INTRODUCTION:

Lacosamide chemically is N, N-acetyl-N-benzyl-D-homoserinamide^[1] and is a functionalized amino acid that has activity in the maximal electroshock seizure test, unlike antiepileptic drugs that are believed to act through voltage-gated sodium channels, lacosamide acts by enhancing slow inactivation only at less depolarized membrane potentials that is it affects only neurons which are depolarized or active for long periods of time, typical of neurons at the focus of an epileptic focus. Lacosamide is not official in any pharmacopoeia. Literature survey revealed methods for estimatiom of lacosamide by RP-HPLC in plasma^[2-4], LCMS/MS in rat plasma^[5] , pharmacokinetic investigation of lacosamide inhuman plasma and veterinary studies^[6], degradation studies^[7,8]and HPLC^[9,10]UV method^[11] in dosage form . The aim of the present study was to develop simple and economical methods for routine analysis of lacosamide.

MATERIALS AND METHODS:

Reagents and Chemicals

Lacosamide was obtained as a gift sample from Torrent Pharm Ltd., Gujarat, India. All chemicals and solvents used were of HPLC/ AR grade. Acetonitrile, Potassium dihydrogen phosphate, O-phosphoric acid (Merck, India).

Distilled water was obtained from a Milli-Q system, (Millipore, Milford, MA, USA). Marketed formulation LACOSAM, available in strength of 50mg was procured from local market.

Instrumentation:

Analysis was performed on integrated system of model HP Hewlett-Packards, Agilent 1200 technology, Chemstation Corporation, Germany. G1310A isocratic pump serial DE62958415, Diode array detector, UV detector and a Welchrom C₈column (250 × 4.6 mm, 5μm) was used for chromatographic separation under suitable conditions. The mobile phase consists of phosphate buffer: acetonitrile (850:150 v/v), pH adjusted to 3.0 with ortho-phosphoric acid at a flow rate of 1.5 ml/min and the run time was 15 min. The detection of the drug was carried out at 215 nm.

Standard solution:

Method-1:

Stock solution of Lacosamide was prepared by dissolving 50 mg of Lacosamide in 100 ml of diluent (water: acetonitrile in ratio of 800:200). Working solution was prepared by further dilution of stock solution suitably with diluent to get a conc. of 50 µg/ml. Appropriate aliquots of the standard stock solution were pipetted out into calibrated 10 ml volumetric flask and the volume was made up to the mark with mobile phase so as on get a concentration of 25-75 μg/ml. Each concentration was prepared in triplicates and injected into the HPLC system and chromatographed separately. Peak areas were plotted against concentration to obtain standard calibration curve.

Analysis of marketed formulation:

Twenty tablets of Lacosamide formulation of strength 50 mg were powdered and weight equivalent to 100 mg and transfer to 200 ml of volumetric flask, about 175 ml of diluents was added and sonicated for about 45 min with intermittent shaking at room temperature then the volume was made up with same diluent i.e. water: acetonitrile (800:200). It was thoroughly mix and filtered through 0.45μ nylon filter. After which 5 ml of solution was transferred to a 50 ml volumetric flask and made up to the mark with diluent.

Method validation:

The HPLC method was validated in accordance with ICH guidelines.^[12-14]

Accuracy:

The accuracy of the method was studied by the recovery study. To the preanlaysed sample solution (50 μg/ml of Lacosamide) a known quantity of Lacosamide was added at the 50%, 80%, 100%, 120% and 150% level and analysed by the proposed RP-HPLC method.

Sensitivity:

Sensitivity of the method was estimated in term of limit of detection (LOD) and limit of quantitation (LOQ). LOD=3.3×ASD/S and LOQ=10×ASD/S, where ASD is the average standard deviation and S is the slope of the line.

System suitability:

Various system suitability parameters were calculated. It was observed that all the values were within the limits. The statistical evaluation of the method revealed its good linearity, reproducibility and its validation of different parameters and led us to the conclusion that it could be used for the rapid and reliable determination of Lacosamide in tablet formulation.

Specificity and Degradation studies:

Specificity of the stability indicating method was established by separation of the principal peak with degradants during forced degradation. The stress conditions utilized were acid hydrolysis, alkaline hydrolysis, oxidation by peroxide and thermal degradation. Overall these studies were aimed to degrade 10%-30% of the drug. Overall summary of degradation studies are presented under [Table 5].

Method-2:

Stock solution of Lacosamide was prepared by dissolving 100mg of accurately weighed drug in to a 100ml volumetric flask with ethanol to give a conc. of 1mg/ml. Fresh aliquots of stock solution 1 to 5ml were transferred into a series of 10ml volumetric flask to provide concentration of 100-500µg/ml. To each volumetric flask, 3.5ml of 2% of vanillin and 1.5ml of concentrated sulphuric acid were added. The solution in each flask was made up to the mark with ethanol. The absorbance of violet colored chromogen was measured at 613nm against reagent blank.

