Developing a single analytical method for estimation of individual drug from a multidrug composition is a very challenging task. A simple, rapid, precise, and reliable reverse phase HPLC method was developed for the separation and estimation of three drugs glimepiride, pioglitazone and metformin in bulk drug mix and pharmaceutical dosage forms. The estimation was carried out using Inertsil ODS-3V (250 mm × 4.6 mm, 5
In the current Indian scenario, most commonly attacking disease to a common man has been found to be diabetes. Recent studies indicate that prevalence of type-2 diabetes is rapidly increasing in the society. Type-2 diabetes is a progressive disorder with a consistent and steady increase in glycosylated hemoglobin (HbA1C) overtime associated with enhanced risk of micro- and macrovascular complications and a substantial reduction in life expectancy. There are three major pathophysiologic abnormalities associated with type-2 diabetes: (i) impaired insulin secretion, (ii) excessive hepatic glucose output, and (iii) insulin resistance in skeletal muscles, liver and adipose tissue. These defects have been treated by use of oral insulin secretagogues (sulphonyl ureas/glinides) or insulin, biguanides, and thiazolidinediones, respectively [
Glimepiride is a medium-to-long acting sulphonyl urea antidiabetic drug. It is chemically 1-[[p-[2-(3-Ethyl-4-methyl-2-oxo-3-pyrroline-1 carboxamido)ethyl]phenyl]sulfonyl]-3-(trans-4-methyl cyclohexyl) urea. The primary mechanism of action of glimepiride in lowering blood glucose appears to be dependent on stimulating the release of insulin from functioning pancreatic beta cells. Metformin hydrochloride is also antidiabetic drug in the biguanide class and it is chemically 1,1-dimethyl biguanide monohydrochloride. It decreases hepatic glucose production, decreases intestinal absorption of glucose, and improves insulin sensitivity by increasing peripheral glucose uptake and utilization. Pioglitazone is a prescription drug of thiazolidinedione class with hypoglycemic (antihyperglycemic, antidiabetic) action; it is chemically (±) 5-[[4-[2-(5-Ethyl-2-pyridinyl)ethoxy]phenyl]methyl]-2,4] thiazolidinedione monohydrochloride. It selectively stimulates the nuclear receptor peroxisome proliferator activated receptor-gamma (PPAR-
The chemical structures of the drugs are as shown in Figure
Chemical structures of the drugs.
For individual estimation of each drug, several methods are available in the literature [
For contributing such a novel cause, through this article, we have tried our best to develop a fast and user-friendly methodology for the simultaneous estimation of glimepiride, pioglitazone, and metformin, using reverse phase-HPLC method in bulk drug mix and pharmaceutical dosage forms.
In the present work, efforts have been made for the simultaneous estimation of glimepiride, pioglitazone, and metformin and its pharmaceutical dosage forms. Several trials have been made with respect to the mobile phase composition, columns, as well as UV detector’s wavelength to develop a suitable and fast method for the analysis of all the three drugs, simultaneously. The ultimate method of analysis has been provided in Section
Samples of glimepiride, pioglitazone, and metformin Hydrochloride Standards were obtained from Startech Labs. Combination drug tablets, Triblend 1, used for the experiment was manufactured by Akesis Pharma Pvt. Ltd. and Zoryl MP2 was manufactured by Intas Pharmaceuticals. HPLC-grade acetonitrile, tetrahydrofuran, dipotassium orthophosphate, trimethylamine, and orthophosphoric acid were obtained from Merck, Darmstadt, Germany.
UV-Visible spectrophotometer used was Shimadzu, Model-2450. The HPLC instrument used was Schimadzu make, model-LC-2010 CHT. Class VP Software was used for data acquisition.
The Chromatographic column, Inertsil ODS-3 V (250 mm × 4.6 mm, 5
Standard solution of 0.4 mg/mL (treat this as 100% for various experimental purpose) was prepared by taking 10 mg each of glimepiride, pioglitazone, and metformin Hydrochloride in 25 mL volumetric flask and 0.5 mL tetrahydrofuran was added and diluted upto the mark with mobile phase.
