Analytical Method Development and Validation of Metoprolol Succinate by High Performance Liquid Chromatography and Ultraviolet Spectroscopy Technique

 

Shashank Soni¹*, Veerma Ram², Divya Verma³, Anurag Verma⁴

¹Department of Pharmaceutics, Amity Institute of Pharmacy, Amity University, Uttar Pradesh, Lucknow Campus, Lucknow, India.

²School of Pharmaceutical Sciences and Technology, Sardar Bhagwan Singh University, Dehradun, India

³Department of Chemistry, University of Lucknow, Lucknow, India.

⁴School of Pharmaceutical Sciences, IFTM University, Moradabad, India.

 

ABSTRACT:

Metoprolol Succinate (MS) is a β 1- blocker and a drug of choice for a patient suffering from cardiovascular diseases. Referable to the highly probable over dosage, a rapid and easy to execute a method for the quantitative determination of MS in biologic matrices would be utilitarian in its therapeutic monitoring as well as in intoxication cases. The report presents two validated methods for detection of MS by UV-VIS and HPLC. The parameters targeted for the validation were: specificity, range, detection and quantification limits, accuracy, and precision and were determined according to International Conference on Harmonization ICH Q2 (R1) guidelines. HPLC was conducted on the Water Spherisorb® analytical column (5µm, 4.6 x 250mm). The mobile phase consisted of a buffer (ACN: orthophosphoric acid: water; pH 3.0) and the flow rate was maintained at 2.0ml/min. MS was monitored using the Water Breeze 2 system equipped with photodiode array detector (λ = 224nm) and also by a UV-VIS spectrophotometer (λ = 224nm) by Shimadzu UV 1800. Linearity was observed in concentration range of 10–50µg/ml by HPLC and 2-10µg/ml by UV spectroscopy method. The correlation coefficient was 0.9992 and 0.9998. All in all, the system suitability parameters were set up inside the range as per ICH. The performed method is rapid, cost-effective and can be used as a quality-control method for routine quantitative analysis of MS in pure and pharmaceutical dosage forms.

 

 

 

INTRODUCTION:

Chemically Metoprolol Succinate (MS) is (Rs)-1-(Isopropylamino)-3-[4-(2-methoxyethyl) phenoxy] propane-2-ol which comes under a category of drugs acting on the cardiovascular system used in the treatment of hypertension and various cardiovascular disorders. It acts as β1- an adrenergic blocking agent used in the treatment of acute myocardial infarction (MI), heart failure, angina pectoris and mild to moderate hypertension (Ptaszynski et al., 2012).

 

It may as well be used for supraventricular and tachyarrhythmia and prophylaxis for migraine headaches. Metoprolol is structurally like bisoprolol, acebutolol, and atenolol in that it has two substitutes in the para position of the benzene ring. The β1-selectivity of these agents is thought to be due in part to the large substituent in the para position. At low doses, Metoprolol selectively blocks cardiac β1-adrenergic receptors with little activity against β2-adrenergic receptors in the lungs and vascular smooth muscle. Receptor selectivity decreases with higher dosages. Unlike, propranolol and pindolol, metoprolol does not exhibit membrane-stabilizing or intrinsic sympathomimetic action. Membrane-stabilizing effects are just noted at doses much more eminent than those needed for β-adrenergic blocking action. Metoprolol possesses a single chiral center and is administered as a racemic mixture (Sharma et al., 2014). Several dosage forms of MS available in commercial markets like tablet, extended release (ER), and injection. MS also comes in combination with hydrochlorothiazide, L-arginine etc. It has an average molecular weight of 267.3639 and monoisotopic of 267.183443671. It shows rapid and complete absorption of 50 % through the gastrointestinal tract, having protein binding of 12%, LD₅₀=5500 mg/kg (orally in rats), toxic effects include bradycardia, hypotension, bronchospasm, and cardiac failure (Sarkar et al., 2008). LD₅₀=2090 mg/kg (orally in mice). MS interacts with Beta 1- adrenergic receptors and cytochrome P450 2D6 enzyme and affects the different alleles like CYP2D6*3, CYP2D6*4, CYP2D6CYP2D6*6*5, CYP2D6*6, CYP2D6*7, CYP2D6*11, CYP2D6*12 and causes the deletion of some base pairs after interacting with alleles. MS shows the several experimental properties like melting point 120 0C, water solubility 1.699 ± 004 mg/L (at 25 °C), log P 1.88, Caco2 permeability -4.59, log S - 2.8, pKa strongest acidic and basic 14.09 and 9.67 respectively, physiological charge 1, hydrogen acceptor and donor count 4 and 2 respectively, polar surface area 50.72 Å, rotable bond count 09, refractivity 76.7 m³·mol^-1 (Brunton et al., 2007; Blomqvist et al., 1988). It shows predicted ADMET properties like human intestinal absorption and Caco2 permeability positive behavior whereas negative effects on crossing the blood-brain barrier. It comes under the category of Biopharmaceutical Class System (BCS) I drug which states that it is extremely soluble and highly permeable (Wikstrand et al., 2003).

