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Received : 13-09-2021

Accepted : 14-10-2021

Available online : 22-10-2021



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Shaikh and Dighe: Simultaneous estimation of pioglitazone, glimepiride & metformin hydrochloride in bulk & tablet dosage form by UV, RP-HPLC method


Introduction

Metformin Hydrochloride is a anti-diabetic drug which belongs to Biguanides drug class. The chemical formula for Metformin hydrochloride is C4H12ClN5. Metformin is prescribed with other medication to control high blood sugar with instruction to follow proper diet and exercise program. It is used to treat patients with type 2 diabetes. Metformin exerts its anti-diabetic action through suppression of generation of glucose in the liver.1, 2, 3 Till date, two important molecular targets of metformin have been identified, both of which are present in mitochondria.2, 3, 4 Metformin acts on mitochondrial respiratory complex I by inhibiting its function, which results in increasing in the cellular ratio of adenosine monophosphate (AMP) to adenosine triphosphate (ATP) as a result of a reduction in the efficiency of ATP production. This increase in the ratio of AMP: ATP triggers the activation of AMP‐activated protein kinase (AMPK), which has alot of effects on energy metabolism, also down regulation of the expression of gluconeogenic genes is controlled by it.2 The activity of adenylate cyclase is also thought to be inhibited by increase in AMP concentration5, it is an important mediator of glucagon action, therefore results in the inhibition of gluconeogenesis. The second target of metformin in mitochondria is mitochondrial glycerol‐3‐phosphate dehydrogenase, 4 which plays a key role in the glycerophosphate shuttle. This oxidoreduction shuttle between the cytosol and mitochondria is necessary for production of the oxidized form of coenzymes required for biochemical reactions, such as nicotinamide adenine dinucleotide in gluconeogenic reactions. The activity of mitochondrial glycerol‐3‐phosphate dehydrogenase is also inhibited by metformin, which suppresses gluconeogenic reactions including the conversion of lactate to pyruvate.4

Figure 1

Structure of metformin hydrochloride

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Figure 2

Structure of pioglitazone

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Pioglitazone hydrochloride is another anti-diabetic drug with chemical formula CHNOS. Pioglitazone hydrochloride belongs to drug class thiazolidinedione that depends on the presence of insulin for its mechanism of action. Pioglitazone hydrochloride decreases insulin resistance in the periphery and in the liver which results in increasing the insulin-dependent glucose disposal and decreasing hepatic glucose output. Pioglitazone isecretagogue. 6Pioglitazone has agonist action for peroxisome proliferator-activated receptor-gamma (PPARgamma). PPAR receptors are found in tissues such as adipose tissue, skeletal muscle, and liver which are important for insulin action.7, 8, 9, 10, 11, 12, 13, 14, 15, 16PPARgammametabolism.

Glimepiride is a potent anti-diabetic drug which belongs to Sulfonylurea drug class. Its chemical formula is C24H34N4O5S.It is used to treat patients with type 2 diabetes. It is prescribed with other medication with proper diet and regular exercise routine. Route of administration is oral it is available in tablet form and the elimination half life of glimepiride is 5-8 hours. Glimepiride exerts its action by increasing insulin release from the pancreas and by improving glucose tolerance. It acts on the "sulfonylurea receptors" on pancreatic β-cell membrane and reduces conductance of ATP sensitive K+ channels and thus causes depolarization. Due to this the Ca2+ influx and degranulation is enhance which in turn increases insulin secretion rate at any glucose concentration.17 It primarily increases 2nd phase insulin secretion and has little effect on 1st phase. The hepatic degradation of insulin and minor action of reducing glucagon release and increasing somatostatin release is also slowed down. It sensitizes the target tissues such as liver to insulin action and exerts strong extrapancreatic action. Number of insulin receptors are increased and by a post receptor action translation of receptor activation is enchanced.

Figure 3

Structure of glimepiride

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Materials and Methods

All the three drugs i.e Pioglitazone, Glimepiride & Metformin Hydrochloride were obtained from RAP Analytical research and training centre and all the chemicals used were of HPLC grade were purchased from Thermofisher pvt ltd. The balance used for weighing is a high precision balance from Wensar with model no. PGB 100. The mobile phase was degassed using Wensar Ultra sonicater and was filtered using Ultra filtration with a membrane filter size of 0.45 microns. For UV spectroscopy, double beam instrument maufactured by Analytical Technologies Ltd. with Model no- UV-2012 was used. The software used for analysis was UV-VIS Analyst.18, 19, 20, 21, 22, 23, 24, 25 The instrument used for the High Performance Liquid Chromatography consisted of binary gradient system. The software used of the HPLC instruments for the method development and the validation purpose was HPLC workstation. The maker of the HPLC system is analytical technologies Pvt. Ltd. with model number HPLC 3000 series. Reciprocating type of pump was used for the analysis of model no.P-3000M. The detector used was UV-VIS detector of single wavelength with the model no. of UV3000M. The C18 type of column was used for the separation which was form cosmosil make having the dimension of 250mm X 4.6mm (ID), particle size of 5micron.

