Glutathione Stability Assay by LC-MS

Glutathione stability was assessed using an assay adapted from a recently reported method27 (link). 250 μL of a 40 mM DMSO stock solution of each compound was added to L-glutathione (15.4 mg, 5 mol equiv.) suspended in 250 μL of DMSO. The resulting mixture was placed in a 37 °C shaker. 10 μL aliquots were removed and quenched in 990 μL of water (containing 0.5% formic acid) at various time points, including at t = 0 min, for analysis by ESI-LC-MS. All ESI-LC-MS analyses were collected on an API2000 LC/MS/MS System (Applied Biosystems) equipped with a turbo-ion spray ESI probe interfaced with a Prominence UFLC (Shimadzu) equipped with a reverse phase BDS Hypersil C18 50 × 2.1 mm column, particle size 3 μm (Thermo Scientific). HPLC/LCMS UV absorption was monitored at 254 nm and 210 nm. Both the compound and the glutathione adduct were identified by MS. Area of the UV peak was recorded for each time point.