Home  |  About JAPTR |  Editorial board  |  Search |  Ahead of print  |  Current issue  |  Archives |  Submit article  |  Instructions  |  Subscribe  |  Advertise  |  Contacts  |Login 
Users Online: 436   Home Print this page Email this page Small font sizeDefault font sizeIncrease font size

 Table of Contents  
Year : 2015  |  Volume : 6  |  Issue : 1  |  Page : 25-28  

Simultaneous determination of linagliptin and metformin by reverse phase-high performance liquid chromatography method: An application in quantitative analysis of pharmaceutical dosage forms

Department of Pharmaceutical Analysis, University College of Pharmaceutical Sciences, Kakatiya University, Warangal, Telangana, India

Date of Web Publication30-Jan-2015

Correspondence Address:
Ciddi Veeresham
University College of Pharmaceutical Science, Kakatiya University, Warangal - 506 009, Telangana
Login to access the Email id

Source of Support: None, Conflict of Interest: None

DOI: 10.4103/2231-4040.150368

Rights and Permissions

To enhance patient compliance toward treatment in diseases like diabetes, usually a combination of drugs is prescribed. Therefore, an anti-diabetic fixed-dose combination of 2.5 mg of linagliptin 500 mg of metformin was taken for simultaneous estimation of both the drugs by reverse phase-high performance liquid chromatography (RP-HPLC) method. The present study aimed to develop a simple and sensitive RP-HPLC method for the simultaneous determination of linagliptin and metformin in pharmaceutical dosage forms. The chromatographic separation was designed and evaluated by using linagliptin and metformin working standard and sample solutions in the linearity range. Chromatographic separation was performed on a C 18 column using a mobile phase of 70:30 (v/v) mixture of methanol and 0.05 M potassium dihydrogen orthophosphate (pH adjusted to 4.6 with orthophosphoric acid) delivered at a flow rate of 0.6 mL/min and UV detection at 267 nm. Linagliptin and metformin shown linearity in the range of 2-12 μg/mL and 400-2400 μg/mL respectively with correlation co-efficient of 0.9996 and 0.9989. The resultant findings analyzed for standard deviation (SD) and relative standard deviation to validate the developed method. The retention time of linagliptin and metformin was found to be 6.3 and 4.6 min and separation was complete in <10 min. The method was validated for linearity, accuracy and precision were found to be acceptable over the linearity range of the linagliptin and metformin. The method was found suitable for the routine quantitative analysis of linagliptin and metformin in pharmaceutical dosage forms.

Keywords: Linagliptin, metformin, reverse phase-high performance liquid chromatography

How to cite this article:
Vemula P, Dodda D, Balekari U, panga S, Veeresham C. Simultaneous determination of linagliptin and metformin by reverse phase-high performance liquid chromatography method: An application in quantitative analysis of pharmaceutical dosage forms. J Adv Pharm Technol Res 2015;6:25-8

How to cite this URL:
Vemula P, Dodda D, Balekari U, panga S, Veeresham C. Simultaneous determination of linagliptin and metformin by reverse phase-high performance liquid chromatography method: An application in quantitative analysis of pharmaceutical dosage forms. J Adv Pharm Technol Res [serial online] 2015 [cited 2023 Mar 20];6:25-8. Available from: https://www.japtr.org/text.asp?2015/6/1/25/150368

  Introduction Top

Despite the various advances in the management of diabetes, it remains to be the major cause of disability and morbidity, including blindness, amputation, heart disease, peripheral neuropathy, and kidney disease. [1] Various studies recommended combination therapy in the treatment of diabetes mellitus to improve glycemic control among which combination of linagliptin with first line of drugs such as metformin was proved to be effective in controlling the metabolic syndrome and resulted in significant reversal of insulin resistance, islet and adipocyte hypertrophy and achieved hepatic steatosis. [2],[3],[4] Linagliptin, 8-[(3R)-3-aminopiperidin- 1-yl]-7-(but-2-yn-1-yl)-3-methyl-1-[(4-ethylquinazolin-2-yl) methyl]-3,7-dihydro-1H-purine-2,6-dione] is a novel dipeptidyl peptidase-4 inhibitor representing a new therapeutic approach by stimulating glucose-dependent insulin release and reduction of glucagon levels. It acts through inhibiting the inactivation of incretins particularly glucagon like peptide-1 and gastric inhibitory polypeptide. Metformin, N, N-dimethylimidodicarbonimidic diamide is a biguanide hypoglycemic agent which stimulates glycolysis in peripheral tissues and is considered to be a vital component in mixed therapies of oral hypoglycemics. [5],[6]

These drugs are generally prescribed in multi-component dosage forms, which are available in the market. In view of this, a simple, precise and accurate method for the simultaneous estimation of metformin and linagliptin in pharmaceutical dosage forms by reverse phase-high performance liquid chromatography (HPLC) has been developed and the developed method was validated as per International Conference on Harmonization guidelines.

