CHAPTER-IV

CELECOXIB

103
 4.1. INTRODUCTION

Celecoxib is a sulfonamide non-steroidal anti-inflammatory drug

(NSAID) and selective COX-2 inhibitor used in the treatment of osteoarthritis,
rheumatoid arthritis, acute pain, painful menstruation and menstrual
symptoms, and to reduce numbers of colon and rectum polyps in patients
with familial adenomatous polyposis. It works by reducing hormones that
[1]
cause inflammation and pain in the body. Celecoxib was discovered and
developed by G. D. Searle & Company and was approved by the FDA on
December 31, 1998.[2-7]

Figure: 4.A. structure of Celecoxib

Systematic (IUPAC) name :4-[5-(4-methylphenyl)-3 (trifluoromethyl)

pyrazol-1-yl] benzenesulfonamide

Formula : C17H14F3N3O2S

Mol. mass : 381.0

Routes : Oral

Celecoxib is licensed for use in osteoarthritis, rheumatoid arthritis,

acute pain, painful menstruation and menstrual symptoms, ankylosing

104
spondylitis and to reduce the number of colon and rectal polyps in patients
with familialr adenomatous polyposis. It was originally intended to relieve
pain while minimizing the gastrointestinal adverse effects usually seen with
conventional NSAIDs. In practice, its primary indication is in patients who
need regular and long term pain relief; there is probably no advantage to
using celecoxib for short term or acute pain relief over conventional NSAIDs,
except in the situation where non-selective NSAIDs or aspirin cause cutaneous
reactions (urticaria or "hives"). In addition, the pain relief offered by celecoxib
is similar to that offered by paracetamol (acetaminophen).[8] Celecoxib is also
used to treat painful menstrual periods and to relieve other types of short
term pain including pain caused by injuries, surgery and other medical or
dental procedures, or medical conditions that last for a limited time.
Celecoxib is also sometimes used with surgery and other treatments to
reduce the number of polyps (abnormal growths) in the colon (large intestine)
and rectum in patients with familialr adenomatous polyposis (a condition in
which hundreds or thousands of polyps form in the colon and cancer may
develop).

Celecoxib may cause common side effects like diarrhea, gas or

bloating, sore throat, cold symptoms. Some side effects can be serious like
unexplained weight gain, nausea, excessive tiredness, unusual bleeding or
bruising, itching, lack of energy, loss of appetite, pain in the upper right part
of the stomach, yellowing of the skin or eyes, flu-like symptoms, blisters,
fever, rash, hives, swelling of the face, throat, tongue, lips, eyes, hands, feet,
ankles, or lower legs, hoarseness, difficulty swallowing or breathing, pale skin,
fast heartbeat, cloudy, discolored, or bloody urine, back pain, difficult or
painful urination, frequent urination, especially at night.
105
4.2. LITERATURE SURVEY

Several analytical methods have been reported for estimation of celecoxib.

[25]
T. E. G. K. Murthy et al developed a simple, accurate, precise and
reproducible method and validated for the estimation of celecoxib in dosage
forms by RP-HPLC. Chromatography was carried out on an ODS column using
a mixture of 0.2% v/v of glacial acetic acid and acetonitrile (32:68 v/v) as the
mobile phase at a flow rate of 1ml/min. Valdecoxib is used as an internal
standard and the detection was done at 260 nm. The retention times of drug
and internal standard were 14.177 and 4.50 min. respectively. The method
produced linear responses in the concentration range of 2- 20 µg/ml of
celecoxib. The method was found to be applicable for determination of the
drug in capsules as well as in tablets.

[26]
Dhabu PM et al described 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. Eluents were monitored at a wavelength of 251 nm using a mixture
(85:15) of methanol and water. Solution concentrations were measured on a
weight basis to avoid the use of an internal standard. The method was
statistically validated for linearity, accuracy, precision, and selectivity. Due to
its simplicity and accuracy, we believe that the method will be useful for
routine quality control analysis.

[27]
Jayasagar G et al described a simple high performance liquid
chromatographic method using UV detection for the determination of

106
celecoxib, a specific COX 2 inhibitor in serum. Serum samples containing the
internal standard, tolbutamide, are eluted through a C18, Wakosil column.
After extracting with dichloromethane, the eluent is monitored at 250 nm.
The mobile phase comprised of 10 mM potassium dihydrogen ortho
phosphate (pH 3.2) and acetonitrile (50:50 v/v) with a flow rate of 1 ml/min.
Retention times of celecoxib and tolbutamide were 9.6 and 3.5 min,
respectively. The mean absolute recovery value was about 70-80%, while the
intra day and inter day coefficient of variation and percent error values of the
assay method were less than 10%. The calibration curve was linear over a
concentration range of 10-1000 ng/ml.

