UV Spectrophotometric Determination of Paracetamol in Presence of Drotaverine Hydrochloride

Objective: The aim of this study is to develop three simple, specific and accurate spectrophotometric methods manipulating ratio spectra for the determination of paracetamol in binary mixture with drotaverine hydrochloride in tablet dosage form. Methods: In these methods, the absorption spectra of paracetamol were divided by 8 μg/ml of drotaverine hydrochloride to get the ratio spectra. In the first method (ratio difference), the difference in peak amplitudes of the ratio spectra were measured at 262 and 272 nm. The second method is a ratio subtraction which is based on determination of paracetamol at 248 nm after subtraction of interference exerted by drotaverine hydrochloride. In the third method (mean centering), the mean centered values of the ratio spectra were measured at 262 nm. Results: The proposed methods were accurate, precise and selective for determination of paracetamol in presence of drotaverine hydrochloride in pure form and in pharmaceutical dosage forms. Beer’s law was obeyed in the concentration range of 1–10 μg/ml in all methods. Conclusion: The developed methods were used to determine the studied drug in bulk powder, laboratory prepared mixtures and pharmaceutical dosage form with good accuracy and precision. All methods were validated according to ICH guidelines and the results obtained were statistically compared to those obtained from a reported method and were found to be in good agreement.


INTRODUCTION
Paracetamol (PAR) is N-(4-hydroxyphenyl) acetamide (Figure 1a).Its molecular weight is 151.16  and its molecular formula is C8H9NO2.It is very slightly soluble in cold water, considerably more soluble in hot water, soluble in methanol, ethanol, dimethylformamide and ethyl acetate.It melts at 169-170.5 °C.It is an analgesic and antipyretic drug with weak antiinflammatory activity.
Drotaverine hydrochloride (DRH) is 1-[(3,4diethoxyphenyl)methylene]-6,7-diethoxy-1,2,3,4-tetrahydroisoquinoline hydrochloride (Figure 1b).Its molecular weight is 433.97 and its molecular formula is C24H31NO4.HCl.It is pale yellow crystals, soluble ethanol.It melts at 197-200 °C.It is commonly prescribed as antispasmodic agent. 1 There are various publications which describe various analytical methods for determination of PAR either present in single or in combination with other active ingredients in pharmaceutical formulations and+ in biological fluids.][13][14][15][16][17][18][19] In this work, three different spectrophotometric methods manipulating ratio spectra were developed namely; ratio difference, ratio subtraction and mean centering of ratio spectra methods, aiming for the determination of PAR in presence of DRH without previous separation.

Apparatus
Shimadzu, UV-Vis 1601PC spectrophotometer (Tokyo, Japan), connected with UV probe program with two matched 1 cm path-length quartz cell.

Samples
Pure samples.Paracetamol pure sample was kindly supplied by Egyptian Co. for Pharmaceutical and Chemical Industries (10 th of Ramadan City), Cairo, Egypt.Its purity was assigned as 99.92% as referred by the supplier.
Drotaverine hydrochloride pure sample was kindly supplied by Alexandria Pharmaceutical Company, Egypt.The purity was assigned as 99.95% as referred by the supplier.
Pharmaceutical preparation.Dropar tablets ® , each tablet claimed to contain 500 mg of paracetamol and 80 mg of drotaverine hydrochloride, manufactured by Fair Deal Corporation (FDC) company, Mumbai, Maharashtra, India), purchased from local market.

Standard solutions
Stock solutions of both drugs (1mg/ml) were prepared separately by dissolving 100 mg of PAR and DRH in 25 ml of methanol and the volumes of each were completed to 100 ml with the same solvent.
Working standard solutions were prepared by further dilution of their stock solutions with methanol.

Construction of calibration graphs
Aliquots of standard PAR solution (100 μg/ml) equivalent to (1-10 μg) were introduced into a series of 10-ml volumetric flasks then completed to volume with methanol.The absorption spectra of the prepared solutions were scanned from 200-400 nm using methanol as a blank and saved in the computer.The retrieved spectra of PAR were divided by that of DRH (8 μg/ml) to get the ratio spectra.

Ratio difference method:
The difference in the peak amplitudes of the ratio spectra was measured at 262 and 272 nm.The measured difference values in peak amplitudes were plotted versus the final concentrations of PAR to get the calibration graph and the regression equation was derived.

Ratio subtraction method:
The constant (amplitude value at 360 nm in the ratio spectra) was subtracted from each ratio spectrum, followed by multiplication of the obtained spectra by the divisor spectrum.The amplitude values were measured at 248 nm in the final spectra.The measured amplitudes were plotted versus the final concentrations of PAR to get the calibration graph and the regression equation was derived.

Mean centering of ratio spectra method:
The ratio spectra (from 200 to300 nm) were mean centered and the mean centered values were measured at 262 nm.The measured mean centered values were plotted versus the final concentrations of PAR to get the calibration graph and the regression equation was derived.