  Materials and methods Top

Chemicals and reagents

Linagliptin and metformin were obtained as a gift samples from MSN Pharmachem Pvt. Ltd. and Aurbindo Pharmaceuticals Ltd. respectively. HPLC-grade methanol and acetonitrile were obtained from Sigma-Aldrich (Bangalore, India). HPLC-grade water was produced with Millipore-DI system coupled with Synergy 185 system by Millipore (Billerica, MA, USA). All the other chemicals used were of analytical grade purchased from Merck Chemicals, Mumbai, India. Fixed-dose combination tablets (Brand name: Jentadueto) containing 2.5 mg of linagliptin 500 mg of metformin manufactured by Eli-lilly., USA, were procured from USA market.

Instrumentation and chromatographic conditions

The HPLC system consisted of a Shimadzu LC-10AT pump, a Rheodyne 7725i sample injector with a 20 μL loop and a Shimadzu SPD-M10Avp diode array detector. The data acquisition was performed by processing software "LC Solution" (Shimadzu Corp, Kyoto, Japan). The method was developed on a LiChrosphere 100 RP 18e (125 mm × 4.0 mm i.d, 5 μm) column maintained at ambient temperature.

The mobile phase was 70:30 (v/v) mixture of methanol and 0.05 M potassium dihydrogen orthophosphate (pH adjusted to 4.6 with orthophosphoric acid) delivered at a flow rate of 0.6 mL/min. The column was maintained at 25°C and the absorption of the elution was measured at 267 nm. The injection volume was 20 μL. The developed method with a simple mobile phase comprising of two components found advantageous over the already reported methods by Swamy and Baba [7] and Kavitha et al., [8] where a mobile phase composed of more than two solvents.

Preparation of standard and sample solutions

Working standard solution

The stock solution of linagliptin and metformin was prepared by dissolving accurately weighed amount of drugs in 25 mL methanol, followed by sonication for 5 min to obtain a final concentration of 100 μg/mL linagliptin and 20,000 μg/mL metformin and further dilutions were prepared in methanol to obtain working standards of different concentrations.

Sample solutions

Twenty tablets, Jentadueto (Eli-lilly., USA) containing 2.5 mg of linagliptin 500 mg of metformin were weighed and finely powdered. A quantity of powder equivalent to 2.5 mg of linagliptin and 500 mg of metformin was weighed and transferred to a standard flask. The drug was diluted using methanol to get a final concentration of 10 μg/mL of linagliptin and 200 μg/mL of metformin.

  Results and discussion Top

0Validation procedures

The validation experiments were performed according "guidance to industry-bioanalytical method validation," recommended by US Food and Drug administration (US Food and Drug administration, 2001). [9]

Specificity and linearity

The chromatograms of the linagliptin and metformin in standard and sample were recorded. In the chromatograms of the formulations, some additional peaks were observed, which however did not interfere with the standard peaks demonstrating the specificity of the study method. The response for the detector was determined to be linear over the range 2-12 μg/mL for linagliptin and 400-2400 μg/mL for metformin. Each of the concentration was injected in duplicate to get reproducible response. The linearity range of the method was found more accurate and precise over the reported methods. [7],[8] The calibration curve was plotted as concentration of the respective drug versus the mean of the response at each level. The proposed method was evaluated by its correlation coefficient and intercept value calculated in the statistical study and were represented by a linear regression equation [Figure 1],[Figure 2] and [Figure 3]. The observed retention times are 4.6 and 6.3 min for metformin and linagliptin respectively, and these retention times were found shorter than the reported methods. [7],[8]
Figure 1: Calibration curve for linagliptin

