A Novel Ultra‐Performance Liquid Chromatography‐Electrospray Ionisation‐Tandem Mass Spectrometry Method for Quantification of Imeglimin in Human Plasma: Application to a Bioequivalence Study

ABSTRACT

The pharmaceutical market has recently witnessed the advent of a novel tetrahydrotriazene molecule belonging to the new pharmacological class “Glimins”, named Imeglimin (IMG). It has been approved in Japan as a safe and highly effective oral diabetic drug for type II diabetic patients. It enhances the function of β‐cells in the human pancreas and increases insulin sensitivity. Hence, this work was directed to provide a fast, sensitive and highly reliable bioanalytical method for its quantification in human plasma. What makes this work greatly distinctive is first: adopting an ultra‐performance liquid chromatography (UPLC) with protein precipitation as a fast and straightforward sample preparation protocol with the highest extraction recovery. Secondly, utilisation of the stable isotope‐labelled molecule, IMG‐d6, rather than a structurally similar analogue, to avoid the interference of any co‐administered drugs in human plasma. Samples were extracted with acetonitrile and then chromatographically separated using an Acquity UPLC BEH HILIC column of 1.7 µm particle size along with a mobile phase solution composed of 70:30 (v/v) acetonitrile and 10 mM ammonium formate buffer acidified with 0.1% formic acid. Upon adjusting the flow rate to 0.3 mL/min, IMG was eluted at 1.3 min with a total run time of only 2.0 min. Mass quantification was performed in positive electrospray ionisation operated in multiple reaction monitoring mode. IMG was quantified at m/z of 156.05 → 112.91 transition pairs while IMG‐d6, at m/z 162.11 → 119.04. The proposed UPLC‐tandem mass spectrometry method was thoroughly validated as per Food and Drug Administration principles over a linearity range of 10.0–3000.0 ng/mL and successfully applied for IMG quantification in human plasma samples. The scope of the work was extended to encompass real analysis of collected human blood samples, calculation of IMG pharmacokinetics parameters and conductance of a bioequivalence study between the IMG generic product versus brand one.

1. Introduction

One of the globally prevalent diseases is diabetes, especially type II, with an increasing risk with obesity and an inactive lifestyle [1, 2, 3]. This evokes an imperious necessity for potent and well‐tolerated medications, able to limit the progression of disease and any other complications. From this perspective, the pharmaceutical market has witnessed the launch of various promising therapeutics for treating type II diabetes over the last few years. Among these is Imeglimin (IMG, Figure 1a), a novel tetrahydrotriazene molecule which belongs to a new pharmacological class of oral antidiabetic drugs named “Glimins” [4, 5]. IMG expresses significant capability in lowering blood glucose levels via unique and versatile mechanisms of action. It enhances the function of β‐cells in the human pancreas, simultaneously with boosting the sensitivity of peripheral cells in skeletal muscles and the liver towards insulin [5, 6]. IMG has been approved in Japan as a safe and highly effective oral diabetic drug for type II diabetic patients, with/without the need for insulin injections [7]. As with any new pharmaceutical drug entity, it is essential to develop a reliable, straightforward and highly selective bioanalytical method for monitoring the behaviour of the drug in the human body in its very low concentrations.

FIGURE 1.

Chemical structures of (a) Imeglimin and (b) the internal standard, Imeglimin‐d6.

During the clinical trials, the development of new pharmaceuticals and even their registration, as well as the establishment of bioanalytical methods, is of great importance. Such methods facilitate the assessment of drug pharmacokinetics and its metabolites [8, 9, 10]. It is considered a main and irreplaceable part during performing a bioequivalence study upon evaluating the pharmacokinetic portfolio of an emerging generic drug in comparison with an already established brand one [11, 12, 13]. As per worldwide regulations, liquid chromatography, coupled with tandem mass spectrometry (LC‐MS/MS), could be considered the highly sensitive and selective bio‐analytical platform, among other methods [14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26], to quantify pharmaceutical drugs & their metabolites in difficult matrices such as plasma, serum, urine and cerebrospinal fluid [27, 28, 29, 30]. Utilising more tiny particles (<2 µm), shorter column and applying very high pressure, a new era of LC, scientifically termed ultra performance LC (UPLC), was introduced [31, 32]. Such amendments aid in better peak shape and resolution as well as massively reduction in analysis time, thus improving method sensitivity and rationalising analysis time and cost [33].

