INTRODUCTION:

Esmolol Hydrochloride (Chemical name: Methyl (RS)-3-{4-[2-hydroxy-3-(propan-2ylamino) propoxy] phenyl} propionate, Molecular formula: C₁₆H₂₅NO₄, Molecular weight: 295.374 g/mol) is an ultra-short acting a beta1-selective (cardio selective) adrenergic receptor blocking agent with rapid onset, a very short duration of action, and no significant intrinsic sympathomimetic or membrane stabilizing activity at therapeutic dosages^[1-5]. Hence, Esmolol widely used in the rapid control of heart rate in patient with arterial fibrillation. Structure of Esmolol Hydrochloride is shown in (fig1). The review of the literature reveals that the present study is to develop and validate a stability indicating HPLC method for Esmolol Hydrochloride according to ICH guidelines. Most HPLC methods reported are useful in estimating Esmolol Hydrochloride in human plasma and biological fluids^[6-8]. One method for estimation of Esmolol hydrochloride by capillary electrophoresis has also been reported^[9,10]. This paper reports a rapid and sensitive HPLC method with UV detection, useful for routine quality control and estimation in pharmaceutical dosage form (injection). The method validated by parameters such as linearity, Accuracy, precision, robustness, LOD and LOQ^[11]

Figure 1: Structure of Esmolol

MATERIALS AND METHODS:

HPLC binary gradient system Model no. HPLC 3000 Series equipped with Column BDS Hypersil grace C18 (4.6 ID x 250mm, particle size-5 micron and pump of P-3000-M reciprocating (40 MPa) with detector UV-3000-M.

Optimization of Chromatographic Conditions:

The chromatographic conditions were optimized to mobile phase of Acetonitrile and Potassium Dehydrogenate Phosphate (Acetonitrile: KH₂PO₄) 80:20 v/v, pH adjusted to 3.0 with O-phosphoric acid with flow rate of 0.8 ml/min and temperature of 40⁰C and wavelength: 224nm. A UV spectrum of Esmolol Hydrochloride (λmax224nm) is shown in (Fig 2).

Figure 2: UV spectra of Esmolol Hydrochloride (λ_max224nm)

Solubility:

Solubility studies were carried to find out suitable solvent in which the drug is completely soluble. Various solvents were tried for checking solubility of Esmolol Hydrochloride. From solubility studies, it was concluded that Esmolol Hydrochloride is soluble in water and freely soluble in alcohol.

Preparation of standard solution of Esmolol hydrochloride (100µg/mL):

An accurately weighed quantity of powder equivalent to 10.0mg of Esmolol Hydrochloride was transferred to 100 mL volumetric flask. The drug was dissolved and diluted up to the mark with water.

Working standard preparation:

Working standard solution containing 10µg/mL of Esmolol was prepared by diluting 1mL stock solution of each of Esmolol, transferred to 10mL volumetric flask. The final Volume made with mobile phase and mixed well.

Marketed Formulation: Esocard

Batch No.: IESLA2602

Expiry Date: 04/2018

Label Claim: Each ml contains Esmolol Hcl IP 10mg

Assay:

Sample solution was prepared by diluting 10ml of Esocard injection equivalent to 100mg of Esmolol Hydrochloride to 100ml with water in 100ml volumetric flask. 3ml of this solution were further diluted to 100ml with mobile phase.

Then solution is injected into HPLC system to obtain the chromatograph for drug solution and sample solution. Measure the area of Esmolol hydrochloride and calculate the percentage of assay. Result of assay is shown in table 1.

Table 1: Assay of marketed formulation

Sr. No.	% Composition	Area of Standard	Area of Sample	% Assay
1	80:20:00	1100926	1102359	100.13

Chromatogram of marketed formulation of Esmolol Hcl RT= 5.858 min Acetonitrile: KH2PO4 (80:20 v/v), pH 3 Grace C18column is shown in (Fig 3)

Figure 3: Chromatogram of marketed formulation of Esmolol Hcl RT= 5.858 min

Acetonitrile: KH2PO4 (80:20 v/v), pH 3 Grace C18column

Chromatogram of Esmolol hydrochloride STD, Acetonitrile: KH₂PO₄ pH 3 (80:20 v/v) Grace BDS C18column) is shown in (fig 4).

Figure 4: Chromatogram of Esmolol hydrochloride std, Acetonitrile: KH₂PO₄ pH 3 (80:20 v/v) Grace BDS C18column)

VALIDATION OF RP-HPLC METHOD:

1. Linearity

Linearity was evaluated for a set of five standard working solutions containing 10-50 µg/ml EsmololHcl respectively. The linearity of calibration graphs and adherence of the system to Beer’s law was validated by determining correlation coefficient.Calibration curve of Esmolol Hcl is shown in (fig5). Linear regression data for calibration curves of Esmolol Hcl and Linearity data is shown in table no 2 and 4 respectively.

