Madhuri Sadu et al., J. Sci. Res. Pharm. 2012, 1(1), 10-12

Journal of Scientific Research in Pharmacy 2012, 1(1) 10-12

Journal of Scientific Research in Pharmacy Research Article Available online through www.jsrponline.com

Estimation of Carbamazepine with Different Forms of Cyclodextrins in Pharmaceutical Preparation by RP-HPLC

Madhuri Sadu1*, Sanjeeva Yarkala1, Vijay Kumar. R2, 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) with 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 hydroxypropyl-βcyclodextrin [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 (α-CD, 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 (990967-906) 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 HPβ-CD 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.

Madhuri Sadu et al., J. Sci. Res. Pharm. 2012, 1(1), 10-12

Journal of Scientific Research in Pharmacy 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.

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.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. .

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.

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.

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

Table 2: Method precision for CBZ

.Precision Injection reproducibility

Method precision

Carbamazepine Limit RSD Limit Average RSD

2.00 0.28% 98-102

100.00 0.96%

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

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

% of Conc.

Resultant solution (ppm)

Area counts

Average SD %

Recovery

80% 80 1150888

1150506 378.06 103.17 80% 80 1150132

80% 80 150498

100% 100 1400278

1402328 2038.

1 100.60 100% 100 1404354

100% 100 1402354

120% 120 1693561

1702029 7529.5

101.75 120% 120 1704556

120% 120 1707970

Average 101.84

SD 1.05

%RSD 1.0

Madhuri Sadu et al., J. Sci. Res. Pharm. 2012, 1(1), 10-12

Journal of Scientific Research in Pharmacy 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 with 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.

References: 1. Letı´cia S. Koester, Clarissa R. Xavier, Paulo Mayorga,

Valquiria L. Bassani, Influence of b-cyclodextrin complexation on carbamazepine release from hydroxypropyl methylcellulose matrix tablets, European Journal of Pharmaceutics and Biopharmaceutics 55 (2003) 85–91.

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3. T. Loftsson, E. Stefa´nsson, H. Friðriksdo´ ttir, J.K. Kristinsson, Novel CD based drug delivery system in: J. Szejtli (Ed.), Proceedings of the Eighth International Symposium on Cyclodextrins, Kluwer, Dordrecht, 1996, pp. 407–412.

4. M.A. Al-Me shal, G.M. El-Mahrook, A.A. Al-Angary, M.W. Gouda, Interaction of carbamazepine with cyclodextrins, Pharm. Ind. 55 (12) (1993) 1129–1132.

5. T. Hoshino, K. Uekama, J. Pitha, Increase in temperature enhances solubility of drugs in aqueous solutions of hydroxypropylcyclodextrins, Int. J. Pharm. 98 (1993) 239–242.

6. M. K. Manoj Babu, and Tapan N. Godiwala, Toward the Development of an Injectable Dosage Form of Propofol: Preparation and Evaluation of Propofol–Sulfobutyl Ether7-b-Cyclodextrin Complex, Pharmaceutical development and technology Vol. 9, no. 3, pp. 265–275, 2004.

7. International Conference on the Harmonization of Technical Requirements for the Registration of Pharmaceuticals for Human Use (ICH) Q2B, Validation of analytical procedures. Methodology, 1996 pp. 1–8. Recommended for Adoption at Step 4 of the ICH process on 6 November 1996 by the ICH Steering Committee. Available on the internet at http://www.ich.org/pdfICH/Q2B.pdf on the 21 January 2002.

Source of support: Nil, Conflict of interest: None Declared