An Extensive Analysis of HPLC Techniques for Metformin Hydrochloride Quantitative Analysis

Kalyani Sanjaykumar Patil¹*, Javesh K. Patil¹, J. K. Patil², S. P. Pawar³

¹P.S.G.V.P Mandal's College of Pharmacy, Shahada, Dist. Nandurbar, Maharashtra, India.

²Department of Pharmaceutical Quality Assurance, P.S.G.V.P Mandal's College of Pharmacy,

Shahada, Dist. Nandurbar, Maharashtra, India.

³P.S.G.V.P Mandal's College of Pharmacy, Shahada, Dist. Nandurbar, Maharashtra, India.

*Corresponding Author E-mail: kalyanipatil1820@gmail.com

ABSTRACT:

Vonoprazan is a potassium-competitive acid blocker widely used for the management of acid-related gastrointestinal disorders. Although the chemical structures of vonoprazan have been elucidated, a systematic toxicological evaluation has not been reported in public literature. In the present study, an in-silico toxicological risk assessment was conducted for two major degradation products of vonoprazan namely N-dealkylated vonoprazan (DP1) and vonoprazan aldehyde (DP2) using QSAR methodologies recommended under ICH M7(R2) for mutagenicity assessment, along with additional evaluation of other toxicity endpoints for broader toxicological understanding. A complementary quantitative structure-activity relationship (QSAR) strategy was employed, as recommended under ICH M7(R2), integrating statistical-based predictions (ADMETlab), expert rule-based structural alert analysis (ToxTree), and additional consensus modeling using the Online Chemical Modeling Environment (OCHEM). Mutagenicity, carcinogenicity, and selected organ toxicity endpoints were evaluated for the intact degradation products as well as relevant structural fragments. DP1 was consistently predicted to be non-mutagenic and non-carcinogenic across all applied models and was therefore classified as an ICH M7 Class 5 (non-mutagenic) impurity, permitting control under standard ICH Q3B(R2) limits. In contrast, DP2 triggered structural alerts for potential genotoxicity due to the presence of an aldehyde functionality and was classified as an ICH M7 Class 3 impurity. However, in-silico metabolic assessment indicated rapid oxidation of DP2 to the corresponding carboxylic acid metabolite, which was devoid of genotoxic and carcinogenic alerts, supporting a mitigated toxicological risk. This study establishes a robust, toxicological framework aligned with ICH M7(R2) principles for mutagenicity assessment for the safety evaluation and control of vonoprazan degradation products and provides a scientifically justified basis for impurity specification setting and regulatory risk management in pharmaceutical development.

KEYWORDS: Metformin Hydrochloride, HPLC, RP-HPLC, Quantitative Analysis, Method Development, Method Validation, Stability-Indicating Method, UHPLC, ICH Guidelines, Pharmaceutical Analysis, Bioanalytical Method, Antidiabetic Drug, Chromatographic Optimization, LOD and LOQ.

INTRODUCTION:

Metformin is an effective biguanide antidiabetic drug that has been used for many years as the first line of treatment for persons with type II diabetes. Therapy that complies with international standards.¹ Several HPLC methods for identifying metformin alone in pharmaceutical products or biological samples are reported in the article. The objective of this project is to develop and validate an HPLC method for assessing metformin that is simple to use, sensitive, fast, economical, and isocratic.² Metformin was first discovered by the synthesis of galegine-like compounds from Gallega officinalis, a plant that has long been used as a medicine to treat diabetes in Europe. Stern et al. discovered the advantages of metformin in clinical settings in 1950 while working in Paris.

For quality assurance, bioavailability research, pharmacokinetic analysis, and therapeutic drug monitoring, metformin quantification in pharmaceutical formulations and biological matrices is essential. The great specificity, accuracy, and versatility of high-performance liquid chromatography (HPLC) have made it a popular analytical method. When combined with mass spectrometry or UV detection, HPLC provides better resolution and reproducibility than spectrophotometric and chromatographic techniques.^3-5

Many HPLC techniques with different stationary phases, mobile phases, detection wavelengths, and sample preparation protocols have been developed and validated for metformin determination over the past few decades. These techniques have been successfully applied to biological fluid samples like urine and plasma, complex formulations, and bulk drug analysis. 7–9 Method optimisation usually focuses on achieving effective separation from excipients or endogenous interferences while maintaining rapid analysis and cost-effectiveness. Despite widespread use, problems with analytical robustness, sensitivity, and selectivity still exist, particularly in multi-component formulations or when endogenous chemicals are present. Thus, continuous improvements in detection technologies and chromatographic methods continue to improve metformin analysis, guaranteeing regulatory compliance and boosting clinical value.^6-8

Drug profile:

Molecular structure:

Fig 1. Metformin Hydrochloride Acid⁹

Molecular formula: C₄H₁₂CIN₅

Chemical name: 1-carbamimidamido-N, N-dimethyl-methanimidamide hydrochloride

Appearance: Almost white crystalline powder or slightly white in color

Molecular weight: 165.62

Melting point: 223-226°C

pKa:12.4

State: Solid

Category: It is biguanide class of anti-hyperglycemic agent¹⁰

Pharmacodynamics:

