Assay Method Development and Validation for Celecoxib Dosage Forms by High Performance Liquid Chromatography

 

O.S.S.Chandana1, R.Ravichandrababu1*

1Department of Chemistry, KITS, Divili, Tirupathi Village, Andhra Pradesh, India

1*Department of Chemistry, Institute of science, GITAM University, Visakhapatnam, Andhra Pradesh, India

*Corresponding Author E-mail: rrcbabu7@yahoo.in

 

ABSTRACT:

The main objective of the work was to develop and validate a stability indicating HPLC method for the determination of celecoxib and to perform forced degradation studies. The method was developed by Agilent HPLC with the column L11 (4.6 x 250mm, 5 μm), it has a mobile phase of monobasic potassium phosphate buffer [pH 3.0], methanol and acetonitrile in the ratio of 60: 30 : 10 v/v/v. The flow rate was set at 2.0 ml/min with a detection wavelength of 215nm using VWD detector. The method was validated for analytical parameters such as specificity, accuracy, precision, robustness and ruggedness as per ICH guidelines.  The linearity was found to be in the range of 25-120 mg/ml with a correlation coefficient value 0.9999. The precision is exemplified by relative standard deviation of 0.3%. Percentage mean recovery was found to be in the range of 97-99, during accuracy studies. The proposed method was found to be simple, accurate, precise, and robust and stability indicating HPLC method. Hence this method can be used for routine analysis.

 

KEYWORDS: Celecoxib, HPLC, Stability indicating, method development, validation.

 

 


INTRODUCTION:

Celecoxib is chemically known as 4-[5-(4-Methylphenyl)-3-(trifluoromethyl) pyrazol-1-yl] benzenesulfonamide. Its chemical formula and molar mass were C17H14F3N3O2S and 381.373 g/mol .It is a COX-2 selective non-steroidal anti-inflammatory drug (NSAID).It is used to treat the pain and inflammation of osteoarthritis, rheumatoid arthritis, ankylosing spondylitis, acute pain in adults, painful menstruation, and juvenile rheumatoid arthritis[1]. Celecoxib is also used for the treatment of colon cancer, ultraviolet light induced skin cancer and breast cancer [2]

 

Figure 1: structure of celecoxib

 

The development and validation of an analytical method is to ensure a specific, accurate and precise method for a particular analyte. The principal objective for that is to enhance the conditions and parameters, which should be observed in the evolution and establishment. Literature review reveals that a few analytical methods are developed for the determination of celecoxib using XRD [3], LC-MS [4] and HPLC [5-7] in bulk and capsules. So far there is no method for stability indicating assay method for celecoxib using HPLC. Hence the author developed a new simple, accurate and stability indicating HPLC method for the determination of celecoxib drug. The method developed was validated as per ICH guidelines [8].

 

MATERIALS AND METHODS:

Chemicals and reagents:

The Samples of Celecoxib and its impurities were obtained from Fortunee Laboratories (P) Ltd, Kakinada, and Andhra Pradesh, India. All other analytical reagents such as Ammonium formate, acetonitrile, hydrochloric acid, sodium hydroxide and hydrogen peroxide (30%) were obtained from Merck specialty chemicals, Mumbai, India. Milli ‛Q’ water is used for the preparation of

Solutions.

 

Instrumentation:

This research was performed on Agilent make HPLC 1100 instrument. It has binary gradient pump, photo diode array detector (UV), column oven with range of 25°C to 60°C with auto injector. The modules are G1310A isocratic pump with solvent cabinet; G1314A variable wavelength detector (VWD) with standard flow cell (10 mm path length, 14 µl volume, 40 bar maximum pressure) and G2220AA 2D-Value Solution Chem Station.

 

Chromatographic conditions:

The separation was achieved on Column,L11,water symmetry (4.6 x 250mm, 5 μm,) Supelcosil DP with a mobile phase of  monobasic potassium phosphate buffer[pH 3.0],methanol and acetonitrile in the ratio of 60: 30 : 10 v/v/v. The flow rate was set at 2.0 ml/min and a detection wavelength of 215nm using VWD detector. The liquid chromatography is equipped with a 215 nm VWD detector. The column oven temperature was maintained at 60oC. The injection volume was 25 µl.