Sample preparation

Accurately 10mg of Pioglitazone, glimepride, metformin hydrochloride drug sample was weighed and transferred to 10ml volumetric flask individually, then it was dissolved using 5ml solvent(mobile phase) and then it was diluted up to the mark which makes stock solution of 1000 μg/ml. Working solution was made as per the requirement.

Method development

In order to obtain resolution of the three drugs that is to develop sharp and well resolved peaks various trials were carried out. By changing the ratio of mobile phase methanol and water various trial were taken and it was observed that all the three drugs were well resolved at the 70% methanol and 30% water at 229 nm. The flow rate of the mobile phase was taken at 0.8 ml/min. Optimised results was shown below of all three drugs

Figure 4

Chromatogram of optimised trail

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Table 0

Rank

Time

Area

Resolut.

T.Plate

Num asymmetry

1

4.033

1618046

6.25

6955

1.03

2

5.523

327268

4.64

7446

1.00

3

6.951

148683

0.00

7768

1.16

Result

All the System Suitability parameters i.e resolution, number of theoretical plates, asymmetry of peaks were found to be within the range which is  indicating the performance of the system.

Linearity

Statistically the response of the analyte  and the concentration were calculated in order to check the linearity of all the three drugs.

Concentration range from 100-500 ug/ml were taken for metformin, 3-15 ug/ml for pioglitazone, 0.4-1.2 ug/ml for glimepiride and the calibration curve was plotted as shown below

Figure 5

Calibrationcurve for metformin

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Figure 6

Calibration curve for pioglitazone

https://s3-us-west-2.amazonaws.com/typeset-prod-media-server/0c7c6cdc-fccb-4b76-8bab-60dd3897ee52image6.png
Figure 7

Calibration curve for glimepiride Regression value for all the three drugs were found within the limit.

https://s3-us-west-2.amazonaws.com/typeset-prod-media-server/0c7c6cdc-fccb-4b76-8bab-60dd3897ee52image7.png

Accuracy

Triplicates of all the three drugs were taken for the recording the data of accuracy and the %RSD for all the drugs were calculated whose data is as follows 

Table 1

Analytical data of accuracy of metformin

Standard Deviation

Accuracy

Precision

Conc.

Conc. (µg/ml)

Area

Mean

SD

%SD

%RSD

 1

100

2046657

 2050075.667

5334.913807

 0.2602301

 0.113609507

2056223

2047347

 3

300

3890897

 3889568.333

 4343.197017

 0.1116627

3884716

3893092

 5

500

5822073

 5819824

 2156.827068

 0.03706

5819626

5817773

Table 2

Analytical data of accuracy of pioglitazone

Standard Deviation

Accuracy

Precision

Comp.

Conc.( µg/ml)

Area

Mean

SD

%SD

%RSD

1

3

150344

149863

443.2527496

0.295772

0.133370602

149471

149774

3

9

295283

295235.6667

108.0755908

0.0366065

295312

295112

5

15

440904

441075.3333

491.9119162

0.1115256

440692

441630

Table 3

Analytical data of accuracy of glimepirideS

Standard Deviation

Accuracy

Precision

Comp

Conc.( µg/ml)

Area

Mean

SD

%SD

%RSD

1

0.4

16831

16780

50.02999101

0.2981525

 0.135143733

16731

16778

3

1.2

39633

 39582.66667

 46.97162264

 0.1186672

39540

39575

5

2

61560

 61538.66667

 20.55075018

 0.0333949

61519

61537

From the above results it is observed that for all the three drugs the % RSD is less than 2% which is within the acceptance limit.

% Recovery

Three concentration range of percentages were injected in order to analyse the recovery of the analyte in the formulation. Following are the results for the same.

Table 4

Statistical data of % recovery of metformin

Sr.no.

% Composition

Area of Standard

Area of Sample

% Recovery

1

50% Recovery

3890897

3881879

99.76822825

2

100% Recovery

4914355

4917149

100.0568538

3

150% Recovery

5822073

5816256

99.90008713

Table 5

SStatistical data of % recovery of pioglitazone

Sr.no.

