Journal of Advanced Research (2010) 1, 323–329

Cairo University

Journal of Advanced Research

ORIGINAL ARTICLE

Spectrophotometric methods for the simultaneous

determination of binary mixture of metronidazole
and diloxanide furoate without prior separation
a,* b
Mohamed R. El-Ghobashy , Nisreen F. Abo-Talib

a
Analytical Chemistry Department, Faculty of Pharmacy, Cairo University, Egypt
b
National Organization for Drug Control and Research (NODCAR), Cairo, Egypt

Received 26 October 2009; revised 1 March 2010; accepted 18 March 2010

Available online 30 June 2010

KEYWORDS Abstract Ratio subtraction and isosbestic point methods are two innovative spectrophotometric
Metronidazole; methods for determining the concentrations of metronidazole (I) and diloxanide furoate (II) in a
Diloxanide furoate; mixture. Metronidazole was determined by direct spectrophotometric method at kmax 314.0 nm in
Binary mixture; the presence of diloxanide furoate in the range of 4–24 lg ml1 with a mean recovery percentage
Isosbestic point; of 99.83 ± 1.41. Two spectrophotometric methods were developed for the spectral resolution of
Ratio subtraction diloxanide furoate when present in mixture with metronidazole without preliminary separation.
The first method depends on measuring the absorbance at the isosbestic point at 277.2 nm in the
range of 5–30 lg ml1 with a mean recovery percentage of 99.96 ± 1.47 for diloxanide furoate.
The second method is the ratio subtraction spectroscopic method for spectral isolation of diloxanide
furoate present in the mixture which can be measured at 251.2 nm in the range of 5–30 lg ml1 with a
mean recovery percentage of 99.73 ± 1.33 for diloxanide furoate determination. The suggested pro-
cedures were validated using laboratory-prepared mixtures and were successfully applied for the
analysis of pharmaceutical preparations. The methods retained their accuracy and precision when
the standard addition technique was applied. The results obtained by applying the proposed methods
were statistically analyzed and compared with those obtained by the reported method.
ª 2010 Cairo University. Production and hosting by Elsevier B.V. All rights reserved.

* Corresponding author. Tel.: +202 33020604.

