Simultaneous Estimation of Metformin Glimeperide and Voglibose by

RP-UPLC

 

Sri Lakshmi D¹, Jane T Jacob², Srinivasa Sastry D¹, Satyanarayana D¹

¹Department of Pharmaceutical Chemistry, Aditya Pharmacy College, Surampalem, Andhra Pradesh India.

²Nitte University, NGSMIPS, Department of Pharmaceutical Chemistry, Mangalore, Karnataka, India.

*Corresponding Author E-mail: dsrilakshmi83@gmail.com

ABSTRACT:

A simple, sensitive and precise RP-UPLC method for estimation of Metformin, Glimeperide and Voglibose has been developed and validated for determination of compounds in commercial tablet dosage form. The compounds were well separated isocratically on a Inertsil ODS column using a mobile phase consisting of buffer (pH 3), and Methanol in the ratio of 70:30 v/v 0.4 ml/min flow rate with PDA detector. Retention time for Metformin, Glimeperide and Voglibose was found to be 0.903, 2.619, 3.818 min respectively. The method was validated in accordance with ICH guidelines. The study showed that the reverse phased liquid chromatography was sensitive and selective for detecting Metformin, Glimeperide and Voglibose using the single mobile phase.

 

 

 

INTRODUCTION:

Metformin:

Metformin is 1-carbamimidamido-N,N-dimethylmethan imidamide and belongs to the class of organic compounds known as biguanides. These are organic compounds containing two N-linked guanidines. Metformin is primarily used for type 2 diabetes, but is increasingly being used in polycystic ovary syndrome

Metformin is a biguanide antihyperglycemic agent used for treating non-insulin-dependent diabetes mellitus (NIDDM). It improves glycemic control by decreasing hepatic glucose production, decreasing glucose absorption and increasing insulin-mediated glucose uptake. Metformin may induce weight loss and is the drug of choice for obese NIDDM patients.

 

Use of metformin is associated with modest weight loss. When used alone, metformin does not cause hypoglycemia; however, it may potentiate the hypoglycemic effects of sulfonylureas and insulin. Its main side effects are dyspepsia, nausea and diarrhea. [1,2]

 

Fig 1 Structure of Metformin

 

Glimeperide:

Glimeperide chemically called as 3-ethyl-4-methyl-N-{2-[4-({[(4-methylcyclohexyl)carbamoyl]amino} sulfonyl)phenyl]ethyl}-2-oxo-2,5-dihydro-1H-pyrrole-1-carboxamide, Glimepiride is the first III generation sulphonyl urea it is a very potent sulphonyl urea with long duration of action. This compound belongs to the class of organic compounds known as benzene sulfonamides. These are organic compounds containing a sulfonamide group that is S-linked to a benzene ring. The mechanism of action of glimepiride in lowering blood glucose appears to be dependent on stimulating the release of insulin from functioning pancreatic beta cells, and increasing sensitivity of peripheral tissues to insulin. Glimepiride likely binds to ATP-sensitive potassium channel receptors on the pancreatic cell surface, reducing potassium conductance and causing depolarization of the membrane. Membrane depolarization stimulates calcium ion influx through voltage-sensitive calcium channels. This increase in intracellular calcium ion concentration induces the secretion of insulin. [3,4]

 

Fig 2 Structure of Glimeperide

 

Voglibose:

Voglibose chemically is (1S,2S,3R,4S,5S)-5-[(1,3-dihydroxypropan-2-yl)amino]-1-(hydroxymethyl) cyclohexane-1,2,3,4-tetrol. Voglibose (INN and USAN) is an alpha-glucosidase inhibitor used for lowering post-prandial blood glucose levels in people with diabetes mellitus. This compound belongs to the class of organic compounds known as aminocyclitols and derivatives. These are cyclitols with at least one hydroxyl group replace by an amino group. Alpha-glucosidase inhibitors are saccharides that act as competitive inhibitors of enzymes needed to digest carbohydrates: specifically alpha-glucosidase enzymes in the brush border of the small intestines. The membrane-bound intestinal alpha-glucosidases hydrolyze oligosaccharides, trisaccharides, and disaccharides to glucose and other monosaccharides in the small intestine. Acarbose also blocks pancreatic alpha-amylase in addition to inhibiting membrane-bound alpha-glucosidases. Pancreatic alpha-amylase hydrolyzes complex starches to oligosaccharides in the lumen of the small intestine. Inhibition of these enzyme systems reduces the rate of digestion of complex carbohydrates. Less glucose is absorbed because the carbohydrates are not broken down into glucose molecules. In diabetic patients, the short-term effect of these drugs therapies is to decrease current blood glucose levels. [5,6]

 

Figure 3 Voglibose

 

Literature  survey  of  Metformin, Glimeperide and Voglibose  revealed few  methods based  on  UV Spectrophotometry [7] , and Chromatography [8-9] have  been reported  for determination  of  both drugs  in  single and  combined  dosage  forms. The  present work describes  the  development and  validation as per ICH guidelines[10]  of  reverse  phase  high performance liquid chromatographic  (RP-HPLC)  method,  which can  quantify  these components simultaneously.

