Agilent 1290 infinity series

Quantification of phenolic compounds was performed according to Coklar and Akbulut (2017) and Demir et al. (2014) with some modifications. For that, an Ultra High Performance Liquid Chromatographic system (UHPLC) Agilent 1290 Infinity Series equipped with a G7167B mutisampler, G7104A flexible pump, G7116B column oven, and a G7117B diode array detector was used. Before the injection, the extracts were filtered through a 0.45 μm pore size x 33 mm syringe filter (Merck, Millex, Germany). The separation was achieved using a reversed-phase C18 column (5 μm, 250 × 4.6 mm i.d.). The mobile phase was (A) water/acetic acid (98:2) and (B) water/acetonitrile/acetic acid (78:20:2). The flow rate was 0.75 mL/min and the gradient was as follows: 10–14 ​% B (5 min), 14–23 ​% B (11 min), 23–35 ​% B (5 min), 35–40 ​% B (14 min), 40–100 ​% B (3 min), 100 ​% B isocratic (3 min), 100–10 ​% B (3 min) and 10 ​% B isocratic (4 min). The detector was set to 280 and 320 nm. The column temperature was 40 °C. According to Sasmaz et al. (2020) and Kelebek (2016) (link), the phenolic compounds were quantified by comparison with peak areas and retention time (Ferreira et al., 2020 (link)) of each standard. The coefficient of correlation (R²) value of the standards phenolic compounds were above 0.9799 in all cases. The data was analyzed using Chemstation software.

For differential metabolomic analyses, 100 μL of urine sample was mixed with 400 μL extract solution (acetonitrile:methanol =1:1) containing an internal standard (L-2-chlorophenylalanine, 2 μg/mL). The samples were sonicated for 10 min in ice-water bath after 60 s vortex. Then the samples were incubated at −40 °C for 1 hour and centrifuged at 10,000 rpm at 4 °C for 15 min. The samples were mixed with 200 μL of 50% acetonitrile after drying, sonicated on ice for ice and centrifuged at 13,000 rpm for 15 min at 4 °C. Furthermore, 10 μL of each sample were mixed to be the quality control (QC) sample.
The Agilent 1290 Infinity series ultra-high performance liquid chromatography (UHPLC) System (Agilent Technologies) works with UPLC BEH Amide column (2.1×100 mm, 1.7 μm). The mobile phase A consisted of ammonium acetate and ammonia hydroxide. The mobile phase B consisted of acetonitrile. Elution gradient conditions were as follows: 0–0.5 min, 95% B; 0.5–7.0 min, 95–65% B; 7.0–8.0 min, 65–40% B; 8.0–9.0 min, 40% B; 9.0–9.1 min, 40–95% B; 9.1–12.0 min, 95% B. The auto-sampler temperature was 4 °C. The column temperature was 25 °C and the injection volume was 1 μL (positive) or 1 μL (negative).