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

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Bilal Ahmad Ghalloo
Hafiz Muhammad Usman Abid
Sobia Abid
Esha Gulfreen
Fatima Aslam
Sana Maryam
Usman Javaid
Tahreem Arshad
Dameesha
Muhammad Usman
Muhammad Ashraf

Keywords

Biological Fluids, Cerebrospinal Fluid, Ascitic Fluid, HPLC, Nitrites.

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. 

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