Spectrophotometric determination of metronidazole and tinidazole via charge transfer complexation using chloranilic acid

Article abstract of DOI:10.1007/BF03246021

A new spectrophotometric method was developed for the determination of two important nitroimidazoles; metronidazole (MZ) and tinidazole (TZ). The method was based on the charge-transfer (CT) complexation reaction of reduced forms of metronidazole and tinidazole as n-electron donors and chloranilic acid (CAA) as π-electron acceptor to form a purple-colored complex with a new absorption band at 520 nm which was adopted as the analytical wavelength. Molar absorptivities of 2.741 × 10² L M^-1 cm^-1 and 2.681 × 10² L M^-1 cm^-1 were obtained for MZ and TZ, respectively. Optimization of reducing agent and time of reduction revealed the superiority of metal hydrides over reducing metals. Reduction of MZ and TZ was completed at 30 °C within 10 min. Optimizations of temperature and time for the complexation reaction revealed that the reaction was completed at 30 °C within 5 min. A 60:40 mixture of 1,4-dioxane: acetonitrile was found to be the best diluting solvent for optimal detector response. The complexes were stable at room temperatures for weeks. Beer's law was observed in the concentration of 5-40 μg ml (MZ) and 4.8-79.2 μg ml^-1 (TZ) with low limits of detection of 1.88 and 0.74 μg ml ^-1, respectively. Overall recoveries of MZ and TZ from quality control samples were 103.19 ± 2.05 (%RSD = 1.99, n = 12) and 101.63 ± 1.41 (%RSD = 1.39) over three days. There was no interference from commonly utilized tablet excipients. No significant difference existed between the results of the new method and the BP titrimetric procedures (p > 0.05). The new CT procedure described in this paper is simple, fast, convenient, accurate and precise and has the novelty of carrying out the reactions at room temperature compared to previously described procedures. The new method could be adopted as an alternative procedure for the quality assessment of MZ and TZ in bulk and dosage forms.

Full text of DOI:10.1007/BF03246021

J. Iran. Chem. Soc., Vol. 7, No. 2, June 2010, pp. 359-370.
JOURNAL OF THE
Iranian
Chemical Society
Spectrophotometric Determination of Metronidazole and Tinidazole via Charge
Transfer Complexation Using Chloranilic Acid
a,
a
b
O.A. Adegoke *, O.E. Umoh and J.O. Soyinka
Department of Pharmaceutical Chemistry, Faculty of Pharmacy, University of Ibadan, Ibadan, Nigeria
Department of Pharmaceutical Chemistry, Faculty of Pharmacy, Obafemi Awolowo University, Ile-Ife, Nigeria
a
b
(Received 27 February 2009, Accepted 11 June 2009)
A new spectrophotometric method was developed for the determination of two important nitroimidazoles; metronidazole (MZ)
and tinidazole (TZ). The method was based on the charge-transfer (CT) complexation reaction of reduced forms of metronidazole
and tinidazole as n-electron donors and chloranilic acid (CAA) as ꢀ-electron acceptor to form a purple-colored complex with a
2
-1
-1
new absorption band at 520 nm which was adopted as the analytical wavelength. Molar absorptivities of 2.741 ꢀ 10 L M cm
2
-1
-1
and 2.681 ꢀ 10 L M cm were obtained for MZ and TZ, respectively. Optimization of reducing agent and time of reduction
revealed the superiority of metal hydrides over reducing metals. Reduction of MZ and TZ was completed at 30 °C within 10 min.
Optimizations of temperature and time for the complexation reaction revealed that the reaction was completed at 30 °C within 5
min. A 60:40 mixture of 1,4-dioxane:acetonitrile was found to be the best diluting solvent for optimal detector response. The
complexes were stable at room temperatures for weeks. Beer’s law was observed in the concentration of 5-40 μg ml (MZ) and
-
1
-1
4
.8-79.2 μg ml (TZ) with low limits of detection of 1.88 and 0.74 μg ml , respectively. Overall recoveries of MZ and TZ from
quality control samples were 103.19 ± 2.05 (%RSD = 1.99, n = 12) and 101.63 ± 1.41 (%RSD = 1.39) over three days. There was
no interference from commonly utilized tablet excipients. No significant difference existed between the results of the new method
and the BP titrimetric procedures (p > 0.05). The new CT procedure described in this paper is simple, fast, convenient, accurate
and precise and has the novelty of carrying out the reactions at room temperature compared to previously described procedures.
The new method could be adopted as an alternative procedure for the quality assessment of MZ and TZ in bulk and dosage forms.
Keywords: Nitroimidazoles, Metronidazole, Tinidazole, Chloranilic acid, Charge transfer complexation
INTRODUCTION
[3]. The most important derivatives contain the 5-
nitroimidazole nucleus with substituents at the N-1 position of
the heterocyclic aromatic ring [4]. The discovery of the anti-
trichomonal properties of metronidazole revolutionised the
treatment of the disease. Although the amoebicidal properties
of metronidazole were studied, it was not clinically tested until
some years later. In laboratory tests, metronidazole is effective
against intestinal amoebiasis in rats and hepatic amoebiasis in
hamsters and is also active against Entamoeba histolytica in
vitro. Initial clinical tests of metronidazole indicated that it
Nitro derivatives of five-membered heterocyles are of
considerable interest. Some are biologically active [1] with
anti-inflammatory or vasodilator activity [2]. Others are useful
synthetic intermediates for many biologically active
compounds; for instance, nitroimidazoles form the basis of
nitro-heterocycles analogous to megazol, an antiparasitic agent
*Corresponding authors. E-mail: ao.adegoke@mail.ui.edu.ng

