–400 atomic mass units.
Mass spectrometry libraries (NIST, FLAVOR, and WILEY) and standard substances were used for the identification of compounds, and the Kovats index was determined using the standard mix (Supelco 44585-U). The results are given as AU × 10⁶ [27 (link)].

Samples for volatile compounds (VOCs) analysis were isolated from the packed fillets of the consumer study. The VOCs were determined by headspace SPME-GC/MS analysis according to Katsouli et al. [18 (link)] based on a modified procedure of Parlapani et al. [20 (link)]. Specifically, volatiles were extracted by homogenizing 5 g of minced fish muscle with 4 mL of saturated saline and incubated at 40 °C for 15 min. The SPME fiber (50/30UM DVB/CARBOXEN-PD) was exposed to the headspace for an additional 40 min, under the same conditions. The headspace was then analyzed using gas chromatography–mass spectrometry (Agilent Technologies, Santa Clara, CA, USA) and the separation was achieved on an Agilent DB-WAX GC Column (30 m 0.25 mm, coated with a 0.25 μm film thickness). 4-methyl-1-pentanol was used as an internal standard; and the identification of the compounds was based on comparing MS data with those of reference compounds and by MS data obtained from the NIST library (NIST/EPA/NIH Mass Spectral Library with Search Program, software version 2.0f) and by semi-quantitative analysis using the method of internal standard. All samples were analyzed in triplicate.

The qualitative and quantitative evaluation of fecal SCFAs and MCFAs was performed by Agilent gas chromatography-mass spectrometry (GC-MS) system composed with 5971 single quadrupole mass spectrometer, 5890 gas chromatograph and 7673 autosampler, through our previously described GC-MS method (18 (link)).
Briefly, just before the analysis, stool samples were thawed and added with sodium bicarbonate 0.25 mM solution (1:1 w/v) in a 1.5 mL centrifuge tube. Then, the obtained suspensions were sonicated for 5 minutes, centrifuged at 5000 rpm for 10 minutes and then the supernatants were collected. The SCFAs were finally extracted as follow: an aliquot of 100μL of sample solution (corresponding to 0.1 mg of stool sample) was added of 50 μL of internal standards mixture, 1 mL of tert-butyl methyl ether and 50 μL of HCl 6 M + 0.5 M NaCl solution in a 1.5 mL centrifuge tube. Subsequently, each tube was shaken in a vortex apparatus for 2 min, centrifuged at 10,000 rpm for 5 min, and lastly the solvent layer was transferred to an autosampler vial and processed three times.