 

MATERIALS AND METHODS:

Experimental:

Materials and Methods: Reagents required Acetonitrile : UPLC grade, Water : UPLC grade, Methanol : UPLC grade, Potassium dihydrogen ortho phosphate: AR grade.

 

Drugs used The gift samples of Metformin, Glimeperide and Voglibose were kindly provided by Reddy’s laboratories and the marketed  formulation containing Metformin (500 mg), Glimeperide (1 mg) and Voglibose (0.2 mg)  were procured from local pharmacy.

 

Instrumentation and Chromatographic Conditions: The developed method  UPLC system with PDA detector data were acquired and processed by Empower software. The separation was carried out at ambient temperature by using a Inertsil ODS (50 x 1.7mm, 3 mm,).The  mobile phase consisting  of  Buffer (pH 3.0): Methanol (70:30v/v). The flow rate was 0.4 ml/min. The injection volume was 5 µL and detection at a wavelength of 232 nm. Mix a mixture of above buffer 700 ml (70%) and 300 ml (30%) of Methanol UPLC degas in ultrasonic water bath for 5 minutes. Filter through 0.45 µ filter under vacuum filtration.

 

Standard Solution Preparation:

Accurately weigh and transfer 500 mg of Metformin,0.2 mg of Voglibose and 1.0 mg of Glimeperide in a 100ml volumetric flask, dissolve in water and  make up with add  Methanol and sonicate to dissolve it completely and  make volume up to the mark with the same solvent. (Stock solution). Further pipette each 1.0 ml of above solutions from the above stock solution into a10ml volumetric flasks and dilute up   to the mark with diluent.  (500 ppm MET,0.2 ppm VOG and 1 ppm of GLIM)

 

Sample Solution Preparation:

Accurately weigh and transfer one tablet tablet powder equivalent to 500 mg Metformin 0.2mg of Voglibose and 1 mg of Glimeperide equivalent weight Tablet powder into a 100ml clean dry volumetric flask and sonicate to dissolve it completely and make volume up to the mark with the same solvent. (Stock solution) Further pipette 1.0 ml of the above stock solution into a 10ml volumetric flask and dilute up to the mark with diluents. (500 ppm MET,0.2 ppm VOG and 1 ppm of GLIM)

 

Linearity of pure standard solution:

The linearity of the samples of Metformin, Glimeperide and Voglibose were prepared by suitably diluting working solution and found to be linear response of drug over a range of 300-700ppm concentration for the Metformin, 0.6-1.4 ppm for Glimeperide and 0.12-0.28 µg/ml for Voglibose respectively. The three such linearities of Metformin, Glimipride and Voglibose were taken for correlation co-efficient and standard deviation calculation.

 

Table 1 Linearity of Metformin, Glimeperide And Voglibose

 

 

Fig 3 Linearity of Metformin

 

Fig 4 Linearity of Glimeperide

 

Fig 5 Linearity of Voglibose

 

Analysis of Formulation:

The developed procedure was extended to formulation of Metformin, Glimeperide and Voglibose, the combination was available in the market of strength 500 mg of Metformin, 1 mg of Glimeperide and 0.2 mg Voglibose respectively. Average weight of twenty tablets were taken and crushed to make  powder, weighed powder containing 500 mg Metformin was transferred to 100ml of volumetric flask and volume was made up to the mark with diluent (Buffer : Methanol) (70:30) and filtered through whatmann filter paper in to another 100ml volumetric flask and make up to mark with same diluent. The same procedure as mentioned for the pure drug was followed for the formulation. The concentrations of Metformin, Glimeperide and Voglibose were determined by measuring peak area at 232 nm.

 

 

Table 2 Assay Results

 

 

Method Validation Summary:

Precision:

Preparation of stock solution:

Accurately weigh and transfer 500 mg of Metformin,2.5 mg of Voglibose and 1.0 mg of Glimeperide in a 100ml volumetric flask, dissolve in water and  make up with add  Methanol and sonicate to dissolve it completely and make volume up to the mark with the same solvent. (Stock solution). Further pipette each 1.0 ml of above solutions from the above stock solution into a 10ml volumetric flasks and dilute up   to the mark with diluent.