Adegoke et al.
was capable of curing invasive amoebic dysentery and
using a charge transfer complexation procedure with
amoebic liver abscess.
chloranilic acid.
Subsequent clinical tests have established metronidazole as
the drug of choice in the treatment of all forms of amoebiasis
in humans [5]. Variation of the structure of metronidazole,
principally to improve trichomonacidal activity and metabolic
stability, led to the discovery of tinidazole. Tinidazole is active
against E. histolytica in vitro, cecal amoebiasis in rats, and
hepatic amoebiasis in hamsters. Clinical tests have established
the value of tinidazole in the treatment of intestinal and
hepatic amoebiasis in humans [5]. Tinidazole has about the
same or slightly greater efficacy than metronidazole in the
treatment of trichomoniasis and in giardiasis. It has been found
to be effective against strains resistant to metronidazole [6].
Chemically, metronidazole is 2-methyl-5-nitroimidazole-1-
ethanol and tinidazole is 1-(2-ethyl-sulphonyl ethyl)-2-methyl-
EXPERIMENTAL
Chemicals and Reagents
Metronidazole brands utilized in this study were Loxagyl
(M & B, Nig. Plc.), Metrodol (Pauco Pharm, Nig. Ltd.),
Metrotabs (SKG Pharm. Ltd.), Tromet 500 infusion
(Microlabs Ltd.) and tinidazole brand was Amtiba 500 (Cadila
Pharm. Dholka, India)
Methanol, ethyl acetate, ethanol, sulfuric acid, chloroform,
acetone, 1,4-dioxan,
hydrochloric acid, sodium nitrite,
sulfamic acid, LiBH , NaBH , Pd-C, zinc dust, acetic
4
4
anhydride, brilliant green, perchloric acid (all analar reagent
grade obtained from BDH Chemical Ltd., Poole, England).
Distilled water, glacial acetic acids, acetonitrile, chloranilic
acid were obtained from Sigma-Aldrich, USA, precoated thin
layer plates GF254 0.2 mm (Merck, Germany). Metronidazole
and tinidazole secondary reference standards were prepared in
our laboratory whose purity was assessed by TLC, melting
point and non-aqueous titration using acetous perchloric acid.
5
-nitroimidazole. The biological activity of nitroimidazoles is
dependent upon the nitro group reduction process due to the
formation of active intermediate species [7-11] that interact
with DNA to produce biochemical damage. The reduction of
nitro group has also been used as a basis for the determination
of these drugs. Several methods have been reported for the
determination of metronidazole and or tinidazole in bulk and
dosage forms or in biological fluids. Such methods include;
non-aqueous titration [12], voltammetric methods [13-17],
HPLC [18,19,20,21] and flow injection analysis [22]. Some
previously described spectrophotometric methods include the
use of vanillin and anisaldehyde [23], p-Benzoquinone [24], ꢀ-
Naphthol [25], ꢁ-naphthol [26], alkaline hydrolysis [27],
Equipment
UV-Vis spectrophotometer (Unicam aurora), analytical
balance H80 (Mettler, UK), ultrasonic bath (Langford
Electronics, UK) Vortex mixer (Griffins and George Ltd.;
Great Britain).
derivative
spectrophotometry
[28],
extractive
Preparation of Chloranilic Acid Stock Solution and
Reduction of Metronidazole and Tinidazole
spectrophotometry using bromothymol blue [29], sodium 1,2-
naphthaquinone-4-sulfonate after condensation reaction [30],
A 0.025 g quantity of chloranilic acid powder was weighed
into a clean 25 ml volumetric flask, a small amount of
acetonitrile; about 5 ml was added to it. This was then shaken
until it had dissolved appreciably. The solution was then made
up to 25 ml mark with acetonitrile to make 0.1% w/v
chloranilic acid solution. A 0.025 g quantity of metronidazole
powder (extracted) was weighed into 7.5 ml hot ethanol with 2
ml 5 M HCl solution and 0.25 mg zinc dust in a clean beaker.
The mixture was heated in a water bath at 90 + 5 °C for 15
min. The residue was cooled, filtered and washed with ethanol
into a 25 ml flask. The solution was then made up to 25 ml
mark with ethanol to make 0.1% w/v metronidazole stock
4
-dimethylamino-cinnamaldehyde [31] and methylbenzo-
thiazolin-2-onehydrazine (MBTH) [32] among others.
A study of the thermodynamic properties of the charge
transfer between metronidazole and chloranilic acid has also
been carried out [33]. These spectrophotometric methods have
their relative merits but majority of them are carried out at
elevated temperatures, are time-consuming, use relatively
expensive reagents, involve extraction, use buffer systems,
only some determine MZ and TZ and many are multi-step
procedures. In this report, we present a simple approach to the
spectrophotometric determination of reduced nitroimidazoles
3
60

