A RP-HPLC Method Development and Validation for the Estimation of Olopatadine in Bulk and Pharmaceutical Dosage forms

 

Saroj Kumar Raul¹*, B.V.V Ravi Kumar², Ajaya Kumar Patnaik³, Nagireddy Neelakanta Rao⁴

¹M .R. College of Pharmacy, Phool-Baugh, Vizianagaram, A.P, India.

²Roland Institute of Pharmaceutical Sciences, Berhampur, Orissa, India.

³Departments of Chemistry, Ravenshaw University, Cuttack, Orissa, India.

⁴Suraksha Pharma Ltd, Suraksh Towers, Hyderabad, A.P, India.

 

ABSTRACT:

A simple, selective, linear, precise and accurate RP-HPLC method was developed and validated for rapid assay of Olopatadine in pharmaceutical dosage form. Isocratic elution at a flow rate of 1.0 mL min -1 was employed on a Kromasil C18 column at ambient temperature. The mobile phase consisted of methanol: phosphate buffer 60:40 (v/v) and the detection wavelength was at 246 nm. Linearity was observed in concentration range of 10-60 μg/mL. The retention time for Olopatadine was 2.4 min. The method was validated as per the ICH guidelines. The proposed method can be successfully applied for the estimation of Olopatadine in pharmaceutical dosage forms.

 

 

 

INTRODUCTION:

The drug Olopatadine (Fig1) generally available as Olopatadine Hydrochloride, is a dibenzoxipine derivative used for systemic treatment of allergic rhinitis, urticaria, and bronchial asthma. Chemically it is described as {(11Z)‐11‐[3‐(dimethylamino) propylidene]‐6,11 ‐dihydrodibenzo [b,e] oxepin‐2‐yl}acetic acid¹. Olopatadine is an anti‐histaminic, with selective H1 ‐receptor antagonist activity as well as anticholinergic and mast cell stabilizer. Its principal effects are mediated via inhibition of H1 receptors. These drugs selectively bind to H1 receptors there by blocking the actions of endogenous Histamine. They act on the bronchi, capillaries, and other smooth muscles. Olopatadine is commercially available as tablets, ophthalmic solution and nasal spray.

 

Literature survey reveals that few spectrophotometric method^2-4 HPLC method⁵, HPTLC methods⁶, LC-MS method ^7-8 has been reported for the estimation of Olopatadine. The aim of the present study is to develop a simple, precise and accurate reversed-phase HPLC method for the estimation of Olopatadine in pharmaceutical dosage form as per ICH guidelines⁹.

 

Figure 1: Chemical structure of Olopatadine

 

MATERIALS AND METHOD:

Instrumental and analytical conditions

The HPLC analysis was carried out on Waters HPLC system (2695 module) equipped with 2487 dual lambda detector with auto Sampler and running on Waters Empower software. The column used is Kromasil C₁₈ (150 × 4.6 mm, packed with 5 µm) and detection was performed at 246 nm. The injection volume of sample was 20 µL and the run time was 5 minutes. An isocratic mobile phase containing methanol and 0.02 M phosphate buffer   at 60: 40 (v/v) at the pH 3.5 was carried with the flow rate at 1.0mL min^-1. The mobile phase was filtered through 0.4µm membrane filter and degassed before use.

 

Reagents and chemicals

Olopatadine working standard was kindly gifted by pharmatrain, Hyderabad. Tablets were purchased from local pharmacy manufactured by Torrent Pharmaceutical Ltd (Allenil). Ultra pure water was obtained from a millipore system. HPLC grade methanol was obtained from Merck (India) limited. All other chemicals used were AR grade. The optimum chromatographic conditions were summarized in table 7.

 

Preparation of mobile phase

Dissolved 2.7218 g of Potassium Dihydrogen orthophosphate in 1000 mL of water and mixed, pH adjusted to 3.5 using ortho phosphoric acid, sonicated to degas the buffer. Transferred 600 volumes of methanol and 400 volumes of buffer into a 1000 volumes mobile phase bottle and mixed. Then sonicated up to 15 minutes for degas the mobile phase and filtered through 0.45 μm filter under vacuum. The same mobile phase was used as diluent.

 

Preparation of Standard Solution

Accurately weighed about 10 mg of Olopatadine and transferred into a 10mL volumetric flask and 7 mL of diluent was added and sonicate to dissolve it completely and the volume was adjusted with the mobile phase to get stock solution of 1000 µg/mL. Then 0.3 mL of stock solution is transferred into 10 ml volumetric flask and make up to volume with mobile phase and filter through 0.45 μm filters, which gives a solution of strength 30 µg/mL. 

 

Preparation of sample solution

Weigh 20 Olopatadine tablets and calculate the average weight. Accurately weigh and transfer the sample equivalent to 50 mg of Olopatadine into a 50 ml volumetric flask. Add about 25ml of diluent, sonicate to dissolve it completely and make volume up to the mark with diluent. Mix well and filter through 0.45 μm filter. Further pipette 0.3 ml of the above stock solution into a 10 ml volumetric flask and dilute up to the mark with diluent. Mix well and filter through 0.45 μm filter.

