Development and Validation of Stability-Indicating RP-HPLC Method for the Determination of Imeglimin Hydrochloride in Bulk Form

Rapid economic growth and urbanization have significantly contributed to the rising global burden of diabetes. Changes in lifestyle, including sedentary behaviour, unhealthy dietary habits, and increased stress levels, have led to higher prevalence rates. Additionally, improved life expectancy and population aging further add to the growing number of diabetes cases worldwide, making it a major public health challenge [1]. The rising prevalence of type 2 diabetes mellitus (T2DM) poses a significant global public health challenge [2,3]. Despite substantial efforts by governmental and non-governmental organizations toward its diagnosis, treatment, prevention, and awareness, the incidence continues to increase. This upward trend is particularly evident among older adults, individuals with obesity, and high-risk ethnic population [4]. Over recent decades, type 2 diabetes mellitus (T2DM) has become increasingly widespread, paralleling the rise in key risk factors such as metabolic dysfunction, obesity, and sedentary lifestyles. Rapid urbanization, unhealthy dietary patterns, reduced physical activity, and increasing stress levels have further accelerated this trend. Additionally, aging populations and genetic predisposition contribute to the growing burden, making T2DM a significant and escalating global health concern [5], [6], [7].

Several oral antidiabetic agents are available for managing the complex pathophysiology of type 2 diabetes mellitus; however, each therapeutic class is associated with inherent limitations [8]. Many of these drugs are linked to notable adverse effects, such as urinary tract infections with sodium–glucose cotransporter-2 (SGLT2) inhibitors (e.g., dapagliflozin); weight gain with sulfonylureas like glipizide; hypoglycemia with sulfonylureas (e.g., glibenclamide), meglitinides, thiazolidinediones, GLP-1 receptor agonists, and insulin; edema with thiazolidinediones such as pioglitazone; and an increased risk of pancreatitis with GLP-1 receptor agonists [9]. Additionally, issues such as declining efficacy over time, patient non-compliance, and cost burden further limit optimal disease management. Despite the wide array of available therapies, none adequately address all three fundamental components of T2DM pathophysiology—excessive hepatic glucose production due to gluconeogenesis, increased insulin resistance, and impaired insulin secretion resulting from pancreatic β-cell dysfunction [10].

Imeglimin is a novel oral antidiabetic agent belonging to the tetrahydrotriazine class, developed for the management of type 2 diabetes mellitus (T2DM) [11]. It is marketed under the brand name Twymeeg [12] and received its first regulatory approval for clinical use in Japan in June 2021, marking a significant advancement in diabetes therapeutics [13]. Notably, imeglimin represents the first approved drug of the emerging “glimin” class of glucose-lowering agents, introducing a new pharmacological category distinct from existing antidiabetic therapies [14]. The mechanism of action of imeglimin is multifaceted and targets the core pathophysiological defects of T2DM. It enhances Glucose-Stimulated Insulin Secretion (GSIS) from pancreatic β-cells in a glucose-dependent manner, thereby reducing the risk of hypoglycemia. Additionally, imeglimin has been shown to preserve and potentially restore pancreatic β-cell mass and function, which are progressively impaired in patients with T2DM. Furthermore, it improves insulin sensitivity in peripheral tissues, particularly in the liver and skeletal muscle, leading to enhanced glucose uptake and reduced hepatic glucose production.

Imeglimin also exerts beneficial effects on mitochondrial function by improving mitochondrial bioenergetics and reducing oxidative stress, which are critical contributors to insulin resistance and β-cell dysfunction. Through these combined actions—enhancing insulin secretion, preserving β-cell integrity, improving insulin sensitivity, and modulating mitochondrial pathways—imeglimin helps restore overall glucose homeostasis. This unique and comprehensive mechanism distinguishes it from traditional antidiabetic agents and highlights its potential as an effective therapeutic option for the management of type 2 diabetes mellitus [11-14].

2. MATERIALS AND METHOD

2.1. DRUG PROFILE:

The structure of Imeglimin Hydrochloride is:

Imeglimin Hydrochloride is classified as an antidiabetic agent used in the management of Type 2 diabetes mellitus. Its chemical name is (R)-6-imino-N,N,4-trimethyl-1,4,5,6-tetrahydro-1,3,5-triazin-2-amine hydrochloride. The molecular formula is C?H??ClN?, with a molecular weight of 191.66 g/mol. For the present study, Imeglimin Hydrochloride was procured from Enaltec Laboratories.

2.2. METHOD:

2.2.1 INITAILISATION OF RP-HPLC METHOD

Table 1: Solubility of Imeglimin HCl in different solvents.

The solubility of the Imeglimin Hydrochloride active pharmaceutical ingredient (API) was systematically evaluated in a range of solvents, including water, acetonitrile, methanol, ethanol, acetone, water–acetonitrile mixtures, orthophosphoric acid, OPA–acetonitrile buffer, and water–methanol mixtures, as summarized in Table 1. This comprehensive assessment was carried out to identify the most suitable solvent system for effective chromatographic analysis. The results indicated that Imeglimin Hydrochloride exhibited better solubility in polar solvents, particularly methanol and HPLC-grade water. Based on these findings, a combination of methanol and HPLC-grade water was selected as the mobile phase. This selection was further supported by their widespread availability, compatibility with the RP-HPLC system, low toxicity, and cost-effectiveness, making them ideal for routine analytical applications and method development.

