ResearchArticle RP-HPLCAnalyticalMethodDevelopmentand...

Hindawi Publishing CorporationJournal of ChemistryVolume 2013, Article ID 726235, 8 pageshttp://dx.doi.org/10.1155/2013/726235

Research ArticleRP-HPLC Analytical Method Development andValidation for Simultaneous Estimation of Three Drugs:Glimepiride, Pioglitazone, andMetformin and ItsPharmaceutical Dosage Forms

Gadapa Nirupa,1 and UpendraM. Tripathi2

1 Department of Chemistry, Jawaharlal Nehru Technological University, Kukatpally, Hyderabad 500085, AP, India2 Startech Labs Pvt. Ltd., 2nd Floor, SMR Chambers, H. Number: 1-58/7 Madinaguda, Hyderabad 500050, AP, India

Correspondence should be addressed to Upendra M. Tripathi; [email protected]

Received 11 February 2012; Accepted 2 May 2012

Academic Editor: Jun Zhang

Copyright © 2013 G. Nirupa and U. M. Tripathi.is is an open access article distributed under theCreativeCommonsAttributionLicense, which permits unrestricted use, distribution, and reproduction in anymedium, provided the originalwork is properly cited.

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 drugsglimepiride, pioglitazone andmetformin in bulk drug mix and pharmaceutical dosage forms.e estimation was carried out usingInertsil 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 of5minutes, 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 validatedmethod was successfully applied to the commercially available pharmaceutical dosage form, yielding very good and reproducibleresult.

1. Introduction

In the current Indian scenario, most commonly attackingdisease to a common man has been found to be diabetes.Recent studies indicate that prevalence of type-2 diabetesis rapidly increasing in the society. Type-2 diabetes is aprogressive disorder with a consistent and steady increase inglycosylated hemoglobin (HbA1C) overtime associated withenhanced risk of micro- and macrovascular complicationsand a substantial reduction in life expectancy.ere are threemajor pathophysiologic abnormalities associated with type-2diabetes: (i) impaired insulin secretion, (ii) excessive hepaticglucose output, and (iii) insulin resistance in skeletal muscles,liver and adipose tissue. ese defects have been treated byuse of oral insulin secretagogues (sulphonyl ureas/glinides)or insulin, biguanides, and thiazolidinediones, respectively[1].

Glimepiride is a medium-to-long acting sulphonylurea antidiabetic drug. It is chemically 1-[[p-[2-(3-Ethyl-4-methyl-2-oxo-3-pyrroline-1 carboxamido)ethyl]phenyl]sul-fonyl]-3-(trans-4-methyl cyclohexyl) urea. e primarymechanism of action of glimepiride in lowering bloodglucose appears to be dependent on stimulating therelease of insulin from functioning pancreatic beta cells.Metformin hydrochloride is also antidiabetic drug in thebiguanide class and it is chemically 1,1-dimethyl biguanidemonohydrochloride. It decreases hepatic glucose production,decreases intestinal absorption of glucose, and improvesinsulin sensitivity by increasing peripheral glucose uptakeand utilization. Pioglitazone is a prescription drug of thia-zolidinedione class with hypoglycemic (antihyperglycemic,antidiabetic) action; it is chemically (±) 5-[[4-[2-(5-Ethyl-2-pyridinyl)ethoxy]phenyl]methyl]-2,4] thiazolidinedionemonohydrochloride. It selectively stimulates the nuclear

2 Journal of Chemistry

Glimepiride

O

NH

SO O

O

O

N

NH

NH

(a)

NH

N

HCl

NH

HN NH2

Metformin hydrochloride

(b)

Pioglitazone

N O

O

O

NH

HCl

S

(c)

F 1: Chemical structures of the drugs.

2000

1000

0

2 4 6 80

MA

U

5.00

0 gl

imep

irid

e

(a)

2000

1000

0

2 4 6 80

MA

U

3.94

2 p

iogl

itaz

on

e

(b)

2000

1000

0

2 4 6 80

MA

U

1.3

42

metf

orm

in H

Cl

(c)

F 2: Individual drugs.

Journal of Chemistry 3

2000

1000

0

2 4 6 80

MA

U

1.34

2 m

etfo

rmin

HC

l

3.95

8 p

iogl

itaz

on

e

5.01

7 gl

imep

irid

e

F 3: Drug mixture.

