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. interchangeably compete for the remaining charged molecular sites. Results Metal complexed phytate subjected to a potential applied to Results demonstrate that for any environmentally relevant pH, at the ESI capillary may be forming MS detectable species that are least one fifth of phytate molecules will not have the same charge uniquely defined by the applied potential and can potentially be as the most abundant phytate species. For example at pH 6, the manipulated to yield structural and kinetic data 111-12 The 12 unit major species of phytate (H 5PA7 ) has a net charge of —7, but difference in mass for phytate in the positive mode (776 versus H(,PA which has a —6 charge and H4PA8 which has a —8 charge 788 in the negative mode) is related to the loss of an additional will also be present. The average charge from both the 20% H6PA6 oxygen atom (16 mass unit) and with four fewer proton losses in and the 20% H4PA" remains —7, but at the molecular level, 40% of the positive mode. Structurally the loss of oxygen is a replacement phytate will have the physicochemical properties of either H6PA6 of P-O-Na with P-Na. Eleven Na ions replace eleven of the P-O or H4PA and not of H5PA7. sites in the positive ion mode (Mass 780) and ten in the negative At pH 7 phosphate buffers are nearly an equimolar ratio of ion mode (Mass 788). With sufficient cone voltage, the monobasic (H7PO4 ) and dibasic (HPO42-) phosphate. The identity fragmentation pattern, mass losses and molecular rearrangements of phosphate speciation present at the molecular level in solution observed occur. Explanation of the distribution of oxygen losses does not change between pH 5 and 7: only the relative abundance among the fragment is quite interesting but outside the scope and between these two species alters. For similar reasons, phytate focus of this manuscript. speciation changes with pH predictably for the each of the twelve

Journal of Food, Agriculture & Environment, Vol.6 (2), April 2008 403 EM

40 ppfTI

UWE

- Polarity

I, J

eKW

8X noo f I r , aoo OO10 o, Ft

Figure 1. ESI-MS spectra in -- and - polarity of ferric chloride and sodium phytate at pH 13 in formic acid mobile phase.

The relationship between n number of Na atoms and m number Since every major fragment contained at least one Fe atom. of H atoms in contrast is direct and unambiguous. In the negative The absence of the second Fe" in most fragments is consistent ion mode, n + m = 14 for all even masses. In the positive ion mode, with lower affinity of iron for the second site. Evidence for phytate n -- m = 18 for all even masses (except for fragments 778 and 780 in chelation with polyvalent cations at basic pH has also recently which the sum is 16). In the negative ion mode in never is less than been reported by He et al. . The binomial/Gaussian type 4; in the positive mode, m never is less than 5. distribution of fragments with variable amounts of Na and W per The presence of iron simplifies interpretation of spectrum. For fragment supports that at this pH cation binding selectivity for z = +1- 1, an odd number of ferric ions in a fragment always phytate is limited. A single I-1 cation could have affinity for any corresponds to an even mass unit. The mass units complexed one of twelve sites. Each one of two H cations would then have with two iron cations have an odd mass. The parent ion is phytate affinity for eleven of twelve sites. The most abundant fragments complexed with two Fe" atom, five Na atoms and one H ion, i.e., at pH 13 have at least half of the sites chelated with cations. No 828. appreciable masses were found below 776 (positive ion mode) or below 788 (negative ion mode).

404 Journal of Food, Agriculture & Environment, V61.6 (2), April 2008 Table 1. Phytic acid and ferric chloride at pH 13 ES I-MS peak assignments. in/z to any portion of the phytate structure would somehow be indeterminate. pH Positive ion mode PH 13 Negative ion mode 13 Cupric chloride (Cu" of 70% and Cu 65 of Peak assignment m/: Charge Peak assignment rn/: Charge 30%) was added to the mobile phase. A cation

C6H6Fe 2Na5021 P6 827 Z= -1 C6H5FeNa5021P6 829 +1 adduct would increase the mass of a parent or

C6H9Fe562Na,026P6 825 Z= -1 C6H11Fe562Na2026P6 827 = +1 daughter ion by 63 and 65. The two masses

