Abstract
Sotagliflozin is a dual SGLT-1 and SGLT-2 inhibitor approved by the FDA in 2023 which has emerged as a novel therapeutic agent for the management of type 2 diabetes mellitus. In this study robust and sensitive LC-MS/MS method was developed and validated for quantification of sotagliflozin in rabbit plasma as rabbit is commonly used non-rodent model for preclinical research. Sample preparation involved protein precipitation for efficient analyte extraction from rabbit plasma. Chromatographic separation was performed utilizing on BDS Hypersil C18 column (100 mm x 4.6 mm, 5 μm) using mobile phase composed of methanol (85%) and 5 mM ammonium acetate in milli-Q water (15%) and rolipram as internal standard. The method employed flow rate of 0.7 mL/min with a total runtime of 4 min and an injection volume of 10 µL. Method validation was carried out in accordance with ICH M10 guidelines, covering precision, accuracy, selectivity, recovery, and stability at different storage conditions. The method was found to be linear over the concentration range of 10-1280 ng/mL with sensitivity of 10.20 ng/mL (LLOQ) in rabbit plasma. Recovery of analyte from the rabbit plasma was found to be > 92% with stability > 99% at different storage conditions (viz., room temperature, autosampler, freeze-thaw and frozen). Overall, the developed LC-MS/MS method offers a simple, precise and reproducible approach for the quantification of sotagliflozin in rabbit plasma and is well-suited for application in pharmacokinetic and other preclinical studies.
Keywords: Sotagliflozin, Rolipram, LC-MS/MS, Protein precipitation, Rabbit plasma
Introduction
Globally millions of individuals are affected by diabetes mellitus. It is prevalent and complex chronic condition characterized by high blood glucose levels. It is anticipated that the number of adults living with diabetes will increase to 152 million by the year 2045, representing a 68% rise [1]. Type-2 diabetes mellitus is a common condition and impacts population of both developed and developing countries. Traditionally oral hypoglycemic agents whether used alone or in combination are typically favored for management and treatment of Type-2 diabetes mellitus [2]. Along with this approach, insulin is generally introduced into treatment regimen when sufficient glycemic control has not been attained [3]. In recent years therapeutic strategies targeted at the renal SGLT-2 have advanced as promising treatment options for Type-2 diabetes mellitus. SGLT-2 is a glucose transporter with low affinity and high capacity located in the S1 and S2 segment of kidneys proximal tubules. Here it plays an important role in the co-transport of sodium and glucose [4]. SGLT-2 receptors account for 60–90% of glucose reabsorption during renal reuptake. Pre-clinical and clinical data suggest SGLT-2 inhibitors are promising glucose lowering agents but still challenge is exist to find the metabolically stable compound which can account for once a day treatment [5]. The C-aryl glycosidase containing compound have been recognized as the most stable class of SGLT-2 inhibitors which subsequently given rise to discovery of remogliflozin, dapagliflozin, canagliflozin, bexagliflozin and sotagliflozin [6,7]. Among all these, in 2023 FDA has approved sotagliflozin as an oral SGLT-2 inhibitor for managing T2DM. Chemically, sotagliflozin is (2S,3R,4R,5S,6R)-2-[4-chloro-3-[(4-ethoxyphenyl)methyl]phenyl]-6-methylsulfanyloxane-3,4,5-triol (Fig. 1(a)) which acts by minimizing glucose reabsorption in the kidney which ultimately increasing glucose excretion from the body [8].
Fig. 1.
Molecular structure of a Sotagliflozin and b Rolipram
Quantitative assessment of drugs and their metabolites across various biological matrices is essential during pre-clinical and clinical development phases. Rabbit plasma was selected as the biological matrix because rabbit is commonly used non-rodent species in regulatory toxicology and safety pharmacology studies. International regulatory guidelines recommend evaluation of drugs in both rodent and non-rodent species during preclinical development. The rabbit model offers practical advantages such as adequate blood volume for serial sampling, established pharmacological characterization and suitability for repeated-dose toxicity studies. Although rat plasma is frequently used in early pharmacokinetic screening and human plasma is relevant for clinical bioanalysis, validated quantification in rabbit plasma is essential for toxicokinetic and safety studies prior to clinical studies.
