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Pawar Anil Raosaheb*1,3 And Chaudhari Pravin Digambar2               
1. Research Scholar, Department of Pharmacy, PRIST University, Thanjavur, Tamilnadu, India.
2. Department of Pharmaceutics, Modern College of Pharmacy, Nigdi, Pune, Maharashtra, India.
3. Department of Quality assurance and PG studies, M.E.S. College of Pharmacy, Sonai, Ahmednagar, Maharashtra, India.


A simple, precise and economical spectrophotometric method has been developed for the estimation of Ezogabine in pharmaceutical formulation. Ezogabine (EZG) is an anticonvulsant drug used for the treatment of partial epilepsy. EZG are available in tablet dosage form in a dose of 50 mg, 200 mg and 400 mg. The purpose of present study is to develop a spectroscopic method for estimation of EZG in tablet formulation. The method is based upon estimation of EZG from pharmaceutical formulation by standard curve method. The absorbance was measured at 218.5 nm. The accuracy and reproducibility results are close to 100% with RSD<2. In proposed method precision was studied as repeatability (RSD<2) and inter and intra- day variations (RSD<2) shows the high precision of the method. The LOD for EZG was found to be 1.047μg/mL. The LOQ for EZG was found to be 3.17μg/mL. The results of proposed method have been validated as per ICH guidelines.

Keywords: Ezogabine, accuracy, precision, LOD, LOQ, ICH guidelines.


Ezogabine is chemically ethyl-N-(2-amino-4-{[(4-fluorophenyl) methyl] amino} phenyl) carbamate (Figure.1). Ezogabine (EZG) is an anticonvulsant drug used for the treatment of partial epilepsy. EZG is official in USP.1 Ezogabine also known as Retigabine is a new potential adjunctive treatment for adults with intractable partial seizures. EZG exerts its anticonvulsant effects through a novel mechanism of action that is unique among existing anti epileptic drugs (AEDs). In particular, EZG activates potassium currents in neurons,2 which should lead to hyper polarization of the membrane potential and decreased neuronal excitability under physiological conditions. Numerous studies have demonstrated that EZG is effective and broad spectrum anti epileptic drug in seizure models. Literature survey reveals that few spectrophotometric 3 and  RP-HPLC analytical method 4,5 was reported for estimation of EZG in formulations, EZG can be also estimated by UV method which is based on formation of colored complex.4 The reported methods for estimation of EZG are time consuming, expensive and which can not applicable for routine analysis.  The aim of the present work is to develop and validate a new spectrophotometric method for estimation of EZG in tablet formulation with good accuracy, simplicity, precision and economy over other chromatographic methods and which can be used for routine analysis.

Figure 1: Structure of Ezogabine

Materials and Methods

Instruments and Apparatus

The instrument used in the present study was JASCO double beam UV/Visible Spectrophotometer (JASCO V-630) with a spectral bandwidth of 1 nm and 10 mm a matched quartz cell was used. All weighing during the experiment was done on an electronic balance (Shimadzu BL 320-H).

Reagents and Chemicals

Analytically pure sample of EZG was obtained as a gift sample from Lupin Research Park Aurangabad. The analytical grade reagent methanol acquired from Loba Chemicals Ltd., Mumbai, India and used as solvent for the experiment. Marketed preparation of EZG was procured from local market.

Determination of appropriate UV wavelength

An accurate wavelength was required for the determination of EZG; the appropriate wavelength for the determination of EZG was determined by scanning the EZG solution over the range of 400-200 nm by UV/Visible Spectrophotometer.

Preparation of Stock Standard Solutions

EZG (100 mg) was accurately weighed and transferred to the volumetric flask (100 mL). It was dissolved properly in methanol (60 mL) and diluted up to the mark with distilled water to obtain a final concentration of 1000 μg/mL and used as a stock solution.  From the stock solution working standard solutions ranging from 2-12 μg/mL was prepared by appropriate dilution with distilled water. They were scanned in the UV region of 400-200 nm. The spectrum (Figure 2) was obtained to determine the maximum absorbance (λ max).

Estimation of EZG from Pharmaceutical Formulation by standard curve method

The content of twenty tablets was accurately weighed and crushed into fine powder. A quantity of powder equivalent to 5 mg of EZG was transferred to 100 mL volumetric flask containing 60 mL methanol, the mixture was shaken manually for 20 min and the volume was made up to the mark with distilled water and filtered through whatmann filter paper (no.41). The solution was further diluted with distilled water to give the concentration within Beer’s Law range. Absorbance of this solution was measured at 218.5 nm and concentration of the drug in the tablet formulation was calculated using a calibration curve.

 Method Validation

Validation of the proposed method was done as per ICH guidelines5 by means of the following parameters.