C6H 11 Fe 6Na3025 P6 810 = = -1 C6H4Fe56Na10016P6 804 = +1 would have a ratio of 70/30. The isotope ratio can reduce the difficulty in interpretation of C6H 10Fe56Na4024P6 800 Z = -1 C6H7Fe56Na9017P6 800 = +1 the data. Fragments 103 and 105 have the right C6H5Fe56Na90 17P6 798 Z = -1 C6H10Fe56Na6021P6 798 = +1 peak ratios but there is no adequate structural C6H8Fe56Na60 1 P6 796 Z = -J C6H13Fe56Na3015P6 796 = +1 interpretation for its mass based upon the C6H 11 Fe56Na3075P6 794 Z = -1 C6H8Fe6Na5018P6 794 = +1 structure of cupric phosphate. The mass of

C6H6Fe56Na80 15P6 792 = = -I C61411Fe56Na5022P6 792 = +1 the fragment minus the mass of the cupric ion

C6H9Fe56Na5022P6 790 Z = -1 C6H6Fe56Na10015P6 790 = +1 (103 minus 65) has a mass of 38. The mass of

C6H4Fe 6Na 100 15P6 788 = -I C61-I14Fe54 Na4023P6 788 +1 38 is undecipherable as a fragment of PA.

C6H9Fe56Na90 1 5P6 786 = +1 Stable forms of phytate present at pH 6 are H5PA7 , H4PA8 and H6PA6 . Thus z in the C6H12Fe56Na6020P6 784 +1 fragments would be 7, 8 and 6 respectively C6 -! Fe6Na3074P6 782 2 +1 (Table 2). With the correct charge on phytate, C6H5Fe56Na 1 I 020P6 780 = +1 the fragments are assignable to chemical

C6H7Fe 6Na 50 1 7P6 778 Z= +1 structure. In each case, accounting is required 56 C6flgFe Na10014P6 776 +1 of both the mass and the net charge on the ion from that mass.

Ciipricpliytate chelatespH 6: At pH 6, significant mass ions are Fully ionized phytate has a charge of-12; thus when = = 7, the observed below m/z 140 (Fig. 2). Six phosphate PO43 groups charge on the cations attached to the molecule must equal 5. For (mass 95) removed from phytate results in inositol C ( H( (OH)( (mass nilz = 105 and 103, 7Na plus 5H and 5Na plus 7H have the 180). No loss of any chemical structure from H,PO4 I- (mass 97) or correct charge and the correct mass. The mass of the parent ion is from H4PO4 (mass 99) will equal the mass 91. The largest mass 733 and 721 respectively. fragments were smaller than the mass for inositol monophosphate For a fragment to have a —6 charge, cation charges to the parent C6H6(OH) 5 [0-PO(OH) 2 ] (mass 224), corresponding to phytate phytate equal +6. For ,n/z 137, 135, 133 and 113, the cations are minus five phosphate groups. Assuming z = 1, neither the mass 2CL121 plus 2Na, 2Cu 2 plus 2H, 2Cu2 and Na plus H and 5Na fragments nor the losses of mass between peaks were attributable plus H, respectively. The loss of an oxygen atom can also be to any specific molecular structure in phytate. A simple present. The mass of the parent compound is simply six times misinterpretation of MS data would be that at this pH, phytate is ni/z. The masses are comparable to those for pH 13. unstable (at least in the vapor phase) and/or that assigning the Fragments with a charge of -8 have cations with a charge equaling +4. Form/z =in sets of two: 97 and 95, 93 and 9l,and 83 and 81, 40 ppm cations are Cu2 plus 2Na, CU21 plus 2H, and 4H. The difference within each of the sets is the loss of one oxygen atom. The mass fragments can be explained without any change in the

Table 2. Phytic acid and cupric chloride at pH 6 ESI-MS peak assignments.

pH 6 Peak assignment = inlz in Charge C6146Cu632Na,024P6 -6 137 820 -12-4+2, C6H6Cu632026P6 -6 135 808 -12--4+2 C6H7Cu632Na024P6 -6 133 798 -12+4+1+1 C6H 11NaO-,4P6 -6 113 676 -12±1+5 C6H 10Na70 19P6 -7 105 733 -12±7+5 C6H12Na502 1 P6 -7 103 721 -12±5+7 C6H6Cu63Na2025P6 -8 97 773 -12+2+2 C6H6Cu63Na,0,4P6 -8 95 757 -12±2±2 C6H6Cu63076P6 -8 93 745 -12±2+2 C6H8Cu63075P6 -8 91 729 -12+2+2 C611 10025P6 83 668 -12+4 Figure 2. ESI-MS spectra of cupric chloride and sodium phytate -8 in - polarity at pH 6 in fornic acid mobile phase. C6H 10024P6 -8 81 652 -12+4

