Simultaneous Determination of Hyoscine N- Butyl Bromide and Paracetamol by RP-TLC Spectrodensitometric Method

Aims: A simple RP-TLC Spectrodensitometric method was developed for determination of Hyoscine N-Butyl Bromide (HBB) and Paracetamol (PAR) either in bulk powder or in their pharmaceutical preparation. Study Design: Validation study. Methodology: In this method, HBB and PAR were separated on RP-18 W/ UV254 TLC plates using developing mobile phase consisting of methanol: citrate buffer (pH=1.5): triflouroacetic acid (70:30:0.1, by volume) at room temperature. Experimental conditions such as band size, slit width, different developing systems and scanning wavelength were carefully studied and the optimum conditions were selected. The obtained bands were then scanned at 210 nm. The two drugs were satisfactorily resolved with RF 0.60 ± 0.02 for HBB and 0.81 ± 0.02 for PAR. The validation of spectrodensitometric method was done regarding linearity, accuracy, precision, and specificity. Results: Linearity of the proposed methods was evaluated and it was found to lie within the concentration range of 2.0-12.0 μg.band for HBB and 2.0-14.0 μg.band for PAR. Research Article British Journal of Pharmaceutical Research, 3(3): 472-484, 2013 473 Conclusion: The proposed method was successfully applied for determination of HBB and PAR in pure form and in their different pharmaceutical formulations. The method proved to be specific, accurate and selective.


INTRODUCTION
Hyoscine N-ButylBromide is a quaternary ammonium anticholinergic agent. It has been used as antispasmodic due to relaxation effect on the smooth muscles of the gastrointestinal, biliary, and urinary tracts [1].
Paracetamol (PAR), 4-acetamidophenol, is an effective analgesic and antipyretic for treatment of minor, non-inflammatory conditions in patients who are prone to gastric symptoms [1]. The structural formulas of HBB and PAR are shown in Fig. 1.
Few methods have been mentioned for analysis of HBB and PAR in binary mixture. In the first method, Erk [18] analysed HBB and PAR mixture by precipitating HBB with ammonium reineckate at pH 6,0 selectively and reading the absorbance of the solution of the precipitate in acetone at 525.0 nm for HBB and by measuring the dA/dλ values at 254.5 nm in the first derivative spectra of the remaining solution for paracetamol.
In the second method [19], solid phase extraction procedure using strong cation exchange cartridges followed by a reversed-phase HPLC assay was applied to the analysis of HBB, PAR and lidocaine hydrochloride in injection forms.
No TLC method has been reported for simultaneous analysis of the two drugs. Therefore, the objective of this work is to develop sensitive and selective RP-TLC method for simultaneous determination of HBB and PAR for routine quality control analysis of these drugs either in bulk powder or in pharmaceutical formulations.

Pure samples
Paracetamol (PAR) and Hyoscine N-Butyl Bromide (HBB) were kindly supplied by CID Co. Chemical Industries Development, Giza, Egypt. Their purity was found to be 99.94±1.537 and 99.21±1.012, respectively, according to the company certificate of analysis.

Reagents
Analytical grade reagents and chemicals were used without further purification:

Preparation OF Standard Solutions
A. A-Paracetamol (PAR) and Hyoscine N-Butyl Bromide (HBB) stock standard solutions (1 mg.mL -1 ). Stock standard solutions of Paracetamol (PAR) and Hyoscine N-Butyl Bromide (HBB) each containing 1 mg.mL -1 were prepared in methanol. B. B-Paracetamol (PAR) and Hyoscine N-Butyl Bromide (HBB) working standard solutions (100 µg.mL -1 ) working standard solutions (100 μg.mL -1 ) of these drugs were prepared by appropriate dilution of the stock solution with methanol.

Linearity and construction of calibration curves
Aliquots equivalent to (2.0 -12.0 μg) of HBB, (2.0-14.0 μg) of PAR were applied accurately from their corresponding stock solutions (l000 μg.mL -1 ) to RP-TLC plates (10x10cm) as band using the Camage TLC sampler. A space of 1 cm was left between each band and1.5 cm from the bottom edge of the plate. The plate was developed in a previously saturated chromatographic tank for one hour with the developing mobile phase consisting of methanol: citrate buffer (pH=1.5): triflouroacetic acid (70.0:29.9:0.1, by volume) by ascending chromatography at room temperature [25].
The bands were detected under UV -lamp and scanned at 210 nm under the specified experimental conditions. The calibration curves were constructed for each compound by plotting the peak area/ 100 versus the corresponding concentration and then the regression equations were computed.

Analysis of laboratory prepared mixtures
The mixtures containing HBB and PAR in different ratios were prepared and analyzed as mentioned under linearity and construction of calibration curves. The concentrations of the two compounds were calculated from their corresponding regression equations.

