CHAPTER -IV

STABILITY INDICATING HPLC METHOD FOR CELECOXIB

RELATED SUBSTANCES IN SOLID DOSAGE FORMS

4.1. INTRODUCTION
An RP-HPLC method was developed and validated for the determination of Celecoxib related
substances in bulk drug. Celecoxib is a COX-2 selective nonsteroidal anti-inflammatory drug
(NSAID) (www.drugbank.ca). 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 in two years or older.

4.1.1. DRUG PROFILE

Celecoxib is chemically known as 4-[5-(4-Methyl-phenyl)-3-(trifluoromethyl) pyrazol-1-yl]
benzene sulfonamide with a molecular formula of C17H14F3N3O2S and its molecular weight is
381.373 g/mol (www.usp.org). The impurities of celecoxib are impurity.
A chemically known as 4-[3-(4-Methylphenyl)-5-(trifluromethyl)-1H-pyrazol-1-yl]
benzensulfoneamide, impurity C chemically known as (4-[5-(3-Methylphenyl)-3-
(trifluromethyl)-1H-pyrazol-1-yl]benzensulfoneamide, impurity D chemically known as 4- [5-
(2-Methylphenyl) -3- (trifluromethyl) - 1H- pyrazol-1-yl] benzensulfoneamide and 4-
methylacetophenone. the structure of celecoxib and its impurities are given in the figure 4.1

Celecoxib
 Impurity A (Regio Isomer) Impurity C (Meta Isomer)

Impurity D (Ortho Isomer) 4-methylacetophenone

Figure 4.1: Structure of Celecoxib and its impurities

4.2. LITERATURE REVIEW

Ambavaram Vijaya Bhaskar Reddy et. al., (2014) reported a selective and sensitive liquid
chromatography-tandem mass spectrometry (LC-MS/MS) method for the simultaneous
determination of (4-sulfamoyl-phenyl) hydrazine hydrochloride and
(4-methyl-acetophenone) para-sulfonamide phenylhydrazine hydrochloride in celecoxib active
pharmaceutical ingredient. The LC-MS/MS analysis was done on symmetry C18 (150 mm
×4.6 mm, 3.5 μm) analytical column, and the mobile phase used was 5.0 mM ammonium
acetate-acetonitrile in the ratio of 30:70(v/v). The flow rate used was 0.7 mL/min

Baboota et. al., (2007) reported a simple, economic, selective, precise and stability-indicating
HPLC method for analysis of celecoxib, both in bulk drug and in microemulsions. Reversed-
phase chromatography was performed on a C18 column with methanol–water, 75:25 (v/v), as
mobile phase at a flow rate of 1.25 mL/min. Detection was performed at 250nm and a sharp peak
was obtained for CXB at a retention time of 4.8 ± 0.01 min.

Bapatu HR et. al., (2014) reported a novel stability-indicating reverse phase high performance
liquid chromatography method for the simultaneous determination of Celecoxib and Diacerein
and its impurities in the capsule dosage form. The method was developed using an L1 column
with a gradient using the mobile phase consist of solution A (pH = 2.3 buffer) and solution B
(methanol and acetonitrile; 50: 50, v/v). The eluted compounds were monitored at 255 nm.

Dhabu PM et. al., (2002) reported a simple and accurate high-performance liquid
chromatographic (HPLC) method to determine Celecoxib in capsule formulations. The drug was
chromatographed on a reversed-phase C-18 column using a mixture (85:15) of methanol and
water with the detection wavelength of 215nm.

Emami J et. al., (2008) reported a simple, rapid, sensitive, reliable, and economic HPLC method
for determination of celecoxib in human plasma which is more feasible than reported celecoxib
HPLC assays. The drug and internal standard were extracted using n-hexane /isoamyl alcohol
(97:3) and analyzed on a C18 µ-Bondapak HPLC column with KH2PO4 (0.01M, pH= 4) -
acetonitrile (60:40) as the mobile phase, at 260 nm.

Jadhav K et. al., (2012) reported a RP-HPLC isocratic separation was achieved on C18 column
(250×4.6 mm i.d., 5µm) utilizing a mobile phase comprising of methanol and acetonitrile in the
ratio of 70: 30(v/v) and the eluents from the column were detected using a variable wavelength
detector at 254nm.

Jayasagar et. al., (2008) reported a simple high performance liquid chromatographic method
using UV detection for the determination of celecoxib, a specific COX 2 inhibitor, in serum
containing the internal standard, tolbutamide, eluted through a C18, Wakosil column. After
extracting with dichloromethane, the eluent is monitored at 250 nm using the mobile phase of 10
mM potassium dihydrogen ortho phosphate (pH 3.2) and acetonitrile (50:50 v/v) with a flow rate
of 1 ml/min.

