Simultaneous Spectrophotometric Estimation and Validation of Metformin Hydrochloride and Glibenclamide in Bulk Drug and Pharmaceutical Dosage Form

 

B Srivastava1, Uttam Singh Baghel*1, Pradeep Swarnkar1 and Shuchi Dave2

1School of Pharmaceutical Sciences, Jaipur National University, Jagatpura, Jaipur-203025 Rajasthan, India.

2Department of Pharmacognosy, VNS College of Pharmacy, Bhopal (M.P.), India.

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

 

ABSTRACT:

Two simple, accurate, economical and reproducible spectrophotometric methods for simultaneous estimation of two-component drug mixture of metformin hydrochloride and glibenclamide in bulk and combined tablet dosage form have been developed. The first method employs formation and solving of simultaneous equations using 237.0 and 229.2 nm as two analytical wavelengths. The second method is absorption ratio method, which uses 237.0 and 225.0 nm as two analytical wavelengths. Both methods were statistically validated according to International Conference on Harmonization and recovery studies confirmed the accuracy of the proposed method.

 

KEYWORDS: Simultaneous spectrophotometric analysis, Absorption ratio method, Metformin hydrochloride and Glybenclamide 

 


 

INTRODUCTION:

A new fixed dose combination containing metformin hydrochloride and glibenclamide is available in the market in tablet dosage form for treatment of Type-2 diabetes. Glibenclamide is an effective, long-acting, second generation sulfonylurea used in the treatment of non-insulin-dependent diabetes mellitus (NIDDM). Glibenclamide is chemically 5-chloro-N-[2-[4-(cyclohexylcarbamoylsulfamoyl) phenyl]ethyl]-2-methoxybenzamide[1]. Metformin Hydrochloride (MET) is a biguanide class of antidiabetic drug, chemically N,N-dimethylimidodicarbonimidic diamide hydro-chloride[2]. Type-2 diabetes is a long-term metabolic disorder wherein the body becomes resistant to the effects of insulin, a hormone that regulates sugar absorption[3]. Oral hypoglycemic drugs prescribe as monotherapy have not provided enough hypoglycemic control for type-2 diabetes patients. For this reason combination therapy is becoming a more prevalent method for achieving satisfactory blood glucose level[4]. Drug monitoring during combination therapy is an important process for titrating the appropriate dosing control and diagnostic purpose[5]. Since combination therapies are becoming wide spread in application it is of importance to develop methods applicable to the pre-clinical development of potential combinations of these classes of drugs.

 

Therefore sensitive rapid and reliable methodology required for simultaneous multianalyte quantization of oral hypoglycemic drugs in tablet doses form. Several method have been reported for the estimation of metformin hydrochloride and glibenclamide individually as well as in combination with other drugs, such as high performance liquid chromatography (HPLC)[6]-[12] liquid chromatography/ tandem mass spectrometry (LCMS/MS)[13]-[16] and  capillary electrophoresis (CE)[17]. Most of these methods are tedious and time-consuming involving complex sample preparation. Even though various methods reported in the literature for estimation of metformin hydrochloride and glibenclamide individual or in combination with other drugs no method had been reported so far for simultaneous estimation of these two drugs using simultaneous equations in bulk and tablet dosage form. The present study was aimed at the simultaneous estimation of metformin hydrochloride and glibenclamide by simultaneous equations method. This method was validated according to the ICH guidelines [18].

 

MATERIAL AND METHODS:

Gift sample of metformin hydrochloride and glibenclamide were procured from Zydus Cadila Pvt. Ltd Ahmedabad (Gujarat), India. The commercial fixed dose combination product (GLINIL-M containing 500 mg metformin hydrochloride and 5mg glibenclamide) was procured from the local Pharmacy. Methanol A.R grade was procured from Qualigens fine chemical Mumbai, India.

 


TABLE 1 : SUMMARY OF OPTICAL CHARACTERISTICS

Parameters

Metformin Hydrochloride

Glybenclamide

Method-I

Method-II

Method-I

Method-II

λmax (nm)

237

237

315

225

Beer’s Law Limit (µg/ml)

2-10

2-10

2 -14

2-14

Molar absorptivity (lit/mole/cm)

1.246 x 104

1.246 x 104

4.4207 x 103

4.4211 x 103

Sandell’s sensitivity (µg / ml / cm-2/ 0.001 )

1.04 x 10-2

1.04 x 10-2

8.08 x10-2

8.0821 x10-2

Correlation coefficient (r2)