[28]
Jadhav AS, et al developed a simple, rapid and accurate Reverse Phase
High-Performance Liquid Chromatography (RP-HPLC) method to determine
assay and known impurity of celecoxib API. The chromatographic separation
was performed on reversed-phase C-18 column. Eluents were monitored on
photo-diode array detector at a wavelength of 254 nm using a mixture (40:60)
of buffer and acetonitrile. Solution concentrations were measured on a
weight basis to avoid the use of an internal standard. The method was
statistically validated for forced-degradation study, linearity and range,
accuracy, precision, stability of analytical solutions, and selectivity. Due to its
simplicity, rapid, and accuracy, it is believed that this is useful to determine
assay and known impurity of celecoxib.

107
 [29]
Xu F et al described a high-performance liquid chromatography (HPLC)-
based method for determining celecoxib concentration in the tongue tissue of
hamsters. Celecoxib mixed with the matrix (final concentration of 6%) was
smeared on the surface of the tongue mucosa of hamsters, and the
concentration and absorption rate of celecoxib in the tongue tissue were
determined by HPLC at 5, 10, 15, 30, 60, 90, 120 min after the application. In
this system, the retention time of celecoxib was 4.4 min. Celecoxib
concentration showed a good linear range within 25-800 microg/L
(R2=0.9991, n=6), with the detection limit for celecoxib of 10 g/L (S/N=3). The
extraction recoveries and method recoveries for celecoxib were 83.75%-
90.01% and 91.98%-99.07%, respectively. The inter-day RSDs were 2.15%,
3.16% and 3.67%, and intra-day RSDs were 3.40%, 4.56% and 4.42%,
respectively. The concentration of celecoxib in hamster tongue tissue within
the first 120 min ranged from 0.685-/+0.019 microg/g to 3.168-/+0.143 g/g,
reaching the peak level at 15 min. Celecoxib can be rapidly absorbed through
the tongue mucosa to reach a high concentration in the tongue tissue,
indicating the possibility of oral COX-2 inhibitors to prevent oral cancer and
precancerous lesions.

[30]
Pramod M Dhabu et al developed 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. Eluents were monitored at a wavelength of 251 nm using a
mixture (85:15) of methanol and water. Solution concentrations were
measured on a weight basis to avoid the use of an internal standard. The
method was statistically validated for linearity, accuracy, precision, and

108
selectivity. Due to its simplicity and accuracy, it is believed that the method is
useful for routine quality control analysis.
[31]
J Emami et al described 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
?-Bondapak HPLC column with KH2PO4 (0.01M, pH= 4) - acetonitrile (60:40)
as the mobile phase, at 260 nm. The method involved simple one-step liquid-
liquid extraction procedure with extraction recovery of greater than 90%. The
standard curve covering 0.01-2.0 g/ml concentration range was linear. The
coefficients of variation and relative errors for inter- and intra-day assay
ranged from 5.67 to 9.83 and 0.35 to 7.89 %, respectively. HPLC assay was
performed isocratically on a reversed-phase column with UV detection. By
this method a limit of quantification of 10 ng/ml of a sample size of 0.5 ml is
achieved which is comparable or even better than the reported methods. The
developed method was applied to the analysis of celecoxib levels in plasma
collected from healthy volunteers who participated in a pharmacokinetic
study.
[32]
S. Baboota et al described a simple, economic, selective, precise, and
stability-indicating HPLC method and validated for analysis of celecoxib (CXB),
a selective COX-2 inhibitor, 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−1.
Detection was performed at 250 nm and a sharp peak was obtained for CXB at
a retention time of 4.8 ± 0.01 min. Linear regression analysis data for the
calibration plot showed there was a good linear relationship between

109
response and concentration in the range 0.27–80 µg mL−1; the regression
coefficient was 0.996 and the linear regression equation was y = 48415x +
54359. The detection (LOD) and quantification (LOQ) limits were 0.086 and
0.2625 µg mL−1 respectively. The method was validated for accuracy,
precision, reproducibility, specificity, robustness, and detection and
quantification limits, in accordance with ICH guidelines. Statistical analysis
proved that the method was precise, reproducible, selective, specific, and
accurate for analysis of CXB. The wide linearity range, sensitivity, accuracy,
short retention time, and simple mobile phase imply the method is suitable
for quantification of CXB with high precision and accuracy.

