Development of Dissolution Test Method for Drotaverine Hydrochloride/Mefenamic Acid Combination Using Derivative Spectrophotometry

Purpose: To develop and validate a dissolution test method for tablets containing 80 mg of drotaverine hydrochloride (DRT) and 250 mg of mefenamic acid (MEF). Methods: Sink conditions, drug stability and specificity in different dissolution media were tested to optimize a dissolution test method using a USP paddle type dissolution test apparatus set at a speed of 50 rpm. The dissolution medium consisted of 900 ml of phosphate buffer (pH 6.8) containing 0.25% w/v cetrimide at 37 ± 0.5 o C and 45 min time-point. To determine both drugs simultaneously, a first derivative UV spectrophotometric method was developed and validated. Drug release was analyzed by first derivative UV method at 253.8 nm and 304 nm for DRT and MEF respectively. The dissolution method was validated as per ICH guidelines. Results: The two brands each showed 98% of drug release for both drugs when the developed dissolution method was used. The regression plot was linear in the concentration range 4 - 24 µg/mL for each of the drugs and regression coefficient (r 2 ) was greater than 0.999 for each drug. Relative standard deviation (% RSD) for precision and accuracy of proposed method was < 2. Conclusion: The proposed dissolution method is simple, cost-effective, precise, accurate and specific. It can be successfully employed in routine quality control of DRT and MEF combination tablets.

Dissolution testing of poorly soluble compounds in immediate release solid dosage forms presents several challenges. Single dissolution method for the analysis of combined dosage forms is preferred for simplification of quality control testing [7][8][9][10][11][12][13]. However, development of a single dissolution method for DRT and MEF is practically challenging due to the pH dependent dissolution of these two drugs. Presently, assay of DRT is not described in any pharmacopoeia and there is no specification for its dissolution test, but a dissolution medium, tris buffer (pH 9.

Selection of wavelength
Standard solutions of DRT and MEF were diluted appropriately with phosphate buffer (pH 6.8) to obtain a solution containing DRT (12 µg/mL) and MEF (12 µg/mL). The spectra of these diluted solutions were scanned in the spectral region of 200 to 400 nm using phosphate buffer pH 6.8 as blank. The zero-order spectra of DRT and MEF were transformed to corresponding firstderivative spectra in the range of 200 to 400 nm.

Derivative conditions
First-order derivative spectra of DRT (12 µg/mL) and MEF (12 µg/ mL) were overlapped. The zero-crossing point (ZCP) values of MEF at which the DRT showed some derivative response were recorded. The wavelength, 253.8 nm, was selected for the quantification of DRT (where the derivative response for MEF was zero) while 304.0 nm was selected for the quantification of MEF (where the derivative response for DRT was zero). The optimized wavelengths of 253.8 nm and 304 nm for DRT and MEF, respectively were confirmed by checking the derivative absorbance of various concentrations of both drugs in the range of 4 -24 µg/mL. [14].

Dissolution test conditions
Dissolution testing of combined DRT and MEF tablet formulation (Doverin-M) was performed in a paddle type USP tablet dissolution apparatus, in 900 mL of various buffer and surfactant media at 50 rpm and 37 ± 0.5 °C for 45 min. Aliquots of 5.0 mL were withdrawn at 5 min interval up to 45 min, and replaced with an equal volume of fresh medium to maintain sink conditions. At the end of the test, the withdrawn samples were filtered, diluted with phosphate buffer pH 6.8 and quantified by the developed and validated spectrophotometric derivative method (n = 3). The amount of dissolved drugs was computed from the respective calibration curves and then plotted against time.
The media in which highest drug release occurred for Doverin-M, and this medium was chosen for in vitro dissolution studies in DROFEM.

Method validation
The dissolution method was validated as per ICH guidelines. The parameters assessed were specificity, linearity, precision and accuracy.

Specificity
Specificity is the ability of the method to accurately measure a compound in the presence of other components such as impurities, degradation products and matrix components. The specificity of the proposed method was evaluated through the analysis of a placebo solution, which it was prepared with the common excipients (lactose, starch, microcrystalline cellulose, magnesium stearate and talc) of the pharmaceutical formulation. Thus, the mixture of component inert was prepared in their usual concentration employed in tablets (concentrations were determined based in Handbook of pharmaceutical Excipients and calculated for medium weight of content). The developed method was applied to in order to check if any component of the formulation could generate a response or a read with absorption band similar to the drugs.

