Quantification of intracellular glutathione, glutathione disulfide, cysteine and cystine levels by HPLC-MS.

In order to accurately quantify the intracellular levels of labile thiol species we applied a modified extraction and sample derivatization procedure optimized to prevent oxidation prior to analysis. Cells cultured as above were quickly washed in 1 mL ice-cold PBS and then extracted once in 500 μL of an ice-cold extraction buffer consisting of 40% methanol, 40% acetonitrile and 20% water containing 100 mM formic acid and 1 mM EDTA; the addition of acid prevents the formation of the highly reactive thiolate anion, while the EDTA prevents oxidation due to metal ions. The levels of glutathione and cysteine were maintained for at least 48 hours under these conditions.

These extracts were derivatized with benzyl chloroformate (which reacts with thiols as well as amines) as above but with a different standard mixture [U-¹³C-¹⁵N-cysteine, 1 μg/mL; U-¹³C-¹⁵N-cystine, 10 μg/mL; 2×¹³C-1×¹⁵N-glutathione (labeled on the glycine carbons and nitrogen), 50 μg/mL; 4×¹³C-2×¹⁵N-glutathione disulfide. Analyte concentrations were quantified by comparison to standard curves prepared by the same method. To determine intracellular concentrations, the total cell volume on replicate plates was determined using packed cell volume tubes.