Identification of phytate in phosphorus-31 nuclear magnetic resonance spectra: The need for spiking

Phosphorus-31 (31 P) nuclear magnetic resonance (NMR) spectroscopy of sodium hydroxide-ethylenediaminetetra-acetic acid (NaOH-EDTA) extracts has recently become a widely used technique for the characterization of soil P. This technique has seemingly enabled easy identification and quantification of phytate (myo-inositol hexakisphosphate), a compound long believed to constitute a major proportion of organic P. Phytate is usually identified by its characteristic pattern of four resonances in the ratio 1:2:2:1. We report that the P-31 NMR spectra of the NaOH-EDTA extracts of four Australian pasture soils contain a set of resonances that bear a striking resemblance to the phytate resonances but that are shown not to be phytate though careful addition (spiking) of pure phytate. The spiking experiments identify a much smaller set of resonances as being phytate. Quantification of these resonances shows that phytate comprises < 5% of organic P and < 3% of total P in these soils. We also show that the P-31 chemical shift of phytate resonances is very sensitive to pH and ionic strength. These results highlight the potential for misassignment of resonances in P-31 NMR spectra of NaOH-EDTA extracts of soil and the possibility that phytate concentrations may be overestimated using this technique and show the value of spiking as a definitive form of identification of P-31 NMR resonances.