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Sharwaree Hardikar. et al. / International Journal of Biological & Pharmaceutical Research. 2013; 4(1): 30-34.

e- ISSN 0976 - 3651

Print ISSN 2229 - 7480

International Journal of Biological

&
Pharmaceutical Research
Journal homepage: www.ijbpr.com

IJBPR

DEVELOPMENT AND VALIDATION OF RP-HPLC METHOD FOR

DETERMINATION OF CARBAMAZEPINE IN RAT PLASMA
Hardikar Sharwaree*, Undale Vaishali, Saindane Rupesh, Garud Mayuresh
Pune District Education Associations Seth Govind Raghunath Sable College of Pharmacy,
Saswad, Dist. - Pune, India - 412 301.
ABSTRACT
Validation of bioanalytical procedure is a regulatory requirement. Selective and sensitive analytical methods for the
quantitative evaluation of drugs (analytes) are critical for the successful conduct of preclinical studies and as a support to
formulation development. During the course of a typical product development program, a defined bioanalytical method
undergoes many modifications. In the present study, to cater the need of employment of bioanalytical method for
carbamazepine using rat plasma was developed and validated. Present bioanalytical method validation included all of the
procedures that demonstrated that, this method used for quantitative measurement of carbamazepine in rat plasma is reliable
and reproducible for the intended use. The fundamental parameters assessed for this validation were accuracy, precision,
selectivity, sensitivity and reproducibility.
Key Words: Carbamazepine, Rat plasma, Validation.
INTRODUCTION
Carbamazepine (CBZ) is a best-selling
antiepileptic, anticonvulsant drug. Its conventional dosage
forms yield variability in peak plasma concentrations from
4 to 32 hours. This is because of its erratic dissolution
particularly at gastric pH. Hence a lot of work is
undertaken by researchers to improve its dissolution since
it is pharmacodynamic as well as pharmacokinetic demand
of CBZ (Anonymous 1). Many of the RP-HPLC methods
for determination of CBZ in human and rabbit plasma are
reported in the literature. These are useful to researchers in
the later phase of product development. To assist
preliminary research work undertaken; there is need to
develop a method to assess concentration of CBZ in rat
plasma if rabbits are unavailable. Hence, the main
objective of the work was to develop and validate a RP-

HPLC analytical method to determine CBZ concentration

in rat plasma.
MATERIALS AND METHODS
Apparatus
The high performance liquid chromatogram used
was Agilent 1120 Compact LC with UV Detector. Column
used was C-18 column. Other equipments used for this
research work were electronic analytical balance
(Schimadzu, Japan), Vortex mixer and table mount high
speed cold centrifuge.
Reagents
Test substance carbamazepine was obtained as a
gift sample from Cipla Ltd, Mumbai. Chromatographic
pure acetonitrile and water were used for mobile phase. All
other reagents used were of analytical pure grade.

Corresponding Author
Sharwaree Rajan Hardikar
Email: sharwareehardikar@gmail.com

Chromatographic conditions
The chromatographic column used was C-18
column with internal diameter of 2.1 and 150 mm of length
with average particle size of 5 m. Mobile phase used was

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Sharwaree Hardikar. et al. / International Journal of Biological & Pharmaceutical Research. 2013; 4(1): 30-34.