Krishnaveni G et. al., (2012) developed a simple, precise and accurate RP-HPLC method for
rapid assay of celecoxib in tablet dosage form. Isocratic elution at a flow rate of 1.5ml/min was
employed on a symmetry chromosil C18 (250x4.6mm, 5µm in particle size) column at ambient
temperature using the mobile phase of methanol: acetonitrile. 60:40 (V/V) with the detection
wavelength of 220 nm.
Srinivasulu Dasari et. al., (2012) reported the development of a new isocratic reverse phase
liquid chromatographic method for the separation and determination of celecoxib in the presence
of its process-related impurities. The successful separation of the drug from the process
impurities was achieved on an inertsil ODS C18 column (250 mm X 4.6 mm, 5 µ) using
phosphate buffer (pH adjusted to 3.5): acetonitrile (45-55v/v) as eluent, at a flow rate of 1.0
mL/min and the detection wavelength was set at 250 nm.

4.3. SCOPE OF THE WORK

The main objective of the research work is to develop a simple, accurate, stability indicating RP-
HPLC method for the quantification of celecoxib and its related substances in celecoxib which
can be able to quantify the degradation products and also to get good baseline separation
between celecoxib and its process related impurities and degradation products. Review of
literature reveals that reported methods were qualitative and instrument methods with high
analysis time. The present study deals with the method development and validation of RP-HPLC
method for celecoxib related substances.

4.4. EXPERIMENTAL
4.4.1. MATERIALS AND REAGENTS
The reference sample of celecoxib and its impurities A, C, D (Simson) and 4-
methylacetophenone (sigma altrich) were received as a gift sample from Fortune laboratories,
Kakinada, Andhra Pradesh. Milli-Q-water was used throughout this research. All other analytical
reagents such as potassium phosphate, acetonitrile, methanol, phosphoric acid, hydrochloric acid,
sodium hydroxide and hydrogen peroxide (30%) were obtained from Merck speciality chemicals,
Mumbai, India.

4.4.2. INSTRUMENTATION
This work has been performed on PEAK Chromatographic (HPLC) instrument. It has binary
gradient pump (G1311A), G1314A variable wavelength detector (VWD), G1329A Auto Sampler
and G1316A column compartment. Chromatogram was analysed using PEAK chromatographic
chemistration version B.02.01.

4.4.3. Preparation of solutions

Buffer preparation
2.7gm of mono basic potassium phosphate was dissolved in 1000mL of HPLC grade water. pH
was adjusted to 3.0 with 10% phosphoric acid.

Mobile phase preparation

Mixture of Buffer, methanol and acetonitrile in the ratio of 60:30:10v/v/v was used. Mobile
phase was filtered through 0.45µM membrane filter.

Diluent preparation
Diluent buffer was prepared by adding 2ml of triethylamine and 2ml of phosphoric acid in
1000ml of HPLC water. Mixture of diluent buffer and acetonitrile in the ratio of 45:55v/v was
used as a diluent.

Impurity stock solution preparation

An accurately weighed amount of about 2.4mg of celecoxib related compound A and related
compound B were transferred into a 20ml volumetric flask individually. 5ml of diluent was
added and sonicated to dissolve. Finally, the volume was made up to the mark with diluent.

System suitability solution preparation:

Accurately weighed amount of about 25mg of celecoxib working standard or reference standard
was transferred into a 50ml volumetric flask. 10ml of diluent was added and sonicated to
dissolve. 1ml of above impurity stock solution was added and then diluted to volume with
diluent.

Standard stock solution preparation

An accurately weighed amount of about 25mg of celecoxib working standard or reference
standard was transferred into a 50ml volumetric flask. 10ml of diluent was added and sonicated
to dissolve. Finally the volume was made up to the mark with diluent. . 5ml of above standard
stock solution was pipetted out and transferred into a 100ml volumetric flask and Made upto the
mark with diluent.

Standard solution
4ml of above standard stock solution was pipetted out and transferred into 50ml volumetric flask
and then made up to the mark with diluent. The sonication bath temperature was maintained at
20 to 25°C while sonication.

Sample preparation
50 mg of Celecoxib capsule powder was weighed and transferred into 100 mL volumetric flask.
50mL of diluent was added and sonicate for about 10 minutes. Finally the volume was made up
to the mark with diluent.

4.5. RESULTS AND DISCUSSION

4.5.1 Method development
Development trials were performed with all neutral buffer salts, using different organic solvents
and using different HPLC columns. Finally chromatographic conditions were optimized with
monobasic potassium phosphate, acetonitrile and methanol using isocratic method .