Click here to view
Figure 2: Calibration curve for metformin

Click here to view
Figure 3: High performance liquid chromatography chromatograms of linagliptin and metformin at different concentrations in the linearity range. (a) 2 μg/mL linagliptin and 400 μg/mL metformin; (b) 4 μg/mL linagliptin and 800 μg/mL metformin; (c) 6 μg/mL linagliptin and 1200 μg/mL metformin; (d) 8 μg/mL linagliptin and 1600 μg/mL metformin; (e) 10 μg/mL linagliptin and 2000 μg/mL metformin; (e) 12 μg/mL linagliptin and 2400 μg/mL metformin

Click here to view

Limits of quantification and detection

Limit of detection was obtained by injecting each standard sample solution at different concentrations to get the signal to noise ratio ≥3 which was found to be 0.07591 μg/mL and 0.0414 μg/mL for linagliptin and metformin respectively. Further, limit of quantification, with a signal to noise ratio ≥10 was found to be 0.2300 μg/mL and 0.1255 μg/mL for linagliptin and metformin respectively.

Precision and accuracy

The intra-day precision expressed as relative standard deviation (RSD %) and accuracy expressed as standard deviation were calculated by analyzing six different standard samples (n = 6) at each of the low, medium and high concentrations on the same day. The inter-day precision and accuracy was evaluated by analyzing six batches of all standard samples on three different days [Table 1] and [Table 2].
Table 1: Intra-day precision data for linagliptin and metformin

Click here to view
Table 2: Inter-day precision data for linagliptin and metformin

Click here to view


The extraction recoveries of linagliptin and metformin were determined by comparing the peak areas of the samples added to standard solutions (n = 6) with those obtained from the direct injection of the standard solutions without any preparations at same concentrations. The recoveries of linagliptin and metformin were determined at low medium and high concentrations with the recoveries ranging from 98% to 102% at different analyte concentrations [Table 3].
Table 3: Recovery data for linagliptin and metformin

Click here to view


The robustness of the method was studied by deliberate changes in the method like alteration in, percentage organic content, pH of the mobile phase and changes in the wavelength. It was observed that there were no distinct changes in the chromatograms demonstrating that the robustness of the developed HPLC method.


The amount of linagliptin and metformin recovered over a period of 30 days in samples stored at −20°C did not differ from the initial concentrations. The stability of these solutions was studied by performing an experiment and looking for changes in separation, retention, and asymmetry of the peaks that were then compared with the pattern of the chromatogram of freshly prepared solutions.

  Conclusion Top

The developed HPLC method is simple, accurate, precise and reliable for the simultaneous estimation of linagliptin and metformin in combined dosage form. The RSD for all parameters was found to be within the prescribed limits. Further, the noninterference of tablet excipients makes the method suitable for routine quantitative simultaneous estimation of both the drugs in multi-component pharmaceutical preparation.

  References Top

Al-Habori M, Al-Mamari M, Al-Meeri A. Type II diabetes mellitus and impaired glucose tolerance in Yemen: Prevalence, associated metabolic changes and risk factors. Diabetes Res Clin Pract 2004;65:275-81.  Back to cited text no. 1
Ponssen HH, Elte JW, Lehert P, Schouten JP, Bets D. Combined metformin and insulin therapy for patients with type 2 diabetes mellitus. Clin Ther 2000;22:709-18.  Back to cited text no. 2
Knowler WC, Barrett-Connor E, Fowler SE, Hamman RF, Lachin JM, Walker EA, et al. Reduction in the incidence of type 2 diabetes with lifestyle intervention or metformin. N Engl J Med 2002;346:393-403.  Back to cited text no. 3
Haak T, Meinicke T, Jones R, Weber S, von Eynatten M, Woerle HJ. Initial combination of linagliptin and metformin improves glycaemic control in type 2 diabetes: A randomized, double-blind, placebo-controlled study. Diabetes Obes Metab 2012;14:565-74.  Back to cited text no. 4
Kirby M, Yu DM, O′Connor S, Gorrell MD. Inhibitor selectivity in the clinical application of dipeptidyl peptidase-4 inhibition. Clin Sci (Lond) 2009;118:31-41.  Back to cited text no. 5
Salsali A, Pratley RE. Does addition of sitagliptin to metformin monotherapy improve glycemic control in patients with type 2 diabetes mellitus? Nat Clin Pract Endocrinol Metab 2007;3:450-1.  Back to cited text no. 6
Swamy AJ, Baba KH. Analytical method development and method validation for the simultaneous estimation of metformin HCL and linagliptin in bulk and tablet dosage form by RP-HPLC method. Int J Pharm 2013;3:594-600.  Back to cited text no. 7
Kavitha KY, Geetha G, Hariprasad R, Kaviarasu M, Venkatnarayanan R. Development and validation of stability indicating RP-HPLC method for the simultaneous estimation of linagliptin and metformin in pure and pharmaceutical dosage form. J Chem Pharm Res 2013;5:230-5.  Back to cited text no. 8
US Food and Drug Administration, 2001. Guidance for industry: Bioanalytical method validation. Available from: http://www.fda.gov/cvm. [Last accessed on 2013 Apr 08].  Back to cited text no. 9


