Targetlynx tools of the masslynx 4

The analyses were performed according to Arapitsas et al. [23 (link)]. Briefly, the separation of the metabolites was achieved on a Waters Acquity UPLC system equipped with an HSS T3 column 1.8 μm, 150 mm × 2.1 mm (Milford, MA, USA). The injection volume of both standard solutions and samples was 10 μL, and the samples were kept at 6 °C during analysis. Mass spectrometry detection was performed on a Waters Xevo TQ-MS (Milford, MA, USA) instrument equipped with an electrospray (ESI) source. Data processing was done using Waters TargetLynx tools of the MassLynx 4.1 software. In order to control the robustness of the system and the stability of the signal, tryptophan deuterated d5 was used as an internal standard (1.33 ppm) and the order of the sample injections was randomized, and to control the instrumental variability, the QC samples were injected every 10 sample injections. The QC samples were prepared as a pooled mix of extracellular and intracellular samples separately.

The UHPLC-MS/MS analysis was carried out on a Waters Acquity UPLC system (Milford, MA, USA) according to Arapitsas et al. [26 (link)] with slight modifications. First, the base and sparkling wine samples were filtered at 0.22 µm by a Millex-GV filtration unit (Merc, Darmstadt, Germany) directly into 2 mL MS certified amber vials and 20 µL of the IS (10 mg 3-nitrotyrosine in 10 mL of MeOH) was added. For the separation, 1.8 µm particle size and 150 × 2.1 mm Waters Acquity HSS T3 column (Milford, MA, USA) were used. Mobile phase A consisted of H₂O containing 0.1% formic acid, and mobile phase B was acetonitrile with 0.1% formic acid. The flow rate was 0.4 mL/min, while the column was kept at 40 °C. The injection volume was 10 µL, and the samples were stored in the autosampler at 6 °C during the analysis. Waters Xevo TQ (Milford, MA, USA) triple-quadrupole mass spectrometer with an electrospray (ESI) source was coupled to the UPLC system to perform the MS analysis. The Waters TargetLynx tools of the MassLynx 4.1 software were used for data processing. Standard solutions at eleven different concentrations were used to construct the calibration curves as adopted from Arapitsas et al. [26 (link)], except for sulfonated indole 3-lactic acid glucoside (Ila-Glu-SO3H) and Ila-Glu, which were quantified as indole 3-lactic acid (Ila).