INTRODUCTION:

Drotaverine Hydrochloride, 1-[(3,4-diethoxy phenyl) methylene]-6,7-diethoxy-1,2,3,4-tetra hydro isoquinolene is an analogue of papaverine (Fig.1). It acts as an antispasmodic agent by inhibiting phosphodiesterase IV enzyme, specific for smooth muscle spasm and pain, used to reduce excessive labor pain¹. Diclofenac Potassium is chemically potassium (o-(2, 6-dichloroanilino) phenyl) acetate, a non steroidal anti-inflammatory drug (NSAID) exhibits anti-inflammatory and analgesic properties² (Fig.1). The exact mechanism of action of DIC is not entirely known, but it is thought that the primary mechanism responsible for its anti-inflammatory, antipyretic, and analgesic action is inhibition of prostaglandin synthesis by inhibition of cyclooxygenase (COX) and it appears to inhibit DNA synthesis³.

Literature survey reveals that various methods have been reported for estimation of DRO such as spectrophotometry, HPLC, HPTLC and voltammetry^1,4-6individually and in combination dosage form. For DIC various analytical methods have been reported for its individual estimation and in combined dosage form and in human plasma which includes spectrophotometry, liquid chromatography with UV detection, HPLC with electrochemical detection^{2,7- 9}.

To our knowledge no spectrophotometric method has been reported for simultaneous analysis of DRO and DIC in combination. Therefore objective of the present work is to develop simple, rapid and accurate Ratio Derivative and AUC spectroscopic methods for simultaneous estimation of DRO and DIC from its formulation. The proposed method was optimized and validated as per the International Conference on Harmonization (ICH) guidelines¹⁰.

MATERIALS AND METHODS:

Instrumentation:

An UV-Visible double beam spectrophotometer (Varian Cary 100) with 10MM matched quartz cells was used. All weighing were done on electronic balance (Model Shimadzu AUW-220D). Ultrasonicator (Model 5.5 150H) was used for sample solution preparation.

DICLOFENAC POTASSIUM

DROTAVERINE HYDROCHLORIDE

Fig.1: Structures of the analytes

Reagents and chemicals:

Pure drug sample of DRO, % purity 99.86 and DIC, % purity 99.91 was kindly supplied as a gift sample by Alkem Pharmaceutical Pvt. Ltd. Mumbai and Aarti Drugs Pvt Ltd., Pune, respectively. These samples were used without further purification. Two tablet formulations (Lot 304 and 308) were supplied by JCPL Pharma Ltd., Jalgaon were used for analysis containing DRO 80mg and DIC 50mg per tablet. Spectroscopy grade methanol and double distilled water was used throughout the study.

Theoretical aspects:

Method A: Ratio Derivative:

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.

Fig.2: Spectra of mixtures of DRO and DIC and the standardized spectra of DRO (24µg mL^-1) and DIC (15µg mL^-1) used as divisor for ratio derivative method.

Standard stock solutions (1000 µg/ml) of DRO and DIC were prepared separately in methanol. The working standard solutions of these drugs were obtained by dilution of the respective stock solution in double distilled water. Using appropriate dilutions of standard stock solution, the solutions were scanned in UV spectrophotometer in the range of 200-400 nm at 0.5 band width and 600 nm/min scan speed parameter. The ratio spectra of different DRO standards at increasing concentrations were obtained by dividing DRO + DIC scans with the stored spectrum of the standard solution of DIC (15 μg mL^-1) as shown in Fig 2 (A). Wavelength 251.96 nm was selected for the quantification of DRO in DRO + DIC mixture Fig.2 (B). The ratio and ratio derivative spectra of the solutions of DIC at different concentrations were obtained by dividing DRO + DIC scans with the stored standard spectrum of the DRO (24 μg mL^-1) [Fig. 3 (A) and (B) respectively]. Wavelength 258.46 nm was selected for the quantification of DIC in DRO + DIC 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 DRO (C_DRO) and DIC (C_DIC) in tablets was calculated by using equations 1 and 2.

At 251.96 nm: C_DRO = (Ratio derivative amplitude for DRO +0.18)/1.3425.... (1)

At 258.46 nm: C_DIC = (Ratio derivative amplitude for DIC - 0.027)/1.9338 .… (2)¹¹

Fig 2(A) Ratio spectra of DRO using 15µg mL^-1 solution of DIC as divisor.

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

Fig 3(A) Ratio spectra of DIC using 24µg mL^-1 solution of DRO as divisor.

