Stability studies on diloxanide furoate: Effect of pH, temperature, gastric and intestinal fluids

Article abstract of DOI:10.1016/j.farmac.2003.11.015

The degradation of the amoebicide diloxanide furoate in alkaline medium at different temperatures was investigated using both a spectrophotometric and a developed HPLC method. In solutions, the drug was found to undergo decomposition, i.e., temperature an

Full text of DOI:10.1016/j.farmac.2003.11.015

IL FARMACO 59 (2004) 323–329
www.elsevier.com/locate/farmac
Stability studies on diloxanide furoate: effect of pH,
temperature, gastric and intestinal fluids
E.A. Gadkariem a, F. Belal a, M.A. Abounassif a, H.A. El-Obeid a,*, K. E.E. Ibrahim b
a Department of Pharmaceutical Chemistry, College of Pharmacy, King Saud University, P.O. Box 2457, Riyadh 11451, Saudi Arabia
b Department of Pharmaceutical Chemistry, Faculty of Pharmacy, University of Khartoum, P.O. Box 1996, Khartoum 11115, Sudan
Received 21 May 2003; accepted 8 November 2003
Abstract
The degradation of the amoebicide diloxanide furoate in alkaline medium at different temperatures was investigated using both a
spectrophotometric and a developed HPLC method. In solutions, the drug was found to undergo decomposition, i.e., temperature and pH
dependent. The pH-rate profile at pH between 7.6 and 9.6 indicated a first-order dependence of Kobs on [–OH]. Arrhenius plot obtained at pH
8 was linear between 40 and 63 °C. The estimated activation energy of hydrolysis was found to be 18.25 kcal degree.mol–1. The effect of
simulated gastric and intestinal fluids on the drug was also investigated. A new thin-layer chromatographic (TLC) procedure for the
fractionation of the drug and its alkaline hydrolysis products has been developed and was found to compare favorably with that of the British
Pharmacopoeia. Three hydrolysis products of a basic methanolic solution of the drug, namely furoic acid, diloxanide and methylfuroate could
be identified by the use of TLC, HPLC, infrared and mass spectrometry.
© 2004 Published by Elsevier SAS.
Keywords: Diloxanide furoate; HPLC; UV spectrophotometry; TLC; Kinetics; Stability
1. Introduction
drug is orally administered and is hydrolyzed in the gut to
release diloxanide, which is considered as the active drug [2].
Methods described for the assay of DF in bulk and/or dosage
forms are compiled in a review by Al-Majed et al. [3].
Procedures for the quantification of the drug are also reported
[4,5]. Recently, we developed a stability-indicating HPLC
method suitable for the assay of the drug in presence of its
photodegradation products [6]. In this piece of work, we
investigated the effects of pH, temperature and simulated
gastric and intestinal fluids on the stability of DF making use
of the stability-indicating power of the developed HPLC
procedure.
During the pharmaceutical development of a new drug, it
is necessary to select as soon as possible the formulation with
the best stability characteristics. Regulations regarding sta-
bility testing for registration application are provided by
current International Commission for Harmonisation (ICH),
which emphasizes the stress testing conditions with the aim
of assessing the effect of severe conditions on the drug [1]. In
practice, the effects of pH and temperature changes on drug
stability are often used in such studies. The well-known
Arrhenius theory is still used to allow a rapid stability predic-
tion. The preformulation pharmacist uses the stress testing to
provide knowledge on the drug’s degradation products, deg-
radation pathways and mechanisms. The results of such stud-
ies are of vital importance in the estimation of a drug product
shelf life during early stages of its pharmaceutical develop-
ment. The results may also serve as guides for better drug
design, drug formulation and drug analysis.
2. Experimental
2.1. Materials
A pure drug sample of DF (Batch No. CDF/DF/175/97)
was kindly provided by Eipico Pharmaceutical Company,
Cairo, Egypt. Ammidin, the internal standard, was obtained
from Memphis Chemical Company, Cairo, Egypt.
2-Furancarboxylic acid was from Merck (Darmstadt, Ger-
many).All other chemicals were of analytical grade reagents.
Diloxanide furoate (DF): 4-[N-methyl-2,2-dichloro-
acetamido] phenyl-2-furoate is a luminal amoebicide. The
* Corresponding author.
E-mail address: humeida@ksu.edu.sa (H.A. El-Obeid).
© 2004 Published by Elsevier SAS.
doi:10.1016/j.farmac.2003.11.015

