Development And Validation Of UV Method For Simultaneous Estimation Of Metoprolol Succinate And Azelnidipine In Pharmaceutical Dosage Form

Azelnidipine is a calcium channel blocker that belongs to the dihydropyridine class. Japan's Daiichi-Sankyo Pharmaceuticals, Inc. is the company responsible for marketing it. When compared to other calcium channel blockers, it has a delayed onset of action and generates a long-lasting drop in blood pressure. Patients who have hypertension can have a progressive reduction in their blood pressure when they take azelnidipine, which is a vasodilator. Because of its vasodilatory effects, azelnidipine does not cause reflex tachycardia, in contrast to other drugs that belong to the same medication class. Metoprolol is a cardioselective beta-1-adrenergic receptor inhibitor that inhibits beta1 receptors in a competitive manner while having limited or no effects on beta-2 receptors when administered orally to humans in dosages of less than 100 mg. Through its negative inotropic and chronotropic actions, it brings to a reduction in cardiac output.

MATERIAL AND METHODS:

1. Method Parameters:

a. Diluent: 0.1% Perchloric acid : Acetonitrile (50: 50%, v/v)

Preparation of 0.1% Perchloric acid:

Add 0.1 ml of Perchloric acid in 100 ml of Water, Mix and filtered.

b. Wavelength: ?1 = 224 nm; ?2 = 260 nm

2. Standard Preparation:

a. Metoprolol Standard Stock Solution-I (MSSS-I):

i. Initially Prepare a Standard Stock Solution (MSSS-I) of by adding  25 mg of Metoprolol in 10 ml volumetric flask & add 5 ml diluent, mix for 2 minutes and make the volume to 10 ml with diluent. (Conc. of Metoprolol = 2500 µg/ml).

b. Azelnidipine Standard Stock Solution-II (ASSS-II):

i. Then prepare a Standard Stock Solution (ASSS-II) of Azelnidipine by adding 8 mg in 10 ml volumetric flask & add 5 ml diluent, mix for 2 minutes and make the volume to 10 ml with diluent. (Conc. of Azelnidipine = 800 µg/ml).

c. Then add 0.1 ml of MSSS-I &0.1 ml ASSS-I in 10 ml volumetric flask and add 5 ml diluent and vortex and make up the volume with diluent. (Conc. of Metoprolol= 25 µg/ml &Azelnidipine = 8 µg/ml).

3. Selection of Wavelength:

25µg/ml of MET Working Standard and 8µg/ml of AZD Working Standardwere scanned in the UV range of 190-400 nm. The overlay of both the spectrum was recorded. From the overlain spectra wavelengths 224 nm (?max of MET) and 260 nm (?max of AZD) were selected for analysis of both drugs using simultaneous method. (?1-224 nm and ?2-260 nm).

The absorbance at ?1 and ?2 was measured and the concentration was calculated using following formula;

Where,

Cx and Cy are the concentrations of Metoprolol and Azelnidipine, respectively,

A1 and A2 are the absorbances of sample at ?1 and ?2, respectively,

ax1 and ax2 are the absorptivity of Metoprolol at ?1 and ?2, respectively,

ay1 and ay2 are the absorptivity of Azelnidipine at ?1 and ?2, respectively.

UV Method Validation

a. Linearity:

1. 5 samples of varying concentrations ranging from 80% to 120% were made.
2. The concentrations are given below
3. The sample preparations are given as below;
4. X ml of BSSS-I and Y ml of MSSS-II was diluted to 10 ml.

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b.  LOD/ LOQ:

Can be calculated by using AVONA Technique.

c. Repeatability :

A single sample was prepared as described and 6 injections were made from same sample; checked for RSD.

d. Accuracy:

1. Samples were made of 80%, 100% and 120% concentration as per Table 1.
2. Samples were injected in triplicate to calculate % RSD.
3. % recovery was also calculated.

d. Intra- & Inter-day Precision:

• The working standard and drug product samples were freshly prepared and analysed in morning and evening for Intra-day precision.
• The same working standard and drug product were used for analysis on 2nd day for inter-day precision.
• % RSD for Assay was calculated for the confirmation of precision.

RESULT :-

UV method  

Selection of Wavelength

The Standard and Sample solution was scanned from 190 to 400 nm by using UV-VIS spectrophotometer against Diluent (0.1% Perchloric acid: Acetonitrile (50:50)) as blank and the maximum absorption of standard and sample solution were recorded.

Result:

1. The maximum absorption for Metoprolol was found to be 224 nm.
2. The maximum absorption for Azelnidipine was found to be 260 nm.

The UV scans for both the drugs is given below:

Figure 1: UV Scan of Metoprolol

Figure 2: UV Scan of Azelnidipine

 UV Method Validation of Metoprolol and Azelnidipine

1. Specificity

It was confirmed with blank and working standard run that there was zero absorbance of blank at set lambda in UV Spectrophotometer.

2. Linearity

The peak response is directly proportional to the concentration of drug and was found to be linear in the range of 8-12µg/ml.

The linearity data for Metoprolol and Azelnidipine is give below:

       
           
       
    Table 2: Linearity data for Metoprolol

Figure 3: Linearity graph of Metoprolol

From the above data it was found that the correlation coefficient was found to be 0.999 for both the drugs i.e. Metoprolol and Azelnidipine respectively, which was found to be within the acceptance criteria of 0.998.

C. LOD and LOQ

Based on the linearity data, LOD and LOQ was calculated and reported as below:

1. The LOD & LOQ for Metoprolol were found to be 1.23µg/ml and 3.72 µg/ml.
2. The LOD & LOQ for Azelnidipine were found to be 0.31 µg/ml and 0.94 µg/ml.

d. Repeatability

Repeatability was performed for both the APIs, the recorded absorbance is shown below:

e. Accuracy

The accuracy was performed at 3 different levels i.e. 80%, 100% and 120%. The accuracy data for Metoprolol and Azelnidipine is given below:

• The %RSD of three replicates of Metoprolol for accuracy level 80%, 100% and 120% was found to be 0.60%, 0.28% and 0.62% respectively.
• The % recoveries for accuracy level 80%, 100% and 120% was found to be 99.81%,    99.95% and 100.85%respectively.

• The %RSD of three replicates of Azelnidipine for accuracy level 80%, 100% and 120% was found to be 0.70%, 0.55% and 0.60% respectively.
• The % recoveries for accuracy level 80%, 100% and 120% was found to be 99.34%,    100.24 % and 99.94% respectively.

f. Intra & Inter day Precision

The Standard solution of Metoprolol and Azelnidipine were examine for Intra and Inter day Precision, the data is shown below:

• The % Assay for Metoprolol for Morning, Evening and Day 2 were found to be 100.20%, 99.76% and 99.72%, respectively.
• The % Assay for Azelnidipine for Morning, Evening and Day 2 were found to be 99.75%, 99.25% and 99.50%, respectively.
• The %RSD for intra and Inter day Precision of Metoprolol and Azelnidipine were found to be 0.22% and 0.25%, respectively.
• Hence, the working standard Metoprolol and Azelnidipine is stable for 2 days as no significant variation was observed.

7.  Assay

The data of the Assay of Metoprolol and Azelnidipine is given below:

Table 10: Assay of Metoprolol and Azelnidipin