A Validated Method for The Simultaneous Estimation of Linagliptin and Metformin in Tablet Dosage Forms by RP-HPLC

 

 B. Sivagami1*, A Purushotham1, Pranabesh Sikdar2, R. Chandrasekar3, M. Niranjan Babu4

¹Associate Professor, Department of Pharmaceutical Analysis, Seven Hills College of Pharmacy,

Venkataramapuram, Tirupati, Chitoor Dist, 517561, A. P. India

²Associate Professor, Department of Pharmaceutical Chemistry, Seven Hills College of Pharmacy,

Venkataramapuram, Tirupati, Chitoor Dist, 517561, A. P. India

³Associate Professor, Department of Pharmacognosy, Seven Hills College of Pharmacy,

Venkataramapuram, Tirupati, Chitoor Dist, 517561, A. P. India

⁴Professor, Department of Pharmacognosy, Seven Hills College of Pharmacy,

Venkataramapuram, Tirupati, Chitoor Dist, 517561, A. P. India

 

ABSTRACT:

A simple, Accurate, precise and rapid method was developed for the simultaneous estimation of the Metformin and Linagliptin in Tablet dosage form. Chromatogram was run through Inertsil ODS C₁₈ (250 x 4.6 mm, 5m). Mobile phase containing Methanol, Acetonitrile and Water in the ratio of 40:40:20 was pumped through column at a flow rate of 1ml/min. Optimized wavelength for Metformin and Linagliptin was 258 nm. Retention time of Metformin and Linagliptin were found to be 2.764 min and 3.162 min %RSD of Metformin and Linagliptin were found to be 0.507 and 0.51 respectively. % Assay was obtained as 99.24% and 99.82% for Metformin and Linagliptin respectively. LOD, LOQ values of Metformin and Linagliptin were 0.08ppm, 0.25ppm and 0.03ppm, 0.08ppm respectively. Regression equation of Metformin was found to be y = 10831x -34273, and for Linagliptin value was found y = 21030x + 31232. Hence the developed method can be successfully employed for the routine analysis of Metformin and Linagliptin in bulk and pharmaceutical dosage forms.

 

 

INTRODUCTION:

Metformin is a biguanide antihyperglycemic agent used for treating non-insulin dependent diabetes mellitus (NIDDM). It improves glycemic control by decreasing hepatic glucose production, decreasing glucose absorption and increasing insulin-mediated glucose uptake. Metformin is chemically 1-carbamimidamido-N, N-dimethylmethanimidamide. Figure 1 ^{[1, 2]}

 

Figure 1 Structure of Metformin

 

Metformin's mechanisms of action differ from other classes of oral antihyperglycemic agents. Metformin decreases blood glucose levels by decreasing hepatic glucose production, decreasing intestinal absorption of glucose, and improving insulin sensitivity by increasing peripheral glucose uptake and utilization. These effects are mediated by the initial activation by metformin of AMP-activated protein kinase (AMPK), a liver enzyme that plays an important role in insulin signaling, whole body energy balance, and the metabolism of glucose and fats. Activation of AMPK is required for metformin's inhibitory effect on the production of glucose by liver cells. Increased peripheral utilization of glucose may be due to improved insulin binding to insulin receptors. Metformin administration also increases AMPK activity in skeletal muscle. AMPK is known to cause GLUT4 deployment to the plasma membrane, resulting in insulin-independent glucose uptake. The rare side effect, lactic acidosis, is thought to be caused by decreased liver uptake of serum lactate, one of the substrates of gluconeogenesis. In those with healthy renal function, the slight excess is simply cleared. However, those with severe renal impairment may accumulate clinically significant serum lactic acid levels. Other conditions that may precipitate lactic acidosis include severe hepatic disease and acute/decompensated heart failure. ^{[3, 4]}

 

Linagliptin is a DPP-4 inhibitor developed by BoehringerIngelheim for the treatment of type II diabetes. Two pharmacological characteristics that sets linagliptin apart from other DPP-4 inhibitors is that it has a non-linear pharmacokinetic profile and is not primarily eliminated by the renal system. Linagliptin is chemically known as 8-[(3R)-3-aminopiperidin-1-yl]-7-(but-2-yn-1-yl)-3-methyl-1-[methylquinazoliyl) methyl]-2, 3, 6, 7-tetrahydro-1H-purine-2, 6-dione. Fig.2 ^[5]

 

 

Figure 2 Structure of Linagliptin

 

