INTRODUCTION:

Celecoxib chemically known as 4-[5-(4-Methylphenyl)-3-(trifluoromethyl)pyrazol-1-yl] benzenesulfonamide[1]. It is a COX-2 selective non-steroidal anti-inflammatory drug (NSAID).It exhibit anti-inflammatory, analgesic and antipyretic activity by selectively inhibiting cyclooxygenase-2 (COX-2) prostaglandin synthesis. It is indicated to relieve the signs and symptoms of rheumatoid and osteoarthritis. Celecoxib is also used for the treatment of colon cancer, ultraviolet light induced skin cancer and breast cancer. [1,5-7]

Fig.1: Chemical structure of Celecoxib

Literature review reveals that a few analytical methods are developed for the determination of Celecoxib using UV-visible spectroscopy [3], HPLC [5-10], LC-MS [14] and Gas chromatography [11] in bulk and capsules [6-18]. But there were no method developed for stability indicating HPTLC method for estimation of Celecoxib. Hence, a simple, specific and sensitive method has been developed for quantification of Celecoxib in capsule formulation and in the bulk drug. As compared to the other analytical techniques HPTLC is the most simple and less time consuming technique and minimal use of solvents and its validation according to ICH guideline.

MATERIALS AND METHODS:

Material and reagents:

Celecoxib was provided as gift samples by Cipla Pharmaceuticals Pvt. Ltd., Marketed formulation Capsule (Mfg. by: Zydus Pharmaceuticals,) with dose of 200mg was procured from local market, Pune. Analytical grade solvents and TLC aluminum plates precoated with silica gel F₂₅₄ used for this study were purchased from Merck Pvt. Ltd, Mumbai

Equipment:

Chromatographic separation of drug was performed by using precoated silica gel TLC plates F₂₅₄, 20x20cm size. Samples were applied on the TLC plates in the form of bands, width 6 mm using Hamilton microliter syringe (100µl). Linear ascending development was carried out in 10× 10cm twin trough glass chamber (CAMAG) using mobile phase which consists of Toluene: Ethyl acetate: Methanol in the ratio of 8:2:1 v/v/v. The slit dimension was kept as 5 × 0.45mm. The optimized saturation time for mobile phase was 30 min. Chromatogram was run till the distance of 8 cm. After development the plate was dried and densitometric analysis was performed on a Camag TLC. Scanner equipped with winCATS software version 1.4.4.6337 at 254 nm. The source of radiation utilized was deuterium lamp.

Preparation of standard solutions (S₁):

10 mg of Celecoxib was weighed accurately and transferred into 10ml volumetric flask, sonicate to dissolve and the volume was made up to the mark with methanol (1000μg/ml). 1 ml of this solution was pipetted out and transferred into a 10ml volumetric flask and the volume was made up to the mark, to get a working standard solution(100μg/ml).

Preparation of sample solution (Formulation) (S₂):

As specified in the label, each tablet contains 200mg of celecoxib. The weight of 20 capsules was noted, empties the capsule and crushed the powder by using mortar and pestle. Weigh quantity equivalent to 10mg of celecoxib and transferred to 10ml volumetric flask and volume was made up to the mark with methanol. 1ml of this solution was pipetted out into a 10ml volumetric flask and the volume was made up to the mark with methanol, to get final concentration(100μg/ml).

Method validation:

The method was validated according to international conference on harmonization (ICH) guidelines [16] for the following parameters: Linearity, precision, the limit of detection(LOD) and limit of quantification(LOQ), repeatability, robustness, recovery and assay.

Linearity:

The calibration curve was plotted between peak areas versus concentration. The linear regression data for the calibration curves (n=6) showed good linear relationship over the concentration range of 100-600ng/spot for Celecoxib with R²=0.9997. The slope and intercept of the regression equation were 1224.2 and 568.09 respectively for the Celecoxib. The linearity was found to be satisfactory and reproducible. The calibration curve for Celecoxib is shown in fig. 2.

Fig.2 Linearity graph of Celecoxib

Table 1: Linearity data of Celecoxib

Sr. No	Parameter	Celecoxib
1	Linearity ng/spot	100-600
2	R²	0.9997
3	Slope	1224.2
4	Intercept	568.09

Precision:

Intraday and interday precision of the method was assessed by developing the plate after application of 3 replicates of different concentration on TLC plate on the same day and the consecutive days respectively. Precision was reported in terms of %RSD. The %RSD values were found to be less than 2% as shown in table 2 and 3.

Table 2: Intraday precision of Celecoxib

Drug	Conc. (ng/spot)	Avg. Area (n=3)	% RSD
Celecoxib	300	1021.36	1.75
	400	1049.53	1.69
	500	1123.03	1.81

Table 3: Interday precision of Celecoxib

Drug	Conc. (ng/spot)	Avg. Area (n=3)	% RSD
Celecoxib	300	1026.26	1.72
	400	1043.89	1.67
	500	1140.15	1.77

Limit of detection and limit of quantitation:

LOD and LOQ were calculated by using standard deviation (σ) and slope (s) from the calibration curve (n=3) by using formula LOD= 3.3 σ/s and LOQ= 10 σ/s. LOD and LOQ were found to be 40ng/spot and 100ng/spot respectively, which indicate the good sensitivity of the method towards the analyte.

