Research & Reviews: Journal of Pharmaceutical Analysis
Menue-ISSN: 2320-0812
e-ISSN: 2320-0812
Sowjanya P*
Department of Pharmaceutical Analysis, CSN Institute of Pharmacy, Bhimavaram, Andhra Pradesh, India
Received date: 20/10/2015; Accepted date: 02/11/2015; Published date: 10/11/2015
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The existing available literature reports do not show any stability indicative analytical method for the estimation of metformin in dosage form. Hence there is a need to develop and validate an analytical method to estimate metformin. Hence the present study aims is to develop and validate a suitable high precision and accurate analytical method for the simultaneous estimation of metformin in dosage form, by reverse phase high performance liquid chromatography (RPHPLC). The main objective of the present work is to develop and validate a suitable high precision and accurate analytical method for the estimation of metformin in dosage form by reverse phase high performance liquid chromatography (RPHPLC).
Metformin; RP-HPLC method
Quantitative measurement was performed on a quaternary HPLC AGILENT series and HPLC pumps, with a 20 μl sample injection loop (automatic), and UV-Visible absorbance detector and photo-diode array detector. The output signal was monitored and integrated using the output signal was monitored and integrated using EZ-Chrome software C-18 column was used for the separation [1,2].
Reagents Used for the Study
Ortho phosphoric acid: AR grade
Acetonitrile: HPLC grade
Water: Milli-Q grade
Methanol: HPLC grade
Diammonium hydrogen phosphate: ARgrade
The objective of this experiment was to optimize the assay method for metformin based on the literature survey made. [3,4].
The retension time was too long and the peak was non symmetric and they do not pass tailing factor in all the above 3 trails and the 4th trail was optimized.
Optimized Method Chromatographic conditions
Flow rate: 1.0 mL per min
Column: Thermosil C18
Detector wave length: 232 nm
Column temperature: 25°C
Injection volume: 20 μl
Run time: 6.0 min
Diluent: mobile phase
Mobile phase: 40:60 (water:acetonitrile)
The retention time was found to be 2.064
It shows the good peak with good symmetry. Hence this method was finalized for the development of metformin.
Optimized Chromatographic Conditions for the Estimation of Metformin (Table 1 and Figure 1).
Figure 1: Standard graph for estimation of metformin
Table 1: Conditions for the estimation of Metformin.
Preparation of Buffer
Weight 2.72 gm of potassium dihydrogen ortho phosphate in 1000 ml hplc water; adjust to pH 2.8 with ortho phosphoric acid.
Mobile Phase
Take HPLC Water 400 mL (40%) and 600 mL of Acetonitrile HPLC (60%) and degas in ultrasonic water bath and mix for 5 minutes and then filter through 0.45 μ under vacuum filtration.
Test Solution Preparation
Weigh 10 mg of metformin and transfer it in to 10 ml of volumetric flask and then add 5 ml of diluent and sonicate dissolve it completely and make up the volume to the mark with the same solvent (Stock solution). Pipette out the 0.4 ml of the above solution and transfer in to volumetric flask make up the solution by adding diluent. Mix the solution thoroughly and filter through by using 0.45 μ filter.
Test Procedure
Inject 20 μL of the standard, sample into the chromatographic system and measure the area for the metformin peak and calculate the%Assay by using the formulae.
System Suitability Requirements
Tailing factor: should not be more than 2.0%. Theoretical plates: should not be less than 2000.
System suitability
For system suitability, two replicates of standard sample were injected and studied the parameters like plate number (N), HETP, peak symmetry of samples, %RSD of areas of reference solution
Evaluated the following system suitability parameters.
Theoretical plates & USP Tailing factor for standard
Acceptance criteria
1. The number of theoretical plates (N) for metformin peaks is NLT 2000.
2. The Tailing factor (T) for metformin peaks is NMT 2.0% (Table 2 and Figure 2).
Figure 2: System Suitability graph.
Table 2: System suitability acceptance criteria.
Precision Preparation of stock solution
Weigh the 10 mg of metformin and transfer in to 10 ml of volumetric flask and add 7 ml of diluent dissolve it completely. By using same solvent make up the volume up to the mark.
Preparation of 40 μg/ml solution
Pipette out 0.4 ml of the above stock solution and transfer into a 10 ml volumetric flask and dilute up to the mark with diluent. Mix thoroughly and then filter through 0.45 μm filter.
Procedure
The standard solution was injected and measured the area in HPLC. This process is repeated to five times. The %RSD for the area of five injections was obtained to be within the specified limits (Table 3).
Table 3: %RSD for the area of injected samples.
Acceptance criteria
The% RSD for the area of five standard injections results should not be more than 2% (Figures 3a-3e).
Figure 3: Precession acceptance criteria graphs: (a): graph for channel 1 and Injection 1.
Figure 3b: Graph for channel 1 Injection 2.