  [Figure 1], [Figure 2], [Figure 3]

  [Table 1], [Table 2], [Table 3]

This article has been cited by
1 A ratiometric fluorescent sensor for detection of metformin based on terbium–1,10-phenanthroline–nitrogen-doped-graphene quantum dots
Masoud Gazizadeh, Gholamreza Dehghan, Jafar Soleymani
RSC Advances. 2022; 12(34): 22255
[Pubmed] | [DOI]
2 Resolving Problems Encountered in Spectrophotometric Analysis of a Ternary Mixture of Linagliptin, Empagliflozin, and Metformin Hydrochloride
Samar H. Elagamy
Analytical Chemistry Letters. 2022; : 1
[Pubmed] | [DOI]
3 A New Stability Indicating Reverse Phase HPLC Method for the Determination of Related Substances of DPP4 Inhibitor Drug: Linagliptin
Appalacharyulu Salapaka, Kishore Babu Bonige, K. Chandrasekhar Reddy, Hemant Kumar Sharma, Uttam Kumar Ray
Asian Journal of Chemistry. 2022; 34(10): 2587
[Pubmed] | [DOI]
4 A Review on HPLC Method Development and Validation for Gliptin Class: New Oral Antidiabetic Agents
Archana B. Gore, Manojkumar K. Munde, Nikita B. Rukhe, Nilesh S. Kulkarni
Research Journal of Pharmaceutical Dosage Forms and Technology. 2022; : 79
[Pubmed] | [DOI]
5 Development and Validation of a Stability-Indicating HPLC Method for Empagliflozin and Linagliptin in Tablet Dosage Form
Wael Abu Dayyih,Israa Al Ani,Ramadan I. Al-Shdefat,Zainab Zakareia,Sarah Ali Hamid,Ashok K. Shakya
Asian Journal of Chemistry. 2021; 33(2): 484
[Pubmed] | [DOI]
6 Quality-by-Design Approach for Chromatographic Analysis of Metformin, Empagliflozin and Linagliptin
Sunitha Gurrala,Shiva Raj,Subrahmanyam CVS,Panikumar Durga Anumolu
Journal of Chromatographic Science. 2021;
[Pubmed] | [DOI]
7 Dual enzymes-mimic activity of nanolayered manganese-calcium oxide for fluorometric determination of metformin
Samaneh Rashtbari, Gholamreza Dehghan, Simin Khataee, Mojtaba Amini, Alireza Khataee
Chemosphere. 2021; : 133063
[Pubmed] | [DOI]
8 Determination of linagliptin and empagliflozin by UPLC and HPTLC techniques aided by lean six sigma approach
Ebrahim A. El-Desouky,Ahmed M. Abdel-Raoof,Ashraf Abdel-Fattah,Ahmed Abdel-Zaher,Ayman O. E. Osman,Ahmed H. Abdel-Monem,Samir Morshedy
Biomedical Chromatography. 2021;
[Pubmed] | [DOI]
9 Application of experimental design in HPLC method optimization and robustness for the simultaneous determination of canagliflozin, empagliflozin, linagliptin, and metformin in tablet
Bahia Abbas Moussa,Marianne Alphonse Mahrouse,Michael Gamal Fawzy
Biomedical Chromatography. 2021;
[Pubmed] | [DOI]
10 Simultaneous Determination of Alogliptin, Linagliptin, Saxagliptin, and Sitagliptin in Bulk Drug and Formulation by UPLC Q-TOF-MS
Ramji Rathod,Faraat Ali,Amrish Chandra,Robin Kumar,Meenakshi Dahiya,Gyanendra Nath Singh
Current Pharmaceutical Analysis. 2020; 17(1): 95
[Pubmed] | [DOI]
11 A rapid, simple and ultrasensitive spectrofluorimetric method for the direct detection of metformin in real samples based on a nanoquenching approach
Sina Azarian,Masoomeh Shaghaghi,Gholamreza Dehghan,Nader Sheibani
Luminescence. 2020;
[Pubmed] | [DOI]
12 Pharmacokinetic interaction between linagliptin and tadalafil in healthy Egyptian males using a novel LC–MS method
