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MICROCHEMICAL JOURNAL ARTICLE NO. 59, 413– 416 (1998) MJ981604 Hydrolysis of Phytic Acid by Microwave Treatment: Application to Phytic Acid Analysis in Pharmaceutical Preparations J. G. March,*,1 F. Grases,* and A. Salvador† *Department of Chemistry, University of Balearic Islands, E-07071 Palma de Mallorca, Spain; and †Department of Analytical Chemistry, University of Valencia, E-46100 Burjassot, Spain Received December 8, 1997; accepted February 13, 1998 The acid hydrolysis of phytic acid in a Teflon reactor using a domestic microwave oven has been studied and compared with other reported procedures. In 0.44 M HCl quantitative hydrolysis was achieved with six heating stages of 2 min each. A lower yield was obtained with H2SO4 and HNO3. The analytical use of this hydrolysis to determine phytic acid by indirect determination of phosphate has been demonstrated by analysis of three pharmaceutical formulations. No sample pretreatment other than obtaining a homogeneous suspension was necessary. © 1998 Academic Press Inositol hexakisphosphoric acid, known as phytic acid, and the lower esters constitute a family of compounds of interest from the life sciences point of view (1). In addition, phytic acid acts as an effective inhibitor of calcium oxalate crystallization, and pharmaceutical preparations containing phytic acid are used to treat relapsed urolitiasic patients (2). There is also evidence suggesting that compounds of phytic acid can prevent cancer in many different organs, such as colon, breast, and prostate (3– 6). On the other hand, the intestinal absorption of multivalent ions, mainly Fe(III) and Zn(II), is inhibited by excessive ingestion of phytic acid (7). In view of this, the interest in pharmacological formulations containing phytic acid and the necessity for suitable analytical methods to assess its quality became evident. A number of methods to determine phytic acid are based on the hydrolysis of phosphoric esters to phosphoric acid and photometric phosphate determination. The hydrolysis is accomplished by heating in HCl medium for 24 h (8) and by phytase catalysis (9). Other methods to determine phytic acid involve HPLC [either with RI detection (10) or with photometric postcolumn derivatization (11)] or GC [after hydrolysis to inositol and its derivatization (8)]. Most of the procedures mentioned require previous purification of dissolved phytic acid through an anion-exchange resin. On the other hand, microwave heating has been widely used for the preparation of samples (12). So, a large number of materials can be digested using a microwave oven before the determination of metallic elements (13). Microwave treatments have been also proposed to achieve the hydrolysis of some compounds, especially proteins (14). The purpose of the present paper, therefore, is to study microwave phytic acid hydrolysis in acid medium to improve its indirect analytical determination through phosphate analysis. The method developed is useful for quality assessment of pharmaceutical products with no sample preparation other than the grinding of a few milligrams of sample (if necessary) and homogeneous suspension in water. 1 To whom correspondence should be addressed. 413 0026-265X/98 $25.00 Copyright © 1998 by Academic Press All rights of reproduction in any form reserved. 414 MARCH, GRASES, AND SALVADOR MATERIALS AND METHODS Apparatus A single-beam spectrophotometer (Shimadzu UV-120-02) with disposable cells of 1-cm optical pathlength, a domestic microwave oven (Sanyo) with rotating turntable, and a 20-ml-capacity PTFE reactor (Nanocolor) with security expansion chamber (MachereyNagel, Duren, Germany) provided by Aquateknica S.A. (Valencia, Spain), sealed with PFA and PTFE dishes, were used in this study. A Perkin–Elmer Inductively Coupled Plasma 2000 emission spectrophotometer was also used for confirmative total phosphorus determinations. Reagents and Pharmaceutical Products All chemicals used were of analytical reagent grade. Inositol hexaphosphoric acid dodecasodium salt from corn was purchased from Sigma (St. Louis, MO). Pharmaceutical products selected to validate the analytical application were commercial formulations from Alter SA (Madrid, Spain), Syntex Latino SA (Madrid, Spain), and Autex SA (Mallorca, Spain). Microwave Treatment and Phosphate Determination A 2.4-ml volume of an aqueous solution containing phytic acid and inorganic acid (HCl, HNO3, or H2SO4) was placed in a PTFE reactor, then sealed and heated at 650 W for 2.0-min intervals. The reactor was allowed to cool between each heating for alternating 20- and 40-min intervals (lower cooling intervals generated excessive pressure in the reactor) and finally allowed to cool to room temperature. Then 0.3 ml of 0.02 M ammonium vanadate (in 0.28 M HNO3) and 0.3 ml of 0.04 M ammonium molybdate ((NH4)6Mo7O24 z H2O) were added, and the mixture was transferred to a cuvette to measure absorbance at 400 nm after 45 min. Free phosphate was determined from the calibration graph with sodium phosphate. The slope of the calibration curve was 3.33 3 1022 liters/mg 3 cm, depending on neither the nature of the acid used nor its concentration. Sample Treatment A tablet or the contents of a capsule were ground and homogenized. Then 15.0 to 20.0 mg was suspended in 50.0 ml water with magnetic stirring. A homogeneous suspension