The Determination of Nitrate and Nitrite in Human Urine and Blood by High-Performance Liquid Chromatography and Cloud-Point Extraction

A simple efficient and practical separation/preconcentration coupled with HPLC method for the determination nitrate and low concentrations of nitrite in human urine and blood was investigated. The method is based on precolumn derivatization using the Griess reaction and cloud-point extraction (CPE) of nitrite anion and direct determination of nitrate using its UV absorbance by ion-pair HPLC. The chromatographic process with detection at two wavelengths (510 and 220 nm) allows the determination of nitrite and nitrate. Decolorization and protein precipitation of urine and blood was applied to overcome the interference of matrix and enhance the sensitivity. The method was validated for linearity, accuracy and precision. Under the optimum conditions, the linear range of nitrite from 10 to 1,000 ng/mL and nitrate from 0.1 to 10 µg/mL. Product recoveries ranged from 92.4 to 99.9%. The limits of detection were 1 ng/mL and 0.1 µg/mL for nitrite and nitrate, respectively. Therefore, the technique was simple and reliable, with potential application in biological sample analysis of nitrate and nitrite.