INTRODUCTION:

Esmolol hydrochloride (ESM), methyl 3-{4-[2-hydroxy-3-(isopropylamino) propoxy] phenyl} propionate hydrochloride, is an ultra-short acting adrenergic receptor antagonist used for the rapid control of heart rate in patients with atrial fibrillation or atrial flutter. Since ESM is widely used in the rapid control of heart rate, it is important to develop and validate analytical methods for its determination in pharmaceutical dosage form. Review of literature has revealed that few methods have been reported for the estimation of Esmolol hydrochloride. Most HPLC methods reported are useful in estimating Esmolol hydrochloride in human plasma^1-5and biological fluids⁶. HPLC⁷ method has been reported for determination of ESM in pharmaceutical injections.

Thorough survey of literature revealed that, although quantification of drugs based on ion-pair complexation is simple, sensitive and accurate, the studies involving any dye towards quantification of Esmolol is has not been reported yet.

In this communication quantitative determination of Esmolol using bromothymolol blue (BTB), bmophenol blue (BPB) and bromocresol green (BCG) is described and the methods have been validated in the lines of ICH.

MATERIALS AND METHODS:

Esmolol hydrochloride is procured from Hetero labs limited, Hyderabad as a gift sample. The dyestuffs viz., BTB, BPB and BCG (AR grade) supplied by SD Fine Chemicals Ltd. Mumbai, are used without any further purification. The dyestuffs were used as 0.025% solutions in doubly distilled water. Sodium acetate-hydrochloric acid buffers⁸ of pH 2.5, 2.8 and 3.5 were prepared by mixing 50ml of 1.0M sodium acetate solution with 50.50, 49.50 or 46.25 ml, respectively, of 1.0 M HCl solution and diluted to 250 ml with doubly distilled water. The pH of each solution was adjusted to an appropriate value with the aid of a pH meter. Chloroform (HPLC grade) supplied by SD Fine Chemicals Ltd. Mumbai is used throughout the work. Stock solutions were prepared for all the dyes and drugs (25mg/100ml).

The spectra (Fig. 1) of ion-pair complexes have been recorded on Shimadzu 140 double beam spectrophotometer, Thermo Nicolet 1000 and also on ELICO 159 UV-Visible single beam spectrophotometer using quartz cells of 10 mm path length. An Elico model Li-120 pH meter was used for pH measurement.

Fig. 1. Absorption spectra of Esmolol hydrochloride-dye complex extracted into 10 ml chloroform: (a) drug = 17.5 mg ml^-1 + 5 ml of 0.025% BCG + 5 ml of pH 2.8 buffer; (b) drug = 17.5 mg ml^-1 + 5 ml of 0.025% BTB + 5 ml of pH 2.5 buffer; (c) drug = 17.5 mg ml^-1 + 5 ml of 0.025% BPB + 5 ml of pH 3.5 buffer

Procedure for the assay of pure drug:

Four different solutions of pure drug in the range of calibration curve were selected and the recovery experiments were performed. The recoveries and their relative standard deviations are tabulated in (Table 2).

Analysis of pharmaceutical formulation:

In order to evaluate the applicability and reliability of the proposed methodology, it was applied to the determination of Esmolol in injections. Satisfactory results were obtained and were found to be in agreement with label claims (Table 3). Three Brevibloc injections were taken and combined diluted further to get required concentrations.

Fig. 2 Calibration graphs for Esmolol hydrochloride with BCG, BPB and BTB ion pair complexes.

Calibration curve:

Different aliquots of drug solution were transferred into 125 ml separating funnel. To this 5 ml of buffer (pH 2.5 2.8 and 3.5), 5 ml of dye were added and total volume was made up to 20 ml with water. 10 ml of chloroform was added and the contents were shaken for 5 min. The two layers were allowed to separate for 5 min. The organic layer was separated and absorbance of yellow colored solution which is stable at least for 3 hrs is measured at 415,412 and 412 nm against blank similarly prepared. The same procedure of analysis is followed either for assay of pure drug or for dosage form. The calibration graphs (Fig. 2) are linear over the concentration ranges are within the permissible range. The optical characteristics and statistical data for the regression equation of the proposed methods are presented in (Table 1).

