INTRODUCTION:

Lacosamide is a functionalized amino acid that has activity in the maximal electroshock seizure test, and is indicated for the adjunctive treatment of partial-onset seizures and diabetic neuropathic pain. Recent studies indicate that Lacosamide only affects those neurons which are depolarized or active for long periods of time, typical of neurons at the focus of an epileptic seizure, as opposed to other antiepileptic drugs such as carbamazepine or lamotrigine which slow the recovery from inactivation and reduce the ability of neurons to fire action potentials.

Fig 1- Chemical structure of Locosamide

Locosamide is sparingly soluble in water and slightly soluble in acetonitrile and ethanol. Lacosamide is indicated for adjunctive therapy for partial onset seizures in patients with epilepsy over 17 years old. Injection is indicated for short term use when oral therapy is not feasible.

MATERIAL AND METHODS:

Apparatus:

HPLC Water 486 Tunable Absorbance Detector Model No HPLC 610 Series Detector UV-visible Software Conquer Column C₁₈ (250mm × 4.6mm), 5μm id. Filtered using 0.45μ membrane filter Nylon 0.45μ and ultra-sonicated Model: WUC 4L Capacity.

Chemicals and solvents:

Locosamide drug were perches from MSN Laboratory Ltd Hyderabad the marketed formulation of tablets of LAC (Label claim 100mg of Lacosamide, Lacasa, Zydus Cadila Healthcare Ltd.) were perches from local market HPLC grade water, methanol were purchased from E. Merck Ltd. Mumbai, India.

Chromatographic condition:

Column: Grace C18 (4.6 ID×250mm; 5μm) column, Mobile phase: Methanol: water (80:20) at pH 3.0 Flow Rate: 1.0ml/min, Detection Wavelength: 210nm, Run time: 7min, Injection volume: 10μl

Detection Wavelength by UV Spectroscopy:

The absorption maximum of LOC was determined using 10μg/ml working solution of LOC. The solution was prepared in mobile phase (Methanol 35: Phosphate buffer 65 at pH 3.0). The absorption spectrum or UV spectrum recorded for LOC was as shown in Figure 2 and the corresponding absorbance values were as illustrated in Table 1. As shown in Table 1, the DGR has showed absorbance at wavelength 210nm. The absorbance at 210nm was found to be 0.43 which found maximum absorbance.

Figure 2. UV spectrum of Lacosamide

HPLC method development:

Preparation of standard stock solution of LOC:

Weighed accurately 10mg of LAC and transferred to 100ml volumetric flask containing a mixture of Methanol: Phosphate buffer (pH 3.0) (35:65). The volume was made up to the mark using same mixture of mobile phase. The resulting stock solution (100μg/ml) was filtered through 0.45µ membrane filter and sonicated for three cycles each of 10 min.

Preparation of working solution of LOC:

1.0ml stock solution was pipetted out from the above standard stock solution of LAC and transferred to 10ml volumetric flask. It was then diluted up to 10ml using mobile phase to obtain resultant solution of 10μg/ml. This working solution was sonicated for three cycles each of 10 min. Furthermore, this solution was used for optimization of chromatographic conditions.

Selection of mobile phase:

To begin with mobile phase composition of Methanol: Phosphate buffer 35:65 was selected for RP-HPLC trial runs. The separation carried out on C₁₈stationary phase (4.6mm x 250mm, 5µ particle size). The results obtained were evaluated for retention time, peak area, no. of theoretical plates and tailing factor. The conditions for trial runs were as given in Table 1 below.

System suitability testing:

System suitability test was carried out by six repeated measurements of 2μg/ml of LAC and observed for the results obtained. The representative chromatogram obtained for this study was as shown in Figure 14. The result were evaluated for peak area, retention time, number of theoretical plates and tailing factor as shown in Table 7. The results obtained were then subjected to determination of statistical parameters like mean, standard deviation and percent relative standard deviation (%RSD). The results of %RSD for retention time and peak area was found to be 0.21 and 0.44 respectively. The results are found in agreement with the standards prescribed for system suitability testing as per ICH Q2R1 guidelines.

Table 1. Table for chromatographic conditions set for the experiment

Chromatographic Conditions
Column	Waters C18 (250mm×4.6mm), 5μm
Mobile phase	Methanol 35: Phosphate buffer 65 (pH 3.0)
Detection Wavelength	210 nm
Flow rate	1.0 ml/min
Temperature	Ambient
Sample size	10 μl
Run Time	7 min

HPLC method validation:

Linearity and Range:

Aliquots of 0.2, 0.4, 0.6, 0.8, 1.0 and 1.2ml standard stock solution (100µg/ml) were pipetted out and transferred to 10ml volumetric flask and diluted up to 10ml using mobile (Methanol 35: Phosphate buffer 65, pH 3.0) to obtain resultant solution of 2, 4, 6, 8, 10, 12µg/ml respectively. Each of this standard working solution of LAC was injected in triplicate to the given set of chromatographic conditions and mean peak area was determined. Calibration curve was constructed between concentration of standard solutions of LAC and mean peak area of each standard solution.

