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A new gas chromatographic method for quantification of Metformin hydrochloride and Vildagliptin in bulk and pharmaceutical dosage form: development and validation



Metformin, an antidiabetic drug, assists in reducing the creation of glucose inside the liver. Vildagliptin, a DPP-4 inhibitor, enhances insulin release from the pancreas and reduces the hormones that elevate blood sugar levels. The combined medications work synergistically to lower blood sugar levels. This study was designed to develop and validate a reliable method of simultaneous assessment of Metformin and Vildagliptin in bulk and pharmaceutical dosage forms. For the chromatographic separation, a Gs-Tek INNOWAX column was utilized. This column has a length of 30 m, an internal diameter of 0.25 mm, and a 1.8 µm film thickness. For the detection, a Flame Ionization Detector was utilized. The ideal conditions included an injection volume of 1 µL with a split mode of 10 to 1 ratio, a flow rate of 1 mL/minute for the nitrogen carrier gas, an injector temperature of 300 °C, a detector temperature of 250 °C, an initial oven temperature of 100 °C that was maintained for seven minutes and then programmed to climb at a rate of 10 °C per minute up to a temperature of 300 °C.


A gas chromatographic method that is simple, precise, accurate, robust, and reliable has been developed and implemented for the simultaneous estimation of Metformin and Vildagliptin in the tablet dosage form. The retention time for Metformin and Vildagliptin was 10.203 and 22.021 min. respectively. Validation studies were performed on the method’s Linearity, detection limit (LOD), and quantitation limit (LOQ), as well as its accuracy, precision, system suitability, and robustness, using the norms established by the International Conference on Harmonization (ICH). The mean recovery value for Metformin and Vildagliptin was 100.31% (% R.S.D. = 0.6743%) and 100.33% (% R.S.D. = 0.6900%). All the results are within the acceptable range.


Validation of the developed method revealed that all the results were within an acceptable range, and techniques can be employed to analyze these two medications in combined dosage forms. It is the first method used for simultaneous estimation of these two drugs.

Graphical abstract


Today's worldwide disease, Type 2 Diabetes Mellitus (T2DM), is treated by using drugs like Metformin (MET) and Vildagliptin (VID) [1]. Metformin, an antidiabetic drug, assists in reducing the creation of glucose inside the liver. Vildagliptin, a DPP-4 inhibitor, enhances insulin release from the pancreas and reduces the hormones that elevate blood sugar levels. In Fig. 1, the chemical structures of drugs are shown.

Fig. 1
figure 1

Chemical structure of a Metformin and b Vildagliptin

The combined medications work synergistically to reduce blood sugar levels. Patients with type 2 diabetes (T2DM) who take Vildagliptin, an orally active, potent, and selective inhibitor of dipeptidyl peptidase-IV (DPP-4), see improvements in their glycemic control, primarily because of improved pancreatic islet function [2]. In summary, Vildagliptin has been reported to enhance insulin levels and reduce the elevated glucagon release in type 2 diabetes people when paired with Metformin, a thiazolidinedione, a sulfonylurea, or insulin. Metformin has been used to treat diabetes for 50 years, and although it has a distinct mode of action and does not resolve ß-cell dysfunction, it is still the primary medication advised by all guidelines [3].

The multiple pathophysiological anomalies and progressive nature of T2DM need various intensification techniques over time. As per recent guidelines, patients need therapeutic combinations much earlier to reach and maintain the ever-stricter glycemic goals. Adverse effects like hypoglycemia can be avoided with careful medicine selection. Metformin and Vildagliptin have merits over conventionally used combinations because they do not raise the probability of weight gain or induce hypoglycemia [4,5,6]. Compared to other diabetic drugs, the safety and tolerability profiles of the combination of Vildagliptin and Metformin are exceptional, and they appear to have positive effects on beta-cell activity [7, 8].

Various analytical methods were used to analyze Metformin when present alone or combined with other medications, including U.V. [9,10,11,12], HPLC [13,14,15,16], HPTLC [17] and GC techniques [18]. The analysis of the relevant literature showed that Vildagliptin, either on its own or in combination with other pharmaceuticals, has been evaluated by U.V. [18], HPLC [19], HPTLC [20, 21], and GC–MS techniques [22]. The literature study also suggests that only a few analytical methods are established for the determination of MET and VID in combined dosage forms, such as RP-HPLC [23], HPLC–MS/MS [24], and HPTLC [25]. This pharmacological combination has not yet been investigated using GC, and no attempts have been made to do so. This study proposes and validates a new gas chromatographic approach for the simultaneous detection of MET and VID in both their bulk form and their pharmaceutical dosage form.