[33]
KG. Jadhav et al described a RP-HPLC method and validated for the
estimation of celecoxib in bulk and pharmaceutical dosage form. 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. The proposed method
has permitted the quantification of celecoxib in the linearity range of
10-100µg/ml and the flow rate was maintained at 1ml/min. The column
was maintained at ambient temperature and the complete separation
was achieved for celecoxib in an overall analytical run time of
approximately 10 minutes. The retention time of celecoxib was found to be
3.2 minutes. The limit of detection and limit of quantification were
found to be 0.69 and 2.12 µg/ml, respectively. The percentage recovery
was found to be in between 99.99 to 100.41. The method was found to
be suitable for the routine quality control analysis of celecoxib in bulk
drug and formulation. The method was validated as per ICH guidelines.

110
 [34]
Ayman K. Hamama et al developed an innovative reversed-phase high-
performance liquid chromatographic method for the simultaneous
determination of rofecoxib and celecoxib in human plasma. The internal
standard was 4-n-pentyl-phenyl-acetic acid. Good chromatographic
separation was achieved using a Zorbax SB-CN (5 µm) analytical column
operated at room temperature and mobile phase consisting of acetonitrile
and water containing 0.1M potassium dihydrogen orthophosphate buffer
adjusted to pH 2.4 with 85% orthophosphoric acid (42:58, v/v). UV detection
is performed at 254 nm, and the flow rate is maintained at 1.0 mL/min.
Plasma samples are extracted into an organic solvent (1-chlorobutane) and
evaporated under an air flow. The calibration curve for rofecoxib was linear
over the range of 10 to 500 µg/L, and the celecoxib calibration curve was
linear over the range of 20 to 2000 µg/L. The lower limit of quantitation for
rofecoxib and celecoxib is 10 and 20 µg/L, respectively, using 1.0 mL of human
plasma. The validation data show that the assay is sensitive, accurate, specific,
and reproducible for the determination of rofecoxib and celecoxib. This
method was therefore considered for appropriate pharmacokinetic studies to
quantitate these therapeutic agents in patients with arthritis conditions.

4.3. EXPERIMENTAL

4.3.1. Instrumentation
Chromatographic separation was performed on a PEAK
chromatographic system equipped with LC-P7000 isocratic pump; Rheodyne
injector with 20μl fixed volume loop, variable wavelength programmable UV
detector UV7000 and the output signal was monitored and integrated by
PEAK Chromatographic Software version 1.06. ElicoSL 159 UV-2301 double
beam UV-Visible spectrophotometer was used to carry out spectral analysis
111
and the data was recorded by Hitachi software. Sonicator (Loba-1.5L)
ultrasonicator was used to sonicating the mobile phase and samples. Standard
and sample drugs were weighed by using Denver electronic analytical balance
(SI-234) and pH of the mobile phase was adjusted by using Systronics digital
pH meter.

4.3.2. Chemicals and Solvents

The reference sample of celecoxib (API) was obtained from Dr. Reddy’s
laboratories, Hyderabad. The Formulation was procured from the local
market. Acetonitrile, methanol, water used were of HPLC grade and
purchased from Merck Specialties Private Limited, Mumbai, India.

4.3.3. The mobile phase

The HPLC grade solvents were used for the preparation of mobile
phase. Mobile phase was prepared by mixing methanol: acetonitrile
60:40(v/v). This mobile phase was filtered through 0.45μ membrane filter and
then it was ultrasonicated.

4.3.4. Standard solution of the drug

About 10 mg of each reference standard was accurately weighed and

transferred to 100 ml volumetric flask. Drug was dissolved in 50 ml of mobile
phase with shaking and then volume was made up to the mark with mobile
phase to get 100µg/ml of standard stock solution of each drug. Then it was
ultra sonicated for 10 minutes and filtered through 0.45μ membrane filter.
Solutions of different concentrations were prepared by diluting the above
stock solution to get the required concentration of the solution.

112
4.3.5. Sample (tablet) solution
The formulation tablets of celecoxib were crushed to give finely powdered
material. From the powder prepared 20µg/ml solution in Mobile phase and
then filtered through Ultipor membrane sample filter paper.