Preparation of Mobile Phase

A precisely weighed quantity of 0.6306 g of ammonium formate was dissolved in 1000 mL of HPLC-grade water to prepare the buffer solution. The pH was carefully adjusted to 4.0 using dilute formic acid. The prepared solution was mixed thoroughly to ensure complete dissolution, followed by sonication to remove any dissolved gases. Finally, the solution was filtered to obtain a clear buffer suitable for chromatographic analysis.

Mobile Phase A: Buffer Solution

Mobile Phase B: Methanol was used as the second Mobile Phase.

Preparation of diluent

A diluent was prepared by mixing HPLC-grade water and acetonitrile in the ratio of 900:100 (v/v). This solution was used both as the diluent for sample preparation and as the blank during analysis.

Preparation of standard stock solution

A standard stock solution of Imeglimin Hydrochloride (2000 ppm) was prepared by accurately weighing 200 mg of the active pharmaceutical ingredient and transferring it into a 100 mL volumetric flask. Approximately 15 mL of the prepared diluent was added to dissolve the drug, and the mixture was thoroughly shaken to ensure uniform dispersion. The solution was then sonicated for about 10 minutes to achieve complete dissolution of the drug. After ensuring clarity, the volume was made up to the mark with the same diluent, resulting in a homogeneous standard stock solution suitable for further analysis.

Preparation of standard solution

A 1000 ppm standard solution of Imeglimin Hydrochloride was prepared by accurately transferring 10 mL of the previously prepared standard stock solution into a 20 mL volumetric flask. About 5 mL of the diluent was added, and the solution was sonicated for approximately 10 minutes to ensure complete mixing. The final volume was then made up to the mark with the diluent, yielding a clear and homogeneous solution suitable for further analytical use.

Preparation of test solution

Test solutions of Imeglimin Hydrochloride at concentrations of 800, 900, 1000, 1100, and 1200 ppm were prepared by transferring 8, 9, 10, 11, and 12 mL, respectively, of the standard stock solution into separate 20 mL volumetric flasks. Each solution was initially diluted with about 5 mL of the diluent, sonicated for 10 minutes to ensure complete mixing, and then made up to the final volume with the same diluent.

Selection of Detection Wavelength

A solution of Imeglimin Hydrochloride was prepared using methanol as the solvent. The UV spectrum was recorded by scanning the solution over the wavelength range of 200–400 nm. The drug exhibited maximum absorbance (λmax) at 240 nm, which was selected as the optimal detection wavelength for further analysis.

Chromatographic conditions

Table 2: Optimized chromatographic conditions used to validate the proposed method.

In accordance with ICH Q2(R1) guidelines, the developed analytical method was validated for key performance characteristics, including specificity, linearity, accuracy, precision, robustness, limit of detection (LOD), and limit of quantification (LOQ) [17], [18]. Validation of optimized method of Imeglimin HCl by RP-HPLC method.

3. RESULT

System Suitability:

System suitability was evaluated using a standard solution of Imeglimin Hydrochloride at a concentration of 100 μg/mL. Key chromatographic parameters, including retention time, theoretical plates, tailing factor, and percentage relative standard deviation (%RSD), were determined to ensure the adequacy of the system.System suitability refers to a set of tests performed to verify that the chromatographic system is functioning properly before or during analysis. It ensures that the system provides acceptable performance in terms of resolution, repeatability, and efficiency, thereby confirming the reliability and accuracy of the analytical results.

Table 3: System Suitability Study Result of Imeglimin HCl

Specificity:

Method specificity was assessed by injecting methanol as the blank and a 100 µg/mL standard solution of Imeglimin Hydrochloride. The chromatograms showed no interfering peaks from the diluent at the retention time of the drug, thereby confirming that the method is specific for the estimation of Imeglimin Hydrochloride.

Specificity is defined as the ability of an analytical method to accurately measure the analyte in the presence of other components such as impurities, degradants, or excipients, without any interference, ensuring reliable and accurate results.

Figure 1: Chromatogram for blank

Figure 2: Chromatogram for standard imeglimin

Accuracy:

The accuracy of the developed method was evaluated at five concentration levels (800, 900, 1000, 1100, and 1200 ppm), with each level analyzed in sextuplicate (six replicates) in accordance with ICH guidelines. Accuracy was determined using the standard addition method. The obtained results were within acceptable limits and were expressed in terms of standard deviation (SD) and percentage relative standard deviation (%RSD), indicating the reliability of the method.

Accuracy is defined as the closeness of agreement between the value found and the true or accepted reference value. It reflects the trueness of the method and its ability to provide results that are free from systematic error.