Spectrophotometric Determination of Metronidazole and Tinidazole
solution [24]. The same procedure was repeated for TZ using
.03 g quantity of tinidazole powder to give 0.12% w/v TZ
stock solution. Four other reducing agents were used for the
preparation of reduced metronidazole and tinidazole namely:
glacial acetic acid and 0.25 mg Pd-C (room temperature) [30],
metronidazole blanks using a mixture of 1,4-dioxan and
0
acetonitrile (60:40) as the blank solvent. These procedures
were repeated for tinidazole (TZ). The wavelength of
maximum absorption (ꢂmax) of the product of the reaction was
noted.
glacial acetic acid and 0.25 mg LiBH (room temperature),
4
glacial acetic acid and 0.25 mg NaBH
4
(room temperature)
Optimization Studies
and glacial acetic acid and 0.25 mg zinc dust (room
temperature). The reduction procedures were quantitatively
monitored by TLC and UV-Vis spectrophotometer.
Temperature and time were optimized using the method of
steepest ascent [34]. Aliquots of reduced metronidazole stock
solution (0.5 ml) was added to the CAA solution (0.5 ml) in a
test tube and the reaction mixture was vortex mixed followed
by incubation at 30 °C and 50 °C for 5 and 20 min. The
procedure was repeated at 60° and 80 °C. Each determination
was done in duplicates. The absorbance readings of the
complex were taken at 520 nm after making up to 5 ml with
1,4-dioxan/acetonitrile mixture. The optimum reaction time
obtained at the optimum temperature was determined by
repeating the above procedure and the reaction was terminated
by making up to 5 ml with the solvent mixture at 0, 2, 5, 10,
15, 20, 25 and 30 min, respectively. The optimal reaction time
was taken as the time corresponding to the maximal
absorbance of the samples. The entire optimization procedure
for temperature and time was similarly carried out for
tinidazole.
Evidence for Formation of Complexes and Thin
Layer Chromatography
A 0.5 ml quantity of the 0.1% Chloranilic acid solution
was measured each into several test-tubes. 0.5 ml quantity of
reduced Metronidazole and reduced Tinidazole were added
from the various reduction procedures. The colours were
formed immediately, and after 5 min and at 80 °C were noted
and recorded. Thin layer chromatographic examination was
conducted using two different solvent systems, Ethyl
Acetate/Methanol (90:10) and Chloroform/Acetone (80:20).
Reduced metronidazole, reduced Tinidazole, their complexes
with chloranilic acid and chloranilic acid solutions were
spotted on a silica gel pre-coated TLC plates and developed in
the mobile phase systems. The plates were viewed under UV
light at 254 and 366 nm and the respective R values were
Stoichiometric Ratio Determination
f
calculated.
Job’s method of continuous variation [35] was used to
determine the stiochiometric ratios of the charge transfer
complexes between CAA and both metronidazole and
tinidazole. Various volumes of 0.12% w/v of reduced
Metronidazole (MZ) (0, 0.25, 0.30, 0.40, 0.50, 0.60, 0.75,
0.80, 1.00), representing different concentrations (0, 60, 72,
Selection of Analytical Wavelength
A test tube containing 0.5 ml of 0.1% of Chloranilic Acid
(
CAA) stock solution was prepared. 4.5 ml of 1,4-
dioxan/acetonitrile (6:4) mixture was added to make up 5 ml
of reaction mixture. Another test tube containing 0.5 ml of
-
1
96, 120, 144, 180, 192, 240 μg ml ) were placed in different
test tubes; respective volumes of 0.1% w/v chloranilic acid
(CAA) in acetonitrile (1, 0.75, 0.70, 0.60, 0.50, 0.40, 0.25,
0.20, 0.00 ml), representing different concentrations (200, 150,
0
.12% reduced Metronidazole (MZ) stock solution was also
prepared. 4.5 ml of 1,4-dioxan/acetonitrile (6:4) mixture was
added to make up 5 ml of reaction mixture. Then the complex
was prepared by placing 0.5 ml of reduced metronidazole
-
1
140, 120, 100, 80, 50, 40 and 0 μg ml ) were then added,
respectively. These mixtures represented mole ratios of
metronidazole (MZ) and chloranilic ccid (CAA) (0:1, 1:3,
1:2.33, 1:1.5, 1:1, 1.5:1, 3:1, 4:1, 1:0). A reaction time of 5
min at 30 °C was allowed. 4 ml of a mixture of 1,4-dioxan and
acetonitrile (60:40) was added to each of the test tubes to
make up 5 ml of the reaction mixture. The same procedure
(
MZ) solution as mentioned above in a clean test tube and 0.5
ml of 0.1% Chloranilic Acid (CAA) (reagent) was added. This
was allowed to stand for 5 min whereupon 4 ml of 1,4-
dioxan/acetonitrile (60:40) mixture was added to make up 5
ml of reaction mixture. The absorption spectra of the
complexes were determined against the CAA and reduced
3
61

