Development and Validation of a Simple Stability- Indicating TLC Method for the Determination of Levamisole in Pharmaceutical Tablet Formulation

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Summary
Asimple, selective, precise, and stability-indicating thin-layer chro-
matographic (TLC) method has been established and validated for
analysis of levamisole (LEV) in pharmaceutical dosage. Normal
phase precoated aluminum TLC silica gel 60F254plates were used as
stationary phase and MeOH–toluene–chloroform (14:36:50 v/v/v)
was used as mobile phase to determine LEV in pharmaceutical
ingredients. The system was found to give compact spot for LEV
(RF= 0.30). Determination was performed in absorbance mode at
223 nm. Regression analysis data for the calibration plots indicated
good linear relationships between response and concentration over
the range 60–2000 ng per spot and 0.998 correlation coefficient.
The slope and intercept of the calibration plot were 4.946 and
267.6, respectively. The recovery of 88.44–102.57% with 2.02%
inter-day and 1.91% intra-day relative standard deviations (RSDs)
shows the validity of the method. Further, the limit of detection
(LOD) and the limit of quantification (LOQ) were 2.2 and 7.5 ng
per spot, respectively. For the stability study of LEV, it was sub-
jected to acid, base, hydrogen peroxide, heat, and photodegrada-
tion (UV) conditions. Statistical analysis proved the method was
repeatable, selective, and accurate forestimation of levamisole. The
proposed method can be successfully used to determine the drug
content of the pharmaceutical formulation.
1 Introduction
Levamisole (LEV) (2, 3, 5, 6-tetrahydro-6-phenyl imidazole
[2,1-b] thiazole) (Figure 1) belongs to the synthetic imidazo-
thiazol derivatives. This compound has been used as an
anthelmintic (anti-worm) agent with immunomodulating prop-
erties, commonly used for large livestock such as cattle, pigs,
and sheep [1, 2].
In 1971, it was found that LEV has immune-stimulatory proper-
ties, following which its use in humans began to expand. Cur-
rently, LEV is used in humans for diseases related to imbalances
in the regulation of immune responses or deficiencies of
immune system including auto-immuno disease, chronic and
recurrent disease, chronic cancer, and infections [3, 4]. Only the
levo-rotatory isomer of this compound has anthelmintic proper-
ties, and since the dextro-isomer has shown more adverse
effects, it has been removed from use in commercial products [5,
6]. LEV, as an inhibitor of lipid peroxidation, is also a radio-pro-
tectant drug [7].
A variety of methods have been reported for analysis of LEV.
These include HPLC determination in biological samples and
tablets [5, 8–11]. Liquid chromatography–mass spectrometry
(LC–MS) and LC–MS/MS are other methods that were used for
its determination [12–14]. Some other methods such as gas
chromatography (GC), gas chromatography–mass spectrometry
(GC–MS), thin-layer chromatography (TLC), capillary elec-
trophoresis, atomic absorption, potentiometry, and spectropho-
tometry were also reported for LEV analysis [7, 15–20].
It is interesting to mention that no stability-indicating TLC
method for analysis of LEV in pharmaceutical dosage forms has
been reported in literature. Therefore, the aim of this work is to
develop a simple, reliable, cost effective, and reproducible TLC
method for the analysis of LEV and study its stability under
stress conditions in its pharmaceutical formulation.
B. Asghari, S.N. Ebrahimi, and F. Mirzajani, Department of Phytochemistry,
Medicinal Plants and Drugs Research Institute, Shahid Beheshti University G. C.,
Tehran, Iran; and H.Y. Aboul-Enein, Pharmaceutical and Medicinal Chemistry
Department, The Pharmaceutical and Drug Industries Research Division, Nation-
al Research Center, Dokki, Cairo 12311, Egypt.
E-mail: haboulenein@yahoo.com
Development and Validation of a Simple Stability-
Indicating TLC Method for the Determination of Levamisole
in Pharmaceutical Tablet Formulation
Behvar Asghari, Samad Nejad Ebrahimi, Fateme Mirzajani, and Hassan Y. Aboul-Enein*
Key Words
Levamisole
Thin-layer chromatography
Pharmaceutical analysis
Validation
Journal of Planar Chromatography 24 (2011) 5, 419–422
0933-4173/$ 20.00 © Akadémiai Kiadó, Budapest
DOI: 10.1556/JPC.24.2011.5.10
419
Figure 1
The chemical structure of levamisole hydrochloride.

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2 Experimental
2.1 Chemicals and Reagents
Analytical grade solvents, chloroform, toluene, and MeOH,
were purchased from Merck Co. (Darmstadt, Germany). The
standard of LEV was a gift obtained from Misr Pharmaceutical
Industries, Egypt. The pharmaceutical tablet of LEV was pur-
chased from local drug store.
