Kinetic study and mechanism of Niclosamide degradation

Article abstract of DOI:10.1016/j.saa.2014.04.050

A spectrophotometric kinetic study of Niclosamide alkaline degradation as a function of drug concentration, alkaline concentration and temperature has been established utilizing double divisor-ratio spectra spectrophotometric method. The developed method allowed determination of Niclosamide in presence of its alkaline degradation products; namely; 2-chloro-4-nitro aniline (DEG I) and 5-chloro salicylic acid (DEG II) with characterization of its degradation mechanism. It was found that degradation kinetic of Niclosamide followed pseudo-first order under the established experimental conditions with a degradation rate constant (k) of 0.0829 mol/h and half life (t_1/2) of 8.35 h. The overall degradation rate constant as a function of the temperature under the given conditions obeyed Arrhenius equation where the activation energy was calculated to be 3.41 kcal/mol.

Full text of DOI:10.1016/j.saa.2014.04.050

Accepted Manuscript
Kinetic Study and Mechanism of Niclosamide Degradation
Hala A. Zaazaa, Maha M. Abdelrahman, Nouruddin W. Ali, Maimana A.
Magdy, M. Abdelkawy
PII:
S1386-1425(14)00623-4
http://dx.doi.org/10.1016/j.saa.2014.04.050
SAA 12014
DOI:
Reference:
Spectrochimica Acta Part A: Molecular and Biomo-
lecular Spectroscopy
To appear in:
Received Date:
Revised Date:
Accepted Date:
2 February 2014
1 April 2014
7 April 2014
Please cite this article as: H.A. Zaazaa, M.M. Abdelrahman, N.W. Ali, M.A. Magdy, M. Abdelkawy, Kinetic Study
and Mechanism of Niclosamide Degradation, Spectrochimica Acta Part A: Molecular and Biomolecular
Spectroscopy (2014), doi: http://dx.doi.org/10.1016/j.saa.2014.04.050
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Kinetic Study and Mechanism of Niclosamide Degradation
Hala A. Zaazaaa , Maha M. Abdelrahmanb , Nouruddin W. Alib,
Maimana A. Magdyb* , M. Abdelkawya
a Pharmaceutical Analytical Chemistry Department, Faculty of Pharmacy, Cairo
University, Kasr-El-Aini 11562, Cairo, Egypt.
b Pharmaceutical Analytical Chemistry Department, Faculty of Pharmacy, Beni-Suef
University, Al-Shaheed Ahmed Hegazy, 26111, Beni-Suef, Egypt.
Abstract
A spectrophotometric kinetic study of Niclosamide alkaline degradation as a function
of drug concentration, alkaline concentration and temperature has been established
utilizing double divisor-ratio spectra spectrophotometric method. The developed
method allowed determination of Niclosamide in presence of its alkaline degradation
products; namely; 2-chloro-4-nitro aniline (DEG I) and 5-chloro salicylic acid (DEG
II) with characterization of its degradation mechanism. It was found that degradation
kinetic of Niclosamide followed pseudo-first order under the established experimental
conditions with a degradation rate constant (k) of 0.0829 mol/h and half life (t1/2) of
8.35 h. The overall degradation rate constant as a function of the temperature under
the given conditions obeyed Arrehenius equation where the activation energy was
calculated to be 3.41 kcal/mol.
Keywords: Niclosamide; Double divisor-ratio spectra spectrophotometry;
Degradation; Kinetic.
*corresponding author E-mail: maimanamagdy@yahoo.com
Telephone: 0201006773868
Fax : 0020822317950
1