 

Procedure:

The standard solution was injected for five times and measured the area for all five injections in HPLC. The %RSD for the area of five replicate injections was found to be within the specified limits.

 

Table 3 Precision results of  Metformin

 

 

Table 4 Precision results of  Glimeperide

 

 

Table 5 Precision results of  Voglibose

 

Acceptance Criteria:

The % RSD for the area of five standard injections results should not be more than 2%.

 

Intermediate Precision/Ruggedness:

To evaluate the intermediate precision (also known as Ruggedness) of the method,   Precision was performed on different day by using different make column of same dimensions. 

 

Preparation of stock solution:

Accurately weigh and transfer 500 mg of Metformin,0.2 mg of Voglibose and 1.0 mg of Glimeperide in a 100ml volumetric flask, dissolve in water and  make up with add  Methanol and sonicate to dissolve it completely and make volume up to the mark with the same solvent. (Stock solution). Further pipette each 1.0 ml of above solutions from the above stock  solution into a 10ml volumetric flasks and dilute up   to the mark with diluent.

 

Procedure:

The standard solution was injected for five times and measured the area for all five injections in HPLC. The %RSD for the area of five replicate injections was found to be within the specified limits.

 

The results are summarized Metformin

 

The results are summarized Voglibose

The results are summarized Glimeperide

 

Acceptance Criteria:

The % RSD for the area of five standard injections results should not be more than 2%.

 

Accuracy:

Preparation of Standard stock solution:

Accurately weigh and transfer 500 mg of Metformin,0.2mg of Voglibose and 1.0 mg of Glimeperide in a 100ml volumetric flask, dissolve in water and  make up with add  Methanol and sonicate to dissolve it completely and make volume up to the mark with the same solvent. (Stock solution). Further pipette each 1.0 ml of above solutions from the above stock solution into a 10ml volumetric flasks and dilute up   to the mark with diluent.

 

Preparation Sample solutions:

For preparation of 50% solution (With respect to target Assay concentration):

Accurately weigh and transfer 500 mg of Metformin,0.2mg of Voglibose and 1.0 mg of Glimeperide in a 100ml volumetric flask, dissolve in water and  make up with add  Methanol and sonicate to dissolve it completely and make volume up to the mark with the same solvent. (Stock solution). Further pipette each 0.5 ml of above solutions from the above stock  solution into a 10ml volumetric flasks and dilute up   to the mark with diluent.

 

For preparation of 100% solution (With respect to target Assay concentration):

Accurately weigh and transfer 500 mg of Metformin,0.2 mg of Voglibose and 1.0 mg of Glimeperide in a 100ml volumetric flask, dissolve in water and  make up with add  Methanol and sonicate to dissolve it completely and make volume up to the mark with the same solvent. (Stock solution). Further pipette each 1.0 ml of above solutions from the above stock  solution into a 10ml volumetric flasks and dilute up   to the mark with diluent.

 

For preparation of 150% solution (With respect to target Assay concentration):

Accurately weigh and transfer 500 mg of Metformin,0.2mg of Voglibose and 1.0 mg of Glimeperide in a 100ml volumetric flask, dissolve in water and  make up with add  Methanol and sonicate to dissolve it completely and make volume up to the mark with the same solvent. (Stock solution). Further pipette each 1.5 ml of above solutions from the above stock solution into a 10ml volumetric flasks and dilute up   to the mark with diluent.

 

Procedure:

Inject the standard solution, Accuracy -50%, Accuracy -100% and Accuracy -150% solutions. Calculate the Amount found and Amount added for Metformin, Voglibose and Glimeperide calculate the individual recovery and mean recovery values.

 

The accuracy results for Metformin

 

The accuracy results for Glimeperide

 

The accuracy results for Voglibose

 

Acceptance Criteria:

The % Recovery for each level should be between 98.0 to 102.0%

 

Linearity:

The linearity of the method was determined by comparing the known concentration Vs response, a series of calibration standards  300, 400, 500, 600, 700 µg/ml of Metformin, 0.6, 0.8, 1.0, 1.2, 1.4 µg/ml of Glimeperide  and 0.12, 0.16, 0.20, 0.24, 0.28 µg/ml of Voglibose  were prepared. The solutions were injected into the chromatographic system and peak area of each peak at each concentration was noted. The calibration curve was plotted using peak area versus concentration of the standard solution.

