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

Development and validation of new analytical methods for simultaneous estimation of Drotaverine hydrochloride in combination with Omeprazole in a pharmaceutical dosage form Academic research paper on "Chemical sciences"

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{"Drotaverine hydrochloride" / Omeprazole / UV-Spectrophotometry / RP-HPLC / Densitometry / "ICH guideline"}

Abstract of research paper on Chemical sciences, author of scientific article — Smita Sharma, Mukesh C. Sharma

Abstract A rapid and precise method (in accordance with ICH guidelines) is developed for the quantitative simultaneous determination of Drotaverine hydrochloride and Omeprazole in a combined pharmaceutical dosage form. Three methods are described for the simultaneous determination of Drotaverine hydrochloride and Omeprazole in a binary mixture. The first method was based on UV-Spectrophotometric determination of two drugs, using Vierordt!s simultaneous equation method. It involves absorbance measurement at 226.8nm (λ max of Drotaverine hydrochloride) and 269.4nm (λ max of Omeprazole) in methanol; linearity was obtained in the range of 5–30μgml−1 for both the drugs. The second method was based on HPLC separation of the two drugs using potassium dihydrogen phosphate buffer pH 5.0: Acetonitrile: Triethylamine (60:40:0.5, v/v) as a mobile phase. Areas were recorded at 260nm for both the drugs and retention time was found to be 2.71min. and 3.87min for Drotaverine hydrochloride and Omeprazole, respectively at 1.0mL/min flow rate. The selected chromatographic conditions were found to determine Drotaverine hydrochloride and Omeprazole quantitatively in a combined dosage form without any physical separation. The method has been validated for linearity, accuracy and precision. Linearity was found over the range of 5–30μgmL−1 for both drugs. The third method was based on HPTLC method for simultaneous quantification of these compounds in pharmaceutical dosage forms. Precoated silica gel 60 F254 plate was used as stationary phase. The separation was carried out using Glacial acetic acid:Cyclohexane:Methanol:(80:15:5 v/v/v) as mobile phase. The proposed method was found to be fast, accurate, precise, reproducible and rugged and can be used for a simultaneous analysis of these drugs in combined formulations.

Academic research paper on topic "Development and validation of new analytical methods for simultaneous estimation of Drotaverine hydrochloride in combination with Omeprazole in a pharmaceutical dosage form"

Arabian Journal of Chemistry (2012) xxx, xxx-xxx

King Saud University Arabian Journal of Chemistry

www.ksu.edu.sa www.sciencedirect.com

ORIGINAL ARTICLE

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

Smita Sharma a *, Mukesh C. Sharma b

a Department of Chemistry, Chodhary Dilip Singh Kanya Mahavidyalya, Bhind, M.P. 477001, India b School of Pharmacy, Devi Ahilya Vishwavidyalaya, Takshshila Campus, Khandwa Road, Indore, M.P. 452001, India

Received 16 July 2012; accepted 15 September 2012

KEYWORDS

Drotaverine hydrochloride;

Omeprazole;

UV-Spectrophotometry;

RP-HPLC;

Densitometry;

ICH guideline

Abstract A rapid and precise method (in accordance with ICH guidelines) is developed for the quantitative simultaneous determination of Drotaverine hydrochloride and Omeprazole in a combined pharmaceutical dosage form. Three methods are described for the simultaneous determination of Drotaverine hydrochloride and Omeprazole in a binary mixture. The first method was based on UV-Spectrophotometric determination of two drugs, using Vierordt!s simultaneous equation method. It involves absorbance measurement at 226.8 nm (k^ of Drotaverine hydrochloride) and 269.4 nm (kmax of Omeprazole) in methanol; linearity was obtained in the range of 530 ig ml-1 for both the drugs. The second method was based on HPLC separation of the two drugs using potassium dihydrogen phosphate buffer pH 5.0: Acetonitrile: Triethylamine (60:40:0.5, v/v) as a mobile phase. Areas were recorded at 260 nm for both the drugs and retention time was found to be 2.71 min. and 3.87 min for Drotaverine hydrochloride and Omeprazole, respectively at 1.0 mL/ min flow rate. The selected chromatographic conditions were found to determine Drotaverine hydrochloride and Omeprazole quantitatively in a combined dosage form without any physical separation. The method has been validated for linearity, accuracy and precision. Linearity was found over the range of 5-30 ig mL-1 for both drugs. The third method was based on HPTLC method for simultaneous quantification of these compounds in pharmaceutical dosage forms. Precoated silica

* Corresponding author. Tel.: +91 9406575528. E-mail addresses: drsmita.sharma@rediffmail.com (S. Sharma), mukeshcsharma@yahoo.com (M.C. Sharma). Peer review under responsibility of King Saud University.