Nonetheless, choice and optimisation of a proper sample preparation protocol is still the main rate‐limiting step during bioanalytical method development. This could be attributed to the severe complexity and existence of various endogenous components interfering with drug quantification via mass detectors. One of the most commonly used sample preparation approaches is protein precipitation, where plasma proteins undergo denaturation through adding organic solvents such as methanol or acetonitrile [34]. This results in removing proteins from the sample matrix as precipitant, leaving the analyte of interest dissolved. Consequently, protein precipitation is considered the most effective protocol that yields the highest extraction recoveries of the drug in comparison to liquid‐liquid and solid‐phase extraction. Furthermore, protein precipitation provides quick and simple procedures with a small volume of blood sample and a few pieces of equipment [35]. Regarding the utilised internal standard (IS), it is highly recommended to utilise the stable isotope‐labelled analyte rather than a structurally similar analogue in mass spectrometric methods [36, 37]. This case is highly demanded, especially during the analysis of blood samples of diabetic patients, where the selected structurally similar drug may be co‐administered by the diabetic patients for other associated health conditions. Thus, the analysis results of IS would express high variability during the analysis of subject samples and disrupt the final analytical results [38].

Our careful review of the literature revealed only one reported high‐performance LC (HPLC) method for determining IMG in spiked plasma samples [39]; however, no bioequivalence assessment has been published yet. In addition, the reported HPLC method suffers from numerous limitations, such as long run time, the dependence on tedious and time‐consuming liquid extraction for drug extraction & sample clean‐up, besides using a different molecule, which is not structurally related to IMG as an IS. The previous work also lacks the real application to withdrawn blood samples from human subjects, as well as not providing any pharmacokinetic manipulation [39].

With full consideration of the points mentioned, this work provided the first LC‐MS/MS method for IMG in human plasma, based on protein precipitation as a fast and straightforward sample preparation protocol. The work was motivated by making good use of UPLC merits during method development rather than conventional HPLC. For satisfactory chromatographic results, a stable isotope of the drug (IMG‐d6, Figure 1b) was employed as an IS instead of using any other drugs that may be present in the blood of diabetic patients. The proposed UPLC‐MS/MS method was applied to quantify IMG in human plasma in nano‐scale concentrations, as well as monitoring pharmacokinetic profiles of IMG in brand & generic products via conducting a bioequivalence study on 29 healthy Egyptian volunteers.

2. Experimental

2.1. Instruments and Software

Waters Acquity UPLC H‐Class‐Xevo TQD system was used for chromatographic analysis of IMG, where a quaternary solvent manager, column heater compartments and an auto‐sample manager of the Flow‐Through Needle type were connected. The device is equipped with a triple quadrupole mass spectrometer of Waters Quattro Premier XE model (Milford, USA), operated with version 4.1 MassLynx software. Drug separation was performed on an Acquity UPLC BEH HILIC column of 1.7 µm particle size and 2.1 × 100 mm dimensions. Other auxiliary instruments were utilised, a five‐digit analytical balance (Kern, ABT‐NM model Balingen, Germany), Elmasonic ultrasonic (EASY 100 H model; Singen, Germany) and Pro‐analytical cooling centrifuge (CR2000 model, United Kingdom). All preparations were conducted using ultra‐pure water, generated from a Merck Milli‐Q purification system (Direct‐8 model; Darmstadt, Germany). Furthermore, all plasma samples were kept at −80°C using a Binder ultra‐low temperature freezer, UF V 500 model (Bohemia, USA).

