Kinetic-Spectrophotometric Determination of Metronidazole Benzoate

Kinetic-Spectrophotometric Determination of Metronidazole Benzoate in
Surfactant Medium
Khalil Farhadi* and Shahriyar Bahar
Department of Chemistry, Faculty of Science, Urmia University, Urmia, Iran
A kinetic method for the accurate and sensitive determination of metronidazole benzoate (MB) has
been described. The method is based on the oxidation of MB with KMnO4 in alkaline medium in the presence
of sodium dodecyl sulphate (SDS). At a fixed time of 10 min, the formed magnate ion is spectrophotometrically
measured at 610 nm. The determination of MB by the fixed-concentration and rate constant
method is feasible with the calibration equation obtained, but the fixed-time method proves to be more applicable.
The proposed method was successfully used for the quantitative determination of MB in suspended
oral syrup after the separation of MB with a simple separation method. Beer’s law was obeyed
from 0.55 mgL -1 to 33 mgL -1 and the RSD% value for syrup was 3.44. The results obtained agreed with
those obtained by the BP method.
Keywords: Metronidazole benzoate determination; Kinetic-Spectrophotometry; KMnO4; SDS.
INTRODUCTION
Metronidazole benzoate (MB), chemically known as
2-(2-methyl-5-nitro-1H-imidazol-1-yl)ethyl benzoate, 1
has particularly high activity in vitro and in vivo against Tvaginalis
and E-histolytica. Metronidazole benzoate has an
extremely broad spectrum of protozoal and antimicrobial activity
which can be used to clinical advantage and also displays
antibacterial activity against all anaerobic cocci and
both anaerobic gram-negative bacilli and anaerobic sporeforming
gram-positive bacilli. It is clinically effective in
trichomoniasis, amebiasis and giardiasis, as well as in a variety
of infections caused by obligate anaerobic bacteria.
NO 2
N
N
Due to the vital importance determination of this drug
in pharmaceutical preparations and biological fluids, sev-
Journal of the Chinese Chemical Society, 2007, 54, 1521-1527 1521
O
Me
O
Metronidazole benzoate (MB)
eral spectrophotometric, 2-7 gas liquid chromatographic, 8,9
high performance liquid chromatographic, 10-15 differential
pulse polarographic 16 and classic 17-21 methods for assay of
metronidazole benzoate have been reported in the literature.
Kinetic methods are becoming of great interest in
chemical analysis. Various kinetic methods have been applied
to the determination of many organic and inorganic
species. 22 This work represents the first attempt at assaying
MB in a pharmaceutical preparation by use of kinetic methods.
The method is based on oxidizing the drug with alkaline
potassium permanganate in the presence of SDS. 23 The
reaction is followed up spectrophotometrically and the rate
of change of absorbance at 610 nm is measured. The fixed
time method is adopted after full investigation and understanding
of the kinetics of the reaction. The proposed
method is simple, accurate, and sensitive and does not require
elaborate treatment and procedures, which are usually
associated with other methods.
EXPERIMENTAL
* Corresponding author. E-mail: kh.farhadi@mail.urmia.ac.ir or khfarhadi@yahoo.com
Materials
All experiments were performed with analytical reagent
grade chemicals. Doubly distilled water was used

1522 J. Chin. Chem. Soc., Vol. 54, No. 6, 2007 Farhadi and Bahar
throughout. Reagent grade metronidazole benzoate and its
syrup were obtained from Tehran Shimi Pharmaceutical
Company, Tehran, Iran.
Reagents and Solutions
Stock solution of MB (10 -3 M) was prepared by dissolving
27.53 mg of pure drug in 100 mL of 0.6 M HCl. The
solution was stable for at least 5 days when kept in a refrigerator
(at 4 �C). The working solutions were prepared by
stepwise dilution as required. Potassium permanganate solution,
10 -2 M, was prepared in distilled water. Sodium hydroxide
solution, 2.5 M, was prepared and kept as a stock
solution. Stock solution of SDS (10 -2 M)was prepared daily
by dissolving 1.4419 g of SDS in water and diluting to 500
mL.
