Validated Analytical Method Development of Celecoxib in Bulk, Tablet and Rat Plasma by UV-Spectroscopy


Praveen Kumar1*, Meenu Chaudhary1, S Bhattacharya1 and Vijay Juyal2

1Division of Pharmaceutical Sciences, Shri Guru Ram Rai Institute of Technology and Sciences, Patel Nagar, Dehradun, 248001, (Uttrakhand), India.        2Department of Pharmacy, Kumaun University, Bhimtal Campus, Bhimtal, Nainital, Uttrakhand.

*Corresponding Author E-mail:



Celecoxib is a nonsteroidal anti-inflammatory drug used in the treatment of pain and inflammation, associated with rheumatoid arthritis, and several other inflammatory disorders. The objective of this study was to develop a simple high sensitive, selective, and rapid quantitative analytical and bioanalytical method for estimation of Celecoxib as API and in biological fluids. The drug shows absorption maxima at 253nm. The linear dynamic response was found to be in the concentration of 2μg-28μg/ml. The slope, intercept and correlation coefficient were found to be 0.0342, 0.0228, and 0.9995 respectively. The percentage recovery of Celecoxib was found to be 100.28%. The extraction of the Celicoxcib from rat plasma was done by Liquid-liquid extraction method. The extraction procedure described by Lee and Min was attempted with some variations. Proposed methods were found to be simple, accurate, precise and rapid and could be used for routine analysis. This method is applied for estimation of Celecoxib as API and in biological fluids. Quantitative recoveries were obtained from plasma. The newly developed methods can be used for routine analysis of Celecoxib for bioavailability of equal significance is the recognition of the immense value of bioequivalence testing as a tool for quality control.


KEYWORDS: Celecoxib, Validation, Rat Plasma, Celebrex Tablets



Celecoxib (CXB) is a selective cyclooxygenase-2 (COX-2) inhibitor used for treatment of rheumatoid arthritis and osteoarthritis1,2. CXB has analgesic, antipyretic, and anti-inflammatory activity as a result of selective inhibition of the enzyme COX-2 and does not inhibit platelet aggregation3. In contrast with other non-steroidal anti-inflammatory drugs (NSAIDs) it has neither acute nor chronic gastrointestinal toxicity4. CXB is also used for treatment of colon cancer5 ultraviolet (UV) light-induced skin cancer6, and breast cancer7.


A thorough literature survey has revealed that a limited number of spectrophotometric, fluorimetric, voltammetric, electrophoretic, and chromatographic methods have been reported for analysis of CXB8. An ultraviolet (UV) spectrophotometric method based on absorption at 251 nm was used for assay of CXB in bulk drugs and capsules9. The method was unsuitable for assay of CXB in microemulsion formulations, because oil peaks interfered with the CXB peak.


It was, therefore, thought worth-while to develop a stability-indicating chromatographic method for assay of CXB in bulk drugs and pharmaceutical dosage forms. Chromatographic methods using acetonitrile and buffer as mobile phase have been reported for assay of CXB in biological fluids10-16, bulk drugs17–20, and pharmaceutical dosage forms21.


The principal objective of this study was, therefore, to develop a new, simple, economical, selective, precise, reproducible, validated analytical and bioanalytical method development of celecoxib in bulk and biological fluids by UV-spectroscopy. In the method proposed the methanol used directly for dilution of the preparations after filtration, and then further used for analysis. Direct use of the solvent methanol as diluents for formulations in quantitative analysis minimizes errors that occur during tedious extraction procedures. The method was validated in accordance with International Conference on Harmonization (ICH) guidelines22.



Preparation of stock solution of Celecoxib:

Accurately weighed Celecoxib (10 mg) was transferred to a 100 ml volumetric flask, dissolved in 10 ml with methanol and made-up the volume up to mark with methanol. The final solution contained 100 mg/ml of Celecoxib.


Determination of wavelength of maximum absorbance of Celecoxib:

5 ml stock solution of Celecoxib was transferred to a 50 ml volumetric flask. It was diluted up to the mark with methanol. The absorbance of the final solution was scanned in the range 230 to 400 nm against methanol as blank. The spectrum is shown in figure 2.