4.5.2 Method development experiment-1

Chromatographic conditions:
Mobile phase : Buffer: methanol: acetonitrile (30:15:5)

Buffer : 8.1243 g of monobasic potassium phosphate was dissolved in

3000mL of Milli-Q water and pH of this solution was adjusted to
3.0 with phosphoric acid.

Diluent : 2 mL of triethylamine and 2 mL of orthophosphoric acid was

added in 1000mL of Milli-Q water. This solution was mixed 450
mL buffer and 550 mL of acetonitrile.

Column : (4.6x250mm, 5µ), Supelcosil DP column

Column oven temperature : 60℃

Flow rate : 1.5 mL per minute

Injection volume : 25 µL

Run time : about 60 minutes

Detection wavelength : 215nm

The separation of celecoxib with its impurities is shown in the figure 4.2

Figure 4.2 Blend Chromatogram for celecoxib and its impurities

Observation
It is evident from fig 4.2 that the resolution between impurity-C and Celecoxib was found to be
2.30; The resolution between Celecoxib and Impurity-A was found to be 1.95.

4.5.3. Method development experiment-2

The experiment was performed by changing the flow rate and keeping the same conditions as
mentioned above.

Chromatographic conditions
Mobile phase : Buffer: methanol: acetonitrile (30:15:5)

Buffer : 8.1243 g of monobasic potassium phosphate was dissolved in 3000mL

of Milli-Q water and pH of this solution was adjusted to 3.0 with
phosphoric acid.

Diluent : 2 mL of triethylamine and 2 mL of orthophosphoric acid was added in

1000mL of Milli-Q water. This solution was mixed 450 mL buffer and
550 mL of acetonitrile.
Column : (4.6x250mm, 5µ), Supelcosil DP column

Column oven temperature : 60℃

Injection volume : 25 µl

Run time : about 60 minutes

Detection wavelength : 215nm

Flow rate : 1.3mL/min; 1.1 mL/min; 1.0mL/min

The separation of celecoxib with its impurities is shown in the figure 4.3.
 Figure 4.3 Blend Chromatogram of celecoxib and its impurities

Table 4.1. Data for experiment-2

Flow rate Retention time Resolution

of celecoxib
Between Impurity C Between Celecoxib
and Celecoxib and Impurity A

1.3 mL/min 23.570 2.38 2.00

1.1 mL/min 28.067 2.42 2.04

1.0 mL/min 31.131 2.48 2.06

Observation:
It is evident from fig 4.7 that the resolution between Celecoxib and Impurity-A was more with
1.3 mL/min flow compared to 1.5 mL/min. The separation of the sample and the impurities was
observed.

4.5.4. Optimized method procedure:

The main aim of this study as to develop a HPLC method to get fine resolutions between AZM
and related substances. Finally better results were obtained employing a Develosil column and
adjusting the pH to 2.5 with potassium dihydrogen phosphate buffer and finally the experiment
was preceded with the following chromatographic conditions

Column : Supelcosil DP (4.6x250mm, 5µ)

Column oven temperature : 60℃

Flow rate : 1.3 mL per minute

Injection volume : 25 µL

Run time : 40 minutes

Detection wavelength : 215nm

Elution mode : Isocratic.

Procedure:
25µL portion of diluent as blank, system suitability solution, standard solution (3 times) and test
solution was injected into the chromatographic system. The chromatograms were recorded and
the peak response was measured. The separation of celecoxib with its impurities is shown in the
figure 4.4.

Figure 4.4. Blend Chromatogram of celecoxib and its impurities

4.6. METHOD VALIDATION:

A Isocratic reverse – phase HPLC procedure was suggested as a suitable method for the analysis
of Celecoxib related substances. From the results of optimized method, 5.348, 19.322, 21.691,
23.501 and 25.114 minutes were the retention times for 4-methyl acetophenone, Impurity D,
Impurity C, Celecoxib and Impurity A respectively. System suitability parameters like
theoretical plate, % relative standard deviation and tailing factor for the 4-methyl acetophenone,
Impurity D, Impurity C, Celecoxib and Impurity A were reported. Mean relative retention factor
values of 4-methyl acetophenone, Impurity A, Impurity C and Impurity D were found to be 1.09,
0.77, 1.05 and 0.88 respectively.
4.6.1. SYSTEM SUITABILITY PARAMETERS:

Studies were performed and reported in the table 4.2.

