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Peroxynitrite has emerged as a major cytotoxic agent implicated in a host of pathophysiological conditions triggered by its multifaceted nitroxidative reactions. Peroxynitrite's very short half-life under physiological conditions reflects both its fast intrinsic decomposition and its notorious reactivity with many cellular targets. Sensitive and accurate measurement of peroxynitrite is crucial to investigations that aim at shedding light on the illusive pathophysiologic roles of this metabolite. The development process of probes and sensors capable of selectively detecting and measuring any analyte depends primarily on the availability of pure authentic amounts of it in order to evaluate sensor performance. There are numerous methods of peroxynitrite synthesis or generation in situ. However, they all come with varying levels of byproducts that may interfere with the characterization of performance of peroxynitrite sensing probes and devices. This chapter gives the background and a brief review of major methods of peroxynitrite synthesis and in situ generation. Particularly, we discuss advantages and drawbacks of the various methods in the context of the development and characterization of peroxynitrite sensors and probes.

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