Development and Validation of RP-HPLC Method for the Estimation of Gemigliptin and Metformin Hydrochloride

 

Vaishali P. Shelke

Saraswati Institute of Pharmacy, Kurtadi, Hingoli.

*Corresponding Author E-mail:

 

ABSTRACT:

RP-HPLC method has been developed for the estimation of Gemigliptin and Metformin Hydrochloride. The quantification was carried out C18 bonded phase i.e. Zorbax Eclipse XDB-C18 (4.6×250mm×5μ) with particle size 5 μ/min an isocratic mode with mobile phase consisting of Methanol: Water (20:80 % v/v). The detection was carried out using UV detector at 233 nm. The solutions of Gemigliptin and Metformin Hydrochloridewere chromatographed at a constant flow rate of 1 ml/min and the retention time of the drug was found to be 2.3 min and5.8 min respectively. The linearity range of Gemigliptin and Metformin Hydrochloride were found to be from 1- 35 µg/ml of Gemigliptin and 2-18 µg/ml of Metformin Hydrochloride. Linear regression coefficient for both drugs was 0.999. As per ICH guideline the method was validated for recovery, Precision, ruggednessand linearity.

 

KEYWORDS: Gemigliptin, Metformin Hydrochloride, RP-HPLC, Validation.

 

 

INTRODUCTION:

Gemigliptin is Dipeptidyl Peptidase-4 inhibitor class of anti-diabetic drug1. Its chemical formula isC18H19 F8N5O22. Gemigliptin adjunct to diet and exercise to improve glycemic control in adults with type 2 diabetes mellitus and can be taken with or without food. It is DPP-4 inhibitors which block the cleavage of the gliptins and thus lead to an increase insulin level and a reduced glucagon level in a glucose-dependent way3. The structure of Gemigliptin is as shown in figure 1. Metformin Hydrochloride is Biguanide Hypoglycaemic agent4. Its chemical formula is C4H11N5. HCl5. It is used in treatment of type- II diabetes in adults. It is Biguanide which suppress hepatic renal output, increases insulin mediated glucose level and decreases fatty acid oxidation6. The structure of Metformin Hydrochloride is as shown in figure 2.

 

Literature survey revealed that very few analytical methods have been reported for estimation of Gemigliptin and Metformin Hydrochloride. Rapid and sensitive RP-HPLC method for analysis of Gemigliptin and Metformin Hydrochloride was available7,8.

 

 

Figure-1

 

Figure-2

 

MATERIAL AND METHOD:

Material

Gemigliptin was received as a gift Sample from Manus Akkteva Biopharma LLP, Ellisbridge, and Ahmedabad and Metformin Hydrochloride was received as gift sample from Swapnroop Pharmaceuticals, Aurangabad. HPLC grade Methanol and Water were procured from Merck India.

 

INSTRUMENT:

The instrument used wasAgilent 1220 LC series HPLC instrument. The instrument is consisting of Agilent 1220 LC pump and variable wavelength programmable UV detector and 20 µl inject port9.

 

Chromatographic conditions:

C18 Column Zorbax Eclipse XDB- C18 (4.6×250mm×5μ) was used for separation. The mobile phase containing Methanol: Water in the ratio 20:80 v/v. was delivered at flow rate 1 ml/ min and elution was monitored at 233 nm. Injection volume was20 µl and analysis was performed at ambient temperature.

 

Preparation of mobile phase:

A mobile phase consisting of Methanol (HPLC grade), water in the ratio of 20:80 v/v was prepared and then filtered through a 0.45 µ membrane filter.

 

Preparation of standard stock solution:

Accurately, about 10 mg of standard Gemigliptin and Metformin Hydrochloride was weighed and transferred to separate 10 ml volumetric flasks. The drugs were dissolved in methanol then volume made up to the mark with same solvent to obtain standard stock solution of each drug of concentration 1000 μg/ml.

 

Preparation of working solution:

Appropriate volume 0.1 ml and 2 ml of standard stock solution of Gemigliptin and Metformin Hydrochloride was transferred into 10 ml volumetric flask, diluted to mark with Distilled Water to give concentration of each drugs. The resulting solution was scanned at 233 nm.