Journal of Fond, Agriculture & Environment, Vol.6(2), April 2008 405 in fragmentation pattern of the parent phytate peak. A portion of the 3 identifies peak assignments corresponding to z, m/z and single peak at 91 also can be the formic dimer present in the mobile pH 2.8 in the presence of ferric ions. The peaks at 202,200, 198 and phase. This peak, however, was not observed at pH 2.8. 196 correspond to ions with a —4 charge. The cations on phytate No peaks found correspond to one or more losses of phosphate add up to +8. The cations are 2Fe 31 plus 2Na, 2Fe3 plus Na and groups or of fragments of phosphate groups. Without the loss of W, 2Fe3 plus Na and W, 2Fe 3 plus 2W and 2Fe 3 plus Na and phosphate groups from phytate, there is no mechanism for H. Consistent with pH 6, the number of oxygen atoms on the formation of fragments smaller than phytate to form. Changes in parent ion can be 24, 25 or 26. charge on the molecule, not changes in fragmentation of phytate, Peak corresponding to a —5 charge are the cluster 167, 165, 163, explain the ESI-MS data. 161 and 142. The cations add up to +7, i.e.,Fe 3 plus 5Na and -1W, 7Na, 2Fe3 plus Nat , Fe 3+ plus 3Na and H, and 6Naplus Ferricphytate chelatespH2.8: The fragments from pH 2.8 (Fig. H. Fragments 128 and 126 correspond to z = -6 and cations 3) could mistakenly be interpreted as from very different molecular equaling +6 are Fe 3+ plus 2Na and W and 4Na plus 2W, source as the spectra taken at pH 13 or pH 6. No mass corresponds respectively. The mass ions at pH 2.8 are similar to those present to the molecular weight of phytate. Evidence of phosphate at pH 13 when corrected for charge and for the mass difference (HPO 3 mass =79 or of fragments of phosphate from phytate from cations present with the parent ion at that pH. are not apparent. The difference between 202 and 126 (high mass and low mass in the data set) itself is only 76. Thus the range is Discussion inadequate to explain fragments of a mass containing six None of the peaks identified would be properly assignable to any phosphate groups. chemical structure without first knowing the pH of the solution Three species of phytate present at pH 2.8 are H7PA, H(PA6 from which the sample was taken. The absence of mass ions in and H8PA4- and z in the fragments is 5, 6 and 4 respectively. Table near the molecular weight of phytate except at strongly basic pH values could be taken as evidence that phytate ions are either too 1&1 complicated or too unstable to detect by mass spectrometry. Instead the parent compound is remarkably resistant to fragmentation. Even at pH 13, the fragments that are lost are the cations that chelate phytate and not phosphate groups. The loss of an oxygen atom attached to phosphate groups occurs more readily than the loss of a phosphate group from the parent ion. The fragmentation pattern for phytate at pH 6 and 2.8 are totally unassignable to phytate without including the correct value of no charge z in the m/z ratio. The presence of even the class of compound as an organic phosphate is indiscernible from the peaks unless the z in the ,n/z ratio is known. Multiple analytical procedures have been developed to measure phytate in soils. Results suggest extracting phytate from soils at any pH except the pH of the soil at which it was found will always alter its charge and therefore its speciation and its affinity for metal adducts. Quantitative extraction of phytate is dependent on the anionic speciation that occurs and the cationic adducts with which they can strongly associate. Figure ESI-MS spectra in negative polarity of ferric chloride and 3. The co-existence of multiply charged species at any ecologically sodium phytate at pH 2.8 in a formic acid mobile phase. relevant pH is an essential part in understanding phytate ligand Table 3. Phytic acid and ferric chloride at pH 2.8 ESI-MS solubility and organic P mineralization. The strength and affinity peak assignments. of any cation associated with a phytate species can be expected to change proportionately to the unit anion charge. Binding will pH 2.8 not necessarily prefer the most abundant phytate species, and Peak assignment Z rn/: in Charge multiple species of phytate can simultaneously support multiple C6H6Fe 62Na2024P6 -4 202 806 -12±6+2 cationic ligands. The multiply charged species of phytate present C6H 7Fe562NaO16P6 -4 200 800 -12±6+1+1 at any one pH often precludes stoichiometric complexation of

