DEVELOPMENT AND VALIDATION OF HPLC METHOD FOR THE DETERMINATION OF NITRITE AND ITS APPLICATION IN THE BIOLOGICAL FLUIDS OF PATIENTS
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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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Bilal Ahmad Ghalloo
Department of Pharmaceutical Chemistry, Faculty of Pharmacy, The Islamia University of
Bahawalpur, Bahawalpur 63100, Pakistan
Hafiz Muhammad Usman Abid
Department of Pharmaceutics, Faculty of Pharmacy, Bahauddin Zakariya University, Multan,60800, Pakistan
Sobia Abid
Department of Zoology, Rahim Yar Khan campus, The Islamia University of Bahawalpur,63100, Pakistan
Esha Gulfreen
Department of Zoology, Rahim Yar Khan campus, The Islamia University of Bahawalpur,63100, Pakistan
Fatima Aslam
Department of Pharmacology, Faculty of Pharmacy, The Islamia university of Bahawalpur,63100, Pakistan
Sana Maryam
Department of Pharmacology, Faculty of Pharmacy, The Islamia university of Bahawalpur,63100, Pakistan
Usman Javaid
Faculty of Pharmacy and Pharmaceutical Sciences, Department of pharmacology, University of Karachi
Tahreem Arshad
Department of pharmaceutics, Faculty of Pharmacy, The Islamia university of Bahawalpur,63100, Pakistan
Dameesha
Department of Pharmaceutical chemistry, Faculty of Pharmacy, Bahauddin Zakariya University, Multan,60800, Pakistan
Muhammad Usman
Department of Pharmaceutical chemistry, Faculty of Pharmacy, Bahauddin Zakariya University, Multan,60800, Pakistan
Muhammad Ashraf
Department of Biochemistry, The Islamia University of Bahawalpur, Bahawalpur, 63100, Punjab, Pakistan.