INTRODUCTION:

Ebastine (fig.1) is 1[4-(1,1-Dimethylethyl)phenyl]-4-[(diphenylmethoxy)piperidin-1-yl]butan-1-one with a molecular weight of 496.7. It is a potent and selective, non-sedating second generation H₁ Receptor antagonist used in the treatment of chronic idiopathic urticaria and allergic diseases. It also blocks the receptors of the platelet-activating factor (PAF). It possesses anti-allergic properties by inhibiting the release of cytokines, particularly of the Tumor Necrosis Factor (TNF) in human mast cells and monocytes by blocking of both PAF and histamine receptors.^4,5,6

Various methods have been reported in the literature for the estimation of Ebastine in bulk drug,⁸ plasma^9,12 and various dosage forms.^10,11 The available methods also suffer from drawbacks such as increased retention time, use of high pump pressure and are less cost effective.

Thus the present study involves the development of a simple reverse phase liquid chromatographic RP-HPLC method for the estimation and quantitation of Ebastine from the bulk drugs. The method developed was found to be rapid, sensitive and cost effective over other reported methods.

MATERIALS AND METHODS:

The liquid chromatographic system consisted of following components: Jasco HPLC model containing PU 1580 Intelligent UV-Vis Detector and Rheodyne injector (7725i) with 20 µ l fixed loop.

The Chromatographic analysis was performed using Borwin chromatography software on a HiQ Sil C₈HS column having 250x 4.6 mm ID and 5 µm particle size. The samples were weighed on Mettler electronic microbalance (MT5).

The analytically pure sample of Ebastine was procured from Bal Pharma Pvt. Ltd., Bangalore, India. HPLC grade Methanol (Purity 99.9%) was procured from Merck (Darmstadt, Germany). The HPLC grade water from Millipore purification system was used. Ammonium dihydrogen orthophosphate (AR grade) was procured from Qualigens Fine Chemicals, Mumbai, India.

Figure.1. Structure of Ebastine

Preparation of mobile phase and stock solution:

10 mM Ammonium dihydrogen orthophosphate was prepared. 100 ml of 1 M solution was diluted upto 1000 ml. 200 ml of prepared buffer was taken and the solution was mixed with 800 ml of Methanol and finally the solution was sonicated for 10 min and filtered using Whatman filter paper No.1.

A stock solution of Ebastine was prepared by accurately weighing 10 mg of drug, transferring to 10 ml of methanol and diluting it up to the mark with methanol. The stock solution thus obtained was of 1 mg/ml. Appropriate aiquot was taken to obtain a standard solution of 100 µg/ml of Ebastine. Resultant solution was filtered through Whatman filter paper No. 1. The calibration curve was prepared in the range of 0.1-2 µg/ml.

Chromatographic Condition:

A reverse phase C₈ column, equilibrated with mobile phase Methanol:10 mM Ammonium dihydrogen orthophosphate buffer (80:20 %v/v) was used. The active principle was eluted isocritically and the mobile phase flow rate was maintained at 1.0 ml/min. The effluents were monitored at 255 nm with the detector. The sample was injected using a 20 µl fixed loop, and the total run time was 8 min.

Calibration Curve:

Appropriate aliquots from stock solution of Ebastine were pipetted out from standard stock solution into series of 10 ml volumetric flasks. The volume was made up to the mark with mobile phase to get solutions having concentration range 0.1, 0.3, 0.5, 0.7, 1.0, 2.0µg/ml for Ebastine. Triplicate dilutions of each concentration were injected in to the RP-HPLC system and chromatogram was generated under the conditions as described above.

VALIDATION OF HPLC METHOD:

The proposed RP-HPLC method was validated as per ICH guidelines.^1,2,3

Specificity:

The specificity of the RP-HPLC method was determined by comparison of the chromatogram of standard and sample solution. The parameters like (tR), resolution (Rs) and tailing factor (Tf) were calculated.

