estimation of piracetam in biologic
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PHARMACIA, vol. 60, No. 4/2013
V. Maslarska
layer densitometric determination
methods were also developed for
ANALYTICAL METHOD DEVELOPMENT AND VALIDATION FOR
Derivative
spectrophotometry
PIRACETAM IN PHARMACEUTICAL
FORMULATION
V. Maslarska
is an
both qualitative and quantitative i
Medical University–Sofia, Faculty of Pharmacy, Department of Chemistry,
2 Dunav st, Sofia 1000, Bulgaria
bands, and for eliminating the effe
Abstract. A simple, rapid and validated HPLC method was developed for determination of Piracetam in
film coated tablets. A Lichrosorb® (RP-18 column with a mobile phase consisting of acetonitrile – phosphate buffer (5:95v/v)) was used. Quantitative evaluation was performed at 205 nm. The HPLC method
is selective, precise and accurate and can be used for routine analysis of preparations in pharmaceutical
industry quality control laboratories.
calculating and plotting one of t
Derivative spectrophotometry is no
Key Words: liquid chromatography, piracetam, tablets, validation
micro computerized UV spectropho
Introduction
Piracetam (2-oxo-pyrrolidin-1-yl)-acetamide is a
nootropic psychopharmacological agent [1-3] having
a variety of physiological effects that may result from
the restoration of cell membrane fluidity. It is used
for treatment of patients suffering from pathological,
neurosensitive [4], and cognitive deficits [5], brainorganic psychosyndromes (e.g., primary degenerative
dementia), vertigo, and myoclonus of cortical origin.
Piracetam is a white powder and is freely soluble in water, soluble in alcohol, and slightly soluble
in methylene chloride [6]. The chemical structure
of the molecule is presented in Figure 1. Literature
survey revealed HPLC methods were developed for
the estimation of piracetam in biological fluids [710]. Capillary electrophoresis [11], thin layer densitometric determination [12], micellar electrokinetic
chromatography [13] methods were also developed
for the estimation of piracetam in biological fluids.
Derivative spectrophotometry is an analytical technique of great utility for extracting both qualitative
and quantitative information from spectra composed
of unresolved bands, and for eliminating the effect of
baseline shifts and baseline tilts. It consists of calculating and plotting one of the mathematical derivatives of a spectral curve. Derivative spectrophotometry is now a reasonably prized standard feature of
modern micro computerized UV spectrophotometry
to develop a simple, economical an
[14-15]. The aim of the present study was to develop
a simple, economical and accurate analytical method
for the estimation of Piracetam in pharmaceutical
dosage forms.
Piracetam in pharmaceutical dosage
Figure 1. Chemical structure of Piracetam
Figure 1: Chemical structure of Pir
Experimental Section
Materials and Methods
Reagents and Chemicals
All chemicals and reagents used were HPLC
grade. Piracetam standard was obtained from Sigma
Aldrich. Tablet formulation containing 400 mg Piracetam was obtained commercially. HPLC grade
Acetonitrile was procured from Merck Ltd. All other
chemical reagents were of analytical grade.
Experimental Section
Instrumentation and Chromatographic Conditions
A Shimadzu HPLC system was utilized consisting of the following components: quaternary pump
Materials and Methods
Reagents and Chemicals
All chemicals and reagents used w
Analytical method development and validation for Piracetam ...
LC – 20 AD, vacuum degasser unit DGU – 20 A5
and a UV/VIS variable detector SPD – 20 A. Separation was carried out on a LiChrosorb C 18 column
(250 x 4.6 mm, particle size 5 µm) under reversed
phase partition chromatographic conditions. The mobile phase consisted of an aqueous solution containing Acetonitrile – 1 g/l solution of Dipotassium hydrogen phosphate (5:95) with pH 6.0 adjusted with
ortho-phosphoric acid. The mobile phase was filtered
through 0.45 µm membrane filter and degassed by using sonicater for about 10 min before use. The sample
solutions were also filtered using 0.45 µm membrane
filters. The mobile phase was delivered isocratically
at a flow rate 1 ml/min. The column was maintained
at 25ºC temperature. The injection volume was a 20
µl and the total run time was 5 minutes. The detection
was carried out at 205 nm.
Preparation of the Standard Solution
About 50 mg of Piracetam were accurately weighed
and transferred into 100 mL volumetric flask and dissolved in mixture of acetonitrile – water (10 : 90 v/v).
The final drug concentration of 50 μg/mL was obtained
by dissolving the appropriate amount from this standard stock solution in the above said mixture. Calibration standards of Piracetam were prepared by making
serial dilutions of the stock solution at concentrations
of 12.5, 25.0, 50.0, 75.0, 100.0 μg/mL.
Sample Preparation
Twenty tablets were accurately weighed (to obtain
the average mass of one tablet) then finely powdered.