Sara S Mourad,Eman I El-Kimary,Magda A Barary,Dalia A Hamdy
Bioanalysis. 2019;
[Pubmed] | [DOI]
13 Biological Safety Studies and Simultaneous Determination of Linagliptin and Synthetic Impurities by LC-PDA
Raquel Balestri Heleno Ferreira,Jonathaline Apollo Duarte,Flávio Dias Ferreira,Luis Flávio Souza de Oliveira,Michel Mansur Machado,Marcelo Donadel Malesuik,Fávero Reisdorfer Paula,Martin Steppe,Elfrides Eva Shermann Schapoval,Clésio Soldateli Paim
Journal of Analytical Methods in Chemistry. 2019; 2019: 1
[Pubmed] | [DOI]
14 A facile nano-iron oxide sensor for the electrochemical detection of the anti-diabetic drug linagliptin in the presence of glucose and metformin
Manal A. El-Shal,Shereen M. Azab,Hassan A. M. Hendawy
Bulletin of the National Research Centre. 2019; 43(1)
[Pubmed] | [DOI]
15 A new stability indicating reverse phase high performance liquid chromatography method for the determination of enantiomeric purity of a DPP-4 inhibitor drug linagliptin
Appalacharyulu Salapaka,Kishore Babu Bonige,Raghu Babu Korupolu,Chandrasekhar Reddy T,Chandrasekhar Reddy K,Sreenivas N,Hemant Kumar Sharma,Uttam Kumar Ray
[Pubmed] | [DOI]
16 A validated LC-MS/MS method for simultaneous determination of linagliptin and metformin in spiked human plasma coupled with solid phase extraction: Application to a pharmacokinetic study in healthy volunteers
Bahia Abbas Moussa,Marianne Alphonse Mahrouse,Michael Gamal Fawzy
Journal of Pharmaceutical and Biomedical Analysis. 2019; 163: 153
[Pubmed] | [DOI]
17 Identification, isolation, characterization, and UHPLC quantification of potential genotoxic impurities in linagliptin
Yiwen Huang,Hui Lu,Fuli Zhang,Chunyan Min
Journal of Separation Science. 2018;
[Pubmed] | [DOI]
18 Determination of Dipeptidyl Peptidase-4 Inhibitors by Spectrophotometric and Chromatographic Methods
P. B. Deshpande,S. R. Butle
Journal of Analytical Chemistry. 2018; 73(4): 303
[Pubmed] | [DOI]
19 Development and Validation of LC–MS/MS Method for Simultaneous Determination of Metformin and Four Gliptins in Human Plasma
Mohammed Al Bratty,Hassan A. Alhazmi,Sadique Akhtar Javed,Keddal G. Lalitha,Mufarreh Asmari,Jessica Wölker,Sami El Deeb
Chromatographia. 2017;
[Pubmed] | [DOI]
20 Novel Contribution of Chromatography in the Development and Analyses of Metformin Hydrochloride in Biological and Environmental Samples
Iqbal Hussain,Imran Ali,Habibur Rahman,Syed Sauban Ghani
Journal of Liquid Chromatography & Related Technologies. 2017;
[Pubmed] | [DOI]
21 A New and Enantioselective Chromatographic Method for Linagliptin on Amylose Coated Chiral Stationary Phase
Cholleti Vijay Kumar,Pasula Aparna,Pavan Kumar Vasa,Y. Ravindra Kumar,Nitin Haridas Dhekale
American Journal of Analytical Chemistry. 2016; 07(07): 556
[Pubmed] | [DOI]


    Similar in PUBMED
   Search Pubmed for
   Search in Google Scholar for
 Related articles
    Access Statistics
    Email Alert *
    Add to My List *
* Registration required (free)  

  In this article
   Materials and me...
   Results and disc...
   Article Figures
   Article Tables

 Article Access Statistics
    PDF Downloaded665    
    Comments [Add]    
    Cited by others 21    

Recommend this journal