Estimation of Levetiracetam in Tablet by UPLC.

Patel Vandana B^1*,Bhallara Harshvardhan² and Patel Bhumika M¹

¹Baroda College of Pharmacy, Limda, Waghodia, Vadodara, India.

²Department of Pharmacy, The Maharaja Sayajirao University of Baroda, Gujarat, India.

*Corresponding Author E-mail: vbpatel04@yahoo.com

ABSTRACT:

A rapid, precise, accurate and specific ultra performance liquid chromatography method was developed for the estimation of levetiracetam in tablet dosage form. Bridged Ethyl Hybrid (BEH), C 18, 100*2.1 mm, 1.7 µ/CLC 253 column, with mobile phase consisting of 0.3 % w/v orthophosphoric acid and methanol in the ratio of 80:20 v/v was used. The flow rate was 1 ml/min and the effluents were monitored at 210 nm. The retention time was 1.55 min and the detector response was linear in the concentration range of 50-150 µg/ml with the linear regression equation being Y=12327x+7411. The limit of detection and limit of quantification was 0.00675and 0.0225μg/ml respectively. The percentage assay of Levetiracetam was 99.57 ± 0.831%. The method was validated in terms of accuracy and precision. The results of the study showed that the proposed ultra performance liquid chromatography (UPLC) method is useful for the routine determination of levetiracetam in its pharmaceutical tablet dosage form.

KEYWORDS: Levetiracetam, Ultra performance liquid chromatography, Estimation, pharmaceutical formulation.

INTRODUCTION:

Levetiracetam is (-)-(S)-α-ethyl-2-oxo-1-pyrrolidine acetamide, is a white to off-white crystalline powder with a faint odor and a bitter taste. It is used as anti epileptic agent. Several techniques viz. HPLC^1-5, gas chromatography⁶, eletrokinetic chromatography⁷ have been reported for the determination of levetiracetam from biological fluids. Few reverse phase HPLC methods have also been reported for determination of levetiracetam in tablet dosage formulation^8-9_.The present research work describes rapid, accurate and sensitive UPLC for determination of levetircetam in bulk drug and pharmaceutical tablet formulation.

EXPERIMENTAL:

MATERIALS AND METHODS:

Levetiracetam was obtained as a gift sample from Alembic Ltd. Vadodara. Methanol and water used were of HPLC grade obtained from Merck, Mumbai.

Instrument:

Quantitative HPLC was perforemed on HPLC system consisting of Waters module with model L-7100 pump, L-7400 for U.V. detector, L-7200 for auto sampler and D-7000 for interface.

UPLC Condition:

The content of mobile phase and diluent were 0.3 % w/v orthophosphoric acid and methanol (80:20). They were filtered before use through a 0.45 µm membrane filter, and pumped from the solvent reservoir to the column at a flow rate of 1.0 ml/min. The run time was set at 5 min. and the column temperature was ambient. Prior to the injection of drug solution, the column was equilibrated for at least 30 min with the mobile phase flowing through the system. The eluents were monitored at 210 nm.

Preparation of Standard Stock solution:

A standard stock solution of the drug was prepared by dissolving 500 mg of levetiracetam in 250 ml volumetric flask containing 50 ml of methanol and 50 ml of water and sonicating for about 15 min. The mobile phase, 120 ml, was added and the flask was sonicated for about 15 min. The volume was made up to 250 ml with diluent to get a 2 mg/ml standard stock solution.

Preparation of calibration curve:

Aliquots of standard levetiracetam stock solution were taken in different 50 ml volumetric flasks and diluted up to the mark with the mobile phase such that the final concentrations of levetiracetam are in the range of 50-150 µg/ml. Each of these drug solutions (20 µl) was injected three times into the column, and the peak area and retention time were recorded. Evaluation was performed with UV detector at 210 nm and a calibration graph was obtained by plotting peak area versus concentration of levetiracetam.

The plot of peak area of each sample against respective concentration of levetiracetam was found to be linear in the range of 50–150 µg/ml with correlation coefficient of 0.9999. The linear regression equation being Y=12327x+7411.

