Abstract

A novel method for analyzing Imeglimin Hydrochloride, an oral antidiabetic agent has been developed and validated using High-Performance Thin Layer Chromatography (HPTLC) for both bulk and tablet forms. The method utilizes a mobile phase of acetone, methanol, toluene, and formic acid in specific proportions. Chromatographic separation was achieved on silica gel TLC plates with densitometry scanning at 244 nm, where the drug shows significant absorbance. Validation followed ICH Q2R1 guidelines, demonstrating satisfactory results for Linearity, Accuracy, Precision (intra- and inter-day), Limit of detection (LOD), Limit of quantitation (LOQ), and Robustness. The calibration curve was linear over the concentration range of 1000-5000 ng/band, with a regression equation of y = 2.9501x + 3834.2 and a correlation coefficient (R²) of 0.9942. Precision studies showed low % RSD values for intra-day and inter-day variations, confirming reliability. The LOD and LOQ were 1074.928 ng/spot and 3257.54 ng/spot, respectively. Recovery studies demonstrated the method's accuracy, with % recovery values near 100% at different spike levels. Robustness testing indicated the method's resilience to small, deliberate changes in experimental conditions, with % recovery within the acceptable limit of 2%. The developed HPTLC method offers a simple, cost-effective, and reliable means for quantitatively analyzing Imeglimin Hydrochloride in pharmaceutical formulations.

Keywords

Introduction

Type 2 Diabetes Mellitus is a chronic condition marked by elevated glucose levels in the blood. The International Diabetes Federation (IDF) estimates that approximately 540 million individuals were affected by diabetes in 2023. Imeglimin is the first in a new tetrahydrotriazine-containing class of oral antidiabetic agents referred to as 'glimins'. Imeglimin Hydrochloride is chemically named (4R)-6-N,6-N,4-trimethyl-1,4-dihydro-1,3,5-triazine-2,6-diamine; hydrochloride. The mechanism of action of imeglimin consists of two main effects: (a) preservation of ?-cell mass and amplification of glucose-stimulated insulin secretion (GSIS); and (b) enhanced insulin action, which includes the potential to inhibit hepatic glucose output and improve insulin signaling in both skeletal muscle and liver[1,2].

Previous analytical studies have focused on HPLC and UV methods for development, validation, and stability [3, 4]. Imeglimin hydrochloride, a novel anti-diabetic drug, is not officially included in any pharmacopeia.  However, there is a gap in the literature regarding High-Performance Thin-Layer Chromatography (HPTLC) methods for determining the amount of imeglimin hydrochloride in tablet dosage forms. This work aims to develop an economical HPTLC method that adheres to International Council for Harmonisation (ICH) guidelines. This approach aims to provide a reliable, cost-effective, and efficient alternative for quantifying imeglimin hydrochloride in tablets, supporting routine quality control and ensuring therapeutic efficacy. The development will include optimization of chromatographic conditions, sample preparation, and validation processes to meet ICH standards for accuracy, precision, and specificity. This method will fill the current gap in analytical techniques for imeglimin hydrochloride, facilitating its broader application and quality assurance in pharmaceutical contexts [5].

MATERIALS AND METHODS

Chemicals and Reagent

Imeglimin hydrochloride was purchased from M/s Jilin Shulan Synthetic Pharmaceutical Co., Ltd., Shulan City, Jilin Province, and China. Imeglimin hydrochloride tablets (500 mg) were obtained from a local pharmacy. All solvents, including acetone, methanol, toluene, and formic acid (AR grade), were purchased from Merck Specialities Pvt. Ltd., Mumbai, India.

Instrumentation and Chromatographic Conditions

Chromatographic separation of the drug was performed on Merck TLC plates precoated with silica gel 60 F254 (10 cm × 10 cm with a 250 ?m layer thickness) from E. Merck (Darmstadt, Germany) using a sample applicator CAMAG Linomat 5 (Switzerland). Samples were applied on the plate as bands with a 5 mm width using a Camag 500 ?L sample syringe (Hamilton, Switzerland). Linear ascending development was carried out in a 20 × 10 cm twin trough glass chamber (CAMAG, Muttenz, Switzerland) using acetone, methanol, toluene, and formic acid (4:3:2:1, v/v/v/v) as the mobile phase. The mobile phase was placed in the chamber for 15 minutes to reach saturation. TLC plates were dried after development using an air dryer. Data were gathered using winCATS software, and densitometric scanning was carried out at 254 nm using the CAMAG thin-layer chromatography scanner III. The radiation source was a deuterium

Preparation of Standard Stock Solution

Imeglimin Hydrochloride 10 mg was accurately weighed and dissolved in 10 mL of methanol to obtain a solution with a concentration of 1000 ?g/mL.

Selection of Detection Wavelength

Bands were scanned in the range of 200-400 nm after chromatographic development, and the drug was found to have significant absorbance at 254 nm, which was chosen as the detection wavelength [6].

Analysis of Marketed Formulation

Twenty tablets were accurately weighed and finely powdered. A tablet powder equivalent to 10 mg of the drug was weighed and transferred into a 10 mL volumetric flask and dissolved in methanol. The mixture was sonicated for 15 minutes, filtered, and then the volume was made up with methanol. A final sample concentration of 1000 ng/band for Imeglimin Hydrochloride was obtained by applying one microliter of this solution to a TLC plate. After chromatographic development, the peak areas of the bands were measured at 244 nm, and the amount of drug present in each sample was calculated by the calibration curve. The protocol was repeated three times for homogeneous sample analysis [7, 8].

