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Electrospray Ionization Mass Spectroscopy Shows Speciation of Phytate to Be Ph Dependent

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Electrospray Ionization Mass Spectroscopy Shows Speciation of Phytate to Be Ph Dependent WFL Publisher ' Science and Technology 0t1t01 hiOildTtOOd. 10Meri-Rastilantie3 B. Fl-00980 Joiii,itil of Food Agriculture & Environment 101.6(2) 402-407. 2008 net Helsinki. Finland c-mail irmibamiom Id-toad Electrospray ionization mass spectroscopy shows speciation of phytate to be pH dependent Lynne Heighton , Walter F. Schmidt 2 Clifford P. Rice 2 and Ronald L. Siefert Che,ni.stri and Biochemistry Department, Univecsitv of Mamyland, College Park, MD 20742, USA.Environmental Management andBvproduct Utilization Laborator USDA/ARS/ANRIBarc-West, 10300 Baltimore Avenue, Beltsville, MD 20705, USA. University of Maryland, Chesapeake Biological Laboratory, One Williams St., Solo/lions, MD 20688, USA. Current address: Chemistry Department (Mail Stop 9B), United States NavalAcademv, 572 MHollowav Rd., Annapolis, MD 21402-5026, USA. e-ntail: schmnidtot@ba. ars. usda.goi heighton(à.unmd. edit Received 5 Jumicirr 2008, accepted 28 Marc!, 2008. Abstract Phosphorus (P) fate and transport is an emergent problem impacting environmental resources. Long time land application of P enriched manure has been implicated in the saturation of available P binding sites in many terrestrial, wetland and sediment systems. Transport of soluble or particle associated P by overland flow and possibly by subsurface leaching has increased eutrophication in waterways. So-inositol hcxkis phosphate or more commonly phytic acid (PA) is an organic phosphate molecule with twelve acidic protons. The acid dissociation constants (pKa) are 1.9(3), 2.4(2), 3.2(1), 5.2(l), 6.3(l), 8.0(1), 9.2( 1) and 9.5(2). The charged species fractions were calculated as a function of pH using the acid dissociation constants. Results predict three different charged species of phytic acid will simultaneously be present at most any environmentally relevant pH. Analysis of the electro-spray ionization mass spectrometry (ESI-MS) solution spectra of iron and copper complexes of PA at pH 2.8, 6 and 13 confirmed multiple charged species of PA occur simultaneously even in the presence of metal cations. Results showed minimal fragmentation ofthe parent phytate anions. Changes in the: of PA anions, not changes in the stability or fragmentation of the parent compound with pH, explain the observed fragmentation pattern. Assigning the correct: is a pre-requisite to identifying the (nil:) composition of PA fragments. Key words: Phytic acid, ESI-MS, organic phosphorus. transport, phosphorus, mnvo-inositol hexkis phosphate. Introduction Phytate is the conjugate base of phytic acid, and phytate is the hexkis phosphate or more commonly called phytic acid (PA) or primary form of organic P found in soil. Each molecule of phytic phytate A fractional speeiation diagram was generated from acid contains six P groups per molecule and is remarkably this data. The pH driven acid/base speciation changes of phytic recalcitrant. The dissociation of phytic acid results in acidic acid are essential to understanding its ability to complex metals protons and a corresponding conjugate base. Each of the six in soil and aqueous ecosystems. Metal-ligand interactions are phosphate groups has two acidic protons disassociating from known to change solubility and mobility in response to changing the phytate anion at progressively higher pH and charge 2. Phytate pH Phytic acid acting as ligand is likely participating in such anions and inorganic P form complexes with metal cations. Spectral reactions. analysis of anionic phytate/cation ratios and speciation has not Electro-spray ionization (ES!) coupled mass spectroscopy (MS) been well characterized. Electro-spray ionization (ESI-MS) methods allow for the mass spectral analysis of large polar non- provides a unique and precise method to investigate the parent volatile biomolecules such as PA. The pH driven charge variability ions of phytate and phytate-cation complexes at multiple pH and multiplicity of charge at any one pH derived from acid values. The procedure could prove useful in identifying phytate dissociation constants of the phytic acid molecule, when speciation in more complicated matrixes, as well as in obtaining combined with soft ionization sample introduction, will produce association constants required for modeling the fate of P in complex corresponding mass spectra with predictable mass to charge environmental systems. More broadly, adding multivalent cations ratios (m/z)9 0, To investigate the charge distribution generated (or anions) to the mobile phase in ESI-MS can assist in identifying by the pH driven change in fractional composition the pH of a previously unassigned fragments of multi-charged anionic (or PA solution was adjusted to 2.8, 6 and 13. Ferric chloride or cationic) species. cupric chloride was added to each pH level to provide complexes Inositol phosphates are metabolically derived organic that would impart added spectral specificity. Predicted (,n/z) peaks phosphates that increasingly appear to be an important sink and and adducts were compared with observed spectral peaks. source of phosphate in the environment . lnositol hexkis phosphates exhibit twelve acid protons in solution. Acid Abbreviations: PA phyirc acid. ESi-MS ciectrospray ionization nias, spccirorrieIrv. 