Oxygen Isotope Analysis of Nanomole Phosphate Using PO₃^- Fragment in ESI-Orbitrap-MS

The oxygen isotope composition of phosphate is a useful tool for studying biogeochemical phosphorus cycling. However, the current Ag₃PO₄ method is not only tedious in PO₄^3- extraction and purification but also requires a large-sized sample at the micromole level, thereby limiting its application. Here, we present an approach to measuring the oxygen isotope composition, δ¹⁸O, of dissolved phosphate at the nanomole level using electrospray ionization Orbitrap mass spectrometry (ESI-Orbitrap-MS). We compared the reproducibility of δ¹⁸O measurements using the H₂PO₄^- ions (m/z = 97 and 99 for H₂P¹⁶O₄^- and H₂P¹⁸O¹⁶O₃^-, respectively) and using the PO₃^- fragment ions (m/z = 79 and 81 for P¹⁶O₃^- and P¹⁸O¹⁶O₂^-, respectively) generated by source fragmentation and by higher-energy collisional dissociation, respectively. The results demonstrate that phosphate δ¹⁸O can be more reliably measured by the PO₃^- ions than by the H₂PO₄^- ions. PO₃^- generated by source fragmentation at 40 V achieved the highest reproducibility for δ¹⁸O based on precision tests. Furthermore, the mass spectrum for a 50:50 μM mixed solution of phosphate and sulfate revealed that PO₃^- ions resulting from source fragmentation at 40 V are the predominant species in the Orbitrap analyzer. Notably, P¹⁶O₃^- ions (m/z: 79) are not interfered with by ³²S¹⁶O₃^- (m/z: 80) ions. This is in contrast to the case for ¹H₂P¹⁶O₄^- ions, which share the same m/z value with ¹H³²S¹⁶O₄^- ions and exhibit much lower signal intensity than HSO₄^- ions. Using the PO₃^- fragment method and six phosphate standards with a wide range of δ¹⁸O values, we obtained a calibration line with a slope of 0.94 (R² = 0.98). The overall uncertainty for ESI-Orbitrap-MS phosphate δ¹⁸O measurement was 0.8‰ (n = 30; 1 SD). With much room for improvement, the PO₃^- fragment method presents a better approach to measuring the phosphate oxygen isotope composition, applicable to nanomole sample sizes in a liquid phase.