Figure 1.The Chemical Structure of Celecoxib


Figure 2: Spectrum of Celecoxcib 200 to 400nm


Preparation of calibration curve for Celecoxib:

Stock solutions of Celecoxib (2 to 14 ml) were pipetted out in to a series of eleven volumetric flasks of 50 ml. The volume in each volumetric flask was made up to the mark with methanol and the mixture was shacked. That produced the concentration range of 2-28 mg/ml of Celecoxib. The absorbance of the solutions was measured at 253 nm against methanol as blank the result was given in table 1. The calibration curve was given figure 3 and linearity, slope, intercept, correlation coefficient and optical characteristics are summarized in Table2.


Table 1:     Data for Calibration Curve of Celecoxcib

Concentration (μg/ml)
















*Average of three reading

Table 2:   Optical parameters and Regression Characteristics of Celecoxcib



Wavelength Maxima

253 nm

Beers law limit (µg/ml)


Molar absorptivity (1 mole-1cm-1)


Sandells sensitivity (mg/cm2/.001absorbance unit)


Regression equation (y = a+ bc)






Correlation coefficient (r2)













Figure:3 Calibration Curve of Celecoxcib at 253 nm.


Recovery Studies and Validation of the Method according to ICH Guidelines:

To study the accuracy of the above proposed method, recovery studies were carried out by the addition of the standard drug solution to the placebo and recovery of drug was calculated. Result of recovery studies are summarized in Table 4. Precision of the method was studied by carrying out interday and intraday analysis and is expressed as relative standard deviation.  Specificity was checked by spiking reference standard by placebo. The results were found to be satisfactory and are reported in Table 3.


Table 3: Summary of validation parameters

Validation Parameters


Linearity and Range (μg/ml)


Correlation Coefficient (r)


Precision (RSD)

Repeatability (n=6)

Intraday (n=3)

Interday (n=3)





% Recovery



No interference found

LOD (μg/ml)


LOQ (μg/ml)












Estimation of Celecoxib in tablet dosage forms:

The twenty tablets (of same respective batch numbers) were accurately weighed and crushed to fine powder. The powder equivalent to 10 mg of Celecoxib was transferred into 100 ml volumetric flask. 10 ml methanol was added and content in flask was sonicated to dissolve and then the volume was made up to mark with distilled water. The solution was filtered through Whatman filter paper no. 40. 10 ml of this solution was diluted to 100 ml with distilled water. The absorbance of these solutions was measured at 253 nm using water as a blank. The concentrations of Celecoxib present in tablet dosage forms were determined and are tabulated in Table 5.

TABLE 4: Recovery of Method From Placebo Solution


Percent of solution in placebo

Amount* Recovered (µg/ml)

Actual amount added (µg/ml)

Percent recovery*

Mean recovery ±Standard

















TABLE 5: Results of Estimation of Celecoxib (Celebrex-200)


Labeled Amount (mg/ml)

Amount found

Percent amount ± Standard Deviation

Relative standard deviation




99.89 ± 0.115



Table 6: Results of Determination of Celecoxib in spiked rat plasma


Theoretical Conc. (μg/ml)

Calculated Conc. (μg/ml)

Amount Found (mg/ml)

%   Assay

Mean % Assay ± SD

























Determination of Celecoxib in spiked rat plasma:

200 μL of rat plasma samples were spiked with 20 - 50 μ of Celecoxib stock solutions and 20 μL of the IS in 1.8 ml disposable Eppendorf tubes. The tubes were vortexed for 30 s after each addition. The solution was mixed with 600 μL of methanol, vortexed at high speed for 1 min, and centrifuged at 20,000 rpm for 15 min. The supernatant was transferred to a 5 ml centrifuge tube and evaporated to dryness under a stream of nitrogen at 37°C. The residue was reconstituted in 100 μg in methanol and taken the absorbance against the reference sample. Each concentration was prepared at least in triplicate. The concentrations of Celecoxib present in rat plasma were determined and are tabulated in Table 6.




1)      UV-Visible spectrophotometer, UV-210 (ELICO)

2)      Weighing balance, HR 200 (Afcoset)

3)      Ultra sonic bath, SW 45 (Toshcon/ Tosniwal)


Methanol AR (Merck Limited), Water; distilled (In house produced)



The method for the estimation of Celecoxib in tablet dosage form was developed. Drug shows absorption maximum at 253 nm. Spectrophotometric method linear response obtained was in the concentration range of 2-28 mg/ml with correlation coefficient 0.9995, recovery of the drug was found to be 99.40% and relative standard deviation was found to be less than 2 % for precision studies.



