Development And Validation of RP-HPLC Method for Estimation of Esmolol HCL from Semisolid Dosage Form

The chemical name of Esmolol Hydrochloride is methyl 3-(4-{2 -hydroxy-3-[(propan-2-yl) amino] propoxy} phenyl) propanoate hydrochloride.

Esmolol Hydrochloride is the salt form of Esmolol, which belongs to the class II anti-arrhythmic drugs. Esmolol hydrochloride blocks β-1 receptors in cardiac muscle and at higher doses, it will occlude β-2 receptors and vascular smooth muscle thus directing to its muscle relaxation 2, 3. It has a molecular formula of C₁₆H₂₅NO₄•HCl with molecular weight 331.8 g/mol. The purpose of this study is to develop a simple, precise, and accurate RP-HPLC method for the estimation of Esmolol Hydrochloride from a semi-solid dosage form and to validate the developed method with study of different parameters as per ICH guidelines as No reported RP-HPLC methods for estimation of Esmolol Hydrochloride in Semi solid dosage forms were found.

Esmolol Hydrochloride

MATERIALS AND METHODS:

Materials

Drug Substance: Working standards Esmolol Hydrochloride (99.9%), were procured from Mehta API.

Esmolol hydrochloride finished product:

Brand name: Diulcus Gel

Product name: Esmolol HCL 14% w/w

Manufacturer: IPCA Lab Ltd.

Instrumentation:

HPLC instrument with UV-visible detector – Agilent Infinity II

Software: Open lab

Column - Intsil C18 column (300 mm x 3.9 mm, 5 µm)

UV-visible Spectrophotometer - SHIMADZU 1601 Software - UV probe, version- 2.34

Digital Analytical Balance - OHSUS

Ultra Sonicator -PS 21

Digital pH meter – Lab India

Controlled temperature water bath

Hot air Oven – Kesar

FTIR – SHIMADZU

Melting Point Apparatus – ANALAB   Model: ThermoCal50

Chemicals and Reagents: Esmolol Hydrochloride API, Acetonitrile HPLC Grade, Phosphate Buffer, Tetrahydrofuran (THF), Ammonium acetate buffer, Hydrochloric Acid, Methanol HPLC Grade, Sodium Hydroxide pellet, Water: Distill water(Milli-Q), HPLC grade water.

Preparation of Mobile Phase: Acetonitrile:6.8g/L Ammonium acetate buffer (15:85) (pH 5)

Preparation of Standard Solution: Weigh accurately 70 mg of Esmolol hydrochloride WS and transfer into 100 ml volumetric flask. Add water about 60 ml then dissolved it then mark up to 100 ml with water. (Concentration of Esmolol hydrochloride is 700 µg /ml)

Preparation of Sample solution: Weigh 500 mg (Equivalent to 70 mg) of topical gel and transfer into 100 ml volumetric flask. Add water about 60 ml then dissolved it then mark up to 100 ml with Mobile Phase.

(Concentration of Esmolol hydrochloride is 700 µg /ml)

Preparation of system suitability solution: Take standard solution for system suitability.

System suitability requirement:

Tailing factor: NMT 2.0 for Esmolol peak

Relative standard deviation: NMT 2.0%   

Chromatographic Conditions:

Column: Intsil C₁₈ column (300 mm x 3.9 mm, 5 µm)

Flow rate: 1.5ml/min

Total run time (min): 20 min.

Injection volume (µl) : 20

Column Temperature(°C): Ambient

Detection Wavelength(nm) : UV 221 nm

IDENTIFICATION AND CHARACTERIZATION

The identification of taken standard API for experimental work had done for confirmation of its identity, standard quality and purity. The identification had done by taking IR and UV spectra, solubility study and melting point determination.

Solubility Study

The solubility of Esmolol Hydrochloride was practically determined by taking 100 mg of the drugs in 100 ml volumetric flasks, adding required quantity of solvent at room temperature and shaken for few minutes. Solubility data for each study was observed and recorded in Table 2.0

Table 1.0 Solubility table as per IP’2014 specification

Table 2.0 Solubility Data of Esmolol Hydrochloride

Melting point of Esmolol Hydrochloride has been determined. The melting points of the compounds were taken by open capillary method.

Table 3.0 Melting Point of Drugs

The IR Spectra of Esmolol Hydrochloride with its functional group identification, were shown in the following graph. IR spectra scanning of sample: Esmolol Hydrochloride

Figure 1.0 IR Spectra of Esmolol Hydrochloride

Table 4.0 IR Spectral interpretation of Esmolol Hydrochloride

SELECTION OF WAVELENGTH:

The RP-HPLC Method used UV detection which is depends on proper selection of detection wavelength. An ideal wavelength is important to get good response for the drugs that can be detected. To determine wavelength for measurement, standard spectra of Esmolol Hydrochloride was scanned between 200- 400nm against diluents. Peak is obtained at 221nm.

Figure 2.0 Overlay UV Spectra of Esmolol Hydrochloride

SELECTION OF COLUMN:

For RP-HPLC method, various columns are available but our main aim is to resolve drug in the presence of excipients. So the C18 column was selected for estimation of Esmolol Hydrochloride. Intsil C18 column (300 mm x 3.9 mm, 5 µm) column was chosen to give good peak shape and high resolution, which also provides high peak symmetry, good retention to drug and facilitates the separation of the drug without the interference of excipients within short run time.

Snapshot of Intsil C₁₈ column (300 mm x 3.9 mm, 5 µm):