15N-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 a15N-labelled nitrite/nitrate tracer analysis using LC-MS/MS following the derivatization with 2,3-diaminonaphthalene. This method allows for the determination of 15N-labelled nitrite/nitrate as 15N-2,3-naphthotriazole (15N-NAT) that can efficiently differentiate newly introduced nitrite/nitrate from the background nitrite/nitrate in biological matrices. We also investigated the contribution of background 14N-NAT isotopomers to 15N-NAT, which has long been overlooked in the literature. Our results indicated that the contribution of background 14N-NAT isotopomers to 15N-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 14N-NAT or the sample matrix measured. An equation has been therefore developed, for the first time, to correct the contribution of background 14N-NAT isotopomers to 15N-NAT. With the proposed 15N-labelled nitrite/nitrate tracer analysis, the amount and percentage distribution of 15NO2- and 15NO3-, both in urine and feces, after oral administration of 15N-labelled nitrite/nitrate are clearly demonstrated. The excretions of 15NO2- and 15NO3- 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 15NO2- and 15NO3-, and it should also be useful to trace the biotransformation of nitrite and nitrate in vivo.