Apparatus
The study was conducted using an LKB 4054 UV/
VIS recording spectrophotometer and 1.00-cm matched
silica cells. All pH measurements were made with a digital
WTW Multilab 540 Ionalyzer (Germany) pH/mV meter using
a combined glass electrode.
Procedures
General Procedure for Kinetic-Spectrophotometric
Determination of MB
Transfer 6 mL of 10 -2 M potassium permanganate, 5
mL of 2.5 M sodium hydroxide and 1 mL of 10 -2 MSDSsolutions
into 25 mL calibrated flasks. Accurate volumes of
working solution of MB, over the concentration range
0.55-33 mgL -1 , were added and the solutions were diluted
to volume with distilled water. At a fixed time of 10 min,
the absorbance was measured directly at 610 nm against an
appropriate blank sample. The calibration graph was constructed
by plotting the final concentration of the drug
against the absorbance values.
Procedure for Determination of MB in Syrup
An accurately measured 2.0 mL aliquot of the mixed
contents of three syrups was transferred into a 5 mL test
tube containing 1 mL 0.5 M NaOH and then centrifuged at
a rate of 5000 rpm for 5 min. The residue was washed at
least three or more times with mild alkaline solution, then
was quantitatively transferred into a 100 mL calibrated
flask, and after the complete dissolution in 0.6 M HCl, diluted
to the mark. The recommended kinetic-spectrophotometric
procedure for the determination of MB was fol-
lowed on appropriate amounts of this solution at room temperature.
RESULTS AND DISCUSSION
Kinetics and Optimization of the Reaction Conditions
MB was found to react with alkaline potassium permanganate
in the presence of SDS producing a green color
as a result of manganate species, which absorbs at 610 nm.
As the intensity of color increases with time, it was deemed
useful to elaborate a kinetically-based method for the determination
of MB. At 610 nm, changing each variable in turn,
while keeping all others constant, optimized the various experimental
parameters affecting the development and stability
of the reaction product.
Effect of the KMnO4 Concentration on the Reaction
The effect of potassium permanganate concentration
on the reaction was studied over the range of 4 × 10 -4 -3.2×
10 -3 MofKMnO4 and five selected concentrations of MB
according to the obtained calibration graph (Fig. 1). The reaction
rate and absorbance increased with increasing
KMnO4 concentration and leveled off at 2.4 × 10 -3 Mfora
maximum concentration of MB in the calibration carve.
Therefore 2.4 × 10 -3 MKMnO4 was chosen as the most
suitable concentration.
Fig. 1. Effect of KMnO4 concentration on the reaction
product of MB (A) 2.0 × 10 -6 M, (B) 2.0 × 10 -5
M, (C) 4.0 × 10 -5 M, (D) 8.0 × 10 -5 Mand(E)1.2
×10 -4 M measured at room temperature after 10
min.

Kinetic Determination of Metronidazole Benzoate J. Chin. Chem. Soc., Vol. 54, No. 6, 2007 1523
Effect of the SDS Concentration on the Reaction
The ability of surfactants to dissolve organic compounds,
which are insoluble or only slightly soluble in water,
is a distinctive feature of solubilization. They are usually
incorporated into a hydrocarbon environment in the interior
of micelles. 24 Therefore, in this study, we used a solution
of surfactants as suitable medium for the complete dissolving
of MB. For this purpose, several anionic, cationic
and non-ionic surfactants were tested. Among the tested
surfactants in an alkaline mixture of MB, the obtained solutions
using SDS were completely transparent, so MB solutions
were prepared in alkaline-SDS media.
The effect of various concentrations of SDS on maximum
absorbance at different amounts of MB was studied
between 4 × 10 -5 –1.2×10 -3 M (Fig. 2). As seen, in all
cases, a maximum absorbance was obtained using concentrations
above 4 × 10 -4 M of SDS. Therefore, the optimum
concentration of SDS was selected as 4 × 10 -4 M in all solutions.
