DG-3 was assessed for its ability to react with GSH in vitro using a slightly modified procedure from previously reported methods (18 (link)–20 (link)). From 10 mM stock solutions in DMSO, 5 μl of aliquots were transferred to an analytical high-performance LC (HPLC) vial containing 495 μl of reaction buffer (50 mM Hepes, buffered to pH 7.4) and with or without 10.0 mM reduced GSH. Samples were taken at 30 min and 18 hours for a Hewlett Packard Series 1100 analytical HPLC-MS system using an ACE C8 column (50 mm by 3.0 mm) with 3-μm particles and flow rate of 1 ml/min. Samples were eluted using a gradient of A, 0.1% trifluoroacetic acid in H2O, and B, acetonitrile, 10 to 90% B over 1.5 min and an additional minute at 90% B. Changes in the absorbance profile at 254 nm were observed, and the HPLC peak areas corresponding to the decay of the parent compound were recorded. Metabolite identification was based on mass/charge ratio values and reported alongside their retention time (tR).