Linagliptin is a competitive and reversible dipeptidyl peptidase (DPP)-4 enzyme inhibitor that slows the breakdown of insulin tropic hormone glucagon-like peptide (GLP)-1 for better glycemic control in diabetes patients. GLP and glucose-dependent insulin tropic polypeptide (GIP) are incretion hormones that increase the production and release of insulin from pancreatic beta cells and decrease the release of glucagon from pancreatic alpha cells. This results in a overall decrease in glucose production in the liver and increase an of insulin in a glucose-dependent manner. ^[6]

 

MATERIALS AND METHODS:

Chemicals and Reagents:

Jentadueto-D2, manufactured by BoehringerIngelheim Pharmaceuticals was purchased from the Apollo pharmacy in Hyderabad. Metformin and Linagliptin tablets were procured from Sigma Aldrich, Pvt. Ltd. Mumbai, distilled water, acetonitrile, phosphate buffer, ammonium acetate buffer, glacial acetic acid, methanol, potassium dihydrogen phosphate buffer, tetra hydrofuran, tri ethyl amine, ortho-phosphoric acid etc. were procured from S. D. Fine Chem. Pvt. Ltd Mumbai.

 

Instrument:

HPLC instrument used was of WATERS HPLC 2695 SYSTEM with Auto Injector and PDA Detector. Software used is Empower 2. UV-VIS spectrophotometer Systronics Instruments and matched quartz was used for measuring absorbance for Metformin and Linagliptin solutions.

 

METHODS:

Standard Preparation:

Accurately Weighed and transferred 250 mg of Metformin and 10 mg Linagliptin of working Standards into a 25ml clean dry volumetric flask, add 3/4^th volume of diluent, Sonicate for 5 minutes and make up to the final volume with diluents. 1ml from the above two stock solutions was taken into a 10ml volumetric flask and made up to 10ml.

 

Sample Preparation:

For analysis of commercial formulation, 20 tablets of Metformin and Linagliptin were weighed the average weight was calculated and powdered. A quantity equivalent to 500mg of Metformin and 2.5mg of Linagliptin was weighed and transferred to a 100ml volumetric flask which contain mobile phase and then shake it for 10mins and sonicate it for 20 min. The solution was allowed to stand at a room temperature for 20-30mins and filtered it through a whatmann filter paper.

 

 

Chromatographic Trials

Trials	Column	Mobile phase ratio	Detection wavelength	Flow rate	Column Temperature	Injection volume	Run time
Trial 1	Inertsil C18, BDS 250 x 4.6 mm, 5m.	Methanol: Water(90:10)	258nm	1ml/min	Ambient	20µl	10min
Trial 2	Kromasil 150 x 4.6 mm, 5m.	Buffer (Kh2po4):  Acetonitrile (30:70A)	258nm	1ml/min	Ambient	20µl	10min
Trial 3	KROMOSIL 150 x 4.6 mm, 5m.	buffer: Acetonitrile (42:58A)	258nm	1ml/min	Ambient	20µl	10min
Trial 4	BDS 150 x 4.6 mm, 5m.	buffer: Acetonitrile (45:55)	258nm	1ml/min	Ambient	20µl	10min
Trial 5	BDS 150 x 4.6 mm, 5m.	Buffer: Acetonitrile (40:60)	258nm	1ml/min	Ambient	20µl	10min
Trial 6	Inertsil -ODS C18(250 x 4.6 mm, 5 µ)	Methanol: Acetonitrile: Water (40:40:20)	258nm	1ml/min	Ambient	20µl	10min

 

 

Figure 3 Optimized chromatogram of Metformin and Linagliptin

 

Optimized Method:

Drugs were eluted with good retention time, resolution; all the system suitable parameters like Plate count and Tailing factor were within the limits.

 

RESULTS AND DISCUSSION ^[7-14]:

_{1. Linearity:}

_{Six Linear concentrations of Metformin (20-80ppm) and Linagliptin (2.5-30ppm) were prepared and injected. Regression equation of the Metformin and Linagliptin was found to be,} y = 10831x - 34273, and y = 213030x + 31232 and regression co-efficient was 0.999.

 

_{Table: 1 Calibration data of Metformin and Linagliptin}

S. No	Concentration Metformin   (µg/ml)	Response	Concentration Linagliptin  (µg/ml)	Response
1	0	0	0	0
2	20	2110652	2.5	560960
3	30	3250149	5	1102034
4	40	4307216	10	2164732
5	50	5320468	15	3171457
6	60	6427385	20	4138838
7	70	7452108	25	5276830
8	80	8769527	30	6343097

 

Figure 4: Calibration curve of Metformin and Linagliptin

 

_{2. Precision:}

_{Intraday precision (Repeatability):}

_{Intraday Precision was performed and % RSD for Metformin and Linagliptin were found to be 0.65% and 0.90% respectively.}

 

