A novel GC-C-IRMS method for determining the carbon isotope composition of inositol hexaphosphate (phytate): A step towards unveiling soil organic phosphorus cycling

In soils, inositol hexaphosphate (phytate) is a recalcitrant form of organic phosphorus (OP), making it a critical component of phosphorus (P) cycling in terrestrial ecosystems. Understanding phytate turnover and the factors influencing it is therefore essential.  However, the lack of multiple stable P isotopes has hindered investigations of phytate dynamics under natural conditions over extended periods. To address this, we propose a novel technique for determining the carbon isotopic composition (δ¹³C) of inositol in phytate at a compound-specific level. For this purpose, phytate was extracted from soil, and purified via ion exchange chromatography, followed by dephosphorylation, derivatization, and analysis using GC-MS and GC-C-IRMS. Pure compounds were also analyzed to assess protocol efficiency, identify isotopic fractionations, and apply isotopic corrections due to derivatization. Phytate extracted from soil samples was identified using GC-MS chromatograms. Replicate analyses of the pure compounds showed that the protocol is highly reproducible. The proposed method was able to identify, quantify, and measure the δ¹³C values of inositol in phytateseparately from other sugar molecules such as glucose and fructose. The δ¹³C values showed high reproducibility, with values varying by less than 0.5‰, and with no detectable isotopic fractionation during sample preparation. The δ¹³C values of phytate in soil samples reflected the dominant vegetation type (C₃ or C₄) at the study site. This study introduces a novel approach to measuring the δ¹³C values of inositol in phytate from environmental samples, offering new opportunities for investigating and quantifying OP dynamics using stable carbon isotopes.