0.9998

0.9998

0.9999

0.9984

Slope

9.498 x 10-2

9.498 x 10-2

1.2394 x 10-2

1.2194 x 10-2

Intercept

1.933 x 10-3

1.933 x 10-3

4.0 x 10-4

 

Method I is the simultaneous equation method while Method II is absorption ratio method and r2 is correlation coefficient

 

TABLE 2: RECOVERY STUDIES

Name of the Drug

Spiking  level(%)

Amount added

(in mg)

Amount recovered (in mg) n=3

Percentage recovery

Average percentage recovery

Method-I

Method-II

Method-I

Method-II

Method-I

Method-II

 

Metformin Hydrochloride

90

450

448.9233

449.23

99.76

99.83

 

99.93

 

99.95

100

500

500.1827

498.9571

100.04

99.79

110

550

549.872

551.3154

99.98

100.24

 

Glybenclamide

90

4.5

4.5767

4.5541

101.70

101.20

 

100.49

 

100.29

100

5.0

5.0015

4.9621

100.03

99.24

110

5.5

5.4867

5.5241

99.76

100.44

Method I is the simultaneous equation method while Method II is absorption ratio method and n is number of replication of experiment.

 

TABLE 3:SUMMARY OF ANALYTICAL METHOD VALIDATION.

Validation parameters

Metformin Hydrochloride

Glybenclamide

Method-I

Method-II

Method-I

Method-II

Recovery (%)

99.93

99.95

100.49

100.29

Intraday precision (%RSD)

0.2654

0.3847

0.1730

0.2541

Interday precision (%RSD)

0.1783

0.1596

0.1979

0.2531

Linearity (r2)

0.9998

0.9997

0.9986

0.9988

Robustness (%RSD)

0.5132

0.3584

0.814

0.3569

LOD(µg/ml)

0.21

0.18

0.72

0.68

LOQ ( µg/ml)

1.21

1.33

2.24

2.50

Specificity

Specific

Specific

Specific

Specific

Method I is the simultaneous equation method while Method II is absorption ratio method, LOD and LOQ are the limit of detection and limit of quantification respectively, RSD is relative standard deviation and r2 is correlation coefficient.

 

 


Shimadzu UV-1800; UV-Vis spectrophotometer with 1 cm matched quartz cells was used for the measurement of the absorbance. Shimadzu -AX -200 electronic balance was used for weighing the samples.

 

Preparation of stock solutions:

Standard stock solution (1000μg/ml) of metformin hydrochloride and glibenclamide wear prepared separately in methanol by transferring accurately weighed 100mg portion of metformin hydrochloride and glibenclamide in separate 100 ml volumetric flasks and volume was made up with methanol. The Working standard solutions were prepared and further diluted in mobile phase to contain a mixture of metformin hydrochloride and glibenclamide in over the linearity range from 2-10 µg/ml and 2-14 µg/ml respectively.

 

Development of the method:

Simultaneous Equation (Method I):

Selection of analytical wavelengths was done by diluting the stock solution of metformin hydrochloride and glibenclamide at a concentration of 10µg /ml. They were scanned in the wavelength range of 200-400nm from the overlain spectra (Fig.1), wavelengths 237.0 and 229.2 nm were selected for the formation of simultaneous equation for construction of calibration curves. Two series of different concentration in range of 2-10 µg/ml for metformin hydrochloride and 2-14 µg/ml for glibenclamide were prepared from the working standard solutions. The calibration curves were plotted at 237.0 and 229.2 nm. The absorptivities (A1%, 1 cm) of both the drugs at both the wavelength were determined. These calculated values were the mean of 5 independent determinations. The absorbance and absorptivity value at the particular wavelength were calculated and substituted in the following equation to obtain the concentrations:

 

                A1 = 972.1 Cx + 460.1Cy ….(1)            and

                A2=  713.2 Cx + 611.5 Cy….(2).

 

Where A1 and A2 are absorbance of sample solution at 237 nm and 229.2 nm respectively Cx and Cy are concentration of metformin hydrochloride and glibenclamide respectively (mole /lit) in sample solution the validity of formed equation was checked by preparing 5 mixed standards measuring there absorbance at respective wavelength and comparing these with the absorbance calculated using above formed equations.

 

 

Absorption ratio method (method II):

From the overlain spectra of metformin hydrochloride and glibenclamide 237.0 nm was taken as λmax for metformin hydrochloride and 225.0 nm as isobestic point for estimation of metformin hydrochloride and glibenclamide. Series of different concentraions in range of 2-10 µg/ml for metformin hydrochloride and 2-14 µg/ml for glibenclamide were prepared from the working standard solutions. The calibration curves were plotted at 237.0 and 225.0 nm. The absorptivities (A1%, 1 cm) of both the drugs at both the wavelength were determined. These calculated values were the mean of five independent determinations.