TABLE 1: Optical Characteristics and Statistical for the Regression Equation of the Proposed Methods

Parameters

Extraction methods with

BTB

BPB

BCG

λ_max (nm)

Beer’s law limit (μg ml^-1)

Molar absorptivity (L mol^-1 cm^-1)

Formation constant, K, M^-1

Sandell sensitivity (μg cm^-2)

Slope (specific absorptivity), b

Intercept (a)

Correlation coefficient (r)

Standard deviation of intercepts (% n=6)

Limit of detection, μgml^-1

Limit of quantification, μgml^-1

Regression equation

415

2.5 - 25

17722

1.12x 10⁶

0.017

0.06

0.083

0.999

0.012

0.65

1.96

Y= 0.06C +0.083

412

2.5 - 25

20971

1.79x 10⁶

0.014

0.071

0.040

0.997

0.013

0.62

1.85

Y= 0.071C -0.040

412

2.5-25

16197

3.26x 10⁶

0.018

0.054

0.101

0.996

0.008

0.48

1.44

Y= 0.054C +0.101

^aWith respect to Y=bc+a, where C is the concentration (μg ml^-1) and Y is absorbance

^bSix replicate samples

TABLE 2: Application of Proposed Methods for the Analysis of Esmolol Hydrochloride in Pure Form

Taken (μg ml^-1)	Proposed methods						Reference method[18]
	Found (μg ml^-1)			Recovery (%)			Recovery (%)
	BTB	BPB	BCG	BTB	BPB	BCG	Recovery (%)
4 8 12 16 RSD (%)	4.01 8.01 12.02 15.98 --	3.98 7.98 11.96 15.99 --	4.01 8.02 11.98 16.03 --	100.31 100.18 100.12 99.90 0.17	99.58 99.81 99.63 99.91 0.15	100.32 100.19 99.87 100.21 0.19	99.98 0.13
Mean±SD t-test F-test				100.13±0.17 1.44 1.78	99.74±0.15 2.47 1.41	100.15±0.19 1.45 2.33	99.98±0.13

TABLE 3: Analysis of Esmolol Hydrochloride from Pharmaceutical Formulations by Proposed Method

Taken (μg ml^-1)	Proposed methods						Reference method[18]
	Found (μg ml^-1)			Recovery (%)			Recovery (%)
	BTB	BPB	BCG	BTB	BPB	BCG	Recovery (%)
4 8 12 16 RSD (%)	4.01 8.03 12.03 16.02 --	3.98 8.02 11.99 16.02 --	3.99 7.98 11.98 15.97 --	100.31 100.36 100.24 100.13 0.11	99.58 100.19 99.88 100.13 0.28	99.68 99.81 99.85 99.19 0.074	100.15 0.20
Mean±SD t-test F-test				100.25±0.11 0.84 0.31	99.95±0.28 1.22 1.89	99.78±0.073 3.46 0.13	100.15±0.20

RESULTS AND DISCUSSION:

Esmolol hydrochloride forms ion-pair complexes in acidic buffer with dyestuffs such as bromothymol blue (BTB), bromophenol blue (BPB) and bromocresol green (BCG) and these complexes are quantitatively extracted into chloroform. Ion-pair complexes of drug with BTB, BPB and BCG absorbed maximally at 415, 412 and 412 nm respectively. The reagent blank under similar conditions showed no absorption.

In order to establish molar ratio between Esmolol hydrochloride and dyestuffs used, the Job’s method of continuous variation⁹ has been applied. In this method, solutions of drug and dyestuff with identical molar concentrations [8 x 10^-5M] were mixed in varying volume ratios in such a way that the total volume of each mixture was the same. The absorbance of each solution was measured and plotted against the mole fraction of the drug, [drug]/ [drug] + [dyestuff] (Fig. 3). This measurement showed that 1:1 complex was formed with each dyestuff. The formation constants^10,11 were also estimated and found to be 1.12x10⁶, 1.79x10⁶ and 3.26x10⁶ M^-1for complexes with BTB, BPB and BCG respectively.

Fig. 3 Continuous-variations study of drug-dye systems: [Drug] = [Dye] = 8x10^-5M

Esmolol hydrochloride contains secondary amine which is protonated in acid medium, while sulphonic acid group is present in BTB, BPB and BCG, that is the only group undergoing dissociation in the pH range 1-5. It is supposed that the two tautomers are present in equilibrium but due to strong acidic nature of the sulphonic acid group. Finally the protonated Esmolol hydrochloride forms ion-pairs with the dyestuffs which are quantitatively extracted into chloroform. The possible reaction mechanisms are proposed and given in (Scheme 1).

Scheme 1 Drug-dye complex

The influence of pH on the ion-pair formation of Esmolol hydrochloride with various dyestuffs has been studied using sodium acetate-hydrochloric acid buffer. The results are shown in (Fig. 4). It is evident that absorbance of complexes with BTB, BPB and BCG was found to be constant within the pH ranges 2.0-3.0, 2.0-3.0 and 3.0-4.0 respectively. Thus, all the absorbance measurements were made at pH 2.8, 2.5 and 3.5 with BTB, BPB and BCG respectively.