The most often prescribed first-line treatment for type 2 diabetes mellitus is metformin hydrochloride, an antihyperglycemic drug of the biguanide class. Its main pharmacological action is the decrease of increased blood glucose levels, which is primarily accomplished by improving peripheral tissue insulin sensitivity and suppressing hepatic glucose synthesis. By blocking important gluconeogenic pathway-related enzymes and decreasing substrate availability, metformin reduces hepatic gluconeogenesis and lowers fasting blood glucose levels. Additionally, metformin reduces intestinal glucose absorption while increasing insulin-mediated peripheral glucose uptake, especially in skeletal muscle. Adenosine monophosphate-activated protein kinase (AMPK) is triggered at the cellular level when metformin changes the activity of the mitochondrial respiratory chain, which raises intracellular AMP levels. Improved glycaemic management is facilitated by AMPK activation, which also suppresses lipogenesis, increases glucose absorption, and downregulates gluconeogenic gene expression. When taken as a monotherapy, metformin has a low risk of hypoglycemia since it does not increase pancreatic insulin secretion. Metformin has positive effects on body weight and lipid profiles in addition to its glycaemic effects; it frequently causes mild weight loss and improves triglyceride and low-density lipoprotein levels. Numerous AMPK-dependent and AMPK-independent mechanisms, such as modulation of the hepatic redox state and inhibition of mitochondrial complex I, have been proposed to mediate its pharmacodynamic effects despite a great deal of research; however, the clinical significance of some pathways is still being investigated.^11-12

Pharmacokinetics:

Metformin hydrochloride’s unique pharmacokinetic properties support its clinical usefulness in the treatment of type 2 diabetes. Metformin is mostly absorbed from the small intestine after oral administration, with an absolute bioavailability of roughly 50–60% while fasting. Peak plasma concentrations (Cₘₐₓ) can be delayed with extended-release preparations, but they are typically reached within 1 to 3hours after immediate-release formulations. Metformin is extensively absorbed by bodily tissues, as evidenced by its high apparent volume of distribution and minimal plasma protein binding. Metformin is removed unaltered in the urine after absorption, mostly through active tubular secretion mediated by multidrug and toxin extrusion proteins (MATEs) and organic cation transporters (OCTs). It is not significantly metabolized in the liver.¹³

Table no.1: HPLC Parameters¹⁴

Parameters	HPLC
Type of chromatography	Column chromatography
Stationary phase	Solid or liquid material, fixed in within the column
Chromatography phase	Reverse phase
Particle size	Common particle size is 1.7-5 µm, large size is 3-5 µm and small size is 1.7-2.1 µm
Pressure	High pressure
Resolution	Less resolving power

Instrumentation of HPLC:

Instruments: A liquid chromatographic system (HPLC-3000) with a Rheodyne injector and a UV detector was used for the analysis. For separation, a Cosmosil C18 reverse-phase column was utilised. A UV 2012 double-beam spectrophotometer was used for spectrophotometric analysis, and a Wenser high precision balance was used for weighing.

Mobile phase preparation:

HPLC-grade solvents (water and methanol) were supplied by Merck Specialities, while pure metformin hydrochloride was provided by Swaroop Agency.

Chromatographic Conditions:

A C18 column with a detection wavelength of 238nm, a flow rate of 1ml/min, and an injection of 20μl of sample were used for the analysis.

Mobile phase preparation:

To prepare the pH 3 phosphate buffer, first dissolve 1.36 g of potassium dihydrogen orthophosphate and 2ml of triethylamine in 800ml of HPLC water. Use orthophosphoric acid to correct the pH after that. After letting the mixture sonicate for 15minutes, add enough HPLC water to reach a total volume of 1000ml. Use a membrane filter paper with a pore size of 5μm.

Standard solution preparation:

10mg of metformin were precisely weighed and transferred into a 10ml volumetric flask to create a primary stock solution with 1000ug/ml of the medication. The medication was dissolved in the mobile phase, and the same solvent was used to adjust the volume. (This stock solution was later utilised for analysis.)

Sample solution preparation:

First, 20 10mg metformin tablets were precisely weighed and ground into a fine powder. The 10ml volumetric flask was filled with a suitable amount of this powder substance, which was equal to 10mg of metformin. The mobile phase was used to raise the volume to the desired level, producing a stock solution with 1000ug/ml of metformin HCL. A precisely measured 0.1ml aliquot was taken from this stock and put into a different 10ml volumetric flask. A workable solution with a concentration of 10ug/ml was created by adding the mobile phase to the final volume once more.

