INTRODUCTION:

Drotaverine hydrochloride, 1-[(3,4-diethoxy phenyl) methylene]-6,7-diethoxy-1,2,3,4-tetra hydro isoquinolene (Fig.1) is an analogue of papaverine¹. It acts as an antispasmodic agent by inhibiting phosphodiesterase IV enzyme, specific for smooth muscle spasm and pain, used to reduce excessive labor pain². Drotaverine hydrochloride is official in Polish Pharmacopoeia³. A few UV spectrophotometric^4-8 and HPLC^9-13 methods have been reported for estimation of drotaverine hydrochloride.

Etoricoxib a newer cyclo-oxygenase-2 inhibitor is mainly used in the management of osteoarthritis, rheumatoid arthritis and acute gouty arthritis^14-17. Chemically, Etoricoxib is a 5-chloro-6’-methyl-3-[4-(methylsulfonyl) phenyl]-2, 3’-bipyridine, and is not yet official in any pharmacopoeia. Its impurities studies and HPLC/MS-MS methods in matrix have been reported¹⁸. The availability of an HPLC method with high sensitivity and selectivity will be very useful for the estimation of Etoricoxib in pharmaceutical dosage forms.

The combination is not included in any pharmacopoeia. The review of the literature revealed that there is no Spectrophotometric method available for determination of this combination. Therefore the aim of the study was to develop simple, rapid, accurate, reproducible and economical spectroscopic methods for both the drugs in combined dosage forms. The proposed method were optimized and validated as per the International Conference on Harmonization (ICH) analytical method validation guidelines.

Fig. 1. Structure of Analytes.

MATERIALS AND METHODS:

Instrumentation:

An UV-Visible double beam spectrophotometer (Varian Cary 100) with 10 mm matched quartz cells was used. All weighing were done on electronic balance (Model Shimadzu AUW-220D). Dissolution apparatus USP Type II was used for Dissolution studies.

Reagents and chemicals:

Pure drug sample of DRT, % purity 98.80 and ETR, % purity 99.92 was kindly supplied as a gift sample by Alkem pharmaceuticals Ltd. Mumbai and Mapro Pharmaceuticals Ltd. Vapi, respectively. These samples were used without further purification. Two Batches of tablet formulations (Batch no.JT901 and JT902) were supplied by JPCL Pharma ltd. Jalgaon were used for analysis containing DRT 80 mg and ETR 90 mg per tablet. Spectroscopic grade Methanol and analytical grade HCL was used throughout the study. All the solvents and reagents used were purchased from LOBA Chemie Pvt. Ltd., Mumbai.

Preparation of standard stock solutions and calibration curve:

Standard stock solutions of both the drugs containing 1mg mL^-1 were prepared separately in the methanol and 0.1N HCL for Method A and B, respectively. The working standard solutions of these drugs were obtained by dilution of the respective stock solution in the methanol for Method A and distilled water for Method B. Ratio Derivative amplitudes of spectrum, by using the above mentioned procedure, were used to prepare calibration curves for both the drugs by Ratio derivative method. Absorbances were measured at selected wavelengths for absorbance corrected method and were used to construct calibration curves. Beer’s law obeyed in the concentration range of 8-40 μg mL^-1for DRT and 9-45 μg mL^-1 for ETR for both the methods.

Preparation of sample stock solution and formulation analysis:

Twenty tablets were weighed accurately and a quantity of tablet powder equivalent to (80 mg of DRT and 90 mg of ETR) was weighed and dissolved in the 80 mL of solvent with the aid of ultrasonicator for 10 min and solution was filtered through Whatman paper No. 41 into a 100 mL volumetric flask. Filter paper was washed with the solvent, adding washings to the volumetric flask and volume was made up to mark. The solution was suitably diluted with solvent to get of 80 μg mL^-1of DRT and 90 μg mL^-1of ETR.

Procedure:

Method A: Ratio Derivative Spectrophotometric Method

Theoretical aspects:

The method involves dividing the spectrum of mixture by the standardized spectra of each of the analyte to get ratio spectra and first derivative of ratio spectrum was obtained which was independent of concentration of divisor (Fig.2). The concentration of active compounds are then determined from calibration graph obtained by measuring amplitude at points corresponding to minima or maxima.

