GSH Adduct Standard and LC-MS Analysis

GSH adduct standard and LC-MS Analysis A glutathionylated 15-oxo-LXA₄ standard was made by incubation of 10 µM 15-oxo-LXA₄-Me with 100 µM GSH in 50 mM potassium phosphate buffer (pH = 8) for 1 hr at 37°C (91 (link),97 (link)). GSH conjugates were extracted from 1 mL of cell supernatant using Oasis HLB 1 cc solid phase extraction columns (Waters) and applied to a Luna C18 column (2 x 100 mm, Phenomenex) at a flow rate of 0.25 mL/min and eluted with a linear consisting of solvent A (H2O + 0.1% acetic acid) and Solvent B (ACN + 0.1% acetic acid). The gradient started at 20% B at 5 min and increasing to 98% B at 25 min. The gradient was held at 100% B for 2 min and equilibrated at 20% B for 35 min. GSH adducts were analyzed on a 6500+ QTRAP coupled to an Exion LC (Sciex) using multiple reaction monitoring (658→308) and positive ionization with the following MS conditions: CUR 40, CAD med, IS 4500, GS1 70, GS2 65, Temp 550°C, DP 80, EP 7, CE 17, CXP 7.