1. Introduction
Niclosamide (Fig. 1), chemically recognized as 5-chloro-N-(2-chloro-4-nitrophenyl)-
2-hydroxybenzamide [1]. Niclosamide is orally administered anthelmintic drug which
is highly effective against cestodes and threadworm. It acts by inhibiting the oxidative
phosphorylation in mitochondria and interfering with anaerobic generation of ATP by
the tape-worm, as a result tape-worm get partially digested in the intestine [2-4].
After extensive literature survey, few analytical methods have been found for
determination of Niclosamide including determination of Niclosamide with
Thiabendazole by ∆A spectrophotometric method [5] or in combination with
Drotaverine hydrochloride by ∆A, second derivative (∆D2) and third derivative (∆D3)
differential ultraviolet spectrophotometric methods[6], or alone using P-benzoquinone
[7]. Also spectrofluorimetric [8], HPLC [9-11], GC [12] and vlotammetric [13-14]
methods were developed for its determination.
Neither stability investigation of Niclosamide nor possible degradation mechanism
has been reported. Niclosamide was subjected to alkaline hydrolysis and the obtained
degradation products were isolated and characterized for their structures. They are
reported as major impurities of Niclosamide namely; 2-chloro-4-nitro aniline (DEG I)
and 5-chloro salicylic acid (DEG II) in British pharmacopoia [2] and the European
pharmacopoia [15]. Additional results from genotoxic studies in rodents and humans
suggest that the drug is absorbed from the gastrointestinal tract, and mutagenic
metabolites 5-chlorosalicylic acid and 2-chloro-4-nitroaniline are excreted as the main
metabolites [16].
The aim of the recommended work is to liberate double divisor-ratio spectra
spectrophotometric method for selective determination of Niclosamide in presence of
its degradation products. The proposed method was directed to reveal the degradation
kinetic and mechanism of Niclosamide under the specified conditions.
2. Experimental
2.1. Instruments
•
A double beam UV-visible spectrophotometer (SHIMADZU, Japan) model
UV-1601 PC with quartz cell of 1 cm pathlength, connected to IBM
compatible computer. The software was UVPC personal spectroscopy
2

software version 3.7.The spectral band width was 2 nm and wavelength-
scanning speed 2800 nm/min.
•
UV lamp with short wavelength 254 nm (USA).
2.2. Materials
(a) Pure standard
ꢀ
Niclosamide was kindly supplied from Alexandria Company for
pharmaceuticals and chemical industries, Alexandria, Egypt. Its purity was
found to be 100.00 % according to the reported method [5].
(b) Pharmaceutical formulations
ꢀ
Yomesan® tablets (Batch No. 1182010) labeled to contain 500 mg of
Niclosamide , manufactured by Alexandria Company for pharmaceuticals and
chemical industries, Alexandria, Egypt.
ꢀ
Niclosan® tablets (Batch No. 103021) labeled to contain 500 mg of
Niclosamide , manufactured by Misr Company for pharmacutical Industry.
S.A.E, Egypt.
(c) Chemicals and reagents
All reagents and chemicals used throughout this work were of analytical grade and
were used without further purification.
ꢀ
Methanol and triethylamine of HPLC grade (CHROMASOLV®, Sigma-
Aldrich Chemie Gmbh, Germany).
ꢀ
Sodium hydroxide (0.5 N and 1N aqueous solution), HCl (1N aqueous
solution), benzene, ethylacetate (El-Nasr Pharmaceutical Chemicals Co., Abu-
Zabaal, Cairo, Egypt).
(d) Degradation of Niclosamide
0.5 gm of Niclosamide powder was transferred into 50 mL glass stoppered flask,
dissolved in 10 mL methanol and completed to the mark with 1N NaOH solution. The
flask was left for 6 h under reflux at 80 ˚C and complete degradation was followed via
TLC using benzene–ethylacetate- methanol- triethylamine (9:1:1:0.1, by volume) as a
developing system. After complete degradation, the solution was filtered, the obtained
precipitate was washed with water where the collected washing was identified as first
3

