Accucore c18

The chromatographic separation was made with an Ultimate 3000 HPLC system (Thermo Fischer Scientific) equipped with a column Accucore C18 (100 × 4.6 mm, 2.6 µm). The mobile phase consisted of: (A) water with ammonium acetate (50 mM) adjusted at pH 4.4 with acetic acid, and (B) acetonitrile 100%. The gradient used is presented in Table 1. To separate the compounds in the peak, the gradient began with a high percentage of the aqueous phase and a low flow; as time passed, the percentage of organic proportion increased along with the flow of the mobile phase.

Following a previous study [24 (link)], analysis of the SCFAs was performed using ultra-high performance liquid chromatography (UHPLC). There was no need for sample pre-treatment prior to injection. SCFA standards of acetic, butyric, lactic, propionic, and succinic acids (all from Merck; Darmstadt, Germany), prepared in the mobile phase at concentrations ranging from 5 to 10,000 ppm, were quickly created. The mobile phase was supplied at a flow rate of 0.250 mL/min and consisted of a mixture of two solutions. The mobile phase consisted of aqueous acetonitrile (1%) and ultrapure water (99%), both acidified with 1% formic acid. After fermentation, 1 mL of the supernatant was centrifuged, filtered through a 0.22 µm nylon filter, and then transferred to a vial for UHPLC analysis. The UV–Vis photodiode array detector (PDA) was set at 210 nm, and the column was a reversed-phase Accucore™ C18 (ThermoFisher Scientific, Waltham, MA, USA) with a particle size of 2.6 µm and length of 150 mm set at 35 °C. The analysis was performed twice, and the information shown represents the mean values of the millimolar (mM) concentration of each SCFA.

Chromatographic separation was accomplished with a Dionex Ultimate 3000RS UHPLC instrument, equipped with Thermo Accucore C18 (100 mm × 2.1 mm i.d., 2.6 μm) analytical column for separation of compounds. Water (A) and methanol (B) containing 0.1% formic acid were employed as mobile phases, respectively. The total run time was 70 min; the elution profile and all exact analytical conditions have been published [67 (link)].

Chromatographic separation was accomplished with a Dionex Ultimate 3000RS UHPLC instrument, equipped with a Thermo Accucore C₁₈ (100 mm × 2.1 mm i. d., 2.6 μm) analytical column for the separation of compounds. Water (A) and methanol (B) containing 0.1% formic acid were employed as mobile phases, respectively. The total run time was 70 min for the elution profile. Mass spectrum analysis was carried out using a Thermo Q-Exactive Orbitrap mass spectrometer (Thermo Scientific, Waltham, MA, USA) equipped with an electrospray ionisation probe interface in positive and negative-ion mode. All detailed analytical conditions have been published [62 (link)].

LC–MS analysis was performed on Acquity Ultra Performance Liquid Chromatography equipped with a XEVO- TQD interfaced via an ESI source (Waters Co., Milford, CT, USA). The compounds were separated on a Thermo Scientific™ ACCUCORE C-18, 150 × 2.1 mm, 2.6 µm reverse-phase column at a constant flow rate of 0.25 mL/min. An applied column and the auto-sampler temperatures were 35 ± 5 °C and 25 ± 5 °C, respectively. The mobile phase consisted of three solvents. Acetonitrile (A) and 0.1% formic acid buffer was prepared in 95:5 v/v, and water/ acetonitrile (B) using a multi-step gradient was applied, as shown in Table 6, with a sample injection volume of 2 μL.