1878-5352 © 2012 King Saud University. Production and hosting by Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.arabjc.2012.09.012

gel 60 F254 plate was used as stationary phase. The separation was carried out using Glacial acetic acid:Cyclohexane:Methanol:(80:15:5 v/v/v) as mobile phase. The proposed method was found to be fast, accurate, precise, reproducible and rugged and can be used for a simultaneous analysis of these drugs in combined formulations.

© 2012 King Saud University. Production and hosting by Elsevier B.V. All rights reserved.

1. Introduction

Drotaverine hydrochloride (DRO) chemically 1-[(3,4-[dieth-oxyphenyl) methylene]-6,7-diethoxy-1,2,3,4-tetrahydroisoqui-nolene (Fig. 1) is a papaverine analog mainly used as an antispasmodic and smooth-muscle relaxant in pain associated with gastrointestinal colic, biliary colic, and postsurgical spasms. It is an antispasmodic agent with smooth muscle relaxant properties. It exerts its action by inhibiting phosphodiesterase enzyme IV which is specific for smooth muscles (Sweetman, 2002; Oneil et al., 2001). Omeprazole (Fig. 2) is chemically known as 6-methoxy-2-[(4-methoxy-3,5-dimethyl-pyridin-2-yl)methylsulfinyl]-1H-benzimidazole. Omeprazole is a used as an antiulcer drug and against other acid-related diseases (Stenhoff et al., 1999). Omeprazole (OME) is the proton pump inhibitor. In the acidic conditions of the stomach, Ome-prazole reacts with a cysteine group in H + /K + -ATPase, thereby destroying the ability of the parietal cells to produce gastric acid (Tripathi, 2008). Literature review reveals that methods have been reported for the analysis of Drotaverine hydrochloride by high-performance liquid chromatography (Bolaji et al., 1993; Lalla et al., 1993; Metwally et al., 2006; Panigrahi and Sharma, 2008; Patel et al., 2007; Topagi et al., 2010; Metwally, 2008), thin layer chromatography (Ayad et al., 2006; Metwally et al., 2006), spectrophotometry (Abdel-latef et al., 2007; Dahivelkar et al., 2007; Kothapalli et al., 2010) and voltammetry (Ziyatdinova et al., 2007). Several analytical methods that have been reported for an estimation of Omeprazole are HPLC (Dubuc et al., 2001; El-Sherif et al., 2006; Murakami et al., 2007; Subramanian and Kumar,2007), LC-MS (Petsalo et al., 2008) and HPTLC (Raval et al., 2008). For Omeprazole methods reported are HPLC-MS and HPLC-UV in biological fluids (Kange et al., 2006; Yuch et al., 2001), capillary electrophoresis (Berzas and Castanda, 2005), HPLC employing electrochemical and coulometric

Figure 1 Structure of Drotaverine hydrochloride.

^ N 'o

Figure 2 Structure of Omeprazole.

detection (Gregory et al., 2001), TLC (Agbaba et al., 2004) and spectrophotometry (Lakshmi and Venkatesan, 2003).

The purpose of this research was to establish and validate, in accordance with International Conference on Harmonization (ICH) guidelines, an accurate, economical, and reproducible procedure for quantitative analysis of Drotaverine hydrochloride and Omeprazole as the bulk drug and in tablet dosage forms. It was thought worthwhile to develop precise, accurate UV spectrophotometric, HPLC and HPTLC methods for simultaneous determination of Drotaverine hydrochloride and Omeprazole in tablets.

2. Materials and methods

2.1. Chemicals and reagents

Commercially available Ranispas-DV (Penta Biotech, India) drug containing 40 mg of Drotaverine hydrochloride and 10 mg of Omeprazole was used. HPLC grade acetonitrile and AR grade potassium dihydrogen phosphate was purchased from Merck, India and were used as received. All other reagents employed are of high purity analytical grade.

2.2. UV-spectrophotometry

SHIMADZU double beam UV/Visible recording spectrophotometer (Model:1700) with 2 nm spectral bandwidth was employed for all spectrophotometric measurements using 10 mm matched quartz cell and Borosil glass wares were used for the study. Calibrated electronic single pan balances Sarto-rius CP 225 D, pH Meter, Enertech Fast Clean Ultrasonicator were also used during the analysis. UV-Spectrophotometric determination of two drugs was done using Vierordt!s simultaneous equation method (Davidson et al., 2001).

