High-Performance Liquid Chromatography Analysis of Phytic Acid on a pH-Stable, Macroporous Polymer Column

Cereal Chem. 66(6):510-515 A procedure was developed that enables up to 60 samples containing sis of the concentrated sample on a macroporous polymer column was phytic acid to be analyzed in one day. Extraction time was reduced to accomplished in 8 min. The column easily resolved mixtures contain1-3 min per sample by using ultrasonic irradiation. Concentration of ing inositol hexaphosphate, inositol pentaphosphate, and inositol tetrathe extract was simplified by using a commercially available silica-based, phosphate. Samples of wheat bran as small as 50 mg were analyzed by anion-exchange column. High-performance liquid chromatography analythis system. Phytic acid (myo-inositol 1,2,3,5/ 4,6-hexakis [dihydrogenphosphate]) is a common constituent of most mature cereal grains and some vegetables and fruits (Oberleas 1973). In grains it often occurs as phytin (a mixed calcium-magnesium salt of phytic acid) and can represent 60-90% of total phosphorous (Lolas and Markakis 1975). Levels can vary with growing conditions, maturity (Makower 1970), type, variety, and mill fraction of the grain. For example, whole wheat can contain 0.32% phytate, the germ fraction 1.1% (O'Dell et al 1972), and the bran fraction 5%. Sesame seed contains 5.18% phytate (deBoland et al 1975), whereas beans (Phaseolus vulgaris), depending on variety, contain 0.541.58% (Lolas and Markakis 1975). Excessive amounts of phytic acid in the diet can have a negative effect on mineral balance because phytic acid forms insoluble complexes with Cu+, Zn+, Fe+, and Ca+ at physiological pH values (Graf 1986, Nolan et al 1987) and, consequently, reduces the bioavailability of these minerals (Morris 1986). Nutritional deficiencies can (Oberleas 1973) but need not necessarily result (Lee et al 1988). Feeding studies utilizing quail (Tao et al 1986) showed that the antinutritive effect can be eliminated by the partial dephosphorylation of phytic acid (IP6) to myo-inositol tetrakisphosphate (IP4) or the lower esters. Additionally, processing (Han and Wilfred 1988) and fermentation during dough making (Nayini and Markakis 1983) can dephosphorylate phytic acid to the lower phosphate esters. However, most of the current convenient analytical methods (Graf and Dintzis 1982a,b; Harland and Oberleas 1986) do not differentiate phytic acid from partially dephosphorylated phytic acids; i.e., the methods will not differentiate grain products that can reduce mineral bioavailability from grain products that cannot. The teleological role of phytic acid also directs our attention to the lower inositol phosphate esters. Currently, phytic acid is considered to be a reserve of phosphorous and cations in plants (Williams 1970), a ballast (i.e., a system to control the level of free phosphate), and possibly an antioxidant to preserve seed viability. Recently it was shown that signal transduction affecting contraction, secretion, proliferation, and photoresponse in animal cells is moderated by myo-inositol (1,4,5) trisphosphate (IP3), myo-inositol (1,3,4,5) tetrakisphosphate (IP4), and calcium (Neher 1987). A similar system appears to exist in plant systems (McMurray and Irvine 1988). A rapid and simple procedure to isolate, identify, and quantify the lower phosphate esters of inositol would facilitate work in this new area. Oberleas and Harland (1986) recently reviewed the various 'U.S. Department of Agriculture, Agricultural Research Service, Northern Regional Research Center, 1815 North University Street, Peoria, IL 61604. The mention of firm names or trade products does not imply that they are endorsed or recommended by the U.S. Department of Agriculture over other firms or similar products not mentioned. This article is in the public domain and not copyrightable. It may be freely reprinted with customary crediting of the source. American Association of Cereal Chemists, Inc., 1989. methods of phytic acid analysis. In general these methods require three steps: extraction, concentration and/ or purification, and analysis. Most current procedures use a 1.5-3 hr extraction with 2.4% HCO (Harland and Oberleas 1986). Subsequent concentration and purification may be by anion exchange on a resin-based column or by precipitation as ferric phytate. Phosphorous analysis of the digested eluent from the anion-exchange column or the digested ferric phytate precipitate permits calculation of the amount of phytic acid in the original sample. In both cases the presumption is that the only source of phosphorous is phytic acid. In ferric phytate precipitation, an additional assumption is that complete precipitation of only IP6 occurs. Unfortunately, the anion-exchange column also retains the lower phosphate esters. The "pure" ferric phytate precipitate can also contain some of these partially dephosphorylated phytic acids. Because these methods do not differentiate IP6, IP5, IP4, etc., calculations can be in error. The indirect method of phytic acid analysis in which phytic acid is added to a standard solution of ferric ions has similar deficiencies. After precipitation of ferric phytate is complete, the remaining ferric ion concentration is determined spectrophotometrically (chromophore varies with investigator). The relationship between Fel and phytic acid in the precipitate is assumed to be stoichiometric (Fe:P, 4:6, Wheeler and Ferrell 1971) as is complete precipitation (Ellis et al 1977, Kikunaga et al 1985). Partially phosphorylated inositols, if present, are incorrectly included in these phytic acid determinations. Partially phosphorylated inositols have been identified and quantified by liquid chromatography on an anion-exchange column utilizing formate, NaCl, or HCO gradients. Dagher et al (1987) followed the sequential hydrolysis of phytic acid in highbran bread using a Dowex 1-X8 column and a linear HCO gradient. This procedure requires large volumes of eluent, long elution times, and large numbers of individual analyses (Bartlett 1982, Phillipy et al 1987, Minear et al 1988, Shayman and BeMent 1988). The solvent gradient precludes the use of ultraviolet (UV) or refractive index (RI) detectors. However a recently described postcolumn detection system circumvents these problems (Mayr 1988, Phillippy 1988). A mass detector or a moving wire flameionization high-performance liquid chromatographic (HPLC) detector might be a suitable alternative to the postcolumn detection system. Previous applications of HPLC to the analysis of phytic acid (Tangendjaja et al 1980; Camire and Clydesdale 1982; Graf and Dintzis 1982a,b; Knuckles et al 1982) used reversed-phase silica columns (C-18 and refractive index detectors). Unfortunately, the solvent front coincided with the phytic acid peak and made quantitation difficult. Lee and Abendroth (1983) recognized the problems associated with quantitating peaks that coincide with solvent fronts and made the quantum leap to the concept of ion pair in order to separate the solvent front from analyte. Sandberg and Ahderinne (1986) and Sandberg et al (1987) extended this application by demonstrating that IP6, IP5, IP4, and IP3 could be separated by adjusting the pH from 7.1 to 4.3. 510 CEREAL CHEMISTRY The official method for phytate in foods (Harland and Oberleas 1986) is elegant but rather lengthy and lacks the ability to identify and measure the lower phosphate esters of myo-inositol. I wanted a procedure that would address these deficiencies and allow samples to be completely analyzed in one day. The method reported herein shortens the extraction and concentration procedures and uses a durable polymer column for HPLC analysis. The method can identify and quantify IP6, IP5, IP4, and IP3. A comparison between the customary phytic acid analytical methodology (method A) and the method presented in this communication (method B) is summarized in Table I. MATERIALS AND METHODS Figure 1 describes the methodology developed for the quantitation of phytic acid in grains and legumes using samples as small