Precision:

Precision of the method was established by defining three quality control standards across the given calibration range of 2 to 12μg/ml. Precision was studied by evaluating repeatability and system precision (intermediate precision).

Accuracy:

% Accuracy was determined from the particulars obtained for precision study. At this point it was determined from the observations of mean peak area obtained in the case of three QC standards of LOC defined for precision study.

Robustness:

Robustness of the method developed for LAC was performed by deliberate changes in method parameters. In this case, the system parameters like organic concentration of the mobile phase (Methanol) and flow rate were varied as per Table 4. The 4μg/ml concentration standard solution of LAC was selected for this study and it was kept constant throughout all system parameters varied. The preferred concentration (4μg/ml) was injected to given chromatographic conditions three times at each level of change and chromatograms recorded.

Preparation of stock from API:

Accurately weighed 10mg of LAC (API) was added in 100 ml volumetric flask containing some amount of mobile phase and volume was made up to the mark using mobile phase to obtain a concentration of 100μg/ml. The resulting solution was filtered through 0.45μ membrane filter and sonicated for three cycles each of 10 min. From the stock solution 0.4ml was withdrawn using micropipette in triplicate and kept in three different 10ml volumetric flasks, cleaned previously and diluted up to 10ml by using mobile phase to obtain resultant solution of 4μg/ml. These solutions were injected for given chromatographic system in triplicate and mean peak area was determined.

Preparation of standard stock solution from dosage form:

Twenty tablets of LAC (Label claim 100mg of Lacosamide, Lacasa, Zydus Cadila Healthcare Ltd.) were weighed, average weight was determined and powdered. Powder equivalent to 10mg of LAC was transferred to 100ml of mobile phase to obtain the main stock sample solution of LAC 100μg/ml. The resulting sample solution was filtered through 0.45μ membrane filter and sonicated for three cycles each of 10 min. From the main stock sample solution aliquot of 0.3ml was withdrawn using micropipette and transferred to 10ml volumetric flask and diluted up to 10ml using mobile phase to obtain resultant solution of 3μg/ml. Similarly, aliquots of 0.4 and 0.5ml were withdrawn from the sample stock solution (100μg/ml) to obtain the working sample solutions of 4 and 5μg/ml. The three sample solutions of LAC viz. 3, 4 and 5 μg/ml were designated as three levels of percent recovery experiment viz. 75, 100 and 125% respectively.

Preparation of test solution for % recovery by spike method:

4μg/ml solution of LAC (API) was spiked into each of above three sample solutions of LAC viz. 3, 4 and 5μg/ml to obtain test solutions at 75%, 100% and 125% respectively. Each of these three levels was injected in triplicate and mean peak area for each level was determined. The mean peak area obtained on API injection (previously determined) was subtracted from the mean peak area of each of these three levels to obtain peak area corresponding to sample solutions. % recovery was determined

Limit of Detection (LOD) and Limit of Quantitation (LOQ):

The LOD and LOQ were calculated from the slope(s) of the calibration plot and the standard deviation (SD) of thepeak areas using the formulae LOD = 3.3 σ/s and LOQ = 10σ/s. The results were given in Table-8.

RESULTS AND DISCUSSION:

Results of system suitability study are summarized in Table 1. Six consecutive injections of the Standard solution showed uniform retention time, theoretical plate count, tailing factor and resolution For both the drugs which indicate a good system for analysis.

Precision:

Table 4 Observation table for Repeatability and Intermediate precision experiment of LAC with three QC standards across range %

Conc. (μg/ml)	Intra-day precision (Repeatability)			Inter-day precision (Intermediate precision)
	Mean area ± SD	% RSD	Inference	Mean area ± SD	% RSD	Inference
03	174507 ± 1756.28	1.01	Pass	175046.00 ± 1596.27	0.91	Pass
07	382643.67 ± 7130.53	1.86	Pass	381550.67 ± 5798.19	1.52	Pass
11	636520.33 ± 8220.03	1.29	Pass	633114.33 ± 11818.93	1.87	Pass

Accuracy:

Table 5 Observations for percent accuracy determined from data obtained in precision study