 

The analytical procedure states the manner of doing the routine analysis. It describes in detail about the steps necessary to perform each analytical experiment or procedures. This may include, but is not limited to the sample, the reference standard and the reagents preparations, use of the apparatus, generation of the calibration plot and use of the formula for the calculation, etc. (ICH Q2B, 1996). The discussion of the validation of analytical procedures is directed to the four most common types of analytical procedures: Identification tests; Quantitative tests for impurities content; Limit tests for the control of impurities; Quantitative tests of the active moiety in samples of drug substance or drug product or other selected component(s) in the drug product (Green., 1996). The main objective of the analytical procedure should be clearly understood since this will govern the validation characteristics which need to be evaluated. Typical validation characteristics which should be as listed like accuracy, precision, repeatability, intermediate precision, specificity, detection limit, quantitation limit, linearity, and range. Furthermore, revalidation may be necessary for the following conditions like changes in the synthesis of the drug substance; changes in the composition of the finished product; changes in the analytical process (Sahoo et al., 2011). Method validation is the procedure applied to affirm that the analytical procedure used for a specific test is suitable for its intended use. Results from method validation can be used to interpret the quality, reliability, and consistency of analytical results; it is an integral part of any good analytical practice (ICH Q2B, 1996). Analytical methods need to be validated or revalidated in the specific conditions like before their introduction into routine use; whenever the conditions change for which the method has been validated (e.g., An instrument with different characteristics or samples with a different matrix); and whenever the method is changed and the change is outside the original scope of the method (Shrivastava et al., 2011).

 

From the literature survey, it was found that various methods were used for the estimation of MS such as spectrophotometric method, High-Performance Liquid Chromatography (HPLC) method (Bhadra et al., 2011), and Gas Chromatographic-Mass Spectrometric (GC-MS) method (El et al., 2005) in laboratory-prepared mixture, pharmaceutical preparation, and biological matrices such as human plasma (Vatsova et al., 1997).

 

However, the aim of the present work is to develop a simple, precise, specific, accurate, cost-effective, and validated HPLC and UV method according to USP and ICH guidelines for the estimation and routine evaluation of MS in pure and pharmaceutical formulations The presented work consists of estimation of MS by HPLC and UV-VIS method and to validate both the equipment used for daily routine analysis of MS for experimental work as per ICH guidelines.

 

EXPERIMENTAL:

Materials, Equipment, and Instruments:

Metoprolol Succinate (MS) was obtained as a gift sample from Akums Drugs and Pharmaceuticals Limited, India. Orthophosphoric acid (OP), Acetonitrile (ACN) used was of analytical grade. 0.45µm membrane filter procured from Rankem. Ultrapure water (Maxima ultrapure water, UK) with a resistivity more than 18 MΩ/cm was used to ensure the absence of undesirable ions during the experimental procedure. Micropipette procured from modern scientific industries, India and high accuracy weighing balance; Shimadzu ATX 224 used during the experimental study. All the other chemicals and reagents used were of analytical grade.

 

For method validation, HPLC used is of the Waters Breeze 2 system, Waters Spherisorb^® analytical column used to have the dimension of 5 µm, 4.6*250mm. Ultraviolet spectroscopy; (Shimadzu-UV-1800) also used for validation purpose. UV used has matched quartz cells (1 centimeter). Both HPLC and UV spectroscopy used integrated with computers having the latest version of Windows 7 home basic for computation of data.

 

Preparation of mobile phase:

Mobile phase consists of ACN and OP was mixed in a ratio of 4:1 maintaining the pH of 3.0. Both the composition is degassed in a sonicator for the time period of 60 minutes. Injection volume was 20.00µl and UV detection was at 224nm.

 

Standard solution preparation for HPLC technique:

Accurately weighed and transferred 50mg of MS into a 50 ml of the calibrated volumetric flask, added about 50 ml of diluent, sonicated to dissolve for 60 minutes, makeup to volume with diluent. Transferred 5.0ml of the above solution into 50ml volumetric flask, diluted to the volume with the mobile phase and mix well. Filter the solution through the 0.45µm filter using vacuum filter.

