DEVELOPMENT AND VALIDATION OF UV SPECTROPHOTOMETRIC ESTIMATION OF DICLOFENAC SODIUM BULK AND TABLET DOSAGE FORM USING AREA UNDER CURVE METHOD

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ABOUT AUTHORS:
Mali Audumbar Digambar*, Jadhav Santosh, Mane Pandurang, Tamboli Ashpak
Department of Pharmaceutics, Sahyadri College of Pharmacy, Methwade,
Sangola- 413307, Solapur, Maharashtra, India
maliaudu442@gmail.com

ABSTRACT
A simple, precise, accurate and economical UV visible spectrophotometric method has  been developed for estimation of Diclofenac sodium drug by AUC method. The standard and  sample solutions were prepared by using double distilled water as a solvent. Quantitative determination of the drug was performed at wavelength range 270-282 nm. The linearity was established over the concentration range of 05,10,15,20&25µg/ml for Diclofenac sodium with correlation coefficient value of 0.9981. Precision studies showed that % relative standard  deviation was within range of acceptable limits. The mean percentage recovery was found to be 99.38%.The proposed method has been validated as per ICH guidelines.

REFERENCE ID: PHARMATUTOR-ART-2323

INTRODUCTION:
Diclofenac Sodium (DS) is chemically Sodium salt of 2-[{2,6-dichlorophenyl}amino]benzene acetic acid. It is having anti-inflammatory and analgesic properties^[1]. Literature survey revealed several analytical methods UV spectrophotometry^[2] and HPLC^[3] have been reported in bulk, pharmaceutical dosage form for determination of Diclofenac sodium. To our notice so far no UV- spectrophotometric method using Area Under Curve (AUC) has been reported for the determination of Diclofenac sodium in bulk and tablets. Hence an attempt has been made to develop new UV-spectrophotometry(AUC) method for estimation of Diclofenac sodium in bulk and pharmaceutical formulations with good accuracy simplicity, precision and economy.

Molecular Formula:C₁₄H₁₀Cl₂NNAO₂.

Molecular weight: 318.130g/mole

Fig. 1: Chemical Structure of Diclofenac sodium.

MATERIALS AND METHODS:

Apparatus and instrumentation:
A shimadzu1800 UV/VIS double beam spectrophotometer with 1cm matched quartz cells was used for all spectral measurements. Single Pan Electronic balance (CONTECH, CA 223, India) was used for weighing purpose. Sonication of the solutions was carried out using  an Ultrasonic Cleaning Bath (Spectra lab UCB 40, India). Calibrated volumetric glassware  (Borosil®) was used for the validation study.

Materials:
Reference standard of Diclofenac sodiumAPI was supplied as gift sample by Marksan Pharmaceutical Ltd.,Verna, Goa. Tablet sample with label claim 500mg per tablet were purchased from local market Pune.

Method development:
Determination of Wavelength Range:
For the selection of analytical wavelength range for area under curve method 20µg/ml solution of Diclofenac sodiumwas scanned in the spectrum mode from 400nm to 200nm  against distilled water as blank. Wavelength range was selected around wavelength maxima (276nm). Different working standards were prepared between 05,10,15,20&25µg/ml. Various  wavelength range were tried and final wavelength range between 270-282 nm was selected on the basis of linear relationship between area and corresponding concentration (Figure 2).

Fig:- 2 UV AUC spectrum of Diclofenac sodium(20µg/ml)

Area under curve (Area calculation):
Area under curve method involves the calculation of integrated value of absorbance with respect to the wavelength between two selected wavelengths such as λ1 and λ2 representing start and end point of curve region. The area under curve between λ1 and λ2 was calculated using UV probe software. In this study area was integrated between wavelength ranges from 227 to 282 nm.

Area calculation: (α+β) =

Where, α is area of portion bounded by curve data and a straight line connecting the start and end point β is the area of portion bounded by a straight line connecting the start and end point on curv data and horizontal axis λ1 and λ2 are wavelength range start and end point of curve region^[4].

Preparation of standard solution:
The  standard  stock  solution  of  Diclofenac sodiumwas  prepared  by  accurately  weighing  & transferring,  10  mg  of  API  to  100  ml  of  volumetric  flask. Then take from that 2ml and add to 10ml volumetric flask and make up with distilled water to get final standard stock solution (20µg/ml) was further diluted with distilled water to obtain 05-25 µg/ml Diclofenac sodiumsolutions.

Calibration curve for Diclofenac sodium:
The dilutions were made from Standard Stock solution to get concentration of 05, 10, 15, 20, and 25µg/ml respectively.  These  solutions  were  scanned  from  400  to  200  nm  and  area  under curve  (AUC)  values  was  integrated in  the  range  of  270-282nm. The calibration curve was plotted between areas under curve values against concentration (Fig. 3).

Fig:-3 Linearity of Diclofenac sodium.

Assay of tablet formulation:
Twenty tablets each containing 500mg of Diclofenac sodiumwere weighed crushed to  powder and average weight was calculated. Powder equivalent to10 mg of Diclofenac sodiumwas transferred in100 ml of volumetric flask. A 50ml of distilled water was added and  sonicated for 15minutes. Then solution was further diluted up to the mark with distilled water. The solution was filtered using Whatmann filte paper no.41, first 5ml of filtrate was discarded. This solution was further diluted to obtain 15µg/mL solution with water, subjected for UV analysis using distilled water as blank. This procedure was repeated three times (Table 1).

