Quantitative determination of nitric oxide from tissue samples using liquid chromatography—Mass spectrometry

Ever since it was found to mediate the endothelium-dependent dilation of blood vessels, nitric oxide (NO) has generated enormous research interest throughout the biological sciences. Over thirty years of research has identified NO as a ubiquitous and versatile regulatory factor utilized by both vertebrates and invertebrates. The short lifetime and low concentration of NO make quantitation difficult. Here we report a method for measuring NO using the selective reaction with 2-​(4-​carboxyphenyl)-​4,​5-​dihydro-​4,​4,​5,​5-​tetramethyl-1H-​imidazolyl-​1-​oxy-​3-​oxide (carboxy-PTIO) to form carboxy-PTI. We used tandem mass spectrometry to verify the validity of this reaction, and liquid chromatography – mass spectrometry to quantitate the amount of carboxy-PTI formed. Using diethylamine nonoate as a NO donor we demonstrate this method can quantitate NO concentrations with a detection limit of 5 nM. We successfully determined the amount of NO generated endogenously by frog heart/aorta when stimulated by carbachol, a non-selective acetylcholine receptor agonist. Based on these results, we suggest that this technique can be useful for the quantitative determination of NO in biological samples. • We report a method to measure NO by reacting it with carboxy-PTIO to form carboxy-PTI. • The carboxy-PTI is quantified by liquid chromatography mass spectrometry (LCMS).