ISSN   0974-3618  (Print)                  www.rjptonline.org

            0974-360X (Online)

 

 

RESEARCH ARTICLE

 

UV-Spectrophotometric Estimation of Olopatadine hydrochloride in Bulk and Pharmaceutical Dosage Form by area under Curve and second Order Derivative Methods

 

Rele Rajan V.*

Central Research Laboratory, D.G. Ruparel College, Matunga, Mumbai 400016.

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

 

ABSTRACT:

Simple and precise UV spectrophotometric methods by second order derivative and area under curve [AUC] have been developed and validated for the estimation of olopatadine hydrochloride in bulk and its pharmaceutical formulation. The standard and sample solutions of olopatadine hydrochloride were prepared in methanol. Olopatadine hydrochloride was estimated at 224 nm for the second order derivative UV-spectrophotometric method (A), while in area under curve (AUC) method (B) the zero order spectrum of olopatadine hydrochloride was measured in between 220 nm to 230 nm. Beer’s law was obeyed in the concentration range of 1 to 16 μg / ml with coefficient of correlation value 0.9994 for second order derivative method. Similarly in AUC method, Beer’s law was obeyed in the concentration range of 1 to 12 μg / ml with coefficient of correlation value 0.9991. These methods were tested and validated for various parameters according to ICH guidelines. The precision expressed as relative standard deviation were of 0.1311 % and 0.5889 % for the above two methods respectively. The proposed methods were successfully applied for the determination of olopatadine hydrochloride in pharmaceutical formulation. Results of the analysis were validated statistically and were found to be satisfactory. The proposed methods are simple, easy to apply, low-cost and require relatively inexpensive instruments.

 

KEYWORDS: Olopatadine hydrochloride, second order derivative spectroscopy, area under curve method, methanol.

 

 

INTRODUCTION:

Olopatadine hydrochloride, chemical name is {(11Z)-11-[3-(di-methyl-amino) propylideine]-6, 11-dihydrodibenzo [b, e] oxepin-2-yl} acetic acid. Olopatadine hydrochloride is a selective histamine H1 receptor-antagonist activity and inhibits the release of histamine from mast cell. It shares many of the pharmacologic effect of mast cell stabilizers. It is used to treat itching associated with allergic conjunctivitis. Its principal effects are inhibition of H1 receptors. The drug selectively binds to H1 receptors there by blocking the actions of endogenous histamine. They act on the bronchi, capillaries, and other smooth muscles1. Literature survey reveals that, HPLC2-4, spectrophotometric5,6, voltametric7 methods for the determination of olopatadine hydrochloride.

 

 

 

Received on 14.01.2015          Modified on 29.01.2015

Accepted on 13.02.2015         © RJPT All right reserved

Research J. Pharm. and Tech. 8(3): Mar., 2015; Page 265-269

DOI: 10.5958/0974-360X.2015.00044.X

 

A new, simple, rapid and reliable UV-spectrophotometric second order derivative and area under curve methods are developed for the determination of olopatadine hydrochloride from pharmaceutical formulation. This method can be used for the routine analysis. In the proposed method optimization and validation of this method are reported.

 

MATERIAL AND METHODS:

Shimadzu UV-1800 was used with 10 mm matched quartz cell to measure absorbance of solution.

A Shimadzu analytical balance with 0.01 mg was used.

 

Chemical and reagents:

Reference standard of olopatadine hydrochloride was obtained from reputed firm with certificate analysis.  All spectral absorbance measurements were made on Shimadzu UV-1800 with 10 mm matched cell.

 

Preparation of standard solution:

About 10 mg of standard olopatadine hydrochloride was weighed accurately and transferred in 100 ml of volumetric flask. About 30 ml of methanol was added and sonicated for 15 minutes. The volume was adjusted up to the mark with absolute alcohol to give concentration as 100 μg /ml.

 

Sample preparation:

About 10 mg of olopatadine hydrochloride sample was weighted accurately and transferred in 100 ml volumetric flask. About 50 ml of methanol was added and sonicated for 10 minutes. The volume was adjusted up to the mark with diluent to give concentration as 100 μg /ml.  Such solution was used for analysis.

 

EXPERIMENTAL:

Method A: second order derivative method

For the selection of analytical wavelength, 10 μg /ml solution of olopatadine hydrochloride was scanned in the spectrum mode from 350 nm to 200 nm by using methanol as blank. The second order derivative spectrum was obtained by using derivative mode by UV probe 2.42  software. From the spectrum, the amplitude of the derivative spectrum was measured at 224. (Fig. 1). Into series of 10 ml graduated flask, varying amount of standard solutions of olopatadine hydrochloride was pipette out and volume was adjusted with methanol as solvent. Solutions were scanned between 350 nm to 200 nm in spectrum mode. The second order derivative spectra were obtained by using derivative mode. Amplitudes of the resulting solutions were measured at between 224 nm by using methanol as blank. The calibration curve was prepared in the concentration range of 1 to 12 μg/ml.  (Fig. 2)

 