Sr. No	Conc. (μg/ml)	Mean Peak Area*	Mean Measured Conc. (μg/ml)	% Accuracy	Inference (Compendial Standard for LAC 95-105 % w/w)
1	3	175046	3.14	104.80	Pass
2	7	381550.67	6.91	98.71	pass
3	11	633114.33	11.50	104.52	pass

Robustness:

Table 6 Observation table for robustness experiment with change in methanol concentration (Mobile phase composition)

Methanol Concentration (%)	Standard Conc. (μg/ml)	Mean peak area*	Mean measured conc. (μg/ml)	% Assay	Inference (Compendial standard 95-105 %w/w)
35	04	217457	3.92	97.94	Pass
38	04	212998	3.84	95.91	Pass
32	04	220327	3.97	99.25	Pass

Table 7 Observations for percent recovery experiment performed for LAC at three levels

% Recovery Level	Conc. of standard spiked (μg/ml)	Conc. of sample (μg/ml)	Mean peak Area of sample conc.*	Amount recovered (μg/ml)	% Recovery	Inference (Standards 95-105% w/w)
75	04	03	167164	3.00	97.95	Pass
100	04	04	211756	3.81	95.37	Pass
125	04	05	269757	4.87	97.20	Pass

Figure 5. Chromatogram of LAC obtained in percent recovery experiment at 125% level

LOD and LOQ:

Table 8 Observations for LOD and LOQ

Standard Drug Solution	LOD (μg/ml)	LOQ (μg/ml)
Lacosamide (LAC)	0.34	1.02

SUMMARY AND CONCLUSION:

A simple, sensitive, precise and accurate RP-HPLC method was productively developed for the quantification of Lacosamide (LAC) as active pharmaceutical ingredient (API). As well, the method was applied for quantification of LAC in marketed tablet dosage form.

Firstly, all reagents and chemicals were procured and LAC was characterized for its purity by determination of melting point and absorption maximum. The optimized and final conditions were set as mobile phase composition, flow rate, wavelength, injection volume and temperature as Methanol 35: phosphate buffer 35 at pH 3.0 percent, 1ml/min, 210nm, 10μl and ambient temperature respectively.

The presented method was further subjected to system suitability testing. The observed results in terms of percent RSD for mean peak area (0.43) and mean retention time (0.21) for this study were found in agreement as per ICH guideline Q2R1.

Additionally, the presented method was validated as per ICH guideline Q2R1 for subsequent parameters in order to establish the presentation of the method throughout schedule analysis of LAC in its life cycle.

1. Linearity and Range

2. Precision

3. Accuracy

4. Robustness

5. Limit of Detection

6. Limit of Quantitation and

7. Percent Recovery

The linearity of the method was established in the concentration range of 02 to 12μg/ml with correlation coefficient of 0.9994. The regression equation was obtained to determine slope and intercept.

The method was evaluated for its indistinguishable performance on frequent measurements. The precision study was carried out in order to confirm the repeatability and intermediate precision of the method. The results obtained showed that the method performed excellent as %RSD obtained on repeated injections of all QC standard were found within acceptance criteria as per ICH Q2R1 guidelines.

Robustness of the method was attained by studying consequence of purposeful changes in system parameters. The flow rate and methanol concentrations were changed to ensure the effect. It was found that the method ascertained to be robust as percent assay of LAC was within acceptance criteria even after deliberate changes up to ±3% in Methanol concentration and ±0.1ml/min in flow rate.

Method was found to be sensitive as the limit of detection and quantitation was 0.34 and 1.02μg/ml in that order.

The applicability of the method for finished product marketed formulation was assessed with tablet dosage form of LAC. The recovery performed at three levels viz. 75%, 100% and 125% was in conformity with the standards prescribed for LAC.

Consequently, ultimately it was concluded that we have approached our objectives by victorious development and validation of RP HPLC method for quantification of Lacosamide. Furthermore, the applicability of the method was also confirmed for routine analysis of LAC in tablet dosage form. The method was also proved specific for determination of LAC in sample matrix.

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Received on 17.08.2021 Modified on 22.11.2021

Asian J. Pharm. Ana. 2022; 12(1):23-28.

DOI: 10.52711/2231-5675.2022.00005

Sr. No.	Parameter	Mean observations	SD	%RSD	Acceptance criteria	Inference
1	Peak Area	112964.83	482.79	0.43	< 2	Pass
2	Retention time	3.83	0.01	0.21	< 0.5	Pass
3	Number of Theoretical plates	5273	--		> 2000	Pass
4	Tailing factor	1.31	--		< 2	Pass

Sr. No.	Conc. of LAC std. solution (μg/ml)	Mean peak Area*
1	02	112887
2	04	217457
3	06	340135
4	08	438102
5	10	547161
6	12	663163