New Spectrophotometric Method Applied to the Simultaneous Determination of Metoprolol Succinate and Hydrochlorthiazide

 

T Venkatachalam¹*, V Kishor Kumar¹, P Kalai Selvi¹, R Srinivasan¹, R Mariammal², KG Lalitha³

¹JKKMMRF College of Pharmacy B. Komarapalayam

²ROVER College of Pharmacy .Perambalur
³ULTRA College of Pharmacy. Madurai

 *Corresponding Author E-mail: venkatachalammpharm@yahoo.co.in

ABSTRACT:

Simple, sensitive, and specific spectrophotometric methods were developed and validated for quantitation of Metoprolol succinate and Hydrochlorthiazide in tablet dosage form. A new analytical method was developed based on the simultaneous estimation of drugs in a binary mixture without previous separation. In simultaneous equation method, Metoprolol succinate and Hydrochlorthiazide were determined by using their absorptivity values at wavelength maxima, viz., 222 nm and 272 nm. The standard deviation value for the different validation parameters was found to be between 0.094 and 1.01 for simultaneous equation method. This method is simple, accurate, rapid, and they require no preliminary separation and can therefore be used for routine analysis of both drugs in quality control laboratories.

 

 

 

INTRODUCTION:

Metoprolol succinate is (MS) Bis [(2rs)-1-{4-(2-methoxy ethyl]-3-[(1-methyl ethyl) amino propan-2-ol] butane dioate. It possesses antihypertensive activity. It is cardio selective β-blocker causing reduction in heart, cardiac output and blood pressure^1-4. Hydrochlorthiazide is (HCT) 6-chloro-3, 4-dihydro-2H-1, 2, 4-benzodiathiazine-7-sulphonamide 1, 1-dioxide. It is having diuretic action, increases renal excretion of sodium and chloride and reduces cardiac load. The two drugs exert additive effects in hypertension^5-7.

 

Literature survey reveals that as such, no analytical method has yet been reported for the analysis of these two drugs in combination. The Indian Pharmacopoeia, British Pharmacopoeia and United States Pharmacopoeia do not specify sepectrophotometric simultaneous determination for Metoprolol succinate and Hydrochlorthiazide

 

As such, no spectrophotometric method for simultaneous estimation of Metoprolol succinate and Hydrochlorthiazide combination has been reported, which prompted to pursue the present work. It was also planned to validate the developed method as per ICH norms^8-10..

 

The objective of the present work is to develop and validate new sepectrophotometric method for simultaneous determination of Metoprolol succinate and Hydrochlorthiazide in a binary mixture without any separation.

 

MATERIALS AND METHODS

Materials:

An UV/Vis recording spectrophotometer (Shimadzu model-U x 1601A) with ±0.5 nm (with auto wavelength correction 1 cm matched quartz cells) wavelength accuracy and 3 nm bandwidth was employed with binary mixture determination. Phosphate buffer pH 6.8 containing 10 % v/v methanol was used as the solvent after considering the solubility factors of both the drugs as well as the interference due excipient matrix present in tablet formulation. Standards (Metoprolol succinate and Hydrochlorthiazide) of purity 98.50 % to 101.50 % were procured from

 

Simultaneous equation method:

This method of analysis is based on the absorption of Metoprolol succinate and Hydrochlorthiazide at the wavelength maximum of other. The quantification analysis of Metoprolol succinate and Hydrochlorthiazide in a binary mixture was performed with the following equation:

The amount of Metoprolol was calculated by the following formula

(AT₂ X A_{1 %} H_{272 nm})  –    (AT₁ X A_{1 %} H_{222 nm}) / (A_{1 %} M_{222 nm} X A_{1 %} H_{272 nm}) – (A_{1 %} M_{272 nm} X A_{1 %} H_{222 nm}) ------(Equation 1).

The amount of Hydrochlorthiazide was calculated by the following formula: (AT₁ X A_1% M_222nm)      –   (AT₂ X A_{1 %} M_{272 nm})/ (A_{1 %} H_{272 nm} X A_{1 %} M_{222  nm})   –  (A_{1 %} H_{222 nm} X A_{1 %} M_{272  nm})………. (Equation 2).