E-mail address: mohamedrefaat73@yahoo.com (M.R. El-Ghobashy). Introduction

2090-1232 ª 2010 Cairo University. Production and hosting by

Metronidazole and diloxanide furoate are formulated together
Elsevier B.V. All rights reserved.
to be highly effective in the treatment of intestinal and extrain-
Peer review under responsibility of Cairo University. testinal amoebic infections. Metronidazole is less effective
doi:10.1016/j.jare.2010.06.001 against parasites in the bowel lumen and is, therefore, used
in combination with a luminal amoebicide, such as diloxanide
furoate in the treatment of invasive amoebiasis.
Production and hosting by Elsevier
Metronidazole 2-methyl-5-nitroimidazole-1-ethanol [1]
was determined individually by short-wave length NIR
324
spectroscopy [2], voltammetry [3–5], NMR spectrometry [6],
gas chromatography [7] and HPLC methods either alone
[8–10] or in the presence of its metabolites [11,12] or in the
presence of its degradation product [13], in addition to its mix-
ture with other drugs [14,15]. First derivative spectrophotom-
etry was used for the determination of metronidazole in
mixture with ciprofloxacin [16].
Diloxanide furoate 2,2-dichloro-N-(4-hydroxyphenyl)-
N-methylacetamide [1] was determined individually by colori-
metric method [17], in the presence of its degradation product
by derivative technique, derivative ratio, TLC-densitometry
[18] and HPLC [18,19] and in mixture with tinidazole and fura-
zolidone by second derivative spectrophotometry [20].
The main problem of spectrophotometric binary mixture
analysis is the simultaneous determination of the two com-
pounds in the same mixture without prior separation. One
spectrophotometric determination method has been used for
resolving such mixture with overlapping spectra, derivative
spectrophotometry [21] and HPLC [22].
The aim of this work is to develop new spectrophotometric
methods for resolving this mixture with spectral interfering
problems, without preliminary separation. The new methods
were very simple, did not require any computer programs
(derivative and derivative ratio) as metronidazole was deter-
mined by direct spectrophotometry and diloxanide furoate
was determined by simple mathematical calculation. Also the
method used did not require any sophisticated instrumentation,
such as HPLC, which requires solvents and time.
Experimental
Apparatus
Spectrophotometer: SHIMADZU UV-1601 PC, dual beam
UV–visible spectrophotometer with two matched 1 cm quartz
cells, connected to an IBM compatible personal computer
(PC) and an HP-600 inkjet printer. Bundled UV-PC personal
spectroscopy software version (3.7) was used to process the
absorption and the derivative spectra. The spectral band width
was 0.2 nm with wavelength scanning speed of 2800 nm min1.
Materials
Pure samples
Metronidazole and diloxanide furoate were kindly supplied by
Egyptian Int. Pharmaceutical Industries Co., E.I.P.I.CO. 10th
of Ramadan City, Area B1 P.O. 149, Egypt. Their purity was
found to be 99.84 ± 1.26 and 100.50 ± 0.71, respectively,
according to the manufacturer’s direct spectrophotometric
method (personal communication).
Market samples
Furazole tablets (E.I.P.I.CO.); batch no. 080435. It was labeled
to contain 200 and 250 mg metronidazole and diloxanide furo-
ate, respectively, per tablet.
Furazole suspension (E.I.P.I.CO.); batch no. 074135. It was
labeled to contain 200 and 100 mg metronidazole and diloxa-
nide furoate, respectively, per 5 ml.
M.R. El-Ghobashy and N.F. Abo-Talib
Chemicals and reagents
All chemicals were of analytical grade and the solvents were of
spectroscopic grade. Methanol, (E-Merck, Darmstadt,
Germany).
Standard solutions
Stock solutions
Metronidazole (I) and diloxanide furoate (II) stock solutions
(1 mg ml1) were prepared by weighing accurately 100 mg of
each powder into two separate 100 ml volumetric flasks. Meth-
anol (50 ml) was added, shaken for a few minutes and com-
pleted to volume with the same solvent.
Working solutions
Four micro litres of the stock solution of (I) and 5 ml of the
stock solution (II) were accurately transferred into two sepa-
rate 50 ml measuring flasks and diluted to the mark with meth-
anol to get a final concentration of 80 lg ml1 and 100 lg ml1
of (I) and (II), respectively.
Laboratory-prepared mixtures
Accurate aliquots equivalent to (40–100 lg) of (I) were trans-
ferred from its working solution (80 lg ml1) into a series of
10 ml volumetric flasks and portions equivalent to (50–
150 lg) of (II) from its working solution (100 lg ml1) were
added to the same flasks and volumes were completed to mark
with methanol and mixed well.
Procedures
Isosbestic spectrophotometric method
Linearity: aliquots from (I) and (II) working solutions (80 lg ml
1
of (I) and 100 lg ml1 of (II), respectively) equivalent to 40–
240 lg of (I) and 50–300 lg of (II) were transferred into two sep-
arate sets of 10 ml volumetric flasks and completed to the mark
with methanol. The zero order absorption spectra were re-
corded for both drugs using methanol as a blank; then the
absorbance was measured at 314.0 nm for (I) and 277.2 nm
(Aiso) for (I) and (II). Two calibration curves were constructed
for each drug relating the absorbance at the selected wavelength
to the corresponding drug concentrations and the regression
equations were computed.
Assay of laboratory-prepared mixtures: Absorbance of the
spectra of laboratory-prepared mixtures containing different
ratios of (I) and (II) were measured at 314.0 nm corresponding
to the contents of (I) only, and at 277.2 nm (Aiso) correspond-
ing to the total content of (I) and (II) in the mixture. The con-
centration of (I) alone and the total concentration of the two
drugs were calculated from their corresponding regression
equations; then by subtraction of (I) concentration from the
total mixture concentration, yielding the actual concentration
of (II) in the mixture.
Ratio subtraction spectrophotometric method
Linearity: Aliquots containing 50–300 lg from (II) working
solution (80 lg ml1) were transferred into a series of 10 ml
volumetric flasks then completed to volume with methanol;
Analysis of metronidazole and diloxanide furoate in binary mixture 325