For linearity 150%, 125%, 100%, 75%, and 50% solutions were prepared. 150% solution was prepared by using 60 mg each of glimepiride, pioglitazone, and metformin hydrochloride was dissolved in 100 mL for 150% solution. 20.83 mL of 150% solution was taken in a 25 mL volumetric flask and make up with mobile phase for 125% solution. 16.67 mL of 150% solution was taken in a 25 mL volumetric flask and make up with mobile phase for 100% solution. 12.5 mL of 150% solution was taken in a 25 mL volumetric flask and make up with mobile phase for 75% solution. 8.33 mL of 150% solution is taken in a 25 mL volumetric flask and make up with mobile phase for 50% solution.
Five different solutions were prepared for performing the accuracy studies. The first solution was prepared by dissolving 10 mg each of glimepiride, pioglitazone, and metformin in 25 mL volumetric flask and make up the solution with 50% linearity solution. The second solution was prepared by dissolving 10 mg each of glimepiride, pioglitazone, and metformin in 25 mL volumetric flask and make up the solution with 75% linearity solution. The third solution was prepared by dissolving 10 mg each of glimepiride, pioglitazone, and metformin in 25 mL volumetric flask and make up the solution with 100% linearity solution. The fourth solution was prepared by dissolving 10 mg each of Glimepiride, pioglitazone, and metformin in 25 mL volumetric flask and make up the solution with 125% linearity solution. The fifth solution was prepared by dissolving 10 mg each of glimepiride, pioglitazone, and metformin in 25 mL volumetric flask and make up the solution with 150% linearity solution.
Two commercial samples were used for batch analysis. Ten tablets were weighed and their average weight was calculated. The tablet was crushed to a homogeneous mixture and 20.19 mg of Triblend 1 tablet and 23.23 mg of Zoryl MP2 tablet have been dissolved in 25 mL each of the mobile phase. To extract the drug in solution, sonicate for 5 minutes followed by cyclomixing for 5 minutes. The resulting solution was filtered using Millipore syringe filter (0.42
The terms selectivity and specificity are often used interchangeably. Specificity is the ability of the method to measure the analyte response in the presence of its potential impurities. This parameter was performed to know the retention time of each drug in a mixture and in the sample to understand if any drug-drug interaction or drug-excipient interaction is present.
System suitability test is used to verify that the resolution and reproducibility of the chromatographic systems are adequate for the analysis to be done. The tests are based on the fact that the equipment, electronics, samples to be analyzed constitute an integral system that can be evaluated as such. The limits for system suitability were set for theoretical plates, resolution, and asymmetry.
Five concentrations of the standard mixture, 50%, 75%, 100%, 125%, and 150%, were injected and chromatogram was recorded. A graph was plotted for the concentration of the corresponding drug versus area. The correlation coefficient
To determine the accuracy in sample preparation method of standard additions was made for measuring the recovery of the drugs. To the standard solution known concentrations of the drug (50%, 75%, 100%, 125%, and 150%) was added. Five different solutions were prepared as mentioned in Section
It is very important that the method developed should be precise. Six replicates of the sample prepared from the commercial tablets were injected and Assay was calculated to measure the repeatability of retention times and peak area of standard and sample.
To verify the robustness of the method, the analysis was done under variable flow rates. The flow rate as per the developed method is 1.7 mL/min. This has been purposely changed to 1.5 mL/min and 1.9 mL/min and the chromatogram was obtained.
To test the ruggedness of the method, the analysis was done on different days and different chemists to check for any changes in the chromatograph. The percentage RSD for the retention time and area was calculated.
The method is said to be reliable if it can be applied for the analysis of glimepiride, pioglitazone, and metformin simultaneously to the pharmaceutical dosage forms or commercial tablets. For this purpose, performance test of the method has been conducted on two market samples manufactured by Akesis Pharma Pvt. Ltd., brand name Triblend 1, and batch number TBD IP0310, and Zoryl MP2 manufactured by Intas Pharmaceuticals, Batch number TF 10D160.