As per ICH guidelines the linearity of an analytical procedure is its ability (within a given range) to obtain test results which are directly proportional to the concentration (amount) of analyte in the sample solution.

The linearity of the response of the drug was verified at 2-12 μg/mL concentrations. The calibration graph was obtained by plotting the absorbance versus the concentration and then linear regression analysis was performed. The equation of the calibration curve for EZG obtained y=0.0125x+0.021, the calibration curve was found to be linear in the concentrations 2-12 μg/mL. The correlation coefficient (r2) was 0.991.

Accuracy and Precision

The accuracy of an analytical procedure expresses the closeness of agreement between the value which is accepted either as a conventional true value or an accepted reference value and the value found. The precision of an analytical procedure expresses the closeness of agreement (degree of scatter) between a series of measurements obtained from multiple sampling of the same homogeneous sample under the prescribed conditions. The accuracy of the proposed methods was checked by recovery studies of the standard addition method. The precision of the analytical method was checked by repeated assay of the tablet formulation of EZG solutions (10μg/ml) without changing the parameter of the proposed spectrophotometric method.


Specificity is the ability to assess unequivocally the analyte in the presence of components which may be expected to be present. Typically these might include impurities, degradants and matrix 6 etc.

Limit of Detection

According to ICH guidelines 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. Limit of detection can be calculated using the following equation as per ICH guidelines.

LOD = 3.3 × N/S

Where, N is the standard deviation of the absorbance of the EZG and S is the slope of the corresponding calibration curve.

Limit of Quantification

The Quantitation limit of an individual analytical procedure is the lowest amount of analyte in a sample which can be quantitatively determined with suitable precision and accuracy. The Quantitation limit is a parameter of quantitative assays for low levels of compounds in sample matrices, and is used particularly for the determination of impurities and/or degradation products. Limit of quantification can be calculated using the following equation as per ICH guidelines.

LOQ = 10 × N/S

Where, N is the standard deviation of the absorbance of the EZG and S is the slope of the corresponding calibration curve.                      


The ruggedness of the analytical method is the degree of reproducibility of test results obtained by the analysis of the same samples under a variety of conditions such as different laboratories, different instruments, different lots of reagents, different temperatures, different days and different analysts, etc. It is normally expressed as the lack of influence on test results of proportional and environmental variables of the analytical method. For ruggedness study, the concentrations of analyte are measured using different parameters such as;

  • Different operator in same laboratory
  • Reagents of different manufacturers
  • Different laboratory


The absorbance of the EZG was measured at 218.5 nm (Figure 2) and calibration curve was plotted as concentrations versus absorbance (Figure 3). The Beers law was obeyed in this concentration range of 2-12 μg/mL with a correlation coefficient (r2) of 0.991 (Table 1). The accuracy was calculated by the standard addition method, the results of the accuracy study are the apparent from the data are close to 100 % and the value of RSD was found to be < 2 (Table 2). The percent recovery was found to be nearly 100% indicating accuracy of the method. The Relative Standard Deviation (RSD) for intra-day analysis of EZG was found in the range of 0.080, RSD for Inter-day analysis of EZG was found to be 0.1098 (Table 3) which shows the high precision of the method. The proposed method was found to be specific as there is no interference from other excipients.

The LOD for EZG was found to be 1.047 μg/mL. The LOQ for EZG was found to be 3.17μg/mL. The ruggedness of the proposed method was studied by different analyst, different reagent manufacturers and different laboratory condition. The results of ruggedness study obtained RSD<2 (Table 4). Marketed brand of tablet (Potiga 50) was analyzed, the amount of EZG determined by proposed method was found to be 98.93%.

                                               Figure 2: Spectrum of EZG in distilled water.

Figure 3: Calibration curve of

Table 1: Absorbance and conc. data of EZG at 218.5 nm

Sr. No. Conc. Absorbance
1 2 0.317
2 4 0.5248
3 6 0.7706
4 8 0.9791
5 10 1.2117
6 12 1.5288

Table 2: Accuracy study data of proposed method in marketed formulation (Victoza)

Level of

Recovery (%)

Amount of EZG



Added conc. of EZG


Total amount

recovered (μg)

% Recovery S.D.* RSD*
80 5 4 8.92 99.11 0.9433 0.3144
5 4 8.99 99.88
5 4 8.82 98.00
100 5 5 9.97 99.70 0.4386 0.1482
5 5 10.04 100.40
5 5 9.96 99.60
120 5 6 10.88 98.90 0.8857 0.2952
5 6 10.85 98.63
5 6 11.03 100.27

*average of three replicates

Table 3: Precision (Repeatability) study results of EZG


Sample number

Assay of EZG as % of labeled amount

(Intra-day precision)


