Sanjeeva Yarkala et al. Journal of Scientific Research in Pharmacy

Madhuri Sadu et al., J. Sci. Res. Pharm. 2012, 1(1), 10-12
Journal of Scientific Research in Pharmacy
Research Article
Available online throug h
www.jsrponline.com
Estimation of Carbamazepine with Different Forms of Cyclodextrins in Pharmaceutical Preparation by
RP-HPLC
Madhuri Sadu1*, Sanjeeva Yarkala1, Vijay Kumar. R 2, and Sameer G. N avalgund1
1Analytical Chemistry Department, ISP India (P) Limited, Somajiguda, Hyderabad – 500 082, INDIA
2. Bright Labs, Kothapet, Dilsukhnagar, Hyderabad, INDIA
Received on: 02-02-2012; Revised on: 25-02-2012; Accepted on: 02-03-2012
ABSTRACT
A new simple, precise, rapid and selective reversed -phase high performance liquid chromatography (HPLC) method has been described for
the determination of assay of carbamazepine (CBZ) with different forms of cyclodextrins in 1:1 mole ratio. The HPLC method depends on the separation
of components of binary mixtures using Zorbax C18 coloumn with mobile phase consisting of water and methanol (40:60% v/v) wit h detection at 285
nm. A part from the simple blends analysis, and also the microwave mixtures were analyzed for CBZ content. The HPLC assay of CBZ was found to be 98
– 102% in all the binary mixtures of alpha, beta, gamma and HP beta cyclodextrins. The accuracy and precision of the method has been determined and
they have been validated by with pure drug analysis.
Keywords: Carbamazepine; Alpha, Beta, Gamma, Hydroxypropyl, Beta Cyclodextrins and RP -HPLC.
INTRODUCTION
Carbamazepine (CBZ) is an anticonvulsant [1] and mood
stabilizing drug, used primarily in the treatment of epilepsy and bipolar
disorder.
Carbamazepine is ideal as a model compound for poorly
soluble drugs, see Fig. 1. It has an aqueous solubility of 0.1 mg/ml, and is
known to complex with cyclodextrins including alpha (α-cyclodextrin),
beta (β-cyclodextrin,), and gamma (γ-cyclodextrin) and hydroxypropylβcyclodextrin [2]. The interaction between carbamazepine and
cyclodextrins is reported to be 1:1 inclusion mixtures [2-5]. Therefore CBZ
samples were micro waved (MW) and physically mixed (PM) with
cyclodextrins (CDs) in 1:1 M ratio. Micro waved and physical mixture
samples were analyzed to determine the assay by HPLC method.
Fig.1: Carbamazepine Chemical Structure
A proper validated HPLC method was developed for the
determination of Carbamazepine in the physical mixtures of
Carbamazepine with Cyclodextrins in 1:1 M ratio to assess the effect of
cyclodextrins on the release of poorly soluble model drug
carbamazepine.
MATERIALS AND METHODS
MATERIALS:
Carbamazepine was purchased from Benzo Chem. Life
Sciences Pvt Ltd. α-CD (Cavamax W6 Pharma), β-CD (Cavamax W7
Pharma), γ-CD ((Cavamax W8 Pharma) and HPβCD (Cavitron W7 HP
Pharma) were obtained from Hyderabad Laboratory, ISP (India) PVT
LTD. All other chemicals and reagents used were of analytical or
Pharmacopeial grade and were used as supplied.
1. Alpha Cyclodextrin: CBZ 1:1M contains 4.358gm of Alpha-CD and
0.642gm of CBZ in 5gm.
2. Beta Cyclodextrin: CBZ 1:1M contains 4.14gm of Beta CD and 0.86gm of
CBZ in 5gm.
3. Gamma Cyclodextrin: CBZ 1:1M contains 4.23gm of Gamma CD and
0.769gm of CBZ in 5gm.
4. Hydroxy Propyl Beta Cyclodextrin: CBZ 1:1M contains 4.02gm of
Hydroxy Propyl Beta CD and 0.976gm of CBZ in 5gm.
*Corresponding author:
Madhu Sadu
Analytical Chemistry Department,
ISP India (P) Limited, Somajiguda, Hyderabad – 500 082,
INDIA
*E-mail:madhu.sadu@gmail.com
Preparation of Physical mixtures:
The physical mixtures of the Drug (CBZ) and each of the excipients ( αCD, β-CD, γ-CD and HPβCD) in 1:1M were prepared by mixing the
suitable given weights of pulverized powders by using mortar and pistol.
CBZ content in the mixtures was further assayed by a HPLC Method at
285 nm.
Preparation of Micro waved mixtures:
Accurately weighed given quantities of drug with respective
cyclodextrins and transfer into a glass beaker. Added 20ml of water into
the glass beaker containing cyclodextrin and the drug. Then the beaker
was kept inside the microwave oven for 2min. Added 1ml of water for
each cycle per minute until to get the clear solution. After getting clear
solution, evaporated the water by using microwave oven to get the yield.
CBZ content in the mixtures was further assayed by a HPLC method at
285 nm.
