Evaluation of an LC–MS/MS assay for 15N-nitrite for cellular studies of l-arginine action

The utility of an LC–MS/MS assay for nitrite determination in studying l-arginine (ARG) cellular action was examined in vitro. EA.hy926 human endothelial cells or cellular fractions (membrane and cytosol) were exposed to 0–500 μM of 15N4-ARG for 2 h. 14N-nitrite and 15N-nitrite in the cell lysate and in the incubation medium were derivatized with 2,3-diaminonaphthalene (DAN) to form 14N- and 15N-naphthotriazole (i.e., 14N-NAT and 15N-NAT). Peak responses of 14N-NAT and 15N-NAT were analyzed by LC–MS/MS with 1H-naphth[2,3-d]imidazole as an internal standard. The calibration curves of DAN-derivatized 14N-NAT and 15N-NAT from 14N-nitrite and 15N-nitrite were linear. Intra- and inter-day variability of the quantification was below 14.2% in quality control samples. Following incubation of EA.hy926 cells with 15N4-ARG, saturable increases of 15N-nitrite accumulation with increasing 15N4-ARG exposure were observed clearly. This increase however could not be detected by the classical fluorescence method, nor were changes in 14N-nitrite level observed. When cellular fractions were exposed to 15N4-ARG, 15N-nitrite formation was only observed in the membrane fragments. The sensitive and selective LC–MS/MS method reported here can be applied to quantify accumulated nitrite levels in human endothelial cells. The selectivity of this stable-isotope labeled LC–MS/MS method offers an advantage over other traditional methods for elucidating cellular ARG action when its stable isotope is employed as a substrate.