1220 infinity lc

Inhibitory compounds were quantified by a 1220 Infinity LC high-performance liquid chromatography system (Agilent Technologies, CA, USA) equipped with a Zorbax Eclipse Plus C18 (4.6 mm x 250 mm, 5 μm) (Agilent Technologies, CA, USA). The mobile phases were A: (methanol 100 %) and B: formic acid-water (2.5 % v/v); the operating conditions were as follows: flow rate of 0.8 ml/min at 30 °C, with a flow gradient during 55 min from 0 % to 48 % of phase B, a pressure of 1200 ± 100 psi, and detection by a diode array detector (DAD) at wavelength screenings at 262, 275, 295 and 342 nm.

Generation of hydroxyl radical was estimated by measuring the conversion of salicylic acid into 2,5-DHBA [30] (link). H9c2 cells were treated with NE for appropriate duration and then loaded with salicylic acid (100 µM). After 2 h, cells were homogenized with ice-cold PBS (0.5 ml) and deproteinized by the addition of 10% perchloric acid containing 1 mM EDTA and 100 mM sodium pyrosulfite (1.5 ml). The deproteinized extracts were then centrifuged at 9000g for 10 min at 4 °C and 1 M HCl (0.4 ml) was added to the supernatant. The resulting solution was then extracted with 3 ml diethyl ether by vertexing for 1 min. The organic phase was separated by centrifuging at 3000g for 5 min, collected and evaporated to dryness under vacuum. The dried residue was dissolved in 0.03 M citrate/acetate buffer, pH 3.6 (the mobile-phase) and analyzed by HPLC using a zobrax C18 column (150×4.6 mm, 1220 Infinity LC, Agilent technologies, USA). The flow rate was kept at 1.0 ml/min. The 2,5-DHBA was detected at a wavelength of 315 nm and its content was expressed as nmol of DHBA/µmol of salicylic acid. The assay was validated by the estimation of hydroxyl radical generated by treating cells with 20 µM CuSO₄ and 1 mM l-ascorbate at 37 °C for 4 h [31] .