development and validation of spectrophotometric method …€¦ · · 2016-06-02 ic value –...

Vol - 4, Issue - 3, Supl - 1 Apr-Jul 2013 ISSN: 0976-7908 Modi et al

www.pharmasm.com IC Value – 4.01 268

PHARMA SCIENCE MONITOR

AN INTERNATIONAL JOURNAL OF PHARMACEUTICAL SCIENCES

DEVELOPMENT AND VALIDATION OF SPECTROPHOTOMETRIC

METHOD FOR SIMULTANEOUS ESTIMATION OF DICLOFENAC

POTASSIUM AND FEBUXOSTAT IN TABLET DOSAGE FORMS.

Modi NR*, Mothalia CP, Prajapati KR, Parmar RR, Shah VN, Shah DA

Department of Quality Assurance, APMC College of Pharmaceutical Education and Research, College campus, Motipura, Himatnagar-383001, Gujarat-INDIA.

ABSTRACT In the present study deals the simultaneous estimation of Diclofenac potassium and Febuxostat in combined tablet dosage form have been developed using 0.1 N NaOH as a solvent. Two method is developed one is simultaneous equation method at 275 nm and 314.5 nm. The second is the Q – analysis (absorption ratio) method, which involves the formation of absorbance equation at 293 nm (isoabsorptive point) and at 314.5 nm the (maximum absorption of Febuxostat). Beer’s law Obeyed in concentration range of 6-30 μg/ mL and 3-15 μg/ mL for Diclofenac potassium and Febuxostat respectively. The accuracy of the methods was assessed by recovery studies was found to be 99.60 ± 0.389 and 99.74 ± 0.213 for simultaneous equation method and 99.74 ± 0.166 and 99.72 ± 0.200 for Q analysis (absorption ratio) method for Diclofenac potassium and Febuxostat respectively. These methods are simple, accurate and rapid; those require no preliminary separation and can therefore be used for routine analysis of both drugs in quality control laboratories. Keywords: Diclofenac potassium , Febuxostat, Simultaneous equation method ,Q–analysis spectrophotometric method. INTRODUCTION

Diclofenac Potassium chemically it is -[(2, 6-dichlorophenyl) amino] benzene acetic acid

monopotassium salt, with a molecular weight of 334.25 g/mol. The empirical formula is

C14H10C12KNO2. It comes under the category of Non-steroidal anti-inflammatory agent

and inhibits prostaglandin synthesis.It is official in British Pharmacopeia, United States

Pharmacopeia and European Pharmacopeia[1-5]. Febuxostat is a xanthine oxidase

inhibitor. The active ingredient in Febuxostat is 2-[3- cyano-4-(2-methylpropoxy)

phenyl]-4-methylthiazole- 5-carboxylic acid, with a molecular weight of 316.38 g/mol.

The empirical formula is C16H16N2O3S. It was recently approved by the European

Medicines Agency on February 21, 2008 and USFDA on Feb 13, 2009[6-9]. Febuxostat is

not official in any pharmacopoeia.The chemical structures of DKP & FEB

shown.Fig.1&2. The combined dosage forms of DKP and FEB are available in the



market for the treatment of GOUT. Deep literature survey reveals that, not a single

analytical method is reported for the determination of these drugs in combined

dosage forms. The present manuscript describes simple, accurate, precise, rapid

and economic spectrophotometric methods for simultaneous estimation of DKP and FEB

in tablet dosage form using 0.1 N NaOH as a solvent.

FIG.1 Chemical structure of Diclofenac Potassium(DKP).

Fig.2 Chemical structure of Febuxostat(FEB).

MATERIALS AND METHODS

Chemicals and Reagents

DKP and FEB bulk powder was kindly gifted by A.P.M.C. Pharmacy College,

Himatnagar, Gujarat, India and Intas Pharmaceuticals, Ahmedabad, Gujarat, India.

Respectively the commercial fixed dose combination XANFEB DSR was procured from

the local market. All other chemicals used were analytical grade. 0.1 N NaOH and

caliberated glass wares were employed throughout the work.

Apparatus

A shimadzu model 1700 (Japan) double beam UV/Visible spectrophotometer with

spectral width of 2 nm, wavelength accuracy of 0.5 nm and a pair of 10 mm matched

quartz cell was used to measure absorbance of all the solutions. A Reptech

electronic weighing analytical balance based on EMFC technology and a Toshcon



ultrasonic bath (Toshniwal process instrument pvt ltd.) was used in the study.

Preparation of standard stock solutions

An accurately weighed quantity of DKP (10 mg) and FEB (10 mg) were transferred to a

separate 100 ml volumetric flask and dissolved in 10 ml methanol and diluted to the

mark with 0.1 N NaOH to obtain standard solution having concentration of DKP (100

μg/ml) and FEB (100 μg/ml).