the spectra of the prepared standard solutions were scanned. A
calibration curve was constructed relating the absorbance of
zero order spectra of (II) at kmax 251.2 nm to the correspond-
ing concentrations and the regression equation was computed.
Aliquot equivalent to 40 lg from the (I) working solution
(100 lg ml1) was transferred into 10 ml volumetric flask and
completed to volume with methanol to be used as a divisor
(4 lg ml1).
Assay of laboratory-prepared mixtures: Absorbance of the
spectra of laboratory-prepared mixtures containing different
ratios of (I) and (II) was scanned; then the (absorbance at each
wavelength) was divided by the spectrum of 4 lg ml1of stan-
dard (I) (divisor) to obtain division spectra and the absorbance
in the plateau region (the constant) was subtracted. By multi-
plication of the obtained spectra (absorbance at each wave-
length) by the spectrum of the divisor the original curves for
direct determination of (II) at 251.2 nm were obtained and
the concentration was calculated from the corresponding
regression equation.
Assay of pharmaceutical formulations
Furazole tablets: Ten Furazole tablets were accurately weighed
and finely powdered. A portion equivalent to 8 mg of (I) and
10 mg of (II) was weighed. The powder was transferred into
a 100 ml beaker, sonicated in 20 ml methanol for 10 min and
filtered into a 100 ml volumetric flask. The residue was washed
three times using 20 ml methanol each time and the volume
was completed to the mark with methanol. Aliquots of 0.5, 1
and 1.5 ml were separately transferred to 10 ml volumetric
flask and diluted with methanol. The general procedures under
linearity were followed.
The validity of the methods was assessed by applying the
standard addition technique.
Furazole suspension: Furazole (0.5 ml) suspension was accu-
rately transferred into a 100 ml beaker, sonicated in 20 ml
methanol for 10 min and filtered into a 100 ml volumetric flask.
The residue was washed using 20 ml methanol and the volume
was completed to the mark with methanol. Aliquots of 0.2, 0.4
and 0.6 ml (for analysis of metronidazole) and 0.5, 1 and 1.5 ml
(for analysis of diloxanide furoate) were separately transferred
to 10 ml volumetric flasks and diluted with methanol. The gen-
eral procedures under linearity were followed.
The validity of the methods was assessed by applying the
standard addition technique.
Results and discussion
Analytical methods for the determination of binary mixture
without previous separation were of interest. Metronidazole

Fig. 1 Zero order absorption spectra of 20 lg ml1 of metronidazole (_____), 20 lg ml1 of diloxanide furoate (- - - - -) and (1:1) mixture
containing 10 lg ml1 of each (. . . . . ..) using methanol as a blank.

Table 1 Method validation for the determination of pure sample of metronidazole and diloxanide furoate by the proposed methods.
Parameter Isosbestic spectrophotometry Ratio subtraction spectrophotometry
Metronidazole Diloxanide furoate Diloxanide furoate
Accuracy mean ± SD 99.83 ± 1.41 99.96 ± 1.47 99.73 ± 1.33
Precision repeatabilitya 100.07 ± 0.76 99.93 ± 0.48 99.87 ± 0.52
Intermediate precisionb 99.95 ± 0.82 100.05 ± 0.60 100.11 ± 0.59
Concentration range (lg ml1) 4–24 5–30 5–30
a
The intraday (n = 3), average of three concentrations (4, 12, 24 lg ml1) for metronidazole and (5, 15, 25 lg ml1) for diloxanide furoate
repeated three times within the day.
b
The interday (n = 3), average of three concentrations (4, 12, 24 lg ml1) for metronidazole and (5, 15, 25 lg ml1) for diloxanide furoate
repeated three times in three successive days.
326 M.R. El-Ghobashy and N.F. Abo-Talib

