Identification and quantification of inositol hexa-kis phosphate (IHP) in environmental samples at neutral pH using electro-spray ionization and raman spectroscopy

Authors: HEIGHTON, L - University Of Maryland; ZIMMERMAN, M - University Of Maryland; Rice, Clifford; Codling, Eton; TOSSELL, J - University Of Maryland; Schmidt, Walter

Submitted to: Journal of Agricultural and Food Chemistry
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 1/31/2012
Publication Date: 3/1/2012
Citation: Heighton, L.P., Zimmerman, M., Rice, C., Codling, E.E., Tossell, J., Schmidt, W.F. 2012. Identification and quantification of inositol hexa-kis phosphate (IHP) in environmental samples at neutral pH using electro-spray ionization and raman spectroscopy. Journal of Agricultural and Food Chemistry. 2:55-63.

Interpretive Summary: The predominant chemical form of organic phosphorus (P) in grain and forage crops and livestock manures is phytate, which contains 6 phosphate groups, each attached to a carbon atom. The fate and transport of phytate added to agricultural soils is poorly understood and very difficult to predict because phytate can be complexed with metals, partially decomposed and adsorbed onto clays or metals or other organic compounds, or totally broken down into inorganic P. Farmers commonly test soils for P, but these soil tests generally require an acidic or basic extraction that can change the forms of soil P, which prevents the identification of which form of P is in the soil. The lack of knowledge about the forms of soil P can lead to under- or over-application of P fertilizers. Excess soil P can increase P runoff into streams and shallow groundwater, which leads to excess microbial growth and eutrophication of the Chesapeake Bay. Our previous research demonstrated: 1) that the forms of phytate present are highly dependent upon the acidity of the water in which it is dissolved, 2) that phytate forms highly stable soluble metal ion complexes, which also depend upon water acidity, and 3) that such forms can be detected and differentiated from inorganic P in natural water using two spectroscopic techniques. Our research shows that these two spectroscopic techniques can detect the difference between phytate and phytate/metal complexes in two types of soils before and after adding organic matter amendments. The spectroscopic techniques can also directly detect very low levels of phytate in soil extracts at the native pH of the soil. These findings will help scientists understand and predict the fate and transport of organic P from crops and manures, which will improve fertilizer P recommendations and reduce P losses to surface waters.

Technical Abstract: Phosphorous (P) is a major contributor to eutophication of surface waters, yet a complete understanding of the P cycle remains elusive. Inositol hexa-kis phosphate (IHP) is the primary form of organic P in the environment and has been implicated as an important sink in aquatic and terrestrial samples. IHP readily forms complexes in the environment due to the 12 acidic sites on the molecule. Quantification of IHP in environmental samples has typically relied on harsh extraction methods. The accuracy of these procedures depends upon which of the molecular properties of IHP and which of the constituent properties of the soil are perturbed. Understanding the interactions, IHP with potential soil and aquatic complexation partners is critical to being able to measure soluble P discrete from soluble IHP. The ability to quantify IHP in-situ or at neutral pH could provide a better understanding of the role of each form in the P cycle. Electro-spray ionization mass spectroscopy (ESI-MS) was used to quantify IHP in two soil samples using a neutral aquatic extraction. Raman spectroscopy was used to collect pH dependent spectra of inorganic P, IHP and soil samples. Density Function Theory (DFT) calculations were used to confirm the pH dependence from the experimental Raman spectra correlates with molecular properties. DFT identifies the 12 acidic sites (and number of sites) in IHP which deprotonate selectively with increasing pH. At neutral pH 7, the eight of twelve, (not six of twelve) will always be deprotonated. These same specific anionic sites are readily available for cationic association and/or mineralization with soil constituents. Measurement techniques presented enable correctly distinguishing between soluble form of P and soluble forms of IHP.