Determination of nitrate and nitrite in rat brain perfusates by capillary electrophoresis

Abstract

A fast and simple method for the direct, simultaneous detection of nitrite (NO(2) (-)) and nitrate (NO(3) (-)) in rat striatum has been developed using a capillary electrophoresis separation of low-flow push-pull perfusion samples. The method was optimized primarily for nitrite because nitrite is more important physiologically and is found at lower levels than nitrate. We obtained a complete separation of NO(2) (-) and NO(3) (-) in rat striatum within 1.5 min. Optimal CE separations were achieved with 20 mM phosphate, 2 mM cetyltrimethylammonium chloride (CTAC) buffer at pH 3.5. The samples were injected electrokinetically for 2 s into a 40 cm x 75 microm ID fused-silica capillary. The separation voltage was 10 kV (negative polarity), and the injection voltage was 16 kV (negative polarity). UV detection was performed at 214 nm. The limits of detection obtained at a signal-to-noise ratio (S/N) of 3 for nitrite and nitrate were 0.96 and 2.86 microM. This is one of the fastest separations of nitrite and nitrate of a biological sample ever reported. Interference produced by the high physiological level of chloride is successfully minimized by use of CTAC in the run buffer.

Measurement of Region-Specific Nitrate Levels of the Posterior Chamber of the Rat Eye Using Low-Flow Push−Pull Perfusion

“…Separation conditions were previously optimized in our laboratory for assaying nitrate and nitrite simultaneously in rat brain perfusates 13 and for clinical vitreous samples. 12 Briefly, electrophoretic assays were performed on a laboratory-built CE system with a commercial high-voltage supply (Spellman, NY) and a VUV-20 variable wavelength ultraviolet/visible absorbance detector (Hyperquan, CO).…”

“…A similar trend was observed in all subjects. Initial NO 3 − levels were likely high due to the insertion of the guide needle 13 ; therefore the basal concentration of nitrate was considered perfusate values corresponding to samples collected during 30-150 minutes of sampling as the signal leveled off during this period. Ideally, the flow rate should remain consistent throughout the time course of the experiment; however, a decrease may be seen during the end of the experiment (Inset of Figure 4).…”

“…Recently, optimized CE conditions for efficiently separating NO 3 − and NO 2 − in various biological systems such as rat extracellular central nervous system (CNS) fluid in fewer than 3 minutes were developed in our laboratory. 13 A further modified run buffer was applied in the clinical analysis of human vitreous. 12 Such a rapid separation allows for multiple analyses of a single sample within the time it takes to collect a 0.5-µL perfusate.…”

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].…”

“…A 1.5-min approach was proposed by Gao et al for the separation and analysis of NO 2 − and NO 3 − in rat striatum [256]. This method was especially optimized for NO 2 − .…”