 

Method Validation

The objective of the method validation is to demonstrate that the method is suitable for its intended purpose as it is stated in ICH guidelines. The method was validated for linearity, precision, accuracy, specificity, limit of detection, limit of quantification, robustness and system suitability.

 

Linearity

From the standard stock solution, the various dilutions of Olopatadine in the concentration of 10, 20, 30, 40, 50 and 60 µg/mL were prepared. The solutions were injected using 20 μL injection volumes in to the chromatographic system at the flow rate of 1.0 mLmin^-1 and the effluents were monitored at 246 nm, chromatograms were recorded. Calibration curve of Olopatadine was obtained by plotting the peak area ratio versus the applied concentrations of Olopatadine, given in table 1. The linear correlation coefficient was found to be 0.999, shown in figure2.

 

Table 1: Linearity of Olopatadine

Concentration (µg/mL)	Average area
10	2154393
20	2844588
30	3540094
40	4339758
50	4998535
60	5694041

 

Figure 2: Linearity curve of Olopatadine

 

Precision

Repeatability of the method was checked by injecting replicate injections of 30 μg/mL of the solution for six times on the same day as intraday precision study of Olopatadine and the % RSD was found to be 0.23, given in table 2.

 

Table 2: Precision  of Olopatadine

Injections	Area
1	3803446
2	3802829
3	3806638
4	3816350
5	3825994
6	3812344
Mean	3811267
SD	8918.3
% RSD	0.2340

 

Accuracy

Olopatadine reference standards were accurately weighed and added to a mixture of the tablets excipients, at three different concentration levels (50, 100 and 150 percent). At each level, samples were prepared in triplicate and the recovery percentage was determined and presented in table 3.

 

 Table 3: Accuracy of Olopatadine

%Conc	Area	Amount added (mg)	Amount found (mg)	% Recovery	Mean Recovery
50%	1792969	5.0	4.94	98.8 %	99.8 %
100%	3639398	10.0	10.0	100 %
150%	5512292	15.0	15.1	100.6 %

 

Table 4: Robustness of Olopatadine

Parameters	Adjusted to	Average Area	Rt	SD	% RSD
Flow rate as per method 1.0mL/min	0.8 mL/min	3816250	2.411	8918.9	0.23
	As it is	3811360	2.409	8993.7	0.24
	1.2ml/min	3809350	2.414	9897.6	0.26
Mobile phase composition Methanol:Buffer (60:40)	Methanol:Buffer (55:45)	3912521	2.412	9972.8	0.25
	As it is	3816350	2.412	9987.6	0.26
	Methanol:Buffer (65:35)	3742312	2.414	8995.8	0.24

 

 

Specificity

Spectral purities of Olopatadine chromatographic peaks were evaluated for the interference of the tablet excipients as per the methodology. In the work, a solution containing a mixture of the tablet excipients was prepared using the sample preparation procedure to evaluate possible interfering peaks and no interference peaks were observed.

 

Robustness

To determine the robustness of the method, two parameters (flow rate, composition of mobile phase) from the optimized chromatographic conditions were varied. Statistical analysis showed no significant difference between results obtained employing the analytical conditions established for the method and those obtained in the experiments in which variations of parameters were introduced. Thus the method showed to be robust which is shown in table 4.

 

Ruggedness

Inter day variations were performed by using six replicate injections of standard and sample solutions of concentrations which were prepared and analyzed by different analyst on three different days over a period of one week. Ruggedness also expressed in terms of percentage relative standard deviation and statistical analysis showed no significant difference between results obtained employing different analyst.

 

Detection and quantitation limits

According to the determined signal-to-noise ratio, Olopatadine presented limits of detection of 0.14 μg/mL and limits of quantitation of 0.5μg/mL, where the compounds proportion found in the sample solutions injected on to the chromatograph. However, the objective of the method is the quantitation of Olopatadine so that the values obtained should be considered as the limit of method sensitivity.

 

System Suitability

System suitability tests were carried out on freshly prepared standard stock solutions of Olopatadine and it was calculated by determining the standard deviation by injecting standards in six replicates at 6 minutes interval and the values were recorded and the system suitability parameters are shown in table 5.

 

Table 5: System Suitability  of Olopatadine

 

Assay of Olopatadine tablet

Three different batches of Allenil were analyzed using the validated method. For the analysis, six replicates of each batch were assayed. Twenty tablets were weighed and finely powdered. An accurately weighed portion of the powder, equivalent to about 50mg of Olopatadine was transferred to a 50 ml volumetric flask followed by the addition of 25 ml of mobile phase. The solution was sonicated for 3 minutes and volume adjusted with the mobile phase then filtered through 0.45 μm membrane filter. Further dilutions were made to get the final concentration equivalent to 30 µg/mL of Olopatadine. The mean peak area of the drug was calculated and the drug content in the tablets was quantified and the results were presented in table 6.

