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 concentra-tions of nitrite in human urine and blood was investigated. The meth-od 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 chromatograph-ic process with detection at two wavelengths (510 and 220 nm) allows the determination of nitrite and nitrate. Decolorization and protein precip-itation of urine and blood was applied to overcome the interference of matrix and enhance the sensitivity. The method was validated for line-arity, 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 mg/ mL. Product recoveries ranged from 92.4 to 99.9%. The limits of detec-tion were 1 ng/mL and 0.1 mg/mL for nitrite and nitrate, respectively. Therefore, the techniquewas simple and reliable, with potential applica-tion in biological sample analysis of nitrate and nitrite