Effect of the NaOH Concentration on the Reaction
The influence of the medium alkalinity was investigated
between 0.1-1 M sodium hydroxide (Fig. 3). It was
observed that increasing NaOH concentration increases the
reaction rate with maximum absorbance being a shorter
time. It was also found that there was no significant difference
in the absorbance of reaction solution at NaOH concentrations
above 0.5 M, while decreasing NaOH concen-
Fig. 2. Effect of SDS concentration on the reaction
product of MB (A) 2.0 × 10 -6 M, (B) 2.0 × 10 -5
M, (C) 4.0 × 10 -5 M, (D) 8.0 × 10 -5 Mand(E)1.2
×10 -4 M measured at room temperature after 10
min.
tration resulted in lower absorbance values. Therefore, 0.5
M NaOH was chosen for all subsequent experiments.
Determination of the Molar Ratio between KMnO4
and MB in the Reaction
The limiting logarithmic method was used for the determination
of the molar ratio between KMnO4 and MB in
the reaction. This method depends on measuring the optical
densities of solution of KMnO4 andMBinwhichtheconcentration
of the two species are varied in turn at a constant
total ionic strength.
The ratio may be found by plotting the logarithms of
the absorbance (A) of the two sets of solutions versus composition,
one with constant KMnO4 concentration and variable
MB concentration, the other with constant MB and
variable KMnO4 concentration (Fig. 4). The slope of the
curve in case 1 yields the number of moles of MB while that
in case 2 gives the number of moles of KMnO4 andsothe
composition of the compound produced can be evaluated.
The molar ratio was found to be 1:2 for MB/KMnO4.
Determination of the Reaction Order
The rate of the reaction was also found to be [MB]dependent.
The rates were followed at room temperature
with various concentrations of MB in the range of 0.55-33
mgL -1 , keeping KMnO4, NaOH and SDS constant at high
concentrations as above.
Fig. 3. Effect of NaOH concentration on the reaction
products of MB (A) 2.0 × 10 -6 M, (B) 2.0 × 10 -5
M, (C) 4.0 × 10 -5 M, (D) 8.0 × 10 -5 Mand(E)1.2
×10 -4 M measured at room temperature after 10
min.

1524 J. Chin. Chem. Soc., Vol. 54, No. 6, 2007 Farhadi and Bahar
The graphs shown in Fig. 5 were obtained, from which
it is clear that the rate increases as the MB concentration increases,
indicating that the reaction rate obeys the following
equation:
Rate = k� [MB] n
Fig. 4. Determination of the molar ratio between
KMnO4/MB by limiting logarithmic method. 1.
Set of solutions with constant MB concentration
and variable KMnO4 concentration. 2. Set
of solutions with co4nstant KMnO4 concentration
and variable MB concentrations.
Fig. 5. Absorbance versus time graphs for the reaction
of MB and alkaline potassium permanganate
showing the dependence of the reaction on MB
concentration. Concentration of MB (A) 0.55
mgL -1 , (B) 5.5 mgL -1 , (C) 11.0 mgL -1 , (D) 16.5
mgL -1 , (E) 22.0 mgL -1 , (F) 27.5 mgL -1 and (G)
33.0 mgL -1 .
(1)
where k� is the pseudo-order constant of the reaction.
Apparently, the reaction proceeds in two steps. The
first step is fast and the second is the rate-determining step.
From Fig. 5, the rate may be estimated by the variable-time
method measurements(25), as �A/�t, taking logarithms
of rates and concentrations; Eq. 1 is transformed
into:
log (rate) = log �A/�t=logk� +nlog[MB] (2)
Regression of log [MB] versus log (rate) by the least
squares method yielded the calibration equation:
log (rate) = 1.5753 + 1.0175 log [MB] (3)
with correlation coefficient r = 0.9896. Hence k� = 37.61 s -1
and the reaction is pseudo first order (n � 1) with respect to
MB.
