INTRODUCTION:

Dobutamine Hydrochloride (DOB)¹ is chemically,(±)-4-(2-{[3-(p-Hydroxyphenyl)-1-methylpropyl] amino}ethyl) pyrocatechol hydrochloride, (Figure 1) and belongs to a series of pyrocatechol derivatives, which exhibit strong sympathomimetic with direct effects on beta₁- adrenergic receptors, giving it a prominent inotropic action on the heart. It is used to the increase the contractility of the heart in acute heart failure, as occurs in cardiogenic shock and myocardial infarction.

Literature survey revealed that several analytical methods have been reported for the determination of Dobutamine hydrochloride in pure drug, pharmaceutical dosage forms and in biological samples using spectrophotometry^2-15, Gas chromatography¹⁶ and liquid chromatography^17-21 either in single or in combined forms. The retention time of the reported HPLC methods is more than five minutes and the flow rate of the mobile phase is 1.0 ml/min, which requires a greater mobile phase for the determination of Dobutamine HCl. Hence, in the present study it was intend to develop a rapid, economical, simple and precise RP-HPLC method for the estimation of Dobutamine HCl in bulk and formulations.

EXPERIMENTAL:

Chromatographic conditions:

The HPLC Agilent 1200 system was equipped with an auto injector (100 µl syringe ) and connected to a variable diode array wavelength detector and recorder. A Luna C₁₈reversed phased column (250 mm x 4.6 mm, 5 μ) bonded on to 5 µm silica gel, manufactured by Agilent, was used for analysis. A mixture of Acetonitrile: Methanol: Tetrahydrofuran (70:20:10 v/v/v, pH 6.5) was used as the mobile phase. The flow rate was 1.0 ml/min, wavelength was 292 nm, and the injection volume was 20 µl. The analysis was carried out at ambient temperature.

Reagents:

All the chemicals were of analytical grade and used without further purification. HPLC grade Acetonitrile (Chromasolv), Methanol (Qualigens), Tetrahydrofuran (Qualigens) and o-Phosphoric acid - ExcelaR grade (Qualigens) were used. The standard drug sample of the Dobutamine HCl was supplied by Sun pharmaceuticals Industries Limited, Mumbai.

Standard and sample preparation:

Standard stock solution at a concentration of 1 mg/1ml Dob was prepared by dissolving a 20 ml of methanol in 100 ml volumetric flask. This solution was suitably diluted with the mobile phase to get a working standard solution of 100 μg/mL of Dobutamine hydrochloride. Five injection vials of Dobutrex were taken. Their aluminium closures were removed. The powders in all the vials were pooled up and the average weight of powder in one vial was calculated. An amount of powder equivalent to 100 mg of Dobutamine hydrochloride was taken in a 100 mL volumetric flask and it was mixed with 20 mL of methanol. The contents of the flask were sonicated for about 20 min for complete solubility of the drug and the volume was made up to 100 mL with mobile phase to get a 1 mg/mL of the solution. Then the mixture was filtered through a 0.45 μ membrane filter. From the above solution a Ten mL aliquot was taken into a separate 100 mL volumetric flask and made up to the volume with mobile phase and mixed well to get a 20 μg/mL of the solution. The above solution (20 μL) was then injected eight times into the column.

Fig 1: Molecular structure of Dobutamine hydrochloride

Method validation:

The method validation parameters, such as, specificity, linearity, accuracy, precision, limit of detection, limit of quantification and robustness were verified as per the International conference on Harmonization (ICH) guidelines²².

Specificity:

The specificity of the method was confirmed by comparison of chromatograms obtained from the standard and the samples.

Linearity and range:

Linearity of the peak area response was determined by taking six replicate measurements at seven concentration points. Working dilutions of DOB in the range of 10-30 μg/mL were prepared by taking suitable dilutions of the standard solutions in different 10 mL volumetric flasks and diluted up to the mark with the mobile phase. Twenty microlitres of the dilution was injected six times into the column.

Accuracy (% Recovery):

The accuracy study was carried out by the standard addition method. Known amounts of standard solution of DOB (10,15 and 20 µg/ml) were added to a sample solution of DOB (20 µg/ml).Each solution was injected in triplicate and the percentage recovery was calculated.