A review of literature has identified multiple HPLC and LC-MS/MS method for quantification of sotagliflozin in bulk and pharmaceutical formulations [9–12]. Although analytical methods have been reported for the estimation of sotagliflozin, these methods are limited by longer run times, complex sample preparation procedures, low sensitivity or lack of validation in non-rodent biological matrices. Furthermore, no fully validated LC-MS/MS method has been specifically developed for quantification of sotagliflozin in rabbit plasma. Unlike selective SGLT-2 inhibitors, sotagliflozin exhibits dual inhibition of SGLT-1 and SGLT-2 transporters, which may result in distinct pharmacokinetic characteristics. The present study addresses these limitations by developing a rapid, sensitive, and reproducible LC-MS/MS method with a short run time of 4 min and simple protein precipitation extraction procedure, thereby enhancing analytical throughput and cost-effectiveness using rolipram as internal standard. The structure of rolipram is illustrated in Fig. 1 (b). This validation was conducted in accordance with the recently revised ICH guidelines (ICH M10) [13].
Materials and methods
Chemicals, reagents and instrumentation
Sotagliflozin (Batch Number: TCI0159) and rolipram (Batch Number: CS-RP-234) with purity of 98% and 98.82% were obtained from Transfo Chem (Maharashtra, India) and Clearsynth (Maharashtra, India), respectively. Methanol, acetonitrile, and ammonium acetate were brought from Merck and deionized ultrapure water was supplied through Milli-Q system. The rabbit plasma samples were obtained from in-house animal laboratory of Jai Research Foundation (Gujarat, India). Shimadzu 8060 NX Triple Quadrupole Mass Spectrometer coupled HPLC system operated in positive ionization mode was employed for analysis. Thermo Scientific BDS Hypersil C18 Column [100 × 4.6 mm, 5 μm particle size] was employed and temperature maintained at 40 °C. Methanol (85%) and 5 mM ammonium acetate in milli-Q water (15%) was used as mobile phase at a flowrate of 0.7 mL/min and 10 µL injection volume. The total run time was 4 min for each injection. Quantification was performed by using MRM mode with transitions of m/z 442.00→359.00 and 442.00→259.10 for sotagliflozin and m/z 276.00→131.10 for rolipram. The ion source parameters; Collision energy was maintained at -13 eV (m/z 442.00→359.00), -20 eV (442.00→259.10) for sotagliflozin and − 28 eV (276.00→131.10) for rolipram. Nebulizing gas flow, heating gas flow and drying gas flow at 2.80 L/minutes, 10 L/minutes and 10 L/minutes, respectively. Interface temperature, DL temperature and heating block temperature at 300 °C, 300 °C and 400 °C, respectively. The LabSolutions software (Shimadzu Corporation) was utilized for data acquisition and instrument control (version number 5.99 SP2).
Preparation of calibration standards and quality control samples
Stock solutions of sotagliflozin and rolipram were prepared separately in acetonitrile. Calibration standards of sotagliflozin in plasma were established in diluent (50% acetonitrile) and spiked into blank rabbit plasma in range of 10 to 1280 ng/mL. Similarly, four QCs samples at 10.20, 30.08, 567.54 & 949.39 ng/mL were prepared in blank rabbit plasma and considered as LLOQC, LQC, MQC and HQC. Stock solution of rolipram (IS) was diluted in diluent (50% acetonitrile) to prepare working solution.
Sample extraction procedure
Simple protein precipitation technique was employed for extraction. Concisely 5 µL standard solution was added in 45 µL of plasma followed by addition of internal standard working solution and vortexed for 2 min. This mixture was precipitated by 1 mL of precipitating solution (90% methanol in milli-Q water) and sample vortexed at 2000 rpm for 10 min. After vertexing, samples centrifuged at 14,000 rpm at 4 °C for 10 min to get supernatant and 10 µL supernatant injected on LC-MS/MS for analysis.