The Star Chromatography Workstation version 6.41 software was used to record the data, and the HSGC—Shimadzu 2010 I GC system, which relates to a flame ionization detector (FID), was used for all experiments.

Gas chromatographic settings

The Gs-Tek INNOWAX column, which has a 30 m length, 0.25 mm I.D., and 1.8 μm df, was used for the separation. FID was used to carry out the detection. Injection volume 1 μL in split mode 10:1, nitrogen used as carrier gas at a constant flow of 1 mL/min, injector temperature 300 °C, detector temperature 250 °C, beginning oven temperature 100 °C kept for 7 min, then scheduled to climb at a rate of 10 °C/min up to 300 °C, were the optimal circumstances.

Preparation of standard stock solutions

The standard stock solution was prepared by dissolving accurately weighed 500 mg of MET and 50 mg of VID, in ethanol in a 100 mL volumetric flask. It is further diluted by taking 1 mL stock solution with 10 mL ethanol to get containing 500 µg/mL MET and 50 µg/mL VID respectively.

Assay procedure for tablet formulation

Twenty commercially available Galvus Met Novartis formulation tablets were ground adequately into powder using mortar and pestle. In a 100 mL volumetric flask, we weighed out powder equal to 500.0 mg of MET and 50.0 mg of VID, added roughly 50 mL ethanol, sonicated for 2 min to ensure appropriate dissolution, and then brought the volume up to the mark using the same solvent. Whatman No.41 filter paper was used to filter the sample solution and 5 mL of the filtrate was placed in a 50 mL volumetric flask, with the remainder of the volume filled with ethanol. The method is developed and validated with the help of this stock solution.

Validation of method

The results of the analysis, which included Linearity, precision, accuracy, detection limit (LOD), quantitation limit (LOQ), system suitability, and robustness, were statistically validated and performed following ICH guidelines Q2 (R1) [26].

System suitability

Three injections of a solution containing 500 µg/mL MET and 50 µg/mL VID were used to evaluate the system's suitability. The subsequent factors like Retention time values, peak areas, tailing factor, theoretical plates, and resolution are considered.

Linearity and range

The stock solution was diluted with ethanol to create standard working solutions with concentrations ranging from 125.0 to 750.0 µg/mL for MET and 12.5 to 75.0 µg/mL for VID. Five different concentrations of each chemical were selected using the previously established gas chromatographic conditions, and three injections were performed for each dilution. Regression equations and correlation coefficients were obtained using calibration curves to develop the Linearity of the suggested method.

Study of LOD and LOQ

The ICH guideline describes multiple methods for estimating detection limits and quantitation limits. The LOD and LOQ This study determined the proposed approach's values using Eqs. 1 and 2.

$${\text{LOD}}= 3.3 x \sigma / s$$
$${\text{LOQ}}=10 x \sigma /s$$

where σ the standard deviation of the Y-intercept. s the slope of the calibration curves.


Six sample solutions (n = 6) were produced and analyzed using gas chromatography, with concentrations of 500.0 µg/mL for MET and 50.0 µg/mL for VID, respectively, to ensure intraday accuracy. The same solution was analyzed over three days to evaluate the Interday precision.


Analysis of standard drug additions at three levels, i.e., multiple-level recovery experiments, were used to determine the reliability of the approach. A predetermined amount of the sample was mixed with a reference standard of varying concentrations (80%, 100%, and 120%) before being evaluated for the substance.


The method robustness was determined by measuring the percentage relative standard deviation (R.S.D.) of retention time and peak area after the oven starting temperature and flow rate were changed by 2.0 °C and 0.1 mL/sec, respectively.



The INNOWAX column effectively separated drugs. As shown in Fig. 2, the retention times for MET and VID were 10.203 and 22.021 min. respectively.