RESULTS AND DISCUSSION

Method Optimization

The main objective was to develop a novel HPTLC method that achieves satisfactory drug resolution. Initially, several trials were performed using different mobile phases to attain better separation. Finally, a mobile phase consisting of acetone, methanol, toluene, and formic acid (4:3:2:1, v/v/v/v) was selected for development. Densitometric analysis was carried out at 244 nm. The retention factor was determined to be 0.61.

Method Validation

By ICH Q2R1 requirements, the proposed method's linearity, accuracy, precision (both intra- and inter-day), limit of detection, the limit of quantitation, and robustness were all validated 9,10].

Linearity

To achieve a concentration range of 1000-5000 ng/band, aliquots of 1, 2, 3, 4, and 5 ?L of the standard stock solutions of Imeglimin Hydrochloride (1000 ng/?L and 2000 ng/?L) were applied onto the TLC plates. Results were reported to be linear, with the regression equation and correlation coefficient shown to be y = 2.9501x + 3834.2 and R?2; = 0.9942. The calibration curve, 3D spectra densitogram, overlay spectrum of Imeglimin Hydrochloride, and densitogram of the standard obtained are represented in Figures 1-9 and Table 1, respectively.

Precision

Intra-day Precision

Six replicates of three different concentrations of a standard solution of Imeglimin Hydrochloride were prepared. To record intra-day differences in the results, all of the stock solutions were examined three times on the same day. For inter-day precision, the stock solutions were examined over three successive days.

Inter-day Precision

The low % R.S.D. values from both intra-day and inter-day observations indicate that the developed method is precise.

Limit of Detection (LOD) and Limit of Quantitation (LOQ)

LOD and LOQ were determined as 3.3?/S and 10?/S, respectively, where ? is the standard deviation of the regression line (y-intercept) and S is the slope of the calibration curve. LOD and LOQ were found to be 1074.928 ng/spot and 3257.54 ng/spot, respectively.

Specificity

The specificity of the method was evaluated by separating the peaks of both the tablet and the API. The spot of Imeglimin Hydrochloride in the sample was confirmed by matching the Rf and spectra of the standard spot. Therefore, the method was considered to be specific. The figure (1-9) shows the chromatogram of Imeglimin Hydrochloride tablets.

Accuracy

The accuracy of the method was assessed through recovery studies. Known quantities of the pure drug were added to re-analyzed sample formulations at 50%, 100%, and 150% levels of the basic concentration of 2000 ng per band from the tablet solution. The drug concentrations were calculated accordingly. The results of the recovery studies indicated that the method is accurate.

Robustness

To establish the robustness of the method, small deliberate changes were made to experimental conditions such as chamber saturation time, the volume of the mobile phase, and the distance run by the solvent front. Under these altered conditions, the stock solution was analyzed, and the results of robustness studies were expressed in terms of % RSD of peak areas. The method was found to be robust as the percentage recovery remained within the acceptable limit of 2%.

DISCUSSION

The method described in this study offers a precise and comprehensive approach for analyzing Imeglimin Hydrochloride using high-performance thin-layer chromatography (HPTLC) in both bulk and pharmaceutical dosage forms. Through meticulous method development, including optimization of the mobile phase composition and chromatographic conditions, clear and symmetrical peaks with a retention factor (Rf) of 0.61 were achieved, indicating satisfactory resolution. Linear regression analysis demonstrated excellent linearity over the concentration range of 1000-5000 ng/spot for Imeglimin Hydrochloride, with a high correlation coefficient of 0.9942. Moreover, the method exhibited remarkable precision, with a percentage relative standard deviation (%RSD) consistently below 2?ross all evaluated parameters. The recovery of Imeglimin Hydrochloride, ranging between 99.78% and 99.98%, further attested to the accuracy and reliability of the developed method [11]. In addition to its analytical performance, the method demonstrated good sensitivity, as evidenced by the low limit of detection (LOD) and limit of quantitation (LOQ) values of 1074.928 ng/spot and 3257.54 ng/spot, respectively. The method was found to be robust because the percentage recovery was within the limit i.e., 2%. The specificity of the method was confirmed by ensuring that the formulation did not interfere with the sample peak. Overall, this HPTLC approach provides a robust and efficient means for quantitatively analyzing Imeglimin Hydrochloride in pharmaceutical formulations. Its accuracy, precision, and sensitivity make it well-suited for routine analysis in quality control laboratories and pharmaceutical industries [12, 13].

CONCLUSION:

The analysis of Imeglimin Hydrochloride in both bulk and tablet forms has been facilitated through the development and validation of a novel HPTLC approach that is simple, reliable, repeatable, and free from excipient influence. The described approach allows for quantitative analysis of Imeglimin Hydrochloride in pharmaceutical dosage forms. Moreover, the method is considered economical as it was developed using affordable and readily available solvents suitable for pharmaceutical studies [14, 15].

ACKNOWLEDGEMENT            

We would like to express our gratitude to the Principal and our Chairman Dr.Nalla G.Palaniswamy and Dr.Thavamani.D D Palaniswamy, Trustee, Dr.N.G.P Research and Educational Trust Coimbatore, for their guidance and support.

AUTHORS CONTRIBUTIONS

All authors have contributed equally.

FINANCIAL SUPPORT

There is no funding to report.

CONFLICT OF INTEREST

The authors declare that they have no conflict of interest.

ETHICAL APPROVALS

This study does not involve experiments on animals or human subjects.

DATA AVAILABILITY

All data generated and analyzed are included in this research article.

REFERENCES

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