C18 nucleosil 100 3 125 4 column

Reactions were performed in 0.1 M potassium phosphate buffer at pH 7.4 and 37 °C and all reactive compounds were handled as described previously [30] (link). Authentic peroxynitrite was rapidly mixed using a Vortex mixer with salicylaldehyde and incubated for 5 min. All reactions of Sin-1, spermine NONOate, NADPH oxidase inhibitor-VAS2870, xanthine oxidase with hypoxanthine as well as horseradish peroxidase/nitrite/hydrogen peroxide were incubated for 90 min under the above described conditions. 50 µl aliquots of the samples were subjected to high performance liquid chromatography (HPLC) analysis. The system consisted of a control unit, two pumps, a mixer, detectors, a column oven, a degasser, an autosampler (AS-2057 plus) from Jasco (Groß-Umstadt, Germany), and a C18-Nucleosil 100-3 (125×4) column from Macherey & Nagel (Düren, Germany). A high-pressure gradient was employed with the organic solvent (90 vv% acetonitrile/10 vv% water) and 50 mM citrate buffer pH 2.2 as mobile phases with the following percentages of the organic solvent: 0 min, 10%; 14.5 min, 42%; 15 min, 10%; 16.5 min, 10%. The flow was 1 ml/min, compounds were detected by their absorption at 280, 330 and 380 nm, and salicylic acid was also detected by fluorescence (Ex. 292 nm/Em. 408 nm). Typical retention times of all standards are shown in Fig. 1.