The method was statistically validated according to ICH guidelines.  The developed validated methods are simple, rapid, precise and accurate. The newly developed methods can be used for routine analysis of Celecoxib in biological fluids.



Authors are sincerely thankful to Department of Pharmaceutical Science S.G.R.R.I.T.S Dehradun for providing necessary facilities and Zydus Cadila, Changodar, Ahmedabad Plant, for providing pure Celecoxib drug sample.



1.       J. Fort, Am. J. Orthop., 28, 13 (1999)

2.       A. Gaurl, A.M. Martel, and J. Castner, Drugs Future, 22, 711 (1997)

3.       G.S. Gies, Scand. J. Rheumatol., 109, 31 (1999)

4.       M.M. Goldberg, Clin. Ther., 21, 1497 (1999)

5.       T. Kawamori, C.V. Rao, K. Seibert, and B.S. Reddy, Cancer Res., 58, 409 (1998)

6.       S.M. Fisher, H.H. Lo, G.B. Gordon, K. Seibert, G. Kellof, R.A. Lubet, and C.J. Conti, Mol. Carcinog., 25, 231 (1999) – 128

7.       R.E. Harris, G.A. Alshafie, H. Asbou-Issa, and K Seibert, Cancer Res., 60, 2101 (2000)

8.       R.N. Rao, S. Meena, and A.R. Rao, J. Pharm. Biomed. Anal., 39, 349 (2005)

9.       R.N. Saha, C. Sajeev, P.R. Jadhav, S.P. Patil, and N. Srinivasan, J. Pharm. Biomed. Anal., 28, 741 (2002)

10.     O.I. Wong, N. Tsuzuki, M. Rhichardson, M.H. Rytting, W.R. Konishi, and T. Higuchi, Heterocycles, 26, 315 (1987)

11.     M.N.E.I. Kousy, J. Pharm. Biomed. Anal., 20, 185 (1999)

12.     R.W. Mathew and F. Jamali, J. Chromatogr., 616, 59 (1993)

13.     C. Giachetti, A. Assandri, G. Zalano, and E. Brembilla, Biomed. Chromatogr., 8, 180 (1994)

14.     H.S. Chow, N. Anavy, D. Salazar, D.H. Frank, and D.S. Albert, J. Pharm. Biomed. Anal., 34, 167 (2004)

15.     E. Stormer, S. Bauer, J. Kirehhiener, J. Brockmoller, and I. Roots, J. Chromatogr B, 783, 207 (2003)

16.     M. Zhang, G.A. Moore, S.J. Gardiner, and E.J. Begg, J. Chromatogr B, 830, 245 (2006)

17.     G. Jayasagar, M.K. Kumar, K. Chandrasekar, P.S. Prasad, and Y.M. Rao, Pharmazie., 57, 619 (2002)

18.     M.A. Hamid, L. Novotny, and H. Hamza, J. Chromatogr. B, 753, 401 (2001)

19.     U. Werner, D. Werner, A. Phal, R. Mundkowski, M. Gllich, and K. Brune, Biomed. Chromatogr., 16, 56 (2000)

20.     U. Satyanarayana, D. Rao, Y.K. Kumar, J.M. Babu, P.R. Kumar, and J.T. Reddy, J. Pharm. Biomed. Anal., 35, 951 (2004)

21.     F. Schoenberger, G. Hienkele, T.E. Murdter, S. Brenner, U. Koltz, and U. Hoffman, J. Chromatogr. B, 768, 255 (2002)

22.     International Conference on Harmonisation (ICH) of Technical Requirements for Registration of Pharmaceuticals for Human Use: Harmonised Triplicate Guideline on Validation of Analytical Procedures: Methodology, Recommended for Adoption at Step 4 of the ICH Process on November 1996 by the ICH Steering Committee, IFPMA, Switzerland





Received on 01.03.2010       Modified on 19.03.2010

Accepted on 07.04.2010      © RJPT All right reserved

Research J. Pharm. and Tech.3 (3): July-Sept. 2010; Page 894-896