Using appropriate dilutions of standard stock solution, the two solutions were scanned separately. The ratio spectra of different DRT standards at increasing concentrations were obtained by dividing DRT+ETR scans with the stored spectrum of the standard solution of ETR (27μg mL^-1) (Fig.2A). Wavelength 338.91 nm was selected for the quantification of DRT in DRT+ETR mixture (Fig.2B). The ratio and ratio derivative spectra of the solutions of ETR at different concentrations were obtained by dividing DRT+ETR scans with the stored standard spectrum of the DRT (24 μg mL^-1) (Fig.2C and 2D, respectively). Wavelength 268.19 nm was selected for the quantification of ETR in DRT+ETR mixture. Measured analytical signals at the selected wavelengths were proportional to the concentrations of the drugs. Calibration curves were prepared from the measured signals at the selected wavelength and concentration of the standard solutions. The amount of DRT (C_DRT) and ETR (C_ETR) in tablets was calculated by using following equations-

At 338.91nm: C_DRT= (Ratio derivative amplitude for DRT-0.055)/ 0.641.... (1)

At 268.19nm: C_ETR = (Ratio derivative amplitude for ETR - 0.68)/ 0.764.… (2)

Fig. 2. Spectrum of mixture of DRT and ETR for ratio derivative method

Fig. 2(A). Ratio spectra of DRT using 27μg mL^-1solution of ETR as divisor

Fig. 2(B). First derivative of the ratio spectra of DRT (8-40μg mL^-1)

Fig. 2(C). Ratio spectra of ETR using 24μg mL^-1solution of DRT as divisor.

Fig. 2(D). First derivative of the ratio spectra of ETR (9-45μg mL^-1)

Method B: Absorption Corrected Method:

λ_max of DRT and ETR was determined by scanning the drug solution in the solvent was found to be at 360.90 nm and 249.10 nm respectively. DRT also showed absorbance at 249.10 nm, while ETR did not show any interference at 360.90 nm. To construct Beer’s plot for DRT and ETR dilutions were made in the solvent using stock solution of 100 µg mL^-1. Also Beer’s plot was constructed for DRT and ETR in solution mixture at different concentration (8:9, 16:18, 24:27, 32:36, 40:45µg mL^-1) levels. Both the drugs followed linearity individually and in mixture within the concentration range 8-40µg mL^-1 and 9-45µg mL^-1 for DRT and ETR respectively (Fig.3)

Fig. 3. Overlay spectrum of DRT and ETR. DRT (8-40µg mL^-1) and ETR (9-45 µg mL^-1)

Determination of Absorption Factor at Selected Wavelengths:

DRT and ETR solution in the solvent of known concentrations were scanned against blank on spectrophotometer. The value of absorption factor was found to be 1.266. Quantitative estimation of DRT and ETR was carried out using following equation:

Corrected Absorbance of ETR at 249.10nm =

Abs_249.10(DRT+ETR) – [(abs_249.10 (DRT)/ abs_360.90(DRT)] × abs_360.90(DRT) or

Corrected Absorbance of ETR at 249.10nm = abs_249.10 (DRT+ETR) – 1.266 × abs_360.90 (DRT)

Where; abs: Absorption value at given wavelengths.

Dissolution Studies: The dissolution study was carried out for the above combination and was validated. A calibrated dissolution apparatus (USP II) paddle 60 rpm and bath temp at 37±1^oC. Nine hundred milliliter freshly prepared and degassed 0.1N HCl solution was used as the dissolution medium. Six tablets were evaluated and dissolution sample were collected at 5, 10, 15, 20, 25, 30, 35, 40 min interval. At each time point, a 5mL sample was removed from each vessel sample, filtered through a nylon filter (0.45μm, 25 mm), 1.8 mL of filtrate was diluted to10 mL with distilled water and analyzed by absorption corrected method (Fig. 4), percentage release of DRT and ETR was calculated by using equations 3 and 4, respectively.

DRT % release = (C_DRT× 900×10×100)/ (1000×80) … (3)

ETR % release = (C_ETR× 900×10×100)/ (1000×90) … (4)

Fig. 4. Dissolution study of DRT and ETR by Absorption corrected method.

