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Accepted 17 September 2010
Revised 17 April 2010
Received 10 February 2009
Indian J. Pharm. Sci., 2010, 72 (5): 625-629
Development and Validation of HPTLC Method for the
Estimation of Almotriptan Malate in Tablet Dosage Form
A. SUNEETHA AND B. SYAMASUNDAR1*
Department of Pharmaceutical Analysis, Hindu College of Pharmacy, Amaravathi Road, Guntur-522 002, 1Department
of Chemistry/Pharmacy, Acharya Nagarjuna University, Nagarjuna Nagar, Guntur-522 510, India
Suneetha and Syamasundar: HPTLC Method of Almotriptan Malate
A new, simple, precise and accurate high performance thin layer chromatographic method has been proposed for the
determination of almotriptan malate in a tablet dosage form. The drug was separated on aluminum plates precoated
with silica gel 60 GF254 with butanol:acetic acid:water (3:1:1) was used as mobilephase. Quantitative analysis was
performed by densitometric scanning at 300 nm. The method was validated for linearity, accuracy, precision and
robustness. The calibration plot was linear over the range of 100-700 ng/band for almotriptan malate. The method
was successfully applied to the analysis of drug in a pharmaceutical dosage form.
Key words: Almotriptan malate, high-performance thin-layer chromatography, tablets
Almotriptan
malate [1] ,
a
selective
5-hydroxytryptamine
1B/1D
(5-HT1B/1D)
receptor agonist, is chemically designated as
*Address for correspondence
E-mail: [email protected]
September - October 2010
1-[[[3-[2-(dimethylamino) ethyl]-1H-indol-5-l]methyl]
sulfonyl] pyrrolidine-(±)-hydroxy butanedioate (1:1),
whose empirical formula is C 17H 25N 3O 2S–C 4H 6O 5,
with a molecular weight of 469.56. Almotriptan
malate is a water soluble white to slightly yellow
crystalline powder, which is used to treat severe
Indian Journal of Pharmaceutical Sciences
629
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migraine headaches and is available as Axert
tablets. Forner et al. [2] and Bosch et al. [3] have
synthesised almotriptan malate and have published
the data in US5565447. A few procedures based
on liquid chromatography have been reported for
the quantitative determination of almotriptan in
human plasma and urine. A HPLC method for
the determination of process-related impurities in
almotriptan malate was reported earlier[4]. Jansat et
al.[5] have published a method based on HPLC for
determining the levels of almotriptan in plasma.
Fleishaker et al.[6] have reported a validated HPLC
method for the determination of almotriptan
concentrations in urine. Literature survey revealed
that no analytical method for determination of
almotriptan malate in pharmaceutical dosage forms
has been reported thus far. This paper describes
a simple, accurate, precise, and sensitive HPTLC
method for determination of almotriptan malate
in tablet dosage form. The proposed method
was optimized and validated in accordance with
guidelines suggested by International Conference on
Harmonization (ICH)[7].
Methanol, butanol, toluene, ethyl acetate, acetic acid,
hexane, acetone and dichloromethane (all are of
Analytical Reagent grade) were obtained from Sisco
Research Laboratories, Mumbai, India. Standard bulk
drug sample of almotriptan malate (99.8% pure) was
obtained as a gift sample from SMS Pharmaceuticals,
Hyderabad, India. The pharmaceutical dosage form
used in this study was Axert tablets with a declared
content of 12.5 mg almotripan malate (Ortho McNell,
USA)
A standard stock solution of almotriptan malate
was prepared by dissolving 10 mg drug in 100
ml methanol to furnish a concentration of 100 μg/
ml. Chromatography was performed on 10×10 cm
aluminum plates precoated with 250-μm layers of
silica gel 60 GF254 (E. Merck, Darmstadt, Germany).
Before use the plates were prewashed with methanol
and activated at 110 for 5 min. Samples were applied
to the plates as bands 6 mm wide and 10 mm apart
by means of a Camag (Switzerland) Linomat V
sample applicator equipped with a 100 μl syringe
(Hamilton, Bonaduz, Switzerland). Linear ascending
development was performed in a 10×10 cm twin
trough glass chamber (Camag), with butanol-acetic
acid-water 3:1:1 (v/v) as mobile phase and the
chamber was presaturated with mobile phase vapour
630
for 10 min. The development distance was 8.5 cm
and the development time was approximately 60 min.
