Development and validation of new analytical methods for simultaneous estimation of Drotaverine hydrochloride in combination with Omeprazole in a pharmaceutical dosage form

2.2.1 Standard stock solution

The standard stock solutions of Drotaverine hydrochloride and Omeprazole were prepared by dissolving accurately weighed 100 mg of drug in 100 ml of a mixture of methanol and double distilled water (50:50) in two separate 100 ml volumetric flasks to get a concentration of 1000 μg/ml. Both were appropriately diluted with a mixture of methanol and double distilled water (50:50) to get a concentration of 100 μg/ml and were kept as stock solutions.

2.2.2 Determination of λ_max

The standard solution of both Drotaverine hydrochloride and Omeprazole (10 μg/ml) were scanned in the wavelength region of 200–400 nm and the λ_max was found to be 226.8 nm (λ_max of Drotaverine hydrochloride) and 269.4 nm (λ_max of Omeprazole), respectively. They were scanned in the wavelength range of 200–400 nm and the overlain spectrum was obtained (Fig 3).

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Figure 3

Overlain spectra of the Drotaverine HCl and Omeprazole.

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2.2.3 Preparation of calibration curve

For each drug, appropriate aliquots were pipetted out from each standard stock solution into a series of 10 ml volumetric flasks. The volume was made up to the mark with methanol and double distilled water (50:50) to get a set of solutions having a concentration range of 5–30 μg/ml for both Drotaverine hydrochloride and Omeprazole. Triplicate dilutions of each concentration of each drug were prepared separately. The prepared working solutions of Drotaverine hydrochloride and Omeprazole were scanned at 226.8 nm and 269.4 nm, respectively. The respective absorbances were recorded and absorbances were plotted against the concentrations to obtain their respective calibration curves. The absorptivity coefficients of each drug at both wavelengths were determined. The concentration of two drugs in the mixture was calculated using equations

2.2.4 Preparation of sample solution

Contents of twenty tablets were weighed accurately and powdered. Powder equivalent to 40 mg of Drotaverine hydrochloride and 10 mg of Omeprazole was weighed. The tablets were triturated with the help of mortar and pestle and made into a fine powder and the residue equivalent to the average weight of a tablet was dissolved in a mixture of methanol and double distilled water (50:50) and sonicated for 10 min in the sonicator. The solution was filtered through Whatman filter paper No. 41 into a 100 mL volumetric flask. Filter paper was washed with methanol, adding washings to the volumetric flask and the volume was made up to the mark with methanol to get the sample stock solution which was further diluted with 0.1 N HCl to get the final concentration of solution (Drotaverine hydrochloride 5 μg mL⁻¹ and Omeprazole 10 μg mL⁻¹) in the linearity range.

2.3.1 Instrument

The HPLC system is equipped with an LC-10 AT VP solvent-delivery system with a universal loop injector (Rheodyne 7725 i) of an injection capacity of 20 μL. Detector consists of photodiode array detector SPD-10 AVP UV–Visible detector. Separation was carried out on a Phenomenex Luna C₁₈ (5 μm × 25 cm × 4.6 mm i.d) under reversed phase partition chromatographic conditions. The equipment was controlled by a PC workstation. The work was carried out in an air-conditioned room maintained at temperature 25 ± 2 °C. Chromatograms were recorded using CLASS-VP software (Shimadzu, Kyoto, Japan).

2.3.2 Chromatographic conditions

The optimal composition of the mobile phase was determined to be potassium dihydrogen phosphate buffer pH 5.0: Acetonitrile: Triethylamine (60:40:0.5, v/v). The flow rate was set to 1 mL min⁻¹ and UV detection was carried out at 260 nm. Ciprofloxacin was used as an internal standard. The mobile phase was filtered through nylon 0.22 μm membrane filter and was degassed before use.

Stock solution was prepared by dissolving Drotaverine hydrochloride and Omeprazole (10 mg each) that were weighed accurately and separately transferred into 100 mL volumetric flasks. Both drugs were dissolved in 25 methanol to prepare standard stock solutions. After the immediate dissolution, the volume was made up to the mark with mobile phase. These standard stock solutions were observed to contain 100 μg ml⁻¹ of Drotaverine hydrochloride and Omeprazole. Ciprofloxacin (internal standard) was taken in a separate 10 mL volumetric flask and dissolved in methanol. Then the volume was made up to the mark with the same solvent. Appropriate volume from this solution was further diluted to get appropriate concentration levels according to the requirement. From the above stock solutions, dilutions were made in the concentration range of 5–30 μg mL^–1 of Drotaverine hydrochloride and Omeprazole, respectively and each concentration contains 5 μg mL^–1 of Ciprofloxacin (internal standard). A volume of 20 μL of each sample was injected into column.