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E.A. Gadkariem et al. / IL FARMACO 59 (2004) 323–329
2.2. Reagents
2.5.1. Effect of NaHCO3 on the stability of DF
Two solutions were prepared as follows:
Solution (a): To 3 ml of the DF solution (200 µg ml–1) in
methanol in a 25-ml volumetric flask, 2.5 ml of 1% w/v
aqueous NaHCO3 solution were added before completing to
volume with methanol.
Solution (b): Three milliliters of the DF solution (200 µg
ml–1) in methanol were transferred into a 25-ml volumetric
flask and completed to volume with 1% NaHCO3 aqueous
solution.
Phosphate and alkaline borate buffers (pH range 7–10)
and simulated gastric and intestinal fluids were prepared
according to USP 1995 [7]. Aqueous sodium bicarbonate
(NaHCO3) (1% w/v) solution was prepared. Acetonitrile
(Hipersolv™, BDH, Poole, England) and water (Chromo-
solv®, Ridel-de Häen, Germany) were also used. Other sol-
vents were of analytical grade and obtained from BDH,
Poole, England.
Both solutions were monitored by UV scanning between
320 and 200 nm and by the HPLC method [6] at 5 min
intervals.
2.3. Standard solutions
Fresh standard solution of DF (200 µg ml–1) in methanol
was prepared and was further diluted with the mobile phase
(30:70 v/v, water/acetonitrile) to the appropriate concentra-
tions of the working solutions.
A stock solution of the internal standard ammidin (300 µg
ml–1) was prepared in methanol and was further diluted as
appropriate.
2.5.2. Effect of temperature on the stability of DF solution
Five milliliters aliquots of the DF solution (200 µg ml–1)
were placed into four 50-ml volumetric flasks. Each flask
was completed to volume with borate buffer (pH 8.0) equili-
brated at the appropriate temperatures of the study (40, 50,
55 and 63 °C). Twenty microliters volumes of each solution
were injected onto the column at appropriate time intervals
ranging between 5 and 20 min, depending on the temperature
of incubation of the solution.
2.4. Apparatus
A Waters Liquid Chromatograph 600E, equipped with
Rheodyne 7161 injector, Waters 486-tunable absorbance de-
tector and Waters-746 data module, was used. The column
used was Lichrosphere 100 RP-18 (5 µm) 150 × 4.6 mm i.d.
(Phase Separation Ltd.). The mobile phase consisted of
water/acetonitrile (30:70 v/v) pumped isocratically at a flow
rate of 1 ml min–1. Degassing of the mobile phase was carried
out by purging pure helium into the solvent reservoir at a rate
of 20 ml min–1; ultraviolet (UV) setting was at 258 nm and
20 µl volumes were injected onto the column at room tem-
perature.
UV spectrophotometric studies were carried out on a
Shimadzu UV 1601 PC Spectophotometer (Kyoto, Japan).
The EI mass spectra were obtained on a Shimadzu GC/MS
spectrometer (Shimadzu Corporation, Japan) operated with
an electron energy of 70 eV and injector temperature of
250 °C. Thin-layer chromatography (TLC) was conducted on
precoated silica gel sheets 60F254, 5 × 10 cm with 0.2 mm
thickness (Riedel-de Häen, Germany). Solvent systems
were; dichloromethane/methanol (24:1 v/v) [8] and
cyclohexane/ethylacetate (3:2 v/v). Visualization was ac-
complished under UV light (254 nm).
2.5.3. Effect of simulated gastric and intestinal fluids on
DF
Five milliliters aliquots of DF solution (200 µg ml–1) were
placed into two 25-ml volumetric flasks. The contents of one
flask was completed to volume with the simulated gastric
fluid and the other with the simulated intestinal fluid; both
being equilibrated at 37 °C.After thorough mixing, the flasks
were incubated at 37 °C. One milliliter aliquots of each
solution were withdrawn at 10 min intervals into 10-ml
volumetric flasks; mixed with 1 ml of the internal standard
solution (60 µg ml–1) and completed to volume with the
mobile phase (duplicates). Twenty microliters volumes were
injected onto the column. Two blanks for each medium were
used, one containing the medium without the enzyme and the
other consisting of the medium containing the boiled inacti-
vated enzyme. The data were treated for log peak area ratio
with time.
2.5.4. Isolation of the hydrolysis products in the basic
methanolic solution of DF
Fifty milligrams of DF reference material were dissolved
in about 15–20 ml of 0.1 M sodium hydroxide in methanol
and allowed to stand for about 15 min. Complete hydrolysis
was checked by both TLC and HPLC methods.
2.5. Procedures
All the solutions for stability studies were prepared by
diluting 200 µg ml–1 of the DF solution in methanol with the
appropriate aqueous buffers (with pH values of 7.6, 7.8, 8.0,
8.2, 8.4, 8.8, 9.2 and 9.6) to obtain a final solution containing
20 µg ml–1 DF and 10% v/v methanol. The study was carried
out at 40 °C. The kinetics of the decomposition of the drug
was monitored adopting the HPLC method [6]. The rate
constant for each pH was calculated from the plot of log
[remaining drug] vs. time.
The base-hydrolyzed drug mixture prepared above was
acidified with concentrated HCl ( 0.5–1.0 ml) and 20 ml of
chloroform were added. The solution was then concentrated
to precipitate out sodium chloride. The mixture was filtered
and the filtrate was allowed to evaporate slowly in the dark in
a fume cupboard. The separated white crystals were charac-
terized by infrared (IR) and mass spectrometry. The absorp-
tion spectrum of the substance was recorded in the UV region
and its kmax was determined.

