INTRODUCTION:

Levetiracetam is a anticonvulsant drug used in the treatment of epilepsy.It is S-enantiomer of etiracetam, structurally similar to the prototypical nootropic drug piracetam.Along with other anticonvulsants like gabapentin it is also sometimes used in the treatment of neuropathic pain Levetiracetam is a white to off-white crystalline powder with a faint odor and a bitter taste. It is very soluble in water. It is freely soluble in chloroform and in methanol, soluble in ethanol, sparingly soluble in acetonitrile and practically insoluble in n-hexane.

The chemical name^1-3 of levetiracetam, a single enantiomer, is (-)-(S)-α-ethyl-2-oxo-1-pyrrolidine acetamide, its molecular formula is C₈H₁₄N₂O₂ and its molecular weight is 170.21.

It is not official in any pharmacopoeia and till now, few liquid chromatography procedures have been reported for the determination of Levetiracetam and their metabolites in biological fluids. However there are no publications concerning the analysis of Levetiracetam in bulk and Pharmaceutical dosage forms^4-11. So it is felt necessary to develop a liquid chromatographic (LC) procedure which would serve as a rapid and reliable method for the determination of Levetiracetam in respective related impurities in bulk and pharmaceutical dosage forms. In the proposed method, related impurities were well separated and eluted before 10min .Finally the method was thoroughly validated for the assay.

EXPERIMENTAL^12-16

Instrumentation : The waters LC system equipped with 2489 pump and 2996 Photodiode detector was used .The output signal was monitored and integrated using waters Empower 2 software

Solutions:

Preparation of pH 2.8 buffer solution:

1.36g of Potassium dihydrogen phosphate and 0.61g of Sodium 1-Heptane Sulphonate was taken in 1000mL of milli-Q water in a suitable container and mixed well , pH of the solution was adjusted to 2.8±0.05 with ortho Phosphoric acid.

Preparation of Mobile phase:

A mixture of pH 2.8 buffer, Acetonitrile in the ratio 90:10 (v/v/v) was prepared and filtered through 0.45µm nylon membrane filter prior to use.

Diluent: Mix Milli Q water and Methanol in the Ratio 70:30(v/v)

Fig-1 HPLC CHROMATOGRAM OF LEVETIRACETAM AND ITS RELATED COMPOUNDS

TABLE-1 SYSTEM SUITABILITY REPORT

Compound	Tailing Factor ^a	Resolution^a	%RSD^a
Levetiracetam	1.1	----	0.8
A	1.1	7.8	1.2

^a Number of samples analyzed are six

Preparation of Standard solution(50µg/ml): About 50mg of Levetiracetam(LEVI) working standard was accurately weighed and transferred in to a 100mL volumetric flask and dissolved in 70ml of diluent and diluted to volume with diluent and mixed well. Further 5mL of the Resulting solution was taken into 50mL volumetric flask and made up to volume with diluent and mixed well. Solution was filtered through 0.45µm nylon membrane filter prior to use.

Preparation of Test Solution: Twenty tablets were taken weighed and powdered .Sample equivalent to About 100mg of Levetiracetam(LEVI) was accurately weighed and transferred in to a 100mL volumetric flask 70ml of diluent was added and sonicated for 30min and diluted to volume with diluent and mixed well. Further 5mL of the Resulting solution was taken into 50mL volumetric flask and made up to volume with diluent and mixed well. Few ml was taken and centrifuged at 2500rpm.5ml of the Clear Centrifuge is taken in to 50ml volumetric flask and made up to volume with diluent. Filtered through 0.45µm nylon membrane filter prior to use.

Preparation of Degradation samples for Specificity Study:

For Acid degradation Levetiracetam sample was refluxed with 0.1N HCl at 70ºC for 10min and then neutralized by adjusting pH to 7.0 with 0.1N NaOH .The Solution was further diluted to required concentration with diluent.

For basic degradation Levetiracetam sample was refluxed with 0.1N NaOH for 5min and then neutralized by adjusting pH to 7.0 with 0.1N HCl .The Solution was further diluted to required concentration with diluent.

For Oxidative degradation Levetiracetam sample was refluxed 1%H₂O₂by heating on mantle at 40ºC for 4 hours. The Solution was further diluted to required concentration with diluent.

