Simultaneous determination of nitrate and nitrite in biological fluids by capillary electrophoresis and preliminary study on their determination by microchip capillary electrophoresis

Abstract

In order to develop a highly sensitive and high-throughput screening method for nitrogen monoxide metabolites in biological fluids, we have investigated the simultaneous determination of nitrite and nitrate, using capillary electrophoresis and microchip capillary electrophoresis. In capillary zone electrophoresis, a running buffer based on human serum components with high ionic strength has been developed for the determination of nitrite and nitrate in human serum and human saliva. We obtained successful separation of nitrite and nitrate in the serum and the saliva within 7 min under optimum analytical conditions. Linear calibration curves for nitrite and nitrate for both peak height and area were obtained by a standard addition method. The limits of detection obtained at a signal-to-noise ratio (S/N) of 3 for nitrite and nitrate in the serum were 2.6 and 1.5 microM, respectively. The values of the relative standard deviation of peak height for the serum with 9.2 microM nitrite and 20.9 microM nitrate were 5.7 and 4.1%, respectively. For on-site analysis with high-throughput screening, a microchip capillary electrophoresis method using a microchip made of quartz with a UV detector was developed. In this high-throughput format, using a running buffer with an electroosmotic flow modifier, the peaks of nitrite and nitrate in an artificial serum sample were obtained within 8 s. In high-resolution mode, using the buffer without electroosmotic flow modifier, the separation of nitrite and nitrate was obtained within 15 s. In high-resolution mode, using an artificial serum sample with 50 microM NO2- and 50 microM NO3-, the limits of detection (S/N = 3) of 41 microM for NO2- and 26 microM for NO3- were obtained. The method was applied to human serum and saliva. We obtained peaks due to nitrite and nitrate in 10-fold diluted saliva.