Rapid and sensitive method for the microassay of nitrosobenzene plus phenylhydroxylamine in blood

An assay method has been developed for the determination of the combined concentration of nitrosobenzene plus phenylhydroxylamine (as nitrosobenzene) in small volumes of blood. The initial step in the procedure consisted of the simultaneous oxidation of phenylhydroxylamine to nitrosobenzene and of ferrous hemoglobin to methemoglobin by ferricyanide. Nitrosobenzene in the ferricyanide-treated blood samples was then extracted into ethyl acetate, and separated and quantitated by reversed-phase high-performance liquid chromatography with electrochemical detection. The sensitivity limit for nitrosobenzene in blood was in the pmol/ml concentration range, less than 100 microliter of blood was required for assay, and the procedure was convenient for routine multisample use. In comparison with previous assays, this method was more sensitive, had a lower coefficient of variation, and required 25-40 fold smaller blood sample volumes. The method was combined with the orbital sinus bleeding technique in order to follow the nitrosobenzene time course in vivo using small serial blood samples from rats treated with intraperitoneal injections of phenylhydroxylamine or aniline.