INTRODUCTION:

Its chemical name is 8-chloro-6,11-dihydro-11-[1-[(5-methyl-3-pyridinyl)methyl]-4-piperidinylidene]-5H-benzo [5,6]cyclohepta[1,2 b]pyridine Fumarate. Rupatadine fumarate is a second generation of antihistamine and platelet activity factor (PAF) antagonist used to treat allergies. Rupatadine possesses anti-allergic properties such as the inhibition of the de-granulation of mast cells induced by immunological and non-immunological stimuli and inhibition of the release cytokines, particularly of the TNF in human mast cell¹.

Structure of rupatadine

Fig.1.

Literature survey reveals the Spectrophotometric^2-4 titration^5,6, HPLC^7-12 methods for the estimation of rupatadine fumarate. Simple, rapid and reliable UV spectrophotometric methods are developed for the determination of rupatadine fumarate. These methods can be used for the routine analysis. In the proposed methods 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 rupatadine fumarate 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 rupatadine fumarate was weighed accurately and transferred in 100 ml of volumetric flask. About 30 ml of absolute alcohol 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.

Fig. 2. Second order derivative spectrum of rupatadine fumarate (10 μg/ml) showing absorbance at 220.5 nm

Fig. 3. Calibration curve for rupatadine fumarate at 220.5 nm by second order derivative Spectroscopy in concentration range of 2 to 12 μg /ml

Estimation from tablets

Twenty tablets were weighed accurately and average weight of each tablet was determined. Powder equivalent to 10 mg of rupatadine fumarate was weighed and transferred in 100 ml of volumetric flask. A 30 ml of absolute alcohol was added and sonicated for 15 minutes and filtered. The filtrate and washing were diluted up to the mark with absolute alcohol to give concentration as 100 μg /ml. Such solution was used for analysis.

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

Parameter	Values
Detection Wavelength (nm)	220.5
Beer Law Limits (µg/ml)	1-30
Correlation coefficient(r²)	0.9989
Regression equation (y=b+ac)
Slope (a)	0.0005
Intercept (b)	0.00001

Table 2: Results of recovery of rupatadine fumarate 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	2.028571	97.14286	0.139971	7.20438
2	2	4.085714	101.4286	0.139971	3.44998
2	4	5.942857	99.04762	0.206032	3.46687
2	6	7.914286	98.92857	0.180702	2.28323
				Mean=0.166669	Mean =4.10111

Experimental Method:

For the selection of analytical wavelength, 10 μg /ml solution of rupatadine fumarate was scanned in the spectrum mode from 300 nm to 200 nm by using absolute alcohol 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 between. 220.5 nm. (Fig. 2).

Into series of 10 ml graduated flask, varying amount of standard solutions of rupatadine fumarate was pipette out and volume was adjusted with absolute alcohol as solvent. Solutions were scanned between 300 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 220.5 nm by using absolute alcohol as blank. The calibration curve was prepared in the concentration range of 1 to 30 μg/ml. (Fig. 3).

Results of analysis are given in table 1.

Validation

Accuracy

Accuracy of the proposed method 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.

Table 3: Precision- method precision

Experiment no.	Weight of rupatadine fumarate taken in mg	Values
1	10	10.005
2	10	10.016
3	10	9.984
4	10	9.991
5	10	10.009
6	10	10.015
	Standard deviation	0.013095
	%RSD	0.130902

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 3.

Inter-day and intra-day precision

An accurately weighed quantity of tablets powder equivalent to 10 mg of rupatadine fumarate was transferred to 100 ml of volumetric flask. A 30 ml of absolute alcohol was added and sonicated for 15 minutes and filtered. The filtrate and washing were diluted up to the mark with absolute alcohol to give concentration as 100 μg /ml. Such solution was used for analysis.

Solution was scanned between 300 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 between 220.5 nm by using absolute alcohol as blank. The amplitude of final solution was read after 0 hr., 3 hrs. and 6 hrs. in 10 mm cell 220.5 nm for second order derivative. Similarly the amplitude of the same solution was read on 1^st, 2^nd and 5^th day. The amount of rupatadine fumarate was estimated by comparison with standard at 220.5 nm for second order derivative, table 4.

