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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
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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.
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