INTRODUCTION:

Carvedilol is official in British pharmacopoeia and European pharmacopoeia (Fig 1). Chemically it is (±)-1-(carbazol-4-loxy)-3[[2-(2-methoxy phenoxy) ethyl] amino] propan-2-ol.It is a white to of white powder with a molecular weight 406.5 and a molecular formula of C₂₄H₂₆N₂O₄. It is freely soluble in dimethyl-sulphoxide, soluble in methylene chloride, sparingly soluble in ethanol, slightly soluble in ethyl ether, practically insoluble in water. Literature survey reveals that there are a few methods reported on HPLC estimation of carvedilol in its tablet dosage form^4-10.The proposed method is accurate, precise and applicable for CONPRES (Torrent) and COREG (Glaxo-Smithkline) tablet formulations.

Fig 1

MATERIAL:

Carvedilol Pure drug was kindly supplied as gratis samples by Torrent Pharmaceuticals. All other reagents were HPLC grade obtained from SD fine chemicals. The common tablets were obtained from local sources. Water purified by reverse osmosis was used for all purposes. Commercial FDCs were sourced from local medical stores.

Received on 03.03.2008 Modified on 10.03.2008

Research J. Pharm. and Tech. 1(1): Jan.-Mar. 2008; Page 18-21

Apparatus

The HPLC system Shimadzu, Japan with LC10AVP, variable wavelength programmable UV-Visible detector SPD-10-AVP, 20 mL injector rheodyne SIL-10AVP and Inertsil C8 column (150 ´ 4.6 mm, 5m) was used. For calibration curve UV-2450 Shimadzu was used. Other instruments included mettler electronic balance AG-245 (Switzerland), branson 3210 sonicator (The hague, the Netherlands) Millipore syringe filtration assembly (Bangalore, India), Remi Centrifuge, Brand autopipette from Merck( Mumbai, India) , dissolution and disintegration apparatus from Electro lab were used.

Pharmaceutical preparation

Commercial FDC formulations from Glaxo-Smithkline (COREG) and Torrent (CONPRES) were procured and assayed. The content declared was 6.25mg per tablet.

CHROMATOGRAPHIC CONDITIONS:

An isocratic high performance liquid chromatograph (Shimadzu) with LC10AVP, variable wavelength programmable UV-Visible detector SPD-10-AVP, 20 mL injector rheodyne SIL-10AVP and Inertsil C8 column (150 ´ 4.6 mm, 5m) was used. Flow rate of mobile phase was maintained at 1.0 ml/min. The detection was carried out by UV detector at 240 nm and injection volume was 20 mL.

A calibration curve for Carvedilol – Stock solution of Carvedilol was prepared by dissolving 10 mg of drug in 10 ml of methanol (1000 µg/ml). From this 5 ml solution was taken and diluted up to 50 ml with simulated gastric fluid (SGF). Further dilutions (1 to10 µg/ml) were prepared and absorbance was measured at 240 nm. Concentrations were prepared 2, 4, 6, 8, 10 mcg/ml and absorbances were found to be 0.242, 0.497, 0.736, 0.967, and 1.203 respectively. Calibration curve shows that carvedilol’s absorbance is proportional to its concentration (Fig 2). Correlation Coefficient was found to be 0.9997.

Preparation of Standard Solution:

About 31.25 mg of pure Carvedilol was weighed into 100ml volumetric flask. 70 ml of mobile phase was added to flask and it was sonicated to dissolve. Volume was made upto mark with mobile phase and 10 ml to 50 ml was made up with mobile phase.

Analysis of Pharmaceutical Formulation:

Sample preparation

Sample	Peak purity index
Blank preparation	-
Standard Preparation	1.00
Unstressed API Preparation	1.00
UnstressedPlacebo preparation	-
Unstressed Sample preparation	1.00

10 tablets were weighed accurately and transferred into 100 ml volumetric flask. About 70 ml of mobile phase was added and sonicated for 30 minutes. Mixture was cooled and volume was made up with mobile phase and mixed. A portion of the solution was centrifuged at 3500 rpm for 15 minutes. Than 5 ml of the clear supernatant was diluted to 50 ml with mobile phase and mixed. This was used for injection.

% Assay = (Test area/Std area)*(Std wt taken/Std wt to be taken)*(Test wt to be taken/test wt taken)* Std assay

Assay procedure

The proposed procedure successfully applied for the analysis of carvedilol in CONPRES and COREG tablet formulations. Results are summarized in Table 1.

Table 1: Assay of Carvedilol Tablets Dosage Form

No.

Brand name of Tablet (mg)

Label amount of Drug (mg)

% Assay found by the proposed method

CONPRES

6.25

100.90

COREG

6.25

101.08

RESULT AND DISCUSSION:

This method is simple, rapid, accurate, and precise for estimation of Carvedilol in pharmaceutical formulation. The retention time of Carvedilol was found 3.775 minutes. Each of samples was injected five times and the same retention times were observed. The percentage RSD of area of five replicates was found 0.37. By the formula percentage assay for Carvedilol was calculated and was found 100.90% and 101.08% for two batches.

To ensure the reliability of method validation parameters; accuracy, linearity, precision, specificity and robustness were performed as per ICH guidelines. The results obtained lies in acceptance criteria. Results of assay method and validation parameters are discussed as follows.

