Novel, Rapid, and Simple Isocratic UPLC-UV Method for Estimating Nitrate and Nitrite Contents in Environmental Water Samples Using an Analytical Quality by Design Approach

In this study, we reported a novel reverse-phase UPLC method for the simultaneous estimation of nitrite and nitrate ions using a Quality by Design (QbD) approach. Nitrite and nitrate ions were separated on an ACQUITY CSH Fluoro-Phenyl column (100 × 2.1 mm, 1.7 μm) in isocratic mode with a mobile phase composition of solvent A and solvent B in a ratio of 980:20 v/v. Solvent A consisted of 100% Milli-Q water, while solvent B comprised 0.5 mL of formic acid in 1000 mL of methanol. The column temperature was maintained at 40 °C, and the flow rate was 0.6 mL/min. Detection was carried out at 214 nm with an injection volume of 5.0 μL. The method was developed and validated in accordance with ICH Q2 (R2) guidelines Validation parameters, including system suitability, sensitivity, precision, linearity, and accuracy, were within acceptable ranges. These findings suggest that the developed RP-UPLC method is highly sensitive and suitable for the estimation of nitrite and nitrate ions in various environmental water samples.