Development and Validation of HPLC Method for Determination of an API in Presence of its Degradation Products

Imeglimin is a novel oral medication used to treat type 2 diabetes (T2D). It's the first in a new class of antidiabetic drugs called glimins, which contain tetrahydrotriazine. Imeglimin's mechanism of action is distinct from older antidiabetic drugs, and it's expected to be used in combination with diet and exercise or as an add on therapy. Imeglimin's effects include: Enhanced insulin secretion imeglimin  increases insulin secretion and protects beta-cell function. the mode of action of Imeglimin is unique and distinct compared with other major classes of therapeutic agents. It involves dual effects, both to enhance insulin action and to reverse pancreatic β?cell dysfunction. Imeglimin primarily works by improving glucose-stimulated insulin secretion (GSIS) and enhancing insulin action, ultimately improving glucose homeostasis. It achieves this by increasing nicotinamide adenine dinucleotide (NAD+) in pancreatic beta cells, which in turn leads to enhanced insulin release and improved mitochondrial function. Imeglimin also helps preserve beta cell mass and potentially reduces hepatic glucose output.[1,2,3] Stability testing forms an important part of the process of drug product development. The purpose of stability testing is to provide evidence on how the quality of a drug substance varies with time under the influence of a variety of environmental factors such as temperature, humidity and light and enables recommendation of storage conditions, retest periods, and shelf life to be established. The assay of Imeglimin API ( Active pharmaceutical Ingradient) in stability test sample needs to be determined using stability indicating method, as recommended by the International Conference on Harmonization (ICH) guidelines and USP. [4,5,6] The present work was designed to develop a simple, precise and rapid analytical LC procedure, which would serve as method for analysis of Imeglimin API in presence of its degradation products. In this method isocratic elution method is selected for the analysis of Imeglimin API because it gave better base line separation and peak width, which is suitable for the routine analysis of Imeglimin. Hydrophilic Interaction Liquid Chromatography (HILIC) is a liquid chromatography technique used for separating polar compounds, particularly those that are difficult to separate using reversed-phase chromatography. It utilizes a polar stationary phase and a mobile phase with a high organic solvent content, typically acetonitrile. The separation mechanism relies on the preferential interaction of polar compounds with the hydrophilic stationary phase in the presence of a highly organic mobile phase. In view of above in the present study we hereby report the development and Validation of HPLC method for determination of Imeglimin API in presence of its degradation products as per ICH guidelines. In order to establish the stability indicating nature of the method, force degradation of Imeglimin was performed under various stress conditions (basic, acidic oxidative, thermal and photo) and stressed samples were analyzed by the proposed method. The proposed LC method was able to separate the drug from degradation products generated during forced degradation studies. The developed method was validated as per ICH guidelines and its updated international convention. The linearity of response, precision, repeatability, accuracy, robustness, ruggedness, specificity, LOD, LOQ of the described method has been checked.[7,8,9,10]

Experimental Conditions

Reagents and reference samples

The API of Imeglimin was purchased from Yarrow Chemicals Ltd. Ammonium acetate was purchased from Merck Ltd. (Mumbai-India) HPLC grade acetonitrile, methanol and HPLC grade water were purchased from Merck (Mumbai, India). 0.20µ and 0.45µ nylon membrane filters were used and purchased from UltraChrom Innovatives Pvt. Ltd. (India). All other chemicals and reagents were used of HPLC grade.

Instrumentation

The high-performance liquid chromatography (HPLC) of Shimadzu SCL-10AVP inbuilt with binary pump (LC-10ATVP), UV detector (SPD-10AVP), Rheodyne 20µl loop capacity manual injector (P/N 77251) was used throughout the analysis. The LC-Solution software was used to interpret the HPLC reports. ZIC-HILIC (5µm; 150 x 4.6 mm ID.) column was purchased from UltraChrom Innovatives (P) Ltd. (India) was used throughout the analysis. Digital weighing balance (ME-204) purchased from Mettler-Toledo (USA), ultra-sonicator Labman® purchased from UltraChrom Ltd, India. Digital pH meter from Mettler-Toledo was purchased from (Mumbai-India). 50 µl micro-syringe was purchased from Hamilton USA. 0.20µ and 0.45µ nylon membrane filters were purchased from Phenomenex® Mumbai, India.

Standard and Sample Preparation

The Standard and sample solutions were prepared separately by dissolving standard and sample in a solvent mixture of Acetonitrile:Methanol:Water (20:60:20 v/v).

Optimization of Chromatographic Conditions

The chromatographic conditions were optimized by different means. (Using different column, different buffer and different modes of HPLC run, Table 1, Fig. a-d.