Fig. 4 Influence of pH [Drug] = [12.5µg ml^-1]

The effect of dyestuff concentrations was also studied by adding different volumes of dyestuff to a constant amount of Esmolol hydrochloride (12.5 µg ml^-1). It is apparent from (Fig. 5). That the maximum absorbance, in each case, was found with 3.0 ml of dyestuff, beyond which absorbance was constant. Thus, 5 ml of each dyestuff was used for ion-pair formation throughout the experiment.

Fig. 5 Influence of the volume of 0.025% Dye [Drug] = [12.5µg ml^-1]

Validation of the proposed method:

All the three proposed methods have been validated in terms of guideline proposed by ICH¹² viz. selectivity, specificity, accuracy, precision, limits of calibration curve, LOD, LOQ, robustness, ruggedness and regression equation. The student t-test and variance F-test have been performed in comparison with a reference method. (Table 1) summarizes the values for Beer’s law limits, molar absorptivity, regression equation, correlation coefficients, relative standard deviation and recoveries. To test the reproducibility of the proposed methods, six replicate determinations of 4µg ml^-1of Esmolol hydrochloride were made. The coefficient of variation was found to be less than 1.2% for all the procedures.

The proposed methods have been successfully applied to the determination of Esmolol hydrochloride in pharmaceutical preparations. The results obtained and shown in (Table 2 and Table 3) were compared to those obtained by a reference method⁸ by means of t-test at 95% confidence level. In all cases, the average results obtained by proposed methods and reference method were statistically identical, as the difference between the average values had no significance at 95% confidence level.

The proposed methods are simple, sensitive and reproducible and can be used for routine analysis of Esmolol hydrochloride in pure form and in formulation.

CONCLUSION:

Esmolol hydrochloride formed ion pair complexes with acidic dyes with 1:1 composition and extractable in to chloroform for assay of the drug. The method is validated and applied to pharmaceuticals.

ACKNOWLEDGEMENTS:

The authors are grateful to Head, Department of Chemistry and Principal, Nizam College for providing facilities.

REFERNCES:

1. Tang Y, He Y, Yao T and Zeng S. Simultaneous determination of the enantiomers of esmolol and its acid metabolite in human plasma by reversed phase liquid chromatography with solid-phase extraction. J Chromatogr B. 805; 2004: 249-254.

2. Tang Y, He Y, Yao T and Zeng S. Stereoselective RP-HPLC determination of esmolol enantiomers in human plasma after pre-column derivatization. J Biochem Biophys Methods. 59; 2004: 159-166.

3. Maurer H H, Tenberken O, Kratzsch C, Weber A A and Peters F T. Screening for library-assisted identification and fully validated quantification of 22 beta-blockers in blood plasma by liquid chromatography–mass spectrometry with atmospheric pressure chemical ionization J Chromatogr A. 1058; 2004: 169-181.

4. Zuppa A F, Shi, Adamson H and Peter C. Liquid chromatography–electrospray mass spectrometry (LC–MS) method for determination of esmolol concentration in human plasma. J Chromatogr B. 796; 2003: 293-301…

5. Achari R, Drissel D and Hulse J D. Liquid-chromatographic analysis for esmolol and its majormetabolite in urine. Clin Chem. 32; 1986: 374-376.

6. Malovaná S, Gajdošová D, Benedik J and Havel J. Determination of esmolol in serum by capillary zone. electrophoresis and its monitoring in course of heart surgery. J Chromatogr B. 760; 2001: 37-43.

7. Vanita somasekhar and. D. Gowri sankar. Development and Validation of a Rapid RP-HPLC Method for the Estimation of Esmolol Hydrochloride in Bulk and Pharmaceutical Dosage Forms. E-Journal of Chemistry. 3; 2010: 807-812.

8. Britton HTS. Hydrogen Ions. Chapman and Hall. London. Vol. I; 1942: 301.

9. Vosburgh WC and Coopper GR. The identification of complex ions in solution by spectrometric measurements. J Am Chem Soc. 63; 1941: 437-442.

10. Likussar W and Boltz DF. Theory of continuous variation plots and a new method for spectrometric determination of extraction and formation constants. Anal Chem. 43; 1971: 1265-1272.

11. Momoki K, Sekino J, Sato H and Yamaguchi N. Theory of curved molar ratio, plots and new linear plotting Method .Anal Chem. 41; 1969: 1286-1299.

12. International Conference on Hormonization (ICH) of Technical Requirement for the Registration of Pharmaceuticals for Human use, Validation of analytical precedures: definations and Terminology Genera 1996.

Received on 14.09.2011 Modified on 05.10.2011

Asian J. Research Chem. 4(11): Nov., 2011; Page 1789-1792