4.4. METHOD DEVELOPMENT

For developing the method, a systematic study of the effect of various

factors was undertaken by varying one parameter at a time and keeping all
other conditions constant. Method development consists of selecting the
appropriate wave length and choice of stationary and mobile phases. The
following studies were conducted for this purpose.

4.4.1. Detection wavelength

The spectra of diluted solutions of the celecoxib in methanol were
recorded separately on UV spectrophotometer. The peak of maximum
absorbance wavelength was observed. The spectra of the both celecoxib were
showed that a wavelength was found to be 220nm.

4.4.2. Choice of stationary phase

Preliminary development trials have performed with octadecyl
columns with different types, configurations and from different
manufacturers. Finally the expected separation and shapes of peak was
succeeded with Zodiac C18 column (250 X 4.6 mm, 5μ).

4.4.3. Flow rate

Flow rates of the mobile phase were changed from 0.6 – 1.5 mL/min
for optimum separation. A minimum flow rate as well as minimum run time
gives the maximum saving on the usage of solvents. It was found from the
113
experiments that 1.5 mL/min flow rate was ideal for the successful elution of
the analyte.

4.4.4. Selection of the mobile phase

In order to get sharp peak with base line separation from interfering peaks
carried out a number of experiments by varying the composition of solvents
and mobile phase flow rate. To have an ideal separation of the drug under
isocratic conditions, mixtures of solvents like methanol, water and acetonitrile
with or without different buffers in different combinations were tested as
mobile phase. A mixture of methanol : acetonitrile 60:40(v/v) was proved to
be the most suitable of all the combinations, since the chromatographic peak
obtained was better defined and resolved and almost free from tailing.

4.4.5. Optimized chromatographic conditions

A mixture of methanol : acetonitrile 60:40(v/v) with C18 column was
proved to be the most suitable of all the combinations since the
chromatographic peak obtained was better defined and resolved and almost
free from tailing. Chromatographic conditions as optimized above were
shown in Table 4.1 These optimized conditions were followed for the
determination of celecoxib in bulk samples and its combined tablet
formulations. The chromatograms of blank, standard and sample were shown
in Figure 4.B, 4.C and 4.D.

114
Table.4.1 : Optimized chromatographic conditions for estimation of
celecoxib

Mobile phase Methanol :Acetonitrile 60:40(v/v)

Pump mode Isocratic

pH 5.2
Diluents Mobile phase

Column Zodiac C18 column (250 X 4.6 mm, 5μ)

Column Temp Ambient
Wavelength 220nm
Injection Volume 20 μL
Flow rate 1.5 mL/min
10 minutes
Run time
3.57 minutes
Retention Time

Figure: 4.B Blank chromatogram

115
Figure: 4.C Standard chromatogram

Figure: 4.D Sample chromatogram

116
4.5. VALIDATION OF THE PROPOSED METHOD

The method was validated for system suitability specificity, linearity,

range, precision, accuracy, sensitivity and robustness as per ICH guidelines.

4.5.1 System Suitability

System suitability tests were carried out on freshly prepared standard
stock solutions of celecoxib and it was calculated by determining the
standard deviation of celecoxib standards by injecting standards in five
replicates at 6 minutes interval and the values were recorded. System
suitability results of celecoxib were shown in Table 3.2

Table: 4.2. System suitability results

System suitability Parameter Result
Retention time 3.57min
Area 77858
Theoretical plates 7387
Tailing factor 1.53

4.5.2. Specificity
The selectivity of an analytical method is its ability to measure
accurately and specifically the analyte of interest in the presence of
components that may be expected to be present in the sample matrix. If an
analytical procedure was able to separate and resolve the various
components of a mixture and detect the analyte qualitatively the method
became a selective method. It has been observed that there were no peaks of
diluents and placebo at main peaks. Hence, the chromatographic system used
for the estimation of celecoxib is very selective and specific. Specificity studies
indicating that the excipients did not interfere with the analysis.
117
 Table 4.3: Specificity study
Name of the solution Retention Time
Blank No peaks
Celecoxib 3.57min

4.5.3. Linearity
Linearity test solutions for the assay method were prepared from stock
solution at five concentrations were prepared and injected. From the peak
area responses and concentration data plots, the correlation coefficient
values were obtained greater than 0.999. The best fit linear equation
obtained. The proposed method was evaluated by its correlation coefficient
and intercept value calculated by statistical study. They were represented by
the linear regression equation (fig-4.E)

