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In this paper, a simple, sensitive, and precise high-performance liquid chromatographic method for the analysis of Naproxen and Pantoprazole has been developed and validated for the determination of compounds in commercial pharmaceutical products. The compounds were well separated on a BDS Hypersil C-18 reversed-phase column by using a mobile phase consisting of Acetonitrile and Mixed Phosphate buffer in 45:55 (V/V%) ratio, at a flow rate of 1.0 mL/min with detection wavelength at 290 nm. The linearity ranges were 20-120 μg. The correlation coefficient was found to be 0.997 and 0.995 for Naproxen and Pantoprazole, respectively. The recovery amount was in the range of 99.67%-101.39%. The high recovery and low relative standard deviation confirms the suitability of the method for determination of Naproxen and Pantoprazole in capsule dosage forms.
ABSTRACT:
A simple, sensitive, and precise High performance liquid chromatographic method for the analysis of Naproxen and Pantoprazole has been developed and validated for the determination of compounds in commercial pharmaceutical products. The compounds were well separated on a BDS Hypersil C-18 reversed-phase column by use of a mobile phase consisting of Acetonitrile and Mixed Phosphate buffer (pH 6.92) in 45:55(V/V%) ratio, at a flow rate of 1.0 mL/min with detection wavelength at 290 nm. The linearity ranges were 20-120µg. The correlation coefficient was found to be 0.997 and 0.995 for Naproxen and Pantoprazole respectively. The recovery amount was in the range of 99.67-101.39%. The high recovery and low relative standard deviation confirms the suitability of the method for determination of Naproxen and Pantoprazole in capsule dosage forms.
KEYWORDS: Naproxen, Pantoprazole.
1. INTRODUCTION:
Naproxen (as shown in Fig: 1) is a propionic acid derivative related to the aryl acetic acid group of non-steroidal anti-inflammatory drugs. The chemical names for Naproxen and Naproxen Sodium are (S)-6-methoxy-a-methyl-2-naphthaleneacetic acid and (S)-6-methoxy-α-methyl-2-naphthaleneacetic acid, sodium salt, respectively1. Naproxen is commonly used for the reduction of pain, fever, inflammation, and stiffness caused by conditions including migraine, osteoarthritis, kidney stones, rheumatoid arthritis, psoriatic arthritis, gout, Ankylosing spondylitis, menstrual cramps, Tendinitis, and Bursitis2,3. It is also used for the treatment of primary dysmenorrhea. Naproxen works by inhibiting both the COX-1andCOX-2 enzymes.Pantoprazole (as shown in Fig:2) is 6-(Difluoromethoxy)-2-[(3,4-dimethoxypyridin-2-yl)methylsulfinyl]-1H-benzimidazole].
Pantoprazole,a proton pump inhibitor drug used for short-term treatment of erosion and ulceration of the oesophagus caused by gastro-oesophageal reflux disease.
Pantoprazole is a proton pump inhibitor (PPI) that suppresses the final step in gastric acid production by forming a covalent bond to two sites of the (H+,K+ )-ATPase enzyme system at the secretory surface of the gastric parietal cell4,5. This effect is dose- related and leads to inhibition of both basal and stimulated gastric acid secretion irrespective of the stimulus.
Few analytical methods were found for this combination. Hence the present work is focused upon new method development and estimation of Naproxen and Pantoprazole.
2. MATERIAL AND METHODS:
2.1. Chemicals:
Naproxen was given as a gift by GlaxoSmithKline, England and Pantoprazole sodium was obtained from Dr. Reddy's, Hyderabad, India. All solvents used were of HPLC -grade and GR grade. Acetonitrile was purchased from J.T. Baker. Potassium di-hydro-O-phosphate (GR), Di-potassium hydrogen phosphate (GR), and Hydrochloric acid (HPLC - grade), Sodium hydroxide (HPLC -grade) was obtained from Merck. Methanol (GR) was obtained from Rankem manufacturers. Water used in the mobile phase was freshly prepared from Milli-Q, NA.
