Chemicals and reagents

Analytical pure Imeglimin was received as gift sample from Exceed Pharmaceuticals, Vapi with batch number-EMV231637, Expiry date-10/09/2026 with purity of 98.4%. Carbamazepine with 99.45% purity was obtained from Cadila pharmaceuticals Pvt., Ltd. Drug free K3EDTA human plasma was procured from Rajkot voluntary blood bank, Rajkot.

Liquid chromatography-mass spectrometry (LC–MS/MS) method development

For mass spectrometry with liquid chromatography, LC–MS/MS of Shimadzu SIL-20AC was utilized, employing a Phenomenex Kinetex PFP column (50 × 4.6 mm, 2.6 μm). Carbamazepine has been used as an internal standard. It is a suitable internal standard for the bio-analytical method validation of Imeglimin hydrochloride due to its structural stability, similar chromatographic behavior, and comparable physicochemical properties. Based on the solubility of the drug and internal standard, multiple trials were conducted using 1% formic acid and acetonitrile in different ratios. After evaluating the separation results, the optimal mobile phase composition was determined to be 0.1% formic acid in acetonitrile (30:70, v/v) ratio, utilizing a flow rate maintained at 0.500 mL/min. The temperature of column oven was set to 40 ± 0.3 °C, while the auto-sampler temperature was controlled around 15 ± 3 °C. An injection volume of 5.0 µL was used for each sample. Mass spectrometers were used for the detection process. The retention time for drug was approximately 2.20 min, while the internal standard (ISTD) showed a retention time of about 1.00 min. The analysis’s overall run time was 4.0 min. All the conditions are shown in Table 1. The ionization was carried out in the positive ion mode employing electrospray ionization (ESI). The parent ion for drug was m/z 156.2, and for the ISTD, it was m/z 237.00. The corresponding daughter ions were m/z 71.1 for Drug1 and m/z 194.100 for the ISTD. Both analytes had a dwell time of 100 ms, with collision energy (CE) set to -30 for Drug1 and – 25 for the ISTD. Additional parameters included a desolvation line (DL) temperature of 250 °C, a 3.0 L/min flow rate of nebulizing gas, a 450 °C heat block temperature, and a drying gas flow rate of 15.0 L/min. The mass conditions are mentioned in Table 2.

Table 1 Preparation of standard calibration curve (CC) spiking solutionsTable 2 Preparation of spiked CC StandardsPreparation of solutions

Stock solution of Imeglimin made by accurately weighing 10 mg of Imeglimin and adding methanol, for final 1 mg/mL concentration. It was kept in storage refrigerator at 5 ± 3 °C and utilized it in 7 days. The internal standard stock solution (ISTD) was made by measuring 10 mg of carbamazepine and diluting it with methanol. The ISTD dilution solution was prepared by dilution 500 ng/mL with methanol. A mix intermediate stock solution of Imeglimin was produced by diluting 0.500 mL to 50 µg/mL of the Imeglimin stock solution. Both diluted solutions were stored at 5 ± 3 °C.

Extraction procedure

The extracted sample preparation involves thawing spiked samples for quality control (QC) and calibration curves (CC), transferring 0.200 mL into pre-labeled tubes, adding 50µL of ISTD dilution and 50µL of 0.1 N NaOH solution, then vortexing. Methyl tert-butyl ether is added, followed by centrifugation for 20 min at 50 RPM. The extracted materials were centrifuged for 10 min at 10,000 RPM and 10 + 2 °C. 6. Using a vacuum oven set to 40 ± 5 °C, 1.0 mL of supernatant was placed into tubes that had already been labeled and evaporated to dryness and samples are injected via HPLC–ESI–MS/MS. Aqueous samples are prepared similarly. Un-extracted sample preparation substitutes spiked samples with blank human plasma, using methanol instead of ISTD dilution. Following the same steps, the procedure is optimized using a precipitation method before LC/MS/MS analysis.

Preparation of standard and quality control sampleCalibration curve (CC) spiking solutions

As stated below in table, CC spiking solutions were made in methanol by successive dilution of the Mix Intermediate Stock Solution. Preparation of standard calibration curve (cc) spiking solutions is described in Table 1. CC standards were created by spiking the appropriate CC spiking solution solutions in drug free k3 EDTA human plasma are described in Table 2.