Calculation of AVG. STD, %RSD of different concentration (800 PPM- 1200 PPM) of Imeglimin HCl on same day:

Table 4: Result of Accuracy of Imeglimin HCl (800-1200 PPM)

(The method pass the test and RSD was found to be less than 2%)

Linearity:

Linearity of the developed method was established by constructing a calibration curve using five concentrations of Imeglimin Hydrochloride (800, 900, 1000, 1100, and 1200 µg/mL). Each concentration was injected into the HPLC system, and the corresponding peak responses were recorded. The calibration curve was plotted with concentration versus peak response, and the regression equation along with the correlation coefficient was determined. The results demonstrated a strong linear relationship between concentration and peak response over the studied range.

Linearity is defined as the ability of an analytical method to produce results that are directly proportional to the concentration of the analyte within a given range, indicating the method’s suitability for quantitative analysis.

Table 5: Result of Linearity of Imeglimin HCl (800-1200 PPM)

Figure 3: Linearity graph of Imeglimin HCl API

RSD value was found less than 2%

A regression coefficient (R²) of 0.9981 obtained between the standard concentrations of Imeglimin Hydrochloride and their corresponding mean peak areas indicated excellent linearity of the calibration curve. Additional regression parameters further supported the linear behavior of the method across the concentration range of 800–1200 µg/mL, confirming its suitability for quantitative analysis.

Precision

Precision of the developed method was evaluated using Imeglimin Hydrochloride solutions at concentrations of 800, 900, 1000, 1100, and 1200 µg/mL. Both intraday and interday precision studies were carried out according to the specified analytical procedures. The results demonstrated consistent and reproducible measurements across different time intervals, indicating the reliability of the method.Precision is defined as the degree of agreement among a series of measurements obtained from multiple sampling of the same homogeneous sample under prescribed conditions. It reflects the reproducibility of the method and is commonly expressed in terms of standard deviation (SD) and percentage relative standard deviation (%RSD).

a.         Repeatability

Repeatability, also known as intra-assay precision, was evaluated by analyzing Imeglimin Hydrochloride solutions in the concentration range of 800–1200 ppm. Each concentration was injected six times under identical operating conditions within the same day. The results demonstrated consistent responses, confirming the method’s repeatability and short-term precision.

Repeatability is defined as the degree of agreement between independent test results obtained under the same operating conditions over a short time interval. It reflects the method’s ability to produce consistent results when performed by the same analyst using the same equipment.

6 Reps of Imeglimin HCl was performed and %RSD was calculated as follows:

Table 6: Result of Repeatability Study (Intra Day) of Imeglimin HCl (800-1200 PPM)

(The method passed the test as the RSD was less than 2%)

b. Intermediate Precision

Intermediate precision of the developed method was evaluated by analyzing Imeglimin Hydrochloride solutions in the concentration range of 800–1200 ppm. Each concentration was injected six times on two different days to assess variability under normal laboratory conditions. The results demonstrated consistent performance, confirming the method’s reliability across different days.

Intermediate precision is defined as the degree of agreement among test results obtained within the same laboratory under varied conditions, such as different days, analysts, or equipment. It reflects the method’s reproducibility under routine analytical conditions.

DAY-1: 6 Reps of Imeglimin HCl was performed for interday precision and %RSD was calculated as follows:

Table 7: Result of Intermediate Precision (Inter Day-1) of Imeglimin HCl (The method passed the test as the RSD was less than 2%)

DAY-2: 6 Reps of Imeglimin HCl was performed for interday precision and %RSD was calculated as follows:

Table 8: Result of Intermediate Precision (Inter Day-2) of Imeglimin HCl

(The method passed the test as the RSD was less than 2%)

c.) Reproducibility

Reproducibility of the method refers to the precision obtained when the analysis is performed under varying conditions across different laboratories. It is typically evaluated through collaborative studies to ensure the method yields consistent and comparable results irrespective of location.Reproducibility is defined as the degree of agreement among test results obtained for the same sample when the analysis is conducted in different laboratories, using different analysts and equipment. It reflects the robustness and transferability of the analytical method for standardization purposes.

Robustness:

Table 9: Result of Robustness of Imeglimin HCl

Force degradation study

Forced degradation studies were conducted under four stress conditions—acidic, alkaline, oxidative (H?O?), and thermal—to evaluate the stability profile of Imeglimin Hydrochloride. The standard drug was subjected to these stress conditions to induce partial degradation. In accordance with ICH Q1A (R2) guidelines, the objective was to develop a stability-indicating analytical method capable of effectively separating the intact drug peak from its degradation products. These studies also aided in identifying conditions that lead to drug instability, thereby providing essential information for developing appropriate formulation, storage, and handling strategies to ensure product stability. Forced degradation studies are defined as stress testing procedures in which a drug substance is subjected to extreme conditions to accelerate its degradation. These studies help in understanding degradation pathways, identifying degradation products, and establishing the stability-indicating nature of an analytical method.

The detailed procedure for forced degradation is described below:

Table 10: Procedure of Force Degradation studies of Imeglimin HCl