INTRODUCTION:

Metoprolol succinate (MS) (Fig. 1) is a cardio selective drug used in the treatment of hypertension and various cardiovascular disorders. The action of Metoprolol succinate is mediated through the beta₁-selective adrenoceptor blockage, thus causing reduction in heart rate and cardiac output. It is a beta₁-selective drug which belongs to the chemical class of beta blockers and is (±) 1-(isopropylamino)-3-[p-(2-methoxyethyl) phenoxy]-2-propanol succinate (2:1) (salt) with molecular formula of (C₁₅H₂₅NO₃)₂. C₄H₆O₄ and molecular weight of 652.81.

Literature survey revealed that various methods have been reported for estimation of MS in bulk drugs ^1-5, plasma ^6-13, serum ¹⁴, urine ^{15, 16} and pharmaceutical dosage forms ¹⁷. The available methods also suffered from drawbacks such as increased retention time, use of high pump pressure and not cost effective. Thus the present study involved development of a simple liquid chromatographic RP-HPLC method for the estimation and quantitation of MS in bulk drugs, which was found to be rapid, sensitive and cost effective over the reported methods.

EXPERIMENTAL:

The Liquid chromatographic system consisted of following components: Jasco HPLC model containing PU 1580 Intelligent HPLC pump, Jasco UV 1520 Intelligent UV-Vis Detector and Rheodyne injector (7725i) with 20 µl fixed loop.

Chromatographic analysis was performed using Borwin Chromatography software on a RP Spherisorb C-18 (Waters) column having 250×4.6 mm ID and 10µm particle size. The Mettler electronic micro balance (MT5) was used for weighing purpose.

An analytically pure sample of MS was procured as a gift sample from Ethypharm Pvt Ltd. (Mumbai, India).HPLC grade acetonitrile and methanol (purity 99.9%) was procured from Merck (Darmstadt, Germany). The HPLC grade water was obtained by double distillation and purification through Milli-Q water purification system. Monobasic potassium phosphate (AR grade) was procured from Qualigens Fine Chemicals (Mumbai, India).

Preparation of mobile phase and Stock Solution:

Monobasic potassium phosphate was weighed (136.1g) and dissolved in 1000 ml of water to get 1 M solution. From this solution 10 ml was taken and volume was made up to 1000 ml to get a solution of 10 mM. 600 ml of the prepared buffer was taken and the solution was mixed with 200 ml of acetonitrile, 200 ml of methanol and finally the solution was mixed well. The solution was sonicated for 10 min and filtered using Whatman filter Paper No.1.

A stock solution of MS was prepared by accurately weighing 10 mg of drug, transferring to 10 ml volumetric flask, dissolving in 5 ml of methanol and diluting it up to the mark with methanol. The stock solution thus obtained was of 1 mg/ml. Appropriate aliquot was taken to obtain a standard solution of 50 µg/ml of MS. Resultant solution was filtered through Whatman filter Paper No.1. The calibration curve was prepared in the range of 30 - 0.85 µg/ml.

Table 1. System Suitability Test Parameters for MS by the Proposed Method

System suitability parameters	Values
Retention time (min)	5.1
Resolution	4.0
Tailing factor (asymmetric factor)	1.30

Chromatographic Condition:

A reverse phase C-18 column, equilibrated with mobile phase acetonitrile: methanol: 10 mM aqueous phosphate buffer (20:20:60%v/v) was used. The active principle was eluted isocratically and the mobile phase flow rate was maintained at 1.0 ml/min. The effluents were monitored at 254 nm with the detector. The sample was injected using a 20 µl fixed loop, and the total run time was 8 min.

Calibration Curve:

Appropriate aliquots were pipetted out from standard stock solution into series of 10 ml volumetric flasks. The volume was made up to the mark with mobile phase to get solutions having concentration range 0.85, 5, 10, 15, 20, 25, 30 µg/ml for MS. Triplicate dilutions of each concentration were injected into the RP-HPLC system and chromatographed under the conditions as described above. Evaluation of drugs was performed at 254 nm.

VALIDATION OF HPLC METHOD:

The proposed RP-HPLC method was validated as per ICH guidelines. ¹⁸

Specificity:

The specificity of the RP-HPLC method was determined by comparison of the chromatogram of standard and sample solution. The parameters like retention time (tR), resolution (Rs) and tailing factor (Tf) were calculated.

Precision:

Precision study was performed to find out intra-day and inter-day variation. It was carried out by estimating the corresponding responses 3 times on the same day and on 3 different day (first, second and fifth day) for 3 different concentration of MS (1, 15, 30 µg/ml) and the results are reported in terms of relative standard deviation (RSD). The repeatability studies were carried out by estimating response of 3 different concentration of MS (1, 15, 30 µg/ml) in triplicate.

