Waters acquity uplc 1 class system

For HILIC analysis, 1 μL of sample was injected onto a Waters ACQUITY UPLC BEH Amide (1.7 μm particle size, 100 × 2.1 mm). Samples underwent a subsequent 1:5 dilution in water for RPLC analysis and 5 μL was injected onto a Waters ACQUITY UPLC HSS T3 column (1.8 μm particle size, 100 × 2.1 mm). Both columns were maintained at 45 °C and mobile phase flow was set to 0.45 mL/min using a Waters ACQUITY UPLC I-Class system (Waters, Milford, MA). The mobile phase consisted of water containing 0.1% formic acid (A) and acetonitrile containing 0.1% formic acid (B). Mobile phase conditions for each column are described in Table S1. Time-of-Flight mass spectrometry was carried out using a Waters Xevo-G2S QTof/MS as previously described^{28 (link),29 (link)}. Briefly, capillary and cone voltage were set at 2 kV and 40 V, respectively. The source temperature was 150 °C and the desolvation temperature was maintained at 600 °C. Nitrogen gas for desolvation and the cone were set at 1200 L/h and 50 L/h, respectively. An MS^E method was used to acquire ions in the range of 50–1200 m/z alternating between MS1 (no collision energy) and MS2 (collision energy ramp of 15–50 V) with a scan time of 0.1 s. Leucine-enkephalin (100 ng/L) was used as a lockmass set to a flow rate of 10 μl/min. The lockmass was acquired every 10 seconds and averaged over 3 scans to ensure mass accuracy throughout the run.

The conditions of UPLC-QTOF-MS/MS are in accordance with the previous study [12 (link), 13 (link)]. Briefly, the analysis was performed on a Waters Acquity UPLC I-Class system (Waters Corp., Milford, United States), coupled with a Waters QTOF-MS/MS Mass System (Manchester, United Kingdom) equipped with electrospray ionization (ESI). Chromatographic separation was performed on a Waters Acquity UPLC HSS T3 column (100 mm × 2.1 mm, 1.8 μm). Mass range, 50-1500 Da; source temperature 100 °C; desolvation temperature, 450 °C; desolvation gas flow, 900 L/h; sampling cone, 40 V; ESI⁻ capillary voltage, 2.5 KV; and ESI⁺ capillary voltage, 0.5 KV. UPLC-QTOF-MS/MS system was controlled by the Masslynx 4.1 platform. The MS^E data collected in a continuum mode were processed using the peak detection and alignment algorithms in UNIFI 1.8, which enabled quasi-molecular ion peaks, adduct ions, and fragment ions to be analyzed as a single entity. RSGB (sugar-free) was filtered through a 0.22 μm microporous membrane before aliquots (2 µL) were transferred to autosampler vials for analysis. Fifteen standards were dissolved in methanol, respectively, and mixed in equal. The final concentration of each standard was 100 ng/mL, and 1µL for analysis.

Two HPLC systems were used for phenolic acid identification and quantification. The phenolic acids were analyzed in a Waters Alliance LC system composed of a Waters e2695 Separations Module and Waters 2998 PDA Detector (Waters Corp., Milford, MA, USA). A Gemini 150 × 4.6 mm 5 μm C18 110 Å LC column (Phenomenex, Torrance, CA, USA) was used for separation, and compounds were detected at 366 nm. The injection volume was 10 μL.
For compound identification of phenolic acids, the samples were analyzed using the method described in Wang et al. [51 (link)] with a Waters ACQUITY^® UPLC I-Class system coupled with a Waters Vion Ion Mobility Quadrupole Time of Flight (IMS QTof) mass spectrometer (MS) (Waters Corp., Milford, MA, USA). The same column, solvent system, and elution gradient as described by Wang et al. [51 (link)] were used with the system for compound identification. In addition, a 1:3 splitter was used to direct one-fourth of the flow (0.25 mL/min) into the MS. Compounds were identified by liquid chromatography tandem mass spectrometry (LC-MS-MS) based on accurate masses, retention times, and UV absorbance at 305 to 390 nm. All solvent systems and elution gradients are summarized in Table 7.