CHIUNG-WEN HUYUAN-JHE CHANGCHENG-CHIEH YENMU-JUNG CHAO2026-10-162026-10-162019-11-01Pure ID: 15423210Pure UUID: 4e35fed7-fba5-4b40-9497-54e618b90e4c10.1016/j.freeradbiomed.2019.08.005https://www.scopus.com/pages/publications/85070518870https://scholars.csmu.edu.tw/handle/123456789/28699Determination 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.10enjournal article