RP-HPLC Analytical Method Development and Validation for Simultaneous Estimation of Three Drugs : Glimepiride , Pioglitazone , andMetformin and Its Pharmaceutical Dosage Forms

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 andmetformin in bulk drug mix and pharmaceutical dosage forms.e estimation was carried out using Inertsil ODS-3V (250mm × 4.6mm, 5 μμm) column; mobile phase consisting of acetonitrile, tetrahydrofuran, and buffer at pH 5; the �ow rate of 1.7mL/min and ultraviolet detection at 228 nm. All the three drugs were properly resolved having run time of 5minutes, 3.9minutes and 1.3minutes for glimepiride, pioglitazone, and metformin, respectively. e method was validated as a �nal veri�cation of method development with respect to precision, linearity, accuracy, ruggedness, and robustness. e validated method was successfully applied to the commercially available pharmaceutical dosage form, yielding very good and reproducible result.


Introduction
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 (HbA 1 C) overtime associated with enhanced risk of micro-and macrovascular complications and a substantial reduction in life expectancy.ere 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.ese defects have been treated by use of oral insulin secretagogues (sulphonyl ureas/glinides) or insulin, biguanides, and thiazolidinediones, respectively [1].
Glimepiride is a medium-to-long acting sulphonyl urea antidiabetic drug.It is chemically 1-[[p-[2-(3-Ethyl-4methyl-2-oxo-3-pyrroline-1 carboxamido)ethyl]phenyl]sulfonyl]-3-(trans-4-methyl cyclohexyl) urea.e 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  receptor peroxisome proliferator activated receptor-gamma (PPAR-) and to a lesser extent PPAR-.It modulates the transcription of the insulin-sensitive genes involved in the control of glucose and lipid metabolism in the muscle, adipose tissue and the liver.As a result, pioglitazone reduces insulin resistance in the liver and peripheral tissues, increases the expense of insulin dependent glucose, decreases withdrawal of glucose from the liver, and reduces quantity of glucose, insulin, and glycosylated hemoglobin in the bloodstream.e combination of glimepiride, pioglitazone, and metformin sustained release complements each other and provides better glycemic control in the management of Type-2 Diabetes and probably in the prevention of its associated macrovascular and microvascular complications [2].e chemical structures of the drugs are as shown in Figure 1.Keeping the medical importance in mind, a group of drugs used for treating/maintaining diabetes, namely, glimepiride, pioglitazone, and metformin has been selected for method development and validation.All the three drugs are antidiabetic drugs.ese drugs are very potent and are normally prescribed either individually or in combinations as per the demand of the situation.ese three drugs are also available in the market as a combination, dosage forms.For individual estimation of each drug, several methods are available in the literature [3][4][5][6][7] even there are couple of methods available for estimation of two drugs at a time [8][9][10][11][12][13][14].ere are some methods where even more than 2 drugs are estimated at a time [15][16][17][18].Very limited work has been done [19] for the simultaneous estimation of all the three drugs, namely, glimepiride, pioglitazone, and metformin.
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.

Experimental Section
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.e ultimate method of analysis has been provided in Section 2 2.3.

Preparation of Standard Solution.
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 �ask and 0.5 mL tetrahydrofuran was added and diluted upto the mark with mobile phase.

Preparation of Linearity Solutions.
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 �ask and make up with mobile phase for 125% solution.16.67 mL of 150% solution was taken in a 25 mL volumetric �ask and make up with mobile phase for 100% solution.12.5 mL of 150% solution was taken in a 25 mL volumetric �ask and make up with mobile phase for 75% solution.8.33 mL of 150% solution is taken in a 25 mL volumetric �ask and make up with mobile phase for 50% solution.
2.6.Sample Preparation for Accuracy.Five different solutions were prepared for performing the accuracy studies.e �rst solution was prepared by dissolving 10 mg each of glimepiride, pioglitazone, and metformin in 25 mL volumetric �ask and make up the solution with 50% linearity solution.e second solution was prepared by dissolving 10 mg each of glimepiride, pioglitazone, and metformin in 25 mL volumetric �ask and make up the solution with 75% linearity solution.e third solution was prepared by dissolving 10 mg each of glimepiride, pioglitazone, and metformin in 25 mL volumetric �ask and make up the solution with 100% linearity solution.e fourth solution was prepared by dissolving 10 mg each of Glimepiride, pioglitazone, and metformin in 25 mL volumetric �ask and make up the solution with 125% linearity solution.e �h solution was prepared by dissolving 10 mg each of glimepiride, pioglitazone, and metformin in 25 mL volumetric �ask and make up the solution with 150% linearity solution.