Fig 3(B) First order derivative ratio spectra of DIC (5-25µg mL^-1)

Method B: Area Under Curve:

For the simultaneous determination using the area under the curve method, standard stock solutions (1000 μg mL^-1) of DRO and DIC were prepared separately in methanol. The working standard solutions of these drugs were obtained by dilution of the respective stock solution in double distilled water. The solutions of drugs were scanned in the range of 200-400 nm at 0.5 band width and 600 nm/min scan speed parameter. For Area under Curve method, the sampling wavelength ranges selected for estimation of DRO and DIC were 237.4-247.2 nm (λ₁-λ₂) and 271.1-282.8 nm (λ₃-λ₄). Mixed standard were prepared and their Area Under the Curve were measured at the selected wavelength ranges^12,13. By using integrated areas, two simultaneous equations (eq. 3 and 4) were constructed and solved to determine concentrations of analytes. Concentration of two drugs in mixed standard and the sample solution were calculated using equation (5) and (6).

A₁ = 3653.54 C_DRO + 1969.8 C_DIC………………………..(1)

at 237.4-247.2 nm………………………………………..(3)

A₂ =1060.20 C_DRO + 3687.33C_DIC......................................(2)

at 271.1-282.8 nm………………………………………..(4)

C_DRO= A₂× a_y1- A₁× a_y2/ a_x2× a_y1- a_x1× a_y2……………(5)

C_DIC= A₂- a_x2× C_DRO / a_y2 ……………………………….(6)

Where,

(a_x1)3653.54 and (a_x2)1060.20 are absortivities of DRO at (λ₁-λ₂) and (λ₃-λ₄) respectively.

(a_y1)1969.8 and (a_y2)3687.33are absortivities of DIC at (λ₁-λ₂) and (λ₃-λ₄) respectively.

A₁ and A₂ are absorbances of mixed standard at (λ₁-λ₂) and (λ₃-λ₄) respectively. C_DRO and C_DIC are the concentrations in g 100mL ^-1.

Fig 4 Overlain zero order absorbance spectra of DRO (24 µg mL^-1) and DIC (15 µg mL^-1)

Preparation of Standard Stock Solutions and Calibration Curve:

Standard stock solutions of pure drug containing 1000 μg mL^-1 of DRO and DIC were prepared separately in methanol. The working standard solutions of these drugs were obtained by dilution of the respective stock solution in distilled water. Derivative amplitudes of spectrum, by using the above mentioned procedure, were used to prepare calibration curves for both the drugs. Beer’s law obeyed in the concentration range of 8-40 μg mL^-1 for DRO and 5-25 μg mL^-1 for DIC by both methods.

Preparation of Sample Stock Solution and Formulation analysis:

Twenty tablets were weighed accurately and a quantity of tablet powder equivalent to 100 mg of DRO (62.5 mg of DIC) was weighed and dissolved in the 80 mL of methanol with the aid of ultrasonication for 5 min and solution was filtered through Whatman paper No. 41 into a 100 mL volumetric flask. Filter paper was washed with methanol, adding washings to the volumetric flask and volume was made up to the mark with methanol. The solution was suitably diluted further with distilled water to get required final concentration of DRO (24μg mL^-1) and DIC (15 μg mL^-1) and proposed methods were followed.

Recovery studies:

The accuracy of the proposed method was checked by recovery studies, 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 DRO and 7.5μg mL^-1of DIC.

Precision of the Method:

Reparability of the methods was studied by repeating the methods six times. To study intraday precision, method was repeated 5 times in a day and the average % RSD was found to be 1.12 and 0.58 for DRO by method A and B respectively; 0.59 and 0.87 for DIC by method A and B, respectively. Similarly the method was repeated on five different days and average % RSD was found to be 1.03 and 0.82 for DRO by method A and B respectively; 1.21 and 1.56 for DIC by method A and B respectively. These values confirm the intra and inter day precision.