2.2. Chemicals and Reagents

The utilised standard drug, IMG with batch number IMG‐P/22004, was bought as a hydrochloride salt from Metrochem Api Private Limited, Telangana, India, where its potency was listed with a certified value of 99.20% and 0.27% for water content. During analysis, IMG‐d6 (Batch number: SL‐AKR‐296‐054) was utilised as IS and was purchased from Simson Pharma Limited, Mumbai, India, labelled with batch number of SL‐AKR‐296‐054 and certified potency of 99.77% and with storage condition at −20°C. The used human plasma, either normal or hemolysed, was obtained from the National Institute of Urology and Nephrology in Cairo, Egypt. On the other hand, ammonium formate, acetonitrile and methanol were acquired as HPLC‐grade from Sigma‐Aldrich (Darmstadt, Germany), while Formic acid was supplied from Fisher Scientific Company (Pennsylvania, USA). The anticoagulant used was heparin sodium in the form of ampoules (5000 IU/mL), manufactured by Chemical Industries Development, Giza, Egypt and were purchased from the local market.

2.3. The Marketed Pharmaceutical Formulations

The utilised test product was Glimcoza 500 mg film‐coated tablets (BN: 243193), manufactured by Atco Pharma for Pharmaceutical Industries, Egypt. Twymeeg 500 mg tablets, manufactured by Sumitomo Dainippon Pharma Co., Ltd, Japan, with batch number 1519C, were used in this study as the reference product. Each tablet was claimed to contain 500 mg IMG.

2.4. Chromatographic Parameters and Mass Quantification

Optimal drug separation was attained upon using an Acquity UPLC BEH HILIC column of 1.7 µm particle size and 2.1 x 100 mm dimensions as a stationary phase with a mobile phase solution composed of 70:30 (v/v) acetonitrile and 10 mM ammonium formate buffer acidified with 0.1% formic acid. The quaternary pump was set at 0.3 mL/min with column and autosampler temperatures of 45°C and ambient temperature, respectively. A run time of 2.0 min was sufficient to separate the drug effectively. The sample was injected at a volume of 2.0 µL and quantified using electrospray ionisation, operated in positive ionisation mode. The dwell time and cone voltage were adjusted at 163 ms and 40 V, respectively, with a collision energy of 25 eV. The parent and daughter ions of IMG were monitored via multiple reaction monitoring (MRM) at m/z 156.05 → 112.91 transition pairs and for IMG‐d6 at m/z 162.11 → 119.04.

2.5. Preparations of Calibrators and Quality Control Samples

Using methanol as a diluent, a stock standard solution of IMG, with a concentration of 200.0 µg/mL, was thoroughly prepared. After that, various volumes were transferred from the prepared IMG stock solution using a micropipette into a set of 10‐mL measuring flasks. The volumes were completed to the final volume with methanol. In this way, working standard solutions with increasing concentrations were obtained as summarised in Table S1. An aliquot of 25 µL was withdrawn from each working solution (0.4, 0.8, 2.0, 8.0, 20.0, 40.0, 48.0, 80.0, 112.0, and 120.0 µg/mL), along with 975 µL blank plasma to get a final volume of 1.0 mL, achieving final concentrations of IMG ranging from 10.0 to 3000.0 ng/mL. It is worth noting that a 3% (w/v) heparin sodium solution was added to the plasma as an anticoagulant, such that each 300 mL blank plasma contains 6.0 mL of heparin solution. Following the same preparation procedures, quality control (QC) samples were prepared except using another standard solution at the concentration levels of 30 ng/mL for low QC (QCL), 700 and 1500 ng/mL for medium QCs (QCM‐1 and QCM‐2) and 2500 ng/mL for high QC (QCH).

2.6. Procedures for Sample Preparation

After allowing samples to be defrosted at ambient temperature, 200 µL of spiked plasma were transferred along with 25 µL of IMG‐d6 methanolic solution (100 ng/mL) into centrifugation tubes. The tubes were vortexed for about 2.0 min, followed by applying the precipitation protocol as the simplest and most straightforward approach for sample clean‐up. This is achieved through the addition of 1.0 mL acetonitrile as the precipitating agent. The tubes were vortexed again for 3.0 min, followed by a 5‐min centrifuge at 5000 rpm and 10°C temperature. A volume of 200 µL of the obtained supernatant was transferred into the UPLC vial with an injection volume of only 2.0 µL.