degradation product DEG I (2-Chloro-4-nitroaniline). Then the filtrate was adjusted to
pH=1.5 using 1N HCl solution, where the second degradation product DEG II (5-
chlorosalicylic acid) precipitated, filtered and then washed with double distilled water.
The obtained degradation products were identified by IR and mass spectrometry.
2.3. Standard solutions
(a) Stock standard solutions of Niclosamide and its alkaline degradation
products (1 mg/mL)
0.1 gm of Niclosamide, DEG I and DEG II were accurately weighed into three
separate 100-mL volumetric flasks, 50 mL of methanol was added to each flask,
shaken to dissolve then the volume was completed to the mark with methanol.
(b) Working standard solutions of of Niclosamide and its alkaline degradation
products (100 µg/mL)
10 mL each of Niclosamide, DEG I and DEG II were accurately transferred from
their stock standard solutions into three separate 100-mL volumetric flasks, then the
volume was completed to the mark with methanol.
(c) Kinetic investigation
Into two separate 100 mL volumetric flasks, standard solution of Niclosamide
(3.06x10-3 and 1.53x10-3 mol/ L) were prepared by dissolving in 10 mL methanol and
then the volume was completed to the mark with 1N NaOH and 0.5N NaOH.
2.4. Laboratory prepared mixtures
Mixtures containing different ratios of Niclosamide, DEG I and DEG II were prepared
using their respective working solutions in methanol. Different aliquots equivalent to
100-300 µg of Niclosamide were transferred into a series of 10 mL volumetric flasks,
then different aliquots of DEG I and DEG II were added in different ratios according
to their molecular weights.
4

3. Procedure
3.1. Spectral characteristics of Niclosamide and its degradation products
Into 10-mL volumetric flasks transfer accurately and separately, aliquots equivalent to
180 µg of Niclosamide and 60 µg of each of DEG I and DEG II from their working
standard solutions (100 µg/mL). Complete the volume with methanol and record the
absorption spectrum over the range of 200-500 nm.
3.2. Double divisor-ratio spectra spectrophotometric method
Different aliquots equivalent to 40-300 µg of Niclosamide were accurately transferred
into a series of 10 mL volumetric flasks and the volume was completed to the mark
with methanol. Divide the absorption spectrum by the spectra of mixture of equal
1
concentrations of both of DEG I and DEG II (10 µg/mL of each). The D spectra of
Niclosamide was obtained using ∆λ = 5 and scaling factor =10, then the peak
amplitude was measured at 243 nm. The calibration curve was constructed relating the
peak amplitudes against the corresponding drug concentrations and the regression
equation was calculated.
3.3. Application to the pharmaceutical formulations
Ten tablets of each Yomesan® and Niclosan® were powdered and mixed well. An
accurately weighed portion of the powdered tablets equivalent to 500 mg of
Niclosamide of each formulation was transferred separately into 100-mL volumetric
flasks. 75 mL methanol was added and then sonicated for 30 minutes, filtered then
completed to the volume with methanol. The solution was diluted to obtain 100 µg/
mL working solution using methanol. The double divisor-ratio spectra
spectrophotometric method was followed and the concentration of Niclosamide was
calculated from its corresponding regression equation.
3.4. Kinetic study
3.4.1. Effect of Niclosamide concentration
Two sets of standard solutions of Niclosamide (3.06x10-3 and 1.53x10-3 mol/L) were
separately prepared in 100 mL glass stoppered flasks after dissolving in 10 mL
methanol then the volume was completed to the mark with 1N NaOH solution,
inserted as rapidly as possible into a thermostatic water bath set at 60 °C, 0.3 mL
from the prepared solutions were quantitatively transferred into 10-mL volumetric
5

