Acquity uplc c8 column

Global metabolite profiling was performed using a Waters ultraperformance liquid chromatography/quadrupole orthogonal acceleration time of flight tandem micro-mass spectrometer (UPLC/Q-Tof Micro MS). Briefly, chromatographic separation was performed on a Waters Acquity UPLC C₈ column (1.8 μm particle size; 1.0 by 100 mm) using a gradient from 5% acidified methanol to 95% acidified methanol at a column temperature of 50°C and a flow rate of 140 μl/min. Column eluent was infused into a Waters Q-Tof Micro MS fitted with an electrospray source, and data were collected in positive ion mode. MS data (including retention times, m/z, and ion intensities) in MassLynx software (Waters) were converted to cdf format using Databridge software. Raw peak areas were normalized to total ion signal in R and subjected to statistical analyses. Identification and alignment of peaks for metabolites that were significantly different between the groups were performed based on accurate mass, isotopic pattern, and tandem MS (MS/MS) information obtained from Massbank (http://massbank.eu), Metlin (http://metlin.scripps.edu), The Human Metabolome Database (http://www.hmdb.ca), and ChemSpider (http://www.chemspider.com) (60 (link), 61 (link)).

LC–MS/MS analyses were performed using an LCMS-8050 triple-quadrupole mass spectrometer (Shimadzu, Japan). Chromatographic analyses were conducted on a Waters Acquity UPLC C₈ column (1.7 μm, 2.1 mm × 50 mm). The column temperature was maintained at 40°C and 2 μL of the sample was injected into the LC-30A UPLC system. Mobile phase A was 0.1% FA in water, and mobile phase B was 0.1% FA in acetonitrile. The flow rate was set to 0.4 mL/min. The gradient was set as follows: B, 5% (0 min) → 5% (0.5 min) → 50% (3 min) → 100% (4 min) → 100% (7 min) → 5% (9 min) → 5% (10 min). The total run time was 10 min. The gradient was shown in Supplementary Table S1.
The following interface settings were employed for sample analysis: ion transfer tube temperature, 300°C; sheath gas flow, 3.0 L/min; auxiliary gas flow, 10.0 L/min; and vaporizer temperature, 400°C. The desolvation temperature was maintained at 250°C. Quantification was performed by multiple reaction monitoring (MRM).

Serum daptomycin concentrations were measured using a high-performance liquid chromatography-tandem mass spectrometry method developed in our laboratory, the lower limit of quantification was 0.05 μg mL⁻¹. Briefly, serum samples were analyzed after protein precipitation. Daptomycin-d5 (Beijing Hager Biotechnology Co., Ltd.) was used as an internal standard. A Waters ACQUITY UPLC C8 column (2.1 mm × 50 mm, 1.7 μm) was used for separation. The mobile phase consisted of phase A: water (0.1% formic acid), phase B: 1:1 methanol: acetonitrile (0.1% formic acid) at a flow rate of 0.4 mL min⁻¹, column temperature of 45°C, injection volume of 2 μL, and analysis time of 4 min. Ion transitions were performed using positive electrospray ionization in the multiple reaction monitoring mode.
Before measurement, the sample was restored to room temperature, 100 μL of 1:1 methanol: acetonitrile was measured into a 1.5 μL EP tube, then 10 μL of daptomycin-d5 with 40 μL of sample/standard/quality control was added, the sample was vortex shaken for 1 min, centrifuged at high speed for 8 min (14,500 r/min), and the supernatant was placed in a 96-well plate for the assay.