2.2. Equipment:
A water 2695 gradient system and column oven, with Empower-2 software and 2487 dual absorbance detector was used. It was manufactured by the company Waters, Alliance, Japan.
2.3. Chromatographic conditions:
BDS Hypersil small C18 column(250 X 4.6mm; 5µ particle size) RP-18 (Thermo Fisher Scientific) was used for analytical separation. The mobile phase consisted of an aqueous solution of mixed Phosphate buffer (pH-6.9) and Acetonitrile in the ratio of 55:45, v/v%. The flow rate was adjusted to 1.0ml/min. The instrument was operated at an ambient temperature. The UV detection was achieved at 290nm and purity analysis was performed over a wavelength range of 200-400nm. The injection volume was 20µL.
2.4 Preparation of Analytical solutions:
2.4.1 Preparation of Mixed phosphate buffer solution:
A weighed quantity of 1.652gm of Potassium dihydrogen-O-phosphate (KH2PO4) and 0.3gm of Dipotassium hydrogen phosphate (K2 HPO4) taken in a 1000ml beaker. To this add 550ml of water and mixed in Ultra-Sonicator, was adjusted to pH=6.9 by using Ortho -phosphoric acid.
2.4.2 Preparation of Standard Naproxen solution:
The standard stock solution of Naproxen prepared by dissolving 50mg of standard drug was weighed and transferred into 50ml volumetric flask to which add 25ml of mobile phase [mixed phosphate buffer (pH=6.9): Acetonitrile (55:45,v/v)] and mixed well. The final volume was made upto50ml with the mobile phase. From the above solution 10ml was taken and diluted to 100ml to get the solution of 100µ g/ml.
2.4.3 Preparation of Standard Pantoprazole sodium solution:
The standard Pantoprazole sodium solution was prepared by taking 4mg of Pantoprazole sodium into 10ml volumetric flask and to this 5ml of mobile phase [mixed phosphate buffer (pH=6.9): Acetonitrile (55:45,v/v)] was added and mixed thoroughly. From this take 2.5ml in 10ml volumetric flask and the volume was made upto 10ml with the mobile phase to get the solution of 100µ g/ml.
2.4.4 Preparation of mixed Standard solution:
Mixed standard solution was prepared by adding 1ml of the standard Naproxen solution to 1ml of standard Pantoprazole sodium solution in 10ml volumetric flask and made the final volume to 10ml with the mobile phase [mixed phosphate buffer (pH=6.9): Acetonitrile (55:45, v/v%)] to get the mixed standard solution of 100µ g/ml and the solution was sonicated for complete solubility.
2.4.5 Preparation of Test Solution (Marketed formulation):
20 capsules were weighed and the average weight (595.1mg) was calculated and the sample weight observed is 120mg which is having a equivalent to 50mg of naproxen and 20mg of pantoprazole, hence 120mg of power (sample) is taken in to 50ml volumetric flask and add 25ml of mobile phase,mix well and finally make up the volume to 50ml with mobile phase and mix thoroughly by using ultrasonicator to get the concentration of 4.8mg/ml. the resultant solution further diluted to get a concentration of 100µg/ml.
Equal volumes of Blank (mobile phase) and six replicate injections of reference solutions were separately injected. Then the test solution was injected in duplicate. The chromatograms were recorded for all injections eliminating the peak due to Blank.
2.5 Validation parameters:
2.5.1. System suitability:
The system suitability studies were done with the100µ g/ml concentrations of standard drug solution.
2.5.2. Selectivity:
The specificity of the method was found out through no interference of mobile phase. The method showed excellent specificity with Naproxen and Pantoprazole eluting at retention time (RT) 3.01 and 4.4 minutes. No interference was observed with mobile phase. The prepared mobile phase was injected in to the system and chromatogram was obtained.