Table no 1. Method Development For HPLC Analysis of Imeglimin API

Validation

For repeatability the freshly prepared solution of Imeglimin was injected 6 times to determine the closeness of results achieved for relative standard deviation (RSD) and data is presented in table no 2. To establish the intra-day and inter-day precision of the method, Imeglimin was analysed on one day and three separate days. Intra day and Inter day precision were calculated and the data are presented in Table no 3.

Robustness of the method was investigated by varying the chromatographic conditions such as changes of flow, changes in mobile phases. changes in wavelength. Robustness of the developed method was indicated by the overall SD between the data at each variable condition and presented in table no 4. Linearity was determined by injecting different concentration of sample solutions (50, 25, 12.5, 6.25, 3.12 and 1.56 ppm) and data is represented in table no 5.

Accuracy of marketed formulation was determined at 80%, 100%, 120% data is represented in table no 6. Specificity of the proposed method was performed by analysing degraded sample solution of Imeglimin. Ruggedness was performed by different analyst. LOD/ LOQ was determined from linearity study. For system suitability freshly prepared solution of Imeglimin was injected 6 times to determine the closeness of results achieved for relative standard deviation (RSD) in percentage.

Forced Degradation Studies

Imeglimin was allowed to Hydrolyse in different strengths of acid (0.1M HCL), base (0.1M NaOH), hydrogen peroxide (3-6% H₂O₂). Imeglimin was also studied for its thermal degradation for 24hrs at 45 degree celcious and photo degradation for six hrs. An accurately weighed 5mg of Imeglimin API was dissolved in 5ml of Acetonitrile:Methanol:Water (20:60:20 v/v) to get 1000 ppm. Pipette out 0.2ml of 1000ppm and dilute it with 3.8 ml of Acetonitrile:Methanol:Water (20:60:20 v/v) to make it 50ppm and samples for forced degradation is prepared. data is presented in table no 8.

RESULTS AND DISCUSSION

Chromatographic conditions: To develop a precise, linearity, specific and suitable stability indicating HPLC (HILIC-Hydrophilic Interaction Liquid Chromatography) method for analysis of Imeglimin, different chromatographic conditions were applied and the results observed are presented in Table 1 and and Figs. 1a-d. In case of HPLC various columns are available, but here Zic-HILIC (150 X 4.6 mm. ID.) column preferred because using this column peak shape, resolution and absorbance were good and Mobile Phase: 15mM Ammonium acetate - Acetonitrile; 50:50 v/v was selected.

Fig a:Trial 1

Fig b:Trial 2

Fig c:Trial 3

Fig d:Trial 4

Repeatability:

calculated RSDs for tested imeglimin was less than 2% which is quite significant in accordance with the ICH guidelines.

Table no 2 : Repeatability Study of Imeglimin

Fig 1: Repeatability

Fig 2: Intraday Study

Fig 3: Interday study

Fig 4: Robustness Study:Flow Rate 1.1 ml/min

Fig 5: Robustness Study: Flow Rate 0.9 ml/min

Fig 6: Robustness Study: 52 %

Fig 7: Robustness Study: 48 %

Fig 8: Robustness Study: 232 nm

Fig 9: Robustness Study: 228 nm

Fig 10 : Calibration Curve of Imeglimin

Table no 3: Intraday Precision Data of Imeglimin

                                     Table 3: Interday/Intermediate Precision Data of Imeglimin

Table no 4: Robustness data of Imeglimin

Table no 5: Linearity Data of Imeglimin

Table no 6: Drug Accuracy Study; Imeglimin

Precision: The results obtained in intraday  precision studies was found within 0.61-1.93 %, peak are found with %RSD NMT 2% which was in agreement with system suitability.  this confirms that the method was precise. The results obtained in interday  precision studies was found within 0.23-0.61%, peak are found with %RSD NMT 2% which was in agreement with system suitability.  this confirms that the method was precise.

Robustness: Overall %RSD of results with change in flow, wavelength and mobile phase composition observed within acceptance criteria, method is robust in terms of slight change in internal method parameters, hence Robustness is justified.

Linearity: Correlation coefficient observed within acceptance criteria hence method is linear,and linearity is justified.

Accuracy: % Mean recovery of Imeglimin observed within acceptance criteria 99.73%-101.16%, also % RSD of recovery observed within acceptance criteria; hence accuracy is justified.

Specificity: This result indicates that the peak of the analyte was pure and it confirmed the specificity of the method.

Ruggedness: By comparing with the optimized chromatogram, the method indicated that by performing with different analyst method was found stable.

LOD/LOQ: They were found to be LOD 1.42 µg/mL and LOQ  4.72 µg/mL

System Suitability Testing:

Table no 7: System suitability of Imeglimin

All the parameters of system suitability are observed within specified limit. Hence system suitability is justified.

Forced Degradation Study:

Table no 8:Results of Stability Studies of Imeglimin