Spiked QC samples

By spiking the appropriate QC spiking solutions, QC samples were created in drug free k3 EDTA plasma as described in Table 3.

Table 3 Preparation of spiked QC samplesBioanalytical validation procedure

Bioanalytical validation is a crucial step in pharmaceutical and clinical research, ensuring the accuracy and reliability of analytical methods used to measure drugs or biomarkers in biological samples. This process involves systematic evaluation of parameters like specificity, accuracy, precision, sensitivity, linearity, and robustness to confirm that the method meets predefined criteria. Regulatory guidelines provide standards for validation, ensuring the consistency and quality of data generated for drug development and clinical applications. US FDA guideline was followed to carry out bioanalytical validation procedure [12].

System suitability

Six successive injections of aqueous MQC-1 with internal standard were used to conduct a system suitability experiment in beginning of the method validation and every day thereafter. The acceptance criteria state that the % CV of the area ratio and retention time for both the drug and the internal standard must be within ≤ 4.00.

Auto-sampler carryover

STD BL, ULOQ, and LLOQ were practice in order to assess auto-sampler carryover. These samples were obtained by auto-sampler based on extracted sample preparation.

Linearity, Accuracy, and Precision of Calibration Curve Standards: Three precision and accuracy batches (P&A) were conducted to assess linearity, accuracy, and precision. The method was evaluated using correlation co-efficient, mean accuracy, and precision. Linearity was detected using a 1/x2 weighted least square regression analysis. All calibration curves analyzed were, in relation to the criteria linear, indicating fitting data points in a straight line and constant proportionality.

Accuracy and precision of quality control samples

Every QC concentration level’s mean accuracy was used to assess accuracy, and every QC concentration level’s CV was used to determine precision. Utilizing six replicate samples at HQC, MQC, LQC, and LLOQ QC samples within batch precision and accuracy was determined. All replicate samples at the HQC, MQC-1, MQC-2, LQC, and LLOQ QC levels for the three precision and accuracy batches will be used to determine the between-batch precision.

Selectivity

Selectivity was done through the determination of two parameters, namely:

Matrix factor

The analytical response to extract with blank matrix spiked sample with an analyte at various levels of concentrations (low, middle, and high) and ISTD in comparison with reference solutions is measured by the matrix factor. The matrix factor of an analyte is done by dividing the matrix factor of peak response ratio, or ISTD, when matrix ions are present to get the ISTD normalization factor. Six batches of screened human plasma will be spiked with an analyte and also ISTD at different concentrations.

Specificity

The specificity of the procedure will be determined by screening 10 distinct batches of standard blank plasma: one lipidemic, one heparinized, one hemolyzed, and one with K3EDTA anticoagulant. In order to assess the specificity, the responses of the extracted LLOQ and AQ LLOQ samples will be compared to the responses of interfering peaks at the drug and ISTD retention times in the standard blank.

Recovery

Recovery for the drug, metabolite and internal standard of method was determined by using six replicates of HQC, MQC-1, MQC-2 and LQC which were analyzed by following the procedure for aqueous sample preparation and compared with same concentration level of QC sample.

Recovery for drug

The percentage of average recovery of an analyte will be determined by comparing the mean peak area of six replicates of extracted plasma quality control samples at different concentrations with the mean peak area of un-extracted samples. The acceptance criteria for this method include a %CV of replicates at each QC level within 15.00%.

Recovery for internal standard

The percentage of average recovery of ISTD was calculated by evaluating the mean peak area of the internal standard in extracted plasma quality control samples at MQC concentration with the mean peak area in samples that were not extracted. The acceptance criteria for ISTD recovery should be within 15.00%.

Stability

Benchtop Stability: Spiked quality control samples were stored in a deep freezer at—78 ± 8 °C for 12 h, then retrieved and kept at ambient temperature for at least 6 h. Benchtop stability was assessed using six replicates of HQC and LQC samples, comparing them to freshly spiked samples. BT stability was evaluated based on % mean stability and % CV of samples, with acceptance criteria of within 15.00% and ± 15.00% from nominal concentration.