Linearity
Appropriate aliquots of standard stock solutions of DRT (100 µg/mL) and MEF (100 µg/ mL) were taken in two different sets of 10 mL volumetric flasks and diluted up to the mark with phosphate buffer (pH 6.8) to obtain final concentrations of 4 -24 µg /mL for both drugs. First-derivative spectra were recorded using the prepared solutions against phosphate buffer (pH 6.8) as blank. The values of the first-derivative absorbance for DRT and MEF were obtained using six different concentrations by measuring each concentration against solvent blank at the chosen wavelength 253.8 nm and 304 nm for DRT and MEF, respectively. The values of firstderivative absorbance were plotted against corresponding concentrations to construct the calibration curves.

Precision
The precision of the method was determined by repeatability (intra-day) and reproducibility (interday precision). It was evaluated based on the relative standard deviation (% RSD) [18]. The intra-and inter-day precision were assessed after subjecting six tablets to the dissolution test conditions, on the same day and on three different days, respectively.

Accuracy
Accuracy was evaluated by adding known amounts of the reference substance to the placebo sample in the dissolution medium at 80 %, 100 % and 120 % of the nominal assay value of DRT and MEF. The accuracy was calculated as the the % drug recovered from the formulation matrix.

Statistical analysis
The dissolution data of the two formulations were subjected to statistical analysis using Student's ttest by Prism 4.0 software, and differences between data were considered significant at p < 0.05.

Development of dissolution test method
Selection of dissolution test method was based on screening studies using USP apparatus 2 at a paddle speed 50 rpm: operating conditions are listed in Table 1.Selection of a dissolution medium to accommodate adequate solubility and stability of both DRT and MEF was critical for the selected dissolution method. Several compendia dissolution media were screened; these include various media -buffer (pH 1.2 to 9.0), surfactant and buffer/surfactant. The addition of surfactants to the buffer solution was also investigated [15,16], Cummulative DRT and MEF release ( Figure 2) in phosphate buffer (pH 6.8) containing 0.25 %w/v cetrimide was 98.8 and 101.3 %, respectively, from Doverin-M tablets. Therefore, the selected dissolution test conditions were: USP apparatus 2 at paddle speed 50 rpm in a medium of phosphate buffer (pH 6.8) containing 0.25 %w/v cetrimide. Dissolution studies on Drofem tablets were performed at these dissolution test conditions. These results, shown in Figure 2, reveal that 99.01 and 101.16 % of DRT and MEF were released, respectively, from Drofem tablets in the optimized medium. Statistical analysis of the dissolution profile data for the two formulations indicate that the profiles were similar (t cal = 4.0 and 0.15 for DRT and MEF, respectively, which is less than t cri = 4.303 (p ≤ 0.05).

Specificity
The derivative spectra of placebo, DRT and MEF in the dissolution medium, shown in Figure 3, reveal that there was no interference from the excipients in the tablets (placebo) with derivative response of either of drugs (DRT and MEF) at their respective analytical wavelengths of 253.8 and 304 nm, respectively. Hence, the method is specific [17].

Precision
The % relative standard deviation values were found to be less than 2 for intra and inter day precision studies and there is no significant difference was observed between intra and inter day values, which indicates that the proposed method was precise and reproducible. Results are reported in Table 2.

Accuracy
The results are reported in Table 3. The %RSD values obtained for DRT and MEF were < 2, indicating that the method is accurate for its intended use [18].

DISCUSSION
In order to develop single dissolution method for simultaneous quantification of the DRT and MEF, initial trials were performed with the objective to accommodate adequate solubility and stability of both drugs. Sink conditions, stability of the drugs and specificity in different dissolution media were tested to optimize a dissolution test. The screening results showed that the dissolution rate of DRT is higher in pH 4.0 tris buffer than in the other buffers and also that if the dissolution rate of MEF increased with increasing medium pH, then a single pH buffer would be unsuitable for the method chosen. Both drugs exhibited higher dissolution rate in 0.25 %w/v cetrimide than in 0.25 %w/v sodium lauryl sulphate and

CONCLUSION
A dissolution test for the simultaneous determination of DRT and MEF in tablets has been successfully developed and validated according to ICH guidelines. The proposed dissolution test conditions should enhance laboratory efficiency and is specific, costeffective, accurate and precise. Thus, the proposed dissolution method can be used in the routine quality control analysis of DRT and MEF when combined in tablets.