Determination of 15 N/ 14 N of Ammonium, Nitrite, Nitrate, Hydroxylamine, and Hydrazine Using Colorimetric Reagents and Matrix-Assisted Laser Desorption Ionization-Time-of-Flight Mass Spectrometry (MALDI-TOF MS).

In the nitrogen (N) cycle, nitrogenous compounds are chemically and biologically converted to various aqueous and gaseous N species. The 15 N-labeling approach is a powerful culture-dependent technique to obtain insights into the complex nitrogen transformation reactions that occur in cultures. In the 15 N-labeling approach, the fates of supplemented 15 N- and/or unlabeled gaseous and aqueous compounds are tracked by mass spectrometry (MS) analysis, whereas MS analysis of aqueous N species requires laborious sample preparation steps and is performed using isotope-ratio mass spectrometry, which requires an expensive mass spectrometer. We developed a simple and high-throughput MS method for determining the 15 N atoms percent of NH 4 + , NO 2 - , NO 3 - , NH 2 OH, and N 2 H 4 , where liquid samples (<0.5 mL) were mixed with colorimetric reagents (naphthylethylenediamine for NO 2 - , indophenol for NH 4 + , and p -aminobenzaldehyde for N 2 H 4 ), and the mass spectra of the formed N complex dyes were obtained by matrix-assisted laser desorption ionization-time-of-flight (MALDI-TOF) MS. NH 2 OH and NO 3 - were chemically converted to NO 2 - by iodine oxidation and copper/hydrazine reduction reaction, respectively, prior to the above colorimetric reaction. The intensity of the isotope peak (M + 1 or M + 2) increased when the N complex dye was formed by coupling with a 15 N-labeled compound, and a linear relationship was found between the determined 15 N/ 14 N peak ratio and 15 N atom% for the tested N species. The developed method was applied to bacterial cultures to examine their N-transformation reactions, enabling us to observe the occurrence of NO 2 - oxidation and NO 3 - reduction in a hypoxic Nitrobacter winogradskyi culture. IMPORTANCE 15 N/ 14 N analysis for aqueous N species is a powerful tool for obtaining insights into the global N cycle, but the procedure is cumbersome and laborious. The combined use of colorimetric reagents and MALDI-TOF MS, designated color MALDI-TOF MS, enabled us to determine the 15 N atom% of common aqueous N species without laborious sample preparation and chromatographic separation steps; for instance, the 15 N atom% of NO 2 - can be determined from >1,000 liquid samples daily at <$1 (U.S.) per 384 samples for routine analysis. This convenient MS method is a powerful tool that will advance our ability to explore the N-transformation reactions that occur in various environments and biological samples.