Recovery Studies:

The accuracy of the proposed methods was checked by recovery study, by addition of standard drug solution to preanalysed sample solution at three different concentration levels (50%, 100% and 150%) within the range of linearity for both the drugs. The basic concentration level of sample solution selected for spiking of the drugs standard solution was 12 μg mL^-1 of DRT and 13.5 μg mL^-1of ETR for both the methods.

Precision of the Method:

To study intraday precision, method was repeated 5 times in a day and the average % RSD was calculated by method A and B, respectively. Similarly the method was repeated on five different days and average % RSD was calculated. These values confirm the intra and inter day precision.

RESULTS AND DISCUSSION:

Practically no interference from tablet excipients was observed in these methods. As their λmax differ more than 20 nm, absorption corrected method was tried for their simultaneous estimation in formulation. Quantitative estimation of ETR was carried out by subtracting interference of DRT using experimentally calculated absorption factor. Both the methods are accurate, simple, rapid, precise, reliable, sensitive, reproducible and economical as per ICH guidelines. The values of % RSD and correlation of coefficient were satisfactory and results of the formulation analysis (Table no.1) and result of the recovery study (Table no.2) indicates that there is no interference due to excipients present in the formulation. It can be easily and conveniently adopted for routine quality control analysis. The absorption corrected method was successfully applied for the dissolution studies.

Table 1: Optical characteristics of the proposed methods and the results of formulation analysis and Precision (%RSD)

Parameter		DROTAVERINE		ETORICOXIB
Parameter		Method A	Method B	Method A	Method B
λ (nm)		338.91	360.90	268.19	249.10
Beer’s law limit (μg mL^-1)		8-40	8-40	9-45	9-45
Regression Equation (y = mx + c)	Slope (m)	0.641	0.026	0.764	0.0005
Regression Equation (y = mx + c)	Intercept (c)	0.055	0.006	0.680	-0.005
Correlation coefficient		0.999	0.999	0.999	0.998
Precision (%R.S.D.)	Repeatability (n=5)	0.76	0.96	1.09	0.92
	Intra-day (3×3 times)	0.58	0.98	1.19	0.95
	Inter-day(3×5 days)	0.65	0.82	1.02	0.93
Formulation Analysis (% Assay, %RSD) n=6	Formulation I	99.01, 0.89	98.91, 1.1	100.89, 1.06	101.72, 1.04
Formulation Analysis (% Assay, %RSD) n=6	Formulation II	100.11, 0.63	99.24, 0.93	99.84, 0.55	101.5, 0.65

R.S.D. is relative standard deviation

Table 2 : Result of the recovery analysis

Compound	Recovery Level (%)	Wt. spiked		Wt. recovered		Recovery (%)		R.S.D. (%) n = 3
Compound	Recovery Level (%)	DRT	ETR	DRT	ET	DRT	ETR	DRT	ETR
Tab I	50	6	6.25	5.96	6.23	99.96	99.68	0.52	0.38
	100	12.0	13.5	12.01	13.54	100.08	100.29	0.17	0.86
	150	18.0	19.75	17.82	19.72	100.22	99.84	0.39	0.18
Tab II	50	6.0	6.25	5.99	6.34	99.83	101.44	0.59	0.28
	100	12.0	13.5	11.98	13.46	99.83	99.70	0.58	0.78
	150	18.0	19.75	18.02	19.80	100.11	100.25	0.33	0.36

Wt. = Weight

CONCLUSION:

The proposed methods are simple, precise, accurate, economical and rapid for the determination of DRT and ETR in combined tablet dosage forms. Analysis of authentic samples containing DRT and ETR showed no interference from the common additives and excipients. Hence, recommended procedure is well suited for the assay and evaluation of drugs in pharmaceutical preparations. It can be easily and conveniently adopted for routine quality control analysis and Absorption corrected method was successfully applied for dissolution study.

ACKNOWLEDGEMENT:

The authors are thankful to m/s Alkem Pharmaceuticals Ltd. Mumbai, India and Mapro Pharmaceuticals Ltd., Vapi, India for providing gift samples of Drotaverine hydrochloride and Etoricoxib, respectively. The authors are thankful to Management of MAEER’s Maharashtra Institute of Pharmacy, Pune for providing necessary facility for the work.

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Received on 08.07.2010 Modified on 23.07.2010

Research J. Pharm. and Tech. 4(2): February 2011; Page 298-301