After several permutation and combinations, above method has been optimized. It is evident from this method that this is a very fast method of analysis compared to the literature available [
The retention times of the standard drugs individually were measured and it was found to be 5.000 min, 3.942 min, and 1.342 min for glimepiride, pioglitazone, and metformin respectively. The drugs were taken as mixture and injected for taking the chromatogram. All the three drugs (glimepiride, pioglitazone, and metformin hydrochloride) were resolved very nicely in a mixture. Retention time of all the three drugs in standard mix was found to be 5.017 min, 3.958 min, and 1.342 min for glimepiride, pioglitazone, and metformin hydrochloride, respectively. This indicates there is no chromatographic interference between the analytes. The sample solution (pharmaceutical dosage form) was then injected and the chromatogram was obtained. The retention time of the drugs in the dosage form (tablet) was found to be 5.358 min, 4.083 min, and 1.308 min for glimepiride, pioglitazone, and metformin hydrochloride, respectively. Respective HPLC chromatograms are represented in Figures
Individual drugs.
Drug mixture.
Dosage forms.
The suitability of the system was studied by performing the experiment and looking for changes in separation, retention times, and asymmetry of the peaks. Five injections of the standard and two injections of the sample were injected for this purpose. The resolution, areas, retention time, theoretical plates values and peak asymmetry were calculated for standard and sample solutions. Results obtained are given in Table
System suitability results.
Standard | Average | % RSD |
---|---|---|
Glimepiride | ||
Retention time | 5.043 | 0.26 |
Area | 14711933 | 0.13 |
Resolution | 4.924 | |
Theoretical plates | 7063 | |
Asymmetry | 0.898 | |
Pioglitazone | ||
Retention time | 3.977 | 0.27 |
Area | 11302936 | 0.1 |
Resolution | 15.52 | |
Theoretical plates | 6782 | |
Asymmetry | 0.94 | |
Metformin | ||
Retention time | 1.342 | 0 |
Area | 11142195 | 0.31 |
Resolution | 0 | |
Theoretical plates | 1347 | |
Asymmetry | 1.17 | |
Dosage form | ||
Glimepiride | ||
Retention time | 5.071 | 0.11 |
Area | 14719434 | 0.1 |
Resolution | 4.99 | |
Theoretical plates | 7142.925 | |
Asymmetry | 0.88 | |
Pioglitazone | ||
Retention time | 3.992 | 0 |
Area | 11285309.5 | 0.11 |
Resolution | 15.775 | |
Theoretical plates | 6893.845 | |
Asymmetry | 0.93 | |
Metformin | ||
Retention time | 1.342 | 0 |
Area | 11109070.5 | 0.31 |
Resolution | 0 | |
Theoretical plates | 1396.245 | |
Asymmetry | 1.205 |
The correlation coefficient
Linearity results.
Linearity range | Correlation coefficient | |
---|---|---|
Glimepiride | 50–150% | 0.995 |
Pioglitazone | 50–150% | 0.9951 |
Metformin | 50–150% | 0.992 |
Graphs for linearity of the drugs.
The percentage recovery of the results obtained is listed in Table
Results for accuracy of the method.
Initial conc area | Sol 1 area | 50% area | Sol 1—50% area | % Recovery | ||
---|---|---|---|---|---|---|
Sol 1 | Glimepiride | 14657264 | 22563825 | 8023992 | 14539833 | 99.2 |
Pioglitazone | 11302419 | 17393625 | 6189621 | 11204004 | 99.13 | |
Metformin | 11072363 | 20056814 | 9030106 | 11026708 | 99.59 | |
Initial conc area | Sol 2 area | 75% area | Sol 2—75% area | % Recovery | ||
Sol 2 | Glimepiride | 14657264 | 26129857 | 11630341 | 14499516 | 98.92 |
Pioglitazone | 11302419 | 20320163 | 8954005 | 11366158 | 100.56 | |
Metformin | 11072363 | 21365397 | 10146889 | 11218508 | 101.32 | |
Initial conc area | Sol 3 area | 100% area | Sol 3—100% area | % Recovery | ||
Sol 3 | Glimepiride | 14657264 | 29367481 | 14657264 | 14710217 | 100.36 |
Pioglitazone | 11302419 | 22539487 | 11302419 | 11237068 | 99.42 | |
Metformin | 11072363 | 22249678 | 11072363 | 11177315 | 100.95 | |
Initial conc area | Sol 4 area | 125% area | Sol 4—125% area | % Recovery | ||
Sol 4 | Glimepiride | 14657264 | 32182741 | 17567370 | 14615371 | 99.71 |
Pioglitazone | 11302419 | 28483584 | 13618562 | 11225022 | 99.32 | |
Metformin | 11072363 | 22950236 | 11878593 | 11071643 | 99.99 | |
Initial conc area | Sol 5 area | 150% area | Sol 5—150% | % Recovery | ||
Sol 5 | Glimepiride | 14657264 | 34362870 | 19549044 | 14813826 | 101.07 |
Pioglitazone | 11302419 | 26320185 | 15120715 | 11199470 | 99.09 | |
Metformin | 11072363 | 23472537 | 12416531 | 11056006 | 99.85 |
The percentage RSD values for the assays in precision study were calculated. The results as shown in Table
Method precision results.
