Oxygen Isotope Analyses of Phosphate and Organophosphorus Compounds by Electrospray Ionization Orbitrap Mass Spectrometry

Orbitrap mass spectrometry with electrospray ionization (ESI-Orbitrap MS) enables ¹⁸O/¹⁶O ratio measurements in phosphate and organophosphorus compounds, which offers promising avenues for the study of metabolic, biogeochemical, and environmental processes. While the instrumental feasibility of such ¹⁸O/¹⁶O ratio measurements has been shown, the applicability of such analyses in aqueous matrices on multipurpose mass spectrometers remains unaddressed. Here, we (i) evaluated the interplay between instrument parameters and the long-term accuracy and precision of ESI-Orbitrap MS for the determination of δ¹⁸O(PO₄) from H₂PO₄^- vs PO₃^- fragments of phosphate, (ii) identified properties of methanolic sample solutions that are critical for accurate and precise measurements, and (iii) propose a sample purification procedure for the elimination of matrix-based interferences. In 10 measurement campaigns over two years, we observed a long-term reproducibility of δ¹⁸O(PO₄) within ±2.8‰. Results of in-source fragmentation experiments of H₂PO₄^- to PO₃^- show excellent agreement of δ¹⁸O and offer promising opportunities to probe for ¹⁸O/¹⁶O ratios in organophosphorus compounds. By investigating the effect of the aqueous matrix and interfering anions on ¹⁸O/¹⁶O ratio measurements, we found that a water content exceeding 50 vol % and the presence of oxyanions such as nitrate and sulfate limit measurement accuracy due to interferences of matrix constituents in the ESI source. To overcome these challenges, we evaluated selective phosphate extraction with a zirconium-based metal-organic framework (MOF) as sorbent. The resulting purification procedure allowed for successful extraction and recovery of phosphate from nitrate- and sulfate-containing aqueous solutions, resulting in methanolic phosphate samples that enabled accurate analyses of δ¹⁸O(PO₄) by ESI-Orbitrap MS.