Acquity uplc beh c8 column

UPLC/MS analysis was performed using a Thermo Scientific™ Dionex™ ΜltiMate™ 3000 Rapid Separation LC (Thermo, Massachusetts, United States) equipped with a Q Exactive system (Thermo, Massachusetts, United States). Plasma samples were separated by a Waters Acquity UPLC BEH C8 column (2.1 × 100 mm, 1.7 μm) kept at 30°C. The mobile phase A was acetonitrile/water (60/40), and the mobile phase B was isopropanol/acetonitrile (90/10). Both A and B contained 0.1% formic acid and 10 mmol/L ammonium formate. The gradient elution condition was as follows: 0−1 min, 98% B; 1−5 min, 98%–30% B; 5−8 min, 30%–0% B; 8−14 min, 0% B. The flow rate was 0.25 mL/min. Mass analysis was carried out in both positive and negative mode with 2.5 kV of spray voltage and 320°C of the capillary temperature. The scan range was m/z 100-1500.

Blood plasma concentrations of INH and its main metabolites AcINH and INA were determined at three time points (pre-dose (0 h), 2 h and 6 h after drug intake) using liquid chromatography-tandem mass spectrometry (LC-MS/MS) method as previously described (Kivrane et al., 2021 (link)). Briefly, plasma samples were analysed using An Acquity UPLC H-Class chromatographic system (Waters, Milford, MA, United States) coupled to a XEVO TQ-S tandem mass spectrometer (Waters, Milford, MA, United States). The chromatographic separation was achieved on the Waters Acquity UPLC BEH C8 column (2.1 mm × 75 mm; 1.7 μm). The multiple reaction monitoring mode (MRM) was used for detection and quantification of the analytes. Data acquisition and analysis were performed using MassLynx software and TargetLynx module (version 4.1., Waters, Milford, MA, United States). Calibration ranges were 0.16–10.0 μg/mL for INH, AcINH and INA, respectively; the lower limits of quantification (LLOQ) were 0.16 μg/mL for INH and both two primary metabolites. The within- and between-run accuracy in all concentration levels ranged from 87.2% to 113.6%, but within- and between-run precision was between 1.6% and 14.9% (at the LLOQ level CV<20%).

Analysis of 3-hydroxybutyric acid (BHB) was done with LC-MS, performed on an I-Class UPLC system (Waters Corp., Milford, MA, USA) coupled to a Thermo Scientific Q Exactive Plus mass spectrometer (Thermo Scientific, Bremen, Germany) operating in negative mode over the mass range 65–600 m/z at a resolving power of 35,000 (at m/z = 200), as previously described [15 (link)]. INS1E cells grown to confluence in 6-well plates were treated with the vehicle (DMSO), the GPR40 antagonist GW1100 (50 nM), the PLCβ inhibitor U73122 (2 μM), the IP3 receptor inhibitor (-) Xestospongin C (3 μM) or the l-type Ca²⁺ channel inhibitor Nifedipine (0.1 μM) for 15 min prior to incubation with 200 µM MCFA for 2 h in RPMI medium with 1 mM glucose. BHB was measured in the media by chromatographic separation performed on an Acquity UPLC BEH C8 column (1.7 mm, 100 × 2.1 mm; Waters Corporation, Milford, MA, USA). BHB was quantified using the generated 8-point external calibration curve with Xcalibur Software 4.0 (Thermo Scientific Inc., Waltham, MA, USA).

Data acquisition was performed using a Q Exactive (Thermo Fisher Scientific, USA). A Waters ACQUITY UPLC BEH C8 column (particle size, 1.7 pm; 100 mm (length) × 2.1 mm (i.d.)) was used for LC separation. Mobile phase A was 40% ethyl cyanide in water, and mobile phase B was 10% ethyl cyanide in isopropanol. Both phases A and B had 0.1% ammonium hydroxide (NH₄OH) and 0.1% ammonium acetate (NH₄OAc). The flow rate was 0.25 mL/min, and the gradient was set as follows: 0-1 min, 98% B; 1-5 min, 98% B to 30% B; 5-8 min, 30% B to 0% B; 8-14 min, 0% B; 14-16 min, 0% B to 98% B. The QC samples were prepared by pooling aliquots of several subject samples and injecting every sample after washing and balancing (total 5 samples and 5 QC samples).
The data acquisition was operated in full MS scan mode and ddMS2 scan mode. The source parameters were set as follows: spray voltage, 2,500 V or -2,500 V for positive or negative modes, respectively; capillary temperature, 320°C; ion source, HESI. The resolution for full MS scan mode was set at 70,000, and the AGC target was set at 3e6 for both positive and negative modes. The maximum IT was set at 100 ms. The mass range was set at 100-1,500 Da. For the dd-MS2 scan mode, the MS resolution was set at 17,500, and the AGC target was set at 1e5. The maximum IT was set at 50 ms. The collision energy was set at SNCE 20-30-40%.

The LC and MS measurement conditions were as follows: Serum samples were subjected to a Nexera UHPLC system and LCMS-8060 triple quadrupole mass spectrometer (Shimadzu Co., Kyoto, Japan). An Acquity UPLC BEH C8 column (1.7 µm, 2.1 × 100 mm; Waters) was used with the following mobile phase compositions: 5 mM NH₄HCO₃/water (mobile phase A), acetonitrile (mobile phase B), and isopropanol (mobile phase C). The pump gradient was programmed as follows [time (%A/%B/%C)]: 0 min (95/5/0), to 8 min (70/30/0), 16 min (30/35/35), 28 min (6/47/47), 35 min (6/47/47), 35.1 min (95/5/0); it was then held for 38 min for equilibration. The flow rate was 0.35 mL/min, and the column temperature was 47 °C. The injection volume was 5 µL. SRM analysis was performed using positive/negative ion-switching mode ESI, with a collision energy of 46 eV. All data were analyzed using Microsoft Excel 2016.

To assess the ceramide species profiling, mass spectrometry-based lipidomics assay was performed as described before^{45 (link)}. Briefly, heart tissues were homogenized in ice-cold chloroform-methanol (2:1). Internal standard (50 pmol CER[N(25)S(18)] (Avanti Polar Lipids, Alabaster)) was added to each sample. The resulting lipid extract was analyzed by ultraperformance liquid chromatography (Acquity UPLC, Waters) coupled to a triple quadrupole mass spectrometer with electrospray ionization (Xevo TQ-S, Waters). Ceramides were separated using an Acquity UPLC BEH C8 column (1.7 µm, 2.1 × 100 mm, Waters) and quantified by multiple reaction monitoring assays. Data are expressed as relative amount (pmol) of ceramide per mg of tissue protein (Protein Assay II, Bio-Rad).

The chemical components of strong powder tea and strong leaf tea were analyzed with LC-MS/MS system (Maxis 3G, Bruker, MA, USA) equipped with ACQUITY UPLC BEH C8 Column (1.7 µm, 2.1 mm × 50 mm, Waters, MA, USA). Column oven temperature was 40 °C. Mass spectra were acquired in ESI negative modes. The tea samples were filtrated with the Acrodisc PVDF membrane filter. The binary gradient 10 mM ammonium formate in water (eluent A) and acetonitrile containing 10 mM ammonium formate (eluent B) in the negative mode was applied with injection volume of 5 μL at a constant flowrate of 0.2 mL/min. The gradient program was as follows: Rate of elute A: 0–10 min: 0%→95%; 10–13 min: 95%; and 13–13.1 min: 95%→0%. The detected molecular masses were found in the Human Metabolome Database [36 ] using MS Search.