E.A. Gadkariem et al. / IL FARMACO 59 (2004) 323–329
325
O
OH
O
C O
(i) NaOH
(ii) HCl
O
+
O
C OH
N CH3
N CH3
C O
C O
F
CHCl2
CHCl2
D
DF
Scheme 1.
3. Results and discussion
products. The Rf value for the drug was 0.6 and those for the
degradation products were 0.75 (volatile), 0.5 and 0.01.
The structure of DF contains both an ester and a substi-
tuted amide linkage; both linkages are liable to hydronium or
hydroxide ion-catalyzed hydrolysis; however, the rate of
[–OH] catalyzed hydrolysis of esters is known to proceed
faster than that of the [H+]-catalyzed process. Amides are
generally much more stable towards alkaline hydrolysis than
esters [9]. Accordingly, the ester group in the structure of DF
is expected to undergo alkaline hydrolysis to yield diloxanide
and furoic acid sodium salts which on acidification give
diloxanide (D) and furoic acid (F) as shown in Scheme 1.
3.3. Characterization of the isolated degradation product
The isolated white crystalline powder obtained from the
acidic methanolic solution (Section 2.5.4) was confirmed by
IR and mass spectrometry to be diloxanide. The IR spectrum
showed the loss of the ester C=O stretch at 1703 cm–1 and the
appearance of O–H stretch at 3300 cm–1. The amide C=O
remained at 1650 cm–1. Table 1 shows the mass spectral
assignments of the product; the presence of two chlorine
3.1. UV spectral changes in alkali-treated DF solution
The absorption spectrum of DF solution shown in Fig. 1,
exhibited a major peak at 258 nm. Treatment of this solution
with alkali resulted in the reduction of its absorption peak
indicating the occurrence of structural changes in its mol-
ecule. Further changes in the spectrum were also observed
when the solution containing the alkaline degradation prod-
ucts was acidified with HCl solution.
3.2. TLC of the acidified basic hydrolysates of DF
Using the TLC method of the British Pharmacopoeia
(BP), the reaction mixture gave three spots. One spot coin-
cided with the parent compound (Rf value 0.90), the other
spots had Rf values of about 0.70 and 0.01; suggesting
incomplete hydrolysis (see Scheme 1). The spots were then
isolated from the TLC plates and further analyzed by UV
spectrometry and HPLC. The results revealed that the spot
with Rf value of 0.9 which, using the BP method, coincided
with that of the parent compound, was actually a composite
of DF and another alkaline degradation product of the metha-
nolic DF solution. It is clear that the TLC system of the BP
failed to resolve the latter alkaline degradate from the parent
drug. A mixed spot of the drug and the degradation products
confirmed this finding. In an attempt to separate this product
from the drug, we developed a TLC method using a mobile
phase consisting of cyclohexane/ethylacetate (3:2) with run-
ning developing time of about 20 min for 8-cm distance. The
method successfully separated the drug from its degradation
Fig. 1. UV spectra for DF solution (24 µg ml–1): (a) before alkaline hydro-
lysis; (b) after alkaline hydrolysis and (c) acidified alkaline hydrolysate.

