Qtof ms

GlcCer (25 mg) was dissolved in 5 mL ethanol and placed in a glass vial tube to which a photosensitizer (0.05 mg RB) was added. After the glass vial tube was completely closed, the sample solution was incubated at 4 °C for up to 120 h under an 18 W LED light of 50 Klux (10 cm vertically above the glass vial tube). The resulting solution was loaded onto a SepPak Plus QMA cartridge (Waters, Milford, USA), equilibrated with ethanol, unoxidized and oxidized GlcCer were eluted with 5 mL of ethanol; RB was retained on the cartridge. The eluate was evaporated and dissolved in 5 mL methanol. A portion of the solution (10 μL) was then dissolved in 1 mL methanol and infused directly into qTOF-MS (Bruker Daltonics GmbH, Bremen, Germany) at a flow rate of 10 μL/min (Table S1) to ensure the formation of oxidized GlcCer.

Chitobiose obtained from quantitative HPLC was further analyzed by quadrupole-time‐of‐flight‐mass spectrometry (QTOF-MS) (Bruker Biospin AG, Bangkok, Thailand). Chitobiose dissolved in water (2 mg.mL⁻¹, 100 μL) was injected into the instrument. A mass range of 50–1000 was selected for data acquisition. Positive ionization mode was chosen using source type Electrospray Ionization (Bruker Apollo II, Thailand). The capillary and charging voltage were set at 4500 V and 2000 V, respectively.

Solutions were collected at seven days from two experimental groups: (1) PSM medium containing 100 mg/L TC with strain M503 and (2) PSM medium containing 100 mg/L TC. Then, the solutions were centrifuged at 10,000 rpm for 10 min. Solid-phase extraction cartridges (Oasis HLB, 6 cc/150 mg, Waters) were used to extract TC and products in the solutions as described by Leng et al. (2016). A UPLC coupled with the Q-TOF-MS (Acquity, Bruker, Bremen, Germany) was used for the analysis of TC degradation products. The conditions of separation and detection were consistent with the previous study [40 (link)]. The mass spectral data were processed by MassLynx (Waters, Milford, MA, USA, version 4.1), and molecular constructions were conducted by Kingdraw (version 2.1).

The analytical method used was liquid chromatography coupled with time-of-flight mass spectrometry (LC-Q-TOF) as described by Taylor et al. (2020 (link)). Briefly extracts were separated using a Dionex Ultimate 3000 UHPLC system (Thermo Fisher Scientific, Bremen, Germany) connected to a Bruker Maxis Impact II electrospray high-resolution time-of-flight tandem mass spectrometer (Q-TOF–MS) (Bruker Daltonics, Bremen, Germany). The resolution of the instrument was 30,000 at m/z 150–200. HyStar software (rev. 3.2) and Target Analysis for Screening and Quantification (TASQ®) 1.4 software (Bruker Daltonics, Bremen, Germany) were used for data acquisition and interpretation, respectively. Suspect compounds (~ 2500 substances included in the PesticideScreener™ 2.1 and ToxScreener™ 2.1 libraries) were identified in the extracts based on the retention time, mass accuracy, isotopic pattern and diagnostic MS/MS fragments.

SolA (10 μM) was analyzed using a QTOF MS equipped with a dual-stage trapped ion mobility separation cell (timsTOF pro Bruker Daltonics Inc, Billerica, MA, USA). Sample injection (40 μl) and LC separation were performed on an Ultimate RS UHPLC system (Thermo Scientific, Germeringen, Germany) with an Acquity UPLC CSH C18 130 Å, 1.7 µm, 2.1 mm × 100 mm protected by a VanGuard 2.1 mm × 5 mm of the same material. A gradient from 1% acetonitrile to 99% acetonitrile in 18 min was applied at 0.4 ml min⁻¹ (solvent A 0.1% formic acid in water, solvent B 0.1% formic acid in acetonitrile), before returning to initial conditions. Eluting compounds were sprayed in positive ion mode by an Apollo II ion funnel ESI source (Bruker Daltonics Inc). Source settings were: capillary voltage 4500 V; end plate offset 500 V; drying temperature 220 °C; desolvation gas (nitrogen) flow 8.0 l min⁻¹; nebulizer gas pressure 2.2 bar. Samples were analyzed in timsoff mode, auto MS/MS settings were: switching threshold 500 cts; cycle time 0.5 s; active exclusion after 3 spectra; release after 0.2 min. Analytes selected for fragmentation were fragmented by collision with nitrogen gas at a collision energy of 30 eV. Precursors and fragments were analyzed by the time of flight analyzer using a range of 100–1350 m/z. Resulting data were analyzed using DataAnalysis ver. 4.3 (Bruker Daltonics).

For the essential amino acid content (EAA) determination, homogenous samples were extracted following the protocol described by Kıvrak et al. [18 (link)] Briefly, 100 mg were weighed into a 2 mL Eppendorf tube and 1 mL of MeOH/H2O (80:20) (v/v) 0.1% HCOOH was added. The mixture was sonicated for 5 min and subsequently vortexed and then immediately centrifuged at 4 °C at 4000 rpm for 15 min. The supernatant was filtered through a 0.22 µm pore-size PTFE membrane filter. The samples were then spiked into the UPLC equipment with a binary pump and an autosampler for up to 96 vials equipped with refrigeration.
The UPLC-QToF-MS/MS instrument consisted of a Waters (Milford, MA, USA) Acquity Ultra Performance LC with a Bruker Daltonics (Billerica, MA, USA) QToF-MS, model maXis. Separation was carried out with a Phenomenex (Torrance, CA, USA) C18 column (Luna Omega Polar C18 50 mm × 2.1 mm, 1.6 μm particle size). Some 3 μL of injection volume was used. The column temperature was set at 40 °C. The solvent system consisted of 0.5% aqueous formic acid (Mobile Phase A) and methanol/water (50:50) containing 0.5% formic acid (Mobile Phase B). The chromatographic conditions gradient is specified in Table 3.