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 lg/ml. Both were appropriately diluted with a mixture of methanol and double distilled water (50:50) to get a concentration of 100 lg/ml and were kept as stock solutions.

CDRO = A2 ay1 — A1 ay2/ax2 ay1 — ax1ay2 Come — A1 ax2 — A2 ax2/ax2ay1 — ax1ay2

(1) (2)

Figure 3 Overlain spectra of the Drotaverine HCl and Omeprazole.

2.2.2. Determination of kmax

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

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 ig/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

where, A1 and A2 are absorbance of mixture at 226.8 nm and 269.4 nm; ax1 and ax2, absorptivities of Drotaverine hydrochloride at 226.8 nm and 269.4, respectively; ay1 and ay2 absorptivities of Omeprazole at 226.8 nm and 269.4 nm, respectively. CDRO and COME are concentration of Drotaverine hydrochloride and Omeprazole in mixture. The absorptiv-ities reported are the mean of six independent determinations (Table 1). Before analyzing the selected tablet formulations the method was validated by analyzing the physical admixtures of Drotaverine hydrochloride and Omeprazole in the ratio of each component as in the formulation in consideration.

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 sonica-tor. 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 igmL-1 and Omeprazole 10 igmL-1) in the linearity range.

2.3. Reverse phase high performance liquid chromatographic method

A reverse phase high performance liquid chromatographic method was developed for the simultaneous estimation of Dro-taverine hydrochloride and Omeprazole in tablet formulation. All the chemical and reagents used were of HPLC grade and purchased from Spectrochem, Mumbai, India.

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 iL. Detector consists of photodiode array detector SPD-10 AVP UV-Visible detector.

Table 1 Optical and regression characteristic parameters for Drotaverine hydrochloride and Omeprazole.

Parameters Absorptivity at 226.8 nm Absorptivity at 269.4 nm

Drotaverine hydrochloride Omeprazole Drotaverine hydrochloride Omeprazole

Beer's law limit (mg/ml) 5-30 5-30 5-30 5-30

Molar absorptivity 3.1 X 10—3 1.6 X 10—3 2.8 X 10—3 1.1 X 10—3

Sandell's sensitivity 2817.36 2014.24 1941.71 1039.22

Correlation coefficient (r2) 0.9996 0.9999 0.9984 0.9991

Relative standard deviation (RSD or%CV) 0.147 0.068 0.181 0.027

LOD (ig/ml) 0.935 0.746 0.136 0.0297

LOQ (ig/ml) 1.173 0.384 0.247 0.0413

Regression equation —0.0386 — 0.0218 — 0.0147 —0.0390

Mean axj = 413.65 ayj = 486.17 ax2 = 366.18 ay2 = 336.96

Standard deviation (SD) 0.0641 0.0318 0.0864 0.0548

Separation was carried out on a Phenomenex Luna C18 (5 im x 25 cm x 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 0C. Chro-matograms 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: Acetoni-trile: 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. Cip-rofloxacin was used as an internal standard. The mobile phase was filtered through nylon 0.22 im 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 igml-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 ig mL-1 of Drotaverine hydrochloride and Omeprazole, respectively and each concentration contains 5 ig mL-1 of Ciprofloxacin (internal standard). A volume of 20 iL 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 chromato-graphic 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 0C. 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.

2.4. TLC Densitometric method

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 iL sample syringe (Hamilton, Bonaduz, Switzerland) on pre-coated silica gel 60 F254 aluminum HPTLC plates (20 cm x 10 cm) with automatic sample applicator LINOMAT V. The plates were prewashed with methanol and activated at 130 0C 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 iL 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 RF 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 0C). 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

Table 2 Analysis of data of tablet formulations.

S.No Parameters UV spectrophotometry HPLC HPTLC

Drotaverine hydrochloride Omeprazole Drotaverine hydrochloride Omeprazole Drotaverine hydrochloride Omeprazole

1 Label claim 40 10 40 10 40 10

2 Drug content 99.48 99.59 100.08 101.06 99.86 98.96

3 ± S.D 0.26 0.48 0.16 0.03 0.14 0.85

4 % COV 0.11 0.35 0.60 0.25 0.19 0.43

S.D. is standard deviation. Value for Drug content (%) are the mean of five estimations.

activated at 120 0C 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 x 0.1 mm and the scanning speed 20 mm/s.