can be determined by direct measurement of absorbance at
314.0 nm, while the absorption spectra of diloxanide furoate
and metronidazole showed severe overlap, which makes the
determination of diloxanide furoate concentration in the mix-
ture more difficult (Fig. 1). By applying the proposed tech-
niques to the spectral data of the mixture, both diloxanide
furoate and metronidazole concentrations could be determined
without any interference.
Isosbestic spectrophotometric method
Erram and Tipnis [23] developed the isosbestic spectrophoto-
metric method. This method was used for simultaneous deter-
mination of (I) and (II) in their binary mixtures. At the
isosbestic point the mixture of drugs acts as a single compo-
nent and gives the same absorbance value as pure drug. Thus,
by measuring the absorbance value at the chosen isosbestic
point 277.2 nm (Aiso) (Fig. 1), the total concentration of both
(I) and (II) could be calculated, while the concentration of (I)
in the mixture could be calculated, without any interference, at
314.0 nm. Thus the concentration of (II) could be calculated by
subtraction.
A linear correlation was obtained between the absorbance
values and the corresponding concentrations of both drugs at
their corresponding wavelengths. The regression equations
were:
Aiso ¼ 0:0134C þ 0:0204 r ¼ 0:9994 at 277:2 nm
A ¼ 0:0324C þ 0:0024 r ¼ 0:9995 at 314:0 nm
where A is the absorbance, C is the concentration of the drug
in lg ml1 and r is the correlation coefficient.
The proposed method was applied for the determination of
(I) and (II) in bulk powder: satisfactory results were obtained
(Table 1).
The laboratory-prepared mixtures were analyzed by
the isosbestic method. The method is valid for determining
the drug in laboratory-prepared mixtures as shown in
(Table 2).

Table 2 Determination of metronidazole and diloxanide furoate in laboratory-prepared mixtures by the proposed methods.
Mixture no. Claimed taken (lg ml1) Isospestic spectrophotometry Ratio subtraction
spectrophotometry
Metronidazole Diloxanide Metronidazole Diloxanide furoate Diloxanide furoate
furoate
Recoverya% Recoverya% at Recoverya% at 251.2 nm
at 314 nm 277.2 nm
1 10 8 101.61 99.00 99.08
2 10 10 98.71 97.26 100.52
3 10 5 101.57 100.86 99.35
4 5 8 97.30 97.45 98.95
5 15 8 100.77 100.29 100.26
6 5 10 98.18 97.07 99.47
Mean ± SD 99.69 ± 1.86 98.66 ± 1.65 99.61 ± 0.64
a
Average of three determinations.

Fig. 2 Division spectra of laboratory prepared mixtures of diloxanide furoate (X) and metronidazole (Y) using 4 lg ml1 of
metronidazole (Y’) as a divisor and methanol as a blank.
Analysis of metronidazole and diloxanide furoate in binary mixture 327

Ratio subtraction spectrophotometric method
The method was applied for determination of mixture of (II)
and (I) when the spectrum of (I) extended than the other
(II), as shown in (Fig. 1). The determination of (II) could be
achieved by scanning the zero order absorption spectra of
the laboratory-prepared mixtures (I and II) in methanol, then
dividing them by a carefully chosen concentration (4 lg ml1)
of standard (I) (I0 = divisor) to produce a new ratio spectra
that represents II/I0 + constant, as shown in (Fig. 2); then,
subtraction of the absorbance values of these constants (I/I0 )
in plateau as shown in (Fig. 3) followed by multiplication of
the obtained spectra by (I0 ) the divisor as shown in (Fig. 4); fi-
nally, the original spectra of (II), which are used for direct
determination of (II) at 251.2 nm, could be obtained and the
concentration from the corresponding regression equation
could be calculated. This can be summarized as follows:
ðII þ IÞ=I0 ¼ II=I0 þ I=I0 ¼ II=I0 þ constant
II=I0 þ constant  constant ¼ II=I0
II=I0  I0 ¼ II
The constant can be determined directly from the curve
(II + I)/I0 by the straight line which is parallel to the wave-
length axis in the region where (I) is extended. The correct
choice of the divisor is fundamental, as, if the concentration
of the divisor increases or decreases, the resulting constant va-
lue will be proportionally decreased or increased [24].
A linear correlation was obtained between the absorbance
and the corresponding concentration of (II) at its correspond-
ing wavelength: the regression equation was:

Fig. 3 Division spectra of laboratory prepared mixtures of diloxanide furoate (X) and metronidazole (Y) using 4 lg ml1 of
metronidazole (Y0 ) as a divisor and methanol as a blank after subtraction of the constant.

