Agilent 6540 q tof

To assess the activity of FAO, cells were treated with vehicle control, etomoxir, scrambled siRNA, or CPT1A siRNA for 48–72 hours. Next, the medium was refreshed with new medium containing 100 μM uniformly ¹³C labeled (U-¹³C) palmitate-BSA and 100 μM natural abundance oleate-BSA. After labeling for 24 hours, cells were harvested, extracted, and analyzed as previously described [17 (link)]. For U-¹³C glucose, U-¹³C glutamine, and U-¹³C palmitate tracing experiments, cells were transferred to media containing ¹³C label and either vehicle control or 200 μM etomoxir for 12 hours, 6 hours, and 24 hours, respectively. The polar portion of the extract was separated by using a Luna aminopropyl column (3 μm, 150 mm × 1.0 mm I.D., Phenomenex) coupled to an Agilent 1260 capillary HPLC system. Mass spectrometry detection was carried out on an Agilent 6540 Q-TOF coupled with an ESI source operated in negative mode. Isotopic labeling was assessed comprehensively by using the X¹³CMS software [20 (link)]. The identity of each metabolite was confirmed by matching retention times and MS/MS fragmentation data to standard compounds. The isotopologue distribution patterns presented were obtained from manual evaluation of the data and calculated by normalizing the sum of all isotopologues to 100%. Data presented were corrected for natural abundance and isotope impurity.

Commercially available reagents were purchased from Sigma Aldrich or Acros and used without further purification. Acyl Meldrum’s acids 9a–f and enaminones 10a–d were prepared according to literature procedures; 9a, 9b, 9c, 9f^{44 (link)}, 9d, 9e^{45 (link)}, 10a–d^{46 (link)}. Analytical thin layer chromatography was performed on aluminum sheets of UV 254 Merck silica gel, and flash chromatography using SilicaFlash P60 silica gel (40–63 µm). ¹H and ¹³C NMR spectra were recorded with Bruker Avance III HD 400 MHz or Varian Gemini 500 MHz and NMR chemic al shifts were reported in δ (ppm) using residual solvent peaks as standards, with the coupling constant J measured in Hz. High resolution mass spectra were recorded with an Agilent 6540 Q TOF system High resolution (HRMS) was recorded on Agilent 6540 QTOF.

HPLC is a chromatographic technique for the separation of multicomponent samples that can process compounds of different molecular weights and polarities. It is widely used for the identification, separation, and purification of chemical components in herbal formulas [30 ]. The sample solutions were injected into the HPLC system (Agilent 6540 Q-TOF and Agilent 1290 UHPLC, Agilent, Santa Clara, CA, USA) and separated using an ultrahigh performance-LC HSS T3 column (2.1 mm × 100 mm, 1.8 μm, Elite, DaLian, LiaoNing, CN). The mobile phase consisted of 100% ultrapure water, 0.1% methanol (a), and 100% acetonitrile (b). The gradient elution program was as follows: 2% B at 0–1 min, 2%–100% B at 1–55 min, 100% B at 55–60 min, and 100%–2% B at 60–61 min. The flow rate was 0.4 mL/min, the injection volume was 10 μL, and the column temperature was maintained at 50°C.