Quantification of glutathione and cysteine by electrospray ionization mass spectrometry, high performance liquid chromatography and ultra performance liquid chromatography
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Three analytical methods, liquid chromatography mass spectrometry (LC/MS), high performance liquid chromatography (HPLC), and ultra performance liquid chromatography (UPLC) were developed for the simultaneous detection and quantitation of glutathione (GSH) and cysteine (CYS) in urine samples. The aminothiols were derivatized with Ellman's reagent. The samples were then analyzed by electrospray ionization negative mode (ESI-) conditions for the LC/MS system and by reversed-phase chromatography with ultra violet (UV) detection for both the HPLC and UPLC systems. Thiols were derivatized with excess Ellman's reagent to ensure an instantaneous and successful derivatization. Ellman's reagent is a thiol specific reagent that blocks the oxidation of thiols to disulfides. Of the three analytical methods studied, the UPLC method showed the best overall performance. A sensitive, reproducible and robust UPLC method has been validated for the simultaneous determination of CYS and GSH in artificial urine. Aliquots (20μL) of artificial urine were injected into a Waters' UPLC instrument equipped with a Waters' AccQ-TagTM Ultra 2.1 X 100 mm column at a flow rate of 0.7 mL/min. The mobile phase consisted of Waters' AccQ-TagTM Ultra Eluent A concentrate: water (1:10), and Waters' AccQ-TagTM Ultra Eluent B. Detection was performed by UV absorbance at 340 nm. No significant interfering peaks were detected at the retention times of CYS and GSH in artificial urine blanks. The calibration curves were linear in the range of 50.0-1000.0 and 0.50-10.0 μM for CYS and GSH, respectively. Both the limit of detection (LOD) and the limit of quantification (LOQ) values for CYS and GSH were established at 50 μM and 0.5 μM respectively. The intra-day and inter-day precision values for CYS were 0.008% and 0.005%, respectively; for GSH they were 0.019% and 0.006% respectively, showing a high degree of reproducibility. The intra-day and inter-day accuracy for CYS were 111%, and107% respectively while for GSH they were 102%, and 107% respectively. Thus, this method will be useful in metabolomics investigations of conditions of oxidative stress by determination of prominent redox-sensitive thiols found in human urine.