Fig. 1. Typical UPLC chromatogram of levetiracetam

Method validation:

Accuracy was determined by recovery studies of levetiracetam, known amount of standard was added to the pre analyzed sample and subjected to the proposed UPLC analysis. The study was done at three different concentration levels. Intra-day precision and inter-day precision of the methods were assessed from the results of triplicate analyses of the pure drug solution. The mean values and relative standard deviation values for replicate analysis at ten different concentration levels were calculated. The limit of detection (LOD) and limit of quantification (LOQ) were calculated according to the ratio of 3.3 and 10 standard deviation of the blank (n = 7), respectively, and the slope of the calibration line.

Estimation of levetiracetam in tablet dosage form:

Average weight of twenty tablets was determined and it was triturated. Weight equivalent to 500 mg of levetiracetam was taken in a 250 ml volumetric flask. Methanol and water was added 50 ml each, and the flask was sonicated for about 15 min. The mobile phase, 120 ml, was added and the flask was sonicated for 15 min. The volume was made up to the mark with diluent. This solution was filtered through 0.45 µ filter. An aliquot of 5 ml of this solution was transferred to a 100 ml volumetric flask and made up to sufficient volume with diluent to get levetiracetam sample solution of 100 µg/ml. Twenty µl of sample solution was injected into the injector of liquid chromatograph. The retention time was found to be 1.5 min. The amount of drug present per tablet was calculated by comparing the peak area of the sample solution with that of the standard solution.

RESULTS AND DISCUSSION:

The UPLC for determination of levetiracetam in its pharmaceutical tablet dosage form were successfully developed. The method showed good linearity in the range of 50 to 150 mg/ml with the correlation coefficient of 0.9999. The regression characteristics and data obtained from the measurements are given in Table 1.

Table 1. Linear Regression Data for Calibration curves.

Parameters	Observations
Concentration range (mcg/ml)	50-150 mg/mi
Slope (m)	12327
Intercept (b)	7411
Correlation coefficient	0.9999
% RSD	0.692

From the typical chromatogram of levetiracetam as shown in fig 1, it was found that the retention time was 1.553 min. A mixture of 0.3% orthophosphoric aid and Methanol in the ratio of 80:20 v/v was found to be most suitable to obtain a peak well defined and free from tailing. In the present developed UPLC method, the standard and sample preparation required less time and no tedious extraction were involved. A good linear relationship (r=0.9999) was observed between the concentration range of 50-150 mg/ml. Low values of standard deviation are indicative of the high precision of the method.

From the recovery studies it was found that about 100.02 % of levetiracetam was recovered which indicates high accuracy of the method. (Table 2) The absence of additional peaks in the chromatogram indicates non-interference of the common excipients used in the tablets.

The limit of detection (LOD) and limit of quantification (LOQ) for levetiracetam were found to be 0.00675and 0.0225μg /ml respectively. The signal to noise ratio is 3 for LOD and 10 for LOQ. The assay of levetiracetam tablets was found to be 99.57 ± 0.831 %.( Table 3)

This demonstrates that the developed UPLC method is simple, linear, accurate, sensitive and reproducible. Thus, the developed method can be successfully used for the routine quality control of bulk and tablet dosage form of levetiracetam within a short analysis time.

ACKNOWLEDGEMENT:

The authors are grateful to Alembic Ltd, Vadodara, for the supply of levetiracetam as a gift sample and for providing the necessary facilities to carry out the research work.

Table 2. Recovery study data

Sample	Amount claimed per tablet	Amount added(%)	Total Amount added (mg)	Concentration recovered^* (mg) ± SD	%recovery± SD	% RSD
1	750	80	600	600.1	100.05 ± 0.155	0.155
2	750	100	750	749.2	99.89 ± 0.345	0.345
3	750	120	900	901.1	100.12 ± 0.225	0.224

^*Indicates that each value is mean ± standard deviations of three determinations; RSD = relative standard deviation

Table 3. Analysis of Commercial tablet formulation

Label claim mg/tablet	Concentration estimated* (mg)	% Concentration estimated*	% RSD
750	746.83	99.57 ± 0.831	0.289

* indicates value for recovery are mean for three determinations, SD is the standard deviation and RSD is relative standard deviation

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Received on 13.10.2009 Modified on 12.12.2009

Asian J. Research Chem. 3(1): Jan.-Mar. 2010; Page 148-150