Weight equivalent to 200 mg of Piracetam (half a tablet) was weighed, transferred into a 100 ml volumetric flask and dissolved with about 50 mL mixture of
acetonitrile – water (10 : 90 v/v). The contents were
sonicated for 5 minutes. The mixture was made up
to 100 ml with the same mixture. The solution was
filtered through a membrane syringe filter (pore size
0.45 µm). The final drug concentration of 50 μg/mL
was obtained by dissolving the appropriate amount
from the filtrate solution. The sample solution was
injected and the peak area was measured for determination of Piracetam in a tablet formulation.
Results and Discussion
After equilibration of column with the mobile
phase indicated by a stable baseline, aliquots of sample (20 µL) were injected. The typical chromatogram
is shown in Figure 2. The amount of Piracetam present in the tablets was calculated using single point
analysis method and results are shown in Table 1.
PHARMACIA, vol. 60, No. 4/2013
43
Figure 2. Chromatogram of Piracetam
Table 1. Results of Assay of Piracetam tablet
Drug #
Piracetam
Label
Mean amount
claim
found (mg/1
(mg/1 tabl) tabl) (n=6)
400
401.7
Mean % Assay RSD
100.4±0.552
The method was developed and validated by using the ICH guideline [16]. The selectivity, limits of
detection and quantification, linearity, precision, and
accuracy were determined. Determination was carried out using tablet formulation. The presented RPHPLC method has been proved to be rapid and was
successfully used for determination of Paracetam.
Linearity
The linearity for Piracetam was determined by
plotting a calibration graph of ratio of peak area of
drug to concentration. The linearity of this method
was found to be in the concentration range 12.5 - 100
μg/mL for Piracetam. Y=5.341E7x +1217.2 which
is linear regression equation with correlation coefficients of 0.999 was determined from linearity curve
(Table 2).
Limit of detection and limit of quantification
In order to estimate the limit of detection and
limit of quantification, mobile phase was injected six
times, and the noise level was determined. The limit
of detection was calculated to be three times the noise
value and ten times the noise, which gave limit of
quantification, and was also cross-checked by formulas given below (Table 2).
LOD=3.3σ/S and LOQ=10σ/S where σ is the
standard deviation of the lowest standard concentration and S is the slope of the standard curve.
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PHARMACIA, vol. 60, No. 4/2013
V. Maslarska
Table 2. Linearity Results, Limit of Detection (LOD) and Limit of Quantification (LOQ)
Compound
r2
Piracetam
0.999
Calibration curve
equation
Y=5.341E7x +1217.2
Accuracy/Recovery
Accuracy of the developed method was confirmed
by performing a recovery study as per ICH norms
at three different concentration levels (50%, 100%,
150%) by replicate analysis (n = 3). The results obtained (Table 3) indicate that recovery is good, not
less than 98% and percentage relative standard deviation is less than 2%.
Precision
The precision of the method was determined by
repeatability, intermediate precision (intra-day, interday) and was expressed as % relative standard deviation (% RSD). Intra-day precision was determined
by performing analysis of triplicate injections of two
different concentrations of Piracetam on the same
day at different time intervals and on two different
days for inter-day precision. The % RSD of the study
was found to be less than 2% as shown in Table 4.
Conclusion
The aim of the present research work was to
achieve highest precision in quantitative estimation
Table 3. Recovery studies of Piracetam
Drug
Piracetam
Concentra- Concentra- R e c o v e r y
tion added tion recov(%)
(μg/mL)
ered
(μg/mL)
25.00
50.00
75.00
Mean
SD
% RSD
% Error
25.05
25.15
24.69
49.85
50.15
49.93
75.22
75.01
75.15
100.2
100.6
98.76
99.70
100.3
99.86
100.3
100.0
100.2
99.99
0.532
0.532
±0.409
LOQ
ng
10
LOD
ng
5
of Piracetam in tablet dosage form. The method was
validated in terms of linearity, precision, accuracy,
limit of detection and limit of quantification. The developed method has a simple procedure for the preparation of the samples, shorter run time for chromatographic analysis (less than 5 min) and a low percent of
organic solvent (acetonitrile 5 %) in the composition
of the mobile phase. Hence the proposed RP-HPLC
method can be considered as simple, rapid, suitable
and easy to apply for routine analysis of Piracetam in
pharmaceutical dosage form.
Table 4. Results of Precision
Sample #
Piracetam
Concentration
(μg/mL)
30
50
RSD (%)
Intra-day
(n=3)
1.0785
0.8543
RSD (%)
Inter-day
(n=3)
1.5223
1.7638
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
Corresponding author:
Medical University – Sofia, Faculty of Pharmacy,
Department of Chemistry,
Tel: +35929236537
Fax: +35929879874
e-mail: [email protected]
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