Figure 5: Calibration curve of Esmolol Hcl

Table 2: Linear regression data for calibration curves of Esmolol Hcl

Parameters	Esmolol Hcl
Linearity range µg/ml	10-50
Correlation coefficient (r²)	0.9987
Slope (m)	35981
Intercept (c)	15702

Table 3: Linearity data

Sr. No.	Conc.	Area(mV)
1	10	355921
2	20	750341
3	30	1100926
4	40	1476586
5	50	1791830

2. Recovery Studies:

Recovery studies were carried out by applying the method to drug sample to which known amount of Esmolol corresponding to 50%, 100%, and 150% of label claim has been added. This samples are analyzed and recoveries of each are calculated. For this study,

· Prepare three preparations of each 50%, 100%, and 150% level and inject in to the chromatography.

· Make the injection lowest concentration to highest concentration.

· Calculate individual recovery, mean recovery and % RSD.\

The results indicate that the recovery of Esmolol Hcl ranges from 98% to 102 % respectively. The recovery by proposed method is satisfactory as % relative standard deviation is not more than ±2.0% and means recovery lies between 98.0 and 102.0%. Recovery data of Esmolol Hcl is shown in table 4.

Table 4: Recovery data of Esmolol Hcl

Sr. No.	% Composition	Amount Taken	Amount Added	Area of Standard	Area of Sample	% Recovery
		(PPM)	(PPM)
1	50% Recovery	20	10	1110926	1106793	99.62796802
2	100% Recovery	20	20	1476586	1474336	99.84762147
3	150% Recovery	20	30	1791830	1787013	99.73116869

3. Limit of Detection and Limit of Quantitation:

The standard deviation of the Y-intercept and average slope of the calibration curve was used to calculate LOD and LOQ using following formulae.

Where, S is average value of slopes of calibration plots and SD is calculated using values of y intercepts of regression equations.The LOD and LOQ are represented in table 5. These results suggest that method is sensitive.

Table 5: LOD and LOQ

Drug	LOD(μg/ml)	LOQ (μg/ml)
Esmolol Hcl	0.4638	1.4055

4. Precision:

Repeatability expresses the precision under the same operating conditions over a short interval of time. The precision of analytical method defined as the closeness of the results between a series of measurements obtained from multiple sampling of same sample under prescribed conditions.

Intra-day precision and Inter-day precision:

Intraday Precision was carried out by preparing test solution of same concentration and analyzing it at two different times in a day. The same procedure was fallowed for two different days to determine interday precision. The result was reported as % RSD. Mean peak value and %RSD were calculated. The precision shows a good reproducibility with % RSD is less than 2.

Result for Intraday precision and interday precision of Esmolol Hcl is shown in table 6 and 7 respectively.

Table 6: Result for Intraday precision of Esmolol Hcl

	Area	Mean	%RSD
Morning	750341	752146.7	0.28%
	752431
	754567
Evening	750251
	750501
	754789

Table 7: Result for Interday precision of Esmolol Hcl

	Area	mean	%RSD
Day1	750341	741254	0.62%
	752431
	754567
Day2	750025
	752586
	741254

5. Robustness:

The effect of small deliberate change in optimized method was studied by robustness evaluation. To evaluate robustness of developed method, parameter was deliberately varied.

Robustness of the method was studied by changing ﬂow rate (±0.2mL/min), change in mobile phase (±2% v/v), and change in pH (±0.2) during analysis. Sample solution of 100% concentration is prepared and injected in triplicate for every condition. Robustness studies of Esmolol HCL are shown in table 8.

Table 8: Robustness studies of Esmolol Hcl

Conc.	Conc.	Area	Mean	SD	% SD
	30	1100926
1	30	1106574	1106172	5057.48	0.45720515
	30	1111017

STABILITY STUDIES:

Table 9: Stability data

Sr. No.	Degradation	Area of Standard	Area of degraded Sample	Degraded up to %
1	H₂O₂ Degradation	1100926	927994	84.29213226
2	Thermal Degradation	1100926	920185	83.58282028
3	Photolytic Degradation	1100926	1100790	99.98764676

1. Hydrogen Peroxide Degradation:

10 mg of pure drug was transferred to round bottom flask. To this 10ml of 3% H₂O₂ was added and this reaction mixture was kept at room temperature for about 24hr. Further 0.3ml of the above solution was diluted to 10mL with diluents to obtain concentration of 30µg/ml. Mixed well and injected onto column under optimized RP-HPLC conditions. Observed for any degradation occurs or not. Chromatogram of Hydrogen peroxide (3% H₂O₂, 24 hrs) is shown in (Fig6).