Table 4.4: Linearity results

Concentration of peak area
Level celecoxib
In µg/ml
Level - 1 20 77858
Level - 2 30 114242
Level - 3 40 165434
Level - 4 50 206673
Level - 5 60 264737
Level - 6 70 298931
Level-7 80 348893

118
 Range: 20µg/ml to Slope 4577
80µg/ml Intercept -17923
Correlation coefficient 0.998

400000

350000

300000

250000

200000

150000

100000

50000

0
0 20 40 60 80 100
-50000

On X axis concentration of sample, On Y axis peak area response

Fig: 4.E: calibration curve of lineary
4.5.4. Precision

Precision is the degree of repeatability of an analytical method under

normal operational conditions. Precision of the method was performed as
intraday precision, Inter day precision.

4.5.4.A. Intra-day precision

To study the intra-day precision, six replicate standard solutions
(80µg/ml) of celecoxib was injected. The percent relative standard deviation
(% RSD) was calculated and it was found to be 1.23, which are well within the

119
acceptable criteria of not more than 2.0. Results of system precision studies
were shown in Table 4.5.

Table 4.5: Intra-day precision results

Conc. RSD
Injection Peak
Sample (in (Acceptance
No. Areas
µg/ml) criteria ≤ 2.0%)
1 368035
2 366761
3 372462
Celecoxib 80 1.23
4 369180
5 365588
6 358893

4.5.4.B. Inter Day precision

To study the inter day precision, six replicate standard solution
(80µg/ml) celecoxib was injected on third day of sample preparation. The
percent relative standard deviation (% RSD) was calculated and it was found
to be 1.14 which were well within the acceptable criteria of not more than
2.0. Results of system precision studies are shown in Table 4.6

4.5.5. Ruggedness
Ruggedness was performed by using six replicate injections of standard
and sample solutions of concentrations which were prepared and analyzed by
different analyst on three different days over a period of one week.
Ruggedness also expressed in terms of percentage relative standard
deviation.

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 Table 4.6: Inter Day precision
Conc. RSD
Injection Peak
Sample (in (Acceptance
No. Areas
µg/ml) criteria ≤ 2.0%)
1 362434
2 361179
3 365395
Celecoxib 80 1.14
4 358615
5 353721
6 357338

Table: 4.7: Ruggedness results

RSD
Conc. Injection (Acceptance
Sample Peak Areas
(in µg/ml) No. criteria ≤
2.0%)
1 363289
2 362558
3 361009
Celecoxib 80 0.387
4 360996
5 364553
6 363297

121
4.5.6. Robustness
Robustness of the method was checked by varying the instrumental
conditions such as organic content in mobile phase ratio (± 2%), wavelength
of detection (± 5 nm), and change in pH of mobile phase (± 0.2). Sample
solution was injected in each condition and assayed for celecoxib.

Table: 4.8: Robustness results

S.NO Parameter Change Area % Of
Change
1 Standard ……….. 348893 …………
2 Mobile phase Methanol:Acetonitrile 345669 0.93
60:40(v/v)
3 Methanol:Acetonitrile 348590 0.09
60:40(v/v)
4 Wavelength 222nm 346007 0.83
5 218nm 347780 0.32
6 Mobile phase 5.4 348095 0.23
7 PH 5.0 349568 0.19

4.5.7. Accuracy:
Accuracy was assessed by determination of the recovery of the method
at three different concentrations (corresponding to 30, 40 and 50% of test
solution concentration) by addition of known amounts of standard to placebo
preparation. The solutions were analyzed in triplicate at each level as per the
proposed method. The percent recovery and % RSD was calculated and
results are presented in Table 4.9. Satisfactory recoveries ranging from 98.4 to
100.8% were obtained by the proposed method. The values of recovery justify
122
the accuracy of the method. The % recovery values were obtained within the
standard limit which confirms that the method is accurate and free from any
positive or negative interference of the excipients. This indicates that the
proposed method was accurate.
4.5.8. Limit of detection and Limit of quantification
To determine the Limit of Detection the sample was dissolved by using
mobile phase and injected until peak was disappeared. After 1.0µg/ml
dilution, Peak was not clearly observed. So it confirms that 1.0µg/ml is limit of
detection and limit of quantification found to be 3.3µg/ml.