 

Mobile phase buffer preparation:

Dissolve about 2.7 g of monobasic potassium phosphate in 1000 ml of Milli-Q water. Adjust the pH to 3.0 with 10% phosphoric acid.

 

Mobile phase preparation:

Mix mobile phase buffer, methanol and acetonitrile in the ratio of 60 : 30 : 10 v/v/v respectively. Filter through 0.45 µm membrane filter. Degas in a sonicator for about 10 minutes.

 

Standard stock solution preparation:

An accurately weighed amount of about 50 mg of Celecoxib working standard or reference standard was transferred into a 50ml volumetric flask. To this 10 ml of diluents were added and sonicated to dissolve for about 5 minutes. Then Diluted to volume with diluent and mixed well.

 

Standard solution preparation:

Pipette 5 ml of above standard stock solution into a 100 ml volumetric flask and make up to the mark with diluent.

 

Test solution preparation:

Twenty tablets were weighed accurately, powdered and the average weight was calculated. A portion of powder, equivalent to exactly 200 mg of Celecoxib was transferred into a 200 ml volumetric flask and 100 ml of diluents was added and sonicate for about 10 minutes with intermediate shaking. Filter the test solution through 0.45 µm PVDF (Smartpor) or Nylon (Millipore, Agela) syringe filter or centrifuge at 3000 rpm for 10 minutes.  5 ml of above clear solution was pipette into a 100 ml volumetric flask and dilute to volume with diluent.

 

Diluent preparation:

2 ml of tri-ethylamine and 2 ml of ortho-phosphoric acid were taken in 1000ml of Milli-Q water. Mix above buffer solution and acetonitrile in the ratio of 45: 55 v/v respectively.

 

Validation:

The proposed method was validated for the analysis of celecoxib using following parameters. System-suitability studies are an intact part of method development and are practiced to ensure satisfactory performance of the chromatographic system. For five replicate injections of the drugs Number of theoretical plates (N) and tailing factor (T) were assessed. Linearity was established by plotting a graph between concentration versus peak area and the correlation coefficient was determined. To obtain proportionality, the slope and intercept of the regression line and correlation coefficient were calculated statistically from the calibration curve of the celecoxib. To find out variations in the test methods precision was studied for celecoxib of spiked test preparation with celecoxib blend solution to get 0.5% of each impurity with respect to test concentration and analyzed as per test method when analysis carried out by Analyst to Analyst, System to System and Column to Column Variation (ruggedness). The mentioned solution was injected six times and the area was measured for all six injections in HPLC. The % relative standard deviation (%RSD) and % content results were used for assessment of precision and ruggedness. The accuracy of the method was demonstrated by analyzing celecoxib of spiked test preparation with LOQ, 100% and 200% of target concentration. After injection, recovery values for individual drugs were estimated. Specificity is the ability of a method to differentiate the analyte(s) of interest from other components in the sample. Placebo was prepared as per the marketed product formulas of drugs. Placebo interference from excipients was studied. Robustness of the method was determined by varying flow rate, and filtration. Bench top stability (250C & 60 % RH) and Refrigerator (80C & 55%RH) stability were determined on the 1st and 2nd day. Forced degradation study was conducted to demonstrate the effective separation of degradants from celecoxib. Celecoxib was exposed to the following stress conditions such as refluxed with 3N HCl solution for about 24 hours at 60ºC (Acid). Refluxed with 3N NaOH solution for about 24 hours at 60ºC (Base). Treated with 10% Hydrogen peroxide (H2O2) for 24 hours at 60ºC (Peroxide). Dry heat at 105° C for about 24 hrs in an oven.

 

RESULTS AND DISCUSSIONS:

Optimization of HPLC conditions:

In order to maximize the resolution and sensitivity of the proposed HPLC method for the separation of celecoxib and its degradation products, different experimental conditions were studied and optimized. This was performed at a detection wavelength of 215 nm which provided the best resolution.

