Agilent 6120 single quadrupole ms

Ferric cyt c (400 µM) or ABTS (400 µM) was mixed with GSSSG or GS³⁴SSG (400 µM), NADPH (400 µM) and GR (1 U ml⁻¹) in a total volume of 100 µl in PBS. After 5 minutes, the reaction was stopped by the addition of 100 µl of MBB in MeOH (10 mM). The samples were incubated for 5 minutes in the dark, and then 200 µl of CHCl₃ was added to remove GR by precipitation. After centrifugation at 300 RCF, the upper phase was removed for further analysis. The sample (10 µl) was injected into an Agilent 1260 Infinity LC system attached to an Agilent 6120 Single Quadrupole MS with ESI source and evaporative light scattering detector (ELSD). Separation was performed on a Kinetex 2.6 μm C18 100 Å LC column (50 × 2.1 mm) at 40 °C using a flow rate of 0.6 ml min⁻¹. Solvent ‘A’ was 0.01% HCOOH in water; solvent ‘B’ was 0.01% HCOOH in MeCN. The method was: 100% A for 2 minutes, then from 100% to 10% A in 10 minutes and then 1% A for another 10–12 minutes. Data were processed with MestReNova (14.2.1) software.