C6H8Fe562026P6 -4 198 794 -12+6+2 phytate with single metal cations. At pH 6 H 6PA" can form

C6H7Fe560Na,014P6 -4 196 784 -12+6+2 neutral complex with two Alcations, but any A1 3 complexes 5 H4 C6H5Fe562NaO27P6 -5 167 837 -12+6-1 with H PA7 or PA8 will simultaneously be charged.

C6H5Fe562028P6 -5 165 823 -12-6-1 Speciation and phytase activity: The pH dependence of phytate C6H6Fe562Na 077P6 -5 163 815 -12+6+1 associations with trace metals, sesquioxides, colloidal clays and C6H7Fe56Na3026P6 -5 161 806 -12+3+3±1 fulvic and humic material has been reported However, at any C6H 12NaO26P6 -5 142 709 -12+1+6 biologically relevant pH, Fig. 2 demonstrates 20% or more of C6H7Fe56Na2025 P6 -6 128 767 -12+3+2+1 phytate will be at one pH value higher or one pH value lower than C6H8Na4O,5 P6 -6 126 758 -12+4+2 the most abundant species. Thus the composition of such phytate-

406 Journal of Food, Agriculture & Environment, Vol.6 (2), April 2008 ligand complexes will always be heterogeneous. Competitive values consistent with the pH dependence of phytate without binding will always occur even among the multiple phytate species metal adducts. In more complicated matrices such as soils, the pH for any particular ligand present. A ligand can more preferentially dependence of the molecular charge on phytate could be similarly bind to less abundant species of phytate with a higher (or lower) important. net charge than to a most abundant phytate species with a lower ESI-MS studies can enable fundamental research on competitive (or higher) net charge. binding of cation with phytate and environmental research on Enzymatic dephosphorylation occurs via two currently forms of phytate and phytate-adducts present in solution without recognized forms of phytase: 3-phytase (E.C.3.1.3.8), which is perturbing its speciation. Traditional soil analysis depends on microbial in origin and is most active at both pH 2.5 and 5.5, and 6- pH-based extraction techniques that are not required for analysis phytase (E.C. 3.1.3.26), which is produced by some plants and is by ESI-MS. ESI-MS detection of multi-charged anions as anion + most active primarily at pH 5 15 . Optimal enzyme activity often is cation adducts (or of multi-charged cations as cation + anion pH dependent. A simpler explanation is that 3- or 6-phytase binds adducts) enables both competitive anion-cation association preferentially to specifically charged sites on the phytate molecule studies that address its molecular properties and distinctive and it is the charges on the phytate which are strongly pH identification of compounds in complicated matrices. The flexibility dependent. From Fig. 2, H 7PA- and H9PA3- are the most abundant inherent in the ability to analyze environmental samples at native forms at pH 2.5. HPA7- is the most abundant form at pH 5 and as pH will facilitate the modeling of environmental systems containing such must have at least one phosphate site on the molecule with phytic acid and other multiply charged environmental ligands. a -2 charge. The electrostatic charge of the phosphate group site with the -2 charge could attract phytate to the proper site that References initiates enzymatic dephosphorylation. Reactions at pH 2.5 may Bar-Yosef, B., Chang, A. C. and Vega, S. 1993. Organic P Transformation be more complex, but phytate speciation phytase. Reactions, and Transport in Soils Monitored by 3 P NMR Equimolar ratios of transition metal-phytate complexes have Spectroscopy. Bard IS-1610-89. been shown to inhibit enzymatic dephosphorylation rates . Champagne, E.T. 1988. Effects of pH on mineral-phytate, protein- Phytate complexation and phytase enzyme activity are competitive mineral-phytate and mineral fiber interactions. Possible consequences reactions. The chemical stability of phytate and of ferric and cupric of atrophic gastritis mineral bioavailability from high-fiber foods. Journal of the American College of Nutrition 7(6):499-508. complexes even in the vapor phase suggests that its chemical Jayasundera, S., Schmidt, W.F., Reeves III, J.B. and Dao, T. H. 2005. stability in soil is also significant. As with the ESI-MS data, Direct P NMR spectroscopic measurement of phosphorus forms in detection of phytate in soil can be inconclusive or misleading. dairy manures. J. Food, Agri. and Environ. 