For Photolightic Stress the samples were exposed to UV at 254nm for 107hrs 30min and visible light for 107hrs 30min s meeting the specification of ICH i.e UV(200watt/m²) and Visible(1.2million Lux hours).

For Thermal Degradation Samples were Exposed to Temperature at 80ºC for 17hrs. The above stressed samples i.e Photolightic and Thermal stress samples solutions were prepared to required concentration with diluent.

Chromatographic Conditions:

A Hypersil gold (150 x 4.6mm;5µm packing) column was used for analysis at column temperature 25ºC. The mobile phase was pumped through the column at a flow rate of 1.0mL/min. The Sample injection Volume was 20µL.The photodiode array detector was set to a wavelength of 220 nm for the detection.

RESULTS AND DISCUSSION

Method development^11-17

Separation of Known degrading impurities

To develop a suitable and robust LC method for the determination of Levetiracetam different mobile phases and columns were employed to achieve the best separation and resolution. The method development was started with a C18 column using a mobile phase of pH 2.8 buffer: Acetonitrile: Methanol in the ratio 75:10:15.In the above condition elution was very broad for Levetiracetam. Early elution with little separation was observed with mobile phase consisting of pH 3.0 buffer: Acetonitrile: Methanol in the ratio 60:25:15.Finally the mobile phase consisting of pH 2.8 buffer: Acetonitrile in the ratio 90:10 was found to be appropriate ,allowing good separation and symmetrical peak at a flow rate of 1.0mL/min using Hypersil Gold 150 x 4.6mm column.. The Chromatogram of Levetiracetam sample spiked with the related compounds using the proposed method is shown in Fig.1.In the proposed method the resolution is more than 2 between the Levetiracetam and Related Compound A .System suitability results of the method are presented in Table 1.Levetiracetam and its related compounds show significant UV absorbance at Wavelength 220 nm. Hence this wavelength has been chosen for detection in the analysis of Levetiracetam.

Column Selection

Based on the retention and separation of the compounds Hypersil Gold (150 x 4.6mm;5µm) column was selected as suitable column for the analysis of Levetiracetam.

METHOD VALIDATION^18-20

The developed LC method extensively validated for assay of Levetiracetam using the following parameters.

Specificity:

Placebo Interference: A study to establish the interference of placebo was conducted. Assay was performed on Placebo in triplicate equivalent to about the weight of placebo in portion of test preparation as per test method. Chromatograms of placebo solutions showed no peaks at the retention time of Levetiracetam peak. This indicates that the excipients used in the formulation do not interfere in estimation of Levetiracetam in Levetiracetam Mesylate tablets..

Interference from degradation products:

A study was conducted to demonstrate the effective separation of degradants from Levetiracetam peak. Separate portions of Drug product, Drug substance and Placebo were exposed to following stress conditions to induce degradation.Stressed samples were injected into the HPLC system with diode array detector by following test method conditions. All degradant peaks were resolved from Levetiracetam peak in the chromatograms of all samples. The chromatograms of the stressed samples were evaluated for peak purity of Levetiracetam using Empower software. In all forced degradation samples, Levetiracetam peak Purity angle is less than purity threshold. From the above results it is clear that the method can be used for determining the stability of Levetiracetam as bulk and pharmaceutical formulations. Fig-2 shows the separation of Levetiracetam from its degradation Products.

FIG-2 HPLC CHROMATOGRAMS OF LEVETIRACETAM AND ITS DEGRADATION PRODUCTS

TABLE-2 RESULTS FOR PRECISION OF TEST METHOD

Sample No.	% Assay
Sample No.	250mg	500mg	750mg
1	101.3	100.9	100.9
2	101.5	101.1	102.0
3	100.1	101.0	101.1
4	101.2	101.0	101.0
5	101.4	100.8	100.2
6	100.6	100.9	101.8
MEAN	101.0	101.0	101.2
%RSD	0.5	0.1	0.6

TABLE-3 ACCURACY IN THE ASSAY DETERMINATION OF LEVETIRACETAM

Sample No.	Spike level	‘mg’ added	‘mg’ found (recovered)	% Recovery	% Average recovery
1	25%	44.055	44.672	101.4	101.0
2		44.104	44.677	101.3
3		44.521	44.699	100.4
4	50%	89.875	90.055	100.2	100.2
5		89.776	89.776	100.0
6		89.578	89.847	100.3
7	75%	135.606	136.148	100.4	100.3
8		135.606	136.013	100.3
9		135.507	135.914	100.3
10	100%	180.842	181.565	100.4	100.6
11		180.842	182.470	100.9
12		180.643	181.366	100.4
13	150%	272.106	274.011	100.7	100.9
14		271.411	274.397	101.1
15		271.312	273.754	100.9