Robustness:

Robustness studies were done by making small, deliberate changes in optimized condition like mobile phase composition; saturation time and scanning wavelength (n=3). The acceptance criterion for %RSD was found to be NMT 2% which indicates the reliability of method. The robustness of the method is shown in table 5.

Recovery:

Recovery also known as accuracy, by spiking the band of formulation with 80%, 100%, 120% of pure drug and then finding out the amount of drug recovered. The mean percentage recovery is shown in table 5.

Table 4: Recovery details of Celecoxib

Label Claim	% level	Initial amount (ng/spot)	Amount added (ng/spot)	% Recovery
Celecoxib	80	200	160	99.3
	100	200	200	100.5
	120	200	240	99.5

Assay:

The % drug found in the formulation of the currently developed method was found to be 99.8%. The densitogram obtained from the formulation shows a single spot at R_f0.63 without any interference from the excipients. The percentage of the drug found with label claims shows the application of this method for the routine analysis of Celecoxib presented in its formulation.

Fig.3 Representative Densitogram of Celecoxib

Forced degradation studies:

Forced degradation studies for various parameters were performed as per international conference on harmonization (ICH) guidelines. According to ICH guidelines the degradation should be 5 to 20%.

Following procedures were applied for the studies after carrying out acidic, basic, oxidative and thermal degradation as per international conference on harmonization (ICH) guidelines. [15]

Acid degradation:

Pipette out5ml of standard solution and 5ml of 1N HCl, was kept at room temperature for 6 hrs. Standard solution of Celecoxib and the degraded sample of Celecoxib were spotted on TLC plate of size 3x10cm and the plate was run with mobile phase consisting of Toluene: Ethyl acetate: Methanol in the ratio of 8:2:1 v/v/v. The plate was dried and scanned at 254nm. Densitogram was recorded and % degradation was calculated.

Base degradation:

Pipette out5ml of standard solution and 5ml of 1N NaOH, was kept at room temperature for 8 hrs. Standard solution of Celecoxib and the degraded sample of Celecoxib were spotted on TLC plate of size 3x10cm and the plate was run with mobile phase consisting of Toluene: Ethyl acetate: Methanol in the ratio of 8:2:1 v/v/v. The plate was dried and scanned at 254nm. Densitogram was recorded and % degradation was calculated.

Oxidative degradation:

5ml standard solution and 5ml of 3% H₂O₂, was kept at room temperature for 2 hrs. Standard solution of Celecoxib and the degraded sample of Celecoxib were spotted on TLC plate of size 3x10cm and the plate was run with mobile phase consisting of Toluene: Ethyl acetate: Methanol in the ratio of 8:2:1 v/v/v. The plate was dried and scanned at 254nm. Densitogram was recorded and % degradation was calculated.

Thermal degradation:

10mg of Celecoxib API put in a petri dish. Petri dish was kept in hot air oven at 80˚C. Powdered sample of 1 mg was withdrawn after 30min, 1hr., 2hr., 3hr. and 4hr. intervals and diluted with methanol. The standard solution and degraded solution was applied on TLC plate and the plate was run with mobile phase consisting of Toluene: Ethyl acetate: Methanol in the ratio of 8:2:1 v/v/v. the plate was dried and scanned at 254nm. Densitogram was recorded and % degradation was calculated.

RESULT:

The developed HPTLC method was validated as per international conference on harmonization (ICH) guidelines. The developed method was found to be linear within the range of 100-600ng/spot (R²=0.9997) for Celecoxib. The accuracy of the method was determined at 80%, 100% and 120% level. The percentage recovery of Celecoxib was found to be in between 99.3-100.5 and The Limit of detection and Limit of quantification of Celecoxib was found to be 40 and 100 ng/spot respectively. The developed method was found to be precise and robust as the % RSD value was not more than 2%.

Table 5: Summary of forced degradation study

Sr No	Degradation condition	Rf of Degradants	% Degradation
1	Acid	0.42	10.89%
2	Base	0.47	8.68%
3	Oxidative	0.32	10.3%
4	Thermal	0.89	8.21%

DISCUSSION:

The reported LOD and LOQ values were 0.086 and 0.2625μg/ml respectively. On comparison, the proposed stability indicating HPTLC method is more sensitive as linearity range is lower 100-600ng/spot. More over LOD (40ng/spot) and LOQ (100ng/spot) values are also much lower as compared to reported HPLC method making the proposed HPTLC method significantly sensitive. Reported HPTLC method is also less time consuming and economic than the HPLC method. Therefore it can be concluded that the developed HPTLC technique is more sensitive, fast, precise, specific, accurate, economic and stability indicating than the reported HPLC method which makes it suitable for the analysis of Celecoxib in bulk drug and its pharmaceutical formulation without any interference from the excipients.