 

Retention time of MS by HPLC technique:

Retention time (RT) is a measure of the time taken for the solute to pass through a chromatography column. It is calculated as the time from injection to detection (CDER, 1994).

 

Linearity:

This is the method's ability to obtain results which may directly or after mathematical changes proportional to the concentration of the analyte in each range. Linearity can be determined by the calculation of the regression line using a mathematical treatment of the results (i.e. least mean squares) versus analyte concentration (Jenke., 1996; Jenke., 1996; Rosing et al., 2000).

 

Accuracy:

Accuracy is a standard of the nearness of the test results that obtained by a method to the true value. Accuracy is the deflection between the found mean value and the true value. It is determined by applying the method to the samples in which known amounts of the analyte have been added to the sample. These values should be analyzed against the standard and the blank solutions to ensure that there is no interference exists. The accuracy can be determined by the test results as a percentage of the analyte recovered by the assay of the sample (ICH Q2B, 1996; Jenke., 1996; Jenke., 1996; Rosing et al., 2000).

 

Precision:

The precision of an analytical method described as the degree of agreement between the individual tests results obtained when the method is applied to multiple sampling of a homogenous sample. Precision is a measure of the reproducibility of the whole analytical method (which includes the process of sampling, sample preparation and analysis) under the normal operating circumstances. Precision can be specified by using the method of assay of a sample for a enough times to obtain statistically valid results. The precision of the method was determined by repeatability and intermediate precision of both standard and sample solutions (ICH Q2B, 1996; Jenke., 1996; Jenke., 1996; Rosing et al., 2000).

 

Ruggedness:

Ruggedness is the degree of reproducibility of results obtained by the analysis of the same sample under a variety of normal test conditions i.e. different analysts, labs, instruments, reagents used, temperatures for assay, small variations in the mobile phase and different days and some others (i.e. from laboratory to laboratory, from analyst to analyst ICH Q2B, 1996; CDER, 1994).For the computation of the Limit of Detection (LOD) and Limit of Quantification (LOQ) limits the following equations were used:

 

LOD=3.3* σ/B…….. (1)

LOQ=10*σ/B……… (2)

Where, σ is the standard deviation of the intercept, and B is the slope of the calibration plot.

 

The recovery coefficient was computed following the equation:

 

% Recovery found = [(an amount found) / (amount known)] * 100 ……… (3)

 

RESULTS:

Section A: Validation method of Metoprolol Succinate by HPLC:

Retention time:

Retention time (RT) was studied for 30µg/ml solution. Total numbers of six injections were run in HPLC column having a flow rate of 2ml/min having run time of 10 minutes. The average RT and % RSD was found to be 3.249 ± 0.23.

 

Linearity:

Linearity was analyzed through the standard plot ranging from 10 - 50µg/ml by diluting appropriate amounts of MS stock solution with ACN and buffer which was prepared in triplicate. Calibration plots were prepared on the same day with the following concentrations (10, 15, 20, 30, 40 and 50µg/ml). The linearity was evaluated by linear regression analysis, which was reckoned by the least-square regression analysis. The regression equation was found to be (y= 20355x + 194992) and r² value 0.9992 was highly significant. It obeys the Beer - Lambert's law in a concentration range of 10 – 50µg/ml (Table 1). The robustness of the assay was verified by means of the ANOVA (Graph-pad prism ®). According to it, there is linear regression and there is no deviation from linearity (P < 0.05).

 

Accuracy by recovery method:

The accuracy of the method was estimated by an addition recovery method. In this, known amount of standard MS was added to the pre-analyzed sample. This was done in 20, 30, 40 µg/ml and reading were performed in triplicate mean peak area ± S.D was found to be 8216289 ± 1.02 with low % RSD value (Table 1).

 

Ruggedness analysis:

The ruggedness of the method was determined by carrying out the analysis by two different analysts in the same day at ambient temperature and the respective RT with peak area was noted. The result was described in (Table 2).

 

Table 1: Linearity and Recovery Data

Linearity No.	Conc. (µg/ml)	Injection No.	Peak Name	RT (min)	Peak Area (mAU)	Mean Peak Area (mAU)
1	10	1	Metoprolol Succinate	3.941	390182	390180
		2		3.941	390181
		3		3.942	390179
2	15	1		3.941	501190	501190
		2		3.941	501131
		3		3.941	501451
3	20	1		3.942	601790	601790
		2		3.941	601790
		3		3.941	601795
4	30	1		3.941	821630	821630
		2		3.942	821629
		3		3.941	821630
5	40	1		3.941	1007406	1007406
		2		3.941	1007405
		3		3.941	1007404
6	50	1		3.941	1205964	1205964
		2		3.942	1205969
		3		3.941	1205966
Recovery %	Injection No.		Peak Name	RT (min)	Peak Area (mAU)	% RSD
50	1		Metoprolol Succinate	3.941	821629	0.89
	2			3.943	821630
	3			3.941	821632
100	1			3.941	821639
	2			3.940	821629
	3			3.941	821629
150	1			3.941	821629
	2			3.941	821629
	3			3.941	821629