Precision:

Standard solutions having concentrations of 15, 20 and 25µg/ml were prepared. For the intra-day studies three repeated measurements of these solutions were carried out within a day and %RSD was calculated. In the Inter-day variation studies, three reported measurements of the same solutions were carried out on three consecutive days and % RSD was calculated.

Limit of detection and Limit of quantification:

The limit of detection (LOD) and limit of quantification (LOQ) were calculated from the standard deviation of responses and slopes using signal-to-noise ratio as per ICH guidelines²².

Robustness:

Robustness of the method was studied by changing the extraction time of DOB, from dosage form by five minutes, composition of mobile phase by 2% of organic phase, flow rate by 0.1 ml/min, and wavelength by 1 nm.

RESULTS AND DISCUSSION:

Chromatographic conditions were developed empirically by selecting various solvents such as water, acetonitrile and methanol in different concentrations, and the retention time and resolution were assessed to get optimum conditions. Under the established chromatographic conditions, DOB was well-resolved. As the system suitability parameters such as tailing factor and theoretical plate numbers were within the acceptable limits, the resolution of peaks were considered optimum. Retention time for DOB for 5.008 minutes indicating the rapid analysis of Dobutamine [Figure 2]. The calibration curves were constructed by plotting the peak areas of DOB versus concentration of DOB, and the regression equation was calculated. Each response was the average of three determinations. The results [Table 1] obtained showed that the linearity range was 10-30µg/ml. The corresponding regression equation was found to be Y=9562.48 X +40.29 (Y is peak area of DOB and X is DOB concentration) with a correlation coefficient of 0.9999.

Table 1: Summary of regression analysis and validation parameters

Parameters

Values

Regression analysis

Slope

Intercept

Correlation

Validation parameters

LOD (µg/ml)

LOQ (µg/ml)

Accuracy (%) ± % RSD

Precision

Intra-day (n=3)

Inter-day (n=3)

System suitability test parameters

Retention time(min) ± % RSD

Tailing factor ± % RSD

Theoretical plates ± % RSD

9562.48

40.29

0.9999

0.34

1.1

99.54 ± 1.2

0.38-0.83

0.38-0.72

5.008 ± 0.03

1.62 ± 0.1

3201± 1.45

The recovery study showed that the mean recovery was 99.54 % and the RSD for three samples was lower than 2.0%. The RSD values of intra-day and inter-day were found to be in the range of 0.38-0.83 and 0.38-0.72, respectively.

As these parameters were within the acceptable range, it was inferred that the method was precise. The accuracy of the developed method was assessed by the standard addition method and the RSD was found to be within the acceptable limit [Table 2]. The limit of quantification (LOQ) of the method, defined as the minimum concentration that could be measured, was found to be 1.1 µ/ml. The limit of detection (LOD) was 0.34 µg/ml [Table 1]. The method was found to be robust as the results were not significantly affected by the slight variation in the composition of mobile phase, flow rate and pH of the buffer. The chromatographic parameters such as selectivity and peak asymmetry are within the acceptable range. The number of theoretical plates and the tailing factor were observed to be satisfactory [Table 1].

Analysis of marketed formulation:

The proposed validated RP-HPLC method was successfully applied to determine DOB in two marketed formulations. The mean percentages of DOB were found to be 99.84 ± 0.92, 100.48 ± 1.01which were comparable to the corresponding labeled amounts [Table 3]. Furthermore, the matrix components, for example, the excipients, did not interfere with the analyte.

Table 3: Assay result for Dobutamine (mg of tablet) in the formulation product

Formulation	Dobutamine in label claim (mg)	Total amount of Dobutamine found (mg)	% of Dobutamine found
1	250	249.6	99.84
2	250	251.2	100.48

CONCLUSION

The validated RP-HPLC method employed here has proved to be rapid, simple, specific, accurate, precise, sensitive and robust. It can be successfully used for the routine analysis of DOB in bulk and in pharmaceutical dosage form.

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Received on 08.11.2010 Modified on 20.11.2010

Asian J. Research Chem. 4(2): February 2011; Page 304-307

Standard Dobutamine concentration (µg/ml)	Sample Dobutamine concentration (µg/ml)	Total amount of Dobutamine from standard graph (µg/ml)	Recovery of standard drug (µg/ml)	% Recovery of standard
20	10	30.01	10.01	99.94
20	15	34.82	14.82	99.54
20	20	40.13	20.13	100.06