flasks for 6 h at 0,1,2,-----,6 h time interval. The flasks were completed to volume
with methanol. The remaining concentration of intact Niclosamide was determined by
applying the previously described double divisor-ratio spectra spectrophotometric
method at 243 nm using its corresponding regression equation. The logarithm of the
remaining Niclosamide concentration was plotted versus time in hours from which the
kinetic order of alkaline degradation process was determined, the degradation rate
constant (k) and the half life (t1/2) were calculated.
3.4.2. Effect of sodium hydroxide concentration on the reaction rate
Two sets of Niclosamide standard solutions (3.06x10-3 mol/L) were separately
prepared in 100 mL glass stoppered flasks after dissolving in 10 mL methanol then
the volume was completed to the mark with 0.5N and 1N NaOH solutions. Then the
prepared solutions were inserted into a thermostatic water bath set at 60 °C. The
procedure was followed as mentioned under 3.4.1.
3.4.3. Effect of temperature on the reaction rate
Two sets of Niclosamide standard solutions (3.06x10-3 mol/L) were separately
prepared in 100 mL glass stoppered flasks after dissolving in 10 mL methanol then
the volume was completed to the mark with 1N NaOH solutions. Then the prepared
solutions were inserted into a thermostatic water bath set at 60 °C and 80 °C. The
procedure was followed as mentioned under 3.4.1. Arrehenius plots were used to
determine the activation energy (Ea), half life of Niclosamide (t1/2) and frequency
factor (A).
4. Results and Discussion
Generally, some amide containing pharmaceutical compounds are liable to hydrolysis
in aqueous solution. Upon refluxing Niclosamide with 1 N NaOH for 6 h at 80 ˚C,
hydrolysis of the amide group occurs and two degradation products were obtained.
The first degradation product is 2-chloro-4-nitro aniline (DEG I) filtered and washed
with water, then the filtrate is adjusted to pH =1.5 with 1 N HCl solution where the
other degradation product, 5-chloro salicylic acid (DEG II) precipitated. The two
degradation products were identified by IR and mass spectrometry where IR
spectrum of Niclosamide showed a characteristic bands at 3576.34 cm-1 (peak 1) ,
6

3088.44 cm-1 (peak 2) and 1679.69 cm-1 (peak 3) corresponding to -OH , -NH , and
C=O groups respectively, while absence of peak 1 and peak 3 in the IR spectrum of
the first degradation product (DEG I) and appearance of a forked characteristic bands
at 3483.78 cm-1 (peak 4) and 3371.92 cm-1 (peak 5) corresponding to -NH2 group
Fig.2. Also the IR spectrum of the second degradation product (DEG II) showed a
characteristic bands at 3226.33 cm-1 (peak 6) and 1671.98 cm-1 (peak 7)
corresponding to -OH and C=O groups of the carboxylic acid and a characteristic
bands at 3081.69 cm-1 (peak 8) corresponding to the phenolic -OH Fig. 2.
The electron impact of Niclosamide showed mass ion peak at 327 m/z corresponding
to the molecular weight of the intact drug while the mass ion peak of DEG I was at
173 m/z and its base peak at 80 m/z corresponding to the main fragment C5H6N
and the mass ion peak of DEG II was at 173 m/z and its base peak at 154 m/z
corresponding to the main fragment C7O2HCl as shown in Fig. 3. Therefore we can
conclude that the alkaline hydrolysis of Niclosamide may be proceeds as shown in
Fig. 1. Pure standards of 2-chloro-4-nitro aniline (DEG I) and 5-chloro salicylic acid
(DEG II) were obtained from Sigma Aldrich [17] and it was compared with the
isolated degradation products with TLC and melting point which confirm the results
or IR and mass spectroscopy.
In accordance with the fact that Niclosamide is susceptible to hydrolysis in alkaline
solution; development of a stability indicating assay method was critical and
information about degradation kinetics under different experimental conditions was
elaborated. According to the method based on the principle of kinetic developed by
Garrett and Carper [18], the k value (reaction rate constant) for the degradation of the
drug in alkaline solution at different elevated temperature was obtained from the
linear expression in equation Log C= logC0 - kt /2.303 [19], where the slope of the
line is –k/2.303. The log of the rates of degradation was then plotted against the
reciprocals of the absolute temperature (Arrehenius plot) [20].
4.1. Double divisor- ratio spectra spctrophotometric method
Double divisor spectrophotometry [21-23], has proved usefulness in the determination
of medicinal substances by eliminating specific interference from degradation
products, co-formulated drugs and also the non-specific irrelevant absorption from the
formulation matrix. The zero order absorption spectra of Niclosamide and its
degradation products show serious overlap that direct determination of Niclosamide is
7