Table 4.2: System suitability results

System suitability parameters Observed value Acceptance criteria

Theoretical Plates Celecoxib 15258 Should be NLT 2000

4-methyl 6781
acetophenone

Impurity A 5789

Impurity C 5687

Impurity D 5127

%RSD Celecoxib 1.91 Should be NMT 5.0

4-methyl 1.72
acetophenone

Impurity A 1.75

Impurity C 1.65

Impurity D 1.22

Tailing factor Celecoxib 1.0 Should be NMT 2.0

4-methyl 1.1
acetophenone

Impurity A 1.1

Impurity C 1.0

Impurity D 1.0
4.6.2 Limit of quantitation:
4.6.2.1. Preparation of impurity stock solution:
20mg, 2.0mg, 2.2mg and 2.2mg of 4-Methyl acetophenone, Impurity-A, Impurity-C and
Impurity-D were accurately weighed individually and transferred into 100ml, 10ml, 10ml and
10ml volumetric flask respectively. Then the volume was made up to the mark using diluent
individually. From each impurity solution 0.75ml was pipetted out and transferred into 50ml
volumetric flask individually. Then the volume was made upto the mark using diluent.

4.6.2.2. Preparation of impurity solution for LOQ:

0.03, 0.02, 0.03 and 0.003ml was pipetted out from the 4-Methyl acetophenone, Impurity-A,
Impurity-C and Impurity-D stock solutions, and transferred into 100ml volumetric flask. Then
the volume was made upto the mark using diluent individually. A series of injections of impurity
solutions for LOQ were injected.

4.6.3. LOQ:
Results of LOQ were reported in the table 4.3. The LOQ values for the impurities was below
reporting threshold (0.05%). The test concentration was optimized as 500PPM.

Table 4.3. LOQ results

Impurity name % LOQ

Celecoxib 0.0001

4-methylacetophenone 0.0041

Impurity-A 0.0111

Impurity-C 0.0083

Impurity-D 0.0111
4.6.4. Linearity:
Linearity was established by plotting a graph between concentration versus peak area and the
correlation coefficient was determined. A series of solutions of Celecoxib related substances
with concentrations ranging from LOQ% to 120% of specification limit prepared and injected
into the HPLC system. Different concentration of Celecoxib and impurities were analysed. A
graph was plotted between concentration and peak area. Correlation coefficient of drugs and its
impurities were above 0.99. The Linearity results were summarized in the table 4.4 & 4.5. The
linearity graphs were shown in figure 4.5. Overlap chromatograms were shown in figure 4.6.

Table 4.4. Linearity data for Celecoxib, Impurity A and C.

Celecoxib Impurity A Impurity C

Mean Conc. Mean± Mean Mean± Mean Conc. Mean±

(µg/mL) SD Conc. SD (µg/mL) SD
(µg/mL)

0.009 3335±9 0.01 3106±7 0.0145 3910±7

0.0253 7237±10 0.0253 35566±87 0.024 4696±8

0.0512 15972±33 0.0511 70836±98 0.049 9806±8

0.083 23792±42 0.081 111882±100 0.079 15240±10

0.1 29223±67 0.1 140434±102 0.099 18939±14

0.12 35177±56 0.12 168862±112 0.122 23603±13

Slope 287036 1468438 185702.5

Intercept 537.0865 -6364.45 705.7122

Correlation
coefficient 0.999188 0.998602 0.999077

Conc. – Concentration; SD – standard deviation

Table 4.5 Linearity data for Impurity D and 4-methyl acetophenone.

 Impurity D 4-methyl acetophenone.

Mean Mean± Mean Mean±

Concentration Standard Concentration Standard
(µg/mL) deviation (µg/mL) deviation

0.015 3990±9 0.0156 3840±9

0.025 4890±9 0.024 4910±9

0.051 10006±12 0.049 9945±11

0.081 15640±15 0.081 15046±12

0.098 19039±35 0.099 18965±34

0.129 24603±41 0.128 24610±21

Slope 185188.7695 184676.8467

Intercept 712.946 678.860

Correlation 0.9992 0.9990

coefficient
Figure 4.5: Linearity curve for celecoxib and its impurities.
 Figure 4.6. Overlap chromatogram of celecoxib and its impurities

4.6.5 Accuracy (% Recovery):

A study of the accuracy of Celecoxib impurities from spiked samples of test preparation was
conducted. Samples were prepared in triplicate at each level by spiking test preparation.

4.6.5.1. Preparation of impurity stock solution:

45.9mg, 4.8mg, 5.1mg and 4.7mg of 4-Methyl acetophenone, Impurity-A, Impurity-C and
Impurity-D were accurately weighed individually and transferred into 100ml, 10ml, 10ml and
10ml volumetric flask respectively. Then the volume was made upto the mark using diluent
individually.

