Characterization of dansylated glutathione, glutathione disulfide, cysteine and cystine by narrow bore liquid chromatography/electrospray ionization mass spectrometry

A method using reversed phase high performance liquid chromatography/electr ospray ionization-mass spectrometry (RP-LC/ESI-MS) has been developed to co nfirm the identity of dansylated derivatives of cysteine (C) and glutathion e (GSH), and their respective dimers, cystine (CSSC) and glutathione disulf ide (GSSG), Cysteine, GSH, CSSC and GSSG are present at low concentrations in rainbow trout (Oncorhynchus mykiss) liver cells. Initially, hepatic cell s were sampled from a suspension culture and disrupted upon addition of 10% perchloric acid. The reduced thiols present in the cell extracts were acet ylated to prevent dimerization and then the C and GSH species were derivati zed,vith dansyl chloride for fluorescence detection, An LC system using a w eak anion exchange column (AE) with fluorescence detection (FLD) was used f or sensitive routine analysis; however, it produced peaks of unknown origin in addition to the expected analytes. Analytes were then separated on a C1 8 RP-LC system using a water/acetonitrile gradient with 0.2% formic acid, a nd detected using LC/ESI-MS at 3.5 KV which produced an intense ion with a minimum limit of detection of less than 0.5 pmole injected (>10:1 signal-to -noise (S/N). Subsequently, fractions of effluent from the AE-LC/FLD system were analyzed by LC/ESI-MS to confirm the presence of the target analytes in routine cell extracts. Monodansylated GSSG was identified as a product t hat could possibly affect the quantification of GSH and GSSG, Copyright (C) 2000 John Wiley & Sons, Ltd.