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E.A. Gadkariem et al. / IL FARMACO 59 (2004) 323–329
Table 1
value = 0.5 corresponded to diloxanide which was isolated
Mass spectral assignments of diloxanide
and characterized as a hydrolytic product of DF. The pres-
ence of furoic acid was also confirmed by TLC where the
degradation product of Rf = 0.01 corresponded to that of
authentic furoic acid. The third degradate (Rf = 0.75) was first
thought to be formed through the cleavage of the amide
linkage. However, studies revealed the product to be methyl-
furoate formed through the alcoholysis of DF by the solvent
methanol resulting in the transesterification reaction shown
in Scheme 2. This was confirmed by the preparation of
methylfuroate by refluxing a mixture of methanol, furoic
acid and sulfuric acid and extracting the product in chloro-
form. The Rf, RT and UV spectrum (kmax at 253 nm) of the
degradate corresponded to those of the prepared methylfu-
roate. The use of ethanol or propanol instead of methanol
lead to the formation of ethylfuroate or propylfuroate having
different chromatographic peaks, which were also resolved
from the drug peak. As expected, when the experiment was
conducted using CHCN/H2O as the solvent, only furoic acid
and diloxanide were identified as the degradation products.
m/z
Species
234
M+
233
[M–H]+
C7H8NO+
122 (100%)
+
150
94
C8H8NO2
[C6H7N]+
+
65
[C5H5
]
41
C2H+O → C2HO+
atoms was evident by the distinct M + 2 and M + 4 peaks (not
shown) due to molecular ions and (M–H) ion at 233 contain-
ing two atoms of the heavy isotope 37Cl. Fig. 2 shows the UV
spectra of the isolated crystals in neutral and acidic methanol
to be identical (kmax at 277 and 228 nm) while that in alkaline
medium exhibited kmax at 247 and 305 nm showing a batho-
chromic shift possibly due to the formation of the phenoxide
ion. In TLC (Section 3.2), the degradation product with Rf
Fig. 3. pH-rate profile for alkaline hydrolysis of DF at 40 °C (pHs 7.6, 8.0,
8.4, 8.8, 9.2 and 9.6).
Fig. 2. UV spectra of isolated hydrolysis product: in methanol (a) and in
alkaline medium (b).
O
O
CH3OH
HO
N COCH2Cl
CH3
+
C O
N COCH2Cl
CH3
O
C OCH3
O
methylfuroate
diloxanide
Scheme 2.

E.A. Gadkariem et al. / IL FARMACO 59 (2004) 323–329
327
Table 2
Values of Kobs of DF at alkaline pHs at 40 °C and their equivalent t1/2 values
pH value
7.6
Kobs (min–1
)
t1/2 (min)
274.35
92.50
38.98
20.80
8.17
2.526 × 10–3
7.448 × 10–3
1.776 × 10–2
3.333 × 10–2
8.485 × 10–2
2.533 × 10–1
8.0
8.4
8.8
9.2
9.6
2.74
Fig. 4. Pattern of the change of UV absorption of DF solution (24 µg ml–1
with the hydrolysis process in 0.1% w/v, NaHCO3 in methanol.
)
3.4. pH-rate profile of the alkaline hydrolysis of DF
The hydrolysis of esters undergoes specific base-catalysis,
and consequently the observed apparent first-order rate con-
stant is expected to vary with the hydroxide ion concentration
[9]:
Fig. 5. Specimen chromatogram of the remaining drug in presence of its
hydrolysis products in 0.1% w/v, NaHCO3 in methanol: (a) DF intact drug;
(b) methylfuroate; (c) diloxanide and (d) furoic acid.
(1) Kobs =KOH [–OH]
where KOH is the rate constant for specific base-catalysis and:
log Kobs = log KOH + log [–OH]
plot gave a slope with a value 1.0, suggesting a first-order
dependence of K on [–OH]. This is clearly evident from the
results of Table 2 which point out that the magnitude of the
observed rate constant (Kobs) increased rapidly at higher pH
values and it is also reflected by the short t1/2 values obtained
at these media. At pHs > 10, fast hydrolysis can take place
even at room temperature. This observation was confirmed
(2) i.e., log Kobs = log KOH + log kw + pH
where kw is the ion product of water.
This indicates that log Kobs should increase linearly with
increase of pH on the alkaline side giving slope of +1.0.
Fig. 3 shows a plot of log Kobs vs. pH at pHs > 7.4. The linear