Precision:

Precision study was performed to find out intra-day and inter-day variation. It was carried out by estimating the corresponding responses thrice on the same day and on three different days (first, second and fifth day) for 3 different concentration of Ebastine (0.1, 0.5, 1 µg/ml) and the results are derived in terms of relative standard deviation (RSD). The repeatability studies were carried out by estimating response of 3 different concentration of Ebastine (0.1, 0.5, 1 µg/ml) in triplicate.

Accuracy (Recovery studies):

Recovery studies were performed by standard addition method at three levels i.e. 80 %, 100 %, 120 %. A known amount of standard Ebastine was added to preanalysed sample and was subjected to proposed HPLC method.

Detection Limit and Quantitation Limit:

A calibration curve was prepared using concentrations in the range of 0.1-2 µg/ml (expected detection limit range). Detection limit for Ebastine was 0.04 µg/ml and quantitation limit was 0.1µg/ml. The standard deviation of y-intercepts of regression line was determined and kept in following equation for the determination of detection limit and quantitation limit. Detection limit = 3.3 x SD/s; Quantitation limit = 10x SD/s where SD is the standard deviation of y intercepts of regression lines and s is the slope of the calibration curve.

Stability:

In order to demonstrate the stability of the standard solution of Ebastine during analysis, the solution was stored over a period of 24 hr at room temperature and then analysed.

Robustness:

Robustness of the method was studied by changing the composition of organic phase by ± 5 % and pH by 0.2, and also by observing the stability of the drug for 24 hr at ambient temperature in mobile phase.

RESULTS AND DISCUSSION:

RP-HPLC method:

The development of the RP-HPLC method for the determination of drugs has received considerable attention in recent years because of its importance in routine quality control analysis. Different analytical columns with various stationary phases were tested.

Excellent separation was achieved by a RP HiQ Sil C₈ column which was finally used for analysis. A RP-HPLC method was proposed as a suitable method for the estimation of Ebastine in bulk drug. The chromatographic conditions were adjusted in order to provide a good performance of the assay. The method involved a mobile phase consisting of Methanol: 10 mm Ammonium dihydrogen orthophosphate (8:2 v/v) accomplished at 255 nm. The retention time was 5.9 min at flow rate of 1 ml/min. The total run time for an assay was approximately 8 min. The mobile phase was chosen after several trials with other solvent combinations. Mobile phase selection was based on peak parameters (symmetry, tailing), run time, ease of preparation and cost. Figures 2 (A, B, C) show a representative chromatogram using the proposed method. As shown in these figures, Ebastine was eluted forming symmetrical peak and well separated from the solvent front. Observed retention time (5.9 min) allowed a rapid determination of the drug (Table 1).

Figure2. HPLC chromatogram of: (A) Blank, (B) 0.1µg/ml Ebastine, (C) 2µg/ml Ebastine

(B)

(C)

Table1. System Suitability Test Parameters for Ebastine by the Proposed Method

System Suitability Test Parameters	Values
Retention time	5.9
Resolution	4.0
Tailing factor (asymmetric factor)	1.30

Linearity:

The linear range was found to be 2-0.1 µg/ml concentration. The regression equation and correlation (r) obtained by least square regression method were 0.998. In the linearity assay, coefficient of variation (C.V) of the response factor was 4.82. The linearity of the calibration graph and conformity of RP-HPLC value to Beer’s law were proven by the high correlation coefficients (r) for the regression equations.

Accuracy and Precision:

Repeatability is given as intra- and inter-day precision and accuracy evaluated by analyzing three different concentrations of Ebastine. The results are given in Table 1. The precision of the RP-HPLC method was demonstrated by the relative standard deviation (RSD %) of lower than 0.62 % for intra-day and 1.26 % for inter-day

(Table 2)

Table2. Summary of Validation Parameters for the Proposed Method

Parameters	Values
Detection limit (µg/ml)	0.04 µg/ml
Quantitation limit (µg/ml)	0.1 µg/ml
Calibration range (µg/ml)	2 - 0.1 µg/ml
Accuracy (%)	98.05-100.6
Precision (RSD %)
Intraday (n=3)	0.03-0.06
Interday (n=3)	0.29-0.90
Repeatability (RSD, n=3)	0.19-0.61
Correlation coefficient (r)	0.998

RSD indicates relative standard deviation.