Method 1 :

The standard solutions of DKP (10 μg/ml) and FEB (10 μg/ml) were scanned separately

in the UV range of 200-400 nm to determine λmax of both the drugs. The λmax of DKP

and FEB were found to be 275 nm and 314.5 nm respectively (Fig.3). Five standard

solutions having concentration 6, 12, 18, 24 and 30 μg/ml for DKP and 3, 6, 9, 12 and 15

μg/ml for FEB were prepared in 0.1 N NaOH using the solutions having concentration

100 μg/ml. The absorbance of resulting solutions was measured at 275 nm

and 314.5 nm and calibration curves were plotted at these wavelengths. The

absorptivity coefficients of these two drugs were determined using calibration curve

equations. The concentration of DKP and FEB in sample solution was determined by

solving the respective simultaneous equations generated by using absorptivity

coefficients and absorbance values of DKP and FEB at these wavelengths. The

absorbance and absorptivities values at the particular wavelength were substituted in

the following equations to obtain the concentration[25].

. . . (1)

. . .(

Where,

A1, A2 –– absorbance of the mixture,

ax1 , ax2 – denotes absorptivities of the x at 275 nm and 314.5nm respectively,

ay1 , ay2 –– denotes absorptivities of Y at 275nm and 314.5 nm respectively,

CX = concentration of DKP.

CY = concentration of FEB.



Fig.3 : Overlain Spectra of Diclofenac potassium(DKP) and Febuxostat(FEB).

Method 2:

The standard solutions of DKP (10 μg/ml) and FEB (10 μg/ml) were scanned in the UV

range of 200-400 nm to determine isoabsorptive point. The isoabsorptive point was

found to be 293nm (Figure.3).Five standard solutions having concentration 6, 12, 18, 24

and 30 μg/ml for DKP and 3, 6, 9, 12 and 15 μg/ml for FEB were prepared in 0.1 N

NaOH using the solutions having concentration 100 μg/ml. The absorbance of resulting

solutions was measured at 293 nm (isoabsorptive point) and 314.5 nm (λmax of FEB) and

calibration curves were plotted at these wavelengths. The absorptivity coefficients of

these two drugs were determined using calibration curve equations. The concentration of

DKP and FEB in sample solution was determined by solving the respective Q-analysis

equations generated by using absorptivity coefficients and absorbance values of DKP and

FEB at these wavelengths. The absorbance and absorptivities values at the particular

wavelength were substituted in the following equations to obtain the concentration[25].

For DKP

. . . (3)

For FEB

. . . (4)



Where,

A1 = Absorbance of sample at isoabsorptive point,

ax1 = Absorptivities of DKP at isoabsorptive point.

Validation of the proposed method:

The proposed methods were validated according to the International Conference on

Harmonization (ICH) guidelines[26].

Linearity (Calibration curve)

The calibration curves were plotted over a concentration range of 6-30 μg/ml and 3-

15 μg/ml for DKP and FEB respectively for Simultaneous equation and Q-analysis.

Accurately measured standard solutions of DKP ( 6, 12, 18, 24 & 30 ml) and FEB (3, 6,

9, 12 and 15 ml) were transferred to a series of 100 ml of volumetric flasks and diluted

to the mark with 0.1 N NaOH for simultaneous equation Method. Accurately measured

standard solutions of DKP ( 4, 6, 8, 10 & 12 ml) and FEB (3, 6, 9, 12 and 15 ml) were

transferred to a series of 100 ml of volumetric flasks and diluted to the mark with 0.1 N

NaOH for Q-analysis Method. The absorbances of the solutions were measured at 275

and 314.5 nm against 0.1 N NaOH as blank for simultaneous equation Method. The

absorbances of the solutions were measured at 293 and 314.5 nm against 0.1 N NaOH as

blank for Q-analysis Method. The calibration curves were constructed by plotting

absorbances versus concentrations and the regression equations were calculated.

Precision

The intraday and interday precision of the proposed methods was determined by

analyzing the corresponding responses 3 times on the same day and on 3 different days 3



different concentrations of standard solutions of DKP and FEB for both methods.

Accuracy (recovery study)

The accuracy of the method was determined by calculating recovery of DKP and FEB by

the standard addition method. Known amounts of standard solutions of DKP and FEB

were added at 80, 100 and 120 % level to prequantified sample solutions of DKP and

FEB. (100 μg/ml for DKP and 40 μg/ml for FEB) The amounts of DKP and FEB

were estimated by applying obtained values to the respective regression line equations.

The experiment was repeated for five times for both methods.

Limit of detection and Limit of quantification

The limit of detection (LOD) and the limit of quantification (LOQ) of the drug were

derived by calculating the signal-to- noise ratio (S/N) using the following equations

designated by International Conference on Harmonization (ICH) guidelines.