not possible Fig.4. The problem of the overlapped spectra of Niclosamide and its
degradation products was solved using the double divisor-ratio spectra
spectrophotometric method.
The absorption spectra of Niclosamide, DEG I and DEG II solutions were divided by
the spectrum of a solution containing DEG I and DEG II (10 µg/mL of each). Then 1D
spectra were obtained using scaling factor=10 and ∆λ=5, where Niclosamide could be
determined at 243 nm without any interefernce from DEG I and DEG II. The first
derivative of the ratio spectra of pure Niclosamide and its ternary mixture with its
degradation products containing the same concentration of Niclosamide as in its pure
spectrum would be coincided in the spectral region corresponding to a maximum
point of the wavelength as shown in Fig. 5.
1
Linear correlations were obtained between peak amplitudes at 243 nm for D spectra
of Niclosamide in the concentration range 4–30 µg/mL from which the regression
equations were calculated and found to be:-
P.A= 0.0369 C + 0.0217
r= 0.9997
Where P.A is the peak amplitude of Niclosamide, C is the concentration in µg/mL
and r is the correlation coefficient.
Results described in Table 1 showed that this method is selective, valid and
applicable for the determination of Niclosamide in presence of its degradation
products in different laboratory prepared mixtures with mean recovery of 100.74
1.528. Method validation was performed according to USP [24] guidelines, Table 2
shows results of accuracy, repeatability and intermediate precision, which shows good
linear relationship for the suggested method as revealed by the correlation coefficient.
Table 3 shows statistical comparison of the results obtained by the proposed method
and those obtained by the reported spectrophotometric method [5]. The calculated t-
and F-values are less than the theoretical ones indicating that there is no significant
difference between them with respect to accuracy and precision. The usefulness of the
proposed method for assay of Niclosamide in its pharmaceutical formulations was
studied by analysis their tablet formulation (Yomesan® and Niclosan® tablets).
Furthermore, the validity of the proposed method was assessed by applying the
standard addition technique, which showed accurate results and there was no
interference from tablet excipients as shown in Table 3.
8

4.2 Kinetic investigation
No kinetic spectrophotometric study has been reported in the literature for the assay of
Niclosamide. The kinetic methods possess some specific advantages like; simplicity
and no need of some experimental steps such as filtration, extraction which are
avoided prior to absorbance measurements. The kinetic methods also are highly
selective methods since they involve the measurement of the absorbance as a function
of the reaction time [25-27]. This work describes a simple and sensitive kinetic
spectrophotometric method for the determination of Niclosamide. The double divisor-
ratio spectra spectrohotometric method was used to determine the order of the alkaline
degradation rate of the reaction by following the decrease in concentration of
Niclosamide within 6 h at 1 h time interval. Alkaline catalyzed hydrolysis is a
bimolecular reaction but one of the reactants is water which is present in large excess
so the change in concentration is negligible. Such reactions where one of the reactants
is present in large excess are considered to follow pseudo-first order kinetics and all
the equation describing first order can be applied for it.
For a given initial concentration of Niclosamide at a constant temperature and
solution normality, by plotting the change of Niclosamide concentration versus time
and a linear relationship between ln (Ct/C0) and time shows that the degradation of
Niclosamide followed a pseudo-first order reaction kinetics. The pseudo first order
rate constant (k) was calculated as follows [20]:
ln Ct/C0= -kt
Where C0 is the initial concentration of Niclosamide, Ct is the time dependent
concentration, t is the time and k is the reaction rate constant
Different variables affecting degradation rate of Niclosamide including, drug
concentration, NaOH normality and temperature have been studied as following:
4.2.1. Effect of Nicloamide concentration
Two different concentrations 3.06x10-3 mol/L and 1.53x10-3 mol/L of Niclosamide
were used for the study. Fig. 6 is a plot of logarithm of the remaining concentration of
Niclosamide versus time. Results in Table 4 shows that the slope of the obtained
curves was nearly the same. The rate of the degradation (k) was almost the same for
9

