Waters acquity uplc beh c18 column

The serum metabolites were analyzed on Waters 2D UPLC (Waters, Milford, MA, USA) using a Waters 2D UPLC (Waters, USA) linked to a Thermo Fisher Scientific Q-;Exactive mass spectrometer with a heated electrospray ionization (HESI) source and the Xcalibur 2.3 software application (Thermo Fisher Scientific, Waltham, MA, USA). Then, we used Waters ACQUITY UPLC BEH C18 column (1.7 μm, 2.1 mm × 100 mm, Waters, Milford, MA, USA) to complete the chromatographic separation and kept the column temperature constant at 45 °C. In the positive mode, the mobile phase was 0.1% formic acid (A) and acetonitrile (B). The mobile phase included 10 mM ammonium formate (A) and acetonitrile (B) in the negative mode. The mass spectrometric settings for the two ionization modes were given in standard methods [18 (link)]. The identification of metabolites meets the levels published by the Metabolomics Standards Initiative (MSI). [19 (link)]

The extracts above were redissolved separately in methanol and passed through a 0.45 µm membrane. A 2 µL of sample was then injected into a Waters ACQUITY UPLC BEH C₁₈ column (2.1 mm × 50 mm, 1.7 µm, 45 °C) connected to a Waters ACQUITY UPLC/Xevo G2 Qtof MS system (Waters Corporation, Milford, MA, USA) equipped with an electrospray source. The column was eluted as follows: 0 min − 95% A + 5% B, 10 min − 0% A + 100% B, where A was water containing 0.1% formic acid and B was acetonitrile. The full scan data were acquired in the positive ion mode from 50 to 1200 Da with a 0.2 s scan time using the following settings: capillary voltage 3.0 kV; de-solvation temperature 350 °C; sample cone voltage 35 V; extraction cone voltage 4 V; source temperature 120 °C; cone gas flow 50 L/h; and desolvation gas flow 800 L/h. The mass spectrometer was calibrated across the mass range of 50–1200 Da using a solution of sodium formate. Data were centroided and m/z values were corrected during acquisition using an external reference consisting of a 0.2 ng/mL solution of leucine enkephalin infused at a flow rate of 5 μL/min via a lockspray interface, generating a reference ion at 556.2771 Da ([M + H]⁺). The lockspray scan time was set at 0.5 s with an interval of 15 s and data were averaged over three scans.

The UPLC fingerprints of the Rhodiola extracts were analyzed using a Waters Acquity UPLC system consisting of Quaternary Pumps Manager, Sample Manager, Column Manager, Photodiode Detector, and Empower 3 software (Waters Corporation, Milford, MA, United States). The UPLC column (Waters ACQUITY UPLC BEH C18, column; 100 mm × 2.1 mm i.d., 1.8 μm, Waters Corporation, United States) solvent system was gradually increasing concentrations (2.5 to 100% in 14 min) of acetonitrile in water with a flow rate of 0.6 mL/min at 75°C. The injection volume was 2 μL, detection was at 221 nm (phenylethanoids tyrosol and salidroside) and 252 nm (phenylpropanoids). All quantitative results were calculated per dry weight of the extracts.

For pharmacokinetic analyses, a cohort of 24 mice were administered a single dose of liposomal EF24 (10 mg/kg) intravenously through tail vein. Serum samples (2–4 mice per time point) were collected at 0.25, 1, 2, 3, 4, 6, 8 and 24 h and stored at −80 °C prior to and after use. LC–MS/MS analyses were carried out on a Waters Xevo TQ-S Triple-quad system equipped with a Waters Acquity I-Class UPLC system, an FTN sample manager, binary solvent manager and TUV detector. A Waters Acquity UPLC BEH C18 column (130 Å, 1.7 µm, 2.1 × 100 mm) together with a Waters Acquity UPLC BEH C18 VanGuard pre-column (130 Å, 1.7 µm, 2.1 × 5 mm) was used for separation. Data recording was achieved with the MassLynx 4.1 software package, data processing and quantification was performed with the integrated TargetLynx software using a quadratic regression model. Detailed LC–MS/MS methodology and data quantification is provided as Additional file 1.

For further investigation on the robustness of QAMS method, the mixed standard solution should be analyzed under different condition. The RCFs and RTT of the other target peaks to IRS were calculated in different flow rates, column temperatures, and columns using the AVG method and assessed by relative standard deviation (RSD, less than 5%) [39 (link)]. The flow rate was set at 0.3/0.4/0.5 mL/min with temperature maintained at 30/35/40°C, respectively. Waters ACQUITY UPLC® BEH C18 column (2.1 × 50 mm, 1.7 μm), Waters ACQUITY UPLC® BEH C18 column (2.1 × 100 mm, 1.7 μm), SHIMADZU Shim-pack GIST C18-HP column (50 mm × 2.1 mm, 3 μm), SHIMADZU Shim-pack GIST C18-HP column (100 mm × 2.1 mm, 3 μm), and SHIMADZU Shim-pack GIST C18-HP column (150 mm × 2.1 mm, 3 μm) were used to study the effect of different columns.

The samples were injected using an autosampler with stack cooler (Open Autosampler UltiMate OAS-3300TXRS, Thermo Fisher Scientific) and eluted through a Waters Acquity UPLC BEH C18 column (130 Å, 2.1 mm × 50 mm, 1.7-µm particles) preceded by a Waters Acquity UPLC BEH C18 VanGuard pre-column, 130 Å, 2.1 mm × 5 mm, 1.7-µm particles) using a Transcend II, LX-2 with UltiMate pumps (Thermo Fisher Scientific). The pressure limits were set at 0.0–1034.0 bar.
The mobile phase consisted of solvent A (97.31% H₂O, 1.25% ACN, 1.25% MeOH, and 0.2% FA) and B (2.49% H₂O, 48.66% ACN, 48.66% MeOH, and 0.2% FA). The Wash1 solvent was mobile phase A and the Wash2 solvent mix was 25:25:25:25 v/v MeOH/IPA/H₂0/ACN + 0.2% FA. The gradient (0.25 mL/min) started with 100% A/0% B. After 0.5 min, we applied a gradient ramp to 0% A/100% B over 8.5 min followed by a 0.5-min flow ramp up to 0.9 mL/min and 5 min of 100% B. At 15 min, the column was equilibrated for 5.5 min with 100% A. At 17.5 min, the flow was changed back to 0.25 mL/min over 0.5 min. The total run time was 20.5 min, including 10-min sample run time and 10.5-min column wash and equilibration. The column temperature was maintained at 60.0 °C using a hot pocket column heater and the samples in the autosampler were kept at 4 °C throughout the analysis. The data was acquired in profile mode from 0.20 to 9.80 min.