Assay 1 | Assay 2 | Assay 3 | Assay 4 | Assay 5 | Assay 6 | Average | % RSD | |
---|---|---|---|---|---|---|---|---|
Glimepiride | 99.55% | 99.05% | 99.21% | 99.15% | 99.21% | 99.72% | 99.31% | 0.21 |
Pioglitazone | 99.37% | 99.44% | 99.31% | 99.54% | 99.41% | 99.26% | 99.38% | 0.099 |
Metformin | 99.20% | 98.95% | 98.65% | 99.63% | 98.85% | 99.05% | 99.06% | 0.34 |
Due to deliberate change in the method, no changes were found in the chromatogram, the method developed is robust. The results are shown in Table
% RSD at different flow rates.
Retention times % RSD | Areas % RSD | Sample area % RSD | |
---|---|---|---|
1.5 mL/min | |||
Glimepiride | 0.05 | 0.14 | 0.4 |
Pioglitazone | 0.04 | 0.09 | 0.22 |
Metformin | 0.06 | 0.52 | 0.22 |
1.9 mL/min | |||
Glimepiride | 0.07 | 0.69 | 0.14 |
Pioglitazone | 0.03 | 0.05 | 0.01 |
Metformin | 0 | 0.21 | 0.18 |
Data acquired and compared, % RSD of area and RT has been calculated and tabulated in Table
RSD of the drugs on different days and different analysts.
Retention time % RSD | Areas % RSD | Sample area % RSD | |
---|---|---|---|
Day 1—Analyst 1 | |||
Glimepiride | 0.03 | 0.1 | 0.04 |
Pioglitazone | 0.08 | 0.1 | 0.04 |
Metformin | 0.03 | 0.01 | 0.02 |
Day 2—Analyst 2 | |||
Glimepiride | 0.04 | 0.04 | 0.05 |
Pioglitazone | 0.06 | 0.01 | 0.03 |
Metformin | 0.08 | 0.1 | 0.06 |
Two market samples have been analysed to see the performance of the method. First tablet taken was Triblend 1 which contains 1 mg of glimepiride, 15 mg of pioglitazone, and 500 mg of metformin hydrochloride; the second tablet ZorylMP2 contains contains 2 mg of glimepiride, 15 mg of pioglitazone, and 500 mg of metformin hydrochloride. Results obtained have been summarized in the Table
Estimation of the drugs in commercial samples.
Label claim | Acquired data | Assay% | |
---|---|---|---|
Triblend 1 | |||
Glimepiride | 1 mg/tab | 1.01 mg/tab | 101% |
Pioglitazone | 15 mg/tab | 15.03 mg/tab | 100.2% |
Metformin | 500 mgltab | 497.33 mg/tab | 99.46% |
Zoryl MP2 | |||
Glimepiride | 2 mg/tab | 2.01 mg/tab | 100.5% |
Pioglitazone | 15 mg/tab | 15 mg/tab | 100% |
Metformin | 500 mgltab | 501.02 mg/tab | 100.2% |
It is concluded from the above study that the current method is fast, reproducible, and simple. By adopting this method one can elute all the three drugs in 5 minutes. Hence this method is definitely time saving to enable the simultaneous estimation of glimepiride, pioglitazone, and metformin. The proposed method is found to be accurate, precise, linear, robust, and rugged.