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 0-500μM of ¹⁵N ₄-ARG for 2h. 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 ¹⁵N ₄-ARG, saturable increases of ¹⁵N-nitrite accumulation with increasing ¹⁵N ₄-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 ¹⁵N ₄-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.