Agilent 6224

Accurate mass measurements were performed on an Agilent 6224 accurate-mass time-of-flight (TOF) mass spectrometer with qualitative Analysis B.04.00 software (All Agilent Technologies, Santa Clara, CA, USA). The operating parameters in the positive ion detection mode were as follows: drying gas (N₂) flow rate, 10.0 L/min; sheath gas temperature, 350°C; capillary, +4000 V; fragmentor, 135 V; skimmer,; collision energy,; and mass range, 100–1500 Da. The ESI-MS/MS spectra was carried out by Thermo-Finnigan TSQ Quantum Ultra tandem mass spectrometer equipped equiped with an electrospray ionization source (ESI), and Xcalibur 1.4 software was used for data acquisition and processing (All Thermo-Finnigan, San Jose, CA, USA). The mass spectra of ESI-MS/MS were recorded in the same ion detection mode to analysis the fragment ions of the related substances. The spray voltage was set at 5000 V. The heated capillary temperature was 350°C. The sheath gas and the auxiliary gas were set at 45 and 10 psi, respectively. The fragment ions were produced by collision-induced dissociation of the selected precursor ions with the collision energy of 35 eV. In the LC-MS/MS measurements chromatographic conditions described in the section of “Chromatographic conditions” were used.

For LC-MS, MG decolorization was performed at 25°C in water instead of buffer. Aliquots (2 μL) were injected into an HPLC system (Agilent 1200 Series, equipped with a Phenomenex Luna C-18 analytical column of 2.0 mm x 150 mm length and 3 μm particle size) coupled with Agilent 6224 Accurate-Mass Time of Flight (TOF) MS. The compounds were resolved by using solvent A: 5 mM ammonium acetate supplemented with 0.5% formic acid and solvent B: acetonitrile. The flow rate was kept at 0.2 mL min^-1. A linear gradient was set as follows: t = 0–2, A = 95; t = 4–5, A = 40; t = 7–11, A = 10; t = 12–15, A = 95. The column effluent was introduced into the electrospray ionization source of the mass spectrometer in positive ion mode. The MS parameters were as follows: capillary voltage 3.5 kV; nebulizer pressure 50 psi; drying gas flow 11 L min^-1; drying gas temperature 360°C; fragmentor voltage 130 V. LC-TOF MS accurate mass spectra were recorded across the range 70–400 m/z. Data processing was carried out with Applied Biosystems/MDS-SCIEX Analyst QS software (Frankfurt, Germany) with accurate mass application-specific additions from Agilent MSD TOF software. Accurate-mass internal mass calibration was performed automatically using a dual-nebulizer ion source. The reference masses were 121.0509 m/z and 922.0098 m/z.

Prior to sample analysis, 120 μL of each H₂O:MeOH extract (1:1.43, vol/vol) was evaporated to dryness using a vacuum concentrator. The dried samples were resuspended in 60 μL of Milli-Q water containing 0.1 mM formic acid (Sigma-Aldrich, Steinheim, Germany) and 0.2 mM methionine sulfone (Sigma-Aldrich) by vortexing for 1 min. After subsequent centrifugation (12,600 × g for 15 min at 4°C), the solution was transferred to CE/MS glass vials which were centrifuged again (4,000 × g for 20 min at 4°C) to precipitate any possible suspended solid particle. Samples were analyzed by CE-ESI(+)-TOF/MS using a CE system (Agilent 7100) coupled to a TOF/MS system (Agilent 6224), following a methodology previously described (64 (link)) (see Text S2 in the supplemental material). CE-TOF/MS data processing consisted of compound deconvolution, annotation of metabolites using an in-house library (65 (link)) and online databases (66 (link)), blank subtraction, internal standard normalization, and data filtering. (Text S2).