different concentrations of Niclosamide, and the half life time were calculated from
the equations [20]:
ln Ct/C0= -kt
t1/2= 0.693/k
4.2.2. Effect of NaOH concentration
To study the effect of NaOH concentration on the reaction rate, experiments were
performed using 0.5 N and 1 N NaOH solutions. Fig. 7 shows that the reaction rate
was increased by increasing the NaOH concentration and the results are shown in
Table 5.
4.2.3. Effect of temperature
The kinetic study of Niclosamide was performed as a function of temperature where
the previous procedure was applied using two different thermostatically controlled
water baths set at 60 °C and 80 °C. The influence of temperature on the reaction rate
constant was given by Arrehenius equation as follows [20]:
ln k = ln A ــ Ea/RT
Where k is the reaction rate constant, A is the frequency factor, Ea is the activation
energy, R is the universal gas constant and T is the absolute temperature, Fig. 8 shows
that increasing the temperature leading to increasing of the reaction rate constant,
while the results in Table 6 shows that the activation energy of the reaction is the
same at different temperature.
Conclusion
The present work provides sensitive, accurate and selective double divisor-ratio
spectra spectrophotometric method for the determination of Niclosamide in presence
of its degradation products in bulk powder or in pharmaceutical formulations. The
developed spectrophotometric method was utilized for determination of Niclosamide
degradation kinetic and all variables affecting degradation rate (k) were studied. The
hydrolysis of Niclosamide obeyed a pseudo-first order kinetics. It was observed that
the degradation rate constants of Niclosamide were increased as a function of
10

increasing the alkaline concentration or the temperature while it was almost the same
at different concentrations of Niclosamide.
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14

Niclosamide
Mol. Formula
C13H8Cl2N2O4
(Mol. Wt. 327)
Reflux for 6 h using 1 N NaOH
+
DEG II
DEG I
5-chloro salicylic acid
Mol. Formula C7H5ClO3
(Mol. Wt. 173)
2-chloro-4-nitro aniline
Mol. Formula C6H5ClN2O2
(Mol. Wt. 173)
Fig. 1. Degradation pathway of Niclosamide.
15

(a)
(b)
.
Fig.2. IR spectrum of (a)Niclosamide, (b) DEG I and (C) DEG
(c)
Fig.2. IR spectrum of (a)Niclosamide, (b) DEG I and (C) DEG
16

(a)
(b)
(c)
Fig. 3. Mass spectrum of (a) Niclosamide, (b) DEG I and (c) DEG II
17

.
Fig. 4: Zero order absorption spectrua of 10 µg/mL of each of Niclosamide (_), Deg I(----)
and Deg II (….) using methanol as a solvent.
Fig. 5: The coincident spectra of the first derivative of the ratio spectra of 18 µg mL-1 pure
Niclosamide (ــــــ) and a ternary mixture (18 µg/mL Niclosamide + 6 µg/mL Deg I +
6 µg/mL Deg II) (-----) using 10 µg/mL of each Deg I and Deg II as a double divisor.
18

Fig. 6: kinetic plot of the alkaline degradation of 3.06x10-3 mol/L and 1.53x10-3 mol/L
of Niclosamide using 1 N NaOH.
Fig. 7: Plot for the effect of sodium hydroxide concentration on the rate of
degradation of 3.06x10-3 mol/L of Niclosamide.
19

Fig. 8: Plot for the effect of temperature on the rate of degradation of 3.06x10-3 mol/L
of Niclosamide.
20

Table 1: Determination of Niclosamide in presence of its alkaline degradation
products in laboratory prepared mixtures by the proposed double divisor-ratio spectra
spectrophotometric method.
Double divisor-ratio spectra
Concentration (µg/mL)
spectrophotometric method
Recovery %**
%
Degradation
products *
DEG
I
DEG
Niclosamide
Niclosamide at 243 nm
II
0.15
0.6
1.2
2.5
4.5
6
0.99
4.11
8.45
15
30
40
30
28
26
25
21
18
15
10
0.15
0.6
1.2
2.5
4.5
6
102.63
99.61
100.19
101.16
101.48
99.83
50
65
7.5
10
7.5
10
98.33
102.70
100.74±1.528
Mean ± SD
* The concentration of the two degradation products is according to their molecular
weight.
** Average of 3 determinations.
1