 

Limit of Detection: 

Preparation of stock solutions:

Accurately weigh and transfer 500mg of Metformin API, 0.2mg of Voglibose, 1 mg of Glimeperide in individual 100 ml volumetric flasks, add about 7mL of Diluents and sonicate to dissolve it completely and make volume up to the mark with the same solvent. (Stock solution).

 

Further pipette 1.0 ml of above solutions into 10 ml individual volumetric flask and dilute up to the mark with diluent.

 

For Metformin:

Preparation of 0.15 µg/ml solution:

From the above stock solution dilutions were made to get the concentration equivalent to 0.15μg/ml

 

For Voglibose:

Preparation of 0.004 µg/ml solution:

From the above stock solution dilutions were made to get the concentration equivalent to 0.004μg/ml

 

For Glimeperide :

Preparation of 0.001 µg/ml solution:

From the above stock solution dilutions were made to get the concentration equivalent to 0.001μg/ml

 

 

Acceptance Criteria:

S/N Ratio value shall be 3 for LOD solution.

 

Limit of Quantification:

Preparation of stock solutions: 

Accurately weigh and transfer 500mg of Metformin API, 0.2mg of Voglibose, 1 mg of Glimeperide in individual 100 ml volumetric flasks, add about 7mL of Diluents and sonicate to dissolve it completely and make volume up to the mark with the same solvent. (Stock solution).

 

Further pipette 1.0 ml of above solutions into 10 ml individual volumetric flask and dilute up to the mark with diluent.

 

For Metformin:

Preparation of 0.45 µg/ml solution:

From the above stock solution dilutions were made to get the concentration equivalent to 0.45μg/ml

 

For Voglibose:

Preparation of 0.012 µg/ml solution:

From the above stock solution dilutions were made to get the concentration equivalent to 0.012 μg/ml

 

For Glimeperide :

Preparation of 0.002 µg/ml solution:

 

From the above stock solution dilutions were made to get the concentration equivalent to 0.002 μg/ml

 

 

Acceptance Criteria:

S/N Ratio value shall be 10 for LOQ solution.

 

Robustness:

As part of the Robustness, deliberate change in the Flow rate, Mobile Phase composition Variation was made to evaluate the impact on the method. The flow rate was varied at 0.3 ml/min to 0.5 ml/min. Standard solution of Metformin, Voglibose and Glimeperide was prepared and analysed using the varied flow rates along with method flow rate.  The results are summarized  On evaluation of the above results, it can be concluded that the variation in flow rate affected the method significantly. Hence it indicates that the method is robust even by change the change of flow rate. The method is robust in change in flow condition.

 

System suitability results for Metformin:

S. No	Flow Rate (ml/min)	System Suitability Results
		USP Plate Count	USP Tailing
1	0.3	2077	1.36
2	0.4	2117	1.39
3	0.5	2526	1.29

* Results for actual flow (0.40 ml/min) have been considered from Assay standard

 

System suitability results for Voglibose

S. No	Flow Rate (ml/min)	System Suitability Results
		USP Plate Count	USP Tailing
1	0.3	7552	1.01
2	0.4	7023	1.03
3	0.5	7242	1.22

 

 

System suitability results for Glimeperide

S. No	Flow Rate (ml/min)	System Suitability Results
		USP Plate Count	USP Tailing
1	0.3	9205	0.99
2	0.4	8438	1
3	0.5	8660	1.1

* Results for actual flow have been considered from Assay standard.

 

Standard solution of Metformin, Voglibose and Glimeperide was prepared and analysed using the varied mobile phase The results are summarized On evaluation of the above results, it can be concluded that the variation in mobile phase affected the method significantly. Hence it indicates that the method is robust even by change in moble phase ratios.

 

System suitability results for Metformin:

S. No	Mobile phase ratio	System Suitability Results
		USP Plate Count	USP Tailing
1	Less org	2455	1.37
2	Actual	2117	1.39
3	More org	2103	1.4

 

System suitability results for Voglibose

S. No	Mobile phase ratio	System Suitability Results
		USP Plate Count	USP Tailing
1	Less org	7955	1.14
2	Actual o	7023	1.03
3	More org	7078	1.25

 

System suitability results for Glimeperide

S. No	Mobile phase Ratio	System Suitability Results
		USP Plate Count	USP Tailing
1	Less org	8094	1.04
2	Actual	8438	1
3	More org	9034	1.11

 

 

Degradation Studies:

The International Conference on Harmonization (ICH) guideline entitled stability testing of new drug substances and products requires that stress testing be carried out to elucidate the inherent stability characteristics of the active substance. The aim of this work was to perform the stress degradation studies on the metformin, Voglibose and Glimeperide  using the proposed method.

