Agilent 7683 series injector

Short chain fatty acids (SCFA) were extracted from frozen fecal samples and analyzed as previously described [18 (link)]. Briefly, fecal aliquots were extracted in acidified water (pH 2.5) containing an internal standard of 5mM of ethylbutyric acid. Suspended samples were homogenized and sonicated, followed by centrifugation to remove particulate matter. Supernatant was analyzed on a Gas Chromatograph with Flame Ionization Detection (GC-FID; Agilent 6890 Plus GC Series, Agilent 7683 Injector series, GC Column: TG-WAXMS A 30mx 0.25mm × 0.25μm). The run program was as follows: Initial temp = 100 °C for 1 min, max temp = 300 °C, equilibration time = 1 min with a ramp rate of 8.0 for 1 min to a final temp of 180 °C, followed by a ramp rate of 20.0 to a final temp of 200 °C for 5 min. Post temperature was 50 °C. The front inlet was split (10:1), 240 °C, 16.57psi. Peak areas were normalized to the internal standard (5 mM ethyl butyric acid, Retention Time (RT) = 9.2) and quantified using standard curves (acetic acid, RT = 5.5; propionate, RT = 6.7; butyrate, RT = 7.7) from dilutions of commercial stocks.

SCFA acids were extracted from fecal samples and quantified by Gas Chromatograph with Flame Ionization Detection (GC-FID) as previously described [41 (link)]. Briefly, one gram of feces was sonicated in acidified, HPLC-grade water (pH brought to 2.5 with HCl) and allowed to sit at RT for 10 min. Samples were then centrifuged, and supernatants removed and frozen overnight at −80 °C. The supernatants were then thawed, centrifuged again to sediment any remaining particulate matter, and filtered through 0.45-micron filters. Collected supernatant was analyzed on a GC-FID (Agilent 6890 Plus GC Series, Agilent 7683 Injector Series, GC Column: TG-WAXMS A 30 m × 0.25 mm × 0.25 μm). Quantities of specific SCFA were determined through generation of standard curves of purified commercial chemicals.

The extracted organic compounds were separated using an Agilent 7890A Series Gas Chromatograph interfaced to an Agilent 5973c Network Mass Selective Detector and an Agilent 7683 Series Injector (Agilent Technologies, Santa Clara, CA, USA). A 5 µL sample was injected with a split ratio of 1:5 by 0.3% standard deviation into an HP-5MS column (30 m × 0.25 mm, 0.25 µm film thickness; Agilent Technologies, USA) using helium as the carrier gas at 1 mL/min. The ion source was maintained at 250 °C. The GC oven was programmed with a temperature gradient starting at 100 °C (for 3 min), which was gradually increased to 300 °C (for 5 min) at 8 °C/min.
MS was carried out in the electron-impact mode at an ionizing potential of 70 eV by selected ion monitoring (SIM). The presence of CoPPIX-specific m/z 621 ion was monitored.
The qualitative and quantitative analyses of CoPPIX were performed using synthetic CoPPIX as a standard (Figure S1). Its concentration was determined based on the standard curve (0.08, 0.8, 1.6, 16, and 32 µM (50, 500, 1000, 10,000, and 20,000 µg/L)). A concentration curve was plotted with the peak areas corresponding to the concentration of CoPPIX tested (Figure S2). The analysis was performed in triplicate. Error bars represented the classical standard deviation for 3 replicates. The statistical significance of the obtained results was tested using the Student’s t-test.