METHODS:
Chromatographic System: A Waters, High Performance liquid
chromatographic system, separation module (Model No. 2690),
consisting of an auto injector and pump (configured to continuous
vacuum) linked to a PDA detector was used. Data was acquired and
processed by using Empower software from Waters. Separation was
achieved by using a Zorbax Eclipse XDB-C18 250 X 4.6 mm, 5 µ (990967906) by Waters.
EXPERIMENTAL ANALYSIS
Standard Preparation:
Ten mg of Carbamazepine working standard was weighed
accurately and transferred to a 100 mL standard volumetric flask. To this
about 20 mL of me thanol was added and mixed well. The solution was
then diluted up to the mark with methanol and mixed well. This yielded a
working standard solution with nominal concentration 100 µg/mL of
Carbamazepine. This working standard solution was analyzed using the
HPLC conditions mentioned above Standard chromatogram (Fig.2).
Internal standard preparation:
Cyclodextrins sample solution preparation (As Control): Accurately
about ten mg each of all Cyclodextrins namely α-CD, β-CD, γ-CD and HPβCD was weighed separately and each was transferred to a separate 100
mL volumetric flask. To each of them about 20 mL of water was added
and mixed well. These solutions were then diluted up to the mark with
water and mixed well. This yielded separate control sample solutions for
all the CDs. These were analyzed using the HPLC method and the
cyclodextrins chroma togram was shown in Fig.3.
Sample preparation:
Micro waved and Physical mixture sample solutions preparation (1:1
M Ratio):Accurately about ten mg each of the sample was weighed and
transferred to a 100 mL volumetric flask. To this about 10 mL of
methanol was added and mixed well. The solution was then diluted up to
the mark with water and mixed well. This yielded the sample solutions.
These were analyzed using the mentioned HPLC method and the sample
chromatogra m was shown in Fig.5.
Journal of Scientific Research in Pharmacy 2012, 1(1)
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Madhuri Sadu et al., J. Sci. Res. Pharm. 2012, 1(1), 10-12
Assay Experiment of CBZ with CD’s: A HPLC method was validated
following ICH guidelines for the quantitative analysis of CBZ with CDs
released during assay analysis. Filtered samples were injected (5µl) in a
Waters (Model No.2690) HPLC coupled to a PDA, A Photo Diode Array
detector. The analytical system consisted of a octadecyl column (Zorbax
Eclipse XDB-C18 250 X 4.6 mm, 5 µm), mobile phase: 60% Methanol and
40% Water, flow rate of 1.0 ml/min. Retention time of CBZ with CDs was
about 7.3 min. Standard curves of CBZ were linear (r2 . 0:999) over the
examined concentration ranges of CBZ: 0.001 - 0.2 mg/ml. Detection and
Quantification limits were determined based on the standard deviation
of the response and the slope of the calibration curve, and they were
found to be 0.0001 and 0.0004 mg/ml, respectively.
Linearity/Range:
Nine solutions were prepared for the linearity test containing
different concentrations of CBZ in the range of 0.001 - 0.2 mg/ml. Each
solution was injected three times and linear regression analysis of CBZ
nominal concentration versus measured concentration was calculated.
The results showed that the method was linear in the range studied and
yielded a correlation coefficient of 0.9999.
RESULTS AND DISCUSSION
HPLC Method Development and Qualification:
Columns with different stationary phases (octyl silica,
octyldecyl silica, and phenyl substituted) were assessed to develop a
method with acceptable separation, reproducibility, and accuracy [6].
The column mentioned in the ‘‘Methods’’ section provided excellent
separation of the active drug substance without any associated
impurities, and degradants. A representative chromatogram, a s shown in
Fig.2, shows the chromatogram of the standard CBZ with retention time
(RT) of around 7.3.
Fig.4: Calibration curve for Linearity
System/Injection Precision:
The system precision was examined by analyzing six
determinations of the same test concentration of CBZ (0.1mg/ml). The
relative standard deviation of the areas of each peak was found to be less
than 2.0% [7] (Table 2).
Method Precision:
The precision of the method was a ssessed by determining the
R.S.D. values of the analysis of 6 different preparations of the same test
concentration of CBZ (0.1mg/ml) of the oral liquid containing 100% of
the theoretical values of the active ingredient and the preservatives. The
R.S.D. values were found to be less than 1.0% [7] (Table 2), which
demonstrates good precision of the me thod
Fig.2: Representative chromatogram for Standard Carbamazepine
(CBZ).
A representative chromatogram, as shown in Fig.3, shows the
chromatogra m of the Cyclodextrins without any peak.
.
.Precision
Table 2: Method precision for CBZ
Injection
Method precision
reproducibility
Limit
RSD
Limit
Average
RSD
0.28%
98102
100.00
0.96%
Carbamazepine
2.00
Accuracy:
To assess accuracy, fresh standard solutions were prepared,
at about 80%, 100%, and 120% of the targeted concentration of CBZ
(0.1mg/ml). Each of the prepared solutions was injected in triplicate and
the peak areas were used to calculated mean and %R.S.D. values and
compared with those obtained with standard solutions. The percentage
recovery of all three solutions was greater than 100.0% as shown in
Table 3.