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E.A. Gadkariem et al. / IL FARMACO 59 (2004) 323–329
by the fact that NaOH solutions caused immediate and com-
plete hydrolysis of the drug compared to solutions of
NaHCO3, which required either a longer time to achieve
complete hydrolysis at room temperature, or heating the
solution at elevated temperatures.
tored by the HPLC method, gave a first-order reaction data
for the plot of log [remaining drug] vs. time. The regression
data obtained was: y = –0.025t + 6.184, r = 0.999. where
y = log area of the [remaining drug] at time t in minutes.
The hydrolysis rate constant was found to be 5.756 × 10–2
min–1 and t1/2 = 12.04 min. This high rate of hydrolysis was
unexpected for solutions in NaHCO3 at room temperature, as
the expected pH of the solution should remain between 8 and
8.5. However, the pH of the final solution was found to be
10.6. This experiment was then repeated using the same
concentration of the drug (24 µg ml–1) in methanol but
completing the solution with 1% w/v aqueous solution of
NaHCO3, i.e., the solution contained 12% methanol and 88%
v/v NaHCO3 (1% w/v). A considerably slow rate of hydroly-
sis was then observed; and the pH of this solution was found
to be 8.7 (pH of 1% w/v NaHCO3 in water was 8.3). Fig. 5
shows high level of methyl furoate, suggesting a driving
effect by the transesterifiction on the DF degradation. Thus,
the unexpected high rate of hydrolysis in NaHCO3 solution
(0.1% w/v in methanol) could be ascribed to the pH and the
transesterification reaction. This aspect is of practical impor-
tance in view of the DF handling and storage.
3.5. Rate-controlled alkaline hydrolysis of the drug with
NaHCO3
A study was conducted using the weak base, NaHCO3, to
obtain a rate-controlled hydrolysis reaction. This goal was
achieved by the addition of 2.5 ml of 1% (w/v) NaHCO3
solution to a solution of DF (24 µg ml–1) contained in 25-ml
volumetric flasks and completing to volume with methanol.
The hydrolysis of the drug was followed by both the UV
spectroscopy and the HPLC methods. The follow up of the
hydrolysis of DF with the UV method conducted with time
revealed a decrease of the intact drug absorbance intensity
with time coupled with change of kmax. An equilibrium state
seems to be reached after about 20 min, beyond which an
increase in absorbance occurred, indicating the existence in
the system of more hydrolytic products other than the intact
drug. Fig. 4 shows the pattern of the change in UV absorption
during the hydrolysis process. These UV results emphasize
that the absorbance measurements at a specific wavelength
may not be adequate for these types of determination if the
degradation products also absorb in the same region. This is
confirmed by the resolved hydrolysis products of the same
alkaline methanol solution monitored by the HPLC method
(Fig. 5). Peak d was identified as furoic acid (RT = 1.38 min)
based on the RT value observed for authentic furoic acid and
peak c was assigned to diloxanide (RT = 2.57 min) corre-
sponding to that of the hydrolysis product isolated and char-
acterized earlier in this study as diloxanide. Similarly, peak b
was identified as methylfuroate (RT = 2.87 min) based on the
RT value observed for the prepared methylfuroate. The hy-
drolysis kinetics of the alkaline methanolic solution as moni-
3.6. Effect of temperature on rate of hydrolysis of DF
The influence of temperature (40, 50, 55 and 63 °C) on the
hydrolysis rate was studied by the HPLC method using
alkaline borate buffer of pH 8. The hydrolysis process fol-
lowed first-order kinetics at all temperatures (Fig. 6). Using
theArrhenius plot (Fig. 7), the calculated energy of activation
was 18.25 kcal degree.mol–1 which lies within the accepted
limits for hydrolysis of esters (8–20 kcal degree.mol–1) [9].
3.7. Effect of simulated gastric and intestinal fluids on
the hydrolysis of DF
DF, being an ester, is considered as a pro-drug, that is
predicted to pass the acidic gastric juice without hydrolysis
Fig. 6. First-order kinetics plots of degradation of DF in alkaline borate buffer, pH 8.0, at various temperatures.

E.A. Gadkariem et al. / IL FARMACO 59 (2004) 323–329
329
4. Conclusion
The results of this study showed that DF solution is de-
graded via a hydrolysis process, which appears to be tem-
perature and [–OH] dependent at constant methanol content.
Three major degradation products were obtained: two were
identified as diloxanide (D) and furoic acid (F), which were
the results of the predicted hydrolysis of the ester group. The
third product was identified as methylfuroate formed as a
result of transesterification.
The TLC procedure developed in this study was success-
fully used to separate the alkaline degradation products from
the parent compound and is recommended to substitute the
official BP TLC method, which failed to accomplish such
separation.
The results of the stability study of DF in simulated gastric
and intestinal fluids confirmed its hydrolysis in the intestines
to its active form diloxanide and furoic acid. It is also estab-
lished that the hydrolytic reaction is an enzyme-catalyzed
process that takes place in the intestines.
Fig. 7. Arrhenius plot for DF at pH 8.
References
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both experiments showed slight hydrolysis, indicating that
the process is mainly achieved through an enzyme-catalyzed
reaction.
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