Alaytical research

Analytical Method Validation of
Stability-Indicating HPLC
Method for Determination of
Assay of Carbamazepine CR
Tablets
Under supervision of:
Dr. Ghada Hadad
Done by:
Ahmed Mohamed El-Gayyar

GRA - GLOBAL RESEARCH ANALYSIS
Volume : 2 | Issue : 11 | Nov 2013 • ISSN No 2277 – 8160
Abstract
This research deals with the developmentand validation of stability
indicating high performance liquid chromatographic method for the
quantitative determination of Carbamazepine CR Tablets form. The
method was developedby using Hypersil ODS V column containing
mobile phase Water: Methanol: Methylene chloride in ratio (30: 30:
3). The flow rate was set at 1.5 mL/minute and the injection volume
was 20μL. The run time of injection is near about 25 min.
Introduction
Carbamazepine is anticonvulsant, which is used to treat seizures in
Epilepsy.It is used to relieve trigeminal neuralgia. Seizure is the
second mostcommonneurological disorderafter stroke that occurs in
humans. It is a white to off-white powderpractically insoluble in water
and soluble in methanol and acetone.
Instruments and Apparatus
The chromatography was performedon a Shimadzu HPLC
instrument equipped with UV detector and LC-solution software,
Mettler Toledo analytical balance and ultrasonic cleaner were used
during the research work. Hypersil ODS V; column was used as
stationary phase. And in mobile phase Water: Methanol: Methylene
Chloride in ratio of 30: 30: 3. The flow rate set to 1.5 ml/minute and
temperature of column 25°C.The injection volume is 20μl and run
time is 25 minute. Shimadzu HPLC instrument equipped with UV
Preparation of Standard Solution

Transfer 200 mg of Carbamazepine WRS into 100mL volumetric
flask. Add 50 ml methanol and sonicate for 5 minutes. Dilute with
methanol, mix and dilute further 5 ml of the solution to 50 ml with
mobile phase until we reach concentration of 200ppm.
Preparation of Sample Solution
20 tablets were weighed and powdered.The powderequivalent to
200 mg Carbamazepine was weighed and transferred in 100 mL
volumetric flask, and add about 50mL of Methanol and sonicate for
15min with intermittent shaking, Coolthe mixture to room
temperature and dilute with Methanol, mix and filter. Again dilute 5mL
of this solution to 50ml volumetric flask and dilute to volume with
Mobile Phase.
Method Validation
The methods were validated in compliance with ICH guidelines.
Accuracy
The accuracy was determined by calculating recoveries of
Carbamzepine by the standard addition method, by spiking
Carbamazepine WRS in placebo at level 80%, 100% and 120% of
concentration in triplicate and then preceded with sample preparation.
The result indicates that the recovery of Carbamazepine from the
sample by the proposed method is satisfactory.
Mean Assay -> 99.01%
Relative standard deviation -> 0.73%
Intermediate Precision (Ruggedness)
The procedure followed for method precision was repeated on a
different day; by different analyst, using a different HPLC system and
different column using same lot of sample. Then we calculated
individual assay value, mean assay value % RSD, and overall %
RSD record.
Method Precision (Repeatability)
The repeatability or reproducibilityof an analytical method refer to en-
ter-assay precision and is expressed as the degree of variation
arising from consecutive and non-consecutive injection analyzed on

the same day. Six replicate injection of the standard preparation were
made into HPLC.
Mean Assay -> 101.36%
Relative standard deviation -> 0.28%
Robustness
The robustness was studied by analyzing the same samples of Car-
bamazepine by variation in the method parameters. The change in
the responses was noted. Robustness of the method was studied by
change in flow rate ± 0.2 ml/min. compositionof mobile phase by ± 5
% of organic solvent, change in pH by ± 0.2 and column oven
temperature by ± 2°C. The parameters used in system suitability test
were asymmetry of the chromatographic peak, peak resolution,
theoretical plates and capacity factor, as RSD of peak area for
replicate injections.
Linearity
Done by preparing a series of standard preparation of
Carbamazepine WRS over the range of 100.50 μg/ml to 301.50 μg/ml
using linearity solution.
Solution Stability
Solution stability was evaluated by injecting initially a freshly prepared
Carbamazepine standard solution and sample solution at different
time intervals.
Results and Discussion
The responses of sample solutions were measured by UV detector
for quantitative of Carbamazepine. The amount of Carbamazepine
present in the sample solutions was determined. The mobile phase
consisting of Water: Methanol: Methylene chloride in ratio (30: 30: 3),
at a flow rate of 1.5 ml/min was found to be satisfactory to obtain
good peak symmetry, better reproducibility and repeatability for
Carbamazepine. Linear correlation was obtained between peak area
and concentration for Carbamazepine in the range of 100.5-301.50

μg/ml. The method was found to be specific as no significant change
in the responses of Carbamazepine was observed after 12 hrs.

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