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

Abstract

A method using reversed phase high performance liquid chromatography/electrospray ionization-mass spectrometry (RP-LC/ESI-MS) has been developed to confirm the identity of dansylated derivatives of cysteine (C) and glutathione (GSH), and their respective dimers, cystine (CSSC) and glutathione disulfide (GSSG). Cysteine, GSH, CSSC and GSSG are present at low concentrations in rainbow trout (Oncorhynchus mykiss) liver cells. Initially, hepatic cells were sampled from a suspension culture and disrupted upon addition of 10% perchloric acid. The reduced thiols present in the cell extracts were acetylated to prevent dimerization and then the C and GSH species were derivatized with dansyl chloride for fluorescence detection. An LC system using a weak anion exchange column (AE) with fluorescence detection (FLD) was used for sensitive routine analysis; however, it produced peaks of unknown origin in addition to the expected analytes. Analytes were then separated on a C18 RP-LC system using a water/acetonitrile gradient with 0.2% formic acid, and 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 that could possibly affect the quantification of GSH and GSSG.

Chromatographic and mass spectrometric analysis of glutathione in biological samples

“…5) or iodoacetic acid [177], which prevents GSH autooxidation. Glutathione ethyl ester [172], thiosalicylic acid [132] or ␥-glutamyl glutamic acid [134,177] have been used as internal standards.…”

Development of Chiral LC-MS Methods for small Molecules and Their Applications in the Analysis of Enantiomeric Composition and Pharmacokinetic Studies

“…The present study demonstrates the efficacy of MS detection for the analysis of underivatized [45][46][47][48][49][50] and derivatized theanine samples. The enantiomeric composition of comercially available theanine samples was also evaluated using this MS method.…”

LC–MS determination of oxidized and reduced glutathione in human dermis: A microdialysis study

“…Derivatization GSH (LOQ) (nM) GSSG (LOQ) (nM) Sample [14] Iodoacetic acid, dansyl choride 25 25 Hepatic cells [15] n-Ethylmaleimide 50 200…”