Figure 6: Chromatogram of Hydrogen peroxide (3 % H₂O₂, 24 hrs)

2. Photolytic degradation:

The Esmolol powder exposed to light to determine the irradiation of light on the stability of powder form of drugs. Approximately 100mg of drug powder spread on a glass dish in a layer that was less than 2mm thickness and were placed in a light cabinet and exposed to UV light for 24hrs. After 24hrs the samples are removed and diluted with diluents to get a concentration of 30µg/ml solution and then injected. Observed for any degradation occurs or not. Chromatogram of photolytic degradation (24hrs at 60°C) is shown in (fig 8).

Figure 8: Chromatogram of photolytic degradation (24hrs at 60°C)

3. Thermal degradation:

Sample powder equivalent to100mg of Esmolol was taken and kept in a controlled temperature oven at 60⁰C for 24 hrs. After 24hrs the powder was diluted with diluents to get a concentration of 30µg/ml solution. Observed for any degradation occurs or not. Chromatogram of Dry heat degradation (24hrs at 60°C) is shown in (fig7).

Figure 7: Chromatogram of Dry heat degradation (24hrs at 60°C)

CONCLUSION:

The present work represents the report that deals with analysis of Esmolol Hcl in bulk and pharmaceutical dosage forms using RP-HPLC. It can be concluded from the results that the proposed method is simple, accurate, robust and precise. Forced degradation is a process that involves degradation of drug products and drug substances at conditions more severe than accelerated conditions and thus generates degradation products that can be studied to determine the stability of the molecule. This method was validated as per ICH guidelines. The method was stability indicating can be used for determination of Esmolol HCl in presence of its degradants.

ACKNOWLEDGEMENT:

The authors are thankful to Swapnroop Drugs and Pharmaceuticals, Aurangabad for providing the pure drug to develop the method and Dr. Kalkotwal R.S. Principal of SND College of Pharmacy, and Dr. Dubey Raghvendra S. for providing support and necessary research facilities.

REFERENCES:

1. Reddy S., Rajan S., Shrinivasaka and Chakravarthy I.E. Structural Identification and characterization of impurities in Esmolol Hydrochloride, Derpharmachemica,2016,8(4): 296-300.

2. Nawal A., Maha F. Ei-Tohamy, Electrochemical system for determination of Esmolol Hydrochloride using square, wave absortive tripping voltammetry, Int J of Electrochemical Sci,2016(11)2709 -2720

3. Sailaja b., Vankteshshwarlu, Pharmaceutical analysis using N- bromo-succinimide methyl orange couple, World J of Pharmacy and Pharmaceutical Sci, 2015,4 (8), 1143-1144

4. Sasikala M., Priyanka P. and Vinodkumar G. T., Quantitative determination of drug by using cerium and rhodamine beta- couple, Int J of Pharmaceutical sci and res, 2015, (12) 5179-5187

5. Thiago V.A., Diago E. N. Takno, Najla M. Kasslo, Fast and Eco-friendly UV. Spectrophotometry and HPLC assay Methods for estimation of Esmolol in Pharmaceutical formulations, Latin American J of Pharm, 2015, 34(9): 1743-48.

6. Anil K. Singh et.al.Fast and Ecofriendly UV. Spectrophotometric and HPLC assay Methods for estimation of Esmolol in Pharmaceutical formulations, Latin American Journal of Pharmacy, 2015, 34(9): 1743-8.

7. Narayana C. Narsimharav, Shivakumar K., spectrophotometric determination of Esmolol hydrochloride using analytical reagent, Ind J of Advanced in chem. Sci,2014, 2(4) 289-93

8. Prashanti, J. Charansingh and G. Vyankteshwarlu, Spectrophotometric study, World J of Pharm and Pharmaceutical sci, 2014, 3(8) 440-445.

9. Kishwarsaleem, Imran Ali, Umma Kulsum and Hasan, Y, Abulenein, Recent development in HPLC analysis of beta blockers in biological sample, J of chromatographic Sci, 2013, 51(8): 807-818.

10. Lei Fang, Bykowski- Turkiewiz, Jeffrey G. Sarver, Paul W. Erhardt, Determination of esmolol and metabolites and enantiomers within human plasma using chiral column chromatography, J of chromatography, 2010, (26) 878

11. Somasekhar Vanita and Gowri Shankar D. Development and validation of rapid RP_ HPLC method for estimation of esmolol hydrochloride in bulk and pharmaceutical formulation, J of chem, 2010, (13), 807-812.

Received on 29.02.2020 Modified on 27.03.2020

Research J. Science and Tech. 2020; 12(2): 136-142.

DOI: 10.5958/2349-2988.2020.00017.0