Dry extract stability

One set of 6 replicates of HQC and LQC were practice (up to drying) as per procedure for extracted sample preparation. Dried samples (before reconstitution) in Eppendorf tube were capped by white cap and stored in deep freezer below − 20 °C for at least 24 h. After specific time period, dried samples were reconstituted with reconstitution solution and injected for analysis along with freshly spiked HQC and LQC in screened biological matrix.

Wet extract stability

Wet extract stability of the spiked quality control samples will be determined by using six replicates of each HQC and LQC samples for at least 2 h and 24 h or as per requirement by storing the samples in auto-sampler or in refrigerator at 5 ± 3 °C. Analyze the WE stability samples along with freshly spiked HQC and LQC and evaluate the WE stability on the basis of % mean accuracy and % CV of WE stability samples.

Freeze–thaw stability

Six sets of HQC and LQC samples were stored in a deep freezer at – 20 ± 5 °C and – 78 ± 8 °C for 24 h. After thawing in a water bath, the samples were analyzed and compared with a freshly retrieved P&A batch. The freeze–thaw stability of the spiked quality control samples was determined during five cycles stored below – 20 ± 5 °C and – 78 ± 8 °C, assessing stability against freshly thawed samples.

Long-term stability

Long-term stability of the spiked quality control samples will be determined by using six replicates of each HQC and LQC samples for at least 25 to 30 days at – 20 ± 5 ºC and – 78 ± 8 ºC and after that the LTM stability samples along with freshly spiked HQC and LQC sample and evaluate the LTM stability on the basis of % mean stability and % CV of LTM stability samples.

Short-term stock solution stability

The study evaluated short-term stock solution stability for Imeglimin and carbamazepine using six injections of aqueous standard equivalent to ULOQ and working concentrations. The stability samples were kept at room temperature for 6 h, and fresh stock solutions were prepared. The mean response ratios of stability samples were compared to comparison samples for analysis.

Long-term stock solution stability

Using six aqueous standard injections, the study assessed the long-term stock solution stability for Imeglimin and carbamazepine. Stability sample preparation and storage at 5 °C were done in order to evaluate stability below 8 °C. These samples’ mean response ratios were contrasted with comparative samples made from recently made stock solutions.

Dilution integrity

An analyte stock solution dilution was made at a concentration that was 1.5–1.8 times higher than the highest standard. For these solutions to reach AUL QC 1/2 and AUL QC 1/10, drug-free plasma was spiked with them. Together with recently spiked CC standards and QC samples, DQC samples were processed and examined. The DI was assessed using precision and percent mean accuracy, and the concentration of DQCs was computed in relation to the CC.

Software used for green analysis

GAPI (Green Analytical Procedure Index) and AGREE (Analytical Greenness) are software tools used to assess the environmental impact of analytical procedures. GAPI evaluates the entire analytical method, including sample preparation, reagents, instrumentation, and waste disposal, using a color-coded pentagram to visually represent environmental, health, and safety factors. AGREE, based on the 12 principles of green analytical chemistry, provides a numerical score (0–1) and a circular pictogram to quantify the greenness of a method. While GAPI focuses on a comprehensive stepwise evaluation, AGREE offers a more quantitative approach, making both tools essential for selecting and developing sustainable analytical methods. A straight forward piece of software that makes it easier to utilize Complex GAPI to evaluate the greenness of analytical processes is included with the suggested tool. It was created in Python with the Tkinter default 28 library. Using drop-down menus, the user selects parameters related to the pre-analysis procedures as well as the sample preparation and analysis phases. A real-time Complex GAPI pictogram is then created for quick reference. When finished, the pictogram may be saved either as a raster image (.png) or vector graphic (.svg) [13]. AGREE—Analytical GREEnness Metric Approach and Software—utilizes standardized greenness metrics to assess and improve the environmental impact of analytical methods. Users input data on chemicals, energy, and waste, which the software evaluates to generate an overall greenness score, offering visual feedback and recommendations for sustainability improvements [14].