Received on 25.07.2025 Revised on 20.08.2025

Accepted on 12.09.2025 Published on 08.10.2025

Available online from October 15, 2025

Asian Journal of Pharmaceutical Analysis. 2025; 15(4):288-294.

DOI: 10.52711/2231-5675.2025.00045

This work is licensed under a Creative Commons Attribution-NonCommercial-ShareAlike 4.0 International License. Creative Commons License.

1. INTRODUCTION:

Metoprolol succinate^1-2is a cardioselective beta1-adrenergic receptor blocker used in the management of hypertension, angina pectoris, and heart failure. Its mechanism of action involves the selective blockade of beta1-adrenergic receptors located primarily in cardiac tissue, which leads to a reduction in heart rate, myocardial contractility, and cardiac output, thereby decreasing oxygen demand. Although metoprolol is beta1-selective, this effect is not absolute, and at higher plasma concentrations it may also inhibit beta2 receptors found in bronchial and vascular smooth muscles. It exhibits no intrinsic sympathomimetic activity and demonstrates membrane-stabilizing properties only at concentrations far exceeding those needed for beta-blockade.

Chemically, metoprolol succinate is the succinate salt of (±)-1-(isopropylamino)-3-[p-(2-methoxyethyl) phenoxy]-2-propanol, with a molecular formula of C₃₄H₅₆N₂O₁₀ and a molecular weight of 652.8g/mol. It appears as a white crystalline powder and is freely soluble in water and methanol, sparingly soluble in ethanol, and practically insoluble in several organic solvents. The drug is formulated as an extended-release tablet designed for once-daily administration, providing consistent plasma levels over a 24hour period. The structural formula includes a phenyl ring, a methoxyethyl group, and an isopropylamino moiety on a propanol backbone. Metoprolol succinate is available in strengths equivalent to 25mg, 50mg, 100mg, and 200 mg of metoprolol tartrate. The chemical structure of metoprolol succinate is shown in Figure1.

Fig No.1.Structure of metoprolol Succinate

Several analytical methods have been reported for the estimation of Metoprolol Succinate, both as a single drug and in combination with other compounds. However, most existing methods focus on its analysis in biological matrices or multi-drug formulations. Very few chromatographic methods are available for the estimation of Metoprolol Succinate as a single active ingredient in tablet dosage forms. This indicates a need for a simple, specific, and validated RP-HPLC method suitable for routine analysis in pharmaceutical quality control. The method developed in this study is designed to be rapid, accurate, precise, and economical, and complies with regulatory validation requirements.

High- pressure liquid chromatography (HPLC):^3-7

High-Performance Liquid Chromatography, also called High-Pressure Liquid Chromatography, is a column chromatography technique used in biochemistry and analysis to separate, identify, and quantify active compounds. This analytical technique is widely used to separate, identify, and quantify each constituent in a mixture. HPLC is a sophisticated column-based liquid chromatography method. In HPLC, the solvent is forced under high pressures of up to 400 atmospheres to separate elements based on relative affinities, rather than flowing naturally through the column. HPLC consists of a stationary phase column, a pump, and a detector to measure molecule retention periods. The retention period is influenced by interactions between the stationary phase, compounds under analysis, and solvents used. Small amounts of samples are added to the mobile phase stream, causing chemical or physical interactions with the stationary phase to delay the process. The amount of retardation depends on the analyte and stationery /mobile phase composition. Retention time refers to the time it takes for an analyte to elute. Solvents are miscible combinations of water or organic liquids. Gradient elution was employed to adjust the mobile phase composition during the study. The gradient separates analyte mixtures according to their affinity for the current mobile phase. The stationary phase and analyte determine the appropriate solvents, additives, and gradients.

Analytical method development:⁸

New procedures are being developed to evaluate novel products in the absence of established techniques. To detect pharmacopoeial or non-pharmacopoeial products, new techniques have been developed to minimize time and increase precision and strength. These approaches are refined and validated by preliminary tests. Alternative methods are developed and implemented to replace the current approach in a comparative laboratory setting, taking into account all available advantages and disadvantages.

2. MATERIAL AND METHODS:

Materials- Metoprolol Succinate was procured from a reliable source. Methanol (HPLC grade), 0.1% OPA, and other reagents were of analytical grade.

