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

Mamoru Oshiki, Komei Nagai, Satoshi Ishii, Yoshiyuki Suzuki, Nobuo Saito, Takashi Yamaguchi, Nobuo Araki, Satoshi Okabe

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In the nitrogen (N) cycle, nitrogenous compounds are chemically and biologically converted to various aqueous and gaseous N species. The 15N-labeling approach is a powerful culture-dependent technique to obtain insights into the complex nitrogen transformation reactions that occur in cultures. In the 15N-labeling approach, the fates of supplemented 15N- 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 15N atoms percent of NH 4 +, NO 2 -, NO 3 -, NH 2OH, and N 2H 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 2H 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 2OH 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 15N-labeled compound, and a linear relationship was found between the determined 15N/ 14N peak ratio and 15N 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 15N/ 14N 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 15N atom% of common aqueous N species without laborious sample preparation and chromatographic separation steps; for instance, the 15N 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.

Original languageEnglish (US)
JournalApplied and environmental microbiology
Issue number7
StatePublished - Apr 2022

Bibliographical note

Funding Information:
This study was supported by JSPS KAKENHI grants 19K05805 to M.O., 20H02290 to N.A., 20H00641 to T.Y., and 19H00776 to S.O., the Institute for Fermentation, Osaka (IFO), and the MnDRIVE Initiative of the University of Minnesota (to S.I.). This study used research equipment shared in the MEXT Project for promoting public utilization of advanced research infrastructure (Program for the SHARE) at Nagaoka University of Technology, grant JPMXS0430300121, and in the Open Facility system managed by the Global Facility Center, Creative Research Institution, Hokkaido University. K.N. was supported by the Japan Public-Private Partnership Student Study Abroad Program.

Publisher Copyright:
© 2022 American Society for Microbiology.


  • 15N atom%
  • colorimetric reagent
  • mass spectrometry
  • nitrogen cycle
  • nitrogen isotope
  • Hydrazines
  • Hydroxylamine
  • Nitrites
  • Colorimetry
  • Nitrates
  • Nitrogen
  • Nitrogen Dioxide
  • Lasers
  • Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization/methods
  • Isotopes

PubMed: MeSH publication types

  • Research Support, Non-U.S. Gov't
  • Journal Article


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