INTRODUCTION:

Ezogabine is chemically known as N-[2-Amino-4-(4-fluorophenyl-methylamino)- phenyl] carbamic acid ethyl ester Ezogabine acts as a neuronal potassium channel opener, for the adjunctive anticonvulsant treatment of partial onset seizures in refractory epilepsy (1-3). Few methods of analysis of ezogabine have been reported like UV and fluorimetric method^[4-6] and HPLC^[7-11]. No High Performance Thin Layer Chromatographic (HPTLC) method reported for Ezogabine in pharmaceutical dosage forms. The present study describes the determination of ezogabine by using a HPTLC method. Unlike HPLC, consumption of mobile phase per sample basis is quite low. This saves cost per analysis and analysis time as well. HPTLC facilitates repeated detection (scanning) of the chromatogram with the same or different parameters. The aim of this study is to develop a simple, precise, rapid, accurate and repeatable HPTLC method for the estimation of ezogabine as per ICH guidelines.

Fig 1: Chemical structure of Ezogabine

MATERIALS AND METHODS^[12-21]

Chemicals and reagents:

Ezogabine was supplied as a gift sample from Torrent pharmaceuticals pvt ltd, Ahmedabad. Reagents used were of analytical grade (MERCK Chem. Ltd., Mumbai). Methanol was selected as the solvent for sample preparation.

HPTLC instrumentation:

Chromatography was performed on 20cm × 10cm aluminum-backed TLC plates coated with 200μm layers of silica gel 60F₂₅₄ (E. Merck, Darmstadt, Germany; supplied by Merck India, Mumbai, India). The plates were prewashed by methanol and activated at 100 – 110 °C for 10 min prior to chromatography. The samples were applied on the plates as 6 mm wide bands, by means of a CAMAG (Muttenz, Switzerland) Linomat-5 sample applicator fitted with a 100μL sample syringe (Hamilton, Bonaduz, Switzerland). Plate was developed to a distance of 8cm using toluene: ethyl acetate: formic acid (3:6:1) as mobile phase in a Camag twin-trough glass chamber previously saturated with mobile phase vapors for 10 min at ambient temperature. Densitometric scanning was performed at 254nm using Camag TLC Scanner 3 equipped with win CATS software version 1.3.0.

Preparation of standard solution:

Standard stock solution containing 1mg/1ml of Ezogabine was prepared in methanol by dissolving 100 mg of Ezogabine in 100ml methanol. Standard solution was further diluted with methanol to obtain working standard solutions in a concentration range of 1000-6000 ng/spot for Ezogabine.

Fig 2. Image of developed plate

Fig 3. Overlay spectrum of Ezogabine

METHOD VALIDATION:

The method was validated as per ICH guide lines ^[12-15] for precision, accuracy, specificity, linearity, reproducibility, LOD and LOQ.

a) Accuracy:

Accuracy of the method was determined by recovery experiments. The reference standards of the respective drug were added to the sample solution at the level of 50%, 100% and 150%. The concentrations of the drugs present in the resulting sample solution were determined by using assay method.

b) Linearity and range:

From the standard stock solutions, a suitable standard solution was prepared. Ezogabine was found to be linear in the range of 1000 to 6000ng/spot. The calibration curve was plotted using peak area Vs concentration of the standard solution. From the calibration curve, the slope and intercept were calculated.

c) Precision:

Precision of the method was determined by:

Intra-day precision

Inter-day precision

1) Intra-day Precision:

Intra-day precision was found out by carrying out the analysis of the standard drug solutions at a concentration of 3000-5000ng/spot of Ezogabine for three times on the same day. The Percentage RSD was calculated.

2) Inter-day precision:

Inter-day precision was found out by carrying out the analysis of the drug solution at a concentration of 2000-4000 ng/spot of Ezogabine for three different days and the Percentage RSD was calculated.

d) Limit of Detection (LOD) and Limit of Quantification (LOQ):

The detection limit of an individual analytical procedure is the lowest amount of analyte in a sample which can be detected but not necessarily quantitated as an exact value. The quantitation limit of an individual analytical procedure is the lowest amount of analyte in a standard which can be quantitatively determined with suitable precision and accuracy. The LOD and LOQ were experimentally verified by the known concentration of a standard solution of ezogabine until the average response approximately 3 or 10 times the standard deviation of the responses for the 6 replicate determinations.

f) Robustness of the method:

The robustness of an analytical procedure is a measure of its capacity to remain unaffected by small changes in the mobile phase composition, mobile phase volume and duration of mobile phase saturation and the effects on the results were examined.