water: acetonitrile (70:30). The column temperature was

kept at 25oC. Flow rate was maintained at 1 mL/min.
Aliquot of 20 L was injected into column for HPLC
analysis. Monitoring was performed at 220 nm (Guidance
for Industry, 2001).
Solution Preparation
Stock solution of CBZ (200 g/mL) was prepared
in a 25 mL amber glass volumetric flask. CBZ working
solutions of concentration 0.25, 0.5, 1.0, 2.5, 5, 10, 15, 20
g/mL were prepared by spiking stock solution
appropriately into rat plasma obtained by procedure
mentioned below. They were all stored away from light
(Dzodi PL et al., 2010).
Treatment to plasma samples
2 mL blood was withdrawn from retro orbital
cavity of Male Sparge Dawlwy rats in 6 X 50 mm glass
test tubes containing disodium edetate. To the blood
sample, fixed amount of CBZ stock solution was spiked,
volume was adjusted to 5 mL by using distilled water and
mixture was vortexed for 30 seconds and then centrifuged
at 4500 rpm for 15 minutes at 4oC. In order to extract the
drug form plasma sample, 0.5 mL of supernatant was
transferred to centrifuge tube and 1.5 mL of mixture of
chloroform and ethyl acetate (1:1) was added, vortexed and
centrifuged at 4500 rpm for 15 minutes at 4 oC. Organic
layer was separated after centrifugation and dried at 60 oC
in water bath. At last, 2000 L acetonitrile was added to
centrifuge tube and 20 L samples was injected for HPLC
analysis. Calibration curves were constructed by analysing
the extracted solutions obtained by procedure mentioned
above. The triplicates of extracted plasma samples with
varying concentrations of CBZ (0.25, 0.5, 1, 2.5, 5, 10, 15
and 20 g/ml) were analysed to construct the calibration
curve (Sachin Ramrao Patil et al., 2012).
RESULTS AND DISCUSSION
Specificity and Selectivity
In order to determine the specificity of current
method, the chromatogram of blank plasma and plasma
sample spiked with CBZ were compared. Figure 1A and
1B represents the chromatograph of blank and plasma
spiked by CBZ respectively. The retention time of CBZ
was found to be 6.37 minutes and no interference of any
plasma component was observed in this range.
Method Validation
Linearity and Sensitivity
Linearity of method was determined by
constructing the calibration curve in triplicate by plotting
area under the curve of peak obtained by analysing various
concentrations of CBZ. Figure 2 represents the calibration
curve of CBZ. The linearity range was found and
confirmed to be from 0.25 to 20.0 g/mL. The regression
equation obtained was y= 4330000x +381373 with

correlation coefficient 0.998. The regression equations

obtained when the procedure was repeated three times are
given in table 1 as standard deviation of slope (Lm) and
intercept (Li) on the ordinate. The linearity of graph was
proved by high correlation coefficient for regression
equation. Limit of detection (LOD) and Limit of
quantitation (LOQ) were also determined from the slopes
of calibration curves by using the equations given below.
LOD =
3.3
S
LOQ =
10
S
where = the standard deviation of the response
S = the slope of the calibration curve
The LOD and LOQ was calculated and reported in table 2.
Accuracy, Precision and Recovery
Accuracy study was carried out to determine the
closeness of the mean test results obtained by the method
to true value. Accuracy is determined by replicate analysis
of samples containing known amounts of the analyte. In
the present work it was confirmed by using three different
concentrations as 5, 10 and 15 g/mL in triplicate. Briefly,
to 10 g/ml solution of CBZ prepared in extracted plasma,
three different concentrations of CBZ 5,10 and 15 g/mL
of CBZ were spiked externally and recovery was
determined. Experiments were performed in triplicate.
Data for accuracy and recovery studies is reported in table
3.
The precision of an analytical method describes
the closeness of individual measures of an analyte when
the procedure is applied repeatedly to multiple aliquots of a
single homogeneous volume of biological matrix.
Precision was assessed by repeated analysis of plasma
specimen containing therapeutic concentration of the drug
being investigated. Repeatability and intermediate
precision of method was confirmed by assessing three
concentrations of CBZ (5, 10 and 20 g/mL) and results
are reported in table 4.Relative standard deviation(RSD)
less than 15% proved accuracy and precision of the
method.
Robustness
The robustness of an analytical procedure is a
measure of its capacity to remain unaffected by small, but
deliberate variations in method parameters and provides an
indication of its reliability during normal usage.
Robustness of current method was determined by changing
the wavelength of detection by 220 2 nm and effect of
change in wavelength on retention time and AUC was
determined. Data for robustness study is shown in table 5.
As the RSD of Retention time and AUC by
changing wavelength were below 15% it was concluded
that method is robust enough to be used to assess in vivo
bioavailability.

