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Research J. Pharm. and Tech. 1(3): July-Sept.

2008,
,

www.rjptonline.org

ISSN 0974-3618

RESEARCH ARTICLE

RP-HPLC Method Development and Validation for Assay of Levetiracetam in


Tablet Dosage Form
J Valarmathy1*, L Samueljoshua2, G Rathinavel1, C Selvin Thanuja1 and T Sivakumar1
1

Department of Pharmacy, Nandha College of Pharmacy, Erode 638052, Tamilnadu, India


Department of Pharmacy, Padmavathi College of Pharmacy, Dharmapuri, Tamilnadu, India

*Corresponding Author E-mail: valarmathyjoshua@rediffmail.com

ABSTRACT
A simple, reproducible and efficient reverse phase high performance liquid chromatography (RP-HPLC) method has been
developed for estimation of Levetiracetam in its tablet dosage form. Separation was done by using mobile phase consists
of buffer solution (pH 2.8) and acetonitrile in the ratio of 90:10. Chromatography separations were carried out on
prontosil C18 column (150X4.6mn; 5m) at a flow rate of 1.2 ml/min and UV detection at 215nm and the retention time
for levetiracetam is 4minutes. The linear dynamic response was found to be in the concentration of 45g-270g/ml. The
slope, intercept and correlation coefficient were found to be 0.9999 respectively. The percentage recovery of
Levetiracetam was found to be 99.08%. Proposed methods were found to be simple, accurate, precise and rapid and could
be used for routine analysis. This condition is applied only for tablet dosage form. The statistical parameters and recovery
studies were carried out and reported.

KEY WORDS : Levetiracetam, Estimation, Tablets, RP-HPLC.


INTRODUCTION:

EXPERIMENTAL:

Levetiracetam (Fig-1) is a single enantiomer of (-)-(s)ethyl-2-oxo-1-pyrrolidine acetamide. Levetiracetam


can prevent myoclonic jerks and generalized
epileptiform activity in patients with photosensitive
epilepsy. A few RP-HPLC methods have been reported
in the literature for the estimation of levetiracetam. The
authors now proposed a high degree of accuracy and
precise HPLC method for the estimation in pure as well
as tablet dosage forms.

A prominence Shimadzu high pressure liquid


chromatographic instrument provided with prontosil C18
column (150X4.6mm, 5m) 10l syringes and supported by
window software was employed in the study. Levetiracetam
was obtained from Orchid Chemicals Pharmaceuticals Ltd;
Chennai.
Potassium
dihydrogen
phosphates,
Sodium-1-heptane sulphonate, Acetonitrile and Water were
of HPLC grade. All other chemicals were of Analytical
Reagent grade.

N
H3CH2C
H2NOC

Received on 29.07.2008
Accepted on 14.10.2008

Modified on 06.08.2008
RJPT All right reserved

Research J. Pharm. and Tech. 1(3): July-Sept.. 2008;Page 395-397

Chromatographic conditions:
The mobile phase was pH 2.8 buffer solution and
acetonitrile in the ratio of 90:10(v/v). Mobile phase was
filtered through 0.45m membrane filter and sonicated
before use. The mobile phase was pumped from the solvent
reservoir in the ratio of 90:10(v/v) to the column at a flow
rate of 1.2ml/min. The run time was set at 8 minutes. The
column was maintained at ambient temperature and the
volume of each injection was 10l. Samples were injected
in to the column and the absorptivity of the mobile phase
was monitored at 215nm using a variable wavelength
detector.
Assay procedure:
Preparation of pH 2.8 Buffer solution
Potassiumdihydrogen phosphate (0.01mol, 1.36gm) and
Sodium-1- heptane sulphonate (0,61gm) in 1000ml of milli

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Research J. Pharm. and Tech. 1(3): July-Sept. 2008,


,

equivalent water was solubilised and adjusted the pH to


2.8 0.05 with ortho phosphoric acid solution.

present in the tablet formulation was calculated by


comparing the peak area from the standard.

Preparation of diluents solution:


Mobile phase consisting of buffer solution (pH 2.8) and
acetonitrile in the ratio of 90:10 was used as diluents
and were filtered through 0.45 membrane filter and
sonicated for 10 minutes.