2.4.2. Preparation of standard and sample solutions Standard stock solution containing 400 lg/ml of Drotaverine hydrochloride and 100 lg/ml of Omeprazole was prepared in methanol. Linearity was performed by applying six times the stock solution to give concentrations of 100-600 and 200600 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 Drota-verine 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 hydro-chloride 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.

3. Results and discussion

Both, UV spectrophotometric, HPLC and HPTLC methods were found to be for routine simultaneous estimation of Dro-taverine hydrochloride and Omeprazole, in tablet dosage forms. The proposed method for simultaneous estimation of Drotaverine hydrochloride and Omeprazole utilizes the spectrum mode of analysis of spectrophotometer. The method utilizes 226.8 nm and 269.4 nm as an analytical wavelength for the estimation of Drotaverine hydrochloride and Omeprazole. The method employing simultaneous equation is a very simple method and can be employed for a routine analysis of Drota-verine hydrochloride and Omeprazole. Once the absorptivity

Table 3 System suitability parameters of HPLC method.

Parameters Drotaverine hydrochloride Omeprazole

Tailing factor 2.63 4.97

Theoretical plates 5249 7491

Resolution factor 3.13 4.95

Asymmetry factor 1.44 1.58

Height 43721 51227

Area 237236 353241

min-XJ 2 A e

Figure 4 Representative HPLC chromatogram of Drotaverine hydrochloride and Omeprazole.

Table 4 Recovery studies.

UV-Spectrophotometry HPLC HPTLC

Excess drug *Recovery % COV Excess drug *Recovery % COV Excess drug *Recovery % COV

Drotaverine hydrochloride

80 100.09 0.53 80 99.82 0.94 80 100.28 0.09

100 100.06 0.38 100 99.92 0.01 100 100.09 0.7

120 100.27 0.17 120 100.26 0.97 120 101.11 0.21

Omeprazole

80 99.91 0.28 80 99.86 0.28 80 99.98 0.15

100 99.98 0.49 100 99.52 0.31 100 100.11 0.39

120 98.83 1.17 120 100.16 0.63 120 99.9 0.68

*Average of six estimations.

Table 5 Summary of repeatability, precision and ruggedness.

Parameter UV-Spectrophotometry HPLC HPTLC

DRO OME DRO OME DRO OME

Repeatability 1.83 0.762 0.88 0.64 0.96 0.38

Precision intra-day 1.07 0.13 0.29 0.43 0.8 0.49

Inter-day 0.29 0.14 0.56 1.55 1 0.72

Ruggedness 0.68 0.52 0.37 0.87 0.34 0.65

Rf - - - - 0.38 0.56

Linearity (ng per band) - - - - 100-600 200-600

Linearity detection (ng/spot) - - - - 68 82

Limit of quantification (ng/spot) - - - - 294 397

n is the number of repetitions; DRO-Drotaverine hydrochloride,OME-Omeprazole.

values are determined very little time is required for analysis, as it would only require the determination of absorbances of the sample solution at two selected wavelengths and few simple calculations. For UV spectrophotometric method, linearity was obtained in the concentration range of 5-30 igmL-1, for both the drugs; with regression 0.9996 and 0.9999, intercept -0.0386 and -0.0218 and slope 0.0854 and 0.0292 for Drotaverine hydrochloride and Omeprazole, respectively. Recovery was in the range of 99 -101%; the values of standard deviation and% R.S.D. were found to be <2%; shows the high precision of the method (Table 4). The low values of these statistical parameters validated the method. LOD and LOQ were found to be 0.935 and 1.173 for Drotaverine hydrochloride and 0.746 and 0.384 for Omeprazole, respectively. Interday and intraday precision studies showed % RSD values <1% that signify the precision of the method.

In HPLC method, an adequate separation of eluted compounds was optimized. Mobile phase and flow rate selection were based on peak parameters (height, tailing, theoretical plates, capacity factor), run time etc. Several aliquots of standard solutions of Drotaverine hydrochloride and Omeprazole were taken in different 10 ml volumetric flasks and diluted up to the mark with a mobile phase such that the final concentration of Drotaverine hydrochloride and Omeprazole is 530 ig mL-1, respectively. The system with potassium dihydro-gen phosphate buffer pH 5.0: Acetonitrile: Triethylamine (60:40:0.5, v/v) with 1mLmin-1 flow rate is quite robust. The optimum wavelength for detection was 260 nm at which better detector response for drugs was obtained. The average retention times for Drotaverine hydrochloride and Omepra-zole were found to be 2.71 and 3.87 min, respectively. The peak