Application of TLC-spectrodensitometric method to pharmaceutical formulations
A) For tablet dosage form: The contents of ten tablets of Buscopan plus® (also for Buscamol®) were thoroughly powdered and mixed then an amount of the powder equivalent to 500 mg of PAR and 10 mg of HBB was weighed accurately in 250-mL beaker, 70 mL of methanol was added, stirred for about 30 min then filtered through filter paper into a 100-mL volumetric flask, the beaker and the funnel were washed and the volume was completed with methanol to get a concentration of 5.0 and 0.10 mg.mL-1 for PAR and HBB, respectively. Appropriate dilutions were made to get a concentration of 100.0 and 2.0 g.mL-1 for PAR and HBB, respectively and a concentration of 5.0 and 0.1 -1 PAR and HBB, respectively.

B) For suppositories dosage form:
The contents of five suppositories of Buscopan plus ® were thoroughly cut to small fragments then an amount of the fragments equivalent to 800 mg of PAR and 10 mg of HBB was weighed accurately in 250-mL beaker, 70 mL of methanol was added, stirred for about 30 min, leave to cool to coagulate the suppository base then filtered through filter paper into a 100-mL volumetric flask, the beaker and the funnel were washed and the volume was completed with methanol to get a concentration of 8.0 and 0.10 mg.mL -1 for PAR and HBB, respectively. Appropriate dilutions were made to get a concentration of 160.0 and 2.0 g.mL -1 for PAR and HBB, respectively and a concentration of 8.0 and 0.1 g.mL -1 PAR and HBB, respectively.

Method Development and Optimization
The aim of this work is to develop an applicable method that can be used successfully for separation and quantification of the studied drugs. Studying of the optimum parameters for maximum separation was carried out by investigating the effect of different variables. Different developing systems with different compositions and ratios were tried, but complete separation of HBB and PAR was achieved by using the reported developing mobile phase [25] consisting of methanol: citrate buffer (pH=1.

Method Validation
Method validation was performed according to ICH guidelines [27]. Linearity of the TLCspectrodensitometric method was evaluated and it was found to lie within the concentration range of 2.0-12.0 µg.band -1 for HBB and 2.0-14.0 µg.band -1 for PAR, Figs. (3)(4). Good linearity was evident by the high value of the correlation coefficient and the low intercept value, ( Table 4). The method can detect low concentrations of the two drugs, the sensitivity of the method is relatively similar to that of spectrophotometric method.
The regression equations were calculated and found to be: Y 1 = 0.155 C 1 + 0.333 r 1 = 0.9998 Y 2 = 0.274 C 2 + 0.169 r 2 = 0.9996 Where Y 1 and Y 2 are the peak area /100, C 1 and C 2 are HBB and PAR concentrations in μg.band -1 respectively and r 1 and r 2 are the correlation coefficients. Precision of the proposed TLC-spectrodensitometric method was evident as shown in Table  4 where the intra-day and inter-day relative standard deviations of the average of concentrations (4.0 ,6.0 and 10.0 μg band -1 for each drug were calculated and found to be 1.014 and 1.182 for HBB , 1.122 and 0.814 for PAR.
Accuracy of the TLC-spectrodensitometric method was checked by applying the method for determination of different samples of pure HBB and PAR. The concentrations of HBB and PAR were calculated from the corresponding regression equations. The results obtained were shown in Table 1. The accuracy for HBB was found to be 100.22±0.733 while it was found to be 99.82±1.048 for PAR.
Accuracy of the TLC-spectrodensitometric method was further assessed by applying the standard addition technique on Buscopan plus ® tablets, Buscamol ® tablets and Buscopan plus ® suppositories where good recoveries were obtained as shown in Table 3 revealing good accuracy of the proposed method.
Specificity of the described method is evident from the TLC-spectrodensitometric chromatogram as shown in Fig. 2 where each drug of the mixture appears at certain R f value (0.61 for HBB and 0.81 for PAR. Specificity of the proposed method is also evident from Table 2 where the accuracy for HBB was found to be 100.25±1.084 while it was found to be 100.53±0.704 for PAR. Robustness of the TLC-spectrodensitometric method was evaluated in the development phase by making small changes in the composition of mobile phase and detection wavelength (209, 211 nm). The low values of %RSD show that the method is robust and that deliberate small changes in the studied factors did not lead to a significant change in R f values, area or symmetry of the peaks.
System suitability tests are based on the concept that the equipment, electronics, analytical operations and samples constitute an integral system that can be evaluated as whole. System suitability is used to ensure system performance before or during the analysis of the drugs. System suitability was checked by calculating the capacity factor(K'), symmetry factor, the selectivity factor(γ) and resolution(Rs), where the system was found to be suitable as shown in Table 5.

CONCLUSION
The proposed method is efficient for providing sensitive and accurate quantitative analysis for simultaneous determination of HBB and PAR in bulk powder and in pharmaceutical formulations. TLC-spectrodensitometric method has the advantages of that several samples can be run simultaneously using a small quantity of mobile phase and provides high sensitivity and selectivity. The separation power of chromatographic methods allows determination of mixture of drugs by any ratio in pharmaceutical formulations.
Statistical analysis was determined by comparing the results of the TLCspectrodensitometric method with those of manufacturer HPLC method. No significant difference was estimated regarding accuracy and precision, as shown in Table 6.

CONSENT
Not applicable.

ETHICAL APPROVAL
Not applicable.