Table 4: Summary of validation parameter for intra-day and inter-day

Sr. no.	Parameters	values
(A)	Intra-day precision ( n=3) Amount found ± % RSD	98.928 % 3.46687
(B)	Inter-day precision ( n=3) Amount found ±% RSD	98.484 % 0.1366
(c)	Ruggedness Analyst to analyst( n= 3) %RSD	0.16789

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 5.

Table 5: Values of results of LOD and LOQ

Parameters	Values
Limit of Detection (μg/ml)	0.02358
Limit of Quantification (μg/ml)	0.0357

Ruggedness

The ruggedness of the method is defined as degree of reproducibility of results obtained by analysis of rupatadine fumarate sample under variety of normal test conditions such as different laboratories, different analysts and different lots of reagents. Quantitative determination of rupatadine fumarate 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 method is useful for routine analysis of rupatadine fumarate 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. The polynomial regression data for the calibration plots showed good linear relationship in the concentration range of 1 to 30 μg/ml and 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 method is 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 method is 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 rupatadine fumarate in pharmaceutical formulation.

ACKNOWLEDGMENT:

Authors express sincere thanks to the Principal, Dr. Tushar M. Desai of D. G. Ruparel College for providing laboratory facilities.

REFERENCES:

1. Picado C. Rupatadine: Pharmacological profile and its use in the treatment of allergic disorders. Expert Opin Pharmacother. (7); 2006 : 1989-2001.

2. P.G. Patel, V.M. Vaghela, S.G. Rathi, N.B. Rajgor, V.H. Bhaskar. Derivative Spectrophotometry method for simultaneous estimation of rupatadine and montelukast in their combined dosage form. Journal of Young Pharmacists. 1(4); 2009: 354-358.

3. A. Goyal, C.S. Sharma, G .Singh. Development of UV and visible spectrophotometric methods for estimation of rupatadine fumarate from tablet formulation. International journal of pharmaceutical research and development. e: 2(4); 2010.

4. R. V. Rele, P.D. Desai, S.A. Sawant. Simple extractive spectrophotometric determination of rupatadine as rupatadine fumarate from pharmaceutical formulation. International Journal of chemical Sciences. 8(1); 2010: 22-28.

5. R. V. Rele, S.A. Sawant, R.N. Mali, A validated non-aqueous potentiometric titration method for the quantitative determination of rupatadine as rupatadine fumarate. Analytical chemistry: An Indian Journal., 8(2); 2009 : 165-167.

6. R. V. Rele, S.A. Sawant, S.A Mahimkar, A validated simple titrimetric method for the quantitative determination of rupatadine as rupatadine fumarate from pharmaceutical dosages. Analytical chemistry, An Indian Journal, 8(4);2009:561-564.

7. Rupali L. Choudekar, M.P. Mahajan, S.D. Sawant, Validated RP-HPLC method for the estimation of rupatadine fumarate in bulk and tablet dosage form. Der pharma chemica. 4(3); 2012: 1047-1053.

8. Kumaraswamy. Gandla, Spandana. R, JMR Kumar, M. Laksmi surekha, JVLN Sheshgiri Rao., Simultaneous RP-HPLC method for estimation of rupatadine fumarate and montelukast sodium in tablet dosage form. Der Pharma Chemica. 4(3); 2012: 1819-1825.

9. Nareshkumar J Malaviya, Rakeshkumar Jat, Rambir Singh, Ketan G. Patel RP-HPLC Method and Its Validation for Assay of Rupatadine fumarate dosage form. Inventi Rapid: Pharm Analysis and quality Assurance. (3);2012.

10. Fu Chuan-shan (Hainan Huatuotianya Pharmaceutical Co., LTD. Hainan, P.R. China) Determination of rupatadine fumarate by HPLC. China Tropical Medicine, 9(7); 2009: 1352-1353.

11. Trivedi HK, Patel M. C.Development of a stability-indicating RP-HPLC method for the determination of rupatadine and its degradation products in solid oral dosage form. Scientia Pharmaceutica. 80(4); 2012: 889-902.

Received on 21.08.2014 Modified on 09.09.2014

Asian J. Research Chem. 7(10): October- 2014; Page 859-862

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

INTRODUCTION: