A high-pressure liquid chromatography (HPLC) assay for measuring picomole quantities of nitrosothiol in biological samples was developed. The assay utilizes the catalytic reduction of nitrosothiol by mercuric cation (Hg2+). Released nitrogen oxide reacts with sulfanilamide (SA) and N-(1-napthyl)ethylenediamine (NNED) to form a stable azo dye. The azo dye is then separated from N-(1-napthyl)ethylenediamine and quantified by reversed-phase HPLC. In addition to nitrosothiol, nitrite and atmospheric nitrogen oxides are sources of nitrogen oxide that react with the reagents, SA and NNED, to form the azo dye. Therefore, a reference sample, which includes the nitrosothiol sample and all reagents except Hg2+, is utilized for the subtraction of nitrite and atmospheric nitrogen oxides which "contaminate" the nitrosothiol sample and reagents. This method is a sensitive (approximately 3 pmol; approximately 10(-1) microM) and accurate means to measure nitrosothiol concentration in biologic samples.
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