4.6.5.2. Preparation of unspiked and spiked test samples:

Below mentioned in table Samples were prepared in triplicate at each level by spiking test
preparation of Celecoxib related substances and reports were given in the table 4.6 and 4.7.

Table 4.6: Preparation of accuracy test spiked samples

Wt. of Volume of Impurity

Sonicatio Made
the diluent stock
S.No Sample Name n time up to
sample added solution
(min) added(mL) (mL)
(mg) (mL)
1 Unspiked sample 67.6 70 15 N/A 100
100% spiked
2 67.7 70 15 0.3 100
sample-1
100% spiked
3 67.3 70 15 0.3 100
sample-2
200% spiked
4 67.6 70 15 0.6 100
sample-1
200% spiked
5 67.7 70 15 0.6 100
sample-2

Table 4.7: Recovery data for Celecoxib impurities.

Mean % Recovery
S.No Sample Name 4-methyl
Impurity-A Impurity-C Impurity-D
acetophenone
1 Unspiked - - - -
2 100% spiked sample-1 96.4 102.1 104.9 105.9
3 100% spiked sample-2 100.3 104.1 108.5 108.2
4 200% spiked sample-1 102.6 105.4 107.9 107.2
5 200% spiked sample-2 101.0 105.6 107.8 107.2

4.6.7. Precision
4.6.7.1. System Precision:
The system precision of test method was evaluated by analyzing six test preparations by spiking
test preparation with Celecoxib related substances blend solution to get 0.2% of each impurity
with respect to test concentration and analyzed as per test method. Results of system precision
were reported in the table 4.8. The Percentage relative standard deviation of system precision
reports was with in 2. From the results, the method has a good system precision. Chromatogram
of system precision was represented in figure 4.7.

Table 4.8: System precision results

4-methyl
Injection N° Celecoxib Impurity-A Impurity-C Impurity-D
acetophenone.
01 1657882.00 3531.29 3813.13 4310.49 4028.65

02 1708883.00 3616.83 3898.38 4329.68 4230.15

03 1659876.00 3701.52 3798.30 4249.28 4115.68

04 1687986.00 3649.13 3697.31 4227.86 4046.01

05 1693897.00 3720.50 3820.50 4366.29 4052.05

06 1753784.00 3690.26 3755.64 4233.69 4151.00

Mean 1693718.00 3651.59 3797.21 4286.22 4103.92

Standard
32404.45 69.89 67.44 57.30 77.46
deviation

% Relative
standard 1.91 1.91 1.78 1.34 1.89
deviation
 Figure 4.7. System Precision

4.6.7.2. Method Precision:

Method precision results were given in percentage content. The individual results of celecoxib
and its impurities were reported in the table 4.9.
 Table 4.9: Method precision data for Celecoxib and its impurities:

Percentage of 4-methyl

Percentage of impurity

Percentage of impurity C

Percentage of impurity
A present in spiked

D present in spiked
acetophenone present in

present in spiked sample

4-methyl acetophenone.

spiked sample
Impurity-A

Impurity-D
Impurity-C
Celecoxib
Injection

sample

sample
1 97.88 98.17 96.71 100.42 100.57 0.238 0.208 0.225 0.254

2 100.90 103.08 99.05 102.66 101.01 0.250 0.214 0.230 0.256

3 98.00 100.29 101.37 100.03 99.14 0.243 0.219 0.224 0.251

4 99.66 98.59 99.93 97.37 98.64 0.239 0.215 0.218 0.250

5 100.01 98.74 101.89 100.61 101.87 0.239 0.220 0.226 0.258

6 103.55 101.15 101.06 98.91 98.77 0.245 0.218 0.222 0.250

Mean 100.00 100.00 100.00 100.00 100.00 0.242 0.216 0.224 0.253

Standard
deviation 1.91 1.72 1.75 1.62 1.22 0.004 0.004 0.004 0.003

%
Relative
1.91 1.72 1.75 1.62 1.22 1.723 1.747 1.621 1.220
standard
deviation

4.6.8. Stability studies

4.6.8.1.Bench top Stability of 100% spiked samples in Accuracy preparation:
Stability of Celecoxib related substances test preparation, which was spiked with impurities at
target concentration level (i.e., 0.3%) and at ambient temperature about (25ºC) at initial and at 24
hours.
 4.6.8.2. Refrigerator Stability of 100% spiked samples in Accuracy preparation:
Stability of Celecoxib related substances test preparation, which was spiked with impurities at
target concentration level (i.e., 0.5%) at refrigerator temperature (8ºC) at initial and at 24 hours.
Percentage concentrations of mentioned impurities results were reported in the table 4.10

Table 4.10 Stability of 100% spiked sample preparation at ambient temperature about
(25ºC) and refrigerator temperature about (8ºC)

Sample Initial B. top Day-1 Difference Refrigerator Day-1 Difference

Name (%
test_1 test_2 test_1 test_2 test_1 test_2 test_1 test_2 test_1 test_2
concentration)

4-methyl
0.2749 0.2805 0.2656 0.2674 0.01 0.01 0.2650 0.2669 0.01 0.01
acetophenone.

