Determination of nitrate in biological fluids by HPLC

Nitrate is the stable product of nitric oxide, which is physiologically active radical, an immunomodulator and a neuromodulator; its quantification in biological fluids is important to study the physiological and biochemical nature. Therefore, the purpose of this study was to quantify nitrate in different biological fluids like serum, cerebrospinal fluid (CSF) and ascetic fluid (ASF) using HPLC technique. A new HPLC method for the estimation of nitrate in serum, CSF and ASF was developed using the mobile phase of 1.0mM each of Na₂CO₃ and NaHCO₃ (1:1, v/v, pH 5 with H₃PO₄) at a flow rate of 1.0mLmin^-1. Eluate was detected at 220nm with the retention time of nitrate 2.55 min. The LOD and LOQ values of nitrate were 0.03μgmL^-1 and 0.098μgmL^-1, respectively. Nitrate was eluted through SAX Hypersil column of 150 × 4.6mm, id, 5μm particle size. Run time was 10min. The method was validated according to the FDA guidelines and was found linear in the range of 0.39 to 50μgmL^-1 and CV was <3%, within limits of FDA guidelines. The method was used successfully for the estimation of nitrate in biological fluids like serum, CSF and ASF of 20 patients each. This is an alternate and reproducible method for the detection of nitrates in biological fluids.