 

Figure 3: Chromatogram of Gemigliptin and Metformin Hydrochloride Mixture

 

Construction of calibration curve:

Appropriate aliquots of the standard stock solutions of Gemigliptin and Metformin Hydrochloride were pipetted out and transferred to a series of 10 ml volumetric flasks respectively. The volume was made up to the mark with water to obtain working standard solutions of Gemigliptin and Metformin Hydrochloride. The concentrations 1-35 µg/ml of Gemigliptin and 2-18 µg/ml of Metformin Hydrochloride. From these solutions, 20 µl injections of each concentration of the drug were injected into the HPLC system three times separately. Evaluation of the drug was performed with the UV detector set at 233 nm and the peak areas were recorded. The standard calibration curve for Gemigliptin and Metformin Hydrochloride was plotted separately as peak area Vs the respective concentration of Gemigliptin and Metformin Hydrochloride as shown in figure 4 and 5. Good linearity was obtained in the concentration range of 1-35 μg/ml for Gemigliptin and 2-18μg/ml for Metformin Hydrochloride.

 

Table No. 1: Calibration Table for Gemigliptin

Concentration in µg/ml

Peak Area of GEM

00

000000

01

538640

05

2503750

10

5385400

15

8130240

20

10964000

25

13717650

30

16452400

35

19196050

-

-

 

Figure No. 4: Calibration Curve of Gemigliptin

 

Table No. 2: Calibration Table for Metformin Hydrochloride

Concentration in µg/ml

Peak Area of GEM

00

0000000

02

1170070

04

2550150

06

3740140

08

5019120

10

6399301

12

7570381

14

8860541

16

10250540

18

11520721

 

Figure No. 5: Calibration Curve of Metformin Hydrochloride

 

VALIDATION OF PROPOSED METHOD:

The proposed method was validated as per ICH guidelines10, 11. The solutions of the drugs were prepared as per the earlier adopted procedure given in the experiment.

 

a) Recovery study

Accuracy of an analytical method is the closeness of the test results obtained by that of the true value. Accuracy of proposed method has been carried out by recovery studies. It was performed by recovery study using standard addition method at 80, 100, and 120 % level; known amount of standard Gemigliptin and Metformin Hydrochloride was added to pre analyzed sample (8, 10, 12 µg/ml) and subjected them to the proposed HPLC method. Results are shown in Table No. 3.

 

b) Precision

Precision of an analytical method is the degree of agreement among individual test results. Precision of the method was verified by using stock solutions in the ratio of 1:20 containing 1 µg/ml Gemigliptin and 20µg/ml of Metformin Hydrochloride. System repeatability was done by repeating the assay three times of the same concentration after every two hours on the same day for intraday precision. Inter-day precision was carried out by performing the assay sample sets after 24 hours and 48 hours, results are reported in Table No. 4 and 5.

 

Table No. 3: Result of Recovery Study

Level of % recovery

Amount present (µg)

Amount of standard added (µg)

Total amount recovered (µg)

% Recovery

GEM

MET HCl

GEM

MET HCl

GEM

MET HCl

GEM

MET HCl

80

100

120

 

1

1

1

20

20

20

430912

538540

646368

9216576

1147570

1382486

9694160

1078432

1182045

2073314

2301941

2534051

Mean

SD

% RSD

SE

99.9850

100.130

99.7490

99.9546

0.19230

0.19239

0.11102

99.9790

100.100

99.9800

100.019

0.06957

0.06955

0.04016

(n=3)

 

Table No. 4: Result of Intraday Precision

Sr. No

Conc. In µg/ml

GEMMET HCl

Peak Area

GEMMET HCl

% Estimation

GEMMET HCl

1

1

20

538540

11475700

99.9810

99.9130

2

1

20

539254

11510700

100.110

100.030

3

1

20

538500

11524100

99.9700

99.9600

 

Mean

100.020

99.9676

SD

0.07784

0.05887

%RSD

0.07783

0.05889

SE

0.04494

0.03399

 

Table No. 5: Result of Inter day Precision

Sr. No

Conc. In µg/ml

GEMMET HCl

Peak Area

GEMMET HCl

% Estimation

GEMMET HCl

1

1

20

538747

11475700

100.010

99.9600

2

1

20

537630

11495710

99.9480

99.7820

3

1

20

538540

11530720

99.9810

100.080

 

Mean

99.9796

0.14993

SD

0.03102

0.15002

%RSD

0.03102

0.08656

SE

0.01790

0.14993

(n=3)

 

c) Sensitivityl:

Sensitivity of the proposed method was estimated in terms of Limit of Detection (LOD) and Limit of Quantitation (LOQ). LOD = 3.3 SD/S and LOQ = 10 SD/S, where SD is the residual standard deviation and S is the slope of the line. LOD was found to be 0.38 ng/mland LOQ was found to be 1 ng/ml for Gemigliptin and LOD was found to be 1.67 ng/ml and LOQ was found to be 1.05 ng/ml for Metformin Hydrochloride.