Specificity

A blank, placebo, API and sample for each degradation were injected and the separation of degraded impurities was checked. The peak purity index for the main peak in all the degraded sample preparation was determined and recorded in Table 2.

Linearity and Range

Linearity was determined at five levels over the range of 60 % to 140 % of sample concentration. A standard stock

solution was prepared and further diluted to attain 60%, 80%, 100%, 120% and 140% of working concentration. Each standard preparation was injected in duplicate. The mean area at each level was calculated and a graph of mean area versus concentration was plotted. The correlation co-efficient (R²), Y intercept, slope of regression line, residual sum of squares were calculated and recorded in Table 3.

Table 2: Specificity

Table 3: Linearity and Range

Parameters
Correlation coefficient (R²)	0.99997
Slope of regression line	164127.7
Y- Intercept	201455.9
Residual Sum of Squares	5628566205.3

Table 4: Accuracy

% concentration	Mean	% RSD
60%	100.2	0.2
100%	99.2	0.21
140%	99.7	0.50

Accuracy (Recovery)

Accuracy was determined over the range 60 % to 140 % of the sample concentration. Calculated amount of Carvedilol working standard was added in placebo to attain 60 %, 100 % and 140 % of sample concentration. Each sample was prepared in triplicate and was analyses for assay of Carvedilol tablets as per test procedure. %Recovery, %RSD, means %recovery was calculated at each Level and recorded in Table 4.

Method Precision (Repeatability)

Method precision was established by assaying six sample preparations under same conditions. Six replicates of sample were prepared at sample concentration by one analyst and analyzed on same day. Individual % assay, mean % assay, % RSD and 95 % confidence interval were calculated and recorded in Table 5.

Intermediate Precision (Ruggedness)

Different analyst, using a different HPLC system, repeated the procedure followed for method precision on a different day using same lot of sample. The mean assay value was calculated and compared with the mean assay value obtained in method precision study. The difference of the mean assays obtained was calculated and recorded in Table 6.

Robustness

The robustness of the method was established by making deliberate minor variations in the following method parameters. Following parameters were changed one by one and their effect on system suitability test and % assay is recorded in Table 7.

1. Changed flow rate by ± 0.1 ml/minute (use flow rate 0.9 ml and 1. 1 ml).

2. Changed mobile phase composition: (a) Buffer: Acetonitrile (63:37), (b) Buffer: Acetonitrile (67:33).

3. Changed pH of buffer by ±0.2 units (1.8 and 2.2)

4. Changed column temperature by ± 5°C. (50°C and 60°C)

5. Changed column lot (different column lots).

Table 5: Repeatability

Parameters
Mean	98.8
SD	0.67
% RSD	0.68
95 % Confidence Interval	98.1; 99.5

TABLE 6: Ruggedness

Parameters
Mean	98.5
SD	0.58
% RSD	0.59
95 % Confidence Interval	97.9; 99.1

CONCLUSION:

The method developed is a very simple reverse phase HPLC method for estimation of carvedilol . This method is applicable for various brands (CONPRES-torrent, COREG-GlaxoSmithKline) of tablets available at market. The mobile phase (buffer: acetonitrile) preparation for this method is very simple for estimation of carvedilol in carvedilol tablet.

The column used for estimation of carvedilol is very simple, inertsil C8 which is easily available at institutional laboratories. This method is also useful for industry for estimation carvedilol.
Method has been validated as per ICH guidelines for all the parameters.

ACKNOWLEGEMENT:

The authors are grateful to Torrent Pharmaceutical for providing facilities and chemicals for research work. Tablet samples were obtained from shekhawat medical store, sikar.

TABLE 7: Robustness

Effect of change in Flow rate

Parameters	1.0 ml/min	0.9 ml/min	1.1 ml/min
Mean	17043635	18874275	15499679
%RSD	0.03	0.08	0.06
Theoretical Plate	5419	5765	5116
Tailing	1.39	1.43	1.35
Retention Time	4.067	4.492	3.692

Effect of change in mobile phase composition

Parameters	Buffer: Acetonitrile (65:35)	Buffer:Acetonitrile (63:37)	Buffer: Acetonitrile (67:33)
Mean	17058283	16974275	17074980
%RSD	0.04	0.04	0.05
Theoretical Plates	5126	4439	4760
Asymmetry	1.33	1.29	1.32
Retention Time (min)	4.025	3.400	4.883

Effect of change in column oven temperature

Parameters	Column oven temperature 55°C	Column oven temperature 50°C	Column oven temperature 60°C
Mean	17058283	17078624	17036973
%RSD	0.04	0.02	0.01
Theoretical Plates	5126	1.39	4380
Asymmetry	1.33	4471	1.31
Retention Time (min)	4.025	4.183	3.825

Effect of change in column lot

Parameters	Lot No. 3CH66819	Lot No. 5F112516
Mean	17058283	16967852
%RSD	0.04	0.02
Theoretical Plates	5126	5030
Asymmetry	1.33	1.34
Retention time (min)	4.025	3.608

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Received on 24.03.2008 Modified on 30.03.2008

Research J. Pharm. and Tech. 1(1): Jan.-Mar. 2008; Page 14-17