2.3.3 Analysis of marketed formulation

Twenty tablets were weighed the average weight was determined and these were powdered. Sample solution was then prepared by dissolving the powdered tablets equivalent to 40 mg of Drotaverine hydrochloride and 10 mg of Omeprazole in a 100 mL of volumetric flask. Then the drugs were dissolved by using 25 mL methanol and the volume was made up to the mark with methanol. 5 mL of this solution was further diluted to 25 mL with the same solvent. With the optimized chromatographic conditions, a steady baseline was recorded, the mixed standard solution was injected and the chromatogram was recorded. Both compounds were identified by comparison of retention times obtained from sample and standard solutions. The work was performed in an air-conditioned room maintained at 25 ± 2 °C. The retention time of Drotaverine hydrochloride and Omeprazole was found to be 2.71 and 3.87 min, respectively. The results are reported in Table 2. The resolution, number of theoretical plates, retention time and peak asymmetry were calculated for the working standard solutions and is as shown in Table 3. The values obtained demonstrated the suitability of the system for the analysis of these drugs in combination. The typical chromatogram of standard solution is as shown in Fig. 4.

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Figure 4

Representative HPLC chromatogram of Drotaverine hydrochloride and Omeprazole.

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2.4.1 Chromatographic conditions

Drotaverine hydrochloride and Omeprazole were simultaneously determined by HPTLC in pharmaceutical formulations. The drugs were separated on silica gel 60 F₂₅₄ plates using a suitable combination of solvents as mobile phase. The validation parameters, tested in accordance with the requirements of ICH guidelines, prove the suitability of methods. The samples were spotted in the form of bands of a width of 4 mm with space between bands of 6 mm, with a 100 μL sample syringe (Hamilton, Bonaduz, Switzerland) on precoated silica gel 60 F₂₅₄ aluminum HPTLC plates (20 cm × 10 cm) with automatic sample applicator LINOMAT V. The plates were prewashed with methanol and activated at 130 °C for 5 min, prior to chromatography. Chromatographic separation studies were carried out on the stock solution of Drotaverine hydrochloride and Omeprazole. Initially on the plates 6 μL of stock solution was applied as a band of 8 mm of width. Plates were developed by linear ascending development using neat solvents like toluene, hexane, methanol, chloroform, dichloromethane, ethyl acetate, acetone, acetonitrile, etc., without chamber saturation. Based on the results of these initial chromatograms binary and ternary mixtures of solvents were tried to achieve optimum resolution between Drotaverine hydrochloride and Omeprazole respectively. After several trials, mixture of Glacial acetic acid: Cyclohexane: Methanol: (80:15:5v/v/v) was chosen as the mobile phase for analysis. Other chromatographic conditions like chamber saturation time, run length, sample application rate and volume, sample application positions, distance between tracks, detection wavelength, were optimized to give reproducible R_F values, better resolution, and symmetrical peak shape for the two drugs. The optimized chamber saturation time for the mobile phase was 30 min at room temperature (28 ± 2 °C). The length of the chromatogram run was approximately 60 mm. The spot appeared more compact and peak shape more symmetrical when the TLC plates were pre-treated with methanol and activated at 120 °C for 5 min. Well-defined spots of standard were obtained when the chamber saturation time was optimized at 20 min at room temperature. After development the plates were dried in current of air by an air dryer. Detection of spot was then performed at 295 nm with a CAMAG TLC Scanner 3 in the absorbance mode operated by winCATS software. The source of radiation was a deuterium lamp. Slit dimensions were 5 mm × 0.1 mm and the scanning speed 20 mm/s.

2.4.2 Preparation of standard and sample solutions

Standard stock solution containing 400 μg/ml of Drotaverine hydrochloride and 100 μg/ml of Omeprazole was prepared in methanol. Linearity was performed by applying six times the stock solution to give concentrations of 100–600 and 200–600 ng/spot of Drotaverine hydrochloride and Omeprazole, respectively. Calibration curve was established by plotting peak area on ordinate and corresponding concentration on abscissa. The developed chromatograms were evaluated by scanning in densitometry mode at 295 nm. The amount of Drotaverine hydrochloride and Omeprazole present per tablet was calculated by comparing peak area of sample with that of standard (Sethi, 1996).

Twenty tablets were accurately weighed, and their average weight was determined. Powder equivalent to 40 mg of Drotaverine hydrochloride and 10 mg of Omeprazole was dissolved in methanol, sonicated for 20 min; solution was filtered and diluted to 100 ml with methanol. The solution was applied on the plate to give 100–600 ng/spot of Drotaverine hydrochloride and 200–600 ng/spot of Omeprazole, respectively. The results of assay are summarized in (Table 5). The method was validated as per the various parameters given in ICH guidelines (ICH guidelines, 1996). The linearity was studied in the concentration range of 100–600 ng/spot of Drotaverine hydrochloride and 200–600 ng/spot of Omeprazole, respectively. Precision of the method is expressed in terms of % RSD. Recovery studies were performed by the standard addition method at 80%, 100% and 120% levels, to the pre-analyzed samples and contents were reanalyzed, using the proposed method (Table 4). Ruggedness of the proposed method was determined by performing assays by two different analysts, using similar operational and environmental conditions. The developed method was validated in terms of linearity, accuracy, limit of detection, limit of quantification, intra-day and inter-day precision and repeatability of measurement as well as repeatability of sample application (ICH guidelines, 1996). The analysis was repeated in triplicate. The content of the drug was calculated from the peak areas recorded.