Tqd instrument

All compounds to be screened are delivered from in-house Sample Management as 10 mM DMSO solutions in 96-well format. The compounds are diluted in a 1:2 AcN/water (0.1% FA) solution. We consider 1 μM optimal for optimization on the Waters Xevo TQS instrument and 5 μM for using the Waters TQD instrument. The QuanOptimize software (Waters) is used as a high-throughput tool for automated method development and batch processing of quantitative bioassays. The QuanOptimize routines can ramp and optimize cone voltage and collision energy. The cone voltage is set to 35 eV for all compounds and only the collision energy is optimized and ramped both in positive and negative mode between 10 and 50 eV in steps of six.
The software identifies the most intense fragment and the corresponding collision energy and creates an MRM transition, which is stored in an MS/MS library. The MS/MS library is shared between the LC-MS/MS instruments. QuanOptimize is also used for creating the sample list by generating MS/MS files from the MS/MS library combining up to 13 different MRM transitions, LC file, MS/MS tune file, and creating quantification methods used for peak integration, calibration, and quantification.

Liquid chromatography-mass spectrometry (LC–MS) analysis were performed to validate whether the active compounds found by network pharmacology are actually existed in SH003. LC–MS analysis was conducted using a Waters TQD instrument. The reference component (four active compounds, 1.0 mg each) was weighed, dissolved in 1.0 mL of methanol to prepare a solution at a concentration of 1.0 mg/mL, then diluted. SH003 powder (10.0 mg) was weighed, sonicated in 1 mL of methanol for 10 min, and filtered through a 0.22 μm syringe filter. Chromatographic separation was achieved on an InfinityLab Poroshell 120 EC-C18 column (100 × 2.1 mm, 2.7 µm, Agilent) with a gradient elution profile using mobile phases A (0.1% Formic acid, 5 mM Ammonium Formate in Water) and B (0.1% Formic acid, 5 mM Ammonium Formate in Methanol). The gradient program commenced with 99% A at 0 min, transitioned to 20% A at 3 min, maintained this composition until 4 min, shifted to 1% A at 5 min, increased to 8% A at 8 min, returned to 99% A at 8.5 min, and persisted until the end of the 12-min analysis. The column temperature was maintained at 40 ℃, and the flow rate was set at 0.5 mL/min. Subsequent network pharmacology analysis was performed for the compounds validated via chromatography analysis.

Preparative scale liquid chromatography was used to purify SFAE products. The crude reaction mixture was purified on VersaPack^TM C18 (SUPELCO Analytical, Bellefonte, PA, USA) preparative column in the MeOH/H₂O system with an increasing MeOH gradient of 50 to 100%. The elution of the reagents was monitored with TLC chromatography performed on C18 Merck silica gel plates (with the same elution system as in preparative separation) and high performance liquid chromatography coupled to mass spectrometer: Waters TQD instrument in scan mode ESI: +100–1000 m/z.