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 (15)N(4)-ARG for 2 h. (14)N-nitrite and (15)N-nitrite in the cell lysate and in the incubation medium were derivatized with 2,3-diaminonaphthalene (DAN) to form (14)N- and (15)N-naphthotriazole (i.e., (14)N-NAT and (15)N-NAT). Peak responses of (14)N-NAT and (15)N-NAT were analyzed by LC-MS/MS with 1H-naphth[2,3-d]imidazole as an internal standard. The calibration curves of DAN-derivatized (14)N-NAT and (15)N-NAT from (14)N-nitrite and (15)N-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 (15)N(4)-ARG, saturable increases of (15)N-nitrite accumulation with increasing (15)N(4)-ARG exposure were observed clearly. This increase however could not be detected by the classical fluorescence method, nor were changes in (14)N-nitrite level observed. When cellular fractions were exposed to (15)N(4)-ARG, (15)N-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.