Method B: Area under curve (AUC) method

Area under curve method involves the calculation of integrated value of absorbance with respect to the wavelength between two selected wavelengths such as λ1 and λ2. The area under curve between λ1 and λ2 were calculated by UV probe 2.42 software. In this method, 10 μg/ml solution of olopatadine hydrochloride was scanned in the spectrum mode from 300 nm to 200 nm. From zero order spectrum the AUC calculation was done. The AUC spectrum was measured between 220 nm to 230 nm (Fig. 3)

 

Fig. 1. Overlay second order derivative spectra of olopatadine hydrochloride (2-12 μg/ml) showing  absorbance at  224 nm

 

Fig. 2. Calibration curve for olopatadine hydrochloride at 224 nm by second order derivative Spectroscopy

Fig. 3. Area under curve spectrum of olopatadine hydrochloride ( 10 μg/ml) showing area from 220  nm to 230  nm.

 

 

 

Fig. 4. Calibration curve for olopatadine hydrochloride by area under curve spectroscopy

 

 

 

Into series of 10 ml graduated flask, varying amount of standard solutions of olopatadine hydrochloride was pipette out and volume was adjusted with absolute alcohol. Solutions were scanned between 350 nm to 200 nm in spectrum mode. The AUC calculations were done and the calibration curve for olopatadine hydrochloride was plotted in the concentration range of 1 to 16 μg/ml  (Fig. 4). Results of analysis are given in table 1.

 

 

 

Table 1: Values of results of optical and regression of drug

Parameter

Second  order derivative method

Area under curve (AUC) method

Detection Wavelength (nm)

224

220-230

Beer Law Limits (µg/ml)

1-12

1-16

Correlation coefficient(r2)

0.9994

0.9991

Regression equation (y=b+ac)

 

 

Slope (a)

0.0006

0.0248

Intercept (b)

0.00005

0.0037

 

 

Table 2: Results of recovery of olopatadine hydrochloride for second order derivative method

Amount of Sample Added in (µg/ml)

Amount of Standard Added in (µg/ml)

Total amount recovered

Percentage recovery (%)

Standard deviation

Percentage of relative standard deviation

(C.O.V.)

2

0

2.0238

101.1905

0.1781

8.8039

2

2

3.9761

99.4047

0.1781

4.4810

2

4

6.1190

101.9841

0.1854

3.030

2

6

7.9761

99.7023

0.1781

2.2338

 

 

 

 

Mean=0.1799

Mean= 4.4373

 

Table 3: Results of recovery of olopatadine hydrochloride for area under curve (AUC) method

Amount of

Sample

Added in

(µg/ml)

Amount of

Standard

Added in

(µg/ml)

Total amount recovered

Percentage recovery (%)

Standard deviation

Percentage of relative standard deviation

(C.O.V.)

2

0

1.9919

99.5996

0.03002

1.5073

2

2

4.0230

100.5758

0.04187

1.02552

2

4

5.9731

99.5521

0.05244

0.8780

2

6

8.07787

100.9734

0.2244

2.7775

 

 

 

 

0.8670

1.5471

 

Validation

Accuracy

Accuracy of the proposed methods was carried as on the basis of recovery studies. It is performed by the standard addition method. Recovery studies were performed by adding standard drug at different levels to the pre-analyzed tablets powder solution and the proposed method was followed. From the amount of the drug estimated, the percentage recovery was calculated. The results of the analysis are shown in table (2, 3).

 

Precision

The method precision was established by carrying out the analysis of homogenous powder blend of tablets. The assay was carried out of drug by using proposed analytical method in six replicates. The values of relative standard deviation lie well within the limits indicated the sample repeatability of the method. The results obtained are tabulated in table 4.

 

Table 4: Precision- method precision

Experiment no.

Weight of olopatadine hydrochloride taken in mg

Content in mg. of olopatadine hydrochloride

Second d order derivative method

Area under curve method

1

10

9.6666

10.1727

2

10

10.666

10.1305

3

10

10.1667

10.1919

4

10

9.8333

10.1382

5

10

10.1667

10.1266

6

10

10.1666

10.1765

 

Standard deviation

0.01311

0.05993

 

%RSD

0.1311

0.5889

 

Inter-day and intra-day precision

An accurately weighed quantity of pharmaceutical formulation equivalent to 10 mg of olopatadine hydrochloride was transferred to 100 ml of volumetric flask. A 30 ml of methanol was added and sonicated for 15 minutes and filtered. The filtrate and washing were diluted up to the mark with methanol to give concentration as 100 μg /ml. Such solution was used for analysis.

 

For second order derivative method

Solution was scanned between 350 nm to 200 nm in spectrum mode. The second order derivative spectrum was obtained by using derivative mode. Amplitude of the resulting solution was measured at 224 nm by using methanol as blank. The amplitude of final solution was read after 0 hr., 3 hrs. and 6 hrs. in 10 mm cell 224 nm for second order derivative (method A). Similarly the amplitude of the same solution was read on 1st, 2nd and 5th day. The amount of olopatadine hydrochloride was estimated by comparison with standard at 224 nm for second order derivative, table 5.