After chromatography the plates were dried
in a current of air with the use of a air drier.
Densitometric scanning was performed with a Camag
TLC Scanner 3 at 300 nm for all measurements. The
scanner was operated by Wincats software version
1.2.3. The source of radiation was a deuterium lamp
emitting a continuous UV spectrum between 200 and
400 nm. The slit dimensions were 5 mm × 0.45 mm
and the scanning speed was 20 mm/s.
After chromatographic development, bands were
scanned over the range 200-400 nm (spectrum scan
speed 20 nm/s) so that the drug could be estimated
at 300 nm fig. 1, which is ascertained by taking the
spectrum at different concentrations between 100–700
ng with 100 ng increment. Further it is also observed
that spectra are similar in their behavior.
The standard stock solution of almotriptan malate
(100 μg/ml) was applied on a TLC plate, in the range
1–7 μl, by use of the Linomat V sample applicator
and 100 μl syringe. The plate was developed and
scanned under the conditions described above. Each
amount was analyzed five times and peak areas were
recorded. A calibration plot of peak area against
respective amount was established for almotriptan
malate.
Twenty tablets were weighed accurately and finely
Fig. 1: Overlain spectra of almotriptan malate from 200 to 400 nm,
recorded in situ on the plate ––––100 ng/band; ––×––200 ng/band;–
–––300 ng/band;–––– 400 ng/band; ––––500 ng/band; ––*––600
ng/band;––+–– 700 ng/band
Indian Journal of Pharmaceutical Sciences
September - October 2010
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powdered. A quantity of powder equivalent to 10
mg almotriptan malate was weighed and transferred
to a standard 100 ml volumetric flask containing
approximately 50 ml methanol. The mixture was
ultra sonicated for 5 min then diluted to volume with
methanol. The solution was filtered using Whatmann
41 filter paper. One to seven microlitres of the filtrate
was applied to a TLC plate. After development
of chromatogram the peak area of the bands were
measured at 300 nm and the amount of drug in each
tablet was determined from the calibration plot. The
analytical procedure was repeated six times for the
homogenous powder sample.
The limit of detection of a compound is defined as
the smallest level of analyte that gives a measurable
response. LOD was determined by using the equation
LOD= 3.3×σ/S. The limit of quantitation is the
lowest concentration of a compound that can be
quantified with acceptable precision and accuracy.
LOQ was determined by employing the relationship
LOQ=10×σ/S, where σ = the standard deviation of the
response and S = the slope of the calibration curve.
To study intra-day and inter-day precision, three
different concentrations of sample solutions were
prepared and applied to the TLC plates. All the
solutions were analyzed in triplicate on the same day
and on three different days to record intra-day and
inter-day variations in the results, respectively.
To check the accuracy of the method, recovery was
measured by the addition of standard drug solution
at three different levels (50, 100 and 150%) to preanalyzed sample solution (200 ng/band for almotriptan
malate was selected so that after standard addition
sample would be in the linear range). Three replicate
estimations were carried out for each concentration
level.
The effect of small, deliberate variation of the
analytical conditions on the peak areas of the drugs
was examined in order to assess the robustness of the
method. The robustness of the method was checked
for 200 and 700 ng/band for almotriptan malate.