 

Precision:

The result of precision and intermediate precision was depicted in (Table 1). The % relative standard deviation for each impurity in six determinations for precision and intermediate precision found below 2.0% confirms the preciseness of the method.

 

Table-1: Precision and Intermediate precision results

S.No.

Precision Results

Intermediate precision results

1

98.4

95.8

2

98.1

95.9

3

98.7

95.8

4

98.6

96.1

5

98.2

95.9

6

98.8

95.6

Average

98.5

95.9

SD

0.2805

0.1643

% RSD

0.3

0.2

 

Linearity:

Linearity parameter was performed from 25ppm to 120ppm and linearity results found to be good. The correlation coefficient value is 0.9999. All results are represented in below table-2 and graph also represented in figure-2.

Table-2: Linearity of Detector Response

S. No.

Celecoxib

Concentration (mg/ml)

Average Area response

1

25

1181778

2

50

2320145

3

75.1

3540116

4

100

4721540

5

110

5183690

6

120

5604899

Correlation Coefficient

0.9999

 

Figure-2: Linearity results

 

Accuracy:

Accuracy parameter was performed from 100% and 200% with test sample and placebo and the results are represented below table-3and table-4.

 

Table-3: Recovery results with test samples.

S.No.

Sample name

% Recovery

% Recovery AVG

1

100 % Sample-1

98.1

98.1

100 % Sample-2

98.2

 

100 % Sample-3

98.1

 

2

200 % Sample-1

97.9

97.9

200 % Sample-2

97.9

200 % Sample-3

97.9

 

Table-4: Recovery results with placebo.

S.No.

Sample name

% Recovery

% Recovery AVG

1

100 % Sample-1

98.1

98.2

100 % Sample-2

98.2

100 % Sample-3

98.2

2

200 % Sample-1

98.2

 

98.0

200 % Sample-2

97.9

200 % Sample-3

98.0

 

Forced Degradation Studies

Peak purity results for forced degradation samples are represented in table- 5. All force degradation chromatographs are represented from figure-3 to 7


 

Table-5: Force degradation results with mass balance.

S.No.

Sample Name

% ofDegradation

% of Assay

Mass balance

1

Unstressed sample

0.0651

97.58

97.65

2

Acid API

0.0651

99.31

99.38

3

Acid RLD

0.0357

98.09

99.12

4

Base API

0.1526

98.83

100.08

5

Base RLD

0.1145

98.85

98.94

6

Oxidation API

0.0586

99.85

99.90

7

Oxidation RLD

0.0166

99.21

98.23

8

Thermal API

0.0757

99.38

99.46

9

Thermal RLD

0.0362

99.98

100.02

 

Figure -3: Test spiked with impurities

 

Figure -4: Forced Degradation-Base Stress

 

Figure -5: Forced Degradation-Peroxide Stress

 

Figure -6: Forced Degradation-Acid Stress

 

Figure -7: Forced Degradation-Thermal Stress

 

Table-6: Robustness results

Condition

Rs_C

Rs_A

RT

Area(mean)

% RSD

Tailig factor

Plates

pH

3.4

1.68

1.91

12.271

2224.61270

0.04

0.9

12234

3.0

1.99

1.92

12.347

2221.12007

0.03

0.9

12189

Organic solvent (Buffer: ACN:MeOH)

60:11:30

1.65

1.85

11.390

2222.40117

0.02

0.9

12203

60:9:30

1.90

2.01

13.515

2184.02637

0.03

0.9

12860

60:10:33

1.65

1.77

10.502

2221.15273

0.02

0.9

11929

60:10:27

1.88

2.14

14.705

2186.46753

0.02

0.9

13164

System suitability

NLT 1.5

NLT 1.5

-

-

NMT 2.0

NMT 2.0

NLT 2000

 

Table-7: Ruggedness results

Sample

Initial

1day refrigerator

Difference

1day bench top

Difference

T1

97.40

96.49

-0.93

97.52

0.12

T2

99.07

98.79

-0.28

97.15

-1.94

 

