Oxygen Isotope Analysis of Nanomole Phosphate Using PO 3 - Fragment in ESI-Orbitrap-MS

The oxygen isotope composition of phosphate is a useful tool for studying biogeochemical phosphorus cycling. However, the current Ag PO method is not only tedious in PO 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, δ O, of dissolved phosphate at the nanomole level using electrospray ionization Orbitrap mass spectrometry (ESI-Orbitrap-MS). We compared the reproducibility of δ O measurements using the H PO ions ( / = 97 and 99 for H P O and H P O O , respectively) and using the PO fragment ions ( / = 79 and 81 for P O and P O O , respectively) generated by source fragmentation and by higher-energy collisional dissociation, respectively. The results demonstrate that phosphate δ O can be more reliably measured by the PO ions than by the H PO ions. PO generated by source fragmentation at 40 V achieved the highest reproducibility for δ O based on precision tests. Furthermore, the mass spectrum for a 50:50 μM mixed solution of phosphate and sulfate revealed that PO ions resulting from source fragmentation at 40 V are the predominant species in the Orbitrap analyzer. Notably, P O ions ( / : 79) are not interfered with by S O ( / : 80) ions. This is in contrast to the case for H P O ions, which share the same / value with H S O ions and exhibit much lower signal intensity than HSO ions. Using the PO fragment method and six phosphate standards with a wide range of δ O values, we obtained a calibration line with a slope of 0.94 ( = 0.98). The overall uncertainty for ESI-Orbitrap-MS phosphate δ O measurement was 0.8‰ ( = 30; 1 SD). With much room for improvement, the PO fragment method presents a better approach to measuring the phosphate oxygen isotope composition, applicable to nanomole sample sizes in a liquid phase.