2.5.3 Specificity:
Specificity is ability to assess unequivocally the analysis in the presence of components which may be unexpected to present. Forced degradation studies are carried out by using 0.1M Hcl,0.1M NaOH and heat.
2.5.4. Accuracy:
Expresses the closeness and agreement between the value which is accepted either as a conventional new value or an accepted reference value and the value found. The solutions were prepared in the concentrations ranging from 80,100 and 120% and standard solution was spiked with sample and % recovery was calculated for the three levels.
2.5.5. Precision and Reproducibility:
Express the closeness of agreement between the series of measurement obtained from multiple sampling of same homogeneous sample under the prescribed conditions.
1) Method Precision:
The solutions were diluted to get a concentration of 10µ g/ml. The sample solution was injected in five into the system and the chromatogram was obtained.
2) System Precision:
The solutions were diluted to get concentration of 10µ g/ml. Inject the sample solution in five replicates injection into the system and the chromatogram was obtained. The percentage RSD was calculated.
3) Intermediate precision (Ruggedness):
It shows the degree of reproducibility of test results obtained by analyzing the sample under variety of normal test conditions such as analyst, instruments. 60µ g/ml solution was injected by two analysts and the chromatogram was obtained.
2.5. 6. Linearity:
Express ability to obtain test results where directly proportional to the concentration of analyte in the sample. The test sample was prepared in different concentrations like 20,40,60,80,100µ g/ml separately and chromatogram was obtained. It is shown in Fig:3 and Fig:4.
2.5.7 Range
The range of an analytical procedure is the interval between the upper and lower concentrations (amounts) of analyte in the sample. The range for Naproxen and Pantoprazole were 80-120µg/ml.
2.5.8 Limit of Detection
Express the detection limit is determined by the analysis of samples with known concentration of analyte and by establishing that minimum level analyte can reliably detected.
2.5.9 Limit of Quantification
Express the detection limit is determined by the analysis of samples with known concentration of analyte and by establishing that minimum level analyte can reliably quantified with acceptable accuracy and precision.
2.5.10 Robustness
It is considered during development phase and depends on the type of procedure under study. Change in flow rate: 80µ g/ml sample was injected at 0.9 ml flow and at 1.1 ml flow rate and change in wavelength: 80µ g/ml was prepared and injected at different wave lengths 258nm and 263nm. The obtained results were tabulated.
3. RESULTS AND DISCUSSION:
The present investigation was aimed to develop analytical method for Naproxen and Pantoprazole and validate the developed method in bulk and pharmaceutical dosage form.
Several trails were carried out to develop an optimize method showing good peak shape, plate count and asymmetry (table: 1). The developed method, was found to be simple, precise, and accurate and rapid by RP-HPLC method, by using BDS Hypersil small C18 (250 X 4.6mm; 5µ particle size) RP-18 (Thermo Fisher Scientific) column in isocratic mode with mobile phase consists of aqueous solution of mixed Phosphate buffer (pH-6.9) and Acetonitrile in the ratio of 55:45v/v, flow rate 1ml/min and effluent monitored at 290nm. The retention time of the compound was shown symmetric peaks at 3.0 and 4.3 (Fig:6 and Fig:7). The percentage purity was calculated and found to be 99.21 and 100.46%w/v.
The developed method was validated as per ICH guidelines for the various parameters. The specificity of the proposed method proven that excipients and blank (Fig: 5) peak do not interfere in the retention time of the analyte. Hence the method showed good specificity at retentiontime 3.0 and 4.3 min.
The accuracy of the method was determined by measuring the drug percentage recovery for 80%, 100% and 120% (table:2). The percentage recovery was determined for Naproxen and Pantoprazole and was found to be99.67-101.39%.
The precision of the method was ascertained from determinations of peak areas of five replicates of standard drug (system precision) and fixed amount of sample drug (method precision). The % Relative Standard Deviation for system precision (table:4) was found to be 0.329316 and 0.171479 and the % Relative Standard Deviation for method precision (table: 3) was found to be 0.50979 and 0.30387 for Naproxen and Pantoprazole respectively. The ruggedness was evaluated by injecting standard solutions of Naproxen and Pantoprazole on different days by using different columns and by different analyst and %RSD was found to be 1.324 and 0.975 for Naproxen and Pantoprazole respectively.