RESULT AND DISCUSSION:

The optical characteristics such as Beer’s law limit, sandell’s sensitivity, molar extinction coefficient, % relative standard deviation (calculated from five measurement containing average of the amount of the Beer’s law limit) were calculated. Regression characteristics like slope, intercept, correlation coefficient and % range of errors (0.05 and 0.01 confidence limit), LOD, LOQ data of HPLC were calculated. Commercial formulation of Lacosamide was successfully analyzed by proposed method and results are calculated. All the proposed method for estimation of Lacosamide are simple, sensitive, accurate and precise and can be used for the routine analysis of this drug in bulk as well as in pharmaceutical formulation. The results are recorded in table-1.

In the method–1, the parameters optimized were, selection of wavelength for detection, effect of ratio of mobile phase, effect of flow rate. A mixture of Water: Acetonitrile in the ratio of 800:200 v/v. was selected as mobile phase and flow rate of 1.5 min/ml seemed to be ideal for the selected drug. The peaks were well resolved in the column of Welchrom C8, 250 × 4.6mm, 5µ. Using the chromatography condition mentioned above, the chromatograms of standard solution of Lacosamide were recorded. A computer controlled data was used to plot the peak area ratio of standard versus conc. in µg/ml. The Lacosamide showed linearity in the range of 25-75µg/ml. The retention time of Lacosamide was found to be 8.460min. The RP-HPLC method was found to be most sensitive and accurate then other developed method and gave reproducible results.

In method-2, Lacosamide was estimated based on the condensation reaction of Vanillin in the presence of Conc. sulphuric acid, resulting in the formation of violet coloured chromogen which showed absorbance maxima at 613nm. The colour was stable more than 1hour 20min. The method obeys Beer’s law in the concentration range of 100-500µg/ml.

CONCLUSION:

Both the methods developed are validated for linearity, precision, reproducible, accuracy, specificity and inter and intraday variations are presented in tables 2-4. The proposed methods are simple, rapid, accurate, precise and specific. Therefore, the developed methods suitable for routine analysis of pharmaceutical dosage forms and for use in laboratory analysis.

Table 1: Optical characteristics

Parameter	Method-1	Method-3
λmax (nm)	215	613
Beer’s law limit (µg/ml)	25-75	100-500
Molar absorptivity ( lit/mol^-1cm^-1)		541.8362
Sandell’s sensitivity (µg/ml/0.001abs units)		0.4619
Regression equation( Y*) Slope (b) Intercept (a)	35.352 6.4142	0.0013 0.0054
Correlation coefficient (r)	0.9981	0.9998
% RSD	0.2155	0.5337
Confidence limits with 0.05 level 0.01 level	1722.498±3.920 1722.498±5.8266	0.6242±0.003176 0.6242±0.005583
LOD ( µg/ml )	0.03855	0.03332
LOQ (µg/ml )	0.127235	0.010097

Table-2: Result of precision studies of lacosamide

Drug	Label Claim	Precision Intraday				Precision Interday
		% Recovery ± SD		% RSD		% Recovery ± SD		% RSD
		Method 1	Method 2	Method 1	Method 2	Method 1	Method 2	Method 1	Method 3
Lacosamide	50mg	93.55 ± 0.007647	101.66 ± 0.002520	2.73952	0.4005	93.55 ± 0.005394	101.66 ± 0.003488	1.7260	0.5538

Table 3: Assay and % recovery

Pharmaceutical

Dosage form

Labelled Amount (mg)

Amount found by proposed method ( mg )

% Recovery of proposed method

Method-1

Method-2

Method-1

Method-2

F-1 (LACOSAM)

49.93

50.83

99.86

101.66

Table 4: Degradation data for method 1

Drug	Degradation	AUC of formulation	AUC of pure drug	% Recovery of pure drug	%Recovery of formulation
Lacosamide	Acid	6942179	6856631	99.60	99.42
	Base	5886201	5823037	83.99	84.38
	Oxidative	6314760	6514840	93.89	89.4
	Thermal	6402678	6659681	95.06	91.76

Table 5: Accelerated data of degradation studies for method 1

Sl. No.	Parameter	No. Of Theoretical Plate		Capacity Factor
1	Acid Degradation	Std.	Test	Std.	Test
1	Acid Degradation	9624	9785	807.00006	807.00006
2	Base Degradation	9792	9589	806.33337	806.33337
3	Oxidative Degradation	8316	11117	807.66675	796.33337
4	Thermal Degradation	11184	11011	793.66675	791.66675

ACKNOWLEDGEMENT:

Authors are thankful to Nitte University for providing the necessary facilities in carrying out the present study.

REFERNCES:

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12. ICH-guidelines Q2A. Validation of Analytical Procedures: Definition and terminology (CPMP III/5626/94). Geneva, Switzerland:1994

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14. ICH-Guidelines Q2(R₁). Validation of Analytical Procedures: Text and Methodology. Geneva, Switzerland: 2005

Received on 19.03.2013 Modified on 02.04.2013

Research J. Pharm. and Tech. 6(5): May 2013; Page 553-555