Sensitivity:

The LOQ is defined as the lowest concentration on the calibration curve at which both accuracy and precision should be within 20 %. LOQ value of the RP-HPLC method was determined as 0.1µg/ml (Table2).

Stability:

The results for stability studies revealed that for the solutions, retention time and peak area of Ebastine remained almost unchanged.

CONCLUSION:

The present study describes a new and simple RP-HPLC method for the estimation of Ebastine in the bulk drugs. The method is suitably validated and is simple, sensitive, accurate and precise. Therefore the proposed method can be used for quantitation of Ebastine in bulk drugs for routine analysis in quality control.

ACKNOLEDGEMENT:

The author greatly acknowledge Bal Pharma Pvt. Ltd., Bangalore, India for providing authentic gift sample of Ebastine.

REFERENCES:

1. ICH 2QA and 2QB Guidelines Validation of Analytical Procedures. March 1995.

2. M Bakshi, S Singh.et al. Development of validated stability-indicating Assay methods- critical review. J Pharma Biomed Anal. 2002; 28: 1011–1040.

3. Guidance for Industry, Bioanalytical Method Validation, U.S. Department of Health and Human Services FDA May 2001.

4. F. Estelle, R. Simons, “Histamine and H1 antihistaminines in allergic disease, Clinical allergy and immunology; 2002, 17(2): 141-148.

5. Welch MJ, Meltzer EO, Simons FER: H₁-antihistamines and the central nervous system. In Simons FER, editor: Histamine and H₁-antihistamines in allergic disease, ed 2, New York, 2002, Marcel Dekker, Pg no: 337.

6. J Sastre et al. Ebastine in allergic rhinitis and chronic idiopathic urticaria. Allergy. 2008;63 ( Suppl 89): 1-20.

7. J.Vincent, R. Liminana, P.A. Meredith and J.L.Reid et al., The pharmacokinetics, antihistamine and concentration effect relationship of Ebastine in healthy subjects; British Journal of Clinical Pharmacol. 1988, 26(5): 497-502.

8. Sunitha P.G, Niramathi V., Aruna A., Vaidhyalingam V.and Shantha A et al. Spectroscopic methods for the determination of Ebastine in tablets. Indian Drugs. 2003; 40(10)2003: 577-579.

9. Mastuda M, Mizuki Y, Terauchi Y et al. Simulteneous determination of the histamine H₁-receptor antagonist Ebastine and its two metabolites, carebastine and hydroxyebastine, in human plasma using high-performance liquid chromatography. J chromatogr B Biomed Sci Appl. 2001; 575: 173-179.

10. Prabhu S L, Kumar C D, Shirwaikar A et al. Determination of Ebastine in Pharmaceutical formulations by HPLC. Indian J. Pharm. Sci. 2008; 70(3): 403-406.

11. Marcela Z. Arend, Simone G. Cardoso, Felipe K. Hurtado, Aline Ravanello, Fibele A.Lanzanova, Clarice M B. Rolim et al. Development and validation of a stability-indicating LC method for determination of Ebastine in tablet and syrup. 2009: 992-994.

12. Sheng Feng, Ji Jiang, Peng Wang, Dongyang Liu,PeiHu; “Simulteneous determination of Ebastine and its active metabolite in human plasma using LC- MS-MS”; Chromatographia; 2009: 1328-1330.

Received on 10.05.2011 Modified on 01.06.2011

Asian J. Research Chem. 4(7): July, 2011; Page 1164-1167