Direct Orbitrap isotopic analysis of nitrate δ15N and δ18O signatures: revealing nitrogen sources in tropical aquatic systems

Nitrate is a key geochemical tracer in aquatic environments, as its isotopic composition provides information about nitrogen sources and the biogeochemical processes affecting nitrogen cycling. The isotopic signatures δ δ¹⁵N and δ δ¹⁸O allow the discrimination of nitrate derived from natural sources and anthropogenic inputs. However, conventional nitrate isotope analysis is mainly based on isotope ratio mass spectrometry (IRMS), which requires prior conversion of nitrate into N₂O through bacterial or chemical processes, making the measurement indirect and destructive. Orbitrap mass spectrometry offers a direct alternative by enabling the measurement of nitrate isotopologues in solution without prior molecular conversion. Nevertheless, reliable isotope ratio determination by Orbitrap depends not only on signal intensity, but ion population statistics, isotopologue abundance, and acquisition conditions. Therefore, this study establishes operational criteria for thedirect determination of δ δ¹⁵N and δ δ¹⁸O by ESI–Orbitrap MS and evaluates its applicability for environmental nitrate tracing in tropical aquatic systems. Method optimization was performed using the isotopic reference material USGS-32 at nitrate concentrations ranging from 1 to 50 µM. Concentration-dependent stability experiments were used to define the optimal analytical range, with 25 µM selected as the working concentration based on isotopic reproducibility and acquisition performance. Matrix effects were evaluated using sulfate, phosphate, and carbonate as representative inorganic constituents of natural waters. Without purification, δ δ¹⁵N showed higher tolerance to matrix contributions, whereas δ δ¹⁸O exhibited progressive positive bias with increasing matrix levels. Solid-phase extraction using a quaternary amine anion-exchange resin and elution with 1 M NaCl provided quantitative nitrate recovery and reduced matrix-induced bias, improving the reliability of isotope measurements. The validated workflow was applied to 14 river and reservoir sites from the Paranaíba River Basin, located in the Brazilian Cerrado. Environmental samples showed δ δ¹⁵N values from 1.72 to 28.56‰ and δ δ¹⁸O values from 2.39 to 16.10‰, revealing spatial variability in nitrate sources and transformation processes. Lower isotopic values were associated with soil-derived nitrogen and nitrified ammonium fertilizers, while intermediate compositions indicated mixed agricultural and organic inputs. Samples enriched in both δ δ¹⁵N and δ δ¹⁸O were consistent with nitrate removal processes, particularly partial denitrification.These results demonstrate that direct nitrate isotope analysis by Orbitrap MS is a reliable approach for investigating nitrogen sources and biogeochemical transformations in natural waters. However, accurate environmental interpretation requires control of ion population during acquisition and purification procedures, especially for δ δ¹⁸O determination in complex aquatic matrices.