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 3 PO 4 method is not only tedious in PO 4 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, δ 18 O, of dissolved phosphate at the nanomole level using electrospray ionization Orbitrap mass spectrometry (ESI-Orbitrap-MS). We compared the reproducibility of δ 18 O measurements using the H 2 PO 4 - ions ( m / z = 97 and 99 for H 2 P 16 O 4 - and H 2 P 18 O 16 O 3 - , respectively) and using the PO 3 - fragment ions ( m / z = 79 and 81 for P 16 O 3 - and P 18 O 16 O 2 - , respectively) generated by source fragmentation and by higher-energy collisional dissociation, respectively. The results demonstrate that phosphate δ 18 O can be more reliably measured by the PO 3 - ions than by the H 2 PO 4 - ions. PO 3 - generated by source fragmentation at 40 V achieved the highest reproducibility for δ 18 O based on precision tests. Furthermore, the mass spectrum for a 50:50 μM mixed solution of phosphate and sulfate revealed that PO 3 - ions resulting from source fragmentation at 40 V are the predominant species in the Orbitrap analyzer. Notably, P 16 O 3 - ions ( m / z : 79) are not interfered with by 32 S 16 O 3 - ( m / z : 80) ions. This is in contrast to the case for 1 H 2 P 16 O 4 - ions, which share the same m / z value with 1 H 32 S 16 O 4 - ions and exhibit much lower signal intensity than HSO 4 - ions. Using the PO 3 - fragment method and six phosphate standards with a wide range of δ 18 O values, we obtained a calibration line with a slope of 0.94 ( R 2 = 0.98). The overall uncertainty for ESI-Orbitrap-MS phosphate δ 18 O measurement was 0.8‰ ( n = 30; 1 SD). With much room for improvement, the PO 3 - fragment method presents a better approach to measuring the phosphate oxygen isotope composition, applicable to nanomole sample sizes in a liquid phase.