 

For area under curve method

Solution was scanned between 350 nm to 200 nm in spectrum mode. The area under curve of resulting solutions was measured at between 220 nm to 230 nm by using methanol as blank. The area under curve of final solutions was read after 0 hr., 3 hrs. and 6 hrs. in 10 mm cell at 220 nm to 230 nm (method B).  Similarly area under curve of the same solution was read on 1st, 2nd and 5th day. The amount of olopatadine hydrochloride was estimated by comparison with standard at 220 nm to 230 nm, table 5.

 

Table 5: Summary of validation parameter for intra-day and inter-day

Sr. no.

Parameters

second order derivative method

Area under curve (AUC) method

(A)

Intra-day precision (n=3)

Amount found ±

% RSD

99.40 %

 

0.1781

99.60%

 

0.03002

(B)

Inter-day precision (n=3)

Amount found ±

% RSD

98.484%

 

0.1866

98.765%

 

0.01765

(c)

Ruggedness

Analyst to analyst

(n= 3)

%RSD

0.16789

0.01823

 

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

The limit of detection (LOD) is defined as the lowest concentration of an analyte that an analytical process can reliably differentiate from back-ground levels. In this study, LOD and LOQ were based on the standard deviation of the response and the slope of the corresponding curve using the following equations-

            LOD = 3.3 σ/S    and   LOQ = 10 σ/S

 

Where σ is the standard deviation of the signal to noise ratio of the sample and S is the slope of the related calibrations graphs.

 

The limit of quantification (LOQ) is defined as the lowest concentration of the standard curve that can be measured with an acceptable accuracy, precision and variability .The values of LOD and LOQ are given in table 6.

 

 

Table 6: Values of results of LOD and LOQ

Parameters

Second order derivative method

Area under curve (AUC) method

Limit of Detection (μg/ml)

0.0006532

0.005248

Limit of Quantification (μg/ml)

0.001975

0.01590

 

Ruggedness:

The ruggedness of the method is defined as degree of reproducibility of results obtained by analysis of olopatadine hydrochloride sample under variety of normal test conditions such as different laboratories, different analysts and different lots of reagents. Quantitative determination of olopatadine hydrochloride was conducted spectrophotometrically on one laboratory. It was again tested in another laboratory using different instrument by different analyst. The assays obtained in two different laboratories were well in agreement. It proved ruggedness of the proposed methods.

 

RESULT AND DISCUSSION:

The second order derivative and area under curve UV-spectroscopic methods are useful for routine analysis of olopatadine hydrochloride in bulk drug and formulation. The derivative spectroscopy method applied has the advantage that it locates hidden peak in the normal spectrum. It eliminates the interference caused by the excipients and the degradation products present, if any, in the formulation. The method was validated according to International Conference on Harmonization guidelines for validation of analytical procedures. Olopatadine hydrochloride has the absorbance maxima at 224 nm (method A) and in the AUC spectrum method areas were measured between 220 nm to 230 nm (method B). The polynomial regression data for the calibration plots showed good linear relationship in the concentration range of 1 to 16 μg/ml  for second order derivative and 1 to 12 μg/ml for area under curve method receptively and values given in table1. Recovery studies were carried out by adding the pure drug to the previously analyzed tablet powder sample and shown in table 2, 3. The percentage recovery value indicates non interference from excipients used in formulation. The reproducibility and accuracy of the method were found to be good, which was evidenced by low standard deviation.

 

CONCLUSION:

The most striking features of two methods are its simplicity and rapidity, not requiring tedious sample solutions preparations which are needed for other instrumental methods.  From the results obtained it can be concluded that the proposed methods are fully validated and found to be simple, sensitive, accurate, precise, reproducible, rugged and robust and relatively inexpensive. So, the developed methods can be easily applied for the routine quality control analysis of olopatadine hydrochloride in pharmaceutical formulation.

 

ACKNOWLEDGMENT:

Authors express sincere thanks to the Principal, Dr. Tushar M. Desai of D. G. Ruparel College, Mumbai.

 

REFERENCES:

1.        http://en.wikipedia.org/wiki/olopatadine

2.        Pawan K Basniwal and Deepti Jain.ICH guideline practice: application of novel RP-HPLC-DAD method for determination of olopatadine hydrochloride in pharmaceutical Products, Journal of Analytical Science and Technology, 4:12; 2013: 2-6.

3.        K. Nageswara Rao, S. Ganapaty  and A. Lakshmana Rao, 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.

4.        Rajan V. Rele and Sandip P. Patil, Reversed phase ultra performance liquid chromatography method for determination of olopatadine hydrochloride from active pharmaceutical dosage form, Der Pharmacia Sinica. 5(1) ;2014:18-22

5.        Jain D and Basniwal P K. spectrophotometric determination of olopatadine hydrochloride in eye drops and tablets, Journal of Pharmaceutical Research, 12(2); 2013: 48-52.

6.        Suddhasattya dey, et al ,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.

7.        N.Y. Sreedhar, A. Sreenivasulu, M. Sunil Kumar and M. Nagaraju. Voltammetric determination of olopatadine hydrochloride in bulk drug form and pharmaceutical formulations, vol. 3(7) ;2012: 2517-2521