Fig. 4 The zero order absorption spectra of diloxanide furoate obtained by the proposed ratio subtraction method for the analysis of
laboratory prepared mixtures after multiplication by the divisor (Y0 ).
328 M.R. El-Ghobashy and N.F. Abo-Talib

Table 3 Determination of metronidazole and diloxanide furoate in their pharmaceutical preparations by the proposed methods and
application of standard addition technique.
Product Isosbestic spectrophotometry Ratio subtraction spectrophotometry
Founda (%) ± SD Added Founda Recovery (%) Founda (%) ± SD Added Founda Recovery (%)
Metronidazole in Furazole tablets 4 3.94 98.50 4 3.94 98.50
(batch no. 080435) 100.23 ± 0.62 8 8.03 100.38 100.23 ± 0.62 8 8.03 100.38
12 11.77 98.08 12 11.77 98.08
Mean ± SD 98.99 ± 1.23 98.99 ± 1.23
Diloxanide furoate in Furazole tablets 5 4.98 99.60 5 4.96 99.20
(batch no. 080435) 99.04 ± 0.98 10 9.81 98.10 98.97 ± 0.23 10 9.86 98.60
15 14.68 97.87 15 14.80 98.67
Mean ± SD 98.52 ± 0.94 98.82 ± 0.33
Metronidazole in Furazole suspension 4 3.95 98.75 4 3.95 98.75
(batch no. 074135) 98.69 ± 0.54 8 7.87 98.38 98.69 ± 0.54 8 7.87 98.38
12 11.90 99.17 12 11.90 99.17
Mean ± SD 98.77 ± 0.40 98.77 ± 0.40
Diloxanide furoate in Furazole 5 4.98 99.60 5 4.97 99.40
suspension (batch no. 074135) 99.20 ± 0.66 10 10.01 100.10 99.13 ± 0.49 10 9.96 99.60
15 14.72 98.13 15 15.08 100.53
Mean ± SD 99.28 ± 1.02 99.84 ± 0.60
a
Average of three determinations.

Table 4 Statistical comparison of the results obtained by applying the proposed methods and the direct spectrophotometric
manufacturer method for the analysis of pure metronidazole and diloxanide furoate.
Manufacturer method Ratio subtraction spectrophotometry Isosbestic spectrophotometry Value
Diloxanide furoate Metronidazole Diloxanide furoate Diloxanide furoate Metronidazole
100.50 99.84 99.73 99.96 99.83 Mean
0.71 1.26 1.33 1.47 1.41 SD
6 6 6 6 6 n
0.504 1.588 1.769 2.161 1.988 Variance
– – 1.251 0.816 0.013 t-Test (2.228)*
– – 3.510 4.288 1.252 F value (5.05)*
*
The values in parenthesis are corresponding to the theoretical values of t and F (P = 0.05).

A ¼ 0:0726C þ 0:0452 r ¼ 0:9997
where A is the absorbance of (II) at 251.2 nm, C is the concen-
tration of (II) in lg ml1 and r is the correlation coefficient.
The proposed method was applied for the determination of
(II) in bulk powder and satisfactory results were obtained see
(Table 1).
The laboratory-prepared mixtures were analyzed by the ra-
tio subtraction method at 251.2 nm. The method is valid for
determining the drug in laboratory-prepared mixtures as
shown in (Table 2).
The proposed methods were successfully applied for the
analysis of both drugs in pharmaceutical dosage form and
the results are shown in (Table 3).
The validity of the proposed methods was assessed by
applying the standard addition technique. The results obtained
were reproducible with low relative standard deviation as
shown in (Table 3).
A statistical comparison of the results obtained by the two
proposed methods and the manufacturer’s method for pure
drugs is shown in Table 4. The values of the calculated t and
F are less than the tabulated ones, which reveals that there is
no significant difference with respect to accuracy and precision
between the proposed methods and the manufacturer’s
procedure.
Conclusion
The aim of this work is to develop simple and new methods for
the simultaneous determination of metronidazole and diloxa-
nide furoate. The isosbestic spectrophotometric and ratio sub-
traction spectrophotometric methods could be applied to the
simultaneous determination of metronidazole and diloxanide
furoate either in their pure powder form or in their combined
preparations. The results demonstrate the usefulness of the
methods, which are simple, safe, sensitive, precise, accurate,
inexpensive and non-polluting. So, the proposed methods
could be used in routine and quality control analysis of metro-
nidazole and diloxanide furoate in pharmaceutical prepara-
tions containing them.
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