1. INTRODUCTION:

Diabetes is one of the costliest health problems in the world. Globally, diabetes is likely to be the fourth leading cause of death [1]. Approximately 90% of people with diabetes have type 2 diabetes. It usually begins as insulin resistance, a disorder in which the cells do not use insulin properly. As the need for insulin rises; the pancreas gradually loses its ability to produce insulin. Type II diabetes is associated with older age, obesity, family history of gestational diabetes, impaired glucose metabolism, physical inactivity and race/ ethnicity [2]. If the glycemic target level is not achieved with one oral agent alone, combination oral and/or insulin therapy is recommended [3, 4]. Combination oral therapy becomes an obvious choice when glycemic control is not achieved with conventional monotherapy [5]. The advantages of oral dose

combinations as compared to their components which are taken alone are lower cost and better patient compliance [6, 7].Combination therapy has been shown to achieve greater blood glucose lowering than monotherapy because different classes have different and complimentary mechanisms of action.

Therefore, it is more logical to add another drug than replace the existing drug. The rapid introduction of combination therapy with two or three complementary oral anti diabetics help in targeting the dual effect and also reduced adverse effects [8]. Pioglitazone is a thiazolidine dione derivative and it is used in patient with NIDDM type of antidiabetic, chemically it is 5-(4-[2-(5-ethypyridin-2-yl] benzyl) thiazolidine-2, 4-dione. The molecular formula is C19H20N2O3S. Chemically, metformin is 1,1-dimethyl biguanide hydrochloride, pioglitazone is (± )-5-[p- [2-(5- ethyl-2-pyridyl)-ethoxy] benzyl]-2,4-thiazolidinedione. Metformin improves hepatic and peripheral tissue sensitivity to insulin without the problem of serious lactic acidosis, pioglitazone has been shown to affect abnormal glucose and lipid metabolism associated with insulin resistance by enhancing insulin action on peripheral tissues.[9] Fig 1 shows that the structures of Pioglitazone [a] and Metformine[b]

[a] Pioglitazone

[b] Metformine

Fig 1: Structures of Pioglitazone [a] and Metformine[b]

2. MATERIAL AND METHODS:

2.1. Apparatus

A double beam UV/Vis spectrophotometer, Shimadzu UV- 1700 Pharmaspec, was employed with a pair of 1 cm quartz cells for all analytical work.

2.2. Reagents and chemicals

Metformine Hydrochloride and Pioglitazone Hydrochloride were obtained from Gen Pharmaceuticals Ltd.Pune, Maharastra, India as a gift sample and were used as working standards. Sodium hydroxide of analytical grade and double distilled water were used throughout the analysis.

2.3. Commercial formulation

A commercial pharmaceutical preparation, Glime P tablet (Metformine Hydrochloride 500 mg and 15 mg Pioglitazone Hydrochloride) was procured from the local market.

2.4. Preparation of standard solution

Standard stock solution of Metformine Hydrochloride and Pioglitazone Hydrochloride was prepared by dissolving 10 mg of each drug separately in 100mL volumetric flask using 0.1N sodium hydroxide as solvent up to 50 ml and volume make up with distilled water and both sample sonicate upto 20 min. Stock solutions of 100 μg/mL were obtained in this manner. From these stock solutions, working standard solutions of concentration were prepared by appropriate dilutions. Working standard solutions were scanned in the entire UV range to determine the λ max. The λ max of Metformine Hydrochloride and Pioglitazone Hydrochloride were found to be 233 nm and 265.5 nm respectively.

2.5. Calibration curves

Seven standard dilutions of each drug were prepared separately having concentrations of 2-20 μg/mL. The absorbances of these standard solutions were measured at 233 nm and 265.5 nm and calibration curve was plotted. The absorptivity coefficients of the two drugs were determined using calibration curve (graph 1 and 2)

Graph 1: Pioglitazone Hydrochloride

Graph 2 :Metformine Hydrochloride

2.6. Preparation of sample solution

Sample solution containing both the drugs was prepared by dissolving 10 mg of each drug in 100mL volumetric flask using 50 ml of 0.1N sodium hydroxide to give stock solutions then both sonicate for 20 min and further with distilled water and made 100 μg/mL stock solution, working standard solution of 10 μg/mL concentration was prepared by appropriate dilution. Seven standard dilutions of concentrations of 2, 4, 8, 10, 12, 16 and 20 μg/mL was prepared from working standard solution. The absorbance of this sample solution was measured at 233 nm and 265.5 nm and their concentrations were determined using proposed analytical methods.

