Simultaneous Estimation of Levetiracetam and its Preservatives In Oral Liquid Dosage form by Rp-Hplc Method

Levetiracetam (LEV) is a novel anti-epileptic agent. The chemical name of LEV, a single enantiomer is (-)-(S)-α-ethyl2-oxo-1-pyrrolidine acetamide. [1] The chemical structure of LEV is shown in Figure 1. Its molecular formula is C8H14N2O2 and its molecular weight is 170.21. It is chemically unrelated with existing anti-epileptic drugs, differing in structure and pharmacology [2, 3]. This is a structural analogue of piracetam, which binds to a synaptic vesicle protein SV2A and is believed to impede nerve conduction across synapses. The exact mechanism by which Levetiracetam shows its antiepileptic effect is still unknown. However, it is believed that it binds to a synaptic vesicle protein, thus slowing down nerve conduction across synapses [4]. Levetiracetam is approved by the U.S. Food and Drug Administration as an adjunct in partial on set seizures, myoclonic seizures and primary generalized tonic-clonic seizures and mono therapy for partial seizures with or without secondary generalization. Levetiracetam has possible benefits for other psychiatric and neurologic conditions4 such as Tourette syndrome, autism, and anxiety disorders [4, 5].


Introduction
Levetiracetam (LEV) is a novel anti-epileptic agent.The chemical name of LEV, a single enantiomer is (-)-(S)-α-ethyl-2-oxo-1-pyrrolidine acetamide.[1] The chemical structure of LEV is shown in Figure 1.Its molecular formula is C 8 H 14 N 2 O 2 and its molecular weight is 170.21.It is chemically unrelated with existing anti-epileptic drugs, differing in structure and pharmacology [2,3].This is a structural analogue of piracetam, which binds to a synaptic vesicle protein SV2A and is believed to impede nerve conduction across synapses.The exact mechanism by which Levetiracetam shows its antiepileptic effect is still unknown.However, it is believed that it binds to a synaptic vesicle protein, thus slowing down nerve conduction across synapses [4].Levetiracetam is approved by the U.S. Food and Drug Administration as an adjunct in partial on set seizures, myoclonic seizures and primary generalized tonic-clonic seizures and mono therapy for partial seizures with or without secondary generalization.Levetiracetam has possible benefits for other psychiatric and neurologic conditions4 such as Tourette syndrome, autism, and anxiety disorders [4,5].

Chemicals and Reagents
Levetiracetam standard drug, Methyl Paraben and Propyl Paraben were obtained as gift samples from Hetero research

Abstract
A rapid, simple, sensitive and accurate RP-HPLC method was developed and validated for simultaneous estimation of Levetiracetam and its preservatives (Methyl paraben and Propyl paraben) in oral liquid dosage form.The chromatographic separations were achieved on a Zorbax CN column (250×4.6 mm inner diameter, 5µm) using a mobile phase consisting of orthophosphoric acid buffer (pH 4.6)acetonitrile (75:25 v/v) at a flow rate of 1.0 mL/min with UV detection at 210nm.This system produced sharp peaks with good resolution, minimum tailing and satisfactory retention time for Levetiracetam, Methyl paraben and Propyl paraben were found to be 4.601, 9.905 and 19.291 respectively.
The method was validated as per USP guidelines which include accuracy, precision, linearity and range, robustness specificity and ruggedness.The described method was linear over the range of 30-90 µg/ml for Levetiracetam with r² of 0.9999 and 60-180 µg/ml for Methyl paraben with r² 0.9997 and 20-60 µg/ml for Propyl paraben with r² 0.9999 .The average recovery of the method was 98.2%, 99.3% and 98.7% for Levetiracetam, Methyl paraben and Propyl paraben respectively.
The developed method is repeatable and selective for the analysis of Levetiracetam and its preservatives in oral liquid dosage form.Hence the method could be successfully applied for routine analysis.The developed method could also applicable in quality control testing in analysis of oral liquid dosage form and in process testing.

Instruments
Waters HPLC system equipped with waters separation module 2695 and auto sampler was employed for analysis.The chromatographic data was acquired by using empower 2 software.Zorbax CN, (250×4.6mm),5µm column was used for separation.Lab India UV 3000/ UV win-Double beam UV-VISIBLE Spectrophotometer, Ultra sonicator-MAN-009-Systronics, Gelman science vaccum lamp, pH meter-µ pH system 361-Systronics were also used.

Selection of Wave Length
In setting up the conditions for development of the assay method, the choice of detection was based on the scanned absorption spectrum for Levetiracetam, Methyl paraben (MP) and Propyl paraben (PP).The U.V spectrum of Levetiracetam, Methyl paraben and Propyl paraben (Figure 2a, 2b, 2c) were obtained separately by scanning the sample solution in methanol over the wavelength range 200-400 nm against the blank.

HPLC Conditions
A mixture of orthophosphoric acid buffer (pH 4.6) and acetonitrile (75:25, v/v) was employed as a mobile phase.Flow rate 1.0 mL/min, injection volume 20 µl and the Detection wavelength was 210 nm.