The linearity was determined as linearity regression of the a claimed analyte concentration of the range 20-120mcg. The b calibration curve obtained by plotting peak area versus concentration (table: 5) was linear and the correlation 5 coefficient was found to be 0.997 and 0.995 for Naproxen 1 and Pantoprazole respectively. Selectivity was performed for hydrolytic degradation studies and the results are shown in Table:6. The robustness (table:7)was carried out with minor but deliberate changes in parameters i.e., detection 2 wavelength, column temperature, and flow rate and theoretical plates and tailing factor were observed and were found to be 4864 and 7656 (theoretical plates ) and 1.12 and 1.1885 (tailing factor) for Naproxen and Pantoprazole 3 respectively. The system suitability parameters like theoretical plates (N), tailing factor (T) were calculated and were found to be more than 2000 and not more than 2 and 4 ascertained that proposed RP-HPLC method was accurate and precise.
4. SUMMARY AND CONCLUSION: 5
The proposed method was found to be simple, precise, and accurate and rapid for determination of Naproxen and Pantoprazole from API and pharmaceutical dosage form. Chromatographic separation was achieved by using symmetry Hypersil C18 (250×4.6mm, 5µ ) column in isocratic mode with mobile phase consisting of mixed Phosphate buffer (pH-6.9) and Acetonitrile in the ratio of 55:45v/v. The flow rate was 1ml/min and effluent was monitored at 290nm. The peaks were eluted at 3.0min for Naproxen and at 4.3min for Pantoprazole. The proposed method was validated for parameters like specificity, accuracy, ruggedness and robustness and ascertained values were found to be within limits.
The method has significant advantages, in terms of shorter analysis time, selectivity, and accuracy than previously reported. The method gave consistent and reproducible recovery for analyte from formulated preparation, with no interferences. The validation study indicates that method can be considered suitable for carrying out quality control and routine determination of Naproxen and Pantoprazole in bulk and pharmaceutical dosage form.
5. REFERENCES:
1 Antonio Segura Carretero, Carmen Cruces-Blanco, Ramirez Garcia M, Canabate B, Alberto Fernandez, Tierrez, Simple and rapid determination of the drug naproxen in pharmaceutical preparations by heavy atom-induced room temperature phosphorescence, Talanta 50 (1999) 401-407.
2 Okramzenitadevi, Validated spectrophotometric determination of Pantoprazole sodium pharmaceutical using ferric chloride and two chelating agents, International Journal of Chem. tech Research., vol.2 (1); 2010: 624-632.
3 Tasnuvahaque, Mesbahuddintalukder, Development and validation of a RP-HPLC method for simultaneous estimation of naproxen and ranitidine hydrochloride, Pak. J. Pharm. Sci. Vol.23, (4); 2010: 379-383.
4 Serenkayiran, sibelbozdag, pehlivan, mustafacelebier, Determination of naproxen sodium from poly (lactide-co-glycolide) corneal scaffolds, Turk j. Pharm. Sci .7(1); 2010: 57-68.
5 Shubhangi, Pawar, Jitendra M, Fegade D and Rajesh, Chaudhari Y, Validated RP-HPLC Method for Simultaneous Quantitation of Domperidone Maleate and Naproxen Sodium in Bulk Drug and Formulation, Scholar Research Library. 2(5); 2010: 229-236.
Rubesh Kumar S.*, Usha Sree G., Jayanthi K., Naga Malleswara Babu B., Duganath N., Devanna N.
Department of Pharmaceutical Analysis, JNTUA - Oil Technological Research Institute, Anantapur -515001,
Andhra Pradesh, India.
*Corresponding Author E-mail: [email protected]
Copyright A&V Publications Aug 2013