 

Preparation of Stock Solution:

Accurately weigh and transfer 500 mg of Metformin, 0.2 mg of Voglibose and 1.0 mg of Glimeperide in a 100ml volumetric flask, dissolve in water and  make up to the mark with  Methanol and  sonicate to dissolve it completely and make volume up to the mark with the same solvent. (Stock solution).

 

Acidic Degradation:

Pipette 1.0 ml of above solution into a 10ml volumetric flask and 3 ml of 0.1N HCl was added. Then, the volumetric flask was kept at 60ºC for 6 hours and then neutralized with 0.1 N NaOH and make up to 10ml with diluent. Filter the solution with 0.22 microns syringe filters and place in vials.

Alkaline Degradation:

Pipette 1.0 ml of above solution into a 10ml volumetric flask into a 10ml volumetric flask and add 3 ml of 0.1N NaOH was added in 10 ml of volumetric flask. Then, the volumetric flask was kept at 60ºC for 6 hours and then neutralized with 0.1N HCl and make up to 10ml with diluent. Filter the solution with 0.22 microns syringe filters and place in vials.

 

Thermal Degradation

Metformin, Voglibose  and Glimeperider  sample was taken in petridish and kept in Hot air oven at 110⁰ C for 24 hours. Then the sample was taken and diluted with diluents and injected into HPLC and analysed.

 

Oxidative Degradation:

Pipette 1.0 ml above  stock solution 2 into a 10ml volumetric flask solution into a 10ml volumetric flask 1 ml of 3% w/v of hydrogen peroxide added in 10 ml of volumetric flask and the volume was made up to the mark with diluent . The volumetric flask was then kept at room temperature for 15 min. Filter the solution with 0.45 microns syringe filters and place in vials.

 

Results:

 

 

RESULTS AND DISCUSSION:

Retention times of Metformin, Glimeperide and Voglibose were found to be 0.903, 2.619 and 3.818 minutes respectively. The developed method was validated according to ICH guidelines, linearity of Metformin was found to be in the range of 300-700 μg/mL, Glimeperide 0.6-1.4 μg/mL and that of Voglibose was found to be  in the range of 0.12-0.28 μg/mL. The percentage recoveries for all the three drugs were found in the range of 99-101%. The limit of detection values for Metformin, Glimeperide, Voglibose were found to be 2.98, 2.97 and 2.95 and the limit of quantification for Metformin, Glimeperide, Voglibose were found to be 9.97, 9.98 and 9.97 respectively.

 

CONCLUSION:

The proposed RP-HPLC method for estimation of Metformin, Glimeperide and Voglibose is simple, rapid, specific, accurate, isocratic and precise with simple mobile phase. The method gives good resolution between the compounds with a short analysis time.

 

REFERENCE:

1.     Metformin HCl IP Vol II, 2186-2187, 2014

2.     http://www.drugbank.ca/drugs/DB00331

3.     Glimeperide IP Vol II, 1864-1865, 2014

4.     http://www.drugbank.ca/drugs/DB00222

5.     Voglibose IP Vol III, 2979-2980, 2014.

6.     http://www.drugbank.ca/drugs/DB04878

7.     Deshpande A S, Nilesh A, Shashikant B B, Development of Novel method for Estimation Of Metformin HCl, Glimeperide and Voglibose in multi Ingredient dosage form by Derivative Spectroscopy, WJPPS, Vol 3, Issue 3, 1812-1823,2014.

8.     Neelima K, Rajendra Prasad Y, Analytical Method Development and Validation of Metformin, Voglibose, Glimepiride in Bulk and Combined Tablet Dosage Form by Gradient RP-HPLC, Pharmaceutical Methods, Vol 5, Issue 1, 27-33, 2014.

9.     Srikanth K, Abdul Rahaman SK, Development and Validation of RP-HPLC method for Simultaneous estimation of Metformin Hydrochloride, Voglibose and Glimepiride in bulk and Pharmaceutical Dosage form, WJPR, Vol 4, Issue 12, 881-891.

10.   International Conference on Harmonization (ICH) of technical requirements for the registration of pharmaceuticals for human use, validation of analytical procedures definitions and terminology, 1996, Geneva.

 

 

 

Received on 01.11.2016       Accepted on 23.11.2016     

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