New Chromophore for Phytic Acid Determination

Cereal Chem. 63(6):475-478 A direct spectrophotometric method was developed to analyze phytic phytate. The new chromophore was used to determine phytic acid in eight acid without acid digestion. The method was based on the precipitation of legume seeds. Values obtained for phytic acid with the new chromophore phytate as ferric phytate followed by conversion to sodium phytate. On agreed with those obtained with Bartlett reagent. Among the legumes heating, phytate reacted with chromogenic reagent and a blue molybdenum investigated, soybean had the highest value (23.35 ± 1.60 mg/g of meal), complex was formed. The same reagent reacted with inorganic phosphate whereas black-eyed pea had the lowest (8.74 ± 0.83 mg/g meal). The and produced a similar blue complex. Both these complexes had maximum advantages of determining phytate from the new chromophore over absorption at 830 nm and were stable for several hours at room methods in the literature are that it eliminates the need for acid digestion of temperature. The color complexes obeyed Beer's Law over a wide range of phytate to inorganic phosphate; it is easy to prepare and is stable; the concentrations of phytic acid and inorganic phosphate. The molar ratio of chromophore reacts with phytate over a wide range of p H from I to 13; and phosphorus to phytate was determined to be 5.84 by comparing moles of it can be used for monitoring chromatographic separation of inositol phosphorus in digested phytate to the moles of phytate in undigested phosphates. Many of the antinutritional factors associated with raw soybean phosphorus or iron in isolated ferric phytate (McCance and can be eliminated or minimized by proper heat treatment (Hafez et Widdowson 1935, Schormuller et al 1956, Samotus and al 1983, 1985). However, poor mineral bioavailability is not Schwimmer 1962, Crean and Haisman 1963, Brooks and Morr directly improved by heating (Erdman 1979). Soybeans, in 1982, Thompson and Erdman 1982). common with other seeds, contain phytic acid, which influences Leucena-Conde and Prat (1957) developed aspectrophotometric the bioavailability of trace metals. Several reviews on phytic acid method for determination of phosphorus. Their reagent gave a deal with the nutritional implications as well as its chemistry and blue color with phosphorus and was easy to prepare, stable in the determination (O'Dell et al 1972, Erdman 1979, Cheryan 1980, air, yielded a low blank, and did not require the use of a reducing Jaffe 1981, Young and Janghorbani 1981). agent. A spray reagent for the detection of phospholipids on To evaluate relationships between phytic acid and the chromatograms, based on the reagent of Leucena-Conde and Prat availability of trace metals in soybean, one must be able to (1957), was prepared by Vaskovsky and Kostetsky (1968). This accurately measure both of them. Several methods are found in the reagent reacted with phospholipids without heating to give blue literature for quantitative determination of phytic acid. Of these, spots. Raheja et al (1973) developed a spectrophotometric method most of the chemical methods are based on the determination of for the quantitative determination of phospholipids without acid. digestion based on the modified spray reagent formulated by Vaskovsky and Kostetsky (1968). 1On sabbatical leave from the Department of Biochemistry, University of Peradeniya, We have developed a new spectrophotometric method for the Peradeniya, Sri Lanka. 2 Nutrition Laboratory, Department of Human Ecology, University of Maryland quantitative determination of phytic acid without acid digestion, Eastern Shore, Princess Anne 21853, from whom reprints should be requested. based on the modified reagent of Raheja et al (1973). The method _______________________________________________ reported here was used to determine phytic acid in eight legume ©1986 American Association of Cereal Chemists, Inc. seeds. Vofl. 63 No.6,1986 475 MATERIALS AND METHODS Extraction of Phytate Several attempts were made to determine the best conditions for All chemicals used were analytical grade and purchased from extracting phytate from legumes. Extractions were carried out Sigma Company (St. Louis, MO). Sodium phytate of 97% purity, with 1.2% HCl, 3% trichloroacetic acid (TCA), and 3% H2SO 4 with containing 12 Na per mole from corn (Type V) was used as a and without 10% Na 2SO 4. The best solvent was found to be TCA. standard. The moisture content was determined and found to be A series of different concentrations of TCA ranging from 1 to 10% 18%. Anhydrous, dibasic sodium phosphate was used as the were tested for extraction of phytate, and no increase in phytate inorganic phosphorus standard. Soybean, black-eyed pea, lima extractability was observed above the 3% level. bean, navy bean, mung bean, winged bean, and black bean were A sample (0.5 g) of legume meal was