Agilent 6550 ifunnel qtof

Serum sample preparation for the metabolic profile followed standard protocols that have been described elsewhere [16 (link), 17 (link)]. Blank samples (75% MeOH in water) and pooled quality control (QC) samples (pooling of aliquots of all samples) were also detected [18 (link)]. In brief, 400 μL of the precooled extraction solvent (V methanol: V acetonitrile = 1 : 1) was added to 100 μL of serum, vortexed (30 s), ultrasound-treated (10 min), and incubated for 1 h at −20°C. The precipitated proteins were centrifuged at 12000 rpm for 15 min at 4°C. The supernatant was transferred to a clean tube, dissolved with a 100 μL water/acetonitrile (1 : 1) solution, then vortexed (30 s), ultrasound-treated (10 min) at 4°C, centrifuged at 12000 rpm for 15 min at 4°C and then separated and subjected to metabolite analysis by ultrahigh-performance liquid chromatography-quadrupole time-of-flight-mass spectrometry (UHPLC-QTOF/MS) using a UHPLC system (1290 series, Agilent Technologies, USA) coupled to a QTOF mass spectrometer (Agilent 6550 iFunnel QTOF, Agilent Technologies, USA). The mobile phase consisted of 25 mM NH₄OH and 25 mM NH₄Ac in water (pH = 9.75). Tandem mass spectrometry (MS) data acquisition was performed using another QTOF mass spectrometer (TripleTOF6600, SCIEX, Canada).

The pigments were identified by UHPLC- High Resolution Electrospray Ionization (HR-ESI) MS analyses according to the method published by Klitgaard et al. [29 (link)], and using the Agilent 1290 Infinity LC system with a DAD detector, coupled to an Agilent 6550 iFunnel Q-TOF with an electrospray ionization source (Agilent Technologies, Santa Clara, CA, USA), and a Poroshell 120 Phenyl–Hexyl column (2.1 mm i.d. × 250 mm, 2.7 μm; Agilent). The analytical conditions used in this study were those earlier reported by Klitgaard et al. [29 (link)]: the separation was performed at 60 °C with a water-acetonitrile gradient (with 20 mM formic acid) going from 10% (v/v) to 100% acetonitrile in 15 min, followed by 3 min with 100% acetonitrile. The flow rate was kept constant at 0.35 mL/min. Mass spectra were recorded as centroid data for m/z 85–1700 in positive and negative ESI-MS mode, with an acquisition rate of 10 spectra/s.

The plasma samples were randomly injected for UHPLC-QTOF/MS analysis in both the electrospray positive and negative modes. Blank samples (75% ACN in water) and QC samples were injected between every eight samples during the acquisition. A UHPLC system (1290 series, Agilent Technologies, USA) coupled to a quadruple time-of-flight (QTOF) mass spectrometer (Agilent 6550 iFunnel QTOF, Agilent Technologies, USA) was employed for the primary metabolic detection and determination. Tandem mass spectrometry (MS/MS) was performed using another QTOF mass spectrometer (Triple TOF 5600+, AB SCIEX, USA), which was better at performing a qualitative metabolite analysis. The QC samples were used for the MS/MS data acquisition. Four mass range segments, 50–300 Da, 290–600 Da, 590–900 Da, and 890–1200 Da, were used to expand the coverage of the MS/MS spectra. The acquired MS/MS spectra were matched with the in-house MS/MS spectral library (Shanghai Institute of Organic Chemistry), which has been verified by the metabolite standards for metabolite identification (details are described in the Supplementary Information).

The secondary metabolites isolated in IC and EC extracts of F. oxysporum were identified by UHPLC-HRMS. Analyses were performed on an Agilent 1290 Infinity LC system with a DAD detector, coupled to an Agilent 6550 iFunnel Q-TOF with an electrospray ionization source (Agilent Technologies, Santa Clara, CA, USA). The separation was performed on a 2.1 mm i.d. × 250 mm, 2.7 μm Poroshell 120 Phenyl-Hexyl column (Agilent) at 60 °C with a water-acetonitrile gradient (both buffered with 20 mM formic acid), according to the method described by Klitgaard et al. (2014 (link)).

Subsequent to the confirmation of implantation, the culture medium samples were
divided according to their implantation outcomes.
The lipids from culture medium were individually extracted using the Bligh and
Dyer method (Bligh & Dyer 1959 (link)), dried
and diluted in 400 µL of MeOH.
Mass spectra were obtained with a direct infusion of both the negative and
positive ion modes into a Q-Tof mass spectrometer (LC-MS, Agilent 6550 iFunnel
Q-TOF) equipped with an automated injector.

Identification of secondary metabolites from Leptadenia reticulata was achieved using an Agilent 6550 iFunnel Q-TOF (Agilent Technologies, Santa Clara, CA, USA), with both positive and negative modes [38 (link)]. Hypersil Gold C-18 (3 µm particle size, 2.1 mm internal diameter, and 100 mm length) was used for the separation of secondary metabolites. A flow rate of 300 µL/min was used. An aliquot of 3 µL was injected independently. 100% acetonitrile with 100% methanol made up mobile phase B, whereas 0.1% formic acid in water made up mobile phase A [39 (link)]. With the following parameters, a complete scan mode was attained in the 100–1000 amu range: capillary voltage (3500 V); nozzle voltage (1000 V); 13 L/min gas flow rate at 300 °C; and nebulization set at 35 psi. Mass Hunter Workstation was used for identification of secondary metabolites based on the m/z (mass/charge) values and spectrum graph.