Preparation of Sample Solution for Batch Analysis.
Two commercial samples were used for batch analysis.Ten tablets were weighed and their average weight was calculated.e 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.e resulting solution was �ltered using Millipore syringe �lter (0.42 ).e resulting clear solution was injected in HPLC in duplicate as per the developed method.

Analytical Method Validation
2.8.�.Speci�city of the Method.e terms selectivity and speci�city are oen used interchangeably.Speci�city is the ability of the method to measure the analyte response in the presence of its potential impurities.is 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.
System suitability test is used to verify that the resolution and reproducibility of the chromatographic systems are adequate for the analysis to be done.e tests are based on the fact that the equipment, electronics, samples to be analyzed constitute an integral system that can be evaluated as such.e limits for system suitability were set for theoretical plates, resolution, and asymmetry.

Linearity.
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.e correlation coefficient ( for each drug was calculated.

Accuracy.
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 2 2.6.e accuracy was expressed as the percentage of the analytes recovery.

Method Precision.
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.
2.8.6.Robustness.To verify the robustness of the method, the analysis was done under variable �ow rates.e �ow rate as per the developed method is 1.7 mL/min.is has been purposely changed to 1.5 mL/min and 1.9 mL/min and the chromatogram was obtained.

Ruggedness.
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.e percentage RSD for the retention time and area was calculated.

Performance Test of the Method/Batch
Analysis.e 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.

Results and Discussion
Aer 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 [19].We have been able to elute all the three drugs within 5 min.In the current days, industries are looking for the methodology which can save sophisticated instruments and chemist's valuable time, and as a result they can release their product analysis report within lesser time.is is the reason why people are more attracted toward ultra-fast liquid chromatography (UFLC) [20,21].In this regard, the current method developed by us is very fast and encouraging.e developed method was validated with a holistic approach according to ICH guidelines and details of �ndings are as below.
3.�.Speci�city of the Method.e 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.e drugs were taken as mixture and injected for taking the chromatogram.All the three drugs (glimepiride, pioglitazone, and metformin  1.
3.3.Linearity.e correlation coefficient ( was calculated, and it was between 0.98 to 1.00 which is well within the acceptance criteria.e results are shown in Table 2. e concentration was found to be proportional to the area, and the response of the detector was determined to be linear over the range of 0.2 to 0.6 mg/mL as shown in the Figure 5. 3.7.Ruggedness.Data acquired and compared, % RSD of area and RT has been calculated and tabulated in Table 6.Based on the data, it is evident that the method is Rugged.

Accuracy. e percentage recovery of the results obtained is listed in
3.8.Performance of the Drug/Batch Analysis.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 7.

Conclusion
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 de�nitely time saving to enable the simultaneous estimation of glimepiride, pioglitazone, and metformin.e proposed method is found to be accurate, precise, linear, robust, and rugged.

T 1 :
System suitability results.
T 3: Results for accuracy of the method.
3.2.System Suitability.e 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.e resolution, areas, retention time, theoretical plates values and peak asymmetry were calculated for standard and sample solutions.Results obtained are given in Table

Table 3 .
e results indicate that the recoveries are well within the acceptance range, therefore, method is accurate and it can be used for the simultaneous estimation of glimepiride, pioglitazone, and metformin.3.5.Method Precision.epercentageRSDvalues for the assays in precision study were calculated.eresultsas shown in Table4indicate that the method developed is precise.
3.6.Robustness.Due to deliberate change in the method, no changes were found in the chromatogram, the method developed is robust.e results are shown in

Table 5 .
T 5: % RSD at different �ow rates.Estimation of the drugs in commercial samples.