(Inter-day precision)

1 98.69 99.63
2 99.65 99.45
3 100.05 98.89
4 98.85 100.54
5 99.12 99.36
6 99.25 98.65
Average 99.343 99.42
RSD±SD 0.080±0.4846 0.1098±0.6589

Table 4: Ruggedness study of EZG

Parameters Amount of EZG

Present (μg)

Amount of EZG*

Found (μg)

Labeled claim*

± SD (%)

Analyst-I 50 49.58 99.16±0.480 0.08
Analyst-II 50 49.12 98.24±0.3623 0.0603
Reagent-I 50 48.99 97.98±0.7962 0.1327
Reagent-II 50 48.48 96.96±1.1476 0.1915
Laboratory-I 50 49.40 98.8±0.4476 0.0743
Laboratory-I 50 48.99 97.98±0.6551 0.1091

* indicates average of six estimation


The wavelength was used for the analysis of the drug was 218.5 nm at which the calibration curves was prepared for the EZG. The linearity was found in the concentration range of 2-12 μg/mL (r2 = 0.991). The accuracy of the method was determined by calculating mean percentage recovery. It was determined at 80, 100 and 120 % level and data are presented in (Table 2) which shows the method accuracy. In proposed method precision was studied as repeatability (RSD<2) and inter and intra-day variations (RSD<2) shows the high precision of the method. The LOD and LOQ for EZG was found to be 1.047 μg/mL and 3.17μg/mL respectively .The ruggedness of the methods was studied by two different analysts, Reagents of different manufacturers and Different laboratory using the same operational and environmental conditions. The developed method for estimation of EZG in tablet dosage form was found to be simple, accurate, reproducible, sensitive, economic, and which can be used for routine analysis and quality control of EZG formulation.


The developed method was found to be simple, sensitive, accurate, precise, reproducible, and can be used for routine quality control analysis of EZG in bulk and pharmaceutical formulation. Thus the reported method is of substantial importance and has great industrial applicability for quality control and analysis of EZG in a dosage form. By observing validation parameter and statistical data, the proposed method was found to be satisfactory over other reported chromatographic methods.


The authors are thankful to Principal Dr. V. K. Deshmukh, M.E.S. College of Pharmacy and secretary, Honorable Prashant Patil Gadakh, Mula Education Society, Sonai, for encouragement and availing of the necessary facilities during the course of investigation. Authors are also gratified to Lupin Research Park, Aurangabad, India for providing a gift sample of Ezogabine.


  • United State Pharmacopeia and National Formulary (USP 29/NF 24). Pharmacopoeial Convention, Rockville MD, US,
  • Rundfelt C. The new anticonvulsant Retigabine (D-23129) acts as an opener of K+ channels in neuronal cells.. Europian Journal of Pharmacology. 1997; 336:243–249.
  • Satyanarayana PVV, Alavala SM. New spectrophotometric methods for the Quantitative estimation of Ezogabine in formulations International Journal of Research in Pharmaceutical Chemistry. 2012; 2(4):1093-1098.
  • V.V. Satyanarayana, and Alavala Siva Madhavi ,”Validated RP – HPLC Method for the Estimation of Ezogabine in Tablet Dosage Form”, IJRPBS volume 2012;3(2), 955-959.
  • Lakshmi , K.Saraswathi , T.V.Reddy, RP-HPLC method development and validation for the analyisis of ezogabine in pharmaceutical dosage forms, Int.J.A.PS.BMS,JAN-MAR.2012;1(1), 7-14.
  • International Conference on Harmonization (ICH), Draft guidelines on validation of Analytical Procedure, Definition and Terminology. Geneva: Federal Register; 1995;11260-62.
  • Beckett AH, Stenlake JB. Practical pharmaceutical chemistry, 4th edition, part -2, NewDelhi, CBS publication, 1997; 285-288.
  • Wang X et al, “Identification and characterization of four process-related impurities in retigabine”, J Pharm Biomed Anal. 2012 Dec, 71:148-51. 13.
  • Damodar Katasani, Srinu Bhogineni and Bala Ramanjaneyulu. New Spectrophotometric methods for the quantitative estimation of Aztreonamin formulations, J.Atoms and Molecules. 2012; 2(1):109–116.
  • Valeant Pharmaceuticals Announces Preliminary Results From Its Phase IIa Retigabine Study for the Treatment of Post herpetic Neuralgia (PHN) (Press release). PRN ewswire. 2009-08-Retrieved 2011;06-13. 15.
  • ICH Q2 (R1). Validation of analytical procedures, Text and methodology, International Conference on Harmonization, Geneva. pp. 2005; 1-17.