Table: 4.9: Recovery results

Concentration Area % of
ppm recovery RSD
50% 30 114601 100.3
30 115119 100.7 1.22
30 112498 98.4
100% 40 166777 100.8
40 162392 98.1 1.35
40 165066 99.7
150% 50 205844 99.5
50 203728 98.5 1.20
50 208709 100.9
Mean:99.65 Mean:1.25

123
 Table 4.10: LOD and LOQ results
Parameter Measured Value
Limit of Quantification 3.3 ppm
Limit of Detection 1.0ppm

4.5.9: Formulation:
For assay of celecoxib (COBIX-200mg ) 20 capsules were weighed and
calculated the average tablet weight. The sample of about 10mg powdered
tablets was accurately weighed and transfered into a 10mL volumetric flask.
5mL of mobile phase was added and sonicated well to completely dissolve the
drug and finally made the volume up to the mark with the same diluent.
Mixed well and filtered the solution through 0.45µ filter. Further pipetted
0.2mL of the above solution into a 25mL volumetric flask and diluted up to
mark with the diluent to get a final solution of 20ppm concentration. An
aliquot of this solution was injected into HPLC system. The peak area of
celecoxib from three independent samples was measured and compared
against the peak area of the standard solution. The proposed method was
able to estimate celecoxib in the tablet formulation with an accuracy of
99.9%.

Table 4.11: LOD and LOQ results

Name Dosa Sample Tab Avg Amount % Of Drug
ge Conc Wehigt Drug Estimated
Estimated
Celecoxib 200 20 460 mg 41.69 96.95
mg ppm/ml

124
4.6. DISCUSSION ON THE RESULTS

In this work an analytical HPLC method for assay and

determination of content uniformity of celecoxib in a tablet formulation was
developed and validated. The basic chromatographic conditions were
designed to be simple and easy to use and reproduce and were selected after
testing the different conditions that affect HPLC analysis, for example column,
aqueous and organic components of the mobile phase, proportion of mobile
phase components, detection wavelength, diluents and concentration of
analyte. The on Zodiac C18 column (250 X 4.6 mm, 5μ), column was used
because of its advantages of high resolving capacity, better reproducibility,
low-back pressure, and low tailing. The proportion of the mobile phase
components was optimized to reduce retention times and enable good
resolution of celecoxib from the degradation products. A detection
wavelength of 220 nm was selected after scanning the standard solution over
the range 200-400 nm by use of the UV detector. Detection at 220nm resulted
in good response and good linearity.

To determine linearity a calibration graph was obtained by

plotting celecoxib concentration against peak area. The method was linear in
the range of 20–80 µg/ml for celecoxib concentration with a correlation co-
efficient 0.998. The result was shown in Table 4.2, Figures 4.E. The accuracy of
the method was assessed by determination of recovery for three
concentrations covering the range of the method. The amount of celecoxib
was recovered, in the presence of placebo interference, was calculated. The
mean recovery of celecoxib was 98.4 to 100.8% respectively which is
satisfactory and result were shown in Table 4.9. Precision of the method were
done in the % RSD for repeatability was found 1.23 and Intermediate
125
Precision was found 1.14. The method precision was found 0.387. From the
data obtained the developed RP-HPLC method was found to be precise. The
accuracy of the method was assessed by determination of recovery for three
concentrations covering the range of the method.

The robustness of the method was assessed by assaying test solutions

under different analytical conditions deliberately changed from the original
conditions. For each different analytical condition the standard solution and
test solution were prepared separately. The result obtained from assay of the
test solution was not affected by varying the conditions and was in
accordance with the true value (Table 4.8). System suitability data were also
found to be satisfactory during variation of the analytical conditions (Table
4.2). The analytical method therefore remained unaffected by slight but
deliberate changes in the analytical conditions. This furnished evidence that
the method was suitable for its intended purpose. The specificity of the
method was determined by checking for interference with the drug from
placebo components. The specificity of the method was also evaluated by
checking the peak purity of the analyte peak during the forced degradation
study were shown (Table 4).

The intensive approach described in this manuscript was used to

develop and validate a liquid chromatographic analytical method that can be
used for both assay and determination of content uniformity of celecoxib in a
pharmaceutical dosage form. This HPLC method for assay and determination
of content uniformity of celecoxib in a tablet formulation was successfully
developed and validated for its intended purpose. The method was shown to
specific, linear, precise, accurate, and robust, because there is no
pharmacopeial method for assay and determination of content uniformity of
126
 celecoxib in pharmaceutical dosage forms, this method is recommended to
the industry for quality control of drug content in pharmaceutical
preparations.

4.7. REFERENCES

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