Faecal volatile compounds
C/min
SEPARATION
Automated headspace solid-phase microextraction (SPME) gas chromatography mass spectrometry (GC–MS) was conducted on an Agilent 7890A gas chromatograph, coupled to a CTC-PAL autosampler and a BencthTOF mass spectrometer (Markes Intl, Laitrisant, UK). Homogenised faecal samples (100–200 mg) were accurately weighed into 10 mL headspace vials, followed by the addition of 2 mL 26% NaCl and 5% metaphosphoric acid prior to sonication (5 min). The samples were then placed into the autosampler tray and incubated at 60 °C for 30 min, followed by a 30 s extraction onto a 75 μm Carboxen/ PDMS fibre (Supelco, Bellefonte, PA) and desorption in the GC injector set at 250 °C for 8 min. The injector was operated in the split mode at a split ratio of 20:1, using helium as a carrier gas at a constant flow of 1.15 mL/min. Chromatographic separation was achieved on VF-WaxMS ((L) 60 m × (D) 0.25 mm × (FT) 0.25 μm) capillary column (Agilent). The oven was held at 40 °C for 8 min followed by a temperature ramp at 4 °C/min to 100 °C, then 15 °C/min to 260 °C and held for 5 min. The mass spectrometer was operated in the total ion scan mode (m/z 30–450) with the ion source and transfer line temperatures held at 250 °C and 245 °C, respectively. The SCFA acetate, propionate, butyrate, valerate and hexanoate, branched chain fatty acids (BCFA) isobutyrate and isolvalerate, as well as cresol and phenol, were quantified using an external calibration curve.