SOJ Materials Science & Engineering

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.

Keywords: Levetiracetam; RP-HPLC; Oral Liquid Dosage Form; Preservatives

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 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 mechanismby 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].

The extensive literature survey revealed few methods are developed to estimate the drug Levetiracetam in raw material, tablets and in biological fluids using UV [5-7], RP-HPLC [2,8-18], LC-MS [19-21], Capillary electrophoresis [22], UPLC [23,24], Gas Chromatography [25,26] and HPTLC [27]. However, there is no method developed yet for the simultaneous estimation of Levetiracetam and its preservatives by RP-HPLC in oral liquid dosage forms. Hence, the present work is aimed to develop a new method for Levetiracetam and its preservatives (Methyl paraben and Propyl paraben) by RP-HPLC in oral liquid dosage form and validation of the developed method

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

labs. Formulation Keppra-1 ml oral solution was purchased from local market. Acetonitrile, Water and Methanol (HPLC grade) from Merck, orthophosphoric acid (AR) from S.D.Fine chemicals were used.

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.

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.

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.

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.

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.

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 =
\[\frac{{{A_T}}}{{{A_S}}}X\frac{{{W_S}}}{{100}}X\frac{5}{{100}}X\frac{{100}}{{{W_T}}}X\frac{{50}}{5}X\frac{P}{{100}}X{\rm{ }}{\mathop{\rm A}\nolimits} \] 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.

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 Figure 2.

The validation of the current method was performed which include accuracy, precision, linearity and range, robustness, specificity and ruggedness [28-32].