RESULTS AND DISCUSSION:

A HPTLC method was developed for the estimation of Ezogabine.

1. Linearity:

Ezogabine was found to be linear in the range of 1000 to 6000 ng/spot. The correlation coefficient of Ezogabine was found to be 0.999. The linearity range of Ezogabine was shown in Table 1 and chromatogram was shown in Fig 4 to Fig 9. The calibration curves was plotted between peak area and concentration of the standard solutions (Fig: 10).

Fig 4. Chromatogram of standard 1 μg / spot of Ezogabine

Fig 5. Chromatogram of standard 2 μg / spot of Ezogabine

Fig 6. Chromatogram of standard 3 μg / spot of Ezogabine

Fig 7. Chromatogram of standard 4 μg / spot of Ezogabine

Fig 8. Chromatogram of standard 5 μg / spot of Ezogabine

Fig 9. Chromatogram of standard 6 μg / spot of Ezogabine

Table 1. Linearity range of Ezogabine

Concentration (ng/spot)	Ezogabine
Concentration (ng/spot)	R_fvalue*	Peak area*
1000	0.66	6037.8
2000	0.67	8282.9
3000	0.67	10466.4
4000	0.67	12424.9
5000	0.67	14596.0
6000	0.67	16952.4

*- each value is the mean of six observations

The calibration curve showed that linear response was observed over the range of concentrations used in the assay procedure. The range demonstrates that the method is linear.

Fig 10. Calibration curve for Ezogabine

2. Accuracy (Recovery studies):

The accuracy of the method was determined by recovery experiments. A known quantity of the pure drug was added to the pre-analyzed sample at 50%, 100% and 150% levels. The recovery studies were carried out 6 times of each level and the percentage recovery and percentage relative standard deviation were calculated and given in Table 2. The percentage recovery of ezogabine was found to be in the range of 98.25-100.25%.

Table 2. Recovery studies of Ezogabine

S. No	Concentration (μg/spot)	Average Peak Area*	% Recovery
1	3	11176.1	100.25
2	4	12931.9	99.16
3	5	14720.0	98.25

*-Each vaue is a mean of six observations.

3. Precision:

The precision of the method was determined by studying reproducibility and repeatability. The area of drug peaks and percentage relative standard deviation of intraday and inter day were calculated and presented in Table 3. The results revealed that the developed method was found to be reproducible in nature.

4. LOD and LOQ of Ezogabine

Table 4. LOD and LOQ

Parameter	Ezogabine (ng /spot)
LOD	0.102
LOQ	0.309

5. Robustness:

The Robustness studies were performed for the standard solutions and were presented in Table 5. The assay values were within the limits that the developed method is robust.

Table 5. Robustness studies

Parameter	Modification	Ezogabine- Recovery (%)
Mobile Phase Ratio	3:6.5:0.5	99.75
Mobile Phase Ratio	3:5.5:1.5	98.92
Development Distance	245 nm	99.9
Detection Wavelength (nm)	252 nm	99.9
Slit Dimension	5.00 x 0.45 mm, Micro	99.72

6. Assay:

Table 6. Assay of Ezogabine

S. No	Concentration (μg/spot)	Peak area	% Assay (%w/w)
1	2	8185.2	98.75
2	3	11147.3	98.15
3	5	14980.7	100.56

*each value is the mean of six observations

CONCLUSION:

Based on the studies conducted and the results obtained, it can be concluded that the developed HPTLC method is simple, accurate, reproducible, robust and cost effective, duly developed and validated for the determination of ezogabine. Statistical analysis proved that method is reproducible and selective for quantitative determination of Ezogabine. HPTLC method can be used for routine analysis of Ezogabine in their dosage form.

ACKNOWLEDGEMENT:

The authors are thankful to Kovai Medical Center Research and Educational Trust and KMCH College of Pharmacy for providing facility to carry out this research work.

CONFLICT OF INTEREST:

Conflict of interest declared none.

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Received on 21.05.2019 Modified on 19.06.2019

Research J. Pharm. and Tech. 2019; 12(12): 5694-5698.

DOI: 10.5958/0974-360X.2019.00985.5

Concentration (ng/spot)	Intraday (n=6)			Inter day (n=3)
Concentration (ng/spot)	Peak area	SD	% RSD	Peak area	SD	% RSD
EZOGABINE
3000	10460.4	25.23	0.24	10157.5	17.00	0.16
4000	12438.3	36.01	0.28	12645.3	27.71	0.21
5000	14564.2	25.38	0.17	14267.4	25.47	0.17