Figure 3c: Graph for channel 1 Injection 3.
Figure 3d: Graph for channel 1 Injection 4.
Figure 3e: Graph for channel 1 Injection 5.
Figure 3f: Graph for channel 1 Injection 6.
Accuracy [5,6] Preparation of stock solution
Weigh the 10 mg of metformin and transfer in to 10 ml of volumetric flask and add 7 ml of diluent and dissolve it completely. By using same solvent make up the volume up to the mark.
Preparation of 40 μg/ml solution
Pipette out 0.4 ml of the above stock solution and transfer into a 10 ml volumetric flask and dilute up to the mark with diluent. Mix thoroughly and then filter through 0.45 μm filter.
Preparation sample solutions For preparation of 50% solution (With respect to Assay concentration)
Weigh the 05 mg of METFORMIN API and transfer in to 10 ml of volumetric flask and add 7 ml of diluent and dissolve it completely. By using same solvent make up the volume up to the mark and use it as stock solution. (with respect to Assay concentration).
Weigh the 10 mg of METFORMIN API and transfer in to 10 ml of volumetric flask and add 7 ml of diluent and dissolve it completely. By using same solvent make up the volume up to the mark and use it as stock solution.
Pipette out 0.4 ml of the above stock solution and transfer into a 10 ml volumetric flask and dilute up to the mark with diluent. Mix thoroughly and then filter through 0.45 μm filter.
For preparation of 150% solution (With respect to target Assay concentration)
Weigh the 15 mg of METFORMIN API and transfer in to 10 ml of volumetric flask and add 7 ml of diluent and dissolve it completely. By using same solvent make up the volume up to the mark and use it as stock solution.
Pipette out 0.4 ml of the above stock solution and transfer into a 10 ml volumetric flask and dilute up to the mark with diluent. Mix thoroughly and then filter through 0.45 μm filter
Procedure
Inject the standard solution, Accuracy -50%, Accuracy -100% and Accuracy -150% solutions.
Calculate the obtained Amount and added Amount for METFORMIN and also calculate the individual and mean recovery values (Table 4).
Table 4: Individual calculations and mean recovery values.
Acceptance criteria
The% Recovery for each level should be between 98.0 to 102.0% (Figures 4).
Figure 4: Graph shows accuracy acceptance criteria.
Linearity: [7,8] Preparation of stock solution
Accurately weigh and transfer 10 mg of METFORMINAPI sample into a 10 mL volumetric flask add about 7 mL of Diluent and sonicate to dissolve it completely and make volume up to the mark with the same solvent (Table 5). (Stock solution)
Table 5: Linearity correlation co efficient calculations.
Preparation of Level – I (20 μg/ml):
0.2 ml of stock solution has taken in 10 ml of volumetric flask dilute up to the mark with diluents
Preparation of Level – I (30 μg/ml):
0.3 ml of stock solution has taken in 10 ml of volumetric flask dilute up to the mark with diluent.
Preparation of Level – II (40 μg/ml):
0.4 ml of stock solution taken in 10 ml of volumetric flask dilute up to the mark with diluent.
Preparation of Level – III (50 μg/ml):
0.5 ml of stock solution taken in 10 ml of volumetric flask dilute up to the mark with diluent.
Preparation of Level – IV (60 μg/ml):
0.6 ml of stock solution taken in 10 ml of volumetric flask dilute up to the mark with diluent.
Procedure
Inject each level into the chromatographic system and measure the peak area. Plot a graph of peak area versus concentration (on X-axis concentration and on Y-axis Peak area) and calculate the correlation coefficient (Table 6).
Table 6: The flow rate analysis in robustness.
Acceptance criteria
Correlation coefficient should be not less than 0.999 (Figures 5 and 6).
Figure 5: Linearity correlation coefficient for different samples.
Figure 6: Linearity graph between the area and the concentration.
Robustness
As part of the Robustness, deliberate change in the Flow rate, Mobile Phase composition, Temperature Variation was made to evaluate the impact on the method.
a) The flow rate was varied.
Standard solution 30 μg/ml was prepared and analysed using the varied flow rates along with method flow rate (Table 7). More flow (Figure 7)
Figure 7: In robustness at more flow rate the graph show.
Table 7: Use of organic composition in the mobile phase.
Less flow (Figure 8)
Figure 8: In robustness at less flow rate the graph show.
The Organic composition in the Mobile phase was varied.
Standard solution 30 μg/ml was prepared and analysed using the varied Mobile phase composition along with the actual mobile phase composition in the method (Table 8 and Figure 9).
Figure 9: In robustness changing the organic phase compositions.
Table 8: Assay results for metformin drug.
Acceptance criteriai
The acceptance criterion for an assay was in between 97% to 103% (Figures 10a, 10b and 11a-11c).
Figure 10: Standard graph for metformin drug.
Figure 11: Graphs for metformin drug by using samples.