40

0

20

1 2 3 40

MA

U

5(minutes)

1.30

8 m

etfo

rmin

4.08

3 p

iogl

itaz

on

e

5.35

8 gl

imep

irid

e

F 4: Dosage forms.

receptor peroxisome proliferator activated receptor-gamma(PPAR-𝛾𝛾) and to a lesser extent PPAR-𝛼𝛼. It modulatesthe transcription of the insulin-sensitive genes involvedin the control of glucose and lipid metabolism in themuscle, adipose tissue and the liver. As a result, pioglitazonereduces insulin resistance in the liver and peripheral tissues,increases the expense of insulin dependent glucose, decreaseswithdrawal of glucose from the liver, and reduces quantity ofglucose, insulin, and glycosylated hemoglobin in the blood-stream. e combination of glimepiride, pioglitazone, andmetformin sustained release complements each other andprovides better glycemic control in the management of Type-2 Diabetes and probably in the prevention of its associatedmacrovascular and microvascular complications [2].

e chemical structures of the drugs are as shown inFigure 1. Keeping the medical importance in mind, a groupof drugs used for treating/maintaining diabetes, namely,glimepiride, pioglitazone, and metformin has been selectedfor method development and validation. All the three drugsare antidiabetic drugs. ese drugs are very potent and arenormally prescribed either individually or in combinationsas per the demand of the situation.ese three drugs are alsoavailable in the market as a combination, dosage forms.

T 1: System suitability results.

Standard Average % RSDGlimepiride

Retention time 5.043 0.26Area 14711933 0.13Resolution 4.924eoretical plates 7063Asymmetry 0.898

PioglitazoneRetention time 3.977 0.27Area 11302936 0.1Resolution 15.52eoretical plates 6782Asymmetry 0.94

MetforminRetention time 1.342 0Area 11142195 0.31Resolution 0eoretical plates 1347Asymmetry 1.17

Dosage formGlimepiride

Retention time 5.071 0.11Area 14719434 0.1Resolution 4.99eoretical plates 7142.925Asymmetry 0.88

PioglitazoneRetention time 3.992 0Area 11285309.5 0.11Resolution 15.775eoretical plates 6893.845Asymmetry 0.93

MetforminRetention time 1.342 0Area 11109070.5 0.31Resolution 0eoretical plates 1396.245Asymmetry 1.205

For individual estimation of each drug, several methodsare available in the literature [3–7] even there are couple ofmethods available for estimation of two drugs at a time [8–14]. ere are some methods where even more than 2 drugsare estimated at a time [15–18]. Very limited work has beendone [19] for the simultaneous estimation of all the threedrugs, 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-friendlymethodology for the simultaneous estimation of glimepiride,pioglitazone, and metformin, using reverse phase-HPLCmethod in bulk drug mix and pharmaceutical dosage forms.


802399211630340.5

14657263.517567370

19549043.5

Are

a

50 100 150

Linearity for glimepiride

Conc CC-0.9950

25

20

15

10

5

0

×106

(a)

6189621

895400511302419

13618561.515120714.5

Are

a

50 100 150

Linearity for pioglitazone

Conc CC-0.9951

1816141210

86420

×106

(b)

9030106 10146889 11072363 1187859312416530.5

Are

a

Linearity for metformin HCl

50 100 150Conc CC-0.9920

1816141210

86420

×106

(c)

F 5: Graphs for linearity of the drugs.

T 2: Linearity results.

Linearity range Correlation coefficientGlimepiride 50–150% 0.995Pioglitazone 50–150% 0.9951Metformin 50–150% 0.992

2. Experimental Section

In the presentwork, efforts have beenmade for the simultane-ous estimation of glimepiride, pioglitazone, and metforminand its pharmaceutical dosage forms. Several trials havebeen made with respect to the mobile phase composition,columns, as well as UV detector’s wavelength to develop asuitable and fastmethod for the analysis of all the three drugs,simultaneously. e ultimate method of analysis has beenprovided in Section 2 2.3.