Evaluation of the Kinetic Methods
Initial-Rate Method (Pseudo-zero-order Method)
In this method, graphs of the rate (at the beginning of
the reaction) versus MB concentration were not easy to obtain,
because the first step of the reaction was too fast to follow,
so tangents of the curve were not easy to draw. This
method was therefore abandoned.
Rate Constant Method
Graphs of log (absorbance) versus time for MB concentration
in the range of 0.55-33 mgL -1 were plotted and
all appeared to be rectilinear. Pseudo first-order-rate constants
(K�) corresponding to different MB concentrations
(C) were calculated from the slopes, multiplied by 2.303
and are presented in Table 1. Regression of C versus k� gave
the equation:
K� = 15.506 C + 0.002 (R 2 = 0.8671) (4)
The value of R indicates poor linearity, which is probably
due to slight changes in the temperature of the reaction.
Variable-Time Method
Reaction rates were recorded for different MB concentrations
in the range of 0.55-33 mgL -1 . A pre-selected
value of the absorbance was fixed and the time was mea-

Kinetic Determination of Metronidazole Benzoate J. Chin. Chem. Soc., Vol. 54, No. 6, 2007 1525
Table 1. Values of k' calculated from slopes of log (Abs.) versus
t (second) graphs multiplied by 2.303 for different
concentrations of MB at a constant concentration of
NaOH, KMnO4 and SDS at room temperature
K' (s -1 ) [MB] (moleL -1 )
1.9 � 10 -3
2 � 10 -6
2.0 � 10 -3
2 � 10 -5
2.8 � 10 -3
4 � 10 -5
3.0 � 10 -3
6 � 10 -5
3.6 � 10 -3
8 � 10 -5
3.5 � 10 -3
1 � 10 -4
3.5 � 10 -3
1.2 � 10 -4
Table 2. Values of reciprocal time taken at fixed absorbance for
the different rates of variable concentration of MB at
constant concentrations of NaOH, KMnO4 and SDS at
room temperature
(1/t) (s -1 ) [MB] (moleL -1 )
3.33 � 10 -4
2 � 10 -6
1.74 � 10 -3
2 � 10 -5
5.86 � 10 -3
4 � 10 -5
8.63 � 10 -3
6 � 10 -5
9.90 � 10 -3
8 � 10 -5
1.10 � 10 -2
1 � 10 -4
1.15 � 10 -2
1.2 � 10 -4
sured next. The reciprocal of time (1/t) versus the initial
concentrations of MB (Table 2) was plotted and the following
equation of the calibration graph was obtained:
1/t = 100.76 C + .00009 (R 2 = 0.9260) (5)
The range of MB concentrations giving the most acceptable
calibration graph with the above equation was
very limited and showed poor linearity, which could be a
disadvantage.
Fixed-Time Method
Reaction rates were determined for different concentrations
of MB. At a pre-selected fixed-time, which was accurately
determined, the absorbance was measured. Calibration
graphs of absorbance versus initial concentration of
MB were established at fixed times of 2, 5, 10, 15, 20 and
25 min with the regression equations assembled in Table 3.
It is clear that the slope increases with time and the most acceptable
values of r and the intercept were obtained for a
fixed time of 10 min, which was therefore chosen as the
most suitable time interval for measurement.
