Clinical assay of four thiol amino acid redox couples by LC–MS/MS: Utility in thalassemia

Abstract

The total concentrations of four sulfur amino acid (SAA) metabolite redox couples (reduced and oxidized forms of homocysteine, cysteine, glutathione, and cysteinylglycine) in human blood are assayed with a simple and sensitive method by liquid chromatography-electrospray positive ionization tandem mass spectrometry. To prevent ex vivo thiol oxidation, iodoacetamide (IAM) is used immediately following the blood draw. To selectively enrich for S-carboxyamidomethylated SAA, and other cationic amino acids metabolites, proprietary strong cation-exchange solid phase extraction tips are used. Analytes are further derivatized with isopropylchloroformate (IPCF) to esterify the amino and the carboxylic groups. Double derivatization with IAM and IPCF improves the reverse phase liquid chromatography separation of SAA metabolites. The use of detection mode of multiple-reaction monitoring (MRM) allows sensitive and specific simultaneous detection of SAA. The internal standards used to account for the matrix effects of human plasma and erythrocytes were plant glutathione analogue, homoglutathione, and stable isotopes of cystine and homocystine. The method was validated for its linearity, accuracy, and precision. Excellent linearity of detection (r2 > 0.98) was observed over relevant ranges for plasma and erythrocyte samples, and the limits of detection was established to be between 5 and 20 nM. Relative standard deviations were <9% for within-day variations and <15% for between day variations. The method was used to assess thiol redox states in plasma and erythrocytes isolated from healthy subjects and thalassemia patients.

Nontraditional Risk Factors and Biomarkers for Cardiovascular Disease: Mechanistic, Research, and Clinical Considerations for Youth

“…196 Quantification of both reduced and oxidized concentrations of glutathione, cysteine, and methionine, the first line of defense of the cell against any rise in oxidants, as well as even more readily oxidized thiol amino acids like homocysteine and cysteinylglycine, permits comprehensive metabolic profiling and may offer more sensitive information on host responses to oxidative stress and inflammation. 197 These techniques, although promising, remain reserved for research protocols.…”

Methodology for a rapid and simultaneous determination of total cysteine, homocysteine, cysteinylglycine and glutathione in plasma by isocratic RP-HPLC

“…Over the years, several analytical methods have been developed for thiols determination such as liquid chromatography (LC) [20,21], gas chromatography [22,23], ion-exchange chromatography [24,25] and capillary electrophoresis [26,27]. High performance liquid chromatography (HPLC) with several detection techniques, such as ultraviolet [4,28,29], fluorescence (FL) [30][31][32], electrochemical [5,6,33] and mass spectrometry [34][35][36], is the most reported methodology. All the referred methods have basic limitations in terms of equipment, reagent costs, complexity, sample preparation, run time, number of thiols simultaneously quantified, and/or validation assessment, which delay their use for high-throughput routine clinical or research purposes [8].…”

Preparation of Intracellular Metabolite Extracts from Liquid Schizosaccharomyces pombe Cultures

“…Unfortunately, there is no good solution for this problem. For the quantification of glutathione using metabolomics, specialized derivatization methods have been developed (Suh et al 2009(Suh et al , 2012 …”