2.7. Simultaneous equations method:

Method was based on simultaneous equation method of Vierodt. The method is applicable in the case of sample containing two drugs, each of which absorbs at the λ max of the other (Beckett et al, 1997). Two equations were constructed based upon the fact that the absorbance of the mixture of Metformine and Pioglitazone at 233nm and 265.5nm is the sum of the absorbances at respective wavelengths. Two equations were developed using absorptivity coefficient values.

CM = A0.0286 - A2 0.01547 / 0.00048×0.0286 – 0.0523×0.01547 -(1)

CP = A1 0.00048 - A2 0.0523 /0.00048×0.0286 – 0.0523×0.01547 (2)

Where CM and C P are concentrations of Metformine Hydrochloride and Pioglitazone Hydrochloride in g/100mL respectively in the sample solution. A 1 and A 2 are absorbances of the mixture at 233 nm and 265.5 nm, respectively. Solving these two equations, the concentrations C H and C T can be readily determined.

Fig:1 Spectra of Mixture (Metformine Hcl and Pioglitazone Hcl)

2.8. Estimation in the marked formulation

Twenty tablets were weighed and crushed to a fine powder. An accurately weighed powder sample equivalent to 10 mg of Pioglitazone Hydrochloride and Metformine Hydrochloride was transferred to a 10 ml volumetric flask, dissolved in 5 ml 0.1N sodium hydroxide, shaken for 10 min and the volume was made up to the mark with 0.1N sodium hydroxide. The solution was then filtered through Whatman filter paper no. 41. The solution was further diluted to get different concentrations in the range of 2-20 μg/mL of both the drugs. For this method the absorbances of the sample solution, i.e., A 1 and A 2 , were recorded at 233 nm and 265.5 nm respectively, and concentration of two drugs in the sample were determined using the equations(1) and (2). The analysis procedure was repeated three times with the formulation. The result of analysis of the formulation is shown in Table 1.

2.9. Method validation

The method validation parameters like linearity, precision, accuracy, repeatability, limit of detection and limit of quantitation were checked as per ICH guidelines.

2.10. Linearity and range

The linearity for Metformine and Pioglitazone were determined at seven concentration levels, ranging from 2- 20μ/mL using working standards shown in table 1-2

2.11. Precision and accuracy

Journal of Applied Pharmaceutical Science 01 (01); 2011: 46-49 The precision of the method was evaluated by inter day and intraday variation studies. In intraday studies, working solutions of standard and sample were analysed thrice in a day and percentage relative standard deviation (% RSD) was calculated. In the inter day variation studies, working solution of standard and sample were analysed on three consecutive days and percentage relative standard deviation (% RSD) was calculated. The data is shown in table 5-6.

The accuracy of the method was determined by recovery studies. The recovery studies were performed by the standard addition method at 80%, 100% and 120% level and the percentage recoveries were calculated and are shown in Table 1-2.

2.12. Limit of detection and limit of quantitation

The Limit of Detection (LOD) is the smallest concentration of the analyte that gives the measurable response. LOD was calculated using the following formula and shown in Table 3-4.

LOD = 3.3 (σ / S)

Where, S = slope of calibration curve, σ = standard deviation of the response.

The Limit of Quantification (LOQ) is the smallest concentration of the analyte, which gives a response that can be accurately quantified. LOQ was calculated using the following formula and shown in Table 3-4.

LOQ = 10 (σ / S)

Where, S = slope of calibration curve, σ = standard deviation of the response.

3. RESULTS AND DISCUSSION:

In the present work, new method, namely, simultaneous equation method (Vierordt's method) was used for the simultaneous spectroscopic estimation of Metformine HCl and Pioglitazone HCl in commercially available tablet dosage form. The concentrations in the range of 2-20 μg/mL of mixed working standard and two sampling wavelengths of 233 nm (λ max of Metformine Hcl), and 265.5 nm (λ max of Pioglitazone HCl) gave optimum accuracy, precision, time, economy, and sensitivity for this method. The proposed procedure was successfully applied to the determination of Metformine Hcl and Pioglitazone Hcl in the commercially available tablets dosage form.