2.7. Bioanalytical Validation of the Proposed UPLC‐MS/MS

Different parameters were assessed in accordance with the Food and Drug Administration (FDA) guidance for “Bioanalytical method validation and study sample analysis” [36]. Firstly, method specificity and selectivity were evaluated through analysis of six different batches of blank human plasma, either normal or hemolysed ones, to ensure the absence of any chromatographic interference (such as concurrent medications or human plasma components) at the retention times of IMG and tested IS. Method linearity was constructed using standard solutions for increasing concentrations ranging from 10.0 to 3000.0 ng/mL. Calibration curves were plotted using peak area ratio (IMG / IMG‐d6) and the corresponding nominal IMG concentrations. Furthermore, within‐run accuracy and precision of the proposed method were evaluated through the analysis of the predetermined QC concentrations in addition to the lower limit of quantification (LLOQ) concentration. The same concentration levels were utilised for assessing between‐run accuracy and precision, but on two successive days.

The efficiency of the adopted precipitation technique in cleaning up the sample and extracting the drug was appraised by comparing the results of four‐level QC samples (extracted samples) versus those of post‐extraction samples, prepared at the same concentration levels but with different procedures, where blank plasma samples were extracted first, then spiked with IMG and IMG‐d6. On the other hand, the effect of plasma matrix on the drug ionisation and response was studied by comparing the results of QCL & QCH samples prepared as per the adopted protein precipitation protocol versus those of other prepared samples of the same concentrations in only methanol without adding plasma.

The effect of dilution of concentrated samples above the upper limit of quantification (ULOQ) level on results integrity was monitored through preparing five replicates of IMG samples at 6000 ng/mL, followed by their four‐fold dilution with blank samples. In addition, triplicate injections of ULOQ samples followed by the injection of blank plasma were conducted to study the carryover validation parameter.

Regarding the stability of samples throughout the whole bioanalytical preparations and analysis, QCL and QCH samples were analysed in five replicates before and after applying the stress condition. The stability of the IMG stock solution and benchtop stability were studied after storing at −20 and 25°C, while the stability of processed samples was studied by storing either in an autosampler at ambient temperature or in a refrigerator at 2−8°C. Furthermore, samples were stored at temperature values of −70°C (±10°C) and 25°C to monitor short‐term and long‐term stability of IMG in plasma samples, respectively. Finally, freeze and thaw stability studies were conducted after four cycles, where QC samples were stored at a temperature of −70°C for 12 h, followed by their thawing at room temperature.

Finally, and as a fundamental part of bioanalytical method validation, an incurred sample reanalysis (ISR) study was conducted where chosen sample subsets of volunteers were re‐analysed and compared to their initial analysis.

2.8. Bioequivalence Study and Pharmacokinetic Analysis

The applicability of the proposed UPLC‐MS/MS method for quantifying IMG in human plasma was further extended to estimate various pharmacokinetic parameters of IMG and evaluate the bioequivalence extent of Glimcoza 500 mg film‐coated tablet as test product, produced by Atco Pharma for Pharmaceutical Industries, Egypt, versus the reference product (Twymeeg 500 mg tablet, manufactured by Sumitomo Dainippon Pharma Co., Ltd, Japan). Herein, a randomised, open‐label, single‐dose, two‐period crossover bioequivalence experiment was adopted under fasting conditions with a washout period of one week. It was worth noting that good clinical practice postulates were carefully followed with complete adherence to the “Declaration of Helsinki” as a record of ethical principles for medical research involving human subjects [40, 41]. First, a study protocol was prepared and then approved on 6 March 2025, with code: IEC_060325_01, by the independent ethics committee at the Advanced Research Centre (ARC), Cairo, Egypt. Peak concentration (C_max), area under the plasma concentration‐time curve from zero to the last time (AUC_0–t) and area under the plasma concentration‐time curve from zero to infinity (AUC_0–∞) were set the primary pharmacokinetic end points of IMG Study. were, while the time of maximum plasma concentration (T_max) was the secondary one. The study also aimed to monitor the subjects’ safety by documenting any raised adverse events, as well as laboratory and clinical examinations at preliminary screening and follow‐up periods. As stated in the IMG study protocol, subjects would be excluded if suffering from known allergies to IMG and/or any other excipients of the tablet dosage form, asthma or atopic constitution. It was not permitted to involve volunteers having any neurological, cardiovascular, haematological, musculoskeletal, gastrointestinal, hepatic, endocrinological, renal, or pulmonary diseases. The exclusion criteria also involved the prohibition of any volunteer administrated within the previous 14 days of the study, any systematic drugs or previously participated in any research in the previous two months. The final number of volunteers involved in the IMG bioequivalence was 29 healthy subjects. In agreement with the study protocol, the involved volunteers were healthy adult Egyptians with their ages ranging from 18 to 52 years old (mean value of 33.13 years old) and body mass index range of 18.73–29.81 kg/m² (mean value of 25.80 kg/m²). Table S2 summarises all demographic data of the volunteers who participated in the study.