Table 1:-Assay of tablet dosage form.        

*n=3,SD=Standard Deviation, % RSD = % Relative Standard Deviation.

Method validation:
The above method was validated for various parameters such as Accuracy, Linearity, Precision, Limit of detection (LOD) and Limit of Quantitation(LOQ) according to ICH  guideline.

Accuracy:
The accuracy for the analytical method was evaluated at 80%, 100% and120% levels of 20µg/ml standard solution.Area under curve (AUC) was measured in wavelength range 270-282 nm and results were obtained in terms of percent recovery. Three determinations at each level were performed and % RSD was calculated for each level.

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Table 2:-Accuracy results for Diclofenac sodium.

Precision: The precision of an analytical procedure expresses the closeness of an agreement (degree of scatter) between a series of measurements obtained from multiple sampling of the same homogeneous sample under the prescribed conditions intraday precision was studied by integrating area of standard solution of 20µg/ml concentration at six independent series in the same day.Inter-day precision studies were performed by integrating area of standard solution of 20µg/ml concentration on three consequent days. The % RSD was calculated.

Table 3:-Precision results for Diclofenac sodium.

Linearity and Range:
The linearity was determined by using working standard solutions between 05,10,15,20&25 µg/ml.The areas under curve (AUC) of these solutions were recorded. Calibration curve of area under curve to concentration plotted on excel sheet and linear regression was  performed.The correlation coefficient, regration Equation was calculated.(Fig. 3)

Fig:-4 Overlay of Diclofenac sodium spectra at diff. Concentration.

Note: (1) Spectra --------shows 05µg/ml.
(2) Spectra -------- shows 10µg/ml.
(3) Spectra--------shows 15µg/ml.
(4) Spectra --------shows 20µg/ml.
(5) Spectra --------shows 25µg/ml.

Limit of Detection and Limit of Quantification:
The Limit of Detection(LOD) is the smallest concentration of the analyte that gives the measurable response.LOD was calculated using the following formula

LOD = 3.3 σ /S

The Limit of Quantification(LOQ) is the smallest concentration of the analyte, which gives response that can be accurately quantified. LOQ was calculated using the following formula

LOQ = 10 σ/S

Where, σ is standard deviation of the response and S is the slope of the calibration curve.

LOD& LOQ of Diclofenac sodium found to be 0.9741µg/ml &2.9311/ml respectively.

Five sets of known concentrations (05-25µg/ml) were prepared and scanned. By using these spectras, regression equations were obtained. By taking average of slopes and standard deviation of y-intercept,LOD and LOQ were calculated. The values of LOD and LOQ are given in table 4.

Table 4:-Summary of validation parameters:

RESULTS AND DISCUSSION:
The UV visible spectroscopic method for the Diclofenac sodium by area under curve was found to be simple, accurate, economical and reproducible. The  drug  concentrations  were  found  to  be linear  in  the  range of 05-25 µg/ml and the correlation coefficient value of 0.9981 indicates that developed method was linear. For Precision the percent relative  standard deviation (% RSD) was found to be 1.8073 while, intra-day and inter-day precision  results in terms of percent relative standard deviation values were found to be 0.21811 and 0.3254 respectively thus the method is observed as precise. The accuracy of the method was assessed by recovery studies at three different levels i.e. 80%, 100%, 120%. The values of standard deviation were satisfactory and the recovery studies were close to 100%. The % RSD value is ≤ 2 indicates the accuracy of the method.The Limit of Detection and Limit of Quantitation values were found to be 0.9741µg/ml & 2.9311µg/ml respectively. The result of  the analysis for pharmaceutical formulation by the developed method was consistent with the  label claim, highly reproducible and reliable. The validation parameters are summarized in Table 4. The method can be used for  routine quality control analysis of Diclofenac sodium in  bulk and pharmaceutical formulations.

CONCLUSION:
The UV spectroscopic AUC method for the analysis of Diclofenac sodium was found to be  simple, precise, and accurate can be used for assay of bulk drug and pharmaceutical dosage formulations.

ACKNOWLEDGEMENT:
The authors sincerely thanks to Sahyadri College of Pharmacy, Methwade, Sangola, Solapur, Maharashtra, India for providing experimental facilities to carry out this work.

REFERENCES:
1. Budavari S Editors, In The Merck Index,13thEdn.Merck & Co.Inc. Whitehouse Station (NJ) 2001.
2. Rajesh Sharma*, Geetam Pathodiya And Ganesh Prasad Mishra, “A Novel Application of Hydrotropic Solubilization In Development and Validation of Spectrophotometric Method For Simultaneous Estimation of Paracetamol and Diclofenac Sodium In Solid Dosage Form,” International Journal of Pharma and Bio Sciences Jul-Sep.2010  1(3), 1-9
3. Aravapalli Vasavi*, D.Visagaperumal “Development and Validation of RP-HPLC method for simultaneous estimation of Eperisone Hydrochloride and Diclofenac Sodium in Pharmaceutical Dosage Form”Adv. Pharm. Edu. & Res. 2013: 3(4), 550- 555.
4. International Conference on Harmonization (ICH) of Technical Requirements for the registration of Pharmaceuticals for Human use, Validation of Analytical Procedures Methodology; ICH-Q2(R1), Geneva, 1996, 1-8.

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