Fig. 2
figure 2

Gas Chromatogram showing the presence of MET (500 μg/mL) and VID (50 μg/mL) in a standard solution

System suitability

It was decided to inject five separate injections for the system suitability study. The acceptance limitations were satisfied for all the system suitability criteria, including the number of theoretical plates, the tailing factor, and the resolution. Also, the relative standard deviation (R.S.D.) of retention time values (Rt) and peak area (drug) for five separate injections does not exceed 2%, indicating that the GC system is suitable for the analysis of MET and VID combinations. Table 1 provides a concise summary of the findings.

Table 1 System suitability parameters

Linearity and range

The parameters of the GC were discussed before they were used, and then the calibration curves for MET and VID were developed by plotting the detector's response against the concentration of the medications. The findings show a strong connection between the detector's response and the drug concentration. The concentration ranges and the slope, y-intercept, and correlation coefficient values for both drugs for MET and VID are provided in Figs. 3 and 4.

Fig. 3
figure 3

Calibration curve for MET for 125, 250, 375, 500, 625 and 750 ppm

Fig. 4
figure 4

Calibration curve for VID for 12.5, 25.0, 37.5, 50.0, 62.5 and 75.0 ppm

LOD and LOQ value

The Detection limit was found to be 12.14 μg/mL for MET and 2.44 μg/mL for VID, while the Quantitation limit was found to be 36.80 μg/mL for MET and 7.37 μg/mL for VID.


Table 2 presents the intraday and Interday precision % R.S.D. values, which all came in at less than 2.0%. These results indicate that the approach has an appropriate level of precision.

Table 2 Study of precision

Study of accuracy

Multiple-level recovery experiments were conducted to conduct the three-level recovery test, consisting of conventional standard additions. The mean recovery value for MET and VID was 100.31% (% R.S.D. = 0.6743%) and 100.33% (% R.S.D. = 0.6900%), respectively. Table 3 shows the findings of the accuracy of the developed method.

Table 3 Accuracy for the determination of M.E.T. and VID


The procedure's reliability was analyzed by purposefully tinkering with the oven's beginning temperature (2.0 °C) and the flow rate (0.1 mL/min.). According to the data presented in Table 4, these adjustments did not substantially impact the retention times or peak areas.

Table 4 Robustness

Analysis of commercial tablet dosage form

The chromatographic approach assessed the two drugs using their combined tablet formulation (500 mg MET, 50 mg VID) as shown in Fig. 5. The analysis was carried out five times with the tablet formulation. The findings obtained using the suggested strategy are presented in Table 5. The assay results demonstrated good accuracy and precision, and the chromatograms of the tablets exhibited no interfering peaks at any point in the analysis.

Fig. 5
figure 5

Gas Chromatogram showing the presence of MET (500 μg/mL) and VID (50 μg/mL) in a sample solution

Table 5 Result of analysis of marketed formulation


In this investigation, a novel gas chromatographic method employing the INNOWAX column was developed to simultaneously detect MET and VID in bulk and pharmaceutical dosage forms. In earlier experiments, the individual GC determination of MET and VID in a variety of samples was shown to be possible. How these two analytes are evaluated simultaneously employing GC in a pharmaceutical binary combination has not yet been documented. The established chromatographic approach mentioned here has greater specificity than methods like spectrophotometry, which does not segregate the chemicals being tested for. The HPLC techniques have a disadvantage. They require a significant amount of organic solvents, making them unfeasible for routine analysis in the pharmaceutical industry because of their high cost.

The primary purpose of this research was to establish a method that could make this determination in a quick, dependable, and direct manner with a minimum amount of sample preparation. This method does not involve any complexation or pretreatment of the medications that are the focus of the research, which contributes to its ease of use and cost-effectiveness. Also, this method supports the goal of the work that was done. The developed analytical technique has successfully passed validation regarding Linearity, range, LOD, LOQ, accuracy, and precision. The robustness of the developed analytical method was also verified.


The current study developed a simple, linear, precise, accurate, and robust gas chromatographic technique to evaluate MET and VID in bulk and pharmaceutical formulations. The resolution between the drugs was improved by the developed method. The procedure's performance was validated statistically per the ICH requirements, and the findings were deemed satisfactory. Also, this is the first instance that the gas chromatography technique has been invented and validated for estimating MET and VID in combination dosage form, to the greatest of our knowledge.