After overnight fasting (10 h before dose administration) and as per the randomised plan, each assigned volunteer had his morning meal and then took a single dose of IMG 500.0 mg, orally, of either test or reference product with a 240‐mL water cup. From each volunteer, a total of 22 blood samples were collected from each volunteer during each period, where a volume of 5 mL blood was withdrawn through a cannula. Immediately, withdrawn blood samples were transferred into tubes pretreated with heparin at the following time intervals: 0 (pre‐dose), 0.25, 0.5, 0.75, 1.0, 1.25, 1.5, 1.75, 2.0, 2.5, 3.0, 3.5, 4.0, 5.0, 6.0, 8.0, 10.0, 12.0, 24.0, 36.0, 48.0 and 72.0 h. Samples were centrifuged for 5.0 min at 3500 rpm and 4°C, followed by their storage at −70°C till time of analysis. After analysis and collecting the results, various pharmacokinetic parameters of IMG were manipulated via SAS analytics Pro 9.4 software and Phoenix WinNonlin (Version 8.3.4, Certara L.P).

3. Results and Discussion

With the proven efficiency of IMG in reducing blood glucose levels in diabetic patients of type II, the need for a reliable and rapid bioanalytical method has been evoked to quantify IMG in their real plasma concentrations (around C_max). From this point, we dedicated our efforts to developing the first LC‐MS/MS method for IMG bioanalysis based on a protein precipitating approach for sample clean‐up rather than the tedious liquid‐liquid extraction or solid phase ones as reported by some previous works [6, 39, 42]. The work was also motivated by using a UPLC chromatographic system rather than a conventional HPLC one to gather its marvellous merits in being faster, more sensitive and greener as it would be illustrated in the lines below. Moreover, the applicability of the proposed method did not limit only spiked human plasma but also extended to encompass real analysis of collected human blood samples, calculation of IMG pharmacokinetics parameters and conductance of bioequivalence study between IMG generic product versus brand one.

3.1. Optimising Sample Preparation and Development of Chromatographic Method

The polar character of IMG and the existence of multiple nitrogenous groups paved the way towards adopting a protein precipitation approach for drug extraction and sample clean‐up in favour of other multi‐step & laborious ones. However, protein precipitation demands a good choice of organic solvent that can attain good drug extraction, leaving behind other cumbersome phospholipids in plasma. Since literature lacks any bioanalytical method of IMG based on protein precipitation, it was challenging to adopt such a sample preparation technique along with achieving sensitive quantification of IMG in concentrations below its C_max. Preliminary trials utilised acetonitrile as a precipitating agent, where satisfactory extraction recovery and sample clean‐up were attained.