 

Where, AT₁ is the absorbance of the solution under test at 273 nm, AT₂ is the absorbance of the solution under test at 222 nm. A_1%   M₂₇₂ is the absorptivity (A ^1%_{1 cm}     at 272 nm) at Metoprolol, A_{1 %}   M₂₂₂ is the absorptivity (A ^{1 %} _{1 cm}    at 222 nm) at Metoprolol. A_{1 %}  H₂₇₂    = is the absorptivity, (A ^{1 %} _{1 cm}    at 272 nm) at Hydrochlorthiazide, A_{1 %}  H₂₂₂    =    is the absorptivity (A ^{1 %}_{1 cm}     at 222 nm) at Hydrochlorthiazide ^{[2, 11]}.

 

Preparation of standard solution:

Weigh accurately 50 mg of Hydrochlorthiazide and 200 mg of Metoprolol succinate into a 250 ml volumetric flask and dissolve using of phosphate buffer pH 6.8 containing 10% v/v methanol. Metoprolol succinate and Hydrochlorthiazide were further diluted to 40 μg/ml and 10 μg/ml using phosphate buffer pH 6.8 respectively. Both solutions were scanned over the range of the 200 nm to 400 nm in the spectrum mode with the scan speed of 480 /min. Overlain spectra exhibit absorbance maxima at 222 nm and 272 nm for Metoprolol succinate and Hydrochlorthiazide respectively, which revealed that the peaks are well resolved, thus satisfying the criteria for obtaining maximum precision based on absorbance (Fig 1).

 

Fig.1: Overlain spectra of Metoprolol succinate, Hydrochlorthiazide, mixed sample in phosphate buffer pH 6.8.

 

Analysis of tablet formulation:

Twenty tablets (brand name- supermet-H, manufactured by The Madras Pharmaceuticals) were taken and their average weight was determined. Tablets were crushed to fine powder containing the equivalent of 40 mg of Metoprolol succinate and 10 mg of Hydrochlorthiazide was taken in 200 ml volumetric flask. It was then dissolved in phosphate buffer pH 6.8 containing 10 % v/v methanol by intermittent shaking for 4-5 min. the volume was made up to 200 ml and the solution filtered through whatman filter paper (no.41). The filtrate was further diluted using phosphate buffer pH 6.8 to get the final concentration of 20 μg/ml of Metoprolol succinate and 5 μg/ml of Hydrochlorthiazide.

 

The analysis of the binary mixture containing Metoprolol succinate and Hydrochlorthiazide was performed from the combined absorbance value at 222 nm and 272 nm (λ _max for Metoprolol succinate and Hydrochlorthiazide) as in the Fig 1 of binary mixture in phosphate buffer pH 6.8 (10 % methanol) by using the formula mentioned above (Equation 1 and 2) and the results were obtained.

 

Fig.2: Overlain spectra of eight mixed samples of Metoprolol succinate and Hydrochlorthiazide in phosphate buffer pH 6.8.

 

Accuracy:

To a pre-analyzed tablet solution having 20 μg/ml of Metoprolol succinate and 5 μg/ml of Hydrochlorthiazide, a definite and different concentration of pure drug was added and then its recovery study was performed.  The result were calculated by using the formula: % label claim =C x D x A/W x 100 /L (Equation 3), % dissolved = C x D x W_s/L x V_m x 100 (Equation 4), where C is concentration from standard curve, D is dilution factor, A is average weight of the tablet, W is a weight of composite taken, W_s is weight in mg of respective standard and V_m is volume of dissolution medium.

 

Linearity:

Hydrochlorthiazide and Metoprolol succinate obeyed Beer-Lambert’s law in the concentration range of 3-10 μg/ml and 12-40 μg/ml respectively. Therefore the linearity of mixed samples of HCT (3, 4, 5, 6, 7, 8, 9 and 10 μg/ml) and MS (12, 16, 20, 24, 28, 32, 36 and 40 μg/ml) were prepared and evaluated.

