6545 qtof ms

The metabolites were analyzed using Agilent 1290 II (Agilent, Santa Clara, CA, USA) with ACQUITY UPLC HSS T3 column (1.8 µm, 2.1 × 100 mm) with mobile phase A: ultra-pure water (0.1% formic acid) and mobile phase B: acetonitrile (0.1% formic acid). The elution gradient was set as: 0 min, 95% A; 0–11 min, 95–10% A; 11–12 min, 10% A; 12–12.1 min, 10–95% A; and 12.1–14 min, 95% A. The flow rate was 0.4 mL/min.
MS was performed using QTOF/MS-6545 (Agilent, Santa Clara, CA, USA). All analyses were performed in electrospray ionization (ESI±) mode under the following conditions: The electrospray voltage was 2.5 kV (positive ion mode) and 1.5 kV (negative ion mode); the heater temperature was 325 °C and the capillary temperature was 350 °C; the sheath gas flow rate was 45 arb; and the auxiliary gas flow rate was 8 arb.

The remaining clinical serum specimens were refrigerated at − 80 °C until tested. Ice-cold methanol was added to the serum. The mixture was then incubated and centrifuged. The supernatant was collected for LC–MS/MS analysis. LC–MS/MS analysis was performed using a QTOF/MS-6545 (Agilent, USA) and 1290 Infinity LC (Agilent, USA). The HPLC conditions were as follows: UPLC: column, ACQUITY UPLC HSS T3 C18, 1.8 µm, 2.1 mm × 100 mm (Waters, USA); column temperature, 40 ℃; flow rate, 0.4 mL/min; injection volume, 2 μL; solvent system, water (0.1% formic acid) and acetonitrile (0.1% formic acid). Information on the specimens was acquired using the LC–MS system, followed by machine orders. The original data were transformed into the mzML format using ProteoWizard software (version 3.0). Peak extraction, alignment, and retention time correction were performed using the XCMS program [27 (link)]. Metabolic identification information was obtained by searching the Pubchem database, KEGG database, the Human Metabolome database [27 (link)]. The differential metabolites were filtered according to P value < 0.05, |log₂FC| > 1, and VIP ≥ 1 (see Additional file 1: Supplementary Methods for details).

Scanning Electron Microscope (SEM) images and energy dispersive X-ray (EDX) spectroscopy were obtained on a field emission scanning electron microscope (FeSEM, ZEISS SUPRA 40VP, Carl Zeiss NTS GmbH, Oberkochen, Germany) at an acceleration voltage of 3 kV. Transmission electron microscopy (TEM) was performed using a JEM 2100F (JEOL Ltd., Akishima, Japan) at room temperature at an accelerating voltage of 200 kV. The crystal information of the samples was collected via an X-ray diffractometer (Bruker, D8-Advance, Karlsruhe, Germany) with Cu Ka radiation. The quantification of fosfomycin was carried out using the Agilent 1290 Infinity II UHPLC apparatus coupled with a 6545 Q-TOF MS (Agilent Technologies, Santa Clara, CA, USA). The optical characterizations were performed using a UV-Vis spectrophotometer (Halo RB-10, Dynamica Pty Ltd., Victoria, Australia). Confocal Laser Scanning Microscopy (CLSM) images were performed using Olympus Fluoview 1000 IX81 confocal laser scanning microscope, operated using 100× oil-immersion objective combined with 5× optical zoom.

Chromatographic analysis was performed on a ultra high performance liquid chromatography (UHPLC) system (1290, Agilent Technologies) equipped with an Acquity UPLC HSS T3 column (2.1 mm × 100 mm, 1.8 μm, Waters, USA) coupled to a quadrupole time of flight mass spectrometer (6545 Q‐TOF MS, Agilent Technologies, USA). Mobile phase A consisted of 0.1% formic acid in positive mode and 0.5 mmol/L ammonium fluoride in negative mode, and mobile phase B consisted of a 9:1 acetonitrile/water (v/v) solution with 0.1% formic acid. The flow rate was maintained at 0.3 mL/min with the following gradient: 0–4.0 min, 100% A, 4–6 min, 100% A, 6–25 min, 75% A, 25–29 min, 100% B, 29–31 min, 100% B, 31–33 min, 100% A. Parameters of mass spectrometry were set as follows: ion spray voltage, 4.0 kV (positive) or 3.5 kV (negative); curtain gas, 40 Pa; source temperature, 550°C; collision energy for collision‐induced dissociation, 30 eV. The MS1 scan range was 50–1000 m/z, and the MS2 scan range was 25–1000 m/z.

The fragmentation of karmitoxin was investigated for potential quantification by SRM, using an Agilent Infinity 1290 UHPLC system (Agilent Technologies, Santa Clara, CA, USA) coupled to an Agilent 6545 QTOF MS and a similar UHPLC coupled to an Agilent 6490 Triple QqQ MS. A range of collision energies were tested (20, 40, 50, 60, 80, 100, and 150 CeV) to determine the optimal fragment ion yields.