High-throughput assay of nitric oxide metabolites in human plasma without deproteinization by lab-on-a-chip electrophoresis using a zwitterionic additive

In order to develop a high-throughput assay for nitric oxide metabolites, nitrite (NO2−) and nitrate (NO3−), in biological fluids, we have investigated the simultaneous determination of them using an electrophoretic lab-on-a-chip (microchip capillary electrophoresis, MCE) technique. In this study, in order to establish an MCE assay process without deproteinization, the addition of a zwitterionic additive into the running buffer to reduce the adsorption of protein onto the surface of channel was investigated. Initially, some zwitterionic additives were investigated by making a comparison of relative standard deviations (RSDs) of the migration times for NO2− and NO3− on capillary electrophoresis. From the results of our comparison of the RSD values, 2% (w/w) N-cyclohexyl-2-aminoethanesulfonic acid (CHES) was selected. As a result of the application of the running buffer with CHES to the MCE process, the complete separation of NO2− and NO3− in human plasma without deproteinization was achieved within 1 min. Since the RSD values of the positions of the peaks were less than 2.3%, beneficial reduction effects on MCE were suggested. When we used an internal standard method in order to correct the injection volume, the RSDs of the peak heights and areas were less than 10%, and the correlation coefficients of spiked calibration curves ranging from 0 to 350 μM were 0.999 and 0.997 for NO2− and NO3−, respectively. The limits of detection (S/N = 3) were 53 μM for NO2− and 41 μM for NO3−. Moreover, the correlation coefficients in excess of 0.99 between the MCE method and a conventional Griess method were achieved for both NO2− and NO3−. Consequently, the possibility of establishing a high-throughput assay process was obtained by utilizing 2% (w/w) CHES to reduce protein adsorption.