Quantitative LC-MS Analysis of Glutathione

Reduced and oxidized glutathione were profiled in negative ionization mode by liquid chromatography tandem mass spectrometry (LC-MS) methods as described previously^{38 (link)}. Data were acquired using an ACQUITY UPLC (Waters Corp, Milford MA) coupled to a 5500 QTRAP triple quadrupole mass spectrometer (AB SCIEX, Framingham MA). Tissue homogenates (30 μL) were extracted using 120 μL of 80% methanol containing 0.05 ng/μL inosine-¹⁵N₄, 0.05 ng/μL thymine-d₄, and 0.1 ng/μL glycocholate-d₄ as internal standards (Cambridge Isotope Laboratories, Inc., Tewksbury MA). The samples were centrifuged (10 min, 9,000 × g, 4ºC) and the supernatants (10 μL) were injected directly onto a 150 × 2.0 mm Luna NH2 column (Phenomenex, Torrance CA). The column was eluted at a flow rate of 400 μL/min with initial conditions of 10% mobile phase A (20 mM ammonium acetate and 20 mM ammonium hydroxide (Sigma-Aldrich) in water (VWR)) and 90% mobile phase B (10 mM ammonium hydroxide in 75:25 v/v acetonitrile/methanol (VWR)) followed by a 10 min linear gradient to 100% mobile phase A. The ion spray voltage was −4.5 kV and the source temperature was 500°C. Raw data were processed using MultiQuant 2.1 software (AB SCIEX, Framingham MA) for automated peak integration. LC-MS data were processed and visually inspected using TraceFinder 3.1 software (Thermo Fisher Scientific; Waltham, MA).