Table 3. Calibration equations at different fixed-times for MB
concentrations in the range of 0.55-33 �gmL -1
Time (min) Calibration equation Correlation coefficient
2 A = 0.0084 C + 0.0669 0.9455
5 A = 0.0252 C + 0.0099 0.9991
10 A = 0.0315 C + 0.0237 0.9989
15 A = 0.0354 C + 0.0286 0.9986
20 A = 0.0376 C + 0.0276 0.9984
25 A = 0.0390 C + 0.0297 0.9981
Table 4. Analytical data for the kinetic determination of MB
Parameter Value
Volume of KMnO4 6mL
Volume of SDS 1 mL
Volume of NaOH 5 mL
Temperature 25 �C
Reaction time 10 min
Concentration range 0.55-33 mgL -1
Molar absorptivity 8669.56
Regression equation A = 0.0315 C + 0.0237
Correlation coefficient 0.9989
Sy/x 0.0225
Sa 0.0160
Sb 0.0009
RSD for syrup 3.44%
Linearity
The kinetic curves obtained at different concentrations
of MB, under the optimized conditions, were processed
by the fixed time method. 26 A calibration graph of
absorbance versus initial concentration of MB was established
at different fixed-time intervals. It was found that the
slopes increase with time and the most acceptable values of
the correlation coefficient (R) and the intercept were obtained
at a fixed-time of 10 min that was, therefore, chosen
as the most suitable time intervals for measurement. The
calibration graph was linear over the concentration range of
0.55-33 mgL -1 . Regression analysis indicated a linear relationship
with negligible intercept. Table 4 presents the analytical
parameters, molar absorptivity and the results of the
statistical analysis of the experimental data regression
equation calculated from the calibration graphs along with
standard deviation of the slope (Sb) and intercept (Sa) on the
ordinate and the standard deviation of residuals (Sy/x). The
high values of the correlation coefficient of regression
equation equations indicate good linearity and conformity
to Beer’s law. The detection limit of the proposed method
was calculated according to a reported method. 27 The de-

1526 J. Chin. Chem. Soc., Vol. 54, No. 6, 2007 Farhadi and Bahar
tection limit was 0.24 µg mL -1 while the quantification
limit was 0.71 µg mL -1 for MB.
Accuracy and Precision
Three replicate determinations at different concentration
levels of MB were carried out to test the precision and
accuracy of the proposed method. The relative standard deviation
(RSD) is shown in Table 4. The figures obtained
point to the good accuracy and repeatability of the method.
The effects of common excipients and other substances
were tested for possible interferences in this assay.
In preliminary experiments, direct application of the proposed
kinetic fixed-time method for the determination of
MB in samples containing some common oxidizable excipients
such as glucose, starch or lactose showed considerable
errors. This problem was easily solved by the quantitative
separation of MB in the alkaline medium.
ANALYTICAL APPLICATIONS
Table 5. Application of the proposed kinetic method to the determination of MB in its
pharmaceutical preparations
%Recovery � SD*
Preparation
Proposed method Reference method 26
Metronidazole Syrup 124 � 4.29 125 � 3.33
(Metronidazole benzoate, 125 mg) t = 0.29, F = 1.66
* Average of three replicate measurements. Tabulated values of t and F are 2.78 and 19.00 at
95% confidence level.
The proposed method has been successfully applied
to determine MB in pharmaceutical preparations. The concentration
of the drug was calculated using the corresponding
regression equation at a fixed time of 10 min. In order to
establish the validity of the proposed analytical method, the
assay of MB in its formulation using the proposed and BP
method 28 was carried out. It should be noted that, in performing
the official method on MB syrup in the first step,
the drug was completely extracted into chloroform and after
evaporating the solvent, the residue was dissolved in anhydrous
glacial acetic acid, titrated with perchloric acid
and determining the end point, potentiometrically. The results
obtained are presented in Table 5. Statistical analysis
of the results obtained by both the proposed method and the
reference non-aqueous titrimetric method revealed no sig-
nificant difference in the performance of the two methods
regarding accuracy and precision as revealed by a t-test and
an F-test, respectively (Table 5). It should be noted that the
reaction medium in non-aqueous titrimetry must be scrupulously
anhydrous and in practice, even a trace amount of
water will affect the results. Therefore, due to the absence
of such errors in the proposed procedure, a convenient and
fast performing method in aqueous media using inexpensive
reagents and direct application of spectrophotometry
in a wide concentration range suggests its application to the
analyses and quality control of the studied compound in
comparison to tedious time consuming official methods.
CONCLUSIONS
The above results obtained from the proposed kinetic
fixed-time method on suspended syrup after the separation
of MB showed that the method is comparable to the official
BP method (see Table 5). The proposed kinetic-spectrophotometric
method is simple, fast and inexpensive, does
not require any toxic organic solvents, and is precise and
accurate. This method was satisfactory for the determination
of MB in drug formulations such as syrup without considerable
interference.