 

d) Ruggedness:

From stock solution, sample solution of Gemigliptin and Metformin Hydrochloride (10 μg/ml) was prepared and analyzed by two different analysts using similar operational and environmental conditions. Peak area was measured for same concentration solutions. The results are shown in Table No. 6.

 

Table No. 6: Result of Ruggedness

Sr. No

Conc. In µg/ml

Peak Area

% Estimation

GEM

MET HCl

GEM

MET HCl

GEM

MET HCl

Analyst 1

1

20

530720

11530720

98.5290

100.080

Analyst 2

1

20

538747

11520380

100.010

99.9970

 

 

 

 

Mean

99.2695

100.0385

 

 

 

 

SD

1.047225

0.05869

 

 

 

 

% RSD

1.054931

0.058667

 

 

 

 

SE

0.604616

0.033888

(n=3)

 

e) Linearity and range:

It was performed using different test concentrations. Response was Linear in the range of 1 to 35 μg/ml for Gemigliptin (Figure no. 3) and 2 to 18 µg/ml Metformin Hydrochloride (Figure no. 4)

 

RESULT:

Developed HPLC for simultaneous determination of Gemigliptin and Metformin Hydrochloride is sensitive, specific, precise, rugged and robust. For this method the retention time were found to be 2.397 min for Gemigliptin and 5.873 min for Metformin Hydrochloride, mobile phase was Methanol: Water 20:80 v/v and flow rate was 1.0 ml/min . % label claim for Gemigliptin and Metformin Hydrochloride was found to be 99.85 and 100.15 indicative of accuracy of the method.

 

Statistical analysis proves developed method is rapid and economicalandcan be used for routine analysis of said drugs in their respective pharmaceutical formulation.

 

ACKNOWLEDGEMENT:

Author is thankful to Manus Akkteva Biopharma LLP, Ellisbridge, Ahmedabad and Swapnroop Pharmaceuticals, Aurangabad for providing gift samples. Author is also thankful to Miss. Khan Hajerafor the project work guidance and support.

 

CONFLICT OF INTEREST:

The authors declare no conflict of interest.

 

REFERENCES:

1.        Dr. KumarGV, Dr. NareshD. Nair AR. Analytical Method Development and Validation for the Simultaneous Estimation of Metformin and Gemigliptin by RP-HPLC Method. IJMPR, 2016;4(6): 321-330

2.        Gemigliptin: Http:// Pubchem. Ncbi.Nlm.Nih.Gov/Ge migliptin

3.        Wei Zeng, Donald MG, Alison FL, Michael SS. Determination of Sitagliptin in human urine and hemodialysate using turbulent flow online extraction and tandem mass spectrometry. J. Pharm. Biomed.Anal.2008; 46(3): 534-542.

4.        Indian Pharmacopoeia, Government of India, Ghaziabad. The Indian Pharmacopoeia Commission. 2010; 6(2): 1657-1658.

5.        Indian Pharmacopoeia, Government of India, Ghaziabad. The Indian Pharmacopoeia Commission. 2014; 7(2): 2186.

6.        The United States Pharmacopoeia. US Pharmacopoeial convention. Inc. Rockville, MD. 31st Revision. (2008); 2640.

7.        Luhar SV, Patel KR, Dr. Narkhede SB. Stability Study of Gemigliptin and Simultaneous Estimation of Gemigliptin and its Degradation Product by RP-HPLC Method. J Pharm SciBioscientific Res. 2016;6(3):338-346.

8.        Dr. Kumar GV, Dr. Naresh D. Nair AR. Analytical Method Development and Validation for the Simultaneous Estimation of Metformin and Gemigliptin by RP-HPLC Method. IJMPR, 2016;4(6): 321-330.

9.        Instruction manual model HPLC-1220 Infinity LC, Agilent Technologies.

10.      ICH, Q2A, Text on validation of analytical products, International conference on Harmonization, Geneva, October 1994: 1-5.

11.      ICH, Q2B, Text on validation of analytical products, International conference on Harmonization, Geneva, November 1996: 1668.

 

 

 

Received on 11.05.2021       Modified on 19.06.2021

Accepted on 24.07.2021   ©Asian Pharma Press All Right Reserved

Asian J. Pharm. Ana. 2021; 11(4):259-262.

DOI: 10.52711/2231-5675.2021.00044