Agilent hp1100 system

Time-point sample extracts were analysed using a Varian Res-Elut C18 reverse phase column (150 x 4.6 mm, 5μm; Varian, Harbor City, CA, USA) by an Agilent HP1100 system (Agilent Ltd, West Lothian, UK) coupled with a photodiode array detector and a binary pump. An isocratic elution using two solvents—solvent A) Water and solvent B) Acetonitrile, both HPLC grade, was applied in order to analyse the two molecules as follows: NAAB-496 (A = 30%; B = 70%) and NAAB-503 (A = 20%; B = 80%). The flow rate was 1 mL/min, and the thermostatically controlled autosampler and column oven were set at 10°C and 25°C, respectively. The detection conditions were set at 280 nm. The UV-Vis spectra were recorded from 200 to 400 nm. Data acquisition was performed using ChemStation A.10.01 software (Agilent, USA). The identification was made according to UV-visible spectra, retention time and co-elution with reference standards. Quantification was carried out by external standard calibration curves (0.039–0.625 μg/mL) and expressed as average ± SD of three independent experiments (n = 3).

Butyrate in culture supernatants was quantified by HPLC coupled to a refracting index detector (RID) and diode array detector (DAD) on an Agilent HP 1100 system (Agilent). Standards of butyric acid (0.09–50 mM) were prepared in 5 mM H₂SO₄ for peak identification and quantification. Samples from four biological replicates were analysed by injecting 20 µL of standard or filtrated (0.45 µM filter) culture supernatant on a 7.8 × 300 mm Aminex HPX-87H column (Biorad) combined with a 4.6 × 30 mm Cation H guard column (Biorad). Elution of was performed with a constant flow rate of 0.6 mL min⁻¹ and a mobile phase of 5 mM H₂SO₄. Standards were analysed as above in technical triplicates.

All the probes were purified on a semi-preparative HPLC Agilent HP1100 system equipped with a reverse-phase column (Phenomenex Aeris 5 μm XB-C18 100 Å, 250 × 10 mm, 5 μm). The flow rate was 2 mL/min eluting with 0.1% HCOOH in H₂O (A) and 0.1% HCOOH in CH₃CN (B), with a gradient of 5 to 95% B over 25 min. Fractions containing the product were combined and the solvent removed via freeze-drying to give probes CatD-P1, CatD-P2 and CatD-P3 as orange solids in >95% purity. Full characterisation of the probes can be found in Supplementary Notes 1–4 and Supplementary Figs. 10–13.