placed in a 125-ml purchased locally. Erlenmeyer flask and extracted with 25 ml of 3% TCA for 45 mm in a shaker of moderate speed at room temperature (230C). Eight Reagents milliliters of the slurry was centrifuged at 20,000 X g for 15 mi at Solution A. Sixteen grams of ammonium molybdate was 230 C. Three milliliters of 1% FeC13"6H 20in L.ONHCI was added dissolved in 120 ml of distilled water. to 5 ml of supernatant and heated in a boiling water bath for 45 Solution B. Forty milliliters of concentrated HCl and 10 ml of min. It was allowed to cool and centrifuged at 20,000 X g for I0 elemental mercury were shaken with 80 ml of solution A for 30 min. The precipitated ferric phytate was suspended in 0.5N HCl min, filtered using Whatman no. 1 filter paper, and the filtrate was and incubated at room temperature (23°C) for 2 hr (Ellis et al used. 1977). The precipitate was washed twice with 0.5 N HCl allowing a Solution C. Two hundred milliliters of concentrated H2SO 4 was 10-min incubation period between each wash. Three milliliters of added carefully to the remainder of solution A and mixed with 1.5N NaOH and 7 ml of distilled water were added to the filtrate of solution B. precipitated ferric phytate and heated in a boiling water bath for 15 Chromogenic solution. Forty-five milliliters of methanol was min. It was cooled and centrifuged. The supernatant was used for mixed with 25 ml of distilled water and 25 ml of solution C. This estimation of phytate. solution is stable for at least three months when stored at 5 C (Raheja et al 1973). Determination of Extracted Phytate Standardphytate solution. The phytate solution was prepared Extracted phytate (0.2 ml) was mixed with 4.6 ml of distilled by dissolving hydrated sodium phytate in distilled water to contain water and 0.2 ml of chromogenic solution and heated in a water 11.740 mg of sodium phytate and the volume adjusted to 100 ml. bath at 95 C for 30 min. It was allowed to cool, and the color was Standard inorganic phosphate solution. Phosphate solution was read at 830 nm against a blank. prepared by dissolving anhydrous Na 2HPO4 in distilled water to Standard phytate solution was used to prepare a series of tubes contain 1.385 mg Na 2HPO4 and the volume adjusted to 100 ml. containing 17.8-177.8 nmol phytic acid per tube. The volumes were adjusted to 5 ml with distilled water and mixed with 3 ml of Preparation of Legume Seeds for Phytate Analysis 1% FeC1 3"6H 20 in LON HCI. These tubes were treated as The legume seeds were ground in a centrifugal grinding mill described for the legume samples; however, the direct (Brinkman, Westberg, NY) and passed through a 0.5-mm pore size determination of phytate in standard solutions gave the same screen. The oil was extracted with hexane using Goldfisch results as the precipitation method. apparatus. The defatted samples were dried overnight at room temperature and stored in airtight bottles in the freezer until Determination of Phytate Phosphorus further experimentation. Phytate extract (2.0 ml) was digested using 0.5 ml of 10NH 2S0 4 . It was further treated with three drops of 30% H20 2 and reheated to Determination of Moisture Content complete oxidation. This solution was neutralized with 5NNaOH The moisture contents of sodium phytate and legume meals were using phenolphthalein as an indicator and adjusted to a known determined according to AOAC methods (1984). volume; 0.2 ml of the neutralized solution was mixed with 4.6 ml of distilled H20 and 0.2 ml of chromogenic reagent in a test tube and heated for 15 min in water bath at 950 C. The tube was cooled and read at 830 nm against a blank. A series of tubes was prepared containing 9.18-97.5 nmol of 0.6 Na 2HPO4. The volume of each tube was adjusted to 4.8 ml with , -o. distilled water, and 0.2 ml of chromogenic reagent was added. "°0P These tubes were treated as described above and the color read at ,. o"830 nm against a blank. .,o ,,The Bartlett method (1959) was also used to determine inorganic <0.4o phosphate in the digested phytate. The amount of phytic acid (mg) m3..)o was calculated by multiplying milligrams of phytic acid c'r" 9")0 • •phosphorus by a factor of 3.553, assuming that 1 mol of phytic acid © 0. ..;o • %contains 6 mol of phosphorus. £L1 -°•'" RESULTS AND DISCUSSION 7•-• •'°'°" •The new feature of the method we developed is that it does not involve acid digestion of the phytic acid. Instead, the chromogenic 0.1 reagent reacts directly with the phytic acid phosphorus, and a molybdenum blue color complex is formed. The chromogenic 0 L.., , ,. , I , I reagent is stable and does not require the use of a reducing agent. When diluted under hot or cold conditions, the reagent itself does 7 00C 750 8 0 0 8 5 0 not produce a molybdenum blue in the acidic medium (LeucenaConde and Prat 1957). The stability of the reagent and the final W AV E L. E N G T H I nq m) color produced with both phytate and inorganic phosphate makes Fig. 1. Spectra of colors given by phytic acid (9.24 jig/ml) and Na 2HPO4 the method very convenient. (0.66 jig/mi) in the reaction mixture: phytic acid determined by the Our data show that the blue color complexes formed with proposed method (--) and Na2HPO 4 by the new method (-) and phytate and inorganic phosphate have maximum absorption at Bartlett method ( .... ). 