HPLC REPORT

Instrumentation
To develop a High Pressure Liquid Chromatographic method for quantitative estimation
of Celecoxib an isocratic PEAK HPLC instrument with Zodiac C18 column (250 mm x
4.6 mm, 5) was used. The instrument is equipped with a LC 20AT pump for solvent
delivery and variable wavelength programmable LC 7000 UV-detector. A 20L
Rheodyne inject port was used for injecting the samples. Data was analyzed by using
PEAK software. A DENVER analytical balance is used to weigh the Drug.

Chemicals and Solvents
The reference sample of Celecoxib (API) was obtained from Reddys
Laboratory,Hyderabad. The formulation samples FINAST-5mg were procured from the
local market.
Acetonitrile: HPLC grade, Merck Specialties Private Limited, Mumbai, India.
Methanol: HPLC grade, Merck Specialties Private Limited, Mumbai, India.

Standard solution of the drug
For analysis Celecoxib 100 ppm standard solution was prepared in Mobile phase and
further required concentrations were prepared from 100 ppm solution by proper dilution.

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



METHOD DEVELOPMENT
For developing the method, a systematic study on 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 wavelength, stationary and
mobile phases. The following studies were conducted for this purpose.

Detection wavelength
The UV absorption spectrum of diluted solution of the Celecoxib was recorded on UV
spectrophotometer. The peak of maximum absorbance was observed at a wavelength of
220nm.

4.2. Choice of stationary phase
Preliminary development trials have been performed with columns of different
configurations from different manufacturers. Finally a peak with proper separation from
solvent front and other sample excepients was succeeded using Zodiac C18 column (250
X 4.6 mm, 5m) column.

4.3. 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.




Flow rate
Flow rate of the mobile phase was changed from 0.5 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 experiments that 1.5 ml/min flow
rate was ideal for the successful elution of the analyte.

Optimized chromatographic conditions
Chromatographic conditions as optimized above were shown in Table. These optimized
conditions were followed for the determination of Celecoxib in bulk samples and in its
tablet formulations.




















Table : 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, 5)
Column Temp Ambient
Wavelength

220nm
Injection Volume 20 L
Flow rate 1.5 mL/min

Run time 10 minutes

Retention Time 3.57 minutes










VALIDATION OF THE PROPOSED METHOD


The proposed method was validated as per ICH guidelines.
Linearity

Linearity was performed by preparing standard solutions of Celecoxib at different
concentration levels including working concentration mentioned in experimental
condition i.e. 20ppml. Twenty micro liters of each concentration was injected in into the
HPLC system. The peak responses were read at 220nm and the corresponding
chromatograms were recorded. Linearity plots of concentration over areas were
constructed individually. Linearity results were presented in Table

Linearity results:

Level
Concentration of
Celecoxib
In ppm
peak area
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


Range: 20ppm to 80ppm

Slope
Intercept
Correlation coefficient


4577
-17923
0.998

Calibration Plot for Celecoxib:




Precision:
To study precision, six replicate standard solutions of Celecoxib (80ppm) were prepared
and analyzed using the proposed method. The percent relative standard deviation (%
RSD) for peak responses was calculated and it was found to be 0.715 which is well
within the acceptance criteria of not more than 2.0%. Results of system precision studies
are shown in Table







-50000
0
50000
100000
150000
200000
250000
300000
350000
400000
0 20 40 60 80 100
Precision Results for Celecoxib:
Intraday precision :


Sample

Conc.
(in ppm)
Injection No. Peak Areas
RSD
(Acceptance
criteria 2.0%)
Celecoxib 80
1
368035


1.23


2
366761
3
372462
4
369180
5
365588
6
358893

Interday precision:


Sample

Conc.
(in ppm)
Injection No. Peak Areas
RSD
(Acceptance
criteria 2.0%)
Celecoxib 80
1
362434
1.14
2
361179
3
365395
4
358615
5
353721
6
357338






Limit of Detection and Limit of Quantification:

To determine the Limit of Detection (LOD) sample was dissolved by using Mobile phase
and injected until peak was disappeared. After 0.03ppm dilution Peak was not clearly
observed, based on which 1.0ppm is considered as Limit of Detection and Limit of
Quantification is 3.0 ppm.




Limit of Detection and Limit of Quantification for Celecoxib:
Parameter Measured Value
Limit of Quantification 3.3 ppm
Limit of Detection 1.0ppm



Accuracy:
The accuracy of the method was determined by standard addition method. A known
amount of standard drug was added to the fixed amount of pre-analyzed tablet solution.
The standard addition method was performed at 50%, 100% and 150% level of 20 ppm.
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.
Satisfactory recoveries ranging from to 98%, to 101% were obtained by the proposed
method. This indicates that the proposed method was accurate.



Recovery Results:
Target Concentration----40ug/ml
Concentration
ppm
Area % of
recovery

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