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A sensitive LC–MS method was developed and validated to quantify glutathione cycle metabolites.2% BSA in PBS was successfully used as surrogate matrix.5-oxoproline, L-glutamate, GSH and GSSG were quantified in tissues and biofluids.5-oxoproline levels were high in all samples from mice with myocardial infarction.Oxidative stress is suggested to play an important role in several pathophysiological conditions. A recent study showed that decreasing 5-oxoproline (pyroglutamate) concentration, an important mediator of oxidative stress, by over-expressing 5-oxoprolinase, improves cardiac function post-myocardial infarction in mice. The aim of the current study is to gain a better understanding of the role of the glutathione cycle in a mouse model of myocardial infarction by establishing quantitative relationships between key components of this cycle. We developed and validated an LC–MS method to quantify 5-oxoproline, L-glutamate, reduced glutathione (GSH) and oxidized GSH (GSSG) in different biological samples (heart, kidney, liver, plasma, and urine) of mice with and without myocardial infarction. 5-oxoproline concentration was elevated in all biological samples from mice with myocardial infarction. The ratio of GSH/GSSG was significantly decreased in cardiac tissue, but not in the other tissues/body fluids. This emphasizes the role of 5-oxoproline as an inducer of oxidative stress related to myocardial infarction and as a possible biomarker. An increase in the level of 5-oxoproline is associated with a decrease in the GSH/GSSG ratio, a well-established marker for oxidative stress, in cardiac tissue post-myocardial infarction. This suggests that 5-oxoproline may serve as an easily measurable marker for oxidative stress resulting from cardiac injury. Our findings show further that liver and kidneys have more capacity to cope with oxidative stress conditions in comparison to the heart, since the GSH/GSSG ratio is not affected in these organs despite a significant increase in 5-oxoproline.