% Composition

Area of Standard

Area of Sample

% Recovery

1

50% Recovery

295283

295340

100.0193035

2

100% Recovery

371066

371107

100.0110492

3

150% Recovery

440904

440600

99.93105075

Table 6

SStatistical data of % recovery of glimepiride Method was found to be accurate by observing the recovery of the analyte.

Sr.no.

% Composition

Area of Standard

Area of Sample

% Recovery

1

50% Recovery

39633

39530

99.74011556

2

100% Recovery

49640

49603

99.92546334

3

150% Recovery

61560

61593

100.0536062

Assay

Assay is the analysis of the marketed formulation which is checked against the standard solution. The results obtained for three drugs are: 

Table 7

tatistical data of assay of metformin

Sr. NO.

% Composition

Area of Standard

Area of Sample

% Assay

1

% Assay

3890897

3896759

100.151

Table 8

Statistical data of assay of pioglitazone

Sr. NO.

% Composition

Area of Standard

Area of Sample

% Assay

1

% Assay

295283

295185

99.9668

Table 9

Statistical data of assay of glimepiride

Sr. NO.

% Composition

Area of Standard

Area of Sample

% Assay

1

% Assay

39633

39730

100.245

It is observed that the assay of the formulation were found within the criteria limit.

Precision

Variations in the results were observed by performing interday and intraday of the drugs. Following are the observed results for the same.

Table 10

Data for intraday precision of metformin

Morning

Evening

Mean

%RSD

3890897

3890160

3884716

3883158

3888863

0.10%

3893092

3891157

Table 11

Data forInterday precision of metformin

Day 1

Day 2

Mean

%RSD

3890897

3893449

3884716

3886600

3892884

0.10%

3893092

3892884

Table 12

Data for intraday precision of pioglitazone

Morning

Evening

Mean

%RSD

295283

294750

295312

295249

295087.2

0.08%

295112

294817

Table 13

Data forinterday precision of pioglitazone

Day 1

Day 2

Mean

%RSD

295283

294668

295312

294876

295017

0.08%

295112

295017

Table 14

Data for intraday precision of glimepiride

Morning

Evening

Mean

%RSD

39633

39610

39540

39576

39586.33

0.08%

39575

39584

Table 15

Data for Interday precision of Glimepiride

Day 1

Day 2

Mean

%RSD

39633

39594

39540

39560

39516

0.08%

39575

39516

As the % RSD in the above all three results were within the range ensures the repeatability of the results.

Robustness

It is performed to check the variations in the experiment i.e. in the chromatographic conditions. The results obtained were as follows.

Table 16

Data of robustness of metformin (Change in flow rate)

Conc. (μg/ml)

Parameter

Condition

Area

Mean

SD

%SD

 200

Change in flow rate

0.65

2905068

2899733

5026.37

0.17333906

0.85

2899046

1.05

2895086

Table 17

Data of robustness of metformin (Change in wavelength)

Conc. (μg/ml)

Parameter

Area

Mean

SD

%SD

 200

Change in Wavelength

2897199

2895475

4677.67

0.16155113

2899046

2890180

Table 18

Data of robustness of pioglitazone (Change in flow rate)

Conc. (μg/ml)

Parameter

Area

Mean

SD

%SD

 6

Change in flow rate

221047

220905

494.122

 0.22368101

221312

220355

Table 19

Data of robustness ofpioglitazone (Change in wavelength)

Conc. (μg/ml)

Parameter

Area

Mean

SD

%SD

 6

Change in Wavelength

221868

221490

327.237

0.14774343

221312

221291

Table 20

Data of robustness ofglimepiride (Change in flow rate)

Conc. (μg/ml)

Parameter

Area

Mean

SD

%SD

0.8

Change in flow rate

28725

28761

38.7427

0.13470582

28802

28756

Table 21

Data of Robustness ofglimepiride (Change in wavelength)

Conc. (μg/ml)

Parameter

Area

Mean

SD

%SD

0.8

Change in Wavelength

28733

28760.7

36.4737

0.12681811

28802

28747

As the RSD is less than 2% showing that the results are unaffected with the experimental changes.

Ruggedness

Wide range of concentration were injected for all the three drugs and their calibration curve was plotted as the response of analyte with the range if concentration.

Table 22

Statistical data of ruggedness of metformin

Conc.

Area

100

2043536

200

2896093

300

3889571

400

4907803

500

5822486

Figure 8

Calibration curve of ruggedness for metformin

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Table 23

Statistical data of ruggedness of pioglitazone

Conc.

Area

3

150608

6

221964

9

295029

12

370883

15

441183

Figure 9

Calibration curve of ruggedness for pioglitazone

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Table 24

Statistical data of ruggedness of glimepiride

Conc.