was obtained after a few minutes. Then 0.5 to 2.2 ml of suspension was transferred to the reactor, and 0.2 ml of 5.3 M HCl and water to 2.4 ml were added. The solution was heated for six 2-min periods, as described earlier. After treatment, a colorless solution was obtained, and sample filtration was not necessary. Finally, vanadate and molybdate reagents were added to the reactor and absorbance was measured. Another aliquot of the suspension was filtered through a disposable 0.45-mm PTFE filter syringe, and the absence of free phosphate was verified using the same analytical reaction. RESULTS AND DISCUSSION Microwave Phytic Acid Hydrolysis The hydrolysis of phytic acid by microwave heating has been studied under several acid conditions to compare the yield of the hydrolysis reaction and facilitate its indirect 415 PHYTIC ACID HYDROLYSIS BY MICROWAVE TREATMENT TABLE 1 Yield of Hydrolysis Reactiona Experiment Inorganic acid Concentration (M) Number of stages Hydrolysis (%) 1 2 3 4 5 6 7 8 9 10 11 HCl HNO3 HCl HNO3 H2SO4 HCl HCl HCl H2SO4 HNO3 HCl 1.77 1.77 0.90 0.90 0.90 0.44 0.44 0.44 0.44 0.44 0.44 3 3 3 3 3 3 4 5 5 6 6 37 15 77 54 76 80 92 98 95 90 99.7 a Initial concentration of phytic acid in Teflon reactor, 14.5 mg liter21; microwaved volume, 2.4 ml. determination through phosphate analysis. The analytical reaction selected for phosphate determination in this work has the advantage that after 45 min, the final absorbance is not dependent on the nature of the acid used and its concentration. Nevertheless, the kinetics of heteropoly acid formation was affected by acidity conditions. In general, the higher the acid concentration, the faster the reaction. At 1.5 M acid, the system reached equilibrium in a few minutes. To propose a general procedure a period of 45 min was recommended, but depending on the working acid concentration this period can be shortened. The yield of hydrolysis has been calculated from the found free phosphate and total phosphate (free and bound to inositol), which were determined theoretically (6 mol phosphate per mole of phytic acid) and confirmed by total phosphorus analysis by ICP-AES. Some of the results obtained are summarized in Table 1. When comparing at the same molar concentration three inorganic acids, it can be observed that HCl gave higher yields, the values being close to those obtained with H2SO4. Nevertheless, HNO3 gave lower yields (see experiments 3–5; 8, 9; and 10, 11). When comparing different acid concentrations, it can be observed that the yield increased at lower acid concentration (see experiments 1, 3, 6 and 2– 4), in agreement with the known fact that the absorption of radiation of aqueous acid solutions is more effective at lower acid concentrations. The more important facts were that the hydrolysis yield increased with the number of heating stages (see experiments 6 – 8 and 11), and that after six stages with hydrochloric acid, hydrolysis can be assumed to be quantitative. This permitted us to determine phytic acid using phosphate solutions as standard, and microwave treatment was not necessary to obtain the calibration function. The analytical use of the proposed microwave hydrolysis can be favorably compared with other reported hydrolysis methods. So, when hydrolysis is carried out at 120°C in 2 M HCl, 24 h is necessary to reach quantitative hydrolysis (8). At 90°C in HNO3, 100 h is necessary. Enzymatic hydrolysis with phytases were interfered with by a variety of compounds normally used in pharmaceutical preparations, such as vitamins A and E (9). As stated, during microwave heating, total dissolution of the samples studied was 416 MARCH, GRASES, AND SALVADOR TABLE 2 Determination of Phytic Acid in Commercial Pharmaceutical Formulations Phytic acid (mg/preparation) Samplea Hydrolysis–photometric methodb ICP-AES method A B C 61.5 28.3 62.0 60.7 27.9 61.4 a Other labeled ingredients: (A) L-glutamine, thiamine, pyridoxine hydrochloride, cyanocobalamine, starch, lactose; (B) cellulose, bran, vitamin A, zinc sulfate, stearic acid, magnesium salt; (C) glucose, citric acid. b n 5 2, RSD 5 2–3%. achieved, and the fact guaranteed total phytic recovery. To date, analyses of real samples have been carried out on solutions obtained from solid–liquid extraction, where total solubilization of phytic acid and phytates was normally assumed. Phytic Acid Analysis in Pharmaceutical Formulations The applicability of the reported procedure has been tested with three pharmaceutical formulations. As indicated under Experimental, phosphate analysis of untreated aliquots was proposed to ensure no degradation due to inappropriate storage or processing conditions. Total phosphorus analysis by ICP-AES was also carried out for comparison with the hydrolysis–photometric results. As can be seen in Table 2, good agreement between both methods is observed. The table includes the other reported components in analyzed samples, to give additional information on non-interfering substances. REFERENCES 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. Sasakawa, N.; Sharif, M.; Hanley, M. R. Biochem. Pharmacol. 1995, 50, 137–146. Grases, F.; Costa-Bauzá, A.; March, J. G. Br. J. Urol., 1994, 74, 298 –301. Kennedy, A. R. J. Nutr., 1995, 125, 733–743. Shamsuddin, A. M.; Yang, G. Y. Carcinogenesis, 1995, 16, 1975–1979. Vucenik, I.; Shamsuddin, A. M. 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