Pal3 rsi 85 autosampler

SCFAs were analyzed by GC-MS, essentially as described before [28 (link)]. Briefly, fecal samples were mixed 1:1 (w:w) with PBS, vortexed for 2 minutes and centrifuged at 14,000×g for 10 min. One hundred and fifty μl of the supernatant were mixed with 550 μl internal standard solution, containing methanol, internal standard (2 mg/ml 2-ethyl butyric acid), and formic acid (20%). The analysis was carried out on a GC-MS (8890 GC System; Agilent Technolgies, Amstelveen, the Netherlands) equipped with a PAL3 RSI 85 autosampler (Agilent) by injecting 1 μl sample on a DB-FATWAX Ultra Inert column (30 m, 0.25 mm, 0.25 μm, Agilent). The temperature settings of the injector port, oven, flame-ionization detector and mass spectrometer detector were 250, 200, 275 and 225°C, respectively. The flow rate over the column was 1.2 ml|/min. The MS Quantitative Analysis (Quant-My-Way) software from Agilent was used to determine concentrations of SCFA. LODs were: 0.95 mmol/L for acetate and 0.91 mmol/L for both propionate and butyrate. The lowest concentrations found in the samples were at least 2-fold higher than the LOD.

SCFAs (acetate, propionate, and butyrate) in the lumen and dialysate samples were analyzed with gas chromatography–mass spectrometry (GC-MS). Samples were prepared for GC-MS as described before [28 (link)]. In short, the samples were centrifuged and formic acid, 2-ethyl butyric acid (internal standard) and methanol were added to the supernatant. The analysis was carried out on a GC-MS (8890 GC System; Agilent Technologies, Amstelveen, The Netherlands) equipped with a PAL3 RSI 85 autosampler (Agilent) by injecting 1 μL sample on a DB-FATWAX Ultra Inert column (30 m, 0.25 mm, 0.25 μm, Agilent). The temperature settings of the injector port, oven, flame-ionization detector, and mass spectrometer detector were 250, 200, 275 and 225 °C, respectively. The flow rate over the column was 1.2 mL/min. With the use of calibration curves of known quantities of standards, quantities of SCFAs in the samples were determined.