Adegoke et al.
Assay of Dosage Forms
was repeated for tinidazole (TZ). The absorbance readings
were recorded at 520 nm.
Weight uniformity test was carried out on the different
brands, and the assay of the active ingredient was then carried
out based on the new method for each brand. Ten tablets were
utilized in each case. Ten tablets of each of the respective
brands of metronidazole were weighed and powered. An
amount of powered tablets of each of the tablet brands
equivalent to 0.025 g of metronidazole were each reduced with
Validation Studies
Six tubes containing varying volumes of reduced
metronidazole (MZ) stock solution, (0, 0.025, 0.05, 0.1, 0.15
and 2.0 ml) with respective concentrations, (0, 5, 10, 20, 30
-
1
and 40 μg ml ) were prepared. 0.5 ml of 0.1% w/v CAA was
added to each of these tubes. These were allowed to stand for
LiBH as mentioned above. 0.1 ml of each of the respective
4
reduced metronidazole (MZ) stock solution equivalent to 20
5
min after which respective volumes of 1,4-dioxan/
-
1
μg ml was then transferred into each of the six test tubes. 0.5
ml of 0.1% w/v CAA was added to each of the tubes. The
reaction was allowed to proceed for 5 min after which 1,4-
dioxan/Acetonitrile (6:4) was added to stop the reaction, and
made up 5 ml of the reaction mixture. The absorbance reading
of the respective reaction mixtures was recorded at 520 nm
using 1,4-dioxan/Acetonitrile (60:40) as blank solvent. Six
determinations were taken in each case. The procedure was
repeated for the single brand of tinidazole using 19.2 μg ml-1
concentration.
acetonitrile (6:4) mixture was added to make up to 5 ml
reaction mixture. The absorbance readings of each of the
mixtures were then recorded at 520 nm. For reduced tinidazole
(
TZ) stock solution, (0, 0.02, 0.04, 0.08, 0.13, 0.17 and 0.33
ml) with respective concentrations, (0, 4.8, 9.6, 19.2, 31.2,
-
1
4
0.8 and 79.2 μg ml ) were prepared. 0.5 ml of 0.1% w/v of
CAA was added to each of these tubes. These were allowed to
stand for 5 min after which respective volumes of 1,4-
dioxan/Acetonitrile (60:40) mixture was added to make up 5
ml reaction mixture. The absorbance readings of each of the
mixtures were then recorded at 520 nm. These processes were
repeated three times and on each occasion freshly prepared
reduced metronidazole and tinidazole (TZ) stock solution was
used. The average absorbance reading was obtained from the
determinations, and used to generate the calibration curves.
Linear regression analysis was used to calculate the slope,
The analysis of all the tablet brands of metronidazole and
tinidazole was repeated using the official BP [12] procedures
while the metronidazole infusion was assayed using a UV-Vis
spectrophotometer. The assay results using the new method
(described in this work) and the official non-aqueous titrations
were compared using the student’s t-test and F-ratio test.
2
intercept and coefficient of determination (r ) of each
Interference Studies
calibration line. The assay precision and accuracy were
determined as documented by the USP [36]. The limit of
detection (LOD) was computed from the calibration graphs
using the equation 3.3 ꢃ/s where ꢃ is the standard deviation of
three blank determinations and s is the slope of the calibration
curve [37]. The limit of quantitation (LOQ) was calculated as
To study the potential interference from the commonly
utilized tablet excipients and other additives such as starch,
lactose, magnesium stearate, talc, gelatin and their mixtures,
recovery studies of reduced metronidazole and tinidazole from
the matrices containing these excipients were carried out.
Under the optimized conditions developed for the formation of
the charge-transfer complex between these drugs and CAA, a
3
ꢀ LOD. Other validation parameters calculated were
Sandell’s sensitivities and molar absorptivities. The formation
constants were obtained from the equation [A ]/[A] = ꢄ/K[D ]
-
1
known amount of 20 μg ml reduced metronidazole and 19.2
0
0
-1
μg ml of reduced tinidazole were added and analyzed.
+
1/ꢄ using the Benesi-Hildebrand plot, where K is the
formation constant, A is the absorbance of the complex and ꢄ
is the molar absorptivity. [A ] and [D ] are the initial
RESULTS AND DISCUSSIONS
0
0
concentrations of acceptor and donor, respectively [38].
Formation constants were obtained from the ratio of intercept
to slope and from the inverse intercept of Benesi-Hildebrand
plots.
The reduced forms of metronidazole and tinidazole formed
immediate purple color with chloranilic acid. This is an
evidence of the formation of a charger-transfer (CT) complex.
Chloranilic acid is a well-known ꢀ-acceptor and the reduced
3
62

Spectrophotometric Determination of Metronidazole and Tinidazole
drugs are n-donors. Chloranilic acid has been used for the
of constituents held together by weak secondary valence
forces of the donor-acceptor type or hydrogen bonds. This
secondary valence force is not a well-defined bond but is
rather an overall attraction between two aromatic molecules.
Important factor in the formation of molecular complexes by
this compound is the presence of steric factors which hinder
this process [39]. The absorption spectra are respectively
presented in Figs. 1 and 2 for MZ and TZ complexes. None of
spectrophotometric determination of drugs containing n-
electron donors such as nitrogen and oxygen. It undergoes two
types of reaction at two different conditions; at normal or
elevated temperature it behaves as acid and undergoes
neutralization reaction forming salt but at low temperature
(
cold condition) it behaves as charge-transfer acceptor thus
forming molecular complex. This molecular complex consists
1
.4
1
0
.05
0
.7
.35
0
1
00
300
500
700
900
Wavelength (nm)
Fig. 1. Absorption spectra of MZ (ꢅ), reduced MZ (ꢆ), CAA (-----) and the new CT complex between CAA and
reduced MZ ( ____).
1
1
0
0
.6
.2
.8
.4
0
1
00
300
500
700
900
Wavelength (nm)
Fig. 2. Absorption spectra of TZ (ꢅ), reduced TZ (------ ), CAA (ꢆ) and the new CT complex between CAA and
reduced TZ ( ____).
3
63