Table 1: Optical characteristics of the proposed methods and results of formulation analysis and precision study

Parameter				DRO		DIC
Parameter				Method A	Method B	Method A	Method B
λ (nm)				251.96	Integrated area between 237.4 - 247.2	258.46	Integrated area between 271.1 - 282.8
Beer’s law Range (μg mL^-1)				8 - 40	8 - 40	5 - 25	5 - 25
Regression Equation (y = mx+c)		Slope (m)		1.3425	-	1.9338	-
Regression Equation (y = mx+c)		Intercept (c)		-0.18	-	0.027	-
Correlation coefficient				0.9998	-	0.9998	-
Precision (%RSD)	Repeatability(n=6)			1.01	0.68	0.79	0.83
	Intra-day(3×5 times)			1.12	0.58	0.59	0.87
	Inter-day(3×5 days)			1.03	0.82	1.21	1.56
Formulation Analysis (% Assay, % RSD)			Tablet I	99.01, 0.41	100.8, 0.32	98.91, 0.51	101.7, 0.33
Formulation Analysis (% Assay, % RSD)			Tablet II	100.11, 0.63	99.24, 0.93	99.84, 0.55	101.5, 0.65

RSD is relative standard deviation

Table2 (A): Result of the recovery analysis by Ratio Derivative Method (Method A)

Formulation	Recovery Level (%)	Amount spiked (μg mL^-1)		Recovery (%)		RSD (%) n = 3
Formulation	Recovery Level (%)	DRO	DIC	DRO	DIC	DRO	DIC
Tablet I	50	6	3.75	100.23	99.06	0.52	0.05
	100	12.0	7.5	101.34	99.97	1.82	0.79
	150	18.0	11.25	100.53	99.70	1.03	0.19
Tablet II	50	6.0	3.75	99.43	100.03	0.59	0.28
	100	12.0	7.5	99.83	99.43	0.68	0.78
	150	18.0	11.25	100.11	100.16	0.33	0.36

Table2 (B): Result of the recovery analysis by Area Under Curve Method (Method B)

Formulation	Recovery Level (%)	Amount spiked (μg mL^-1)		Recovery (%)		% RSD (n = 3)
Formulation	Recovery Level (%)	DRO	DIC	DRO	DIC	DRO	DIC
Tablet I	50	6	3.75	99.99	99.56	1.52	0.75
	100	12.0	7.5	100.12	100.45	1.72	1.12
	150	18.0	11.25	99.72	99.21	0.85	1.45
Tablet II	50	6.0	3.75	100.75	100.13	0.75	0.29
	100	12.0	7.5	99.87	99.91	0.72	0.58
	150	18.0	11.25	100.13	100.33	0.63	0.32

RSD is relative standard deviation

RESULTS AND DISCUSSIONS:

The proposed methods for simultaneous estimation of DRO and DIC in combined dosage form were found to be accurate, simple and rapid. Since none of the method is reported for simultaneous analysis of the two drugs earlier, the developed methods can be used for routine analysis of two drugs in combined dosage forms.

Method A involves dividing the spectrum of mixture into the standardized spectra for each of the analyte and deriving the ratio to obtain spectra that is independent of analyte concentration used as divisor. Method B involves formation and solving of simultaneous equation. Once the equations are formed, then only measurement of the area of sample solution at two wavelength ranges and simple calculations are required. Under experimental conditions described, calibration curve, assay of tablets and recovery studies were performed. Using appropriate dilutions of standard stock solution the two solutions were scanned separately. The zero order overlain spectra are shown in Fig 4. A critical evaluation of proposed method was performed by statistical analysis of data where slope, intercept, correlation coefficient is shown in Table 1. As per the ICH guidelines, the method validation parameters checked were linearity, accuracy and precision. Beer’s law obeyed in the concentration range 8 - 40 μg mL^-1 for DRO and 5 - 25 μg mL^-1 for DIC with correlation coefficient of > 0.999 for both the drugs. The proposed methods were also evaluated by the assay of commercially available tablets containing DRO and DIC (n = 5). The results of formulation analysis are also presented in Table 1. For DRO, the recovery study results ranged from 99.72 to 101.34% with % RSD values ranging from 0.52 to 1.82 % for both the methods. For DIC, the recovery results ranged from 99.06 to 100.45 %, with % RSD values ranging from 0.05 to1.45% for both the methods. Results of recovery studies are shown in Table 2. The accuracy and reproducibility is evident from the data as results are close to 100 % and standard deviation is low.

CONCLUSION:

The validated spectrophotometric methods employed here proved to be simple, economical, precise and accurate. Thus it can be used as IPQC test and for routine simultaneous determination of DRO and DIC in tablet dosage form.

ACKNOWLEDGEMENT:

The authors wish to express their gratitude to Alkem Pharmaceutical Pvt. Ltd. Mumbai and Aarti Drugs Pvt Ltd., Pune, India for the gift sample of pure DRO and DIC respectively. The authors are also thankful to the management of MAEER’s Maharashtra Institute of Pharmacy for providing necessary facilities.

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Received on 03.04.2010 Modified on 28.04.2010

Asian J. Research Chem. 4(1): January 2011; Page 35-39