 

FIGURE 1: A TYPICAL OVERLAIN SPECTRUM OF METFORMIN HYDROCHLORIDE AND    GLYBENCLAMIDE

Abs. is absorption and nm is wavelength.

 

Estimation from formulation:

Twenty tablets were weighed and finely powdered. A quantity of powder equivalent to 500 mg metformin hydrochloride and 5mg glibenclamide was weighed and transferred to a 500 ml volumetric standard flask. To it 495 mg of pure glibenclamide was added and sonicated for 10 min with 200ml methanol. The solution was made up to the mark with methanol. The contents were mixed thoroughly and filtered through a 0.45µ filter.  Solution was further diluted to achieve final concentration of 5µg/ml for metformin hydrochloride and 5µg/ml for glibenclamide. The absorbance’s were noted at respective wavelength. The concentration of each drug in tablet formulation was determined using above methods.

 

RESULTS AND DISCUSSION:

The proposed UV- Spectrophotometric methods required fewer reagents and materials, and it is simple and less time consuming. This method could be used in quality control test in pharmaceutical industries. The Overlain spectra of metformin hydrochloride and glibenclamide were shown in (figur1). The molar absorptivity and Sandell’s sensitivity values show the sensitivity of metformin hydrochloride and glibenclamide at respective point wavelengths. The overlain spectra of                 metformin hydrochloride and glibenclamide showed that the peaks are well resolved thus satisfactory criteria for obtaining maximum precision based on absorbance ratios (A2 A1 / aX2 aX1) for drug Y and (aY2 aY1 / A2 A1) for drug X should lie outside the range of 0.1-2.0 where A1 and A2 represent absorbance of tablet solution at λ1 and λ2, aX1 and aX2 represents absorptivities of X at λ1 and λ2 and aY1 and aY2 denote a absorptivities of Y at λ1 and λ2, respectively. In the present contest, present criteria found to be satisfied for metformin hydrochloride (X) and glibenclamide (Y). Where λ1 is 237.0nm and is λ2 229.2nm. In overlain spectra, glibenclamide shows two distinct  peaks one at around 229.2nm and other at 300.0 the peak at 229.2 nm was found to be prominent hence for simultaneous equations method; the peak was used for determination of glibenclamide. Since only one prominent peak exists for metformin hydrochloride at 237.0 nm, the same was used for its determination. Absorbance was determined for both the wavelength. Calibration curves were plotted and regression analysis was carried out (Table 1). The absorbitivity was than calculated and substituted in equation 1 and 2 along with absorbance values to obtain to concentration of drugs.

 

In absorption ratio method, two wavelengths are selected from overlain spectra out of which one is isobestic point and another is λmax of one of the drugs. The spectra of metformin hydrochloride and glibenclamide when overlaid indicated that the isobestic point was at 225.0 nm at which estimation of glibenclamide was done and estimation of metformin hydrochloride was done at its λmax 237.0 nm.

 

To determine the accuracy of method, recovery study was performed using the method of addition. To the preanalysed marketed formulation an accurately raw material is added at 3 levels viz., 90%, 100%, 110% for both the drug and total drug contents determined as described for formulation. The percentage recovery was determined and results are given   (Table 2).

 

By observing the validation parameters (Table 3), both the methods found to be specific, accurate, precise, repeatable and reproducible. However, Absorption ratio method has an advantage of simpler calculations over the simultaneous equations method; both methods can be employed for routine analysis of tablet for assay.

 

These methods are simple, specific and easy to perform and require short time to analyze the samples. Low limit of quantification and limit of detection makes these methods suitable for use in quality control. This method is enables for quantitative determination of metformin hydrochloride and glibenclamide because of good molar absorptivity and sandell’s sensitivity. The methods were found to be accurate, precise, linear, robust and rugged.

 

ACKNOWLEDGEMENTS:

The authors are thankful to Zydus Cadila Pvt.Ltd Ahmedabad (Gujarat), India for providing the gift samples of metformin hydrochloride and glibenclamide.

 

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Received on 25.07.2009          Modified on 23.09.2009

Accepted on 12.10.2009         © RJPT All right reserved

Research J. Pharm. and Tech. 3(1): Jan.-Mar. 2010; Page 109-112