THERMAL EXCHANGE OF OXYGEN ISOTOPES BETWEEN WATER AND INOSITOL PHOSPHATE ISOMERS MEASURED BY ORBITRAP MASS SPECTROMETRY

Inositol phosphates (IP_x) represent the largest portion of organic phosphorus in the environment. Enzymatic hydrolysis of IP_x liberates orthophosphate which can enter water bodies, leading to algae proliferation and overall degradation of water quality. Source tracking of IP_x and other organic P compounds in the environment is currently impossible due to a lack of appropriate analytical techniques. It means development of suitable tracer can fill critical knowledge gaps on the environmental loading, retention time, and water quality impacts of IP_x compounds. In this study, we thermally incubated phytate (inositol hexakisphosphate salt) in ¹⁸O-labeled water to induce oxygen isotope exchange between the water and phosphate in the intact IP_x molecules. We employed direct infusion method to achieve high mass resolution in Orbitrap mass spectrometry and quantified isotope values of three intact IP_x isomers (IP₆, IP₅, and IP₃) as well as PO₃ fragment ions generated from IP₆, IP₅, and IP₃ using HCD. Our results indicate that at high temperature, ¹⁸O-isotope in water exchanged with the IP_x isomers. This requires further validation because the exchange was not found to be uniform for each isomer. Further analyses of isotopes of PO₃ and PO₄ fragment Oxygen exchange occurred between the water and phosphate moieties but was also observed to occur on the hydroxyl group where phosphates were thermally hydrolyzed. Relative standard errors of measurement were sub 0.75 permil for ¹³C and ¹⁸O in intact phytate ions and sub 1.5 permil for PO₃ fragments after 30 minutes of measurement. Though precision does not reach the level of conventional IRMS, the streamlined sample preparation methods and adequate precision of the generated data indicates that Orbitrap MS is a valid method of measurement for stable isotopes of phytate. The novel methods of analyzing isotopes in intact IP_x compounds is transformative as this can be adapted to measure isotopes in other organic P compounds. This opens new opportunities to advance our understanding of the role of organic P in nutrition and water quality in the environment.