Impurity A 0.2547 0.2643 0.2516 0.2541 0.00 0.01 0.2516 0.2533 0.00 0.01

Impurity C 0.3313 0.3378 0.3289 0.3281 0.00 0.01 0.3280 0.3305 0.00 0.01

Impurity D 0.3334 0.3443 0.3286 0.3306 0.00 0.01 0.3325 0.3284 0.00 0.02

Total impurity 1.1943 1.2269 1.1747 1.1802 0.01 0.04 1.1771 1.1791 0.01 0.05

4.6.9. Robustness
4.6.9.1. Filter Validation:
Two diluted standard preparations were made and filtered through above mentioned filters. Area
ratio was calculated for filtered diluted standard solutions against unfiltered diluted standard
solution and reported in the table 4.11. The 0.45 µm Nylon and 0.45 µm PVDF filters were
suitable for performing related substances test sample preparation

Table 4.11: Robustness for filter variation

 Sample Centrifuge Nylon Difference PVDF Difference
Name (%
test_1 test_2 test_1 test_2 test_1 test_2 test_1 test_2 test_1 test_2
concentration)

Impurity A 0.2547 0.2643 0.2538 0.2656 0.00 0.00 0.2533 0.3162 0.00 -0.05

Impurity C 0.3313 0.3378 0.3332 0.3379 0.00 0.00 0.3314 0.3373 0.00 0.00

Impurity D 0.3334 0.3443 0.3328 0.3403 0.00 0.00 0.3360 0.3451 0.00 0.00

4-methyl
0.2749 0.2805 0.2759 0.2821 0.00 0.00 0.2741 0.2797 0.00 0.00
acetophenone

Total impurity 1.1943 1.2269 1.1957 1.2259 0.00 0.00 1.1948 1.2783 0.00 -0.05

4.6.9.2. Effect of Variation in organic solvent, pH, Column temperature and flow Rate:

The effect of variation in organic solvent, pH, column temperature and flow rate were conducted
with effect of variation in mentioned. Resolution between impurity C and celecoxib & resolution
between impurity A and celecoxib were evaluated and the results were shown in table 4.12.
Results were satisfied with criteria.
 Table 4.12: Robustness data for effect of Variation in organic solvent, pH, Column
temperature and flow Rate

Resolution
Parameter Range Condition
Impurity C Celecoxib and
and celecoxib Impurity A
pH 3.0 Buffer : ACN :
Initial N/A MeOH = 60 : 10 : 30, 2.38 2.00
60℃, 1.3 mL/min
Buffer : ACN :
MeOH
60 : 9 : 30 2.53 2.24
Organic ±10%
60 : 11 : 30 2.25 1.98
60 : 10 : 27 2.40 2.19
60 : 10 : 33 2.19 1.80
2.8 2.32 2.03
Ph ±0.2 3.0 2.37 2.06
3.2 2.33 2.00
55℃ 2.42 2.02
Column
Oven ±5℃ 60℃ 2.38 2.00
temperature
65℃ 2.29 1.98
1.0 2.48 2.06
1.1 2.42 2.04
Flow rate ±0.2mL/min
1.3 2.38 2.00
1.5 2.30 1.95

4.6.10. Specificity:
Blank and Placebo were injected with same chromatographic conditions. Chromatograms of
specificity were present in figure 4.8. From the report of chromatogram, there was no
interference of blank and placebo. Overall results of all parameters expressed the good resolution
of celecoxib from the impurities.
 Figure 4.8: Blank, Placebo and Celecoxib chromatogram for specificity.

4.7. FORCE DEGRADATION STUDIES:

The drug product samples were stressed under conditions and analyzed along with unstressed
drug product using an HPLC equipped with a variable wavelength detector. A reasonable attempt
was made to degrade the drug product but not to exceed 50% of degradation. The interference
from any unknown degradation product to celecoxib peak was checked. In the present study the
celecoxib drug substance was subjected to stress conditions as per ICH guidelines. The study
was performed by subjecting the drug substance to acidic, alkaline, oxidizing, thermal and
photolytic conditions.
4.7.1. Acid hydrolysis
Accurately 49.2mg of celecoxib test sample was weighed into 50ml volumetric flask. It was
dissolved and diluted to volume with diluents. 5ml of this (first solution) solution was pipetted
out and added with 5ml of 3N HCl and kept for 24 hrs at 60o C in a water bath and then it was
neutralized with 5 ml of 3N NaOH. Finally, it was diluted to 100 ml volumetric flask. From this
(second solution) 5 ml of solution was pipetted out and transferred into 100 ml volumetric flask.
Finally, it was diluted to volume with diluents.