Table 2: Ruggedness analysis by analyst I and II

Analyst I						Analyst II
Standard No.	Injection No.	Peak Name	RT (min)	Peak Area (mAU)	% RSD	Injection No.	RT (min)	Peak Area (mAU)	% RSD
1	1	Metoprolol Succinate	3.941	821629	0.78	1	3.941	821629	0.78
	2		3.942	821630		2	3.941	821629
	3		3.941	821629		3	3.942	821630
	4		3.942	821630		4	3.941	821629
	5		3.941	821629		5	3.941	821629
	6		3.941	821629		6	3.942	821630
1	1		3.941	821629	0.86	1	3.941	821629	0.86
	2		3.942	821630		2	3.941	821629
2	1		3.941	821629	0.86	1	3.941	821629	0.86
	2		3.941	821629		2	3.941	821629
3	1		3.941	821629	0.86	1	3.941	821629	0.86
	2		3.942	821630		2	3.942	821630
4	1		3.943	821630	0.89	1	3.941	821629	0.89
	2		3.941	821629		2	3.941	821629
5	1		3.941	821629	0.89	1	3.941	821629	0.89
	2		3.941	821629		2	3.941	821629
6	1		3.942	821630	0.89	1	3.941	821629	0.89
	2		3.941	821629		2	3.941	821629

Method Precision:

The precision of the assay was determined by repeatability (Intraday) and intermediate precision (Interday) and reported as % relative standard deviation (RSD). For this, 20, 30, 40 µg/ml concentration solutions were measured three times in a day and the same was measured in the next 3 days. The %RSD was calculated (Table 3).

 

Table 3: Method Precision by Intraday and Interday precision

Intraday precision						Interday precision
Standard No.	Injection No.	Peak Name	RT (min)	Peak Area (mAU)	% RSD	Injection No.	RT (min)	Peak Area (mAU)	% RSD
1	1	Metoprolol Succinate	3.941	821629	0.78	1	3.941	821629	0.78
	2		3.941	821629		2	3.942	821630
	3		3.941	821629		3	3.941	821629
	4		3.942	821630		4	3.942	821630
	5		3.941	821629		5	3.941	821629
	6		3.942	821630		6	3.941	821629
2	1		3.941	821629	0.86	1	3.941	821629	0.86
	2		3.941	821629		2	3.941	821629
3	1		3.942	821630	0.86	1	3.943	821630	0.86
	2		3.941	821629		2	3.941	821629
4	1		3.941	821629	0.86	1	3.941	821629	0.86
	2		3.941	821629		2	3.942	821630
5	1		3.942	821630	0.89	1	3.941	821629	0.89
	2		3.941	821629		2	3.941	821629
6	1		3.941	821629	0.89	1	3.941	821629	0.89
	2		3.941	821629		2	3.941	821629

 

Section B: Validation method of Metoprolol Succinate by UV-VIS spectroscopy method:

 

Standard stock solution of MS in 0.1 M HCl solution:

Standard drug solution of MS was prepared by dissolving 10 mg of MS in 5 ml 0.1 M HCl in a 10 ml volumetric flask, shaken well, followed by vortexing for 5 minutes and final volume was adjusted to get a solution of the concentration of 1 mg/ml. This 1 mg/ml solution was used as a stock solution.

 

Calibration plot of MS in 0.1 M HCl solution:

Five milliliters of 1 mg/ml aliquot solution were further diluted up to 50 ml of 0.1 N HCl (pH 1.2) in a 100 ml volumetric flask and the final volume was adjusted up to 100 ml. This was scanned spectrophotometrically in the wavelength region 200–400 nm to determine the wavelength of the absorption maximum (λmax). The λmax was found to be 224 nm against the blank. From 1 mg/ml stock solution, the serial dilution pattern was followed to obtain aliquots of 2–10µg/ml concentration. The regression equation was found to be (y=0.0544x + 0.1381) and r² value 0.9998.

 

Repeatability analysis:

Repeatability analysis was performed with concentration range 6µg/ml. Mean concentration was found to be 6.03 ± 0.78 having % RSD 0.53. The repeatability analysis was performed in triplicate.