Adegoke et al.
the drugs or their reduced forms or CAA has any significant
The interaction of CAA with both reduced MZ and TZ
gave rise to a bathochromic shift and a new absorption band at
520 nm for each of the complexes. This can be attributed to
the formation of new molecular complexes. The reaction is
illustrated in Scheme 1. The formation of a radical anion in
absorbance at 520 nm selected as the analytical wavelength.
The obtained wavelength is significantly higher than some of
the previously reported procedures, offering the advantage of
simple colorimetric analysis of these nitroimidazoles.
N
N
MZ
TZ
NO2
H C
H C
3
3
N
NO2
N
hydride transfer in three steps to reduce
CH CH SO C H
CH CH OH
2
2
2
2
5
2
2
NO to NH in both MZ and TZ
2
2
3Li+ + 3AlH3+3 H-
2
e
3
LiAlH4
LiAlH4
LiAlH4 2e
N
N
H C
3
N
NO
H3C
N
NO
CH CH SO C H
2 2 5
CH CH OH
2
2
2
2
LiAlH4
2e
2e
LiAlH4
N
N
H C
3
H C
N
NHOH
3
N
NHOH
CH CH OH
2
2
CH CH OH
2
2
2
e
LiAlH4
LiAlH4 2e
N
charge acceptor
N
N
O
H C
3
N
NH2
CH CH SO C H
2 2 5
H C
3
HO
Cl
Cl
NH2
2
2
reduced TZ
CH CH OH
2
2
reduced MZ
Charge-donor
OH
Charge-donor
O
CAA
O
O
-
Cl
+
HO
Cl
N
H C
3
N
NH2
OH
CH2CH2SO2C2H5
Tinidazole-CAA CT complex
(purple, 520 nm)
N
+
O
O
-
HO
Cl
Cl
H C
3
NHOH
CH CH OH
N
2
2
OH
metronidazole-CAA CT complex
purple, 520 nm
Scheme 1. Mechanism for the reduction of MZ and TZ by LiAlH and formation of charge transfer complexes with CAA
4
3
64

Spectrophotometric Determination of Metronidazole and Tinidazole
such molecular interactions has been established by electron-
nature of LiBH , it was adopted as the reducing agent for both
4
spin resonance measurements [40]. The stability of the ion-
pair produced expectedly was affected by several factors
which were optimized in this study. The result of the tests
using different reducing agents is presented in Table 1, where
varying results were obtained. Optimization of reduction time
revealed that the reduction was completed within 10 min using
the metal hydrides which was found to be convenient since
room temperatures were adopted. Intense and immediate
purple color was obtained when Pd-C, LiBH and NaBH were
used as reducing agents in glacial acetic acid. The inability of
Zn in ethanol to effectively reduce the compounds and then
generate the purple-coloured complex with CAA might be due
to the interaction of Zn with CAA preventing it from being
available for charge-transfer reaction. Due to the homogenous
MZ and TZ, as this would not require filtration unlike Pd-C
and Zinc reducing systems. This is the first reported procedure
adopting the metal hydrides as convenient reducing agents. Its
other advantage is the ability to carry out the reduction
procedure at room temperature. The reduction of
metronidazole has been reported to be a complex process
involving 6 electrons for complete reduction of the nitro group
to the amine derivative via hydroxylamine intermediate [41].
The purple colors produced with CAA however became
slightly discharged at elevated temperatures. This is typical of
most CT complexes as an increase in temperature destroys the
intermolecular binding forces of attraction between ion pairs.
The result of the TLC is presented in Table 2. Single well-
defined spots were obtained for the CT complexes as distinct
4
4
Table 1. Result of the Charge-Transfer Complexation of Reduced Metronidazole and Tinidazole with Chloranilic Acid
Analyte
MZ
TZ
Reducing agentsa
Observations
Comments
Color at elevated
temperature (80 °C)
No color
Zn/HCl in ethanol at 90 ± 5 °C for No significant
5 min color formed
Zn/HCl in ethanol at 90 ± 5 °C for No significant
5 min color formed
Pd-C/GAA in ethanol at room Purple color was
temperature for 10 min formed
Pd-C/GAA in ethanol at room Purple color was
temperature for 10 min formed
LiBH /GAA in ethanol at room Purple color was
1
No color
1
MZ
TZ
More intense
with time (5 min) discharged
More intense Color
with time (5 min) discharged
Instantaneous and Color
intense
Instantaneous and Color
intense
Instantaneous and Color
intense
Color
slightly
slightly
slightly
slightly
slightly
slightly
MZ
TZ
4
temperature for 10 min
formed
discharged
LiBH /GAA in ethanol at room Purple color
4
temperature for 10 min
formed
discharged
MZ
TZ
NaBH /GAA in ethanol at room Purple color
4
temperature for 10 min
formed
discharged
NaBH /GAA in ethanol at room Purple color
Instantaneous and Color
4
temperature for 10 min
GAA = Glacial acetic acid.
formed
intense
discharged
a
Table 2. R Values for Thin Layer Chromatographic Analysis
f
Mobile phase systems
Reduced
metronidazole
0.89
MZ-CAA
complex
0.84
CAA
Reduced
tinidazole
0.90
TZ-CAA
complex
0.87
0.04
Ethyl acetate:methanol (9:1)
Chloroform:acetone (8:2)
0.06
0.71
0.39
0.36
0.63
3
65