TABLE-4 STABILITY DATA OF LEVETIRACETAM IN STANDARD AND TEST SOLUTIONS

BENCH TOP STABILITY
Time in Days		% Assay of Standard preparation		Difference from Initial		% Assay of test preparation				Difference from Initial
						Test - 1		Test - 2		Test - 1		Test – 2
Initial		99.57*		NA		101.3		101.5		NA		NA
1		99.43		0.1		101.3		101.1		0.0		0.4
2		98.59		1.0		99.8		100.0		1.5		1.5
REFRIGERATOR STABILITY
Time in Days	% Assay of Standard preparation		Difference from Initial		% Assay of test preparation				Difference from Initial
					Test - 1		Test - 2		Test - 1		Test – 2
Initial	99.57*		NA		101.3		101.5		NA		NA
1	99.44		0.1		100.6		100.9		0.7		0.6
2	97.99		1.6		99.7		100.1		1.6		1.4

* Potency of Levetiracetam on as is basis

FIG-3 LINEARITY OF DETECTOR RESPONSE GRAPH

Fig -4 TYPICAL LC CHROMATOGRAM OF FORMULATED LEVETIRACETAM

Linearity of Detector Response: Linearity of detector response was established by plotting a graph to concentration versus average area and determining the correlation coefficient. A series of solutions of Levetiracetam standard were prepared in the concentration range of about 43.444µg/mL to 289.629µg/mL A graph was plotted to concentration in µg/mL on X- axis versus response on Y-axis. The detector response was found to be linear with a correlation coefficient of 0.999. Linearity graph is shown in Fig-3.

Precision of test Method: The precision of test method was conducted by assaying six samples of Levetiracetam tablets. The Average % assay of Levetiracetam in Levetiracetam tablets was found to be 101.0% ,101.0% and 101.2% respectively for 250mg, 500mg and 750mg tablets and RSD was found to be 0.5%,0.1% and 0.6% respectively. The results were given in Table-2.A Typical LC Chromatogram is Shown in Fig-4

Accuracy: A study of recovery of Levetiracetam from spiked placebo was conducted at Six different Spike levels i.e. 25%, 50%, 75%, 100%, 125% and 150% .Samples were prepared by mixing placebo with Levetiracetam raw material equivalent to about of the target initial Concentration of Levetiracetam. Sample solutions were prepared in triplicate for each spike level and assayed as per proposed method. The Slope ,intercept ,% recovery and Correlation Coefficient were Calculated and given in Table-3.The mean Recoveries of Levetiracetam from spiked were found to be in the range of 100.2-101.0%`

Ruggedness: A study to establish the stability of Levetiracetam in standard and test solutions were conducted on bench top and Refrigerator at Initial, 1 day and 2 days. The assay of Levetiracetam i in standard and test solutions were estimated against freshly prepared standard each time. The difference in % assay of Standard and Test solutions from initial to 1 day and 2 days was calculated and given in Table-4. From the above study, it was established that the standard and test preparation was stable for a period of 2days on bench top and Refrigerator.

ROBUSTNESS: A study to establish the effect of variation in mobile phase composition, Flow, Temperature and pH of buffer in mobile phase was conducted. Standard and test solutions prepared as per proposed method were injected into HPLC system. The System suitability parameters and % Assay were evaluated. From the above study the proposed method was found to be Robust.

ACKNOWLEDGEMENTS

The authors wish to thank the Orchid Healthcare for providing the samples of Levetiracetam.

REFERENCES

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Received on 30.03.2009 Modified on 22.06.2009

Asian J. Research Chem. 2(3): July-Sept. 2009 page 253-257

TABLE-2 RESULTS FOR PRECISION OF TEST METHOD

% Assay

250mg

500mg

750mg

101.3

100.9

100.9

101.5

101.1

102.0

100.1

101.0

101.1

101.2

101.0

101.0

101.4

100.8

100.2

100.6

100.9

101.8

101.0

101.0

101.2

0.5

0.1

0.6

99.57*

99.57*