 

Method precision by Interday and Intraday analysis:

The precision of the assay was determined by repeatability (Intraday) and intermediate precision (Interday) and reported as % relative standard deviation (RSD). For this, 2µg/ml, 4µg/ml, and 6 µg/ml concentration solution were measured three times in a day and the same was measured in the next 3 days. The % RSD was calculated and %RSD was found to be least (Table 4).

 

Table 4: Data for Intraday and Interday analysis

Interday Analysis	Absorbance(unit)				Concentration found (µg/ml)			Mean Conc. (µg/ml)	S.D	%RSD
Concentration(µg/ml)	1	2		3	1	2	3
2	0.246	0.245		0.246	1.98	1.96	1.96	1.96	0.67	0.89
4	0.356	0.356		0.355	4.00	4.00	3.98	3.99	0.78	0.98
6	0.467	0.466		0.466	6.04	6.02	6.02	6.02	0.89	0.97
Intraday Analysis	Absorbance (unit)				Concentration found (µg/ml)			Mean Conc. (µg/ml)	S.D	%RSD
Concentration(µg/ml)	1		2	3	1	2	3
2	0.246		0.245	0.246	1.98	1.96	1.98	1.97	0.11	0.49
4	0.356		0.355	0.356	4.00	3.98	4.00	3.99	0.13	0.19
6	0.467		0.467	0.467	6.04	6.04	6.04	6.04	0.25	0.45

 

 

Recovery Studies:

Recovery study (spike method) was performed at 80%, 100%, 120%, and the mean recovery was found to be 98.19 ± 0.43 and % RSD were found to be 1.12 in limits as mentioned in ICH guidelines (Table 5).

 

Ruggedness:

The ruggedness of the method was determined by carrying out the analysis by different analysts and the respective absorbance of 2µg/ml was noted. The result was indicated as % RSD (Table 5).

 

Sensitivity:

The limit of detection (LOD) and limit of quantification (LOQ) for MS were determined by using the standard deviation of response and slope (ICH Q2 (R1), 2005; USP, 2009). The LOD and LOQ values are 0.0955 µg/ml and 0.2896 µg/ml respectively.

 

 

Table 5: Data for Recovery studies and Ruggedness Analysis

Concentration (µg/ml)	Absorbance (unit)	Concentration found (µg/ml)	% Recovery	% RSD	Ruggedness Analysis
					Concentration (µg/ml)	Absorbance by analyst I	Absorbance by analyst II	% RSD for Absorbance by analyst I	% RSD for Absorbance by analyst I
5 (80 %)	0.466	6.02	99.66	1.12	2	0.246	0.246	0.98	0.87
5 (100 %)	0.476	6.21	99.10		2	0.246	0.246
5 (120%)	0.483	6.34	99.90		2	0.246	0.246
					2	0.246	0.245
					2	0.245	0.246

 

CONCLUSION:

The development of HPLC and UV-VIS methods for the determination of drugs has received considerable attention over the years because of their reliability in the quality control of drugs and drug products. The objective of this study was to develop a rapid HPLC and UV-VIS method for the estimation of Metoprolol Succinate from its bulk form using a commonly employed reverse phase C-18 column with UV detector. The proposed method is simple, rapid and statistically validated for its accuracy. The runtime by HPLC method is relatively short, i.e. 10.00 minute, which helps in fast determination of many samples. The results obtained from the validation parameter met the requirements as specified by the ICH. The calibration curve showed linearity over a concentration range of 10 - 50 µg/ml with a regression coefficient of 0.9992 for HPLC and linearity range of 2-10 µg/ml with a regression coefficient of 0.9998 for UV-VIS. The carried study reflects that the method was found to be simple, specific, precise, linear and proportional i.e. it follows the Beer's - Lambert law. Recovery test (spike method) which was performed for concentration 80, 100 and 120 % was found with an average value of 98.19 ± 0.89 with low % RSD value, all the analytical findings by HPLC and UV-VIS method indicates that the proposed method for the analysis of drugs is highly accurate. The method was found to have a suitable application in routine laboratory analysis with a high degree of accuracy and precision. The developed method was strictly based on the USP and ICH guidelines (ICH Q2B, 1996; ICH Q2 (R1), 2005; USP, 2009). Hence, this method can be employed for the routine determination of MS in pure and pharmaceutical formulations. It also infers that the equipment (HPLC and UV-VIS) used during the study gives the accurate results and equipment is validated for the routine experimental work for estimation of MS from its dosage form.

 

CONFLICT OF INTEREST:

None.

 

ACKNOWLEDGEMENT:

This research work was supported by Sardar Bhagwan Singh University, Dehradun, India.

 

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Received on 17.02.2020            Modified on 23.04.2020

Accepted on 29.06.2020         © RJPT All right reserved