Adegoke et al.
0
.282
0.28
.278
.276
.274
.272
0
0
.35
0
0
0
0
0
.3
0.27
0
.268
.25
0
10
Time (min)
20
30
3
0
60
Temperature ( C)
90
o
Fig. 5. Optimization of time required for the formation of
MZ (ꢅ) and TZ (ꢆ) intermolecular complexes with
CAA.
Fig. 3. Optimization of reaction temperature for MZ-CAA
CT complex: (ꢅ) 5 min, (ꢆ) 20 min.
0
.3
0
.3
.25
.2
.15
0
0
0.2
0
0
.1
0
3
0
60
Temperature ( C)
90
o
0
0.5
1
Mole ratio of CAA
Fig. 4. Optimization of reaction temperature for TZ-CAA CT
Fig. 6. Stoichiometric ratio determination for the new method
complex: (ꢅ) 5 min, (ꢆ) 20 min.
for MZ (ꢅ) and TZ (ꢆ).
from CAA and reduced MZ and TZ. The formation of new
compounds is therefore corroborated by the TLC study.
The effect of solvents on the absorptivity of the CT
complexes revealed that optimal detector response was
obtained using a 60:40 mixture of 1,4-dioxane:acetonitrile as
solvent to stop the reaction after each time interval. CAA
solution in acetonitrile gives an absorption spectrum with an
absorption maximum at 430 nm. Other common solvents such
as water, methanol and ethanol have significant interaction
with CAA (purple color) since they all possess n-electrons on
their oxygen atoms.
Optimizations of temperature and time required for the
complex formation were carried out at four temperature levels.
The results are respectively presented in Figs. 3 and 4 and the
optimization of time is presented in Fig. 5 for MZ and TZ. The
optimum reaction temperature and time for the complexation
reaction between CAA and both compounds were found to be
at 30 °C and 5 min, respectively.
Adopting Job’s method of continuous variation, equimolar
concentrations of the charge-transfer acceptor (CAA) and the
reduced n-electron donors (MZ, TZ) gave optimal absorbance
values at mole ratio 1:1. This implies that the reduced drug
substances combine with the reagent on one mole basis. This
further corroborates the single spot observed on TLC. The
stiochiometric ratio determination is presented in Fig. 6 for the
two CT complexes.
For metronidazole analysis, Beer’s law was observed in
-
1
the concentration range of 0-40 μg ml with linear regression
2
equation of y = 0.0041x + 0.0569 (r = 0.9973), where y is the
-
1
absorbance and x is the concentration in μg ml . The limit of
-
1
detection is 1.439 μg ml . This limit of detection compares
favourably with the previously reported methods for the
3
66

Spectrophotometric Determination of Metronidazole and Tinidazole
analysis of metronidazole in dosage forms. Sandell’s
sensitivity was found to be 23.49 ng cm per 0.001A. The95%
confidence limit for the slope and intercept is respectively
0
0
.008
.006
-
2
0
.0041 ± 0.0036 and 0.0569 ± 0.0049. The molar absorptivity
0.004
2
-1
-1
is 2.741 ꢀ 10 L M cm . The linear calibration for tinidazole
0
.002
0
-
1
(
range 0-79.2 μg ml ) analysis was established as y =
2
-1
0
.00211x + 0.05242 (r = 0.9970) with LOD of 0.7407 μg ml
-
2
and Sandell’s sensitivity of 47.39 ng cm per 0.001A.
The molar absorptivity for the TZ-CAA complex was
obtained as 2.681 ꢀ 10 L M cm . The 95% confidence limit
for the slope and intercept is respectively 0.00211 ± 0.00128
and 0.05242 ± 0.000698. Using the Benesi-Hildebrand plot,
profoundly large formation constants were obtained for the CT
0
20
40
60
80
-1
[
Do] ꢀ 1000
2
-1
-1
Fig. 7. Benesi-Hildebrand plot for the estimation of formation
constants: TZ (---), MZ (___).
1
0
9
complexes of reduced MZ and TZ; 2.62 ꢀ 10 and 7.14 ꢀ 10 ,
respectively. These high formation constants explain the
instantaneous nature of the formation of the complexes and
thus lend credence to the ability of the formed n-electrons in
reduced MZ and TZ to donate electrons avidly to the charge-
deficient CAA. The Benesi-Hildebrand plot is presented in
Fig. 7. The analytical and validation parameters are
summarised in Table 3.
concentration levels. The result is presented in Table 4. The
overall recoveries for MZ and TZ are respectively 103.19 ±
2.05 (%RSD = 1.99, n = 12) and 101.63 ± 1.41 (%RSD =
1.39).
The new methods were eventually applied to the assay of
metronidazole and tinidazole in their dosage forms. The
results obtained are presented in Table 5. There was no
significant difference in the content of the two drugs when the
new methods were compared with official non-
aqueous titrations for MZ and TZ tablets and UV-Vis
The accuracy and precision for the assay of metronidazole
and tinidazole via charge transfer complexation with CAA
were studied on three successive days using three
Table 3. Analytical and Validation Parameters for the CT Complexes of MZ and TZ with CAA
Parameter
Beer’s law limits (μg ml )
Limit of detection (μg ml )
Metronidazole
Tinidazole
-
1
5-40
1.439
4.8-79.2
0.7407
-
1
-
1
Limit of quantitation (μg ml )
4.317
2.221
-
1
-1
2.74112 ꢀ 102
23.49
2.6811 ꢀ 102
47.39
Molar absorptivity (L M cm )
-
2
Sandell’s sensitivity (ng cm per
0
.001 absorbance unit)
a
Regression equation
Intercept, a
0.0569
0.0041
0.9986
0.00498
0.0036
0.05242
0.00211
0.9985
0.0006984
0.0012812
7.1395 ꢀ 109
Slope, b
Correlation coefficient, r
Confidence interval of intercept, ꢀ
Confidence interval of slope, ꢁ
b
10
Formation constant , K
2.6173 ꢀ 10
a
-1
b
Y = bX + a, where Y is the absorbance for concentration X μg ml . estimated from Benesi-
Hildebrand plot.
3
67

