Simultaneous Determination of Nitrite and Nitrate by Ultra-performance Liquid Chromatography in Rat Plasma

Objective: To develop an ultraperformance liquid chromatography (AcquityTM UPLC) method for simple, fast, accurate and simultaneous determination of nitrite and nitrate ions as stable forms of nitric oxide (NO) production in rat plasma. Methods: The ion-pairing AcquityTM UPLC method with reverse phase BEH C18 column was used along with photodiode array (PDA) UV-Vis detector to separate and detect nitrite and nitrate ions at an absorbance of 212 and 208 nm, respectively. Due to small particle size (1.7 μm) of the BEHC18 column, a narrow peak was achieved with an improved sensitivity and separation at the cost of pressure. The anions were analyzed in ultra-filtered deproteinized rat plasma sample (10 kDa, cut-off). Results: The UPLC bioanalytical method was linear (r=0.9998, 0.9988) over wide ranges of 4-500 μM (nitrite) and 6-400 μM (nitrate) concentration. The most important advantages of the present method are; short time run time, no sample pre-treatment (unlike enzymatic reaction and pre-column treatment), low sample volume and simultaneous estimation of nitrite and nitrate in rat plasma. Conclusion: It was concluded that the developed UPLC method for determination of simultaneous nitrite and nitrate in rat plasma was rapid, sensitive, accurate and linear over the wide ranges of concentration. This method can be used for simultaneous determination of nitrite and nitrate in rat plasma with various advantages over the reported methods.