Vanquish flex uhplc system

Standard sugars and compounds 1–4 (each 1.0 mg) were dissolved in 6 mol L⁻¹ CF₃COOH (1 ml) and heated at 90°C for 2 h and cooled to room temperature. The hydrolysate was extracted with CHCl₃ for three times, and the aqueous layer was concentrated to obtain the residue containing sugars. The obtained residues were dissolved in pyridine (200 μl), and L-cysteine methyl ester hydrochloride (1 mg) was added and heated at 60°C for 1 h. Subsequently, o-tolyl isothiocyanate (10 μl) was added and heated at 60°C for another 1 h (Mitaine-Offer et al., 2010 (link)). After the reaction, the supernatants were filtrated and subjected to UPLC analysis (Vanquish Flex UHPLC system equipped with CAD detector) (Thermo Fisher Scientific, Waltham, United States) using a 100 mm × 2.1 mm, 1.8 μm, HSS T3 column (Waters Corporation, Milford, United States). The elution program consisted of a linear gradient of CH₃CN in water (containing 0.1% formic acid, v/v) from 20% to 30% for 8 min (flow rate: 0.6 ml/min). The atomization temperature and wave filtering time were set at 35°C and 1 s, respectively. For compound 2 and 4, derivatives of l-rhamnose and d-glucose were detected. However, for compound 3, d-xylose was also observed. For compound 1, only derivative of d-glucose was detected [t_R 4.43 min for d-glucose, t_R 6.31 min for l-rhamnose, and t_R 4.79 for d-xylose].

The non-targeted metabolic profiling analysis was performed by an ultra-HPLC (Vanquish Flex UHPLC system; Thermo Scientific, Bremen, Germany) system coupled with high-resolution MS (Q Exactive Focus; Thermo Scientific).

The UHPLC-Q-Exactive Orbitrap-MS conditions were performed according to our previous study [30 (link)]. Chromatographic separation was carried out on a Vanquish Flex UHPLC system (Thermo Fisher Scientific Inc., Waltham, MA, USA) equipped with an ACQUITY HSS T 3 column (100 mm × 2.1 mm, 1.8 μm; Waters, Milford, MA, USA) at 35 °C. The mobile phase consisted of 0.1% aqueous formic acid (A) and acetonitrile (B). The gradient elution conditions were set as follows: 0–1.5 min, 5–8% B; 1.5–3 min, 8–12% B; 3–4.5 min, 12–30% B; 4.5–10 min, 30–35% B; 10–12 min, 35–40% B; 12–15 min, 40–65% B; 15–18 min, 65–85% B; 18–21 min, 85–95% B; 21–21.1 min, 95–5% B and 21.1–25 min, 5–5% B. The flow rate was 0.4 mL/min, and the volume of injection was 3 μL.
Mass data were obtained using a Q-Exactive Orbitrap Mass technology (Thermo Fisher Scientific Inc., USA) equipped with an electrospray ionization (ESI) source. Full-scan MS spectra were monitored in the range of m/z 100–1500 Da. The capillary voltage was set at 2.8 kV. The source and desolvation temperatures were maintained at 100 and 400 °C, respectively. The cone and desolvation gas flow rates were 20 and 800 L/h, respectively. Xcalibur 2.1 software (Thermo Fisher Scientific Inc., USA) was used to analyze all the mass data.