Adegoke et al.
Table 4. Accuracy and Repeatability for the New Spectrophotometric Method
Concentration Day 1*
Day 2*
Day 3*
Inter-day statistics
-
1
(
μg ml )
%Mean
%RSD
%Mean
%RSD
%Mean
%RSD
%Mean
%RSD
recovery
recovery
recovery
recovery
Metronidazolea
5
1
3
104.1
100.6
105.2
2.3
2.4
1.9
104.1
100.6
105.4
2.3
2.4
1.3
103.1
100.2
105.0
0.4
1.6
1.5
103.8
100.4
105.2
1.7
2.1
1.6
5
0
Tinidazoleb
1.9
4
1
4
.8
9.2
0.8
101.2
99.5
103.2
2.2
0.9
1.1
102.2
99.8
102.6
102.5
99.8
102.9
0.8
0.5
1.6
102.0
99.7
102.9
1.6
0.8
1.3
1.0
1.1
a
b
n = 12, overall recovery for MZ is 103.2 (%RSD = 2.0); overall recovery for TZ is and 101.6 (%RSD = 1.4).
Table 5. Analysis of Metronidazole and Tinidazole Dosage Forms*
Drug formulation
New method
%±S.D.)
95% C.I.b
Official method
p-Valuea
(
F-test
t-test
Metronidazole
20.44 ± 0.21
20.49 ± 0.21
20.56 ± 0.29
20.53 ± 0.21
Metrodol tablet
Loxagyl tablet
Metrotab tablet
Tromet-500
102.07 ± 1.14
102.44 ± 1.20
102.81 ± 1.79
102.07 ± 1.14
103.23 ± 1.10
103.80 ± 1.25
104.44 ± 0.36
104.04 ± 1.22
0.916
0.970
0.036
0.934
0.231
0.135
0.077
0.063
infusion
Tinidazole
Amtiba-500 tablet
101.41 ± 1.24 19.50 ± 0.19
102.73 ± 0.79
0.498
0.107
a
*
Content of metronidazole tablet and that of infusion stipulated by BP = 95-105%. statistical analyses
done between the results obtained from the proposed method and the official non-aqueous titration.
b
-1
-1
confidence interval calculated for 20 μg ml and 19.2 μg ml for MZ and TZ, respectively.
spectrophotometric method for MZ infusion (p > 0.05). This
therefore establishes the equivalence of the two methods. The
chemical equivalence of the brands of MZ tablets was also
established as there was found to be no significant difference
in the contents of all brands using ANOVA (p > 0.05). In the
presence of the commonly adopted tablet excipients, the
recoveries of MZ and TZ were respectively: lactose (101.95 ±
and 101.32 ± 1.14) and for the combination of all the
excipients (103.05 ± 0.69 and 102.80 ± 0.18). This implies that
there was no interference from the commonly used excipients,
thereby confirming some measure of selectivity in the new
method in question.
This new spectrophotometric method enjoys some well-
defined advantages compared with the previously reported
methods: the reactions can be carried out at room temperature,
colouration of formed charge-transfer complexes is instant and
stable, reaction time is short and the reducing agent chosen
0
0
0
.86 and 101.32 ± 1.14), gelatin (103.05 ± 0.69 and 102.80 ±
.18), starch (101.95 ± 0.86 and 101.32 ± 1.14), talc (101.95 ±
.86 and 101.32 ± 1.14), magnesium stearate (101.95 ± 0.86
3
68