Method validation
The current methodology was validated as per ICH guidelines (ICH M10) by assessing system suitability, specificity and selectivity, determining linearity, evaluating precision and accuracy, stability, matrix effect, autosampler carryover and recovery [13].
System suitability
System suitability tests play an essential role to ensure reliable and consistent performance of the bioanalytical method. Extracted plasma samples spiked with internal standards were prepared and injected six times from the same vial. Signal to noise ratio and % RSD of the peak area ratio were calculated.
Selectivity
Six separate drug free rabbit plasma samples were selected to examine interference of drug and internal standard at respective retention times. Selectivity of method was determined based on the chromatograms of both blank plasma and spiked plasma with sotagliflozin and internal standard.
Specificity
Blank plasma fortified with analyte at ULOQ and IS individually to assess specificity of the method. The interference at analyte retention time was monitored in the presence of sample of internal standard and vice versa.
Autosampler carryover test
Autosampler carryover was assessed by processing and analyzing standard blank, LLOQ, and ULOQ standard in sequence followed by reinjection of the initial blank sample to verify carryover. The second injection in series (LLOQ) was injected to find if there was any interference at retention time of sotagliflozin in standard blank.
Linearity
Total of eight calibration standards, including ULOQ and LLOQ were utilised to prepare calibration standard curves spanning from 10 to 1280 ng/mL. Peak area ratio of the sotagliflozin relative to internal standard against sotagliflozin nominal concentrations was plotted to generate regression equation with weighting factor (1/x2).
LLOQ determination
Method sensitivity was evaluated by LLOQ determination. LLOQ samples were processed by spiking sotagliflozin into six individual lots of rabbit plasma at LLOQ level with a working internal standard solution. These samples were analyzed with precision & accuracy samples and under a calibration curve along with batch quality control (QC) samples. Three batches of LLOQ determination were evaluated.
Precision and accuracy
Six replicates of QCs (LLOQC, LQC, MQC, and HQC) representing the entire calibration curve range with concentrations at LLOQC (slightly higher than LLOQ concentration), LQC (approximately 3 times the LLOQ concentration), MQC (approximately 40–60% of ULOQ) and HQC (approximately 75–85% of ULOQ) were used for determination of precision and accuracy. To assess the ruggedness and potential transferability of the developed LC-MS/MS method, additional evaluations were carried out under deliberately varied conditions. The method was tested using two different analysts and columns on different days.
Reinjection reproducibility
To verify the validity of the processed samples and to ensure proper sample storage before injection, reinjection reproducibility was evaluated. Accepted precision and accuracy batch samples were stored for a period of 24 h in an autosampler at 2 to 8 °C and the entire batch was re-injected for estimation of reinjection reproducibility.
Matrix effect
The matrix effect for sotagliflozin was investigated at LQC and HQC. Six separate lots of blank rabbit plasma were extracted and the extract was spiked at LQC and HQC, separately. Analyte peak area in the samples was compared with neat standard solutions at the same theoretical concentrations (LQC and HQC). IS normalized factor and matrix factor were calculated.
Recovery
The recovery of sotagliflozin was assessed at three extracted levels (LQC, MQC, and HQC). The results were compared with the mean analyte response from post-extracted sample along with the internal standard at its working concentrations.
Stability
Stability of sotagliflozin in rabbit plasma was established under following storage conditions: (1) Bench top stability (samples stored to room temperature) (2) Autosampler stability (samples stored in autosampler), (3) Freeze-thaw stability (samples after five cycles of freeze and thaw) (4) Long term stability (samples stored for 20 days at -70 °C).