Instrumentation:

· HPLC System: Agilent 1260 Infinity II

· Detector: UV Detector

· Column: Phenomenex C18 (250 × 4.6mm, 5µm)

· UV Spectrophotometer: Jasco UV 550

Chromatographic Conditions

· Mobile phase: Methanol: 0.1% OPA (60:40)

· Flow rate: 1.0mL/min

· Detection wavelength: 222nm

· Injection volume: 20µL

· Column temp: 35°C

· Run time: 6min

HPLC method development:

Preparation of Standard Solution:

· Accurately weighed 25mg of Metoprolol Succinate and transferred it into a 25mL volumetric flask.

· Added 15–20mL of distilled water and sonicated to dissolve completely.

· Diluted to the mark with distilled water to prepare a 1000PPM stock solution.

· Pipetted 0.5mL of the stock solution into a 25mL volumetric flask and diluted to the mark with water to obtain a 20PPM solution.

Preparation of Standard Stock Solution for chromatographic development:

Metoprolol Succinate Standard stock solution was prepared by dissolving 25 mg Metoprolol Succinate into a 25 mL clean and dried volumetric flask added about 15-20 mL of Water to dissolve it completely and made volume up to the mark with Water (1000 PPM). Further diluted 2 ml of stock solution to 20 mL with mobile phase. (100 PPM).

Preparation of System suitability test (Metoprolol Succinate standard solution):

Weighed about 25mg of Metoprolol Succinate and transferred in 50mL volumetric flask, added 35 mL of Water, sonicated to dissolve it, made volume up to the mark with Water. Pipette out 0.5Ml from standard stock solution and transferred into 25ml volumetric flask and made volume up to the mark with mobile phase (10 µg/mL working concentration), chromatograms were Recorded.

Analysis of marketed Test sample:

Marketed test sample Having Name Met XL 50 mg tablets are selected for analysis and for Doing validation.

Sample preparation of Marketed test sample:

Weighed 20 tablets transferred in mortar pestle and crushed to fine powder. Mixed the contents with butter paper uniformly. Weighed the powder material equivalent to 2mg of Safinamide (81.20mg of powder material) and transferred to clean and dried 50mL of volumetric flask. Added 35 mL of Water, sonicated for 10minutes with intermittent shaking. After 10 minutes allow to cool the solution to room temperature and made volume up to the mark with Water. Filtered the solution through suitable 0.45µ syringe filter discarding 3-5mL of initial filtrate. Further diluted 1.5ml of filtered stock solution to 25ml with mobile phase (30mcg of Safinamide), injected the resultant solution and chromatograms were recorded and results are recorded.

Table No.1. Sample Prepared in duplicate. Summary of sample preparation as follows:

Sample	Sample (mg)	Diluted to (mL)	Volume taken	Diluted to (mL)
Sample 1	128.2	50	0.5	25
Sample 2	128.8	50	0.5	25

Formula for % Assay calculation:

₁₀₀

So, Placebo prepared at lab level by using formula as follows:

Table No.2 Placebo Preparation of metoprolol succinate

Sr. No.	Ingredients	Role	Qty (mg)
1	Lactose	Filler	80
2	Starch	Binder	5
3	Magnesium stearate	Lubricant	5
4	Talc	Glidant	5
5	crospovidone	Disintegrants	5
Total			100 mg

Total 10 gm of placebo prepared

3. VALIDATION OF RP-HPLC METHOD:^9-13

The developed method for estimation of Metoprolol Succinate was validated as per ICH guidelines for following parameters.

1. Filtration Study:

Filtration study of an analytical procedure checks the interference of extraneous components from filter, deposition on filter bed and compatibility of filter with sample. this study was conducted with Metoprolol Succinate Test sample (Tablet solution). filtration study carried out with unfiltered and filtered test solution. During filtration activity 0.45 µm PVDF and 0.45µm Nylon syringe filters used by discarding 5 m). filtration sample.

2. Stability Of Analytical Solution

Stability study was conducted for standard and test sample solution. Stability study was performed at normal laboratory conditions. the solution was stored at normal illuminated laboratory conditions and analyzed after 12 Hours and 24hours. Standard and Test solution stability study was perform hours. Standards the difference between results of test solution at each stability time point to that of initial.

3. Specificity:

Specificity is the ability to access unequivocally the analyte in the presence of components Which may be expected to be present. Following solution shall be prepared and injected to prove the specificity nature of the Method.