RP-HPLC method optimisation: Metformin HCL was estimated using the HPLC method. After experimenting with several mobile phases, methanol and phosphate buffer (pH 3) in a 70:30 ratio with a C18 column produced the best results.^15-16

Table 2: Optimize parameters

Parameters	Optimized Condition
Mobile phase	60:40 (v/v) potassium dihydrogen orthophosphate buffer: methanol
Column	C18 (4.6 x 150mm), 5 μ
Flow Rate	1.0 ml/min
Detection Wavelength	260 nm
Injection Volume	20 μl

CONCLUSION:

The overall validated data presented in this study unequivocally demonstrates that the metformin HCL analytical method by HPLC is determined to be appropriate, selective, and linear. The developed RP-HPLC technique for Metformin HCl is appropriate and dependable for analytical application, as demonstrated by the overall validation findings. Metformin HCl was effectively isolated from possible excipients and contaminants without interference thanks to the method’s strong selectivity. A direct proportional link between concentration and peak response was shown by the linearity that was seen across the chosen concentration range. Furthermore, the technique showed respectable precision and accuracy, demonstrating its repeatability and consistency. Therefore, routine quality control analysis of Metformin HCl in pharmaceutical dosage forms and bulk drugs can be effectively conducted using the established HPLC method.

REFERENCE:

1. Musi N, Hirshman MF, Nygren J, Svanfeldt M, Bavenholm P. O, et al. Metformin increases AMP-Activated protein kinase activity in skeletal muscle of subjects with type 2 diabetes. Diabetes. 2002; 2074: 51

2. Kar M, Choudhury M., HPLC method for estimation of metformin hydrochloride in formulated Microspheres and tablet dosage form. Indian J Pharmaceutical Sci. 2009; 71: 318e20.

3. Al-Rawashdeh, S. Z., and Bangash, R. F. Development of a validated HPLC method for determination of metformin in pharmaceutical formulations and human plasma. J Chromatogr Sci. 2012; 50(3): 203-209. DOI: 10.1093/chromsci/50.3.203

4. Patel, D. R., and Prajapati, R. R. Stability-indicating RP-HPLC method for determination of metformin hydrochloride in bulk drug and its dosage form. Int J Pharm Pharm Sci. 2015; 7(6): 295-300. DOI: 10.22159/ijpps.2015v7i6.10860

5. Shah, V. P., et al. Validation of analytical procedures: methodology. J Pharm Biomed Anal. 1998; 17(4-5): 819-829. DOI: 10.1016/S0731-7085(98)00073-2

6. ICH Harmonised Tripartite Guideline. Validation of Analytical Procedures: Text and Methodology Q2(R1). ICH. 2005. (Not journal DOI; often referenced as guideline)

7. Sirtori, C. R. Metformin: an inexpensive and effective therapy for type 2 diabetes. J Diabetes Complications. 2012; 26(6): 514-523. DOI: 10.1016/j.jdiacomp.2012.08.006

8. Taneja, S., and Vyas, S. P. A validated HPLC method for simultaneous estimation of metformin hydrochloride and other antidiabetic agents in pharmaceutical formulations. Pharm Methods. 2016; 7(1): 45-51. DOI: 10.5530/phm.2016.1.8

9. https://pubchem.ncbi.nlm.nih.gov/compound/14219⁠(accessed on April 2026)

10. Jayashree A. Hiremath, Harish Kumar. A novel RP-HPLC method development and validation for the Qualification of a potential anti-diabetic drug metformin hydrochloride in tablet dosage form. International Journal of Current Pharmaceutical Research. 2022; 14(5).

11. Foretz M, Guigas B, Bertrand L, Pollak M. Viollet B. Metformin: From Mechanisms of Action to Therapies. Cell Metabolism. 2014; 20(6): 953–966.DOI: 10.1016/j.cmet.2014.09.018

12. Rena G, Hardie DG, Pearson ER. The Mechanisms of Action of Metformin.Diabetologia. 2017; 60(9): 1577–1585 DOI: 10.1007/s00125-017-4342-z

13. Graham GG, Punt J, Arora M, et al. Clinical pharmacokinetics of metformin. Clinical Pharmacokinetics. 2011; 50(2): 81–98.doi: 10.2165/11534750-000000000-00000

14. Maheshwari Choudhari, Vishal Rasve, Shilpa Khilare, Poonam Ghorpade. Research Journal of Pharmacognosy and Phytochemistry. 2023:15(3): 241–248 DOI: 10.52711/0975-4385.2023.00038

15. Nilesh Nikam, Avish Maru, Anil Jadhav, Prashant Malpure. Analytical Method Development and Validation of Metformin Hydrochloride by using RP-HPLC with ICH Guidelines. International Journal of Trend in Scientific Research and Development (IJTSRD).  2019; 3(3).

16. Maruthur NM, Tseng E, Hutfless S, Wilson LM, Suarez-Cuervo C, Berger Z, Chu Y, lyoha E, Segal JB, Bolen S. Diabetes Medications as Monotherapy or Metformin- Based Combination Therapy for Type 2 Diabetes: A Systematic Review and Meta- analysis. Annals of Internal Medicine.  2016; 164 (11): 740-51

Received on 21.05.2026      Revised on 05.06.2026

Accepted on 17.06.2026 Published on 10.07.2026

Available online from July 25, 2026

Asian Journal of Pharmaceutical Analysis. 2026; 16(3):229-232.

DOI: 10.52711/2231-5675.2026.00035

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