Results and discussion
Design and optimization of method
A reliable and simple liquid LC/MS-MS method developed for identification of sotagliflozin in rabbit plasma. Although stable isotope-labeled analogs are typically favored for their enhanced capability to correct matrix effects and ionization variability, a deuterated internal standard for sotagliflozin was not easily accessible and is linked to considerably higher costs. Therefore, rolipram was selected as an alternative internal standard based on its consistent extraction recovery, similar chromatographic retention behavior and stable ionization response under the optimized conditions. Previous studies have pointed out specific challenges arising from the structural and physicochemical properties of gliflozins [14–17]. Sotagliflozin possesses intermediate polarity, allowing it to be effectively retained on a column with a moderately polar stationary phase. During the optimization of the method, various stationary phases were tested alongside a mobile phase composed of varying amounts of acetonitrile, methanol, and buffer (ammonium acetate and ammonium formate). Initially, a mobile phase that included equal volumes of aqueous buffer and organic solvent was employed to attain optimal chromatographic separation. However, significant recovery for sotagliflozin was not realized. Further optimization was accomplished by adjusting the buffer concentrations to enhance response and achieve an acceptable peak shape. It has been noticed that buffers (5–8 mM) improve peak shape and after rigorous optimization of various parameters, an optimal compromise among the retention time, resolution, analyte response, and peak symmetry was attained using mobile phase compromising methanol (85%) and 5 mM ammonium acetate in milli-Q water (15%) BDS Hypersil C18 column. The retention time recorded for sotagliflozin was 2.23 within 4 min run time.
Extraction procedures and pre-conditioning techniques plays crucial role in bioanalysis by eliminating unwanted interferences [18]. Sample clean-up is essential before injection of samples on hyphenated technique. Over the year, various methods have been developed which are versatile, specific and selective. Selection of the sample preparation technique relay on the nature of matrix to be cleaned and physicochemical properties of the drug. Due to simplicity and ease of use, liquid-liquid extraction, solid phase extraction and protein precipitation are still popular for the extraction of drug moiety from biological matrices. The selection of the solvent is critical and aqueous methanol as a precipitating agent was effective in getting good recoveries for sotagliflozin and rolipram.
Method validation
System suitability
System suitability tests were performed each day before analytical run at ULOQ and LLOQ level. Signal to rise (S/N) at LLOQ level for sotagliflozin in plasma was found to greater than 5. The % RSD of area ratio (analyte area/IS area) at ULOQ level for sotagliflozin was found less than 5.
Selectivity
Among six different lots of rabbit plasma, no interference noticed at the retention time of analytes. Spiked LLOQ concentration was also found to be within ± 20% of the nominal concentration.
Specificity
Specificity data indicated that the method developed was highly specific for sotagliflozin showing no endogenous interference at the retention time of both, sotagliflozin and rolipram.
Autosampler carryover test
The autosampler carryover experiment showed absence of carryover of sotagliflozin in standard blank and reinjected standard blank after injection of LLOQ and ULOQ (Table 1).
Table 1.
Autosampler carryover test
| Standard blank | Analyte peak area | %Carryover (Analyte) | IS peak area | %Carryover (IS) |
|---|---|---|---|---|
| Standard Blank | 0 | 0 | 0 | 0 |
| LLOQ | 13,758 | Nil | 197,689 | Nil |
| ULOQ | 1,674,380 | Nil | 180,971 | Nil |
| Standard Blank (Reinjection) | 0 | 0 | 0 | 0 |
Linearity
The calibration curve for sotagliflozin was linear and spanning in the range of 10 to 1280 ng/mL which is demonstrated by equation Y = 0.0656981 x – 0.00220110 with a correlation coefficient greater than 0.99. All calibration points satisfied the acceptance criteria. Linearity data are depicted in Table 2, chromatograms are presented Figs. 2, 3 and 4 and calibration curve is presented in Fig. 5. 1/x² weighting factor was applied to the regression model to minimize relative error across the concentration range particularly at lower concentration levels. Also based on residuals plot, the residuals are scattered approximately randomly around zero and there is no trend in the spread of residuals with concentration.
Table 2.
Representative data of linearity for sotagliflozin
| Nominal concentrations (ng/mL) |
Recovered concentrations (ng/mL) |
Accuracy (%) | Regression equation |
|---|---|---|---|
| 10 | 9.884 | 96.8 |
Y = 0.0.0656981 x – 0.00220110 (r2 = 0.998) |
| 20 | 19.960 | 99.8 | |
| 40 | 40.040 | 100.1 | |
| 80 | 79.321 | 99.15 | |
| 160 | 160.833 | 100.5 | |
| 320 | 326.630 | 102.0 | |
| 640 | 624.798 | 97.6 | |
| 1280 | 1293.128 | 101.0 |
Fig. 2.
