INTRODUCTION:

Chemically, Drotaverine (DV) is (1-(3,4-diethoxybenzylidene)-6,7-diethoxy-1,2,3,4 tetrahydroisoquinoline) hydrochloride. It is a benzylisoquinoline derivative¹. It is a highly potent spasmolytic agent and has excellent smooth muscle relaxant properties². Aceclofenac (AF) is 2-[(2,6-Dichlorophenyl) amino] benzene acetic acid carboxymethyl ester³. It is used as anti-inflammatory drug. Structures of both the drugs (DV and AF) are shown in figure 1. Literature survey reveals that assay of AF in bulk and dosage form is official in Indian Pharmacopoeia 2007⁴and British Pharmacopoeia 2008³. Several analytical methods have been reported for estimation of DV are spectrophotometry^5-7, HPLC⁸, thin layer chromatography^9,10and voltametry¹¹. The analytical methods reported for estimation of AF are spectrophotometry^12-14, HPLC^15-17, LC-MS¹⁸ and fluorimetry¹⁹. The present paper describes simple, accurate, specific and precise methods for simultaneous estimation of DV and AF in their combined tablet dosage form. The proposed method is optimized and validated as per the ICH guidelines^20,21.

In the present work, a successful attempt has been made to estimate both these drugs simultaneously using two UV spectrophotometric methods (a) Absorbance Correction method and (b) First order Derivative method²².

MATERIALS AND METHODS:

Instrument used was an UV-Visible double beam spectrophotometer, make: SHIMADZU (model UV-1800) with a pair of 1 cm matched quartz cells. All weighing was done on Shimadzu analytical balance (Model AU-220). Pure drug samples of DV and AF were obtained as gift samples from Astran Labs, Ahmedabad. Combined tablet formulation (ESNIL) was procured from local market. Methanol AR was used as solvent.

Preparation of standard stock solution:

Accurately weighed quantity of DV (80 mg) and AF (100 mg) was transferred to two separate 100 ml volumetric flasks, dissolved in little amount of methanol and diluted to the mark with methanol (stock solutions: 800 μg/ml of DV and 1000 μg/ml of AF).

Preparation of working standard solution:

100 mg/ml of AF solution was prepared by diluting 10 ml of stock solution with methanol in 100 ml volumetric flask up to the mark. 80 mg/ml of DV solution was prepared by diluting 10 ml of stock solution with methanol in 100 ml volumetric flask up to the mark.

rotaverine

Aceclofenac

Figure 1: Chemical structures of the analytes.

Figure 2: Overlain zero order spectra of DV and AF in methanol.

Absorbance correction method (method I):

Absorbance correction method uses the absorbances at two selected wavelengths, one at λmax of one drug where other drug also shows considerable absorbance and other being the wavelength at which the first drug has practically nil absorbance. From the stock solutions, working standard solutions of DV (16μg/ml) and AF (20μg/ml) were prepared by appropriate dilution and were scanned in the entire UV range to determine the suitable wavelengths. DV and AF have λmax at 360 nm and 276 nm respectively. Both the drugs were found to have considerable absorbance at 276 nm while at 360 nm only DV has absorbance. The wavelengths selected for analysis were 276 nm and 360 nm for AF and DV respectively as shown in figure 2. A series of standard solutions ranging from 8-36 μg/ml of DV and 10-40 μg/ml of AF were prepared separately and the absorbances of solutions were measured at 276 nm and 360 nm. A calibration curve was prepared by plotting absorbance versus corresponding concentration of drug. The concentration of two drugs in sample solution was calculated by using following equations:

C_DV= A₁/ a_x1……………..………….......……………. (1)

A₂ –a_x1C_DV

C_AF = ------------- ………………….(2)

a_y2

Where, A₁and A₂ are the absorbances of mixture at 360 nm and 276 nm respectively, a_x1and a_x2 are absorptivities of DV at 360 nm and 276 nm respectively, a_y2isabsorptivity of AF at 276 nm, C_DVis concentration of DV, ^CAFis concentration of AF.

Figure 3: Overlain first order derivative spectra of DV (16 µg/ml) and AF (20 µg/ml) in methanol.

Table 1: Validation Parameters for Absorbance Correction Method

PARAMETERS	DV	AF
Linearity range	8-32 µg/ml	10-40 µg/ml
Correlation Coefficient	0.9992	0.9990
Precision	% RSD
Repeatability Intraday Interday	0.67-0.95% 0.40-1.68% 0.17-1.68%	0.34-0.52% 0.07-0.85% 0.10-0.84%
% Recovery	99.16%-100.31%	98.15%-100.46%
Ruggedness	0.11-0.74%	0.05 – 0.44%

*DV- Drotaverine; AF-Aceclofenac; RSD-Relative Standard Deviation.

Table 2: Validation Parameters for First order derivative Method

PARAMETERS	DV	AF
Linearity range	8-32µg/ml	10-40µg/ml
Correlation Coefficient	0.9990	0.9990
Precision	% RSD
Repeatability Intraday Interday	2.17-3.15% 1.92-4.72% 2.15-4.72%	2.34-3.53% 1.79-4.35% 1.74-4.08%
% Recovery	99.28%-101.11%	99.08%-100.71%
Ruggedness	2.32-5.24%	2.40-5.66%.

*DV- Drotaverine; AF-Aceclofenac; RSD-Relative Standard Deviation.