Availability of data and materials

All data and materials are available upon request.







International conference of Harmonization


Limit of detection


Limit of quantification


High performance liquid chromatography


High performance thin layer chromatography


Liquid chromatography/Mass spectroscopy


Liquid chromatography–Mass spectroscopy/Mass spectroscopy


Relative standard deviation


United Sate pharmacopoeia


  1. Mohan V, Zargar A, Chawla M, Joshi A, Ayyagari U, Sethi B, Gaurav K, Patted UR, Bhagat SV, Mane AI (2021) Efficacy of a combination of metformin and vildagliptin in comparison to metformin alone in type 2 diabetes mellitus: a multicentre, retrospective, real-world evidence study. Diabetes Metab Syndr Obes 29:2925–2933

    Article  Google Scholar 

  2. Halimi S, Schweizer A, Minic B, Foley J, Dejager S (2008) Combination treatment in the management of type 2 diabetes: focus on vildagliptin and metformin as a single tablet. Vasc Health Risk Manag 4(3):481–492

    CAS  PubMed  PubMed Central  Google Scholar 

  3. Group UKPDS (1998) Effect of intensive blood-glucose control with metformin on complications in overweight patients with type 2 diabetes (UKPDS 34). Lancet. 352(9131): 854–65

  4. Ahrén B (2006) Glucagon secretion in relation to insulin sensitivity in healthy subjects. Diabetologia 49:117–122

    Article  PubMed  Google Scholar 

  5. Wu T, Zhang X, Trahair LG, Bound MJ, Little TJ, Deacon CF et al (2016) Small intestinal glucose delivery affects the lowering of blood glucose by acute vildagliptin in type 2 diabetes. J Clin Endocrinol Metab 101(12):4769–4778

    Article  CAS  PubMed  Google Scholar 

  6. Ayalasomayajula SP, Dole K, He Y-L, Ligueros-Saylan M, Wang Y, Campestrini J et al (2007) Evaluation of the potential for steady-state pharmacokinetic interaction between vildagliptin and simvastatin in healthy subjects. Curr Med Res Opin 23(12):2913–2920

    Article  CAS  PubMed  Google Scholar 

  7. Guarino E, Nigi L, Patti A, Fondelli C, Dotta F (2012) Combination therapy with metformin plus vildagliptin in type 2 diabetes mellitus. Expert Opin Pharmacother 13(9):1377–1384

    Article  CAS  PubMed  Google Scholar 

  8. Schnaars Y, Gaikwad S, Gottwald-Hostalek U, Klingberg U, Vadla HKC, Prathap VR (2022) Bioequivalence studies of new generic formulations of vildagliptin and fixed-drug combination of vildagliptin and metformin versus respective originator products in healthy volunteers. Diabetes Ther 13(6):1215–1229

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  9. Babu GR, Kumar GV, Kalyani M, Roshna M, Rani PJ, Kumar PV et al (2017) Stability-indicating simultaneous estimation of vildagliptin and mirabegron in bulk and pharmaceutical dosage form by using UV spectroscopy. World J Pharm Pharmac Sci 6(5):912–925

    CAS  Google Scholar 

  10. Kumari B, Khansili A (2020) Analytical method development and validation of UV-visible spectrophotometric method for the estimation of vildagliptin in gastric medium. Drug Res 70(09):417–423

    Article  CAS  Google Scholar 

  11. Banik S, Karmakar P, Miah MAH (2015) Development and validation of a UV-spectrophotometric method for determination of vildagliptin and linagliptin in bulk and pharmaceutical dosage forms. Bangladesh Pharmac J 18(2):163–168

    Article  Google Scholar 

  12. Sarangi RR, Panda SN, Panda SK, Sahu KC (2011) Simultaneous UV-spectrophotometric estimation of glipizide and metformin in bulk and its dosage form. Int J Pharmac Biol Arch 2(4):1137–1145

    Google Scholar 

  13. Satheeshkumar N, Pradeepkumar M, Shanthikumar S, Rao VJ (2014) Development of validated stability indicating assay method for simultaneous estimation of metformin hydrochloride and vildagliptin by RP-HPLC. Drug Res 64(03):124–129