· Blank (Mobile phase as a diluent)

· Placebo

Placebo Sample solution preparation:

Weighed 103.74mg of placebo material (Which is equivalent to 25mg of Metoprolol Succinate) and transferred to clean and dried 50mL of volumetric flask. Added 35 mL of Water, sonicate for 10minutes with intermittent shaking. After 10 minutes allow to cool the solution to room temperature and made volume up to the mark with Water. Filtered the solution through suitable 0.45µ Nylon syringe filter discarding 3-5mL of initial filtrate. Further dilute 0.5ml of filtered stock solution to 25ml with mobile phase, injected the resultant solution and chromatograms were recorded.

4. Linearity and Range:

Preparation of linearity solution:

The linearity of an analytical procedure is its ability (within a given range) to obtain test results Which are directly proportional to the concentration (amount) of analyte in the sample. 5 levels of Linearity was performed from 50% to 150% of working concentration

linearity metoprolol succinate stock solution:

Weighed 25mg of Metoprolol Succinate and dissolved in 50mL with Water. Further diluted 5ml of stock solution to 50mL with Water (50µg/mL).

5. Limit of detection (lod) and limit of quantitation (loq):

Detection limit:

The detection limit of an individual analytical procedure is the lowest amount of analyte in a Sample which can be detected but not necessarily quantitated as an exact value.

Quantitation limit:

The quantitation limit of an individual analytical procedure is the lowest amount of analyte in A sample which can be quantitatively determined with suitable precision and accuracy.

As per ICH Q2R1 guidelines LOD and LOQ was determined by using the approach Based on The Calibration Curve in which residual standard deviation of a regression line was calculated And determined the LOD and LOQ by using following formula:

LOD = 3.3 σ / S

LOQ = 10 σ / S

Where,

σ = residual standard deviation of a regression line

S = Slope of regression line

6. Accuracy (% Recovery):

The accuracy of the analytical procedure expresses the closeness of agreement between the Value which is accepted either as a conventional true value or an accepted reference value and the value of the value found, Accuracy will be conducted in the range from 50 % to 150% of working concentration. Solution of each accuracy level was prepared in triplicate. Calculated % Recovery for each Sample, Mean %recovery for each level and overall recovery and also calculated %RSD for Each level and % RSD for overall recovery.

7. Precision:

Precision of an analytical procedure expresses the closeness of agreement between a series of Measurements obtained from multiple sampling of the same homogeneous test under the Prescribed conditions. Precision is of two types, Repeatability and Intermediate precision.

8. Robustness:

The robustness of an analytical procedure is a measure of its capacity to remain unaffected by Small, but deliberate variations in method parameters and provides an indication of its reliability During normal usage.

Determination: Standard solution was injected under different chromatographic conditions as Shown below.

a) Changes in flow rate by ±10%. (±0.1ml/min)

b) Change in column oven temperature. (±2ºC)

c) Change in wavelength (±3nm)

Table No.3.Optimized Chromatographic Condition

Parameter	Description
Mode	Isocratic
Column Name	Phenomenex C18, 250 mm X 4.6mm ID, 5 μm
Detector	UV Detector
Injection Volume	20 µl
Wavelength	222 nm
Column Oven temp	35ºC
Mobile Phase	Methanol: 0.1% OPA in Water (60:40%V/V)
Flow Rate	1.0 ml/min
Diluent	Mobile phase
Run time	6 Minutes

Fig. No.2 Chromatogram of optimization trial

4. RESULT AND DISCUSSION:

1) Filtration Study:

Filtration study of an analytical procedure checks the interference of extraneous components from filter, deposition on filter bed and compatibility of filter with sample. Performed on tablet test sample.

Table No.4. Results of Filter study

Sample description	Area	% Absolute difference
Unfiltered	4230205	NA
0.45 µ PVDF filter	4199825	0.72
0.45 µ Nylon filter	4209304	0.49

2) Solution Stability:

Stability study was conducted for Standard as well as Test Sample. Stability study was performed at normal laboratory conditions. The solution was stored at normal illuminated laboratory conditions and analyzed at initial, after 12 hours and 24 hours.

Table No. 5. Results of Solution stability.