Area

0.4

16812

0.6

28766

0.8

39607

1.0

49589

1.2

61511

Figure 10

Calibration curve of ruggedness for glimepiride

https://s3-us-west-2.amazonaws.com/typeset-prod-media-server/0c7c6cdc-fccb-4b76-8bab-60dd3897ee52image10.png

Results are showing that the developed method is stable as regression coefficient of all are within the limit hence change doesn't affects the results.

Table 25

Statistical data of degradation studies of metformin

Sr. no.

Degradation

Area of Standard

Area of degraded Sample

Degraded up to %

Actual % degradation

1

Acid Degradation

5822073

5566418

95.60886646

4.391133536

2

Basic Degradation

5822073

5445290

93.52837039

6.471629607

3

H2O2 Degradation

5822073

5818895

99.94541463

0.054585368

4

Thermal

5822073

5819926

99.9631231

0.036876899

5

Photolytic

5822073

5821547

99.99096542

0.009034583

Table 26

Statistical data of degradation studies of pioglitazone

Sr. NO.

Degradation

Area of Standard

Area of degraded Sample

Degraded up to %

Actual % degradation

1

Acid Degradation

440904

426845

96.81132401

3.188675993

2

Basic Degradation

440904

413594

93.80590786

6.194092138

3

H2O2 Degradation

440904

440145

99.82785368

0.172146318

4

Thermal

440904

440233

99.84781268

0.152187324

5

Photolytic

440904

440374

99.8797924

0.120207574

Table 27

Statistical data of degradation studies of glimepiride

Sr. NO.

Degradation

Area of Standard

Area of degraded Sample

Degraded up to %

Actual % degradation

1

Acid Degradation

61560

59446

96.56595192

3.434048083

2

Basic Degradation

61560

58265

94.64749838

5.352501624

3

H2O2 Degradation

61560

59546

96.72839506

3.271604938

4

Thermal

61560

61541

99.9691358

0.030864198

5

Photolytic

61560A

60563

98.38044185

1.619558155

Limit of detection (LOD) and limit of quantification (LOQ)

It is calculated by slope and the standard deviation. Following are the formulas used to estimate the LOD and LOQ:

LOD= 3.3σ/S

  1. Where, σ = the standard deviation of the y-intercept

  2. S = slope of calibration curve of analyte

LOQ= 10σ/S

  1. Where, σ = the standard deviation of the y-intercept

  2. S = slope of calibration curve of analyte

  3. The result of LOD and LOQ of respective drugs are as follows:

Metformin

  1. LOD =1.3608,

  2. LOQ = 4.1239

Pioglitazone

  1. LOD = 0.04710,

  2. LOQ = 0.14274

Glimepiride

  1. LOD = 0.00468,

  2. LOQ = 0.01421

Degradation studies

Five degradation parameters (acidic, basic, thermal, photolytic and peroxide) were performed to analyse the stability of all three drugs. Drugs are forcefully degraded under specified conditions and results were calculated to ensure the % degradation.

It was observed that all the three drugs are stable under the degradation parameters also. All the three drugs were carried out under five stressed conditions and the results obtained showing that the drugs are not affected that much i.e. all the results are under the limit criteria of degradation only.

Discussion

For simultaneous estimation of metformin, pioglitazone and glimepiride, an analytical method was developed and validated by UV spectroscopy and reverse phase chromatography. The proposed method was found to be specific, accurate and precise. The method was found to be linear which was performed by linearity study for all the three drugs which was studied as per their respective concentration. Around more than 0.99 regression coefficient value was observed which was within the limit. The retention time for metformin was observed at around 4.0 min, for pioglitazone it was observed 5.5 min and for glimepiride it was observed around 6.8 min. The recovery for the method was also accurate within the limit as per the guidelines only which was observed within 98% to 102%. The percent relative standard was also observed below 2 value which falls under the guidelines. No degradation interference was observed while developing and validating the analytical method. It was also observed that the results are unaffected for the small variations in the method showing the repeatability of the developed analytical method. The method was found to be simple, rapid and robust for the analysis of metformin, pioglitazone and glimepiride.

Conclusion

For the simultaneous estimation of metformin, pioglitazone and glimepiride of stability indicating analytical method development and validation in the formulation by RP-HPLC technique, the method was found to be accurate, sensitive, reproducible, linear and precise. The results which was observed was found to be relevant that are carried out as per International Council on Harmonisation guidelines. Hence, for the quality control analysis the proposed method was carried out for the routine analysis in the tablet dosage form.

Source of Funding

None.

Conflict of Interest

None.

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