3(2):335-340. For example, enzymatic phytase methods may systematically Hansen, J.C., Cade-Menton, B. and Strawn, D.S. 2004. Phosphorus underestimate the amount of phytate present in soils due to the speciation in manure-amended alkaline soils. J. Environ. Qual. formation of stable cationic adducts and/or due to the multiplicity 33:1521-1527. of phytate speciation. -Turner, B.J. and Haygarth, P. M. 2001. Biogeochemistry - Phosphorus solubilization in rcwetted soils. Nature 411(6835):258-258. Conclusions 6Brinch-Pedersen, H., Sorensen L. D. and Holm, P.B. 2002. Engineering crop plants: Getting a handle on phosphate. Trends in Plant Science The mass balance of phytate innately includes the multiplicity of 7(3):118-125. its speciation. The accuracy of monitoring and predicting phytate 7Stumm, W. and Morgan, J.J. 1996. Aquatic Chemistry. John Wiley and fate and transport over time and space is thus seriously incomplete, Sons, Inc., New York. if phytate is assumed to be present as a single species. At usual Morel, F.M.M. and Hering, J. G. 1993. Principals and Applications of water or soil pH values, from 20-40% of phytate has a molecular Aquatic Chemistry. John Wiley and Sons, Inc., New York. charge either one unit lower (or one unit higher) than the major Wan Berke], G.J. and Zhou, F. 1995. Characterization of an electrospray component. Phytate, like phosphate, is its own buffer (except that ion source as a controlled-current electrolytic cell. Anal. Chem. it forms both a conjugate acid and a conjugate base). The charge 67:2916-2923. on phytate species is predictably and systematically pH dependent. "Fenn, J.B., Mann, M., Meng, C. K., Wong, S.F. and Whitehouse, C.M. 1989. Electrospray ionization for mass-spectrometry of large The multiplicity of speciation and metal adducts to any of the biomolecules. Science 246(4926):64-71. components of that multiplicity can be a significant reservoir of "Ikonomou, M. G., Blades, A. T. and Kebarle, P. 1991. Electrospray-ion phytate in the soil and simultaneously in the variability and lack spray: A comparison of mechanisms and performance. Anal. Chem. of reproducibility in analysis of soil phosphorus. Accurate 63(18):1989-1998. environmental mass balance and kinetic calculations for organic 2Blades,A. T., lkonomou, M. G. and Kebarle, P. 1991. Mechanism of and inorganic phosphorus in soils requires that speciation of electrospray mass spectrometry. Electrospray as an electrolysis cell. phytate is explicitly part of the accounting. Anal. Chem. 63(19):2109-2114. ESI-MS analysis demonstrates z in the mass/charge m/z ratio "He, Z., Honeycutt, C.W., Zhang, T. and Bertsch, P.M. 2006. Preparation is critical for the detection and identification of phytate species. and FT-JR characterization of metal phytate compounds. J. Environ. Qual. 35:1319-1328. Indeed, if z values were not known, even the class of compounds "Stewart, J.W.B. and Tiessen, H. 1987. Dynamics of soil organic (organic phosphate) would have been barely if at all discernable phosphorus. Biogeochemistry 4:41-60. from the fragmentation data. In spectral data at pH 13,6 and 2.8, 5Pallauf, J. and Rimbach, G. 1997. Nutritional significance of phytic no evidence was found of the loss of one or more phosphate acid and phytase. Arch. Anim. Nutr. 50:301-319. groups from any phytate parent ion. In addition, stable metal 16Dao, T. 2003. Polyvalent cation effects on myo-inositol hexakis adducts were routinely present even in the vapor phase. Ferric dihydrogen phosphate enzymatic dephosphorylation in dairy waste- and cupric ions form stable phytate-metal adducts at multiple pH water. J. Environ. Qual. 32:694-701.

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