2.2 TLC Instrumentation
Sample solution for TLC analyses was spotted as 8-mm bands
with a CAMAG 100-μL syringe on precoated silica gel alu-
minum plate 60GF254(10 × 10 cm) with 200-mm thickness from
Merck Co. (Darmstadt, Germany) using a CAMAG Linomat V
(Muttenz, Switzerland) sample applicator. A constant applica-
tion rate of 200 nLs–1was employed and the space between two
bands was 10 mm. Zones were quantified by linear scanning at
223 nm with a CAMAG TLC scanner 3 equipped with a deu-
terium source in the reflection mode. Slit dimension settings
were 8-mm length and 0.45-mm width, with a scanning rate of
20 mm s–1. Evolution of the peaks was performed using the
winCATS software version 1.2.2.
2.3 Sample and Standard Solution Preparation
For the extraction of LEV, 0.2 g of powdered tablet was sonicated
in 20 mLof MeOH for 10 min at room temperature. The solution
was centrifuged for 10 min at 6000 rpm and filtered through 0.45-
mm Millipore filter. TLC sample solution was dried under the
reduced pressure using rotary-evaporator at room temperature and
finally dissolved in 2-mLMeOH and kept in fridge until use.
2.4 Optimization of the Chromatographic Condition
In order to evaluate development method for the analysis of
LEV, various mobile phases such as CHCl3–toluene–acetone
(70:50:20 v/v/v), CHCl3–toluene (50:50 v/v), and CHCl3–
toluene–MeOH (40:40:20 v/v/v) were investigated in our labora-
tory for reproducible retention factor (RF). Among the different
solvent mixtures, the one consisting of MeOH–toluene–CHCl3
(14:36:50 v/v/v) gives a sharp and well-defined peak (Figure 2).
Linear ascending development was carried out in a twin-trough
glass chamber that was saturated with mobile phase. The opti-
mized saturation time was 15 min at room temperature
(25 ± 2°C). The development process was performed for 80 min
and finally the plates were dried with a stream of cool air.
2.5 Method Validation
The method was validated via several parameters including lin-
ear dynamic range (LDR), limit of detection (LOD), limit of
quantification (LOQ), precision, accuracy, recovery, and sample
stability.
2.6 Linear Dynamic Range (LDR)
Stock solutions of LEVwere prepared in MeOH. Different solu-
tion volumes were spotted on TLC plate to obtain the concentra-
tions of 50, 80, 100, 150, 200, 350, 500, 750, 1000, 1150, and
TLC Method to Determine Levamisole in Tablets
420
Journal of Planar Chromatography 24 (2011) 5
Figure 2
Chromatogram of the extract of Levamisole; RF= 0.3; mobile phase:
MeOH–toluene–CHCl3(14:36:50 v/v/v); (A) standard of levamisole,
(B) extract from tablet, and (C) UV-degradation products.

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2000 ng per spot. The peak area values were plotted versus the
corresponding concentrations. In order to decrease the environ-
ment variations, the paired comparison method was performed,
in which the sample and standard solutions were spotted and
analyzed on the same plate.
2.7 Accuracy and Recovery Study
Previously analyzed samples were spiked with an extra 100%,
150%, and 200% of LEV standard and the mixtures were reana-
lyzed. The experiments were repeated five times.
2.8 Precision
System inter-day and intra-day repeatability were determined by
applying the LEV sample solution nine times to give 700 ng per
spot.
2.9 Stability in Solution and during Chromatographic
Process
In order to study the stability of LEV in solution, two different
sample preparations were performed. The samples were spotted
just after their preparation and 3 h later. The analysis of the plate
was accomplished after its development at optimum condition.
The stability of the extracted sample during the chromatograph-
ic analysis was studied using 2D chromatography method.
2.10 Force Degradation Study
Force degradation of LEV tablets was carried out under ther-
molytic, photolytic, acid and base hydrolytic, and oxidative
stress conditions. Sample solutions were prepared by extracting
appropriate amount of LEV tablets, as mentioned previously. In
all studies, 1 mL of prepared stock solution was used for the
force degradation studies. Thereafter, the solutions were dried
under reduced pressure and dissolved in 300 mL MeOH. Final-
ly, they were applied three times on TLC plate. After chro-
matography, the average peak area of LEV was calculated.
2.11 Thermally Induced Degradation
In order to evaluate the effect of temperature on LEV degrada-
tion, the solution and dry powder of the samples were studied.
One milliliter of sample solution, which was kept at 90°C for
6 h, and also 0.2 g of the powder sample, which was held at
100°C for 48 h, were prepared and reanalyzed by the proposed
TLC method.
2.12 Photochemically Induced Degradation
The photochemical stability of the drug was studied by exposing
the stock solution extracted from powdered tablet to direct UV
light (at 254 nm) for 24 h at room temperature.
2.13 Acid- and Base-Induced Degradation
To study the amount of acid- and alkali-induced degradation,
1 mLof LEV solution was mixed with 1 mLHCl or NaOH solu-
tion in the concentration of 0.1 M and kept for 48 and 2 h, respec-
tively. The process was performed at 25 ± 2°C and dark condi-
tion in order to exclude the possible degradative effect of tem-
perature and light, respectively.
