Fecal samples were freeze-dried (FreeZone®4.5 L, Labconco, Kansan City, MO, USA) at −107 °C, 0.2 mbar for 24 h and then pooled and mixed as previously described by Martias et al. [28 (link)].
For each extraction protocol, the extraction was repeated 10 times. Five extraction procedures were tested on 50 mg for each of a pool of dried feces: H2O:MeOH:CHCl3 (1:1:1), ACN:H2O (1:1), MeOH:H2O (1:1), MeOH:H2O (4:1), MeOH:H2O:ACN (1:1:1). Samples were vortexed during 10 min and centrifuged (10 min at 4 °C, 15,000 g). Supernatants were collected in 2 aliquots (one for the NMR analysis and the other for MS analysis) for further solvent evaporation in a SpeedVac (ThermoFisher). A graphical visualization of all sample preparation protocols is given inFigure S1c .
For the MS analysis, dried-residues were dissolved in 150 µL of ACN:H2O (4:1). 75 µL were used for HILIC and the remaining phase were evaporated in a SpeedVac (ThermoFisher). Then, dried-residues was dissolved in 75µL of MeOH:H2O (1:9) for RP-LC. For the 1H-NMR analysis, dried-residues were dissolved in 200 μL of a deuterated buffer (0.2 M potassium phosphate buffered deuterium oxide (pH = 7.44 ± 0.5) and 10 μL of deuterium oxide (D2O) with external reference [3-trimethylsilylpropionic acid (TSP) at 3.2 mM]).
For each extraction protocol, the extraction was repeated 10 times. Five extraction procedures were tested on 50 mg for each of a pool of dried feces: H2O:MeOH:CHCl3 (1:1:1), ACN:H2O (1:1), MeOH:H2O (1:1), MeOH:H2O (4:1), MeOH:H2O:ACN (1:1:1). Samples were vortexed during 10 min and centrifuged (10 min at 4 °C, 15,000 g). Supernatants were collected in 2 aliquots (one for the NMR analysis and the other for MS analysis) for further solvent evaporation in a SpeedVac (ThermoFisher). A graphical visualization of all sample preparation protocols is given in
For the MS analysis, dried-residues were dissolved in 150 µL of ACN:H2O (4:1). 75 µL were used for HILIC and the remaining phase were evaporated in a SpeedVac (ThermoFisher). Then, dried-residues was dissolved in 75µL of MeOH:H2O (1:9) for RP-LC. For the 1H-NMR analysis, dried-residues were dissolved in 200 μL of a deuterated buffer (0.2 M potassium phosphate buffered deuterium oxide (pH = 7.44 ± 0.5) and 10 μL of deuterium oxide (D2O) with external reference [3-trimethylsilylpropionic acid (TSP) at 3.2 mM]).
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