830 nm. This is in agreement with the results of Bartlett (1959), 476 CEREAL CHEMISTRY which gave the same spectral curve with inorganic phosphate (Fig. after digestion using Bartlett reagent and these data are shown in 1). Table I. The results obtained with chromogenic reagent without The blue complex for phytate obeys Beer's Law in the range digestion of phytate agreed favorably with those obtained with 17.8-177.8 nmol of phytic acid (Fig. 2). For inorganic phosphate, Bartlett reagent. Reported values for phytate in navy beans (Lolas this method and the Bartlett method are highly sensitive and could and Markakis 1975), lima beans (Chang et al 1977), and red kidney detect 9.8 nmol of phosphorus in 5 ml of final reaction mixture. beans (Iyer et al 1980, Tabekhia and Luhn 1980) agreed with those Further, solutions with high color value could be diluted with by the new method. Soybean was found to have the highest phytate distilled H20 and read in the most accurate range for content, and this value agreed with those reported by Camire and spectrophotometry without losing proportionality. Clydesdale (1982). However, Erdman (1979), Lolas et al (1976), In the method reported here, 0.2 ml of chromogenic solution was and de Boland et al (1975) reported lower values. Tabekhia and added directly to the purified sodium phytate or inorganic Luhn (1980) reported a higher phytate content for black-eyed peas phosphate, and the mixture heated was at 95' C. A 15-min heating and a lower value for mung beans compared to our values. The time was selected for inorganic phosphate, the time at which a differences in phytate content between our values and those constant color was obtained (Fig. 3). The heating time of 30 min reported may be attributable to variation in cultivars, varieties, or was selected for phytate, the time at which color was high and environmental conditions. hydrolysis of phytate was minimum. Refluxing of phytate with The average value for the major ratio of phosphorus to phytate chromogenic solution at 1000 C produced a linear increase in color in the phytate extracts of eight legumes was 5.67 ± 0.09. This ratio absorbance at 830 nm up to 30 min, and thereafter an exponential increase in absorbance was observed. This indicated the great TABLE I sensitivity of the chromophore towards high temperature and Phytic Acid Content of Legume Meals Determined longer incubation time. by the New Chromophore and Compared to the Bartlett Methoda The effect of pH of the phytate solution on color production was mg Phytic Acid/g Defatted Mealb studied. When the pH was between 1 and 13 the color production mg Phytic Aatte Mealo was not affected; however, any change in the pH beyond these Legume New Chromophorec Bartlett Method limits inhibited the production of color. Mung bean 9.48 ± 0.32 9.25 ± 0.21 To prove that standard sodium phytate used had six atoms of Black bean 8.85 ± 0.56 8.61 ± 0.45 phosphorus per molecule of phytate, the 'phytate was first Navy bean 12.54 ± 0.07 11.39 ± 0.78 precipitated as ferric phytate according to Ellis et al (1977) and Red kidney bean 11.80 ± 0.28 11.32 ± 0.43 then converted to sodium phytate. This phytate was then digested Large lima bean 10.77 ± 0.79 10.07 ± 0.15 to inorganic phosphate as described in Materials and Methods. Soybean 23.35 ±4 1.60 21.47 ± 2.03 The molar ratio of phosphorus to phytate in standard sodium Winged bean 14.32 ± 0.16 13.94 ± 0.98 phytate was found to be 5.84. This value is in agreement with those Dried legume seeds were ground and defatted with hexane as solvent.T obtained by de Boland et al (1975) for inositol hexaphosphate and solvent was completely evaporated at room temperature. The dried is a good indicator of the purity of the phytate standard. The defatted meal was used for assay as described in Materials and Methods. presence of any inorganic phosphate as an impurity in sodium bThe analysis was done on nine replicates, and the average is reported ± phytate will increase the ratio above 1:6, whereas the presence of SEM. inositol phosphates other than hexaphosphate will decrease the c Phytic acid was determined without digestion using chromogenic reagent. ratio below 1:6 (de Boland et al 1975). As an application of our method, the phytate content of eight legumes, mung bean, black bean, navy bean, red kidney bean, large lima bean, black-eyed pea, soybean, and winged bean were determined using the new chromophore, and the data are shown in Table I. The phytate content of these legumes was also determined E 0.4