The student t-test and F-test values for the 95% confidence
level did not exceed the theoretical values of 2.78
and 19.0 for t and F-tests, respectively, indicating no significant
difference between the accuracy and precision of the
two methods. Therefore, this makes the method applicable
for the determination of MB in pharmaceutical formulations.
Received March 16, 2007.

Kinetic Determination of Metronidazole Benzoate J. Chin. Chem. Soc., Vol. 54, No. 6, 2007 1527
REFERENCES
1. British Pharmacopocia, IIMSO, London, 1993.
2. Sastry,C.S.P.;Aruna,M.;Vijaya,D. Indian J. Pharm. Sci.
1987, 49, 190.
3. Sastry,C.S.P.;Aruna,M.;Rao,A.R.M. Talanta 1988, 35,
23.
4. Avramova, Y.; Zaikov, K.; Chokhadzhieva, D. Khig.
Zdraveopaz. 1990, 33, 77.
5. Sastry, C. S. P.; Aruna, M.; Rao, A. R. M.; Tipirneni, A. S. R.
P. Chem.Anal.(Warsaw)1991, 36, 153.
6. Sanyal,A.K. Analyst 1992, 117, 93.
7. Maheshwari, R.; Chaturvedi, S.; Jain, N. Indian J. Pharm.
Sci. 2006, 68(2), 195.
8. Gorog, S.; Futo, M.; Lauko, A. Acta Pharm. Hung. 1976, 46,
113.
9. Gutierrez, C. R.; Garzon, A. Rev. Soc. Quim. Mex. 1979, 23,
129.
10. Mishal, A.; Sober, D. J. Pharm. Biomed. Anal. 2005, 39,
819.
11. Sasa, S. I.; Khalil, H. S.; Jalal, I. M. J. Liq. Chromatogr.
1986, 9, 3617.
12. Bempong, D. K.; Manning, R. G.; Mirza, T.; Bhattacharyya,
L. J. Pharm. Biomed. Anal. 2005, 38, 776.
13. Pashankov, P.; Kostova, L. J. Chromatogr. A 1987, 394, 382.
14. Avramova, Y. Pharmazie 1988, 43, 436.
15. Ali, M. S.; Chaudhary, R. S.; Takieddin, M. A. Drug. Dev.
Ind. Pharm. 1999, 25, 1143.
16. Dalkara, S.; Ong, H.; Braun, J.; Plourde, R. Anal. Lett. 1984,
17, 793.
17. Gajewska, M. Acta Pol. Pharm. 1972, 29, 393.
18. Gajewska, M. Acta Pol. Pharm. 1973, 30, 167.
19. Inamdar, M. C.; Mody, S. B. Indian J. Pharm. Sci. 1977, 39,
106.
20. Parimoo, P. Indian J. Pharm. Sci. 1986, 48, 186.
21.Zhang,J.;Tan,Y.L.;Chen,D.H.;Yuan,Y.H.;Tang,W.J.;
Hu,X.A. Zhongguo Yaoke Daxue Xuebao 2000, 31, 309.
22. Crouch, S. R.; Cullen, T. F.; Scheeline, A.; Kirkor, E. S.
Anal. Chem. 1998, 70, 53.
23. Sykes, P. A Guide Book to Mechanism in Organic Chemistry;4
th ed.; Longman: London, 1987; p 186.
24. Shinoda, K.; Nakagawa, T.; Tamamushi, B.; Isemura, T.
Collodial Surfactants; Academic Press: New York, 1963; pp
97-178.
25. Yatsimirskii, K. B. Kinetic Methods of Analysis; Pergamon
Press: Oxford, 1996.
26. Perez-Bendito, A.; Silva, M. Kinetic Methods in Analytical
Chemistry; Eilis Harwood: London, 1988; p 40.
27. Miller, J. C.; Miller, J. N. Statistics for Analytical Chemistry;2
nd ed., Ellis Hrwood: Chichester, 1988.
28. United States Pharmacopia, 16 th ed.; Easton. Pa. 180420,
1985; p 580.