Table 3: Accuracy for CBZ
Fig.3: Representative chromatogram for Cyclodextrins (CDs).
This HPLC method was qualified by assessing system
Suitability, LOD, LOQ and Linearity, Precision, Accuracy.
System Suitability:
The %CV of peak area and retention time for the drug is
within 2% [7] indicating the suitability of the system (Ta ble 1).
Table 1: System Suitability of Carbamaz epine
S.No
Retention time
Peak Area
1
7.329
1481611
2
7.33
1488451
3
7.322
1494805
4
7.328
1490347
5
7.325
1489113
6
7.321
1490364
Average
7.325833
1489115
STD.Dev
0.003764
4294.295
% RSD
0.051378
0.288379
LOD & LOQ:
The results indicated that the method was sensitive enough
to detect a concentrati on of 0.0001 mg/ml and able to quantify at a
concentration of a bove 0.0004 mg/ml.
% of
Conc.
Resultant
solution
(ppm)
Area
counts
80%
80
1150888
80%
80
1150132
80%
80
150498
100%
100
1400278
100%
100
1404354
100%
100
1402354
120%
120
1693561
120%
120
1704556
120%
Average
120
1707970
SD
Average
SD
%
Recovery
1150506
378.0
6
103.17
1402328
2038.
1
100.60
1702029
7529.
5
101.75
101.84
1.05
%RSD
1.0
Application of the method to Pharmaceutical mixtures:
The HPLC method developed is sensitive and specific for the
quantitative determination of CBZ. Also, the method is validated for
Journal of Scientific Research in Pharmacy 2012, 1(1)
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Madhuri Sadu et al., J. Sci. Res. Pharm. 2012, 1(1), 10-12
different parameters, hence has been applied for the estimation of drug
in pharmaceutical mixtures. CBZ of 10 mg strength from the different
mixtures (Physical mixtures and Micro waved mixtures of CBZ with
different forms of CDs, ISP INDIA Pvt. Ltd., Hyderabad, India) were
evaluated for the amount of CBZ present in the mixtures. Each sample
was analyzed in triplicate after extracting the drug as mentioned in the
sample preparation of the experimental section. The amount of CBZ
present in ‘Physical mixtures’ and ‘Micro waved mixtures’ was
mentioned in the Table 4. None of the mixtures was interfered with the
analyte peak as seen in Fig. 4. The spectrum of CBZ extracted from the
mixtures was matching with that of standard CBZ showing the purity of
peak of CBZ in both the mixtures.
Table 4: Assay values for Carbamazepine w ith CDs
Serial No.
Sample Details
% Assay
1
CBZ
100.00
2
α-CD
NA
3
β-CD
NA
4
γ-CD
NA
5
HP β-CD
NA
6
Micro waved CBZ
98.81
7
Micro waved CBZ + α-CD
101.34
8
Micro waved CBZ + β-CD
111.28
9
Micro waved CBZ + γ-CD
111.13
10
Micro waved CBZ + HP β-CD
106.70
11
Physical mixture CBZ + α-CD
97.82
12
Physical mixture CBZ + β-CD
98.73
13
Physical mixture CBZ + γ-CD
98.44
14
Physical mixture CBZ + HP β-CD
95.93
Note: NA: Not Applicable, CBZ: Carbamazepine, CD: Cyclodextrin
Fig.5: Representative chromatogram for the Sample (Mixture of CBZ
& CDs)
CONCLUSION:
A rapid, specific isocratic HPLC method has been developed
for the determination of Carba mazepine in the Physical and Micro waved
mixtures of Cyclodextrins using a PDA detector. The me thod was
validated for system suitability, LOD & LOQ, Linearity, Precision and
Accuracy. The method uses a si mple mobile phase composition, easy to
prepare with little or no variation. The rapid run time of 15 min and the
relatively flow rate (1.0 ml/min) allows the analysis of large number of
samples with less mobile phase that proves to be cost-effective.
The CDs were analyzed to check for any interference in the
HPLC analysis, specifically at the retention time of Carbamazepine. No
peaks were observed at the retention time of Carbamazepine for the
analysis of the CDs, indicating that they did not show any interference
when analyzed individually. The typical chromatograms for the CDs were
found to be similar to that of the blank chroma togram. Thus it may be
concluded that the CDs may not contribute to the assay values for
Carbamazepine in the samples. Hence, this method can be successfully
applied for the identification, quantitative analysis of Carbamazepine in
the Physical and Micro waved mixtures of Cyclodextrins.
The assay of the micro waved and physical mixture samples
were found to be in the range of 95.9 % to 111.3 %.
ACKNOWLEDGEMENTS
This work was supported by ISP (INDIA) PVT LTD located at
Hyderabad.
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Source of support: Nil, Conflict of interest: None Declared
Journal of Scientific Research in Pharmacy 2012, 1(1)
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