Initial

2days refrigerator

Difference

2days bench top

Difference

T1

97.40

99.59

2.25

99.18

1.83

T2

99.07

100.60

1.54

98.98

-0.09

 


Robustness:

Robustness is performed by injecting the diluent as blank, system suitability solution, standard solution (3 times), and test solutions into the chromatographic system, recorded the chromatograms and measured the peaks responses. The results are depicted in the table-6

 

Ruggedness:

Ruggedness was performed for test solutions stability for day-1 and day-2 on bench top and refrigerator stability. All results found to be good. All results are listed in below table-7.

DISCUSSION:

The developed method can be used for routine analysis because the linearity found in Celecoxib is nearing 1 that is 0.9999 which shows the good regression for linearity. Maximum recovery is obtained by this developed method and the mean percentage recovery for each component is nearing 100%. Therefore this method can be used for the routine analysis and one most important reason is that the developed method does not involve the use of expensive reagents. The spectrophotometric assay methods employed in our study indicated less interference from excipients used in formulation by the percent recoveries values. Most of the existing methods consumed expensive reagents for individual drug analysis. But the method we developed involves chemicals like acetonitrile and buffer, which are easily available. Also, our proposed method requires less time for the determination of celecoxib compared to other methods.

 

CONCLUSION:

A validated HPLC analytical method has been developed for the determination of Celecoxib in bulk and dosage form. The proposed method was simple, accurate, precise, specific and suitable to use for the routine analysis of Celecoxib in either bulk API powder or in pharmaceutical dosage forms. Method validation parameters results are evaluated and found to be acceptable and this stability indication method can be used for regular analysis.

 

ACKNOWLEDGEMENT:

The authors would like to thank Department of Chemistry, Institute of science, GITAM University, Visakhapatnam for providing necessary facilities. The authors are also grateful to Fortune labs, Kakinada for providing gift sample of celecoxib drug.

 

CONFLICT OF INTERESTS:

Declared None

 

REFERENCES:

1.        KG. Jadhav, NM. Gowekar, SN. Gowekar, et al., A Validated RP-HPLC Method for the Determination of Celecoxib in Bulk and Pharmaceutical Dosage Form, International Journal of Research in Pharmaceutical and Biomedical Sciences, 2012, 3 (3), 1312-1316.

2.        S. Baboota, S. Faiyaz , A. Ahuja1 , J. Ali , S. Shafiq ,  S. Ahmad , et 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, 18, 116-129.

3.        Choubey Pravir, Manavalan , Dabre Rahul , Jain Girish, et al., Pre-formulation Studies for development of a generic capsule formulation of Celecoxib comparable to the branded (Reference) Product, Innovations in Pharmaceuticals and Pharmacotherapy, 2013, 1 (3), 230-243.

4.        Ambavaram Vijaya Bhaskar Reddy, Nandigam Venugopal and Gajulapalle Madhavi, et al., 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, 2-18.

5.        Emami J., Fallah R., Ajami A, et al., A rapid and sensitive HPLC method for the analysis of Celecoxib inhuman plasma: application to pharmacokinetic studies, DARU 2008, 16 (4), 211-217.

6.        Jayasagar G, Kumar MK, Chandrasekhar K, Prasad PS, Rao YM, et .al., Validated HPLC method for the determination of Celecoxib in human serum and its application in a clinical pharmacokinetic study, Pharmazie. 2002, 57 (9), 619-21.

7.        Sharma Tejal, Solanki N.S., Mahatma O.P., SinghaiS., et.al., Statistical Assurance of Process Validation by Analytical Method Development and Validation for Celecoxib capsules, 2012, 4 (1), 68-72.

8.        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 26.07.2016             Modified on 02.08.2016

Accepted on 08.08.2016           © RJPT All right reserved

Research J. Pharm. and Tech 2016; 9(11): 1951-1956.

DOI: 10.5958/0974-360X.2016.00400.5