Formic acid b

The analysis of phenolic acids and flavonoids content was carried out with the LC-MS 8045 apparatus (Shimadzu, Kyoto, Japan) equipped with ESI type ion source. The separation of analytes was performed with a Prominence-I LC-2030C 3D Plus (Shimadzu, Kyoto, Japan) unit equipped with a Kinetex 2.6 µm C18 100A 100 × 3.0 mm column with a Security Guard ULTRA 3 mm (Phenomenex, Torrance, CA, USA).
We used 0.1% aqueous formic acid (A) and methanol with 0.1% of formic acid (B) (Sigma-Aldrich, Steinheim, Germany) as mobile phases. The gradient programme was as follows: from 10% to 20% B in 0–5 min; from 20% to 60% B in 5–10 min; from 60% to 10% B in 10–13 min; 10% B up to 17 min. The mobile phase flow was 0.35 mL·min⁻¹ at 35 °C.
The screening of non-volatiles was carried out with polyphenols: standard mixture of phenolic acids and alcohols and polyphenols: standard flavonoids mixtures (MetaSci, Toronto, ON, Canada).
The identified compounds were quantified using the MRM mode (Table 1) referring to the calibration curve. The analyses were performed in three repetitions.

Sample reconstitution was performed in 300 μl of water. In all, 250  μl aliquot of reconstituted sample material were used for individual sample preparation of 96-well plates including the addition of 25 μl of full RP 2× labelled standard mix (l-glutamine-¹³C₅; l-glutamic acid ¹³C₅; creatinine-methyl-D₃; cytidine-5,6-D₂; citric acid ¹³C₆; l-isoleucine-¹³C₆¹⁵N; l-leucine-13C₆; l-phenylalanine-¹³C₉¹⁵N; hippuric acid-D₅, benzoic acid-¹³C₆, octanoic acid-¹³C₈, l-tryptophane-¹³C₁₁¹⁵N₂). In addition, 50 μl aliquots of each sample were used for pooling and generation of QC sample. For chromatographic separation a 2 μl aliquot of extracted metabolites from each sample was injected onto a reverse-phase 150 × 2.1 mm ACQUITY 1.8-μm High Strength Silica (HSS) column (Waters Corp.) kept at 45 °C using an ACQUITY UPLC system (Waters Corp.). The mobile phase consisting of 0.1% v/v formic acid (Fisher Scientific) in water (A) and acetonitrile containing 0.1% formic acid (B, Sigma-Aldrich). Each sample was resolved for 12.65 min at a flow rate of 0.5 ml/min. The gradient consisted of 99% A and 1% B for 0.1 min, a ramp of curve 6–100% B from 0.1 to 10.70 min.