78

FABAD J. Pharm. Sci., 30, 78-82, 2005

RESEARCH ARTICLE

INTRODUCTION

Carbamazepine (CBZ), 5-H-Dibenz [b.f] azepine-5-

carboxomide (Fig.1), is widely prescribed as an anti-

convulsant, antiepileptic and antimanic drug1,2. In

the body, CBZ is metabolized to an active metabolite,

CBZ-10.11 Epoxide (CBZ-EP), which also displays

anticonvulsant properties similar to those of the parent

compound3,4. CBZ is poorly soluble in aqueous media

and has a high oral bioavailability in humans3.

Determination of Carbamazepine UsingRP-HPLC Method in Pharmaceutical Preparations

Carbamazepine (CBZ), which was developed as an anticonvulsant drug, is also used as an antiepileptic, antidepressive, antianalgesic and antimanic drug. A reversed-phase (RP)-HPLC method for the quantitative determination of CBZ in pure forms and in pharmaceutical preparations was developed and validated in the present study. Parameters such as linearity, precision, accuracy, specificity, stability, limit of detection and limit of quantitation were studied according to the International Conference on Harmonization Guidelines. Different analytical columns with various stationary phases were tested. Good separation was achieved using Bondolone C18 column (150 x 3.9 mm. 5 µm). RP-HPLC was carried out using a mobile phase of acetonitrile-Milli-Q grade water (30:70 v/v) pumped at a flow-rate of 1 ml min-1. The injection volume was 10 µl and the peaks were detected at 220 nm. Linearity was obeyed in the range 0.25-25 µg ml-1. The developed method was successfully applied to assay of CBZ in pharmaceutical preparations. This method is suitable for practical routine analysis of commercially produced pharmaceutical preparations.KeyWords : Carbamazepine, RP-HPLC, pharmaceutical preparationsReceived : 15.05.2006Revised : 21.06.2006Accepted : 10.07.2006

º*

Atatürk University, Faculty of Pharmacy Department of Analytical Chemistry, 25240, Erzurum, TURKEY

Corresponding author e-mail: yucelkadi@yahoo.com or yucel@atauni.edu.tr

79

Demirkaya, Kad›o¤lu

Several methods have been reported for the determi-

nation of CBZ standards and in pharmaceutical prep-

arations including spectrophotometric methods4-7,

spectrofluorimetry methods8,9, gas-liquid chromatog-

raphy (GC)10,11, FT-Raman spectroscopy12 and liquid

chromatography13-18.

Therefore, the purpose of this investigation was to

develop and validate a reversed-phase (RP)-HPLC

method that could be used for determination of CBZ

in pure forms and in pharmaceutical preparations.

In the present paper, a simple, rapid, sensitive, precise,

accurate and specific RP-HPLC-DAD method is de-

scribed and applied to pharmaceutical preparations,

which require high sensitivity and selectivity.

MATERIALS AND METHODS

Materials and chemicals

Carbamazepine (CBZ) was kindly supplied by No-

vartis (Turkey). HPLC and analytical grade solvent

(methanol, acetonitrile) were purchased from Merck

(Germany). The water of HPLC was prepared by

double glass distillation and filtration through Milli-

pore 0.45 µm. white nylon HNWP 47 mm filters.

Three commercial preparations-Tegretol tablet (pro-

duced by Novartis Pharm. Ind., Turkey, containing

200 mg CBZ/tablet), Karberol tablet (produced by

Munir Sahin Pharm. Ind., Turkey containing 200 mg

CBZ/tablet) and Karbelex tablet (produced by Liba

Pharm. Ind., Turkey, containing 300 mg CBZ/tablet)-

were assayed.

Chromatographic system and conditions

A Thermoseparations Spectra HPLC system was

applied to perform the analyses. Detection was ac-

complished using UV 6000 LP photodiode array

detector. The Series P 400 gradient auto pump and a

Thermoseparations As 3000 autosampler were condi-

tioned at 25°C. Chromatographic software

Chromoquest30 was used for data collection and

processing.

Three different analytical columns with various sta-

tionary phases were tested: Phenomenex Bondolone

RP C18 column (150 x 3.9 mm. 5 µm.), Ace 5 C8 column

(150 x 4.6 mm. 5 µm.) and Nucleosil 5 NH2 100A (250

x 4.0 mm 5 µm).

Standard solutions

Standard stock solution of CBZ was prepared with

methanol to a concentration 50 µg ml-1 and stored at

4°C. Working solutions (0.25-25 µg ml-1) were prepared

by diluting with water appropriate volumes of stock

solutions at 10 ml as needed to construct the calibration

curves. The working solutions were prepared freshly

in analysis daily. The solutions were filtered through

a phenomenex membrane of 0.45 µm pore size (25

mm filter) and transferred to an autosampler vial for

analysis. Calibration graphs were prepared using

peak area versus concentrations of working solutions

in mobile phase. Six replicate 10 µl injections were

made for each concentration. The final concentrations

of CBZ in the samples were calculated by comparison

with peak area obtained with an average of six injec-

tions of standard solutions.

Analysis of pharmaceutical preparations

Twenty tablets (Tegretol® Karberol® and Karbelex®

tablets) were accurately weighed and finely powdered

in a mortar. An amount of tablet mass equivalent to

content of a tablet (200 mg for Tegretol® and

Karberol®, 300 mg for Karbelex®) was transferred to

a 50 ml volumetric flask and dispersed in 40 ml

methanol. The flask was placed in ultrasonic bath for

15 min. The resulting suspension was diluted to

volume with methanol and then filtered. Further

dilutions were performed to constitute the calibration

graphs for the RP-HPLC method.