012 dissociation constants have been published for myo-inositol charge raito, ppm part per nnlhon. a , fraction of charged species 402 Journal of Food, Agriculture & Environment, Vol.6(2). \ pi \ I atrials and Methods :,cid dioLj:I1 ion co, 1:i,1t (1)1\ ) of 1:ne. Bct\\ CCO 11 5 and The calculations at unit intervals from pH 1 to 14 and graph ofthe 7, four species of phytate (H ,PA0 , H5PA, H4PA and HPA) PH driven fractional species change of phytate 78 were generated account for nearly all the phytate present. However, unlike on an Excel spreadsheet. The acid dissociation constants (pKa) phosphate, the relative amounts of all four species, not just two, govern the pH of the proton loss. Thus the species fraction (a) change systematically as the pH is altered. was generated by the sequential dissociation of each single For confinning phytate speciation Figs 1- 3 are data from ESI- proton. The number designation with (a) represents the fraction MS spectra at pH values of 13, 6 and 2.8 respectively. The of phytate molecules in which only the first proton has dissociated. assignment of peaks in each spectrum to chemical structure is For the fraction a7 seven protons have dissociated. At every pH presented in Tables 1-3. value: a 0 + a+a+a+a4 +a5 +a+a7 +a9 a9 a a,, Ferricphyrate chelates atpH 13: At pH 13 the potential parent a9 = fraction of species in the form H12A ion of phytate predicted would be C 6H( [OPO(0) ]9 = 648.08 or a, = fraction of species in the form H A- C9H6[OPO(ONa)2]( = 924.08. a = fraction of species in the form The spectrum of sodium phytate and ferric chloride at pH 13 is The general form of speciation for a polyprotic acid HA is shown in positive and negative modes (Fig. 1). At pH 13 the a = [HI/Di charge on phytate is —12, so in the absence of chelation, a mass a, =Kl[Hj"/D, fragment of 55 would be present and that fragment was not found; a =(K1K2 K[H1")/D therefore these multiply charged species were not detected in the whereD =[H]+Kl[HII +KlK2[H] 2 + .......+ KIK2K3 Kn electrospray interface and chelated species with charges of+ I or - 1 were found as the most abundant species. A cluster of ,n/z Identfjing speciation: The speciation of the charged fractions peaks for the negative polarity begins at 788 and ends with 827. In of PA at pH 2.8. 6 and 13 was investigated using electrospray positive mode the cluster begins at 776 and ends at 829. The peak ionization mass spectroscopy (ESI-MS). A Waters Quattro LC assignments and mass to charge ratios are presented in Table I. with MassLynx software was used to generate the spectra. These can best be explained as chelated forms of the phytate Instrumental parameters such as cone and capillary voltages were molecule as described in Table 1. adjusted to obtain robust spectra. Instrumental set points varied Van Berkel and Zhou " sited lkonornou and co-workers 11.12 in with the pH and analyte. A solution of 40 ppm PA was prepared Postulating the controlled-current electrolytic nature of the electro- and aliquots were adjusted to the desired pH using hydrochloric spray ion source suggesting that in positive mode or high positive acid and/or sodium hydroxide. voltage the build up of negative ions in the capillary is balanced by electrochemical oxidation reactions that produce neutral or Speciation with iizeta/ adducts: At each specified pH, ferric or positive ions and in a negative polarity the positive ion build up is cupric chloride was added to aliquots of PA at up to three times counter balanced by reduction reactions and emission of neutral the stoichiometric concentration of PA. The stoichiometry is one or negative ions -n The cone voltage provides the activation mole of metal adducts per 1/6 mole of PA. For ferric chloride at pH energy to develop a molecular charge and/or for molecular 13, the actual concentration in solution was constrained by Fe3 rearrangements. Aphytate solution at PH 13 is fully deprotonated. solubility. Samples were introduced by direct injection using an In the vapor phase, ferric ions, sodium ions and proton anions infusion pump to meter at a rate of 10 il/minute of analyte in the from the solution are ever present and multiple combinations will presence of 1% formic acid:methanol (70:30) mobile phase result in the same charge. The peaks and assignments delivered at a rate of 0.3 ml/minute using a high pressure liquid corresponding to the molecular ion are listed in Table 1. The large chromatography pump (HPLC). The mass to charge ratios (m/z) of array of fragments demonstrates at least one ferric ion is part of the generated spectral peaks were then analyzed in relation to the every fragment and that sodium and proton cations phytate charge corresponding to phytate speciation present.
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