    CAS  Google Scholar 

  14. Jayaprakash R, Natesan SK, Lalitha K (2017) Stability indicating RP-HPLC method development and validation for the simultaneous determination of vildagliptin and metformin in pharmaceutical dosage form. Int J Pharm Pharm Sci 9(3):150–157

    Article  CAS  Google Scholar 

  15. Mahrouse MA, Lamie NT (2019) Experimental design methodology for optimization and robustness determination in ion pair RP-HPLC method development: application for the simultaneous determination of metformin hydrochloride, alogliptin benzoate and repaglinide in tablets. Microchem J 147:691–706

    Article  CAS  Google Scholar 

  16. Shakoor A, Ahmed M, Ikram R, Hussain S, Tahir A, Jan BM et al (2020) Stability-indicating RP-HPLC method for simultaneous determination of metformin hydrochloride and vildagliptin in tablet and biological samples. Acta Chromatogr 32(1):39–43

    Article  CAS  Google Scholar 

  17. Havele S, Dhaneshwar S (2010) Estimation of metformin in bulk drug and in formulation by HPTLC. J Nanomedic Nanotechnolo 1(102):1–3

    Google Scholar 

  18. Tao Y, Zhang B, Chen B (2019) Central composite design for optimizing derivatization of metformin and guanylurea in water samples detected by gas chromatography-mass spectrometry. pp. 12–5.

  19. Gore AB, Munde MK, Rukhe NB, Kulkarni NS (2022) A review on HPLC method development and validation for gliptin class: new oral antidiabetic agents. Res J Pharm Dosage Forms Technol 14(1):79–6.

    Article  Google Scholar 

  20. Shirode AR, Maduskar PD, Deodhar MS, Kadam VJ (2014) RP-HPLC and HPTLC methods for simultaneous estimation of metformin hydrochloride and vildagliptin from bulk and marketed formulation: development and validation. Br J Pharmac Res 4(20):2370–2386

    Article  Google Scholar 

  21. Butle SR, Deshpande PB (2024) Validated stability-indicating HPTLC method development for determination of vildagliptin as bulk drug and in tablet dosage form. Eur J Pharm Med Res 02:234–237

    Google Scholar 

  22. Uçaktürk E (2015) Development of sensitive and specific analysis of vildagliptin in pharmaceutical formulation by gas chromatography-mass spectrometry. J Anal Methods Chem 2015:707414

    Article  PubMed  PubMed Central  Google Scholar 

  23. Varma D, Lakshmana RA, Dinda SC (2013) Simultaneous determination of metformin and vildagliptin in solid dosage form by stability indicating RP-HPLC method. Int Res J Pharm 4:122–128

    Google Scholar 

  24. Uber CP, Pontes FLD, Gasparetto JC, Francisco TMG, Piantavini MS, Cardoso MA et al (2014) HPLC-MS/MS method for simultaneous quantification of vildagliptin, metformin, and metformin-related compounds in tablets. Int J Pharm Pharm Sci 6:203–207

    CAS  Google Scholar 

  25. El-Kimary EI, Hamdy DA, Mourad SS, Barary MA (2016) HPTLC determination of three gliptins in binary mixtures with metformin. J Chromatogr Sci 54(1):79–87

    CAS  PubMed  Google Scholar 

  26. Guideline ICH (2005) Validation of analytical procedures: text and methodology. Q2 (R1), pp 1-13

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The authors thanked the Management, Mula Education Society, Sonai and AETs St. John Institute of Pharmacy and Research, Palghar for providing the necessary tools to conduct this research.


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“Conceptualization, P.M. methodology, Y.K., R.P., A.P.; software, P.M.; validation, P.M., Y.K., and R.P.; formal analysis, P.M., R.B., R.P.; investigation, Y.K.;P.M..; resources, Y.K., R.P; data curation, Y.K., R.P.; writing—original draft preparation, Y.K., R.P.,R.B.; writing—review and editing, P.M., supervision, P.M., All authors have read and agreed to the published version of the manuscript.”

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Mohite, P., Bhusal, R., Khandre, Y. et al. A new gas chromatographic method for quantification of Metformin hydrochloride and Vildagliptin in bulk and pharmaceutical dosage form: development and validation. Futur J Pharm Sci 10, 62 (2024).

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