Esquire 6000

To study the formation of GSNO from NO₂-Ln and GSH, we incubated several concentrations of NO₂-Ln (0.1 and 1 mM) with 1 mM GSH in 50 mM phosphate buffer, pH 7.4, containing 0.1 mM DTPA (diethylenetetraminepentaacetic acid) for 1 h at RT^a with a gentle agitation. Reactions were conducted in darkness. Formation of GSNO was analyzed by LC-ES/MS (Bruker Esquire 6000, HPLC Agilent 1100) in negative ion mode. The different analytes were separated in a Waters Spherisorb ODS2 C18 column (3 mm × 125 mm, 5 μm). The mobile-phase composition was water (A) and acetonitrile (B) both with 1% of formic acid at a flow rate of 0.6 ml min⁻¹. The gradient profile was as follows: 2–5% B (0–5 min); 40–95% B (6–22 min); and 95–2% B (22–25 min). MS/MS/MS (M3) analysis from GSNO was conducted in 0.40 V (335) and 0.60 V (305). The desolvation temperature was set at 400°C. In all cases, the data were collected, analyzed, and processed using Data Analysis Mass Spectrometry Software (Bruker, Daltonics).

Mass spectra before and after electrolysis were recorded using a Bruker Esquire6000 mass spectrometer fitted with an Agilent electrospray (ESI†) ion source.