Table 2: Results of assay validation parameters of the proposed method for
determination of Niclosamide.
Double divisor-ratio
spectra
Parameters
spectrophotometric
method
Range (µg/mL)
4-30
Slope
Intercept
Correlation coefficient
Accuracy
0.0369
0.0217
0.9997
100.14±1.283
100.74±1.528
(mean ± SD)
Selectivity
(mean ± SD)
Precision (%RSD)
Repeatability*
Intermediate precision*
LOD** (µg/mL)
1.31
1.67
0.93
2.78
LOQ** (µg/mL)
* The intraday precision (n=3), average of three different concentrations (6, 8 and 10
µg/mL) repeated three times within day. The interday precision (n=3), average of
three different concentrations repeated three times in three successive days.
** Limit of detection and quantitation are determined via calculations LOD = (SD of
the response/slope) × 3.3; LOQ = (SD of the response/slope) × 10.
2

Table 3: Statistical comparison of the results obtained by the double divisor-ratio
spectra spectrophotometric method and the reported spectrophotometric method for
the determination of Niclosamide in its pharmaceutical formulations.
Yomeasn ® tablets
Double divisor-
Niclosan® tablets
Double divisor-ratio
spectra
ratio spectra
spectrophotometric
method
Items
Reported
method [5]*
Reported
spectrophotometric
method
method [5]*
98.00
1.252
97.17
1.456
96.26
1.523
96.17
1.472
Mean
SD
Standard
addition
technique
**(Mean± SD)
%RSD
100.86±
1.077
99.83±
1.230
-
-
1.277
6
1.498
6
1.582
6
1.531
6
N
1.567
2.119
2.319
2.167
Variance
Student's
t-test
1.067
1.352
-
-
0.109
1.078
-
-
(2.228) ***
F-value
(5.050) ***
* Spectrophotometric determination of Niclosamode by measuring the ΔA values at
405.8 nm
** Average of 3 determinations .
*** Figures between parenthesis represent the corresponding tabulated values of t and
F at p= 0.05.
3

Table 4: kinetic data for the effect of Niclosamide concentration on its alkaline
degradation process.
Concentration
(mol)
Regression
K (mol/h)
0.0829
t1/2 (h)
8.35
equation
Y=0.037C-4.0289
r=0.9798
3.06X10-3
Y=0.036C-4.3286
r=0.9899
1.53X10-3
0.0825
8.13
Table 5: kinetic data for the effect of sodium hydroxide concentration on the alkaline
degradation of Niclosamide.
Sodium hydroxide
concentration
Concentration
(mol)
K (mol/h)
0.0829
t1/2 (h)
8.35
1N NaOH
3.06X10-3
0.5 N NaOH
0.0622
11.14
3.06X10-3
Table 6: Activation energy, Frequency factor and half life for Niclosamide in solution
of 1N NaOH.
Ea
kcal/mol
Temperature
60°C
K (mol/h)
0.0829
t1/2 (h)
8.35
A
3.41
0.084
0.113
80 C
0.111
6.24
3.41
4

25

1. No kinetic spectrophotometric study has been reported for the assay of Niclosamide
2. spectrophotometric kinetic study of Niclosamide alkaline degradation as a function
of drug concentration, alkaline concentration and temperature has been established
utilizing double divisor-ratio spectra spectrophotometric method.
3. Niclosamide followed pseudo-first order under with a degradation rate constant (k)
of 0.0829 mol/h and half life (t1/2) of 8.35 h
4. The overall degradation rate constant as a function of the temperature under the
given conditions obeyed Arrehenius equation where the activation energy was
calculated to be 3.41 kcal/mol.
26