 

Precision:

Repeatability: Weigh accurately 50 mg of Hydrochlorthiazide and 200 mg of Metoprolol succinate into a 250 ml volumetric flask and dissolve using of phosphate buffer (pH 6.8) containing 10% v/v methanol. Metoprolol succinate and Hydrochlorthiazide were further diluted to 40 μg/ml and 10 μg/ml using phosphate buffer (pH 6.8) respectively. The readings were taken for 6 replicates of the same at 222 nm and 272 nm for Metoprolol succinate and Hydrochlorthiazide respectively.

 

TABLE 1: RESULTS OF ANALYSIS OF TABLET FORMULATION

Label claim (μg/ml)	MS*	40	40	40	40	40	40
	HCT*	10	10	10	10	10	10
Simultaneous equation method (%)*	MS	101.82	101.7	101.21	100.92	101.37	100.92
	HCT	100.08	99.15	98.68	97.95	98.68	99.15

 

TABLE 2: STATISTICAL VALIDATION OF TABLET FORMULATION AND RECOVERY EXPERIMENT RESULTS OBTAINED FOR METOPROLOL SUCCINATE AND HYDROCHLORTHIAZIDE

Contents	Drug	Standard deviation	Coefficient of        variation
Tablet formulation	MS*	0.3818	0.145
	HCT*	0.707	0.500
Recovery studies	MS	0.427	0.182
	HCT	0.425	0.366

 

TABLE 3: RECOVERY EXPERIMENT RESULTS OBTAINED FOR METOPROLOL SUCCINATE AND HYDROCHLORTHIAZIDE

Amount of pure drug added (μg/ml)	MS*	5	10	15	20	25
	HCT*	25	20	15	10	5
Simultaneous equation method (%)*	MS	99.85	98.9	99.15	99.83	99.22
	HCT	97.67	99.13	98.42	97.9	97.77

 

Reproducibility: It was determined by quantifying the amount of Metoprolol succinate and Hydrochlorthiazide in the formulation. The method for analyzing tablet formulation was carried out repeatedly for six different sets of samples having the same concentration (20 μg/ml of Metoprolol succinate and 5 μg/ml of Hydrochlorthiazide). The absorbance was observed at 222 nm and 272 nm for Metoprolol succinate and Hydrochlorthiazide respectively in order to calculate the standard deviation.

 

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

The LOD and LOQ of standards of HCT and MS (2, 3, 4, 5, 6 and 7 μg/ml) were prepared and evaluated.

Limit of detection was calculated from the given formula:

 

RESULT AND DISCUSSION:

In the present work simultaneous equation method were developed for the simultaneous spectroscopic estimation of Metoprolol succinate and Hydrochlorthiazide in commercially available tablet dosage form using phosphate buffer pH 6.8 containing 10 % v/v methanol as solvent. From the overlain spectra of two drugs, 222 nm and 272 nm were selected as the sampling wavelength for Metoprolol succinate at a concentration of 40 μg/ml and Hydrochlorthiazide at a concentration of 10 μg/ml (Fig 1).

The use of 8 mixed samples and two sampling wavelengths of 222 nm and 272 nm (λ_max for Metoprolol succinate and Hydrochlorthiazide) gave optimum accuracy, precision, time, economy, and sensitivity (Fig.2). The purpose of the procedure was successfully applied to the determination of Metoprolol succinate and Hydrochlorthiazide in the commercially available tablet dosage form, and the results are presented in table 1 and 2.

 

The values of standard deviation for the validation parameters (linearity, accuracy, repeatability, intermediate precision and reproducibility) of Metoprolol succinate and Hydrochlorthiazide in tablet formulation were found to be between 0.094 and 1.01.

 

A critical evaluation of the proposed method was performed by the statistical analysis of the experimental data. In order to demonstrate the validity and applicability of the proposed method, recovery studies were conducted by analyzing the mixture of Metoprolol succinate and Hydrochlorthiazide in different composition ratios.

 

The percentage recoveries were found to be 99 – 101 %, and results summarized in tables 2 and 3, agreed with the labeled content. The percentage recoveries of Metoprolol succinate and Hydrochlorthiazide from spiked excipients are summarized in table 3.

