DEVELOPMENT AND VALIDATION OF HPLC METHOD FOR THE DETERMINATION OF NITRITE AND ITS APPLICATION IN THE BIOLOGICAL FLUIDS OF PATIENTS

Abstract

Purpose of study: The study's goal was to measure nitrite using the high-performance liquid chromatography (HPLC) technique in numerous biological fluids i.e. serum, ascetic fluid, and cerebrospinal fluid (CSF).

Methodology: Using distilled water (pH 6.5 with H₃PO₄) as the mobile phase and a flow rate of 0.9 ml per min., an HPLC method was developed for the quantitative determination of nitrite in serum, CSF, and ascetic fluid. With a nitrate retention time of 3.5 minutes, the eluate was detected at 220 nm. The LOD and LOQ values of nitrite were 0.006 μg/ml and 0.012 μg/mL, correspondingly. A 250 × 4.6 mm, i.d. C18 BDS Hypersil column containing particles with a size of 5 µm was used to elute nitrite. Ten minutes was the run time. The method was validated in compliance with FDA guidelines, and the results showed that the % CV was less than 3% and the method was linear in the range of 0.012 to 100 μg/mL.

Conclusion: The developed technique was successfully used to measure the amount of nitrite in biological fluids, including serum, CSF, and 20 patients' ascetic fluids. The work discusses the results.