Identification of lower-order inositol phosphates (IP₅ and IP₄) in soil extracts as determined by hypobromite oxidation and solution ³¹P NMR spectroscopy

Inositol phosphates (IP) are a major pool of identifiable organic phosphorus (P) in soil. However, insight on their distribution and cycling in soil remains limited, particularly of lower-order IP (IP5 and IP4). This is because their quantification typically requires a series of chemical extractions, including hypobromite oxidation to isolate IP, followed by chromatographic separation. Here, for the first time, we identify the chemical nature of organic P in four soil extracts following hypobromite oxidation using solution 31P NMR spectroscopy and transverse relaxation (T2) experiments. Soil samples analysed include the A horizon of a Ferralsol from Colombia, of a Cambisol from Switzerland, of a Gleysol from Switzerland and of a Cambisol from Germany. Solution 31P NMR spectra of the phosphomonoester region on soil extracts following hypobromite oxidation revealed an increase in the number of sharp signals (up to 70), and an on average 2-fold decrease in the concentration of the broad signal compared to the untreated soil extracts. We identified the presence of four stereoisomers of IP6, four stereoisomers of IP5, and scyllo-IP4 (using solution 31P NMR spectroscopy). We also identified for the first time two isomers of myo-IP5 in soil extracts: myo-(1,2,4,5,6)-IP5 and myo-(1,3,4,5,6)-IP5. Concentrations of total IP ranged from 1.4 to 159.3 mg P/kgsoil across all soils, of which between 9 % and 50 % were comprised of lower-order IP. Furthermore, we found that the T2 times, which are considered to be inversely related to the tumbling of a molecule in solution and hence its molecular size, were significantly shorter for the underlying broad signal compared to the sharp signals (IP6) in soil extracts following hypobromite oxidation. In summary, we demonstrate the presence of a plethora of organic P compounds in soil extracts, largely attributed to IP of various order, and provide new insight on the chemical stability of complex forms of organic P associated with soil organic matter.