2.1.Materials, Reagents, andChemicals. Samples of glimepir-ide, pioglitazone, and metformin Hydrochloride Standardswere obtained from Startech Labs. Combination drug tablets,Triblend 1, used for the experiment was manufactured byAkesis Pharma Pvt. Ltd. and Zoryl MP2 was manufacturedby Intas Pharmaceuticals. HPLC-grade acetonitrile, tetrahy-drofuran, dipotassium orthophosphate, trimethylamine, andorthophosphoric acidwere obtained fromMerck, Darmstadt,Germany.

2.2. Equipments. UV-Visible spectrophotometer used wasShimadzu, Model-2450. e HPLC instrument used was

Schimadzu make, model-LC-2010 CHT. Class VP Sowarewas used for data acquisition.

2.3. Chromatographic Conditions. eChromatographic col-umn, Inertsil ODS-3V (250mm × 4.6mm, 5 𝜇𝜇m), columnwas used as a stationary phase. Mobile phase was preparedwith buffer, acetonitrile, tetrahydrofuran (40 : 50 : 10). Bufferwas prepared by dissolving 7.1 g of dipotassium hydrogenorthophosphate in 1000mL of water. e pH was adjusted to5.0 with orthophosphoric acid. Injection volume was 20𝜇𝜇L.e pump �ow rate was 1.7mL/min.e eluent was detectedat 228 nm at 25∘C.

2.4. Preparation of Standard Solution. Standard solution of0.4mg/mL (treat this as 100% for various experimentalpurpose) was prepared by taking 10mg each of glimepiride,pioglitazone, and metformin Hydrochloride in 25mL vol-umetric �ask and 0.5mL tetrahydrofuran was added anddiluted upto the mark with mobile phase.

2.5. Preparation of Linearity Solutions. For linearity 150%,125%, 100%, 75%, and 50% solutions were prepared. 150%solution was prepared by using 60mg each of glimepiride,pioglitazone, and metformin hydrochloride was dissolved in100mL for 150% solution. 20.83mL of 150% solution wastaken in a 25mL volumetric �ask and make up with mobilephase for 125% solution. 16.67mL of 150% solution wastaken in a 25mL volumetric �ask and make up with mobilephase for 100% solution. 12.5mL of 150% solution was takenin a 25mL volumetric �ask and make up with mobile phasefor 75% solution. 8.33mLof 150% solution is taken in a 25mL


volumetric �ask and make up with mobile phase for 50%solution.

2.6. Sample Preparation for Accuracy. Five different solutionswere prepared for performing the accuracy studies. e�rst solution was prepared by dissolving 10mg each ofglimepiride, pioglitazone, and metformin in 25mL volu-metric �ask and make up the solution with 50% linearitysolution. e second solution was prepared by dissolving10mg each of glimepiride, pioglitazone, and metforminin 25mL volumetric �ask and make up the solution with75% linearity solution. e third solution was preparedby dissolving 10mg each of glimepiride, pioglitazone, andmetformin in 25mL volumetric �ask and make up thesolution with 100% linearity solution. e fourth solutionwas prepared by dissolving 10mg each of Glimepiride,pioglitazone, and metformin in 25mL volumetric �ask andmake up the solution with 125% linearity solution. e�h solution was prepared by dissolving 10mg each ofglimepiride, pioglitazone, and metformin in 25mL volu-metric �ask and make up the solution with 150% linearitysolution.

2.7. Preparation of Sample Solution for Batch Analysis. Twocommercial samples were used for batch analysis. Ten tabletswere weighed and their average weight was calculated.e tablet was crushed to a homogeneous mixture and20.19mg of Triblend 1 tablet and 23.23mg of Zoryl MP2tablet have been dissolved in 25mL each of the mobilephase. To extract the drug in solution, sonicate for 5 minutesfollowed by cyclomixing for 5minutes.e resulting solutionwas �ltered using Millipore syringe �lter (0.42𝜇𝜇). e result-ing clear solution was injected in HPLC in duplicate as perthe developed method.

2.8. Analytical Method Validation

2.8.�. Speci�city of the Method. e terms selectivity andspeci�city are oen used interchangeably. Speci�city is theability of the method to measure the analyte response inthe presence of its potential impurities. is parameter wasperformed to know the retention time of each drug in amixture and in the sample to understand if any drug-druginteraction or drug-excipient interaction is present.