shapes of both the drugs were symmetrical and the asymmetry factor was lesser than 2.0. The proposed method was validated as per the standard analytical procedures. Each of the samples was repeated 6 times and the same retention time was observed in all the cases. Precision of proposed HPLC method was found to be 0.0178 (RSD) for Drotaverine hydrochloride and 0.0048 for Omeprazole that indicate good precision of the samples analyzed. The correlation coefficient 'r' values (n = 6) for both Drotaverine hydrochloride and Omeprazole were P0.999. Accuracy of the method was calculated by recovery studies (n = 3) at three levels. The method was found to be accurate and precise as indicated by results of recovery studies and % RSD not more than 2%. LOD and LOQ for Omeprazole were found to be 0.63 and 0.98 ig/ml respectively and for Drotaverine were 1.41 and 1.79 ig/ml, respectively. The mean recoveries obtained for Drotaverine hydrochloride and Omeprazole were 99.36% and 101.03%, respectively.

In HPTLC method, Chromatographic separation of the drugs was performed on aluminum plates precoated with silica gel 60 F254, with Glacial acetic acid: Cyclohexane: Methanol: (80:15:5v/v/v) as mobile phase. Chromatographic evaluation of the separated zones was performed at 295 nm. The linear regression data for the calibration plots showed good linear relationship with r2 0.9997 and 0.9984 in the concentration range of 100-600 ng/spot and 200-600 ng/spot for Drotaverine hydrochloride and Omeprazole, respectively were applied, in duplicate, to a TLC plate, and after development peak height and peak area data and drug concentration data were treated by linear least-squares regression to determine linearity. The accuracy and reliability of the method was assessed by an evaluation of linearity (100-600 ng/spot for Drotaverine hydro-

chloride, and 200-600 ng/spot for Omeprazole), precision (in-tra-day RSD 0.48-0.82% and inter-day RSD 1.16-1.64% for Drotaverine hydrochloride, and intra-day RSD 0.48-0.63% and inter-day RSD 0.18-0.53% for Omeprazole), accuracy (99.61 ± 0.14% for Drotaverine hydrochloride and 99.08 ± 0.31% for Omeprazole), and specificity, in accordance with ICH guidelines (Table 5). The LOD and LOQ were determined from the slope of the lowest part of the calibration plot. The LOD and LoQ were 36 ± 0.16 and 57 ± 0.25 ng, respectively, for Drotaverine hydrochloride and 18 ± 1.01 and 72.0 ± 0.26 ng, respectively, for Omeprazole, which indicate the sensitivity of the method is adequate. Rf Spots at Rf 0.38 ± 0.82 for Drotaverine hydrochloride and 0.56 ± 0.12 for Omeprazole were observed in the chromatogram obtained from Drotaverine hydrochloride and Omeprazole extracted from tablets (Table 6). There was no interference from excipients commonly present in the tablets. The Drotaverine hydro-chloride and Omeprazole content were found to be 100.0100.53% and 99.02-99.89%, respectively, of the label claim. The low values of% RSD are indicative of the high repeatability of the method. The low values of% RSD obtained after introducing small changes in mobile phase composition and volume were indicative of the robustness of the method. There was no significant variation of the slopes of the calibration plots.

4. Conclusion

The developed UV Spectrophotometric method, RP-HPLC and HPTLC technique is precise, specific, and accurate. The method was simple and had a short runtime of 10 min, which makes the method rapid. The results of the study indicate that the proposed HPLC method was simple, precise, highly accurate, specific and less time consuming. Statistical analysis proves that the method is repeatable and selective for the analysis of Drotaverine hydrochloride and Omeprazole as bulk drug and in pharmaceutical formulation without any interference from the excipients. The method can be used to determine the purity of the drug available from various sources by detecting the related impurities. The results of the recovery studies performed show the high degree of accuracy of the proposed methods.

Acknowledgements

We are grateful to Prof. D.V. Kohli and Prof. Abhay Kumar Singhai Department of Pharmaceutical Sciences Dr. H.S. Gaur, Sagar University Sagar (M.P) India, and Head, School

of Pharmacy, Devi Ahilya Vishwavidyalaya, Indore (M.P) 452001 India for the facility given and valuable suggestion.

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Table 6 Robustness of the HPTLC method.

Parameter ±S.D. of peak % COV

area (n = 6) (n =6)

Mobile phase composition 48.62 0.82

Time from application to 36.83 0.56

development (±10 min)

Time from development to 31.70 0.64

scanning (±10 min)

Activation of TLC plates 33.64 0.72

n-number of determinations.