A VALIDATED ULTRA-PERFORMANCE LIQUID CHROMATOGRAPHY METHOD FOR NITRATE AND NITRITE MEASUREMENT

Objective: The objective of this study was to develop a simple and sensitive ultra-performance liquid chromatography (UPLC) method for determination of nitrate and nitrite in plant extracts and biological samples.Methods: Nitrate was analyzed directly by injecting the filtered sample into UPLC and monitoring the chromatogram at 222 nm whereas, for nitrite analysis, the samples were derivatized using Griess reagent, and chromatogram was monitored at 520 nm. The method was validated for accuracy and precision.Results: The calibration curve was obtained in the range of 0.05–500 μg/ml for nitrate and 5–3200 ng/ml for nitrite which were extremely linear, where both correlation coefficients were >0.9990. Accuracy of the method was found as 99.87% and 100.24% for nitrate and nitrite, respectively. High degree of precision and repeatability was obtained since coefficient of variation values were <2.1%. Among the three commonly used anticoagulants, i.e., K2EDTA, Li-Heparin, and sodium citrate, Li-Heparin did not show any interference with nitrate or nitrite analysis.Conclusion: Results clearly indicate the sensitivity and suitability of the developed method for estimation of nitrate and nitrite in plant extracts and biological samples. Our findings also indicate that Li-Heparin does not interfere with analysis and thus can be used as anticoagulant while collecting blood.