Method validation

The methods were validated according to the Inter-

national Conference on Harmonization Guidelines19

for validation of analytical procedures.

N

COH N

Chemical structure of carbamazepine.Figure 1.

80

RESULTS AND DISCUSSION

RP-HPLC method

The development of the RP-HPLC method for the

determination of drugs has received considerable

attention in recent years because of its importance in

routine quality control analysis. Different analytical

columns with various stationary phases were tested.

Good separation from these three columns was

achieved using a Phenomenex Bondolone RP C18 column. The latter was finally used for analysis. A

RP-HPLC method was proposed as a suitable method

for the estimation of CBZ in pharmaceutical dosage

form. The chromatographic conditions were adjusted

in order to provide a good performance of the assay.

The method involved a mobile phase consisting of

acetonitrile- Milli-Q grade water (3:7 v/v) accom-

plished at 220 nm. The retention time was 8.2 min at

a flow-rate of 1 ml min-1 and the injection volume

was 10 µl. The total run time for an assay was approx-

imately 9 min. The integrator attenuation was 8 and

the chart speed was 0.2 cm min-1. The mobile phase

was chosen after several trials with other solvent

combinations. Mobile phase selection was based on

peak parameters (symmetry, tailing), run time, ease

of preparation and cost. Figures 2 and 3 show a typical

chromatogram obtained from the analysis of a stan-

dard and solution of commercial CBZ using the pro-

posed method. As shown in these figures, CBZ was

eluted forming symmetrical peak and well separated

from the solvent front. Observed retention time (8.2

min) allowed a rapid determination of the drug.

System suitability parameters calculated under the

optimized experimental conditions were capacity

factor (k’) 4.46; symmetry factor 1.05 and column

efficiency (n) 4303 plates/m.

Linearity

To determine the linearity of the RP-HPLC method,

calibration standard solutions of CBZ were prepared

as in the text. The linear ranges were found to be 0.25-

25 µg ml-1 concentration. The regression equation

and correlation coefficient (r) obtained by least square

regression method were y=449820x+115048 (y: peak

area, x: concentration) and 0.9995, respectively. The

regression equation calculated from calibration curves

given with the standard deviations of slope (Sb) and

intercept (Sa) on the ordinate are given in Table 1. The

linearity of the calibration graph and conformity of

RP-HPLC value to Beer’s Law were proven by the

high correlation coefficients (r) for the regression

equations.

Accuracy and precision

Repeatability is given as inter- and intra-day precision

and accuracy evaluated by analyzing three different

concentrations of CBZ. Accuracy of the method was

checked for six days at three concentration levels at

2.5, 7.5 and 15 µg ml-1 in six replicates. The results

are given in Table 2. The precision of the RP-HPLC

method was demonstrated by the relative standard

derivation (RSD %) of lower than 5.88% for intra-day

and 6.68% for inter-day. Recovery experiments were

FABAD J. Pharm. Sci., 30, 78-82, 2005

a) HPLC chromatogram of obtained calibration graph point using the assay spiked procedureb) Calibration graphs (n=6)

Figure 2.

HPLC chromatogram of 10 µg ml-1 CBZ: a) Standard b) Tegretol c) Karbelex d) Karberol

Figure 3.

Table 1. Results of regression analysis of data for the quantitation of CBZ by the proposed methods (n=6)

81

Demirkaya, Kad›o¤lu

performed via standard-addition technique. To fixed

and known amount of drug in the pre-analyzed tablet

extracts, pure CBZ (standard) was added at three

levels and the total amount was found by method.

The experiment at each level was repeated six times.

The developed RP-HPLC method was applied to

three serials of tablets and the percent recoveries

obtained are given in Table 3. These results indicated

a very good reproducibility of this method.

Sensitivity

Limit of detection (LOD) and limit of quantitation

(LOQ) were determined by an empirical method that

consisted of analyzing a series of standard solutions

containing decreased amounts of CBZ. The LOQ is

defined as the lowest concentration on the calibration

curve at which both accuracy and precision should

be within 20%. LOQ value of the RP-HPLC method

was determined as 0.07 µg ml-1, and its precision and

accuracy were well within the proposed criteria (Table

1). The LOD for CBZ determination was approximate-

ly 0.05 µg ml-1.

Stability

For the determination of the stability of CBZ in solu-

tion at ambient 4ºC and -20ºC refrigeration tempera-

ture, three sets of low (2.5 µg ml-1), medium (7.5 µg

ml-1) and high (15 µg ml-1) concentrations were di-

vided into 12 tubes. One set was assayed immediately

and taken as standard (100%). One of the sets was

stored at 4ºC for 24 h and for 1 week. The other set

was stored at – 20ºC in a deepfeezer for 24 h. The

remaining set was allowed to stand at ambient tem-

perature for 24 h. The stability measurements were

then carried out. The results were evaluated by com-

paring these measurements with those of standards

and expressed as percentage deviation. They are

summarized in Table 4.

CONCLUSION

This paper describes a reversed-phase HPLC method

for determination of CBZ in pure forms and in phar-

maceutical preparations with minor sample treatment.

The validation studies show good recoveries, precision

and accuracy. This method was successfully applied

to assay of CBZ in tablets (Tegretol® Karberol® and

Karbelex® (Table 5)). In summary, the reported method

can be used for the routine quality control analysis

of the investigated drug in pharmaceutical prepara-

tions.

Bradley MK, Rene H, Levy RR, Matson R,

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