Reactive oxygen species are free radicals capable of damaging the cellular components in a process called oxidative stress. Among the different biomarkers that are used to determine level of oxidative stress is the ratio between reduced and oxidized thiols, such as glutathione and oxidized glutathione. The use of glutathione ratio as a biomarker of oxidative stress is possible because the thiols are responsible for reducing the oxidizing species in a process that oxidizes the thiols into their disulfides. Under normal conditions, the cells can regenerate the reduced thiols by the action of reductases, which keeps the ratio constant. However, under oxidative stress, the cell cannot regenerate the reduced thiols rapidly enough. This in turn increases the concentration of the disulfide, and the ratio decreases. The ratio can also be inadvertently altered during sample manipulation because thiols can autoxidize. Therefore, for their accurate determination, thiols should be derivatized prior to analysis. The existing protocols using liquid chromatography-mass spectrometry (LC-MS) for thiol analysis largely focus on urine or plasma analysis, and do not consider exposure to oxidation during sample handling, while the few studies on intracellular thiol concentrations employ derivatization after cell lysis. The main objective of this thesis was to develop a LC-MS method to accurately measure individual thiols and disulfides, and their ratios in Jurkat cells. 
To achieve this goal, the selectivity and efficiency of two different derivatizing agents that are able to permeate the cell membrane were first compared in detail: N-ethyl maleimide (NEM) and N-phenyl ethyl maleimide (NPEM). They were compared in terms of their derivatization efficiency, electrospray ionization enhancement, stability and selectivity/side product formation with focus on four abundant intracellular thiols: cysteine (CYS), homocysteine (HCY), N-acetyl cysteine (NAC), glutathione (GSH) and their corresponding disulfides. While NPEM provided greater ionization efficiency than NEM (NPEM/NEM varies from 2.1x for GSH to 5.7x for CYS), it was also more unstable, forming more side-products. The instability of its maleimide ring led to reaction with amines, as well as double derivatization and cyclization reactions, which corresponded to about 10% of the signal of CYS. NEM showed only minor contribution of side reactions (about 1.5% of the signal of CYS), so it was chosen as the derivatizing reagent for the protocol. The derivatizing conditions with NEM were further optimized to minimize side product formation, and pH 7.0 was selected for further assay development while being compatible with cell handling. 
In the next step, a full cell extraction protocol was developed to quantify the thiol ratios in Jurkat T cells. Briefly, the optimized protocol required 1 × 106 cells and combined NEM derivatization prior to cell lysis, cell lysis and extraction using 20% methanol (v/v) and protein precipitation by methanol. The thiols were then chromatographically separated using a biphenyl, reversed-phase, separation in combination with Quadrupole Time of Flight Mass Spectrometry (QToF-MS) analysis. Protocol optimization included evaluation of different lysis solvents, recovery, matrix effects, and evaluation of the number of washes required to ensure as complete removal of extracellular metabolites as possible without compromising cellular integrity. The final method was tested for its capacity to evaluate oxidative stress in cells stimulated by hydrogen peroxide, a known inducer of oxidative damage. The results show that the method was capable of differentiating between the control, mild and intense oxidative stress conditions. 
To the best of my knowledge, this is the first cellular protocol that combines NEM derivatization prior to cell lysis with LC-MS determination of individual thiol ratios. An innovative aspect of this procedure is the protection of reduced thiols prior to lysis, which minimizes changes in the ratio caused by sample manipulation, as opposed to the typical procedure which has the derivatization after extraction. This work is also the first systematic comparison of NEM versus NPEM derivatization for LC-MS analysis and shows clearly the propensity of NPEM for side-product formation under conditions commonly used for maleimide derivatization. In summary, this research contributes towards more accurate measurement of thiol ratios as readouts of oxidative stress.