Figure. 1. Structure of Metoprolol succinate

Figure 2. HPLC chromatogram of: (A) Blank, (B) 0.85 μg/ml MS, (C) 30 μg/ml MS

(A)

(B)

(C)

Accuracy (Recovery studies):

Recovery studies were performed by standard addition method at three levels i.e. 80%, 100% and 120%. A known amount of standard MS was added to preanalyzed sample and was subjected to proposed HPLC method.

Detection Limit and Quantitation Limit:

A calibration curve was prepared using concentrations in the range of 30 - 0.85 µg/ml (expected detection limit range). Detection limit for MS was 0.25 µg/ml and quantitation limit was 0.75 µg/ml. The standard deviation of y-intercepts of regression line was determined and kept in following equation for the determination of detection limit and quantitation limit. Detection limit = 3.3σ/s; Quantitation limit = 10σ/s, where σ is the standard deviation of y-intercepts of regression lines and s is the slope of the calibration curve.

Stability:

In order to demonstrate the stability of the standard solution of MS during analysis, the solution was stored over a period of 24 hr at room temperature and then analyzed.

Robustness:

Robustness of the method was studied by changing the composition of organic phase by ±5% and pH by 0.2, and also by observing the stability of the drug for 24 hr at ambient temperature in mobile phase.

Table 2. Summary of Validation Parameters for the Proposed Method

Parameters	Values
Detection limit (µg/ml)	0.25 µg/ml
Quantitation limit (µg/ml)	0.75 µg/ml
Calibration range (µg/ml)	30 - 0.85 µg/ml
Accuracy (%)	98.05-100.59
Precision (RSD^a, %)
Intraday (n=3)	0.30-0.62
Interday (n=3)	0.29-1.26
Repeatability (RSD^a, n=3)	0.18-0.59
Correlation coefficient (r)	0.9988

^aRSD indicates relative standard deviation

RESULTS AND DISCUSSION:

RP-HPLC method:

The development of the RP-HPLC method for the determination of drugs has received considerable attention in recent years because of its importance in routine quality control analysis. Different analytical columns with various stationary phases were tested.

Good separation was achieved using a RP Spherisorb C-18 column and was finally used for analysis. A RP-HPLC method was proposed as a suitable method for the estimation of MS in bulk drug. The chromatographic conditions were adjusted in order to provide a good performance of the assay. The method involved a mobile phase consisting of acetonitrile-Methanol-10mM aqueous phosphate buffer (2:2:6 v/v) accomplished at 254 nm. The retention time was 5.1 min at a flow-rate of 1 ml/min. The total run time for an assay was approximately 8 min. The mobile phase was chosen after several trials with other solvent combinations. Mobile phase selection was based on peak parameters (symmetry, tailing), run time, ease of preparation and cost. Figures 2 (A, B, C) show a representative chromatogram using the proposed method. As shown in these figures, MS was eluted forming symmetrical peak and well separated from the solvent front. Observed retention time (5.1 min) allowed a rapid determination of the drug (Table 1).

Linearity:

The linear range was found to be 30 - 0.85 µg/ml concentration. The regression equation and correlation coefficient (r) obtained by least square regression method were 0.998. In the linearity assay, the coefficient of variation (C.V) of the response factor was 4.82. The linearity of the calibration graph and conformity of RP-HPLC value to Beer’s Law were proven by the high correlation coefficients (r) for the regression equations.

Accuracy and precision:

Repeatability is given as inter- and intra-day precision and accuracy evaluated by analyzing three different concentrations of MS. The results are given in Table 1. The precision of the RP-HPLC method was demonstrated by the relative standard derivation (RSD %) of lower than 0.62% for intra-day and 1.26% for inter-day. (Table 2)

Sensitivity:

The LOQ is defined as the lowest concentration on the calibration curve at which both accuracy and precision should be within 20%. LOQ value of the RP-HPLC method was determined as 0.75 μg/ml. The LOD for MS determination was approximately 0.25 μg/ml. (Table 2)

Stability:

The results for stability studies revealed that for the solutions, retention time and peak area of MS remained almost unchanged.

CONCLUSION:

Proposed study describes new and simple RP-HPLC method for the estimation of MS in bulk drugs. The method was validated and found to be simple, sensitive, accurate and precise. Therefore the proposed method can be used for quantification of MS in bulk drugs as well as for routine analysis in quality control.

ACKNOWLEDGEMENT:

The authors greatly acknowledge Ethypharm Pvt. Ltd., (Mumbai, India) for providing authentic gift sample of MS.

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Received on 21.02.2009 Modified on 15.04.2009

Asian J. Research Chem. 2(2): April.-June, 2009 page 119-122