For optimization of method, different mobile phase
compositions were employed to achieve good
separation. The method development was initiated by
using different proportions of mobile phase consisting
variety of solvents like: toluene:methanol:ethylacetate,
September - October 2010
TABLE 1: ANALYSIS OF MARKETED FORMULATION OF
ALMOTRIPTAN MALATE BY HPTLC METHOD
Drug
Almotriptan
malate
Label claim
12.5 mg
Amount found* Recovery*
%
12.42 mg
99.36
RSD %
0.52
*Average of six determinations
TABLE 2: RESULT FROM RECOVERY STUDIES OF
ALMOTRIPTAN MALATE
Sample
Excess of drug Theoretical
added to the content (ng)
analyte (%)
Almotriptan
50
300
malate
100
400
150
500
Recovery* %
RSD %
99.54
99.82
101.1
0.21
0.35
0.61
*Average of three determinations
TABLE 3: METHOD VALIDATION PARAMETERS
Parameters
Linearity (ng/band)
Correlation coefficient
LOD (ng/band)
LOQ (ng/band)
Precision
Interday (% RSD)
Intraday (% RSD)
Specificity
Results of almotriptan malate
100-700
0.9994
28.19
93.99
0.51
0.23
specific
Fig. 2: Densitogram obtained from a sample solution of almotriptan
malate
dichloromethane:methanol, butanol:aceticacid:water,
toluene:methanol:acetone, toluene:methanol:ethyla
cetate:aceticacid, hexane:ethylacetate. Of these the
mixture butanol-acetic acid-water 3:1:1 (v/v) was
found to be suitable for the studies. The RF value
of almotriptan malate was 0.53±0.01. The proposed
HPTLC method was validated in terms of linearity,
precision, accuracy, specificity and robustness. The
calibration plot was found to be linear over the
range 100–700 ng/band for almotriptan malate, with
a correlation coefficient of 0.9994±0.0002. The LOD
Indian Journal of Pharmaceutical Sciences
631
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and LOQ were found to be 28.19 and 93.99 ng/
band, respectively. The values of percent relative
standard deviations were found to be 0.23 and
0.51 for Intraday and inter day precision studies,
respectively which indicate that the method is
precise. The method was also evaluated by assay
of commercially available tablets (Axert) containing
almotriptan malate. The resulting densitogram for a
sample solution of almotriptan malate is presented
in fig. 2 indicating a concentration of 700 ng/band.
Six replicate analyses were performed on accurately
weighed amount of the tablets (Table 1). The percent
assay was found to be 99.36±0.522 for almotriptan
malate. To study the accuracy of the method, recovery
studies were performed. For almotriptan malate,
recovery ranged from 99.54 to 101.1% with values
of percent RSD ranging from 0.21 to 0.61 indicating
that the proposed HPTLC method is highly accurate
(Table 2). To confirm the specificity of the proposed
method, the solution of formulation was spotted on
TLC plate, developed and scanned. It was observed
that the excipients present in the formulation did
not interfere with the sample peak. Study of the
robustness of the method revealed that the peak areas
were unaffected (RSD < 2%) by small changes in
composition and volume of mobile phase indicating
appreciable robustness of the method. The method
validation parameters are presented in Table 3.
ACKNOWLEDGEMENTS
The authors wish to express their gratitude to SMS
Pharmaceuticals, Hyderabad, India for the gift
sample of pure almotriptan malate. One of us (A.S)
is thankful to management of Hindu College of
Pharmacy, Guntur, India for providing free access to
the facilities and to carry out research work.
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tripartite guidelines (Nov 2005), Validation of Analytical Procedures‫׃‬
Text and Methodology Q2 (R1).ICH, Geneva.
The validated HPTLC method for almotriptan malate
is simple, rapid, accurate, precise, sensitive, specific
and robust and can thus be used for routine analysis
of almotriptan malate in a tablet dosage form.
Accepted 18 September 2010
Revised 03 May 2010
Received 30 November 2009
Indian J. Pharm. Sci., 2010, 72 (5): 629-632
Spacer/Linker Based Synthesis and Biological Evaluation
of Mutual Prodrugs as Antiinflammatory Agents
V. S. VELINGKAR*, D. R. JAIN AND D. C. AHIRE
Department of Pharmaceutical Chemistry, Prin. K. M. Kundnani College of Pharmacy, Jote Joy Building, Rambhau
Salgaonkar Marg, Cuffe parade, Mumbai-400 005, India
Velingkar, et al.: Synthesis and Biological Evaluation of Antiinflammatory Mutual Prodrugs
Mutual prodrugs of some antiinflammatory agents were synthesized with the aim of improving the therapeutic
*Address for correspondence
E-mail: [email protected]
632
Indian Journal of Pharmaceutical Sciences
September - October 2010