Reverse Phase High Performance Liquid Chromatography for Validation of Epinastine Hydrochloride in Bulk and Pharmaceutical Dosage Form

 

Rajan V. Rele. Prathamesh P. Tiwatane

Central research laboratory, D.G. Ruparel College, Matunga, Mumbai, 400 016.

*Corresponding Author E-mail: drvinraj@gmail.com

 

ABSTRACT:

The validation method of epinastine hydrochloride from combined dosage form i.e. eye drops was described by high performance liquid chromatography method with separation of drugs on Water symmetry RP18 (150 x 4.6 mm i.d.) and 5 µ particle size.  A mixture of buffer and acetonitrile (73:27 % (v/v)) was constituted as mobile phase. The chromatograms were studied at 220 nm as wavelength. The mobile phase was also used as a diluent.  A validated of method was studied for linear regression, accuracy, method as well as system precision. The robustness study was done for change in wavelength, mobile phase composition and flow rate as per ICH guidelines. The method has been successfully used to analyze epinastine hydrochloride from dosage form i.e. eye drops.

 

Keywords: Epinastine hydrochloride, Tri-ethyl amine, Ortho phosphoric acid.

 

INTRODUCTION

Epinastine hydrochloride is, 3-amino-9, 13-b-dihydro-1Hdibenz[c, f] imidazo [1, 5-a] azepine hydrochloride, is an antihistamine and an inhibitor of histamine release from the mast cell for topical administration to the eyes.

 

HPLC1-5, UV spectrophotometric methods6,7 were reported for determination of epinastine hydrochloride in combined dosage form in literature. This new work presents reproducible reverse phase high performance liquid chromatographic method for assay of epinastine hydrochloride in eye drops as dosage form.  

 

Chemical and reagents:

Standard of epinastine hydrochloride were used which are validated as per pharmacopeia. All chemicals of analytical grade were used such as tri-ethyl amine, acetonitrile and ortho phosphoric acid. The HPLC grade water was used from Millipore.

 

For preparation of standard and sample solutions, the diluent was mobile phase i.e. mixture of buffer of pH 4.22 and acetonitrile (73:27 % v/v).

 

Instrumentation:

The Merck Hitachi HPLC system equipped with

a) D 7200 separation module as auto sampler

b) D- 7400) as UV detector

c) PC based EZ Chrom Elite software.

A analytical balance (0.01 mg) made of Shimadzu was used.

 

Preparation of Standard preparation:

Standard solution:

A 500 μg /ml of epinastine hydrochloride standard solution was prepared by using diluent [mixture of buffer of pH 4.22 and acetonitrile [73:27 % (v/v)] respectively. 

 

Sample preparation:

A eye drop solution equivalent to 5 mg of standard epinastine hydrochloride was used for preparation of sample solution to give concentration as 500 μg /ml of epinastine hydrochloride respectively.  

 

Chromatographic condition:

Chromatographic study was performed on Water symmetry RP18 (150 x 4.6 mm i.d.) with 5 µ particle size column. The mobile phase was buffer of pH 4.22 and acetonitrile [73:27 % (v/v)]. The buffer constituted as 0.01% (v/v) tri-ethyl amine. The pH 4.22 of buffer solution was made with ortho-phosphoric acid. The flow rate of 1.0 ml /min was maintained in study at wavelength 230 nm. and injection volume as 10.0 µl.

 

Method validation:

System suitability:

For study of System and method precision six replicates were used.  From such data, Parameter i.e. theoretical plates (N), asymmetry, resolution and area were calculated. The results are shown in table 1 which indicates system suitability of the system.

 

Table 1: parameters of system suitability

Parameter

Epinastine hydrochloride

Retention time

3.4

Theoretical plates (N),

2251

Area

7150097

Asymmetry

1.68727

 

Specificity:

Specificity study was performed by injecting blank, standards. The chromatogram of the standard and sample assayed are given in figure 1 and 2 respectively.

 

Fig.1: Chromatogram of epinastine hydrochloride (standard)

Fig.2: Chromatogram of epinastine hydrochloride (sample)

 

Linearity:

 From the given data of 50% 80%, 100% 120% and 150%, linear calibration was drawn as peak area (y) of different concentration v/s concentration (x). The data is tabulated in table no. 2. The linearity graph is given in fig no.3.

 

Table 2: parameters of linear graphs

Parameter

Epinastine hydrochloride

Range of linearity

25 -75μg /ml

Slope

138624

Intercept

 52279

Coefficient of correlation

0.9999

 

Fig. 3: linearity graph of epinastine hydrochloride

Table 3: Accuracy - %Recovery –Epinastine hydrochloride

Level

Test

Weight in mg

Area

Quantity added in

 μg /ml

Quantity recovered in μg /ml

% Recovery

Mean recovery

80%

1

5.11

5658033

40.96

40.47

98.81

97.70

2

5.12

5677391

40.96

40.61

99.15

3

5.13

5447422

40.96

38.97

95.13

100%

1

5.10

7108260

51.2

50.85

99.31

99.35

2

5.14

7110400

51.2

50.86

99.34

3

5.17

7114679

51.2

50.89

99.40

150%

1

5.12

8527847

61.44

61.00

99.29

99.41

2

5.14

8535864

61.44

61.06

99.38

3

5.11

8550847

61.44

61.17

99.56

Mean recovery of all level

98.82

 

Accuracy:

For the study of accuracy 80 %, 100 % and 120 % levels were used.  The accuracy was determined as the percentage recovered by the assay. The results of the percentage recovery are enclosed under table no.3.