No significant degradants have been observed in the acidic conditions and it is shown in figure
4.9

Figure 4.9.Forced Degradation-Acid Stress

4.7.2. Base hydrolysis

Accurately 49.8mg of celecoxib test sample was weighed into 50ml volumetric flask. It was
dissolved and diluted to volume with diluents. 5ml of this (first solution) solution was pipetted
out and added with 5ml of 3N NaOH and kept for 24 hrs at 60 o C in a water bath and then it was
neutralized with 10 ml of 3N HCl. Finally, it was diluted to 100 ml volumetric flask. From this
(second solution) 5 ml of solution was pipetted out and transferred into 100 ml volumetric flask.
Finally, it was diluted to volume with diluents.

No significant degradants have been observed in the basic conditions and it is shown in figure
4.10.
 Figure 4.10. Forced Degradation-Base Stress

4.7.3. Thermal degradation

Accurately 49.8mg of celecoxib test sample was weighed into 100ml volumetric flask. It was
dissolved and diluted to volume with diluents. 5ml of this (first solution) solution was pipetted
out and transferred into 100 ml volumetric flask. Finally, it was diluted to 100 ml volumetric
flask. From this (second solution) 5 ml of solution was pipetted out and transferred into 100 ml
volumetric flask. Finally, it was diluted to volume with diluents. The solution was kept at oven
for 24 hrs at 105oC.

No significant degradants have been observed in the thermal conditions and it is shown in figure
4.11.

Figure 4.11. Forced Degradation-Thermal Stress

4.7.4. Oxidation degradation :
Accurately 49.8mg of celecoxib test sample was weighed into 100ml volumetric flask. It was
dissolved and diluted to volume with diluents. 5ml of this (first solution) solution was pipetted
out and transferred into 100 ml volumetric flask. 5 ml of 10% H2O2 was added. The mixture was
diluted to 100 ml volumetric flask. From this (second solution) 5 ml of solution was pipetted out
and transferred into 100 ml volumetric flask. Finally, it was diluted to volume with diluents. The
solution was kept at oven for 24 hrs at 60oC.

No significant degradants have been observed in the oxidative conditions and it is shown in
figure 4.12.

Figure 4.12.Forced Degradation-Peroxide Stress

4.7.5. Humidity stress studies:

Celecoxib was placed into a humidity chamber under conditions. Accurately 49.8mg of
celecoxib test sample was weighed into 100ml volumetric flask. It was dissolved and diluted to
volume with diluents. 5ml of this (first solution) solution was pipetted out and transferred into
100 ml volumetric flask. Finally, it was diluted to 100 ml volumetric flask. From this (second
solution) 5 ml of solution was pipetted out and transferred into 100 ml volumetric flask. Finally,
it was diluted to volume with diluents. The solution was kept at oven for 7 hrs at 72RH.

No significant degradants have been observed in the humidity conditions and it is shown in
figure 4.13.
 Figure 4.13. Forced Degradation-UV Stress

4.7.6. UV degradation studies:

Celecoxib was placed exposed to near ultraviolet lamp in photostablity chamber providing
illumination of not less than 1.2 million lux hours. Accurately 49.8mg of celecoxib test sample
was weighed into 100ml volumetric flask. It was dissolved and diluted to volume with diluents.
5ml of this (first solution) solution was pipetted out and transferred into 100 ml volumetric flask.
Finally, it was diluted to 100 ml flask. From this (second solution), 5 ml of solution was
pipetted out and transferred into 100 ml volumetric flask. Finally, it was diluted to volume with
diluents.

No significant degradants have been observed in the UV conditions and it is shown in figure
4.14.