Our next objective was to optimise chromatographic conditions to well resolve IMG and the IS from any endogenous plasma matrix impact, in parallel with shortening total run time and saving time & resources. Regarding the utilised IS, the FDA and World Health Organisation (WHO) Expert Committee recommend the utilisation of the stable isotope‐labelled analyte rather than a structurally similar analogue in mass spectrometric methods [36, 37]. Consequently, IMG‐d6 was chosen as an IS, mimicking the physiochemical properties, extraction behaviour, and mass detection parameters of the main analyte except for molecular weight. Following numerous trials for settling the chromatographic conditions, the Acquity UPLC BEH HILIC column offered better separation with enhanced peak shape than the traditional BEH column. This may be attributed to the matching of column HILIC character with highly polar compounds, rapid equilibrium and enhanced sensitivity on MS detection. Efficient separation of IMG and the IS was attained upon using a mobile phase solution composed of acetonitrile and 10 mM ammonium formate buffer acidified with 0.1% formic acid in a ratio of 70:30 (v:v) and pumped isostatically at 0.3 mL/min. Consequently, IMG and IMG‐d6 were eluted at 1.3 min with a total run time of only 2.0 min, Figure 2. The mass detection was run in positive electrospray ionisation mode, where MRM mode was assigned to the drug, and IS were monitored via MRM mode at m/z 156.05 → 112.91 transition pairs for IMG and at m/z 162.11 → 119.04 for IMG‐d6 as demonstrated in Figure 3.

FIGURE 2.

Representative chromatograms of (a) blank human plasma, (b) spiked plasma at the lower limit of quantification (LLOQ), and (c) plasma sample from a volunteer at 3.5 h after administration of one tablet containing 500.0 mg Imeglimin.

FIGURE 3.

The positive ion ESI‐MS/MS spectra for the parent (a, c) and daughter (b, d) ions of Imeglimin and the internal standard, Imeglimin‐d6.

3.2. Bioanalytical Method Validation

Guidance for industry M10 bioanalytical method validation and study sample analysis was followed for complete validation of the proposed UPLC‐MS/MS method for IMG quantification in human plasma [36].

3.2.1. Specificity and Selectivity

Upon analysis, six batches of normal blank human plasma and a hemolysed one showed no significant co‐interference from plasma endogenous components at the retention times of IMG and IMG‐d6. This is reflected by exemplary chromatograms of drug/IS‐free (blank), drug‐free (zero) human plasma and spiked samples with IS and drug at the LLOQ level, Figure 2. No plasma batches revealed interfering reactions greater than 5% of IS response or 20% of LLOQ, which meet the FDA acceptance criteria.

3.2.2. Linearity Range and Calibration Curve

Calibration curves were prepared and validated for linearity, along with injecting blank and zero samples for each calibration running batch. Acceptable recoveries (±15%) were obtained for all calibration points with the exception of LLOQ (±20%) as stated by FDA guidance [36]. The results revealed an LLOQ value of 10.0 ng/mL, with a linearity ranging from 10.0 to 3000.0 ng/mL.

3.2.3. Accuracy and Precision

The suggested LC‐MS/MS method was tested for accuracy and precision using spiked human plasma with ELO at LLOQ and the four settling QC samples as prescribed in the experimental section. Table 1 shows recovery% ranges from 90% to 114% for within‐run and 96% to 106% for between‐run, confirming method accuracy, Table 1. In addition, the precision of the proposed method was assured, where the coefficient of variation (CV%) was within the acceptance limit (± 15%) as stated by FDA bioanalytical guidance, Table 1. The results of within and between run precisions were in ranges of 5%–13% and 5%–14%, respectively [36].

TABLE 1.

Results of within and between runs recovery% and CV% for assessing accuracy and precision of the proposed method for Imeglimin determination.

Concentration (ng/mL)		Within run			Between run
		Recovery%	CV%		Recovery%	CV%
LLOQ	10	114%	5%		106%	5%
QCL	30	94%	10%		96%	10%
QCM‐1	700	111%	9%		104%	7%
QCM‐2	1500	96%	9%		100%	12%
QCH	2500	90%	13%		98%	14%

3.2.4. Extraction Recovery

To evaluate the extraction efficiency of the adopted protein precipitation approach in analysing the drug, spiked samples were prepared at the four settled QC levels. The obtained peak area of the drug and IS was compared to the obtained results for a sample prepared by adding the drug and IS standards directly to post‐extracted blank samples. Sufficient recovery values of extraction were obtained for IMG drug (83.91%, 92.03%, 88.59% and 72.77%) and IMG‐d6 (99.29%, 90.88%, 87.98% and 88.43%) at QCL, QCM‐1, QCM‐2 and QCH samples.