LOD = 3.3 × σ/S

LOQ = 10 × σ/S

Where, σ = the standard deviation of the Intercept of Caliberation curve and S = slope of

the calibration curve.

Analysis of DKP and FEB in combined Dosage Form (Tablet)

Twenty tablets were accurately weighed and average weight was calculated. The tablets

were triturated to a fine powder. An accurately weighed quantity of powder equivalent

to 100 mg DKP & 40mg FEB was dissolved in 10 ml methanol and sonicated for 20

min and volume was made up to 100ml. The solution was filtered through Whatman

filter paper No 41 and aliquot portion of filtrate was diluted to produce solution having

concentration of 10 μg/ml of DKP and 4 μg/ml of FEB. The absorbance of sample

solution was measured at selected wavelengths and the concentrations of the two drugs

were estimated using equations (1) and (2) for simultaneous equation method

and equations (3) and (4) for absorbance ratio method. The analysis procedure was

repeated six times and the results are depicted in Table 2.

RESULTS AND DISCUSSION



TABLE 1: REGRESSION ANALYSIS DATA AND SUMMARY OF VALIDATION

PARAMETER OF THE CALIBERATION CURVES

Sr No

Parameters Method 1 Method 2

DKP FEB DKP FEB 1 Wavelength(nm) 275 314.5 275 314.5 293 314.5 293 314.5 2 Beer’s law limit

(μg /ml) 6-30 6-30 3-15 3-15 6-30 6-30 3-15 3-15

3 Regression equation (y = mx + c)

y = 0.031x - 0.011

y = 0.003x- 0.004

y = 0.016x- 0.014

y = 0.063x - 0.031

y = 0.020x - 0.001

y = 0.003x- 0.004

y = 0.040x - 0.020

y = 0.063x - 0.031

4 Slope (m) 0.031 0.003 0.016 0.063 0.020 0.003 0.040 0.063 5 Intercept (c) 0.011 0.004 0.014 0.031 0.001 0.004 0.020 0.031 6 Correlation

coefficient(r2) 0.9997 0.9985 0.9984 0.9991 0.9997 0.9985 0.9985 0.9991

7 LOD (μg/ml) 0.25 0.80 0.02 0.16 0.23 0.80 0.12 0.16 8 LOQ (μg/ml) 0.77 2.43 0.07 0.49 0.70 2.43 0.38 0.49 Precision(% RSD) 9 Interday(n=3) 0.77 1.38 0.95 0.55 0.71 1.38 0.61 0.55 10 Intrady(n=9) 0.88 1.69 1.42 1.04 1.23 1.69 1.10 1.04

TABLE 2: RESULTS OF RECOVERY STUDIES

Level of recovery

Amount of pure drug added (ml)

Simultaneous equation Method

% recovery

Q-Absorbance

method %Recover

DKP (100ug/ml)

FEB (100ug/ml)

DKP FEB DKP FEB

80 % 18 7.2 99.915 99.803 99.551 99.848

100 % 20 8.0 99.169 99.917 99.850 99.833

120 % 22 8.8 99.739 99.504 99.827 99.493

Mean % recovery 99.60767 99.74133 99.74267 99.72467

SD 0.389956 0.213294 0.166386 0.200769

RSD 0.391492 0.213847 0.166815 0.201324



The overlain spectra of DKP and FEB exhibit λ max of 275 nm and 314.5 nm for DKP

and FEB respectively which are quite separated from each other. Additionally one

isoabsorptive point was observed at 293nm. This wavelength was selected for

simultaneous estimation of DKP and FEB for Q value analysis and it is assumed to be

sensitive wavelength. The criteria for obtaining maximum precision by Simultaneous

equation method were calculated and found to be out side the range 0.1-2 and for Q-

analysis ratios of absorbances at 2 different Wavelengths were found to be constant.

Standard calibration curves for DKP and FEB were linear with correlation coefficients (r)

values in the range of 0.9984 – 0.9997 at all the selected wavelengths and the values were

average of three readings with standard deviation in the range of 0.0015 – 0.0045. The

calibration curves were repeated three times in a day and the average % RSD was found

to be 0.86 for DKP and 0.70 for FEB; similarly the method was repeated for three

different days and average % RSD was found to be for 1.26 DKP and 1.18. for FEB . The

accuracy of the methods was confirmed by recovery studies.

CONCLUSION

The proposed methods for simultaneous estimation of DKP and FEB were found to be

simple, accurate, economical and rapid. Due to accuracy, precision and sensitivity, the

developed simultaneous equation method and Q-ratio method could be used for routine

analysis of DKP and FBE in tablet dosage forms.

ACKNOWLEDGEMENT

The authors are thankful to Intas Pharmaceutical, Ahmedabad, Gujarat, India for providing

DKP and FEB for research.

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For Correspondence: Modi NR Email: [email protected]

development and validation of spectrophotometric method …€¦ · · 2016-06-02 ic value –...

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