 

Precision

The study of method precision was carried out in six replicates. The relative standard deviation was calculated and it was in given limits. The results of the same are tabulated in the table no.4.

 

Table 4: Statistical evaluation of the data

Test

Epinastine hydrochloride

 

Weight of test

Area

% assay

Test-1

5.21

7150097

99.13

Test-2

5.22

7160919

99.48

Test-3

5.18

7192857

100.11

Test-4

5.15

7180190

99.35

Test-5

5.23

7160985

100.63

Test-6

5.17

7169780

99.59

 

Mean Assay

99.71

 

SD

0.552

 

RSD

0.553

 

Study of robustness:

It was carried out by Variation in the flow rate by + 0.2 ml /min, mobile phase composition by + 2 % and wavelength by + 5 nm. The results are studied of the analysis of the samples from the results it was interpreted the robustness of the method.

 

RESULT:

The validation of drug by RP-HPLC has great importance in checking the quality of drugs. In reported method, the time of retention for epinastine hydrochloride were 3.4 min. The linear range for epinastine hydrochloride was 25-75 μg / ml respectively

The coefficient of co-relation was 0.9999 (Table no. 2) hence interference peaks of diluent was not observed at the retention time.  Hence method has specificity and suitable in the linear range applied. It gives that a good correlation in the linearity applied. The values relative standard deviations was found to be less than one.  The   mean recovery was 99.71%. %. The study of Variation in the flow rate by + 0.2 ml /min, mobile phase composition by + 2 % and wavelength by + 5 nm has no effect diluents on the drug study.

 

DISCUSSION:

The acceptable limit was found in average recovery and % RSD for assay values of epinastine hydrochloride. The stability of the proposed method was confirmed by study of method and system precision. The acceptable limits were followed in robustness parameters like wavelength, flow rate as well as composition of mobile. The relative standard deviations for replicates were within the acceptable limit.  The retention times were 4.1 minute for epinastine hydrochloride respectively. Hence method required less time to complete one sample validation compare the methods suggested in literatures.

 

CONCLUSION:

The proposed HPLC method is as per ICH guidelines for validation for epinastine hydrochloride respectively. It was found to be accurate and precise. The replicate analysis showed repeatability of results. It showed the robustness and system suitability of method. The Value of retention times was 4.1 minutes for epinastine hydrochloride hence the method is time saving as compared literature methods.  From the above parameters it is observed that the epinastine hydrochloride was validated accurately in less time and with more economy by using the proposed RP-HPLC method in formulation and in bulk. Hence it is strongly recommendation for the quality control to adopt such economical and time saving method for assay of drugs in combined dosage as well as individual assay of both drugs in raw material.

 

ACKNOWLEDGMENT

Authors express sincere thanks to the principal, of D.G. Ruparel College, Mumbai for encouragement and providing laboratory facilities.

 

REFERENCES:

1.      Aparajita Malakar, Bishwajit Bokshi. Development and validation of stability indicating RP-HPLC method for the determination of Epinastine Hydrochloride in Pharmaceutical dosage form, International Current Pharmaceutical Journal. 2012;1(3): 50-55.

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5.      Ramzia I. El-Bagary, Amal Boshra, Maha M. El-Hakeem, Amira M. Abdelra’oof . Three analytical methods for determination of Epinastine hydrochloride in bulk and in ophthalmic solutions, Journal of Chemical and Pharmaceutical Research. 2012; 4(2):1361-1369.

6.      Vkds. Sai, Ritu Kimbahune, Mubeen G, Preeti Karwa, S.Nandini. Application of derivative spectroscopy for quantitative estimation of Epinastine hydrochloride in presence of benzalkonium chloride as preservative in eye drops, International Journal of Biopharmaceutics. 2013; 4(2) : 104-109.

7.      K. Harinadha Baba, C. Rambabu, Riyaz Ahmed Khan and K. Anil Kumar, Assay of epinastine in bulk and its pharmaceutical formulations by extractive spectrophotometry, Rasayan Journal of Chemistry. 2014; 7 (4):359 – 364.

 

 

 

 

 

 

 

Received on 28.02.2023                    Modified on 27.03.2023

Accepted on 13.04.2023                   ©AJRC All right reserved

Asian J. Research Chem. 2023; 16(3):237-240.

DOI: 10.52711/0974-4150.2023.00039