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 PO43- 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, delta O-18, of dissolved phosphate at the nanomole level using electrospray ionization Orbitrap mass spectrometry (ESI-Orbitrap-MS). We compared the reproducibility of delta O-18 measurements using the H2PO4- ions (m/z = 97 and 99 for (H2PO4-)-O-16 and (H2POO3-)-O-18-O-16, respectively) and using the PO3- fragment ions (m/z = 79 and 81 for (PO3-)-O-16 and (POO2-)-O-18-O-16, respectively) generated by source fragmentation and by higher-energy collisional dissociation, respectively. The results demonstrate that phosphate delta O-18 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 delta O-18 based on precision tests. Furthermore, the mass spectrum for a 50:50 mu M 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, (PO3-)-O-16 ions (m/z: 79) are not interfered with by (SO3-)-S-32-O-16 (m/z: 80) ions. This is in contrast to the case for (H2PO4-)-H-1-O-16 ions, which share the same m/z value with (HSO4-)-H-1-S-32-O-16 ions and exhibit much lower signal intensity than HSO4- ions. Using the PO3- fragment method and six phosphate standards with a wide range of delta O-18 values, we obtained a calibration line with a slope of 0.94 (R-2 = 0.98). The overall uncertainty for ESI-Orbitrap-MS phosphate delta O-18 measurement was 0.8 parts per thousand (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.