2.14 Oxidation-Induced Degradation
One milliliter of LEV solution was mixed with 1 mL of hydro-
gen peroxide in the concentration of 2.5% (v/v) and kept at 25 ±
2°C and dark condition for 48 h to evaluate the oxidation-
induced degradation.
3 Results and Discussion
3.1 Linear Dynamic Range (LDR)
The obtained standard curve of the peak area values plotted
against corresponding concentrations was linear in the range of
50–2000 ng per spot (n = 3). The developed TLC method for
determination of LEV showed a high correlation coefficient
of determination (R2= 0.998) by the linear equation of
Y = 4.946X + 267.6 (Table 1).
3.2 Limits of Detection and Quantification (LOD and LOQ)
LOD and LOQ were calculated by the method based on the stan-
dard deviation of response (s) and the slope of calibration plot (S),
using the formulae LOD = 3.3s/S and LOQ = 10s/S. The LOD and
LOQ were 2.29 and 7.57 ng, respectively, as shown in Table 1.
3.3 Accuracy, Recovery, and Precision Study
The recovery and accuracy of the known amounts of added ana-
lyte were calculated for each sample. The results shown in
Table 1 indicate the good accuracy of the method. The inter-day
and intra-day variability or precision data are summarized in
Table 1. The low values of RSD indicate the repeatability of the
method. Method of recovery of LEV was obtained according to
the comparison between the peak areas of the spiked sample, at
the concentration of 1356, 1698, and 2040 ng mL–1for LEV, and
those of the unspiked solutions. The chosen concentrations were
equal to 100%, 150%, and 200% of the calculated amount of
LEV in each sample (Table 2).
3.4 Stability in Solution and during Chromatographic
Process
The sample was stable during preparation process, in solution,
and on TLC plate. To evaluate the stability of LEV on the chro-
TLC Method to Determine Levamisole in Tablets
Journal of Planar Chromatography 24 (2011) 5
421
Table 1
Statistical evolution of linear part of calibration dependence of levamisole.
CompoundRange [ng per spot] EquationR2
LOD [ng per spot] LOQ [ng per spot]RSD%
Intra-dayInter-day
Levamisole50–2000Y = 4.946X + 267.6 0.9982.29 7.571.912.02

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matographic condition, 2D chromatography was performed.
The sample will be stable if the zone is located on the diagonal
connecting the application position with the intersection of two
solvent fronts [21]. The result demonstrates that the extracted
real sample was stable during the chromatographic treatment.
3.5 Force Degradation Studies
The percentage of LEV decomposition on thermolytic condition
for solution and solid sample was 9.16% and 7.66%, respective-
ly. As it can be seen from Table 3, LEV in solution and solid
form that decomposed under photolytically induced degradation
condition was 35.58% and 7.74%, respectively. The results
demonstrate that under thermal and photolytic conditions the
solid form of LEV was more stable. The alkali degradation that
can be ascribed to the thiol ring opening was 18.19% [22]. Fur-
ther, the amount of acidic hydrolysis was much higher than that
in alkaline condition. The 45.35% degradation in acidic condi-
tion demonstrates its low stability. Finally, Table 3 shows
77.24% stability for LEV under hydrogen peroxide oxidation
condition.
4 Conclusion
The present study describes a fully validated TLC analytical
method for the analysis of LEV in its tablet formulation. Fur-
thermore, the stability study of LEV was investigated when the
drug was subjected to acid, base, peroxide, dry heat, wet heat,
and photodegradation (UV). The proposed method reported is
simple, rapid, precise, accurate, and cost effective. This pro-
posed TLC method can be used for the determination of LEV in
bulk and its pharmaceutical formulation in quality control labo-
ratories.
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Ms received: August 10, 2010
Accepted: March 14, 2011
TLC Method to Determine Levamisole in Tablets
422
Journal of Planar Chromatography 24 (2011) 5
Table 2
The recovery studies of levamisole.
SampleExtra amount Theoretical Recovery [%]
added concentration
[ng per spot][ng per spot]
1684135690.38 ± 1.69
21026169899.80 ± 1.61
3 13682040100.94 ± 1.92
Table 3
Results of forced degradation study samples using proposed
method, indicating percentage degradation of levamisole.
Stability condition/duration/state (%) Degradation of levamisole
Thermal/1001C/48 h/solid7.66 ± 2.63
Photo/UV/24 h/solid 7.74 ± 0.87
Acidic/0.1 M HCl/48 h/solution45.35 ± 0.45
Alkaline/0.1 M NaOH/2 h/solution18.19 ± 1.86
Oxidative/2.5% H2O2/48 h/solution22.76 ± 2.24
Photo/UV/24 h/solution35.58 ± 3.43
Thermal/neutral/901C/6 h/solution9.16 ± 1.95