2.8.2. System Suitability. System suitability test is used toverify that the resolution and reproducibility of the chro-matographic 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 canbe evaluated as such.e limits for system suitability were setfor theoretical plates, resolution, and asymmetry.

2.8.3. Linearity. Five concentrations of the standard mix-ture, 50%, 75%, 100%, 125%, and 150%, were injected andchromatogram was recorded. A graph was plotted for theconcentration of the corresponding drug versus area. ecorrelation coefficient (𝑟𝑟𝑟 for each drug was calculated.

2.8.4. Accuracy. To determine the accuracy in sample prepa-rationmethod of standard additions wasmade formeasuringthe recovery of the drugs. To the standard solution knownconcentrations of the drug (50%, 75%, 100%, 125%, and150%) was added. Five different solutions were prepared asmentioned in Section 2 2.6. e accuracy was expressed asthe percentage of the analytes recovery.

2.8.5. Method Precision. It is very important that the methoddeveloped should be precise. Six replicates of the sampleprepared from the commercial tablets were injected andAssay was calculated tomeasure the repeatability of retentiontimes and peak area of standard and sample.

2.8.6. Robustness. To verify the robustness of themethod, theanalysis was done under variable �ow rates. e �ow rateas per the developed method is 1.7mL/min. is has beenpurposely changed to 1.5mL/min and 1.9mL/min and thechromatogram was obtained.

2.8.7. Ruggedness. To test the ruggedness of the method, theanalysis was done on different days and different chemists tocheck for any changes in the chromatograph. e percentageRSD for the retention time and area was calculated.

2.8.8. Performance Test of the Method/Batch Analysis. emethod is said to be reliable if it can be applied for the analysisof glimepiride, pioglitazone, and metformin simultaneouslyto the pharmaceutical dosage forms or commercial tablets.For this purpose, performance test of the method has beenconducted on two market samples manufactured by AkesisPharma Pvt. Ltd., brand name Triblend 1, and batch numberTBD IP0310, and ZorylMP2manufactured by Intas Pharma-ceuticals, Batch number TF 10D160.

3. Results and Discussion

Aer several permutation and combinations, above methodhas been optimized. It is evident from this method that thisis a very fast method of analysis compared to the literatureavailable [19]. We have been able to elute all the three drugswithin 5min. In the current days, industries are looking forthe methodology which can save sophisticated instrumentsand chemist’s valuable time, and as a result they can releasetheir product analysis report within lesser time. is is thereason why people are more attracted toward ultra-fast liquidchromatography (UFLC) [20, 21]. In this regard, the currentmethod developed by us is very fast and encouraging. edeveloped method was validated with a holistic approachaccording to ICH guidelines and details of �ndings are asbelow.

3.�. Speci�city of the Method. e retention times of thestandard drugs individually were measured and it was foundto be 5.000min, 3.942min, and 1.342min for glimepiride,pioglitazone, and metformin respectively. e drugs weretaken as mixture and injected for taking the chromatogram.All the three drugs (glimepiride, pioglitazone, andmetformin


T 3: Results for accuracy of the method.

Initial conc area Sol 1 area 50% area Sol 1—50% area % Recovery

Sol 1Glimepiride 14657264 22563825 8023992 14539833 99.2Pioglitazone 11302419 17393625 6189621 11204004 99.13Metformin 11072363 20056814 9030106 11026708 99.59







Initial conc area Sol 5 area 150% area Sol 5—150% % Recovery


T 4: Method precision results.

Assay 1 Assay 2 Assay 3 Assay 4 Assay 5 Assay 6 Average % RSDGlimepiride 99.55% 99.05% 99.21% 99.15% 99.21% 99.72% 99.31% 0.21Pioglitazone 99.37% 99.44% 99.31% 99.54% 99.41% 99.26% 99.38% 0.099Metformin 99.20% 98.95% 98.65% 99.63% 98.85% 99.05% 99.06% 0.34

