U3000 chromatography system

The metabolites were resuspended in 100 μL methanol–water solution (v/v, 50:50), and the supernatant was analyzed using a U3000 chromatography system (Thermo Fisher Scientific, CA, USA) coupled with Orbitrap Fusion Lumos apparatus. Two replicates were used for each sample. The resuspended sample (1 μL) was injected into an Agilent Poroshell 120 SB-C18 chromatographic column (2.1 × 100 mm, 2.7 μm) using an autosampler for separation at 30 °C. Separation was performed with 0.2 % formic acid eluent A and acetonitrile mobile phase B at 0.2 mL/min. Gradient elution was set and performed within 2 min with 100 % solution A, 2–12 min at 100–0 % A, and maintenance for 4 min, which was followed by return to the starting conditions in 1 min.
Mass spectrometry analysis was performed in the positive ion mode. The following conditions were used: spray voltage, 3.5 kV; sheath gas flow rate, 35 arb; auxiliary gas flow rate, 10 arb; purge gas flow rate, 2 arb; ion transfer tube temperature, 320 °C. Parent ion full-scan mass spectrum acquisition was performed in the range of 100–1000 mass/charge (m/z).

Functional
groups and concentrations of specific compounds present within the
supernatant were characterized via a combination of pH testing with
a handheld pH meter, FTIR, and liquid chromatography–mass spectroscopy
(LC–MS).
A drop of the filtered sample supernatant was
placed on a PerkinElmer spotlight 400 FTIR attenuated total reflectance
(ATR) crystal, and absorption was measured between 4000 and 650 cm^–1. To better display absorption characteristics of
the mixed products, DI water was measured, and its spectrum was subtracted
from those of the supernatants.
LC–MS was performed on
a Q-Exactive HF mass spectrometer
with a U3000 chromatography system (Thermo Fisher Scientific); samples
were prepared at 20 μg/mL in 1:1 acetonitrile/water and run
for 30 min at 35 °C through a Waters BEH Amide UHPLC column (2.1
× 100 mm). Mobile phase A was composed of 80:20 MeCN/H₂O with 0.1% NH₄OH, and mobile phase B was identical except
for a 70:30 ratio of MeCN/H₂O. Commercially available standards
for glucose, fructose, galactose, mannose, xylose, ribose, arabinose,
deoxyglucose, deoxyribose, and glutaric acid were analyzed as external
standards, and a ¹³C-labeled glucose compound was run across
all samples as an internal standard.