Lc tofms

HUVECs were seeded into 6-cm dishes (1 × 10⁵ cells per dish) and incubated with EGM-2 containing 50 μM 5-HETE, 50 μM 5-HEPE, or 5 μM 5-oxo-ETE for 6 h. After incubation, a portion of the medium was collected and mixed with three times the volume of acetonitrile containing 0.1% formic acid. The diluted media were centrifuged, and the supernatant was collected. Cells were washed twice with PBS and harvested using a scraper. The cell suspensions were centrifuged, and the pellets were sonicated with 0.1% formic acid in acetonitrile. 5-HETE, 5-HEPE, and 5-oxo-ETE were separated on an InertSustain ODS-3 column (2.0 mm diameter. × 250 mm; GL Science Inc., Tokyo, Japan) with gradient elution (10 mM ammonium acetate solution/acetonitrile, 55/45 to 5/95 in 25 min) at a flow rate of 0.2 mL/min. The compounds were identified and quantified by liquid chromatography-time of flight mass spectrometry (LC-TOFMS) (Agilent Technologies, Santa Clara, CA, USA) using Agilent Mass Hunter Workstation Software (version B.07.00 Service Pack 2, Agilent Technologies, Santa Clara, CA, USA). The velocity of drying gas was 10 L/min. The temperature of the gas was 325 °C. The voltages of the Vcap, fragmenter, and skimmer were 3500, 125, and 65 V, respectively. The pressure of the nebulizer was 30 psig.

Optical rotations were measured on a Rudolph Research Analytical AUTOPOL IV digital polarimeter at 589 nm. UV absorptions were acquired with an Agilent Cary 60 UV-vis spectrophotometer. IR spectra were recorded with an Agilent Cary FTIR 630 spectrometer and PerkinElmer Spectrum Two equipped with a UATR (single reflection diamond) sample introduction system. NMR spectra were recorded on Varian Direct Drive 500 MHz and Varian Inova 500 MHz spectrometers. Chemical shifts are reported with the use of the residual CDCl₃ signals (δ_H 7.27 ppm; δ_C 77.0 ppm) as internal standards for ¹H and ¹³C NMR spectra, respectively. COSY, HSQC, HMBC, and ROESY experiments corroborated the ¹H and ¹³C NMR assignments. Analytical LC/MS with a Phenomenex Kinetex C18 column (50 × 2.1 mm, 2.6 μm) on an Agilent 6230 LC/TOF-MS with electrospray ionization detection provided the high-resolution masses. Semi-preparative and analytical HPLC separations were performed on a Shimadzu LC-20 AT system equipped with an ultraviolet (UV) detector using a Luna silica column (5 μm, 250 × 10 mm), and a YMC C-18 column (10 μm, 150 × 4 mm). MPLC was performed on a Teledyne Isco CombiFlash Rf 200i equipped with an evaporative light-scattering detector (ELSD) and a multiwavelength UV detector using a RediSep Rf silica 80 g flash column, and silica gel 230–400 mesh was used to load samples.