Assay of laboratory prepared mixtures
Aliquots of standard PAR solution (100 μg/ml) and standard DRH solution (100 μg/ml), in the specified range, were introduced into a series of 10-ml volumetric flasks and diluted to volume with methanol.Procedure for each method was applied and the concentrations of PAR in the prepared mixtures were determined from the corresponding regression equation for each method.

Application to pharmaceutical preparation
The contents of five Dropar tablet ® , each labeled to contain 500 mg of PAR and 80 mg of DRH, were weighed, powdered and mixed well.An accurately weighed quantity of the powder equivalent to one tablet was introduced into a 100-ml volumetric flask and volume was adjusted up to the mark with methanol.The flask was sonicated for 15 minutes with vigorous shaking then filtered.The clear filtrate was claimed to contain 5mg/ml of PAR and 0.8 mg/ml of DRH.One ml of the clear filtrate was transferred to 100-ml volumetric flask and diluted to volume with methanol to obtain a solution labeled to contain 50 μg/ml of PAR and 8 μg/ml of DRH.Procedure for each method was repeated using aliquots covering the working concentration range.PAR concentrations were calculated from the corresponding regression equation for each method.

Spectral characteristics and methods development
Zero-order absorption spectra of PAR and DRH (Figure 2), show severe overlap, which does not permit direct determination of PAR in presence of DRH.Ratio difference method [20][21][22] In this method, absorption spectra of PAR were divided by that of DRH (8 μg /ml), as a divisor, to get the ratio spectra, as shown in Figure 3.The difference in peak amplitudes between 262 and 272 nm in the ratio spectra is proportional to the concentration of PAR without interference from DRH.
The method comprises two critical steps; the first is the choice of the divisor concentration and the selected divisor concentration should compromise between minimal noise and maximum sensitivity.The second critical step is the choice of the wavelengths at which measurements are recorded.] In this method, the absorption spectra of binary mixtures of PAR and DRH were divided by that of DRH (8 μg/ml) to get the ratio spectra, as shown in Figure 4, then subtraction of the absorbance values in plateau region at 360 nm (the constant), as shown in Figure 5, followed by multiplication of the obtained spectra by the spectrum of the divisor, as shown in Figure 6 to get the final spectra.The peak amplitude at 248 nm in the final spectra is proportional to the concentration of PAR without interference from DRH.    22 In this method, the obtained ratio spectra (from 200 to 300 nm) were mean centered and the mean centered values were measured at 262 nm to allow determination of PAR without interference from DRH.

Linearity and range
Under the described experimental conditions, the calibration graphs for the methods were constructed by plotting the response versus PAR concentrations in μg/ml.The regression plots were found to be linear over the range of 1-10 μg/ml for the three proposed methods as shown in Figures 8-10.Linearity ranges, intercepts, slopes and coefficients of determination for the calibration data were listed in Table 1.

Limits of detection (LOD) and limits of quantification (LOQ)
LOD and LOQ were calculated according to ICH guidelines from the following equations: LOD = 3.3 σ / S LOQ = 10 σ / S Where σ is the residual standard deviation of a regression line and S is the slope the calibration curve.The small values of LOD and LOQ, as listed in Table 1 indicate good sensitivity.

Accuracy
Accuracy of the proposed methods was assessed using three different concentrations of pure samples of PAR covering the linearity range, each in triplicate, then accuracy as percent recovery (%R) was calculated.The good %R, listed in Table 1, confirms excellent accuracy.

Precision
Precision of the proposed methods was assessed using three different concentrations of pure samples of PAR covering the linearity range, each in triplicate, within one day for repeatability and in three successive days for intermediate precision, then precision as percent relative standard deviation (%RSD) was calculated.The small values of %RSD, listed in Table 1, indicate high precision of the methods.2.

Application to pharmaceutical preparation
The proposed methods were successfully applied for determination of PAR in Dropar ® tablets without interference from DRH or the additives present.Standard addition technique was applied and the good recoveries of the pure added amounts of PAR, indicates no matrix interference, as shown in Table 3. Statistical analysis of the results obtained by the proposed methods and the reported method 2 by applying t-test and F-test at 95% confidence level 25 , revealed no significant difference between the proposed and reported methods, as shown in Table 4.

CONCLUSION
The proposed methods are simple, rapid, accurate and precise and can be used for the selective determination of paracetamol in presence of drotaverine hydrochloride in pure form and in pharmaceutical dosage forms.

Figure 4 .
Figure 4. Ratio spectra of laboratory prepared mixtures of PAR and DRH using 8 μg/ml of DRH as a divisor.

Figure 5 .
Figure 5. Ratio spectra of laboratory prepared mixtures of PAR and DRH after subtraction of the constant.

Figure 6 .
Figure 6.Final spectra of laboratory prepared mixture of PAR and DRH after multiplication by the divisor spectrum.

Figure 10 .
Figure 10.Calibration curve of paracetamol by mean centering method.