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Sharwaree Hardikar. et al. / International Journal of Biological & Pharmaceutical Research. 2013; 4(1): 30-34.

Figure 1A. Chromatograph of blank rat plasma

Figure 1B. Chromatograph of CBZ spiked in rat plasma

Figure 2. Calibration curve of CBZ

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Sharwaree Hardikar. et al. / International Journal of Biological & Pharmaceutical Research. 2013; 4(1): 30-34.

Table 1. Slope and Y-intercept of calibration curves

Slope of Line (Lm)
5.00 X 1006
4.00 X 1006
4.00 X 1006
Table 2. LOD and LOQ of CBZ
Slope of lines
Mean slope
5.00 X 1006
4.33X 1006
4.00 X 1006
06
4.00 X 10

Y-intercept (Li)
72109
1.0 X 1006
2.0 X 1006

Standard Deviation of slopes

LOD(g/mL)

577350.3

LOQ(g/mL)

0.44

1.33

Table 3. Data for accuracy study

% Recovery
CONC.
OF STOCK SOL.*
10
10
10
*All conc. in g/mL

CONC
Of
SPIKED
SOL.*
5
10
15

TOTA
L
CONC
*
15
20
25

AUC

OBSERV
ED
CONC*

%
RECOVER
Y

MEAN
RECOVE
RY

SD

RSD

69930325
86865763
1.08x 1008

16.1
20.05
24.89

107.33
100.25
99.56

102.3811

4.30

4.01

Table 4. Repeatability and Intermediate Precision study

CONC. (g/ml)
AUC1
AUC2
AUC3
MEAN
REPEATABILITY
5
21306544
20445255
20950929
20900909
10
48368568
46565564
48925992
47953375
20
89302903
83749233
87049561
86700566
INTERMEDIATE PRECISION
5
22388027
21306544
22053136
21915902
10
46512623
48368568
46697883
47193025
20
90298695
89302903
87091681
88897760
Table 5. Data for Robustness study
WAVELE
RETENTION
MEAN
NTIME(RT)(min) RT(min)
GTH(nm)
218
6.08
219
6.06
220
6.06
6.11
221
6.16
222
6.18

SD
OF RT

0.06

CONCLUSION
Simple reversed-phase HPLC method for analysis
of the carbamazepine in rat plasma had been developed
and validated. The optimized chromatographic conditions
gave optimum baseline separation. The method was found
to be linear, precise, accurate and sensitive; thus being
suitable for direct analysis of carbamazepine in the
laboratory. The method was found to be robust and will

SD

RSD

432817.7
1233772
2793235

2.070808
2.572858
3.221703

553648.1
1022256
1641445

2.526239
2.166116
1.846441

RSD
OF RT

AUC

MEAN
AUC

SD
OF AUC

RSD OF
AUC

0.94

61540011
64299617
70032643
65424292
59390594

6413743
1

4053236

6.319611

enable quality-control analyst to perform preclinical

bioavailability studies.
ACKNOWLEDGMENTS
The authors are thankful to Tatyasaheb Kore
Institute of Pharmacy, Warna Nagar for the technical
support.

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Sharwaree Hardikar. et al. / International Journal of Biological & Pharmaceutical Research. 2013; 4(1): 30-34.

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and iminodibenzyl in solid dosage form by column high-performance liquid chromatography. J AOAC Int. 2010;
93(4): 1059-68.
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and Drug Administration, Center for Drug Evaluation and Research (CDER), Center for Veterinary Medicine (CVM),
2001.
Sachin Ramrao Patil, Lokesh Kumar, Gunjan Kohli, and Arvind Kumar Bansal. Validated HPLC Method for Concurrent
Determination of Antipyrine, Carbamazepine, Furosemide and Phenytoin and its Application in Assessment of Drug
Permeability through Caco-2 Cell Monolayers. Sci Pharm. 2012; 80(1): 89100.