Sample solution			Standard solution
Time point	Area	% Absolute difference	Time point	Area	% Absolute difference
Initial	4231978	NA	Initial	4269302	NA
12 Hours	4203628	0.67	12 Hours	4245745	0.55
24 Hours	4191503	0.96	24 Hours	4235978	0.78

3) Specificity:

Specificity is the ability to access unequivocally the analyte in the presence of components which may be expected to be present.

Blank and Placebo solution prepared and injected to check interference at R.T. of Metoprolol succinate.

Table No.6 Results of Specificity.

Description	Observation
Blank	No interference at R.T. of Metoprolol succinate due to blank
Placebo	No interference at R.T. of Metoprolol succinate due to placebo

4) Linearity and Range:

Linearity of an analytical method is its ability to elicit test results that are proportional to the concentration of analyte in samples within a given range.

Fig No.3 Calibration curve of Metoprolol succinate

Table No. 7 Linearity Data for Metoprolol succinate

Level	Conc (µg/mL)	Area	Mean	% RSD
50%	5.00	2086036	2083752	0.114
		2081304
		2083915
75%	7.50	3176097	3173468	0.092
		3170305
		3174001
100%	10.00	4282016	4279304	0.060
		4278985
		4276910
125%	12.50	5322973	5328138	0.086
		5329680
		5331761
150%	15.00	6379971	6384673	0.072
		6384950
		6389097

Table no. 8. Data of linearity of Metoprolol succinate:

Sr No	Parameter	Result value	Acceptance criteria
1	Beer's linearity range	5.0-15µg/mL	NA
2	Correlation coefficient (R²)	0.99994	NLT 0.98
3	Intercept	-52737.8	To be report
4	Slope	430260.48	To be report
5	% RSD for area at each level	NA	NMT 2.0

The respective linear equation for Metoprolol succinate was

Y = M X + C

Y = 430260.48 X + -52737.8

Where, X = concentration of Analyte in µg/mL

Y = is area of peak.

M = Slope

C= Intercept

Conclusion:

From the calibration curve it was concluded that the Metoprolol succinate shows linear response in the range of 5.0-15.0μg/ml. The Regression value was found well within the limit.

5) Limit of Detection (LOD) and Limit of Quantitation (LOQ):

σ = 18476.36232 (Residual standard deviation of a regression line)

s = 430260.48 (Slope)

Detection limit (LOD):

LOD = 3.3 σ / S

LOD = 3.3 x 18476.36232 / 430260.48

LOD = 0.142 µg/mL

Quantitation limit (LOQ):

LOQ = 10 σ / S

LOQ = 10 x 18476.36232 / 430260.48

LOQ = 0.429µg/mL

6) Accuracy (Recovery):

The accuracy of an analytical method is the closeness of test results obtained by that method to the true value. The accuracy of an analytical method is determined by applying the method to analyzed samples to which known amounts of analyte have been added.

Table no .9 Result and statistical data of Accuracy of Metoprolol succinate

Level (%)	Area	Recovered conc (µg/mL)	Added conc (µg/mL)	% Recovery	Mean Recovery	% RSD
50	2136025	5.00	5.04	99.21	99.61	1.0473
	2145781	5.02	5.08	98.82
	2169810	5.08	5.04	100.79
100	4263025	9.98	10.12	98.62	99.37	0.7976
	4279685	10.02	10.00	100.20
	4260045	9.97	10.04	99.30
150	6396197	14.97	15.12	99.01	99.80	1.1586
	6478502	15.17	15.00	101.13
	6377150	14.93	15.04	99.27

Overall Recovery: 99.40 %

% RSD for Overall Recovery: 0.644

Sr.No	Parameters	Interday Precision	Intraday Precision
1	Mean	98.588	98.91
2	SDT	1.13309	1.139889
3	%RSD	1.149	1.152

Change in Parameter	R.T.	Standard area	Asymmetry	Theoretical plates
Wavelength by +3 NM (225 NM)	3.04	4008250	1.04	8424
Wavelength by -3 NM (219 NM)	3.04	4260369	1.01	8196
Flow rate by +10% (1.1mL/min)	2.78	3725036	1.01	7942
Flow rate by -10% (0.9mL/min)	3.38	4823910	1.05	9013
Column oven temp by +2ºC (37 ºC)	3.05	4240692	1.03	8172
Column oven temp by -2ºC (32 ºC)	3.06	4246978	1.06	8360