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.

Simultaneous analysis of nitrate and nitrite in a microfluidic device with a Cu-complex-modified electrode

“…These methods suffer from either time-consuming and complicated chemical reactions or the need of large amounts of reagents/analytes [4]. To overcome these drawbacks, CE with a UV-absorbance detector (CE-UV) has been used for analyzing these ions [5][6][7][8]. CE-UV methods, however, have some common drawbacks, such as bulky and sophisticated instruments, dependence on the optical path length, and sample turbidity.…”

Bioanalytical profile of the l-arginine/nitric oxide pathway and its evaluation by capillary electrophoresis

“…This sensitivity was sufficient to quantify nitrate and nitrite in the mammalian brain extracts, but was insufficient to detect the nitrite ion in biological fluids with apparently low nitrite content. Other bioanalytical examples and improvements of this technique have been reported subsequently [250][251][252][253][254][255][256][257][258][259][260][261].…”

“…In particular, a large number of methods were developed to assay nitrite and nitrate in human serum [255,257,258,264,405,406] and urine [253,[407][408][409] as indicators of systemic and whole body NO synthesis, respectively. Other biochemical parameters of the L-arginine/NO pathway became increasingly used in assessing disease risk factors and in laboratory diagnostics of samples from patients and laboratory mammalian models.…”

Unconventional detection methods for microfluidic devices

“…The instrument was utilized in testing the applicability of transforming affinity CE (ACE) techniques to microchip format [50], in the rapid separation of antimicrobial metabolites with high efficiency [51], and in chiral separations of pharmaceuticals by CD EKC [52,53]. The same system was employed in developing a method to test for nitrogen monoxide metabolites in biological fluids [54], along with a single-step quantitation of DNA [55], and chip gel electrophoresis method for high-sensitivity DNA detection [56], as well as a monosaccharide derivative separation [57].…”