Figure 4.14. Forced Degradation-Humidity Stress

4.7.7. Sunlight stress studies:
Celecoxib was placed exposed to sunlight not less than 1 hour. Accurately 49.8mg of celecoxib
test sample was weighed into 100ml volumetric flask. It was dissolved and diluted to volume
with diluents. 5ml of this (first solution) solution was pipetted out and transferred into 100 ml
volumetric flask. Finally, it was diluted to 100 ml volumetric flask. From this (second solution) 5
ml of solution was pipetted out and transferred into 100 ml volumetric flask. Finally, it was
diluted to volume with diluents. No significant degradants have been observed in the sunlight
stress conditions and it is shown in
figure 4.15

Figure 4.15. Forced Degradation-Sunlight Stress

4.7.8. Hydrolysis studies:

Celecoxib was placed exposed to sunlight not less than 1 hour. Accurately 49.8mg of celecoxib
test sample was weighed into 100ml volumetric flask. This solution was added with 10ml of
distilled water and kept for 6 hrs at 60o C in a water bath. Finally it was diluted to 200 ml
volumetric flask. Finally, it was diluted to 200 ml volumetric flask. 5ml of this (first solution)
solution was pipetted out and transferred into 100 ml volumetric flask. Finally, it was diluted to
100 ml volumetric flask. From this (second solution) 5 ml of solution was pipetted out and
transferred into 100 ml volumetric flask. Finally, it was diluted to volume with diluents. No
significant degradants have been observed in the hydrolysis conditions and it is shown in figure
4.16.
 Figure 4.16. Forced Degradation-Hydrolysis Stress

Forced degradation studies reports have shown little deviation in Celecoxib. Purity factor of
Celecoxib by forced degradation studies was mentioned in table 4.13 and 4.14. It was found to
be within the threshold level in all forced degradation studies. Main peak was separated from
known impurity and unknown impurities in forced degradation. Mass balance values were within
the acceptance limit. (NLT 95.0). The peak purity of Celecoxib was passed in all degradation
samples. Celecoxib was very stable in acid, base, oxidation, and thermal condition. Figure 4.14
to 4.21 shows the chromatograms of degradation studies, drug spectra and peak purity factor
graph.

Table 4.13. Forced degradation studies

Celecoxib Purity factor Threshold limit Criteria

Unstressed 999.984 990.000 Accepted
Acid stressed 999.983 990.000 Accepted
Base stressed 999.982 990.000 Accepted
Thermal stressed 999.982 990.000 Accepted
H2O2 stressed 999.980 990.000 Accepted
Humidity stressed 999.881 990.000 Accepted
UV stressed 999.901 990.000 Accepted
Under sunlight 999.907 990.000 Accepted
By Hydrolysis 999.904 990.000 Accepted
 Table 4.14. Forced degradation studies

% of % of Mass
S.No. Celecoxib
Degradation Assay balance

1 Unstressed sample 0.0488 100.01 100.06

2 Acid stressed 0.0879 100.85 100.94

3 Base stressed 0.1598 99.85 100.01

4 Thermal Stressed 0.0438 101.38 101.42

5 H2O2 stressed 0.4084 101.28 101.69

6 Humidity stressed 0.1475 97.50 97.65

7 UV stressed 0.0485 97.45 97.49

8 Under sunlight 0.0671 102.01 102.08

9 By Hydrolysis 0.0891 98.09 98.18

4.8 CONCLUSION
Validation was performed in the developed analytical method for its acceptable performance to
ensure suitability of indent purpose. The validation parameters like accuracy, precision,
specificity, detection limit, quantitation limit, linearity, range, ruggedness and robustness were
executed and established method conditions to meet the requirements to execute the analysis of
celecoxib and its impurities. Under the specificity experiment samples were stressed various
stress conditions and analyzed along with unstressed samples. Celecoxib was found to be very
stable under all degradation conditions . The developed method can be used for routine analysis
because the linearity found in Celecoxib, 4-methyl acetophenone, Impurity A, Impurity C and
Impurity D was approximately equal to 1 that is 0.9991, 0.9986, 0.9990, 0.9992 and 0.9990
respectively which shows the good regression for linearity. The results from solution stability
experiments confirmed that standard and sample solutions were stable up to 24 h for both assay
and related substances analysis. Maximum recovery is obtained by this developed method and
the mean percentage recovery for each component was almost 100%. Data of repeat experiment
were showed <2% RSD (relative standard deviation) for assay and <2% RSD for impurities. In
all the deliberate varied chromatographic conditions like flow rate (±0.2 mL/min), column
temperature (±5°C), composition of organic solvent (±10% of method organic solvent) and pH of
mobile-phase buffer (±0.2), all analyte and impurities were adequately resolved and elution
orders remained unchanged. The resolution between all pair compounds was >2.0. These results
are conforming good precision of the method. 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. Also, our proposed method requires less time for the determination of
Celecoxib and its known impurities simultaneously when compared to other methods. The
developed method is uncomplicated, accurate, sensitive and precise for the determination of
related substances in the Celecoxib. The satisfying percent of recoveries and low percent RSD
Values were confirmed the suitability of the developed method for the usual analysis of
Celecoxib related substances in pharmaceuticals.
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