 

Table 6: Contents of Olopatadine in tablets (n=6)

Sample tablet	Batch	Labelled amount (mg)	Amount found (mg)±SD	%Amount found
Allenil (5mg)	1 2 3	5 5 5	4.99±0.14 4.96±0.05 5.02±0.11	99.8 99.2 100.4

S.D=Standard Deviation

 

All the analyzed batches presented Olopatadine were very close to the labeled amount. The Olopatadine content in the tablets samples varied from 99.2 to 100.4%.

 

RESULTS AND DISCUSSION:

The nature of the sample, its molecular weight and solubility decides the proper selection of the stationary phase. The drug Olopatadine was preferably analyzed by reverse phase chromatography and accordingly C₁₈ column was selected. The elution of the compound from the column was influenced by polar mobile phase. The ratio of the methanol to phosphate buffer was optimized to give symmetric peak with short run time.

 

 

Figure 3: Standard Chromatogram of Olopatadine.

 

 

Table7: Developed Chromatographic Conditions

Parameters	Method
Stationary phase (column)	Kromasil C18 (150 × 4.6 mm, packed with 5 µm)
Mobile Phase	60:40 (Methanol : Phosphate Buffer)
pH	3.5 ± 0.02
Flow rate (ml/min)	1.0
Run time (minutes)	5.0
Column temperature (°C)	Ambient
Volume of injection loop (ml)	20
Detection wavelength (nm)	246
Drugs RT (min)	2.4

 

Different mobile phases were tried but satisfactory separation, well resolved and good symmetrical peaks were obtained with the mobile phase of methanol: phosphate buffer at the ratio of 60:40 (v/v).The retention time of Olopatadine was found to be 2.4 min, which indicates a good base line. The RSD values for accuracy and precision studies obtained were less than 2% which revealed that developed method was accurate and precise. The system suitability parameters are given in Table 5. Developed chromatographic method was applied for the determination of Olopatadine in tablet formulation, given in table 7. A typical chromatogram showing the separation of Olopatadine is shown in figure 3.

 

CONCLUSIONS:

A validated RP-HPLC method has been developed for the determination of Olopatadine in tablet dosage form. The proposed method is simple, rapid, accurate, precise and specific. Therefore, it is suitable for the routine analysis of Olopatadine in pharmaceutical dosage form.   

 

ACKNOWLEDGEMENTS:

The authors are thankful to Pharmatrain, Kukatapally, Hyderabad for providing gift sample of Olopatadine and for providing necessary facilities to carry out the research work.

REFERENCES:

1.       http:/www.rxlist.com

2.       Suddhasattya D, Vikram Reddy Y, Swetha B, Sandeep Kumar D, Murthy PN, Sudhir KS, Dhiraj KS, Patro S and Subhasis M, Method development and validation for the estimation of Olopatadine in bulk and pharmaceutical dosage forms and its stress degradation studies using UV-VIS spectrophotometric method. International Journal of Pharmacy and Pharmaceutical Sciences.2 (4); 2010:212-218.

3.       Mathrusri Annapurna M, Hima Bindu G and Divya I. New analytical methods for the determination of Olopatadine (an anti-allergic drug) in eye drops. Drug Invention Today. 4(8); 2012: 441-443.

4.       Khan SP, Pathi PJ, Reddy RP, Reddy KV and Appalaraju N. Extractive Spectrophotometric estimation of Olopatadine using acid dyes in tablet dosage forms. Journal of Pharmacy Research. 5 ( 4); 2012: 2104-2106.

5.       Rao NK, Ganapaty S and Rao LA. Validated RP-HPLC method for the determination of Olopatadine in bulk drug and in pharmaceutical dosage form. International Journal of Pharmaceutical, Chemical and Biological Sciences. 2(4); 2012: 712-717

6.       Mahajan A, Gandhi P, Pandita N, Gandhi SV and Deshpande P. Validated high performance thin layer chromatographic method for estimation of Olopatadine hydrochloride as bulk drug and in ophthalmic solutions. International Journal of ChemTech Research. 2(3); 2010: 1372-1375.

7.       Liang W, Zhou H, Liu D, Hu P and Jiang J. Quantitative determination of Olapatadine in human plasma by high performance liquid chromatography and tandem mass spectrometry. Journal of Chinese Mass Spectrometry. 27;2006:193-197.

8.       Fujita K, Magara H and Kobayashi H. Determination of Olopatadine, a new antiallergic agent, and its metabolites in human plasma by high performance liquid chromatography with electrospray ionization tandem mass spectrometry. Journal of Chromatography B. 731(2); 1999: 345-352.

9.       Validation of analytical procedures: methodology (Q2B), ICH Harmonized Tripartite Guidelines, Geneva, 1996.

 

 

 

 

Received on 17.11.2012        Modified on 20.11.2012

Accepted on 23.11.2012        © AJRC All right reserved