3.2.5. Matrix Effect

To assess the influence of plasma matrix on the response of both drug and IS, two matrix factors (MF) were calculated at QCL and QCH levels, utilising six distinct batches of normal plasma alongside one hemolysed sample. MF could be defined as the ratio of the peak area of the drug or IS spiked with human plasma to that prepared without adding the plasma matrix at an equivalent concentration. After that, IS‐normalised MF for each plasma batch was obtained by dividing the MF of the main analyte by that of IS. The obtained CV% values were 3% and 1% at QCL and QCH levels, respectively, which matched the acceptability requirements of FDA guidelines (not exceeding 15%).

3.2.6. Dilution Integrity

Dilution integrity was thoroughly examined to see how dilution with blank plasma, if needed, affected the accuracy and precision of the proposed method. Upon performing dilution of a sample with a concentration above ULOQ by 4‐fold with blank samples of human plasma, the accuracy was 95.16% with a CV% of 0.25%, demonstrating that sample dilution had no effect on study sample accuracy and precision.

3.2.7. Carryover Effect

The add‐on effect of the remaining drug, previously injected in the past runs, was carefully examined, where the results of blank samples after several injections of ULOQ injection did not exceed 20% of the IMG drug at the LLOQ concentration level or 5% of the IS reading. Thus, such results shed light on the absence of carryover effect on the performance of the proposed UPLC‐MS/MS method.

3.2.8. Stability Studies

To determine how storage conditions might affect the concentration of the analyte, IMG stability was examined at several phases of preparation and analysis. Table 2 shows the results where stock solutions showed no changes upon storage for 64 days at −20°C and no changes in IMG working solutions upon keeping on the bench for up to 23 h. Moreover, processed samples showed high stability for 47 h in the autosampler compartment (25°C) and for 46 h upon storage in a refrigerator (2–8°C). Long‐term and short‐term stability of IMG plasma samples were 64 days and 27 h, respectively. Drug instability was negligible in spiked plasma samples after the fourth freeze‐thaw cycle.

TABLE 2.

Stability results of Imeglimin at different conditions.

Examined condition	QCL (30 ng/mL)			QCH (2500 ng/mL)
	Accuracy% a	CV%		Accuracy% a	CV%
Stock Solution Stability (at −20°C for 64 days)	96.42%	3.23%		100.09%	2.49%
Benchtop solution stability (at 25°C for 23 h)	92.26%	7.56%		101.99%	6.39%
Autosampler stability (at 25°C for 47 h)	99.97%	3.49%		104.09%	2.05%
Processed sample stability (at 2–8°C for 46 h)	99.03%	2.90%		101.11%	2.98%
Short‐term stability (at 25°C for 27 h)	94.92%	6.07%		100.06%	3.33%
Long‐term stability (at −70°C for 64 days)	86.18%	2.85%		86.71%	2.74%
Freeze and Thaw stability at −70°C (4 cycles)	97.44%	0.97%		98.68%	1.90%

^a Analysis of five replicates (n = 5).

3.2.9. Incurred Sample Reanalysis

ISR is useful to analyse time‐dependent variations, such as back‐conversion to other forms or protein binding that may change the main analyte concentration in investigated samples. The total number of samples collected during the bioequivalence study was 1294, where 120 samples were selected for the ISR study. The number of accepted samples was 117, achieving an acceptance rate of 97.50%. Thus, the proposed UPLC‐MS/MS was proven to be reliable for IMG analysis in human plasma.

3.3. Pharmacokinetic and Bioequivalence Study

A clinical bioequivalence study was performed on twenty‐nine healthy Egyptian volunteers under fasting conditions to predict the pharmacokinetic characteristics of IMG, as well as to assess the bioequivalence extent between the brand and generic product. After conducting the study as described in the experimental section, the gathered human blood samples were effectively analysed using the suggested UPLC‐MS/MS method. This is followed by calculating different pharmacokinetic parameters for both the test and reference medication products. Full data for the bioequivalence study, including study design, study population, demographic data, ethical consideration, eligibility criteria, inclusion/exclusion criteria and safety evaluation, were described in a previous publication by our team [43]. The calculated pharmacokinetic parameters were collectively summarised in Table 3. The estimated C_max was manipulated and equalled to 101.75%, with about 90% confidence intervals ranging from 94.42% to 109.64%. The values for AUC_0‐t and AUC_0‐∞ were also calculated and found to be 100.66% and 100.13%, respectively, with corresponding 90% confidence intervals of 94.28%–107.46% and 93.77%–106.92%. Moreover, Figure 4 demonstrates comparable outcomes profiles between Glimcoza 500 mg film‐coated tablet and Twymeeg 500 mg tablets. In summary, the compared products were determined to be bioequivalent and well‐tolerated by the research participants, with no significant adverse events observed.