 

Beer-lamberts law obeyed in the range of 3 – 10 μg/ml and 12 – 40 μg/ml for Hydrochlorthiazide and Metoprolol succinate, respectively. Five mixed samples 3, 4, 5, 6, 7, 8, 9 and 10 μg/ml, for Hydrochlorthiazide and 12, 16, 20, 24, 28, 32, 36 and 40 μg/ml for Metoprolol succinate were obtained from separate sample solutions (Fig 2). The data showed that the method was linear with correlation coefficient of 0.9972 and 0.9933 for Metoprolol succinate and Hydrochlorthiazide (Fig 3 and Fig 4).

 

The relative standard deviations were found to be within the limit, indicating good accuracy, precision and repeatability of the proposed methods (Table 4). The standard deviation and coefficient of variation values of validation parameter are within the limit.

 

Fig 3: Linearity curve for Metoprolol succinate

 

TABLE 4: RESULTS OF VALIDATION PARAMETERS (μg/ml)

Validation parameter	Drug	Standard deviation	Coefficient of        variation
Linearity	MS*	0.085	0.64
	HCT*	0.91	0.83
Accuracy	MS	1.01	0.99
	HCT	0.58	0.50
Repeatability	MS	0.19	0.71
	HCT	0.31	0.94
Reproducibility	MS	0.21	0.52
	HCT	0.11	0.61
Robustness	MS	0.15	0.35
	HCT	0.09	0.34

 

Fig 4: Linearity curve for Hydrochlorthiazide

 

The LOD and LOQ calibrated curve for Metoprolol succinate and Hydrochlorthiazide is shown in (Fig 5 and Fig 6). The LOD and LOQ values for Metoprolol succinate was found to be 0.57 μg/ml and 1.85 μg/ml and for Hydrochlorthiazide was found to be 0.612 μg/ml and 1.87 μg/ml respectively (Table 5).

 

TABLE 5: RESULTS OF LOD and LOQ

Parameters	Drug	μg/ml
LOD*	MS*	0.57
	HCT*	0.672
LOQ*	MS	1.85
	HCT	1.87

Fig 5: Calibration curve of LOD and LOQ for Hydrochlorthiazide

 

Fig.6: Calibration curve of LOD and LOQ for Metoprolol succinate

 

With respect to the validation results obtained, the developed simultaneous equation method is found to be more accurate, precise, simple with limited errors and could be used for rapid and reliable determination of Metoprolol succinate and Hydrochlorthiazide in routine laboratory analysis.

 

REFERENCES:

1.       Indian Pharmacopoeia. New Delhi: The Controller of Publication; 1996. 2: 370 - 486.

2.       United States Pharmacopoeia NF-25, Rockville: United States Pharmacopoeial Convention, 2007. 30: 1123 - 1263.

3.       Budavari S. The Merck Index. 12^th ed. New Jersey: Whitehouse Station; 1996, p. 6072.

4.       Rang HP, Dale MM. Pharmacology. 2^nd ed. ELBS and Churchill Livingstone; 1991, p. 405.

5.       Physicians’ Desk Reference, 59^th ed. Rockville: Thomson Publishers; 2005.

6.       Anthony CM. Clarke’s analysis of drugs and poisons, 3^rd ed. London, Pharmaceutical Press; 2: p. 1109 – 1110.

7.       ICH Harmonized Tripartite Guidelines: Code Q2A. Text on Validation of Analytical procedure, Step 3. Consensus Guidelines; 1994.

8.       ICH Harmonized Tripartite Guidelines: Code Q2A. Text on Validation of Analytical procedure, Step 4. Consensus Guidelines; 1994.

9.       Validation of Analytical Procedures-Definition and Terminology FDA’s Center for Veterinary Medicine Guidance Document, 63, 1999.

10.     Prasad CVN. Derivative spectrophotometric analysis of metoprolol and Hydrochlorthiazide. J AOAC Intl., 1997; 325-330.

11.     Davidson AG. Ultraviolet – visible absorption spectrophotometry. In: Beckett AH, Stenlake JB, editors. Practical Pharmaceutical chemistry. 4^thed. New Delhi: CBS Publishers and Distributors; 2005. 2: p. 275 - 337.

 

Received on 24.12.2009        Modified on 29.01.2010

Accepted on 17.02.2010        © AJRC All right reserved