N-labelled nitrite/nitrate tracer analysis by LC-MS/MS: Urinary and fecal excretion of nitrite/nitrate following oral administration to mice.

Determination of the modulation of nitrite and nitrate levels in biological samples usually poses a major challenge, owing to their high background concentrations. To effectively investigate the variation of nitrite/nitrate in vivo, in this study, we developed aN-labelled nitrite/nitrate tracer analysis using LC-MS/MS following the derivatization with 2,3-diaminonaphthalene. This method allows for the determination of N-labelled nitrite/nitrate as N-2,3-naphthotriazole (N-NAT) that can efficiently differentiate newly introduced nitrite/nitrate from the background nitrite/nitrate in biological matrices. We also investigated the contribution of background N-NAT isotopomers to N-NAT, which has long been overlooked in the literature. Our results indicated that the contribution of background N-NAT isotopomers to N-NAT is significant. Such contribution is constant (~2.2% under positive ion mode and 1.1% under negative ion mode), and does not depend upon the concentration of N-NAT or the sample matrix measured. An equation has been therefore developed, for the first time, to correct the contribution of background N-NAT isotopomers to N-NAT. With the proposed N-labelled nitrite/nitrate tracer analysis, the amount and percentage distribution of NO and NO, both in urine and feces, after oral administration of N-labelled nitrite/nitrate are clearly demonstrated. The excretions of NO and NO were significantly increased with the increasing dose implying that the dietary nitrite/nitrate intake is an important source in urine/feces. The present method allows for the simple, reliable and accurate quantification of NO and NO, and it should also be useful to trace the biotransformation of nitrite and nitrate in vivo.