TABLE 3.

Pharmacokinetic parameters of Imeglimin following administration of one tablet of Glimcoza 500 mg film‐coated tablet (test product) and Twymeeg film‐coated tablet (reference product) under fasting conditions.

Parameters	Test product	Reference product
C max (ng/mL)
Mean ± SD	783.53 ± 240.99	751.52 ± 170.84
Range	258.33 − 1244.51	414.38 − 1118.00
T max (h)
Median	3.50	3.50
Range	0.50 − 5.00	1.00 − 6.00
AUC0‐t (ng.h/mL)
Mean ± SD	6419.20 ± 2043.07	6270.69 ± 1682.86
Range	2264.78 − 10,267.71	2936.68 − 10,808.07
AUC0‐∞ (ng.h/mL)
Mean ± SD	6554.91 ± 2059.57	6434.08 ± 1670.28
Range	2370.22 − 10,424.70	3124.70 − 10,945.50
Ke (h−1)
Mean ± SD	0.17 ± 0.05	0.16 ± 0.05
Range	0.08 − 0.34	0.09 − 0.36
T 1/2 (h)
Mean ± SD	4.51 ± 1.29	4.61 ± 1.22
Range	2.06 − 8.17	1.92 − 7.42

FIGURE 4.

Mean plasma concentration along with their ± SD after a single oral dose administration of 500.0 mg Imeglimin test and reference products to 29 healthy volunteers.

4. Conclusion

This work was regarded as a fruitful attempt to provide a novel, simple, productive and reliable UPLC‐MS/MS method for IMG bioanalysis in real human plasma samples. This is owing to adopting a protein precipitating approach for sample clean‐up and IMG extraction rather than the tedious, time and money‐costing multi‐step procedures as reported by some previous work. The study was prompted by the utilisation of a UPLC chromatographic system for its superior speed, sensitivity, and environmental benefits. Finally, the applicability of the proposed method showed success in drug analysis in human plasma with the conduct of a bioequivalence study between the IMG generic product and the brand one.

Author Contributions

Mina Wadie: conceptualization, methodology, writing – original draft, and visualization, Kamal A. Badr: conceptualization, data curation, resources, and writing – review and editing, Ahmed A. Hosny: conceptualization, methodology, resources, and writing – review and editing, Liliya Logoyda: conceptualization, methodology, investigation, validation, visualization, and writing – original draft, Omnia M. El Sebai: methodology, validation, and supervision, Mamdouh R. Rezk: investigation, project administration, supervision, and writing – review and editing.

Ethics Statement

Human subjects participated in this research as a study protocol was prepared and then approved on 6 March 2025, with code: IEC_060325_01, by the independent ethics committee at the Advanced Research Centre (ARC), Cairo, Egypt. All study procedures adhered to the ethical principles of the institutional ethics committee and the International Conference on Harmonisation's Good Clinical Practice (ICH‐GCP) guidelines. Additionally, informed consent is available.

Conflicts of Interest

The authors declare no conflicts of interest.

Clinical Trial Registration

Full data for the bioequivalence study, including study design, study population, demographic data, ethical consideration, eligibility criteria, inclusion/exclusion criteria and safety evaluation were registered at ClinicalTrials.gov under the title of “Bioequivalence Assessment Between Two Imeglimin Hydrochloride Film Coated Tablet Formulations (BEGliTabATCO)” with the following ID: NCT07127094.

Supporting information

Supporting File: ansa70079‐sup‐0001‐SuppMat.docx.

Data Availability Statement

All data generated or analysed during this study are included in this published article.