CONCLUSION:

A simple, specific, precise and accurate HPTLC method has been developed as stability indicating method for estimation of Celecoxib in bulk drug and pharmaceutical formulation. ICH guidelines were followed throughout the study for method validation and stress testing. Developed HPTLC method effectively separate principle drug peak from degradation product peaks thus it can be employed as a stability indicating method.

ACKNOWLEDGMENT:

Authors are thankful to Cipla Laboratory, Mumbai for providing API as gift sample. We are also thankful to Principal and Management of Bharati Vidyapeeth Poona College of Pharmacy, Pune for providing all facilities for carryout research work.

AUTHORS CONTRIBUTIONS:

All the authors have contributed equally.

ABBREVIATION:

RSD = Relative standard deviation.

CONFLICT OF INTERESTS:

The authors report no conflict of interest.

REFERENCE:

1. Khopkar S.M. Basic concept analytical chemistry. 5^th edition. New Age International Ltd Publishers, New Delhi, 1998; 1:178-79.

2. Green M. J. A Practical Guide to Analytical Method Validation.1996; 305-09.

3. R. Revathi, R. Venkatesha Perumal, S. Sudharshini, A.Mohammed Ansar, A. Thilagalakshmi, and A. P. Dinesh et al. Simple UV Spectrophotometric Determination of Celecoxib in Pure Form and in Pharmaceutical Formulations. International Journal of Pharmaceutical Sciences 2011 ; Vol. 1 (2) :49-50.

4. Vijaygeeta Ragupathy, Shantha Arcot. Simultaneous spectrophotometric determination of diacerein and Celecoxib in capsule by chemometric method. International Research Journal of Pharmacy. 2013.

5. S. Baboota, S. Faiyaz, A. Ahuja, J. Ali, S. Shafiq, and S. Ahmadet al. Development and validation of a stability-indicating HPLC method for analysis of Celecoxib (CXB) in bulk drug and micro emulsion formulations. ACTA Chromatographica. 2007.

6. Dalapathi B Gugulothu and Vandana B Patravale. A New Stability-Indicating HPLC Method for Simultaneous Determination of Curcumin and Celecoxib at Single Wavelength: an Application to Nano particulate Formulation. Pharmaceutica Analytica Acta. 2012.

7. Pramod M. Dhabu and Krisnacharya G. Akamanchi. A Stability-Indicating HPLC Method to Determine Celecoxib in Capsule Formulations. Drug Development and Industrial Pharmacy. 2002; Vol. 28: 815–21.

8. Chandana OSS, Ravichandrababu R. Stability indicating HPLC method for Celecoxib related substances in solid dosage forms. Int J Res Pharm Sci 2017; 7(1):10 – 18.

9. Ayman K. Hamama, John Ray, Richard O. Day and Jo anne E. Brien. Simultaneous Determination of Rofecoxib and Celecoxib in Human Plasma by High-Performance Liquid Chromatography. Journal of Chromatographic Science. August 2005; Vol. 43.

10. L.I. Bebawy A.A. Moustafa, N.F. Abo-Talib. Stability-indicating methods for the determination of doxazosinmezylate and Celecoxib. Journal of Pharmaceutical and Biomedical Analysis. 2002; 779–93.

11. S. Chandran, P. R. Jadhav, P. B. Kharwade and R. N. Saha. Rapid and Sensitive Spectrofluorimetric Method for the Estimation of Celecoxib and Flurbiprofen. Research Paper Indian Journal of Pharmaceutical Sciences. January February 2006.

12. Małgorzata Starek and Marek Rejdych.Densitometric Analysis of Celecoxib, Etoricoxib and Valdecoxib in Pharmaceutical Preparations. Journal of Planar Chromatography 2009; 6: 399–403.

13. M. Starek, J. Krzek, and P. Rotkegel. TLC Determination of Piroxicam, Tenoxicam, Celecoxib and Rofecoxib in Biological Material. Journal of Analytical Chemistry 2015; Vol. 70: 351–359.

14. Ambavaram Vijaya Bhaskar Reddy, Nandigam Venugopal and Gajulapalle Madhavi. A selective and sensitive LC-MS/MS method for the simultaneous determination of two potential genotoxic impurities in Celecoxib. Journal of Analytical Science and Technology. 2014; 5-18.

15. ICH, Stability testing of new drug substances and products Q1A (R2), International Conference on Harmonization, IFPMA, Geneva, 2003.

16. ICH, Q2 (R1): Validation of Analytical Procedures: Text and Methodology, in proceeding of International Conference on Harmonization. Geneva, Switzerland; 2005;1:11

Received on 16.09.2019 Modified on 17.11.2019

Research J. Pharm. and Tech. 2020; 13(8):3661-3665.

DOI: 10.5958/0974-360X.2020.00647.2