1290 infinity binary pump

The chemical profiling of wMMM was carried out using a LC–MS instrument consisting of the Agilent 6200 series Time-of-Flight/6500 series (Agilent, Palo Alto, CA, USA) connected to the Agilent high performance liquid chromatography (HPLC) 1290 Infinity Binary Pump (Agilent) with an Electrospray ionization (ESI) interface. Zorbax Eclipse C18 column (5 µm, 150 mm × 2.1 mm) was used for chromatographic separation at a flow rate of 0.2 mL/min with two separate mobile phases. The mobile phase was a gradient system of high A in the first 3 min and high B in the next 15 min, followed by high A again (A: 0.1% formic acid in water and B: 0.1% formic acid in acetonitrile). The temperature of the column was maintained at 40 °C and the injection volume was 3 µL, with a total run time of 30 min. The spectra were obtained in ESI+ and ESI-modes and were acquired using a PDA detector. An Agilent Database Library was used to verify the compounds.

TRIS and TRIS-IBCF were separated using an Agilent 1290 HPLC system (Palo Alto, CA, USA), which consisted of a 1290 Infinity Binary Pump containing a Jet Weaver V35 Mixer, 1290 Infinity Autosampler and a 1290 Infinity Thermostatted Column Compartment.
HPLC conditions: Gradient elution was performed on a reverse-phase separation column (Geminutesi C18, 3.0 µM, 150 mm, 2.1 mm ID by Phenomenex, Switzerland) at a flow rate of 0.3 mL/minute. The column was maintained at 400 °C in all experiments. The mobile phase consisted of water (Solvent A) containing 1 mM ammonium formate and 5% MeOH and MeOH containing 1 mM ammonium formate (Solvent B). The following elution gradient was used: 0% B/100% A was converted to 95% B over 8 min, and the initial conditions were returned to over 4 min (0% B/100% A).

Maternal blood samples were taken during or soon after delivery. Samples were collected using venous puncture into 5 mL tubes (Vacutainer; Becton-Dickinson). The serum was then taken to the laboratory and was directly separated out from the whole blood and frozen at −80 °C until all samples were analyzed. Amino acid levels were analyzed using liquid chromatography-tandem mass spectrometry (LC-MS/MS) 6460 Triple Quad with 1290 Infinity Binary Pump (Agilent Technologies^®, USA) and converted into µM (µmol/L).

Macrophages (5 × 10⁵ cells/mL in 24-well plate) were infected with A. fumigatus conidia at a 1:2 effector-to-target ratio for 6 h at 37°C in 5% CO₂. Cells were detached with accutase (GRiSP), and the resulting cell suspensions were pooled from three different wells. The resulting cell suspensions were centrifuged at 4°C, the supernatant discarded, and the resulting pellet was immediately frozen in liquid nitrogen to quickly quench the cell metabolism. The analysis of the metabolites was performed with liquid chromatography-mass spectrometry (LC-MS/MS). The analysis was performed in an Agilent 1290 Infinity using a 1290 Infinity Binary Pump (1200 bar) and a 1260 Infinity Quaternary Pump (400 bar). The LC system was coupled to an Agilent 6470 triple-quadrupole mass spectrometer using an electrospray ionization (ESI) interface working in multiple reaction monitoring (MRM) mode. The chromatographic method used HILIC interaction chromatography (Agilent InfinityLab Poroshell). MRM transitions were chosen according to the Metabolomics dMRM library and method from Agilent Technologies (p/n G6412AA).

Dangguisu-san powder (10.0 g) was dissolved in 100 mL methanol (100%, v/v) and subjected to ultrasonic extraction at room temperature for 60 min. The extract was filtered through qualitative filter paper (5–8 µm, 150 mm) and concentrated under reduced pressure until dry. The dried samples were dissolved in methanol (100%, v/v) to prepare 10 mg/mL of a final concentration. HPLC analysis was performed using an Agilent 1290 HPLC system (Agilent Technologies, Santa Clara, CA, USA) equipped with a 1290 Infinity Binary Pump, autosampler, and photodiode array (PDA) detector. Separation was performed on a YMC-Pack Pro C18 RS (2.0 mm i.d. × 150 mm, 3.0 µm). The mobile phase consisted of H₂O (containing 0.1% formic acid; solvent A) and CH3CN (containing 0.1% formic acid; solvent B) with the following gradient elution profile: 0.00–20.00 min, 10–100% B; 20.10–25.00 min, 100% B; 25.10–30.00 min, 10% B. The flow rate was set at 0.300 mL/min and the injection volume was 10.00 µL. Mass spectrometry was performed on an Agilent 6530 Q-TOF mass spectrometer (Agilent Technologies, Santa Clara, CA, USA) equipped with an electrospray ionization (ESI) interface and analyzed in the negative mode. All MS data were acquired using the MassHunter Data Acquisition Software and the “Find by Formula” function was used to detect the four key components.

An Agilent 1290 Infinity liquid-chromatography system was used, consisting of a 1290 Infinity binary pump (G4220A) with a Jet Weaver V35 mixer, a 1290 Infinity Flexible Cube Solvent Management module (G4227A), a 1290 Infinity HiP sampler (G4226A), a 1290 Infinity Thermostated Column compartment (G1316C) with an IM-qTOF-MS (Agilent 6560), equipped with a Dual Agilent Jet Stream electrospray ionization (AJS ESI) Source. The instrument was used in qTOF-only mode; that means the trapping gate is off and ions are just transferred through the drift tube to the rear funnel, so that there is no separation by ion mobility and the system is used like a conventional qTOF instrument. For detailed information about the chromatographic and mass spectrometric setup, we refer to the supporting information S1 File.

After confirming the higher biocompatibility of wPB compared to ePB, we identified the chemical compositions of wPB in accordance with our previous protocol [31 (link)]. In summary, an LC–MS instrument (Time-of-Flight/6500 series, Agilent, Palo Alto, CA, USA) connected to a high-performance liquid chromatography (HPLC) system (1290 Infinity Binary Pump, Agilent) with an electrospray ionization (ESI) interface was utilized for chemical profiling. Chromatographic separations were carried out using a C18 column (5 µm, 150 mm × 2.1 mm; ZORBAX Eclipse Plus C18, Agilent) at a flow rate of 0.2 mL/min, with two mobile phases. The gradient system consisted of high A for the first 3 min, high B for the next 15 min, and then high A again (A: 0.1% formic acid in water and B: 0.1% formic acid in acetonitrile). The column temperature was maintained at 40 °C, and the injection volume was 3 µL, with a total run time of 30 min. Spectra were obtained in ESI- modes using a photodiode array detector. An Agilent Database Library was used for compound verification.