Spectrophotometric Determination of Metronidazole and Tinidazole
reduces both analytes considered for this study.
[13] A.N. Papas, M.F. Delaney, Anal. Lett. 15 (1982) 739.
[
14] A.Z.A. Zuhri, S.I. Al-Khalil, M.S. Suleiman, Anal.
Lett. 19 (1986) 453.
CONCLUSIONS
[
15] G.J. Patriarche, J.C. Vire, Anal. Chim. Acta
(1987) 193.
196
The spectrophotometric determination of metronidazole
and tinidazole through charge transfer complexation with
chloranilic acid as described in this report is simple, fast,
accurate and precise. It is as accurate as official non-aqueous
titrimetric procedures with the advantage of using a small
sample size. Similar advantage of sensitivity over the
previously reported analytical methods for nitroimidazoles is
also observed. The possibility of adopting metal hydrides for
the prior reduction of nitroimidazoles is also shown. It could
adopt as an alternative method in the assay of MZ and TZ in
tablets and infusion as well as in in-process quality control of
MZ and TZ tablets and infusion.
[16] C. Yarnitzky, W.F. Smith, Int., J. Pharmac. 75 (1991)
161.
[17] G.O. El-Sayed, Microchem. J. 55 (1997) 110.
[18] P.D. Panzade, K.R. Mahadlik, East. Pharm. 43 (2000)
115.
[19] K.B. Mustapha, M.T. Odunola, M. Garba, O. Obodozie,
African J. Biotechnology 5 (2006) 1188.
[20] K. Lanbeck, B. Lindström, J. Chromatogr. B 162
(1979) 117.
[21] L.P. Hackett, L.J. Dusci, J. Chromatogr. A 175 (1979)
347.
[
22] S.S Simoes, P.M. Everado, N.G. Everado, S.L
Wellington, N.T. M Pablo, C.U.A. Mario, C.S Edvan,
B.N. Valberes, J. Braz. Chem. Soc. 17 (2006) 609.
REFERENCES
[1]
[2]
[3]
S.C. Cherkofsky, (1980). US Pat. 4 199 592.
[23] B.H. Ahmed, J.O. Onah, Glob. J. Pure Appl. Sci. 9
(2003) 359.
[24] A. Rehman, A.S. Ijaz, A. Raz, J. Iran. Chem. Soc. 2
(2005) 197.
T. Kusumi, K. Nakanishi, (1981). US Pat. 4 288 445.
A.R. Katritzky, E.F.V. Scriven, S. Majumder, R.G.
Akhmedova, N.G. Akhmedova, A.V. Vakuienko,
(
2005). 179 ARKAT USA, Inc.
P. Zuman Z. Fijalek, J. Electroanal. Chem. 296 (1990)
38.
R.B. Khambatta, Ann. Trop. Med. Parasitol. 62 (1968)
39.
[25] N. Saffaj, T. Saffaj, M. Charrouf, B. Ihssane, A.
Abourrche, Y. Abboud, A. Benanamara, Indian J. Anal.
Chem. 4 (2007).
[26] T. Saffaj, M. Charrouf, A. Abourriche, Y. Aboud, A.
Bennamara, M. Berrada, Dyes & Pigments 70 (2006)
259.
[27] L. Lopez Martine, F.J. Luna Hernandez, P.L. Lopez
Alba, Anal. Chim. Acta 340 (1997) 241.
[28] C.V. Prasad, V. Sripriya, R.N. Saha, P. Parimoo, J.
Pharm. Biomed. Anal. 5 (1999) 961
[4]
[5]
[6]
5
1
A. Gennaro, Remington’s Pharmaceutical Sciences,
1
1
th
8 ed., Mack Publishing Co., Easton, 1993, pp. 1228-
230.
[
[
[
[
[
[
7]
8]
9]
R.J. Knox, R.C. Knight, D.I. Edwards. Br. J. Cancer 44
1981) 741.
(
D.I. Edwards, R.C. Knight, A. Zahoor, Int. J. Rad.
Oncol. Biol. Phys. 12 (1986) 1207.
P.J. Declerck, C.J. De Ranter, Biochem. Pharmacol. 35
[29] R.G. Bhatkar, S.K. Chodankar, Indian J. Pharm. Sci. 42
(1980) 127.
[30] N.D. Dinesh, P. Nagaraja, K.S. Rangappa, Turk. J.
Chem. 28 (2004) 335.
[31] O.S. Kamalapurkar, J.J. Chudasama, East Pharm. 26
(1983) 207.
[32] P. Nagaraja, K.R. Sunitha, R.A. Vasantha, H.S.
Yathirajan, J. Pharm. Biomed. Anal. 28 (2002) 527.
[33] J.O. Onah, Global J. Pure & Appl. Sci. 10 (2004) 125.
[34] H.T. Karnes, C. March, Pharm. Res. 10 (1993) 1420.
(1986) 59.
10] P.J. Declerck, C.J. De Ranter, J. Chem. Soc., Faraday
Trans. I 83 (1987) 257.
11] J.H. Tocher, D.I. Edwards, Biochem. Pharmacol. 48
(1994) 1089.
12] British Pharmacopoeia, Her Majesty Stationery Office,
London, 1998, pp. 98, 1814 and 1815.
3
69

Adegoke et al.
rd
[
[
35] J. Rose, Advanced Physico-Chemical Experiments,
Pitman, London, 1964, p. 54.
36] United States Pharmacopoeia (USP 24/NF 19), US
Pharmacopoeial Convention, USA, p. 819.
[39] M. Alfred, S. James, C. Arthur, A.H.C. Chun, 3 ed.,
Lea and Febiger Philadelphia, USA, 1993, pp. 319-320.
[40] M. Abdel-Salam, A.S. Issa, M. Mahrous, M.E. Abdel-
Hamid, Anal. Lett. 18 (1985) 1391.
[
[
37] K. Basavaiah, V.S. Charan, Sci. Asia 30 (2004) 163.
38] H.A. Benesi, J.H. Hildebrand, J. Am. Chem. Soc. 71
[41] M.A. La-Scalea, S.H.P. Serrano, I.G.R. Gutz, J. Braz.
Chem. Soc. 10 (1999) 127.
(1949) 2703.
3
70