Combined measurement of 15N-NH4+, NH2OH, NO2- and NO3- via derivatization and UPLC-MS

¹⁵N tracing is an important tool to study N transformations in soils, cultures and aquatic systems. The most commonly used methods for quantification of ¹⁵N in NH₄⁺, NH₂OH, NO₂^- and NO₃^- rely on conversion to N₂O and subsequent analysis by GC-IRMS. The disadvantage of these methods is that all four compounds are converted to the same product and thus cannot be measured in one run. Here, we convert each N species into a unique derivatization product that can be distinguished by high resolution mass spectrometry. For application in soils, the workflow includes the following steps: (1) extraction, (2) derivatization, (3) solid phase extraction (SPE) and (4) UPLC coupled to ESI-MS, in our case a Q-Exactive Orbitrap. NH₄⁺, NO₂^- and NO₃^- can be extracted together with 1 M KCl. NH₂OH, however, is not stable under these conditions and requires a different, newly developed extraction procedure. Steps (2) and (3) are carried out separately for each N compound. For the derivatization we employ reagents commonly used for the spectrophotometric detection of the respective N species: ortho-phthalaldehyde for NH₄⁺, quinoline-8-ol for NH₂OH, and n-naphthylethylenediamine with sulfanilamide or sulfanilic acid for NO₂^- and NO₃^-, respectively. Before reversed phase separation by UPLC, the derivatization products need to be cleaned up by SPE (step 3) to remove salts originating from extraction and derivatization that would harm the mass spectrometer. After clean-up, the SPE eluates can either be measured individually to achieve maximum sensitivity or be combined to measure all four compounds in one run on the UPLC-MS system. So far, we have optimized steps (1) to (3) and are currently working on optimizing the UPLC-MS method for concurrent separation and (isotope) quantification of all four N forms.