The amount of Levetriacetam present in the sample =

Preparation of standard solution:


About 90mg of pure sample of levetiracetam was
weighed accurately and transferred to a 50ml
volumetric flask and dissolved in 35ml of diluent. The
solution was sonicated for 30min and then the volume
was made up with a further quantity of diluent to get
1.8mg/ml solution. From this 5ml of the solution was
transferred in to 50ml volumetric flask and the volume
was made up to mark with diluent to get 180g/ml.
Subsequent dilution of this solution ranging from 45270g/ml were made in 10ml volumetric flask. The
prepared solutions were filtered through 0.45
membrane filter and then 10l of filtrate was injected
each time in to the column at a flow rate of 1.2ml/min.
Each concentration was injected 6 times into the
column and corresponding chromatograms were
obtained. Detection of the drug was one at 215nm from
the chromatogram, the retention time and mean peak
area rations were recorded for all the concentration.
These determine the linearity by applying the
mentioned chromatographic conditions. From the data
obtained, correlation coefficient, Y-intercept and slope
were calculated to provide mathematical estimation of
the degree of linearity. The results are shown in table-1
Table1- Analytical
Levetiracetam
Parameters

performance

Linear dynamic range g/ml


Correlation coefficient
Y-intercept
Slope of regression line

parameters

of

Average area
45-270g/ml
0.9999
12438
6474.3x

Estimation of Levetiracetam in tablet dosage forms:


20 tablets were weighed and powdered. Accurately
weighed portion of the tablet powder equivalent to
180mg was taken in 100ml volumetric flask and 75 ml
of diluent was added, shaken well and allowed to stand
for 30min with intermittent sonication to ensure
complete solubility of the drug. The mixture was then
thoroughly mixed and made up to the mark with
diluent. Centrifuged at 2500rpm for 10min. From the
above supernatant solution, pipetted out 5ml into a
50ml volumetric flask, diluted to volume with diluent,
mixed well and filtered through a 0.45 membrane.
10l of filtrate was injected into the column. The
concentration was injected 6 times. The amount of drug

Sample peak area standard dilution P


--------------------- --------------------- -- Average weight of tablet
Standard peak area Test dilution
100

P = % potency of Levetiracetam working standard


The HPLC method developed in the present study has also
been used to quantify Levetiracetam in tablet dosage form.
Levetiracetam tablets (containing 750mg) were quantified
using the proposed analytical method. No interfering peaks
were found in the chromatogram indicating that the tablet
excepients did not interfere with the estimation of drug by
the proposed HPLC method. The tablets were found to
contain 97.00-103.00% of drug. A known amount of drug
solution was added to the sample of tablet dosage form and
subjected to estimation of drug by proposed method. There
was a high recovery of (Table 2) levetiracetam (97.15101.41%) indicating that the proposed procedure for
determination of levetiracetam in tablet dosage form is
highly accurate.

RESULTS AND DISCUSSION:


The aim of this study was to develop a simple, accurate and
precise HPLC method for the analysis of levetiracetam in
bulk and tablet dosage forms using mobile phase (pH 2.8
buffer and acetonitrile 90:10 v/v) and commonly employed
RP- C18 column with UV detector at 215nm.
Results of recovery study -Table 2

Spike
level
%

25
50
75
100
150

Amount
of
Levetirac
etam
187.5
375
562.5
750
1125

Recovery
from
drug solution

Recovery
formulation

Mean
amount

Mean
amount

189.5
378.6
567.01
755.6
1133.88

Table 3 -Peak Area


Name
Standard
Levetiracetam
Sample Levetiracetam

Mean
(%)
recove
ry
98.34
97.09
97.51
99.13
97.70

185.52
368.21
576.85
745.63
1120.57

from

Mean(%)
recovery

97.22
97.15
101.41
98.42
97.06

Peak area
1173908
1172850

The run time of the method was set at 8mintues. Retention


time was found to be around 4minutes and levetiracetam
appeared on chromatogram at 4.170min. This indicates that
the present HPLC method is rapid which in turn shows that
the method consumes less volume of HPLC solvents. When
the same drug solution was injected 6 times, the retention
time of the drug was found to be same. The retention time of

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Research J. Pharm. and Tech. 1(3): July-Sept. 2008,


,

the standard levetiracetam was observed to be


4.174minutes. The quantitative estimation was carried
out on tablet by taking the same concentration as for
standard solution and corresponding sample peak area
were represented in Table 3. The average percentage
purity was found to be 99.08%Hence, the proposed
HPLC method is simple, precise, accurate and rapid for
the determination of levetiracetam in dosage forms. It
can be easily and conveniently adopted for routine
quality control analysis of the drug.

ACKNOWLEDGEMENTS:
The authors thank Orchid Chemicals and
Pharmaceuticals Ltd; Chennai, India for providing a
gift sample of levetiracetam. The authors are also
thankful to Mr. V. Shanmugam, Chairman, Nandha
College of pharmacy, Erode-52, Tamilnadu, India for
providing facilities for the research work

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