Method Development and Validation of Metoprolol Succinate by RP-HPLC Method

Metoprolol succinate is a selective β-adrenergic receptor blocker widely used in the management of various cardiovascular disorders. It is primarily indicated for the treatment of hypertension, angina pectoris, heart failure and for the prevention of myocardial infraction. By selectively inhibiting β1 receptors in the heart, metoprolol reduces heart rate, myocardial contractility and cardiac output thereby reducing blood pressure and decreasing the oxygen demand of the heart. Metoprolol succinate is well absorbed after oral administration and undergoes hepatic metabolism, mainly by cytochrome P450 enzyme. This drug is commonly prescribed in long term cardiovascular therapy.

DRUG PROFILE:

Figure.no:1 Chemical structure of Metoprolol succinate

IUPAC Name: ?4-(2-methoxy ethyl) phenoxy?-3-?1-methyl ethyl amino?-2-propane butanedioate.

Molecular formula: C34H56N2O10                       

Molecular weight: 652.82g/mol

Solubility: Methanol and  Purified water

MECHANISM OF ACTION:

Metoprolol succinate is a cardio selective, β1-adrengeric receptor blocker that lowers heart rate, blood pressure and cardiac workload by competitively inhibiting catecholamine binding in the heart. As a long-acting extended-release formulation, it provides sustained 24-hours antagonism, reducing renin secretion and reducing oxygen demand making it effective for the hyper tension, angina and heart failure.

RP-HPLC METHOD

Chromatographic Condition

The isocratic mobile phase consisting of methanol and  0.1%Triethylamine in the ratio of (70:30), flowing through the column and constant flow rate of 1.0ml/min. The mobile phase was filtered through 0.45µm membrane filter and was degassed before used 30 mins. C18 Column (250 mm x 4.6 mm,5µ) as used as a stationary phase.

MATERIALS AND METHODS:

Drug Sample and Formulation used

Metoprolol Succinate bulk  drug  were Gifted By Madras Pharma,  Chennai.

Chemicals and Solvents used

All the Chemicals Used were of analytical Grade as well as HPLC grade procured from Great scientific Laboratory and Sumison Laboratory from Tiruvannamalai and Chennai.

The Chemical used for the Study were,

• Methanol (HPLC GRADE)
• Milli-Q Water (HPLC WATER)
• Triethylamine Buffer (Analytical grade)

Apparatus and Glassware’s

• 100ml volumetric flask
• 10ml volumetric flask
• Beakers
• Glass rod, etc.

Instruments

Instruments employed for the study were,

1. SHIMADZU AUX – 220 DIGITAL BALANCE
2. SIMADZU prominence HPLC iLC2030 plus
3. Sonica Ultra Sonic Cleaner-Model 2200 plus
4. ELICO PH Meter (Model LI -120)
5. Melting Point Apparatus.

METHOD DEVELOPMENT:

Prepartion of Mobile Phase

1. For Trail Chromatogram of Metoprolol Succinate:

Methanol and 0.1 % v/v of Triethylamine in milli-q water(60:40) was taken into a 1000 ml volumetric flask and mixed properly. Then, this mobile phase is sonicated for 15 min and used as the mobile phase by isocratic elution method.

2. For Optimized Chromatogram of Metoprolol Succinate:

Methanol and 0.1 % v/v of Triethylamine  in milli-q water (70:30)  was taken into a 1000 ml volumetric flask and mixed properly. Then, this mobile phase is sonicated for 15 min and used as the mobile phase by isocratic elution method.

Preparation of Standard Solution

Accurately weighed 25 mg of metoprolol succinate and transferred into 100 ml volumetric flask and dissolved with (methanol and 0.1 % v/v of Triethylamine Solution (70:30) ) are added and to adjusted the volume up to the mark of volumetric Flask. From this 1ml was taken and added into 10ml volumetric flask with mobile phase are added and to adjusted the volume upto the mark of the volumetric flask.

Initial Separation Condition: Trail 1

For Method Development of Metoprolol Succinate Using to conducted the First trial injections were performed with mobile phase of  Methanol and 0.1 % v/v of Triethylamine in milli-q water (60:40) . 

Chromatogram Of Trail 1

Metoprolol Succinate – Optimized Chromatogram:

We get Optimized Chromatogram with 70:30 ratios of mobile phases of Methanol and 0.1 % v/v of Triethylamine in milli-q water to identify the best chromatographic conditions.

Optimized Chromatogram

VALIDATAION PARAMETERS:

A. Linearity

Linearity studies were carried out in the concentration range of 0.5μg/ml, 0.75μg/ml, 1μg/ml, 1.25μg/ml and 1.5μg/ml.  The sample solution was made from the stock solution the readings were obtained by measuring the absorbance at 234 nm and presented in table and linearity curve.

Linearity Chromatogram in 50%

Linearity Chromatogram in 75%

Linearity Chromatogram in 100%

Linearity Chromatogram in 125%

Linearity Chromatogram in 150%

B. Accuracy

Accuracy of the method was evaluated by the standard addition technique at three concentration levels of 80%, 100%, and 120%. Known amount of standard drug solution were added to the pre - analyzed sample to be applied final concentration 20µg/ml, 25µg/ml and 30µg/ml. The solution were diluted with mobile phase composed of Methanol and 0.1%Triethylamine in Water. In the ratio of 70:30 and analyzed  under optimized condition.    

Accuracy Chromatogram in 80%

Accuracy Chromatogram in 100%

Accuracy Chromatogram in 120%

The  %RSD value less than 2% was found.

C. Precision

1ml of stock solution was pipetted out into 10ml volumetric flask diluted upto mobile phase with the mark. Mixed well and filtered through 0.45µm filter. Therefore, the concentration of the resultant solution was 25µg/ml. The solution was taken and injected for five times within the same day and the chromatogram was recorded. The peak area was measured to calculate the % Relative standard deviation value.

Chromatogram of Precision

Table: Interday Precision

Table: Intraday Precision

The %RSD Value less Than 2% was Found. So the  method is highly precise and reproducible.

D. Robustness:

The robustness of the method was determined by introducing small changes in HPLC parameters such as changing in wavelength.

E. RUGGEDNESS:

The ruggedness of the proposed method was evaluated by applying the developed procedure assay of 25μg/ml of Metoprolol Succinate using the same instrument by two different analyst under the same optimized conditions at different days.