Table 3: Recovery studies

_{Name of Drug}	_{Amount of Drug Added (}µg/ml)	_Method I(AC)		Method II(FD)
_{Name of Drug}	_{Amount of Drug Added (}µg/ml)	_%Recovery*	_SD	_%Recovery*	_SD
DV	12	100.21	0.003	100.25	0.000
AF	15	100.46	0.004	100.23	0.000
DV	16	99.16	0.005	99.28	0.001
AF	20	98.15	0.003	99.08	0.001
DV	20	100.31	0.002	101.11	0.001
AF	25	99.98	0.005	100.71	0.001

*Mean of Three estimations, AC-Absorbance Correction; FD-First Order Derivative; DV- Drotaverine; AF-Aceclofenac; SD-Standard Deviation.

Table 4: Results of simultaneous estimation of DV and AF in marketed Formulation by Method I & II.

Method	mg/tablet		%of label claim* ± S.D.
Method	DV	AF	DV	AF
Method-I(AC)	80	100	99.25+0.003	99.40+0.004
Method-II(FD)	80	100	100.19+0.000	101.40+0.000

*Average of five determinations; AC-Absorbance Correction; FD-First Order Derivative; DV- Drotaverine; AF-Aceclofenac; SD-Standard Deviation.

First order derivative method (method II):

From the stock solution of 1000 μg/ml AF and 800 μg/ml DV, working standard solutions of the drugs were prepared by appropriate dilution and were scanned in the range of 200-400 nm. Then their first order derivative spectra were scanned using the spectrum mode with medium scan speed using derivative wavelength difference(∆λ) one. From their first order derivative spectra, AF was determined at 306.5 nm (Zero Crossing Point of DV) and DV was determined at 249 nm (Zero Crossing Point of AF) as shown in figure 3. Standard solutions were prepared having concentration 10-40 μg/ml for AF and 8-32 μg/ml for DV. The absorbances of solutions were measured at 306.5 nm and 249 nm. A calibration curve was prepared by plotting absorbance against respective concentration.

Assay of tablet formulation by method I and II:

Ten tablets were weighed and crushed to obtain a fine powder. An accurately weighed tablet powder equivalent to about 80 mg of DV and 100 mg of AF was transferred to 100 ml volumetric flask and dissolved in 50 ml of methanol. The volume was made up to the mark using methanol as solvent. The resulting solution was filtered through Whatmann filter paper and 10 ml of this filtrate was appropriately diluted to get concentration of 80 μg/ml of DV and 100 μg/ml of AF. This solution was further diluted to get concentration of 16 μg/ml of DV and 20 μg/ml of AF. Absorbance of sample solutions was measured at 276 nm and 360 nm and the concentration of two drugs in the sample were determined using Equations (1) and (2) (Method-I). The tablet sample solution was also analysed by first _{order derivative method}. Absorbance of sample solutions was measured at selected wavelengths and concentration of two drugs in the sample were determined.

Method validation:

Linearity and range:

Aliquots of standard stock solutions of DV and AF were taken in volumetric flasks and diluted with methanol to get final concentrations in range of 8-32 μg/ml for DV and 10-40 μg/ml for AF. This calibration range was prepared five times and absorbances were measured at respective wavelengths for each drug separately.

Precision:

Precision of the methods were determined by performing interday variation, intraday variation and method repeatability studies. In interday variation, the absorbance of standard solutions of DV (8-32 μg/ml) and AF (10-40 μg/ml) were measured on five consecutive days. In intraday variation the absorbances were measured five times in a day. In repeatability study, three concentrations of both the drugs were analysed in triplicate.

Recovery studies:

To study the accuracy of the proposed methods, recovery studies were carried out by standard addition method at three different levels. A known amount of drug was added to preanalyzed tablet powder and percentage recoveries were calculated.

Ruggedness:

The data for ruggedness obtained from two different analysts for DV and AF.

RESULTS AND DISCUSSION:

The proposed methods were validated as per ICH guideline. The plot of absorbances versus respective concentrations of DV and AF were found to be linear in the concentration range of 8-32 μg/ml and 10-40 μg/ml respectively with correlation coefficient 0.9992 at 360 nm and 0.9990 at 276 nm for absorbance correction method (Method I).For first order derivative method (method II) linearity range was same as for method I with correlation coefficient 0.9989 at 249 nm (ZCP of AF) and 0.9990 at 306.5 nm (ZCP of DV). Precision was calculated as interday and intraday variations and % RSD was found to be less than 2 for method I and less than 5 for method II for both the drugs shown in Table 1 and Table 2. The accuracy of method was determined at 75, 100 and 125 % level. The % recovery ranges from 98.15 to 101.11 for both the methods as shown in Table 3. The % RSD of ruggedness for DV ranges from 0.11 to 0.74%, while for AF it was found to be 0.05 to 0.44% for method I and for method II 2.32 to 5.24% and 2.40 to 5.66% for DV and AF respectively. Results for all validation parameters are presented in table 1, 2 and 3. The two _methods can be successfully _used for simultaneous estimation of DV and _{AF in
their combined tablet dosage form.}Marketed tablets were analyzed and results obtained were in the range of 98-102% (Table 4).

CONCLUSION:

The proposed methods give accurate and precise results for determination of DV and AF in marketed formulation (tablet) without prior separation and are easily applied for routine analysis. The most striking feature of both the methods is its simplicity and rapidity. Method validation has been demonstrated by variety of tests for linearity, accuracy, precision and ruggedness. The developed methods have several advantages, as they are simple, accurate and precise. The proposed methods were successfully applied to determination of these drugs in commercial tablets.

ACKNOWLEDGEMENT:

The authors are thankful to Astran Lab, Ahmedabad for providing pure gift samples of Drotaverine hydrochloride and Aceclofenac. The authors are also thankful to the Principal, Maliba Pharmacy College for providing necessary facilities.

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Received on 19.05.2010 Modified on 02.06.2010

Asian J. Research Chem. 3(4): Oct. - Dec. 2010; Page 969-972