Standard Solutions
Stock standard solution was prepared by dissolving 50 mg Levetiracetam, 50 mg Methyl paraben and 50 mg propyl paraben separately in 100 ml mobile phase.
Working standard solution was prepared to get a final concentration of 60 µg/ml Levetiracetam, 120 µg/ml Methyl paraben and 40 µg/ml propyl paraben.

Sample Solution
Weigh and transfer accurately about 5ml of Levetiracetam oral solution into 100ml clean dry volumetric flask.Dissolve and dilute to volume with mobile phase and filter.Transfer 2ml of above filtered solution into 100 ml volumetric flask and dilute to volume with mobile phase and mix.

Method Development and Optimization of HPLC Conditions
The HPLC conditions were optimized by using trials with different columns, several mobile phase compositions, flow rate and pH.The mobile phase containing orthophosphoric acid buffer (pH 4.6) and acetonitrile (75:25, v/v), Zorbax CN (250×4.6mm)5µm column at a flow rate of 1.0 ml/minute and 210 nm wavelength detection was selected because it gave sharp peaks with good resolution, minimum tailing and satisfactory retention time.

System Suitability
The system suitability parameters were evaluated with the help of standard chromatogram which is shown in Figure 2. Plate counts (N), Tailing factor (T) and resolution (R s ) are determined from replicate injection of standard compared with method specification Levetiracetam and its preservatives solutions were prepared and injected.All the system suitability parameters were within the acceptance criteria, which is listed in Table 1.

Accuracy Precision
The precision of the developed method was studied by performing intraday and interday variations.Intraday variation was determined by six replicate injections of standard in the same day and the interday was determined in six consecutive days.The precision was evaluated by calculating the %RSD of peak areas of six replicate injections of LEV, MP and PP.The % RSD of intraday precision was found to be 0.54, 0.55 and 0.81 for LEV, MP and PP respectively and the % RSD of interday precision was found to be 0.68, 0.74 and 0.98 for LEV, MP and PP respectively.The lower % RSD values showed excellent precision of the method.

Linearity
Five different concentrations of standard solutions were selected to demonstrate the linearity of the method, i.e. 30-90 µg/mL for LEV, 60-180 µg/mL for MP and 20-60 µg/mL for PP.The calibration curve was plotted using concentration versus peak area.An excellent linearity was obtained with correlation coefficient value 0.9999, 0.9997 and 0.9999 for LEV, MP and PP respectively.

Robustness
The robustness was evaluated by making slight variations in the optimized conditions such as mobile phase composition, pH of mobile phase, flow rate and temperature.The results obtained (Listed in Table 3) after the small deliberate changes in the method parameters has proven that the developed method is robust.

Specificity
The specificity of the proposed method was demonstrated by interference study.It was found that presence of some common exicipients did not cause any interferences at the retention time of LEV, MP and PP (Figure 3).Thus the developed method can be successfully applied for simultaneous determination of LEV, MP and PP in oral liquid dosage form.

Ruggedness
Ruggedness (Intermediate precision) of the method was evaluated by analyzing six samples by two analysts in the same laboratory by using different HPLC systems.From the Ruggedness data, the %RSD of the peak area of LEV, MP and PP were found to be 0.67, 0.74, 0.98 and 0.53, 0.55, 0.81 for the analyst 1 and 2 respectively, which is well within the acceptance criteria indicating that the present method is rugged.

LOD and LOQ
LOD and LOQ were calculated by using the standard deviation of the regression line of the calibration curve and its slope.The

Application of the Method for Estimation of LEV in Marketed Oral Liquid Dosage Form
Using the proposed HPLC method, the determination of LEV in its commercial oral liquid dosage form was carried out.Satisfactory results were obtained, which are in good agreement with the label claim.The results are shown in Table 4.

Conclusion
A simple, specific, reliable method was developed for simultaneous estimation of Levetiracetam and its preservatives in marketed oral liquid dosage form.The method offers good resolution between the proposed components within a suitable analysis time.Based on the results obtained, it was found that the developed method is accurate, precise and sensitive.The proposed method has an advantage over the published methods for analyzing Levetiracetam in presence of the preservatives used in commercial formulation.Therefore the applied method could be conveniently adopted for the routine quality control analysis of Levetiracetam and its preservatives from its oral liquid dosage form.
20µl of blank, placebo, standard preparation (5times), and sample preparation were injected into the chromatographic system separately.Chromatogram was recorded and peak responses were measured.The placebo chromatogram was examined for any extraneous peaks observed in the chromatogram of sample preparation.The concentration of drug was calculated by using the following formula.Concentration of drug = Where A T = Area count of Drug peak in sample solution.A S = Average area count of five replicate injections for drug W S = Weight of drug working standard taken in mg W T = Weight of sample taken in mg P = Purity of drug working standard used A = weight per ml of solution.

00133 Simultaneous Estimation of Levetiracetam and its Preservatives In Oral Liquid Dosage form by Rp-Hplc Method
LOD values were found to be 0.937, 1.612, 0.077µg/mL for LEV, MP and PP respectively.The LOQ values were found to be 2.841, 4,887, 0.234µg/mL for LEV, MP and PP respectively.

Table 4 :
Application of developed method for assay of marketed formulation