Acquity uplc h class plus

All of the samples were analyzed by reverse phase ultra-performance liquid chromatography (RP-UPLC) using a Watters ACQUITY UPLC H-Class PLUS (Waters Corporation, Milford, MA, USA) system adjusted to 215 nm.
The hydrolysis fractions were then injected into a C4 Colum (2.6 µm 150 × 2.1 mm) after filtration at 0.22 µm with polyvinylidene difluoride (PVDF) filters. The injection volume was 2.5 μL. The flow rate was set at 0.3 mL·min⁻¹.
The elution program was as follows: The mobile phases were ultrapure water/trifluoroacetic acid (99:1, v/v) as solvent A and acetonitrile/trifluoroacetic acid (99:1, v/v) as solvent B. A gradient was applied with solvent B increasing from 5% to 30% in 30 min, then to 60% for 10 min and held until 47min at 95%, then back to initial conditions. UV absorbance scans were performed between 200 and 390 nm at a rate of one spectrum per second with a resolution of 4.8 nm. Chromatographic acquisition and analysis were performed with Empower 3 software (Version 3 Waters). Each sample analysis was performed in triplicate to ensure technical reproducibility.

CES1
knockout cells
as well as their parental THP-1-IFNAR2KO cells (10⁶) were
treated with 50 μM BDW568 for the indicated time. Cells were
collected and washed by 1× PBS once, and then 50% acetonitrile
was added and vortexed for 1 min. Samples were centrifuged at 17,000 g for 10 min to remove insoluble cell debris. Compound 4 (0.5 μL at
1 mM; see Supplementary Information) was
added to the supernatant of each sample (19.5 μL) as an internal
standard for UPLC-MS analysis. Ten microliters of the mixed solution
were injected into UPLC-MS for analysis with the following conditions:
instrument: Waters ACQUITY UPLC H Class Plus in tandem with a Qda
mass detector; column: ACQUITY UPLC BEH C18 1.7 μm (21 ×
50 mm); mobile phase: acetonitrile and 0.1% formic acid water solution;
gradient: 0 min, 2% acetonitrile; 3 min, 98% acetonitrile; 5 min,
2% acetonitrile; UPLC-MS results were analyzed using Waters Empower
3 software.

(S)-Warfarin 7-hydroxylation by CYP2C9 was measured as previously reported, with several modifications [23 (link)]. The reaction mixture, in a total volume of 150 μL, consisted of the following components: the microsomal fraction (25 μg), (S)-warfarin (0.2, 0.5, 1, 2, 5, 10, 20, or 40 μM), and 100 mM potassium phosphate buffer (pH 7.4). Following pre-incubation at 37 °C for 3 min, reactions were initiated by the addition of 10 mM NADPH, with incubation at 37 °C for 60 min. Reactions were terminated by adding 150 μL of acetonitrile containing 25 nM 7-ethoxycoumarin as an internal standard. After protein removal by centrifugation at 15,400× g for 10 min, 10 μL of the supernatant was injected into an ultra-high performance liquid chromatography (UPLC)-fluorescence system consisting of an ACQUITY UPLC H-Class PLUS (Waters, Milford, MA, USA), ACQUITY UPLC FLR Detector (Waters), and an ACQUITY UPLC HSS C18 column (2.1 × 50 mm, 1.8-μm particle size; Waters) maintained at 40 °C. The mobile phase was a mixture of acetonitrile and water (40:60, v/v) containing 0.1% formic acid at a flow rate of 0.5 mL/min. (S)-7-Hydroxywarfarin content was measured at an excitation wavelength of 320 nm and an emission wavelength of 415 nm. Standard curves were constructed in the 12.5–6400 nM range using metabolite standards, with a quantification limit of 10 nM for (S)-7-hydroxywarfarin.

The lyophilized culture broth samples were dissolved in methanol–water mixture (1:1, by vol.) and centrifuged (15 min, 15,000 rpm; centrifuge ELMI CM-50, Latvia). The samples were chromatographed on an ACQUITY UPLC H-Class PLUS (Waters, Santa Clara, CA, USA) equipped with a time-of-flight mass-selective detector Xevo G2-XS TOF (Waters, USA). An 0.5 µL sample aliquot was injected on a Titan C18 (100 × 2.1 mm, 1.9 µm; Supelco, Bellefonte, CA, USA) column maintained at 50°C and eluted with a gradient of 10 mM solution of ammonium acetate in deionized (Simplicity UV, Millipore, France) water (solvent A) and LC-MS grade (Panreac, Barcelona, Spain) acetonitrile (solvent B) delivered at a rate of 0.5 mL/min. The following gradient program was used (vol. % of the solvent B): 0–1 min, from 5% to 15%; 1–2 min, from 15% to 65%; 2–3 min, from 65% to 85%; 3–5 min, from 85% to 95% (see also [33 (link)]). The analysis was carried out in the negative ion detection mode (m/z range of 100–1900), the ion source parameters were: ion source temperature—150 °C, desolvation temperature—650 °C; capillary voltage—3.0 kV; sample injection cone voltage—30 V; nitrogen (desolvation gas) flow rate—1101 L/h. The recorded data were processed with MassLynx v. 4.2 software (Waters, USA) and CTA was quantified using external calibration method.

Sphingolipids were measured using the LC–MS/MS system (LC: Acquity UPLC^® H-class PLUS; MS: Xevo TQ-S micro, WATERS Corporation). Briefly, 5.0 μL samples were injected and the LC separation was performed using a general-phase column [U-InertInertSustainSwift Amide 3um column: 2.1 × 100 mm (UP), GL Sciences Inc, USA ] with a gradient elution of solvent A (10 mM Ammonium acetate, 95% acetonitrile, 5% water) and solvent B (10 mM Ammonium acetate, 50% acetonitrile) at 0.6 mL/min. The conditions were as follows: A gradient run was performed at 99% solvent A and 1% solvent B for 1 min, followed by an at 90% solvent A and 10% solvent B for 1 min, next 3 min performed at 60% solvent A and 40% solvent B which followed by an at 20% solvent A and 80% solvent B for 2 min. Total run time 11 min, target column temperature 50.0a°C, target sample temperature 10.0 °C.
The mass spectrometer was operated in electrospray ionization-positive ion mode and the analytical conditions were as follows: the cone gas flow was set at 50 (L/Hr), the desolvation gas flow at 1000 (L/Hr), the source temperature at 150.0 °C, desolvation temperature at 600.0 °C.
The analyses were performed in multiple reaction monitoring (MRM) mode in the positive ion mode for sphingolipids. The MRM settings are described in Supplemental Table S1. The data were analyzed by MassLynx, TargetLynx XS (WATERS Corporation).