Search

In order to develop a high-throughput screening method for the nitrogen monoxide metabolites, nitrite and nitrate, in biological fluids, we have investigated the simultaneous determination of these metabolites using microchip capillary electrophoresis (MCE). In this study, the control of applied voltage to obtain higher sensitivity by increasing the sample injection volume was investigated. Also, the improvement of reproducibility by correcting the injection volume using the internal standard was investigated. By increasing the sample volume, the limits of detection achieved for nitrite and nitrate were 24 and 12 microM, respectively. Because we used a 10-fold diluted sample when detecting nitrite and nitrate in human serum, it was necessary to increase the sensitivity by a factor of 10-50. The run-to-run and day-to-day relative standard deviations achieved were improved to less than 10% by using an internal standard to correct the injection volume. Moreover, we obtained successful separation of nitrite and nitrate in spiked human serum within 6.5 s under optimum analytical conditions. As a result, although it is necessary to obtain greater sensitivity, it was concluded that determination of the amount of NO metabolites in biological fluids using MCE is possible.

In order to improve NO2− peak height and obtain a convenient buffer system for the assay of nitrogen monooxide metabolites, we developed a novel running buffer for the simultaneous determination of nitrite and nitrate in human serum by capillary electrophoresis. The addition of cetyltrimethylammonium chloride to the running buffer resulted in high-speed separation using reverse electroosmotic flow. Highly sensitive determination was also achieved using stacking with 10-fold diluted sample solutions. The samples were injected hydrodynamically for 100 s into a 50 cm×75 μm I.D. capillary. The separation voltage was 10 kV (negative polarity). UV detection was performed at 214 nm. We obtained complete separation of nitrite and nitrate in deproteinized human serum within 6 min with optimum analytical conditions. Linear calibration curves for nitrite and nitrate for both peak height and peak area were obtained with standard addition method. The limits of detection obtained at a signal-to-noise ratio of 3 for nitrite and nitrate were 4.1 and 2.0 μM, while the values of relative standard deviation of peak height were 2.4 and 2.6%, respectively. [Copyright &y& Elsevier]

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 7min 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 1.5μM, respectively. The values of the relative standard deviation of peak height for the serum with 9.2μM nitrite and 20.9μM 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 8s. In high-resolution mode, using the buffer without electroosmotic flow modifier, the separation of nitrite and nitrate was obtained within 15s. In high-resolution mode, using an artificial serum sample with 50μM NO2- and 50μM NO3-, the limits of detection (S/N = 3) of 41μM for NO2- and 26μM 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. [Copyright &y& Elsevier]