The recovery studies were carried out at different concentrations by spiking a known concentration of standard drug to the preanalysed sample and contents were reanalysed by proposed methods. The results of marketed formulation analysis and Recovery studies are depicted in Table 1-2. The method was validated Statistically for range, linearity, precision, accuracy, repeatability, LOD, and LOQ Table 3-4. Accuracy was ascertained on the basis of Recovery studies. Precision was calculated as inter and intraday Variation for both the drugs. The percentage recoveries for of Metformine HCl and Pioglitazone HCl were found to be 97.69±.098-99.44±0.95 and 98.93±0.32- 99.93±0.14 for this method respectively. The contents estimated using the proposed method was found in agreement with the labeled amount Table 1-2.The relative standard deviations was found to be within the limit, indicating good accuracy, precision, and repeatability of the proposed method.

Table 1: Determination of Accuracy by percentage recovery method for Pioglitazone HCl

Ingredients

Tablet amount (µg/ml)

Amount added

(µg/ml)

Level of addition

Amount recovered (µg/ml)

Percentage recovery

Average % recovery

Pioglitazone

50(µg/ml)

2(µg/ml)

2.5(µg/ml)

3(µg/ml)

80%

100%

120%

4.5(µg/ml)

5(µg/ml)

5.5(µg/ml)

99.77%

99.98%

100.24%

99.99±0.7662

Table 2: Determination of Accuracy by percentage recovery method for Metformine HCl

Ingredients

Tablet amount (µg/ml)

Amount added

(µg/ml)

Level of addition

Amount recovered

(µg/ml)

Percentage recovery

Average % recovery

Metformine

50(µg/ml)

2(µg/ml)

2.5(µg/ml)

3(µg/ml)

80%

100%

120%

4.5(µg/ml)

5(µg/ml)

5.5(µg/ml)

99.98%

99.99%

100.90%

100.29%±1.7891

Table 3 :Validation parameters for Pioglitazone

Sr. No

Parameters

Results

Absorption (nm)

Linearity range (µg/ml)

Standard regression equation

Correlation coefficient (r²)

A (1%, 1cm)

Accuracy (% recovery± SD)

Precision (% CV)

Specificity

LOD

LOQ

265.5nm

5-25 µg/ml

y=0.016x

r²=0.995

15324

99.99±0.1315

101.5 %,100.9%

A 50 µg/ml solution of candidate drug in solvent (0.1 N NaOH and distilled water mixture in the ratio of 50:50 respectively ) at UV detection ʎ of 265.5 nm will show an absorbance value of 0.7662

0.0077

0.0235

Table 4 :Validation parameters for Metformin

Sr. No

Parameters

Results

Absorption (nm)

Linearity range (µg/ml)

Standard regression equation

Correlation coefficient (r²)

A (1%, 1cm)

Accuracy (% recovery± SD)

Precision (% CV)

Specificity

LOD

LOQ

233nm

2-20 µg/ml

r²=

35781

100.29%±0.2567

100.7 %,101.3%

A 25 µg/ml solution of candidate drug in solvent (0.1 N NaOH and distilled water mixture in the ratio of 50:50 respectively ) at UV detection ʎ of 233 nm will show an absorbance value of 1.7891

0.067

0.2055

Table 5: Precision data for the developed method Assays of Pioglitazone as % of labeled amount

Sample number

Analyst –I(Intra- day precision)

Analyst –II (Inter- day precision)

101.6

101.9

101.5

101.4

101.1

100.3

101.2

100.1

100.8

101.0

100.6

99.9

Average ±S.D.

101.5 ±

0.324

100.9±

0.493

Table 6: Precision data for the developed method Assays of Metformine as % of labeled amount

Sample number

Analyst –I (Intra- day precision)

Analyst –II (Inter- day precision)

100.4

100.6

100.8

100.4

100.7

101.2

101.5

101.1

101.5

101.0

101.1

Average± S.D.

100.7±

0.444

101.3±

0.343

4. CONCLUSION:

The Vierordt’s method permits simple, rapid and direct determination of of Metformine HCl and Pioglitazone HCl in commercially available tablet dosage form without previous separation. The results of analysis of two drugs from tablet formulation using method was found close to 100%, Standard deviation was satisfactorily low indicating accuracy and reproducibility of the method. Recovery studies were satisfactory which showed that there is no interference of excipients.

5. ACKNOWLEDGEMENT:

The authors wish to thank Gen Pharmaceuticals Ltd Pune, Maharashtra for providing the gift samples of of Metformine HCl and Pioglitazone HCl.

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Received on 30.12.2011 Accepted on 28.02.2011

Asian J. Pharm. Ana. 2(1): Jan.-Mar. 2012; Page 05-09