_{Inter day precision:}

_{Inter day precision was performed with 24 hrs time lag and the % RSD obtained for Metformin   and Linagliptin were 0.38% and 0.72%.}

 

_{Table: 2 Repeatability results for Metformin and Linagliptin}

Sr. No.	Metformin	Linagliptin
1	4196762	2245703
2	4237539	2291408
3	4219201	2278639
4	4278401	2239286
5	4235847	2267407
6	4219201	2278639
Mean	4231159	2266847
Std. Dev.	27435.85	20436.91
%RSD	0.648424	0.901557
Inter day precision results for Metformin and Linagliptin
Sr. No.	Metformin	Linagliptin
1	4219201	2278639
2	4237216	224732
3	4235847	2267407
4	4195611	2254490
5	4226557	2231236
6	4237216	2267407
Mean	4225275	2260652
Std. Dev.	16219.94	16338.36
%RSD	0.383879	0.722728

Average of six determinations.

 

3. Accuracy:

Three concentrations 50%, 100%, 150%, were injected in a triplicate manner and amount recovered and % recoveries are displayed in Table 3.

 

Table: 3 Table of Accuracy

Sample	Concentration (%) (µg/ml) (µg/ml)	Amount Recovered (µg/ml)	Recovery (%)	%RSD
Metformin	50	49.83	100.75	0.92
	100	100.03	99.70	0.41
	150	150.07	100.21	0.31
Linagliptin	20	20.04	100.22	0.18
	40	39.78	100.02	0.091
	60	59.88	99.98	0.09

 

4. LOD:

Limit of detection was calculated by standard deviation method for Metformin and Linagliptin and LOD for Metformin and Linagliptin were found to be 0.491and 0.522 respectively.

      

 

5. LOQ:

Limit of Quantification was calculated by standard deviation method for Metformin and Linagliptin and LOQ for Metformin and Linagliptin were found to be 1.48 and 1.58 respectively.

 

6. Robustness:

Small deliberate changes were made in the method like flow rate, mobile phase ratio, and temperature but there was no recognized change in the result and the obtained results were within the limits as per ICH Guide lines. [15]

 

Table: 4 Robustness data _{of Metformin and Linagliptin}

S.NO	Robustness condition	Metformin  %RSD	Linagliptin %RSD
1	Flow minus	0.5	0.3
2	Flow Plus	0.1	0.7
3	Mobile phase minus	0.3	0.5
4	Mobile phase Plus	0.5	0.2
5	Temperature minus	0.4	0.5
6	Temperature Plus	0.1	0.2

 

Figure 5: Robustness Chromatograms _{of Metformin and Linagliptin}

 

Assay:

Standard preparations were made from the API and sample preparations were prepared from formulation. Both sample and standards were injected six times. Drug in the formulation was estimated by taking the standard as the reference. The Average %Assay was calculated and found to be 99.24% and 99.82% for Metformin   and Linagliptin respectively.

 

Table 5 Assay of Tablet

S. No.	Metformin %Assay	Linagliptin %Assay
1	98.54	99.55
2	99.58	99.88
3	98.86	99.40
4	99.56	100.30
5	99.86	100.53
6	99.06	99.28
AVG	99.24333	99.82333
STDEV	0.503812	0.505754
%RSD	0.507653	0.506649

 

CONCLUSION:

Finally with the above experimental data and results the developed RP-HPLC method is suitable for determination of Metformin and Linagliptin. The developed method is having the following advantages. Requires less run time for recording chromatograms were less than10 min.  Percentage of recovery shows that the proposed method is free from interferences of excipients used in the formulation. Therefore the proposed method is simple, precise, accurate and rapid that can be effectively applied for routine analysis in quality control of bulk drugs and pharmaceutical dosage form.

 

LIST OF ABBREVIATIONS:

RP-HPLC: Reverse Phase High performance Liquid Chromatography; HPLC: High Performance Liquid Chromatography; GC-MS-Gas: Chromatography Mass Spectroscopy; LC-MS: Liquid Chromatography Mass Spectroscopy; LC: Liquid Chromatography; RSD: Relative Standard Deviation; SD: Standard Deviation; RT: Retention Time; UV: Ultraviolet Spectroscopy; T: Tailing factor; N: Theoretical Plates; nm: Nanometer; ppm: Parts Per Million; LOD: Limits of detection; LOQ: Limits of Quantification; R2: Correlation Coefficient

 

ACKNOWLEDGEMENTS:

The authors are thankful and grateful to the Management of Seven Hills College of Pharmacy, Tirupati for providing us with the required facilities to carry out this research work.

 

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Accepted on 05.10.2019           © RJPT All right reserved

Research J. Pharm. and Tech 2020; 13(3):1266-1270.