A fast and sensitive method for the determination of nitrite in human plasma by capillary electrophoresis with fluorescence detection

Abstract

Analysis of nitrite, the indicator of nitric oxide (NO) generation in vivo, provides a useful tool to study NO synthesis in vivo. A fast and sensitive fluorometric CE method was developed for determination of nitrite in human plasma through its derivatization with 2,3-diaminonaphthalene (DAN). Nitrite in human plasma was easily reacted with DAN under acid conditions to yield the highly fluorescent 2,3-naphthotriazole (NAT). Fluorescence detection was optimized to achieve subnanomolar detection which allows a direct analysis of plasma samples unlike most CE-UV methods using sample stacking. Acetonitrile was used to remove the protein. Short-end injection and a high voltage (-30 kV) were used to shorten the analysis time. The good separation was achieved with 20 mM borate buffer at pH 9.23. The separation of NAT was obtained within 1.4 min. The deproteinized plasma sample was injected hydrodynamically for 5s at -50 mbar into a 60 cm × 75 μm internal diameter uncoated fused-silica capillary. Excitation wavelength was selected with a broad-band filter (240-400 nm), and the emitted light was measured at 418 nm by the use of a cutoff filter. A good linearity (R(2)=0.9975) was obtained in the range from 2 to 500 nM. The detection limit of nitrite was 0.6 nM in original plasma samples, which is 750 times lower than our previous CE-UV method. The developed fluorometric CE method offers the advantages of more simple system and lower cost compared with the current fluorometric HPLC methods without losing sensitivity. The detected mean nitrite concentration in human plasma by this method was consistent with the most frequently reported values.