Simultaneous determination of nitrite and nitrate anions in plasma, urine and cell culture supernatants by high-performance liquid chromatography with post-column reactions

A high-performance liquid chromatographic method for the determination of nitrite and nitrate anions derived from nitric oxide in biological fluids is presented. After separation on a strong anion-exchange column (Spherisorb SAX, 250x4.6 mm I.D., 5 mu m), two on-line post-column reactions occur. The first involves nitrate reduction to nitrite on a copper-plated cadmium-filled column. In the second, the diazotization-coupling reaction between nitrite and the Griess reagent (0.05% naphtylethylendiamine dihydrochloride plus 0.5% sulphanilamide in 5% phosphoric acid) takes place, and the absorbance of the chromophore is read at 540 nm. This methodology was applied to biological fluids. Before injection into the chromatographic system, the samples were diluted and submitted to suitable clean-up procedures (urine and cell culture supernatant samples are passed through C-18 cartridges, and serum samples were deproteinized by ultrafiltration through membranes with a molecular mass cut-off of 3000). The method has a sensitivity of 30 pmol for both anions, as little as 0.05-0.1 ml sample volume is required and linearity is observed up to 60 nmol for each anion.