hydrochloride) were resolved very nicely in amixture. Reten-tion time of all the three drugs in standard mix was foundto be 5.017min, 3.958min, and 1.342min for glimepiride,pioglitazone, and metformin hydrochloride, respectively.is indicates there is no chromatographic interferencebetween the analytes. e sample solution (pharmaceuticaldosage form) was then injected and the chromatogram wasobtained. e retention time of the drugs in the dosage form(tablet) was found to be 5.358min, 4.083min, and 1.308minfor glimepiride, pioglitazone, and metformin hydrochloride,respectively. Respective HPLC chromatograms are repre-sented in Figures 2, 3, and 4. ere is no speci�c changein the chromatogram. is indicates that there is no drug-excipient interference and the drugs are properly resolvedby this method. erefore, this is a suitable method for thesimultaneous estimation of glimepiride, pioglitazone, andmetformin in drug mixture and dosage forms.

3.2. System Suitability. e suitability of the systemwas stud-ied by performing the experiment and looking for changesin separation, retention times, and asymmetry of the peaks.Five injections of the standard and two injections of thesample were injected for this purpose. e resolution, areas,retention time, theoretical plates values and peak asymmetry

were calculated for standard and sample solutions. Resultsobtained are given in Table 1.

3.3. Linearity. e correlation coefficient (𝑟𝑟𝑟 was calculated,and it was between 0.98 to 1.00 which is well within theacceptance criteria. e results are shown in Table 2. econcentration was found to be proportional to the area, andthe response of the detector was determined to be linear overthe range of 0.2 to 0.6mg/mL as shown in the Figure 5.

3.4. Accuracy. e percentage recovery of the results ob-tained is listed in Table 3. e results indicate that therecoveries are well within the acceptance range, therefore,method is accurate and it can be used for the simultaneousestimation of glimepiride, pioglitazone, and metformin.

3.5. Method Precision. e percentage RSD values for theassays in precision studywere calculated.e results as shownin Table 4 indicate that the method developed is precise.

3.6. Robustness. Due to deliberate change in the method,no changes were found in the chromatogram, the methoddeveloped is robust. e results are shown in Table 5.


T 5: % RSD at different �ow rates.

Retention times % RSD Areas% RSD Sample area % RSD1.5mL/min

Glimepiride 0.05 0.14 0.4Pioglitazone 0.04 0.09 0.22Metformin 0.06 0.52 0.22

1.9mL/minGlimepiride 0.07 0.69 0.14Pioglitazone 0.03 0.05 0.01Metformin 0 0.21 0.18

T 6: RSD of the drugs on different days and different analysts.

Retention time % RSD Areas % RSD Sample area % RSDDay 1—Analyst 1

Glimepiride 0.03 0.1 0.04Pioglitazone 0.08 0.1 0.04Metformin 0.03 0.01 0.02

Day 2—Analyst 2Glimepiride 0.04 0.04 0.05Pioglitazone 0.06 0.01 0.03Metformin 0.08 0.1 0.06

T 7: Estimation of the drugs in commercial samples.

Label claim Acquired data Assay%Triblend 1

Glimepiride 1mg/tab 1.01mg/tab 101%Pioglitazone 15mg/tab 15.03mg/tab 100.2%Metformin 500 mgltab 497.33mg/tab 99.46%

Zoryl MP2Glimepiride 2mg/tab 2.01mg/tab 100.5%Pioglitazone 15mg/tab 15mg/tab 100%Metformin 500 mgltab 501.02mg/tab 100.2%

3.7. Ruggedness. Data acquired and compared,%RSDof areaandRThas been calculated and tabulated in Table 6. Based onthe data, it is evident that the method is Rugged.

3.8. Performance of the Drug/Batch Analysis. Two marketsamples have been analysed to see the performance of themethod. First tablet taken was Triblend 1 which contains1mg of glimepiride, 15mg of pioglitazone, and 500mg ofmetformin hydrochloride; the second tablet ZorylMP2 con-tains contains 2mg of glimepiride, 15mg of pioglitazone, and500mg of metformin hydrochloride. Results obtained havebeen summarized in the Table 7.

4. Conclusion

It is concluded from the above study that the current methodis fast, reproducible, and simple. By adopting this methodone can elute all the three drugs in 5 minutes. Hence this

method is de�nitely time saving to enable the simultaneousestimation of glimepiride, pioglitazone, and metformin. eproposed method is found to be accurate, precise, linear,robust, and rugged.

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