Representative chromatogram of blank rabbit plasma
Fig. 3.
Representative chromatogram of rabbit plasma with IS only (Standard Zero)
Fig. 4.
Representative chromatogram of sotagliflozin (with IS)
Fig. 5.
Calibration curve of sotagliflozin
LLOQ determination
LLOQ of sotaglifloin in rabbit plasma was established at 10.20 ng/mL. S/N ratio surpassed 5 with the recovered concentrations of the samples within ± 20% of the nominal values. Furthermore, %CV for the samples was below 20% (Fig. 6).
Fig. 6.
Representative chromatogram of LLOQ Sample
Precision and accuracy
Precision and accuracy results for sotagliflozin detailed in Table 3 which includes data for each day. The percentage coefficient of variation (%CV) was found below 2.78 which is less than 15% of guideline limit. Accuracy levels ranged from 98.99% to 106.46% remaining within ± 15% of nominal concentration across QC levels. These results confirm that the method is both robust and reliable for routine bioanalysis and potentially transferable across laboratories with minimal variation in performance (Figs. 7, 8 and 9).
Table 3.
The detection of precision and accuracy of sotagliflozin
| Interval | Recovery levels | Fortified concentrations (ng/mL) | Recovered concentrations (ng/mL) | Accuracy (%) | Precision (%CV) |
|---|---|---|---|---|---|
| Day 1 | LLOQ | 10.20 | 10.86 | 106.46 | 2.71 |
| LQC | 30.08 | 30.03 | 99.82 | 1.36 | |
| MQC | 567.540 | 567.60 | 100.01 | 1.78 | |
| HQC | 949.390 | 939.66 | 98.99 | 1.82 | |
| Day 2 | LLOQ | 10.20 | 10.42 | 102.15 | 2.78 |
| LQC | 30.08 | 30.01 | 99.76 | 2.69 | |
| MQC | 567.540 | 562.77 | 99.16 | 1.32 | |
| HQC | 949.390 | 950.99 | 100.52 | 1.43 | |
| Day 3 | LLOQ | 10.20 | 10.37 | 101.65 | 1.12 |
| LQC | 30.08 | 29.90 | 99.41 | 2.05 | |
| MQC | 567.540 | 561.97 | 99.02 | 1.10 | |
| HQC | 949.390 | 954.33 | 100.52 | 1.03 |
Fig. 7.
Representative chromatogram of LQC sample
Fig. 8.
Representative chromatogram of MQC sample
Fig. 9.
Representative chromatogram of HQC sample
Reinjection reproducibility
Reinjection reproducibility was assessed over a 24 h period, demonstrating accuracy range of 99.41% to 101.65%, with precision values remained 1.19.
Matrix effect
The results obtained indicate that there were no significant matrix effects. The variation in the IS-normalized matrix factor for the analytes was calculated and are in between 0.94 and 1.12 indicating that the matrix components effect on the response of the analyte was insignificant.
Recovery
A simple protein precipitation method produced highly precise (%CV ≤ 2.1) and quantitative recovery of sotagliflozin (92.70-98.54%) across QC levels depicted (Table 4). The mean recovery for the internal standard was around 98%.
Table 4.
The data of % recovery of sotagliflozin from rabbit plasma
| Recovery level | Concentrations (ng/mL) | Recovery (%) |
|---|---|---|
| LQC | 30.08 | 98.54 |
| MQC | 567.54 | 92.70 |
| HQC | 949.39 | 95.32 |
Stability
Drug stability was performed at various experimental storage condition. Percentage recoveries for the stability samples ranged between 99.78% and 105.32% at defined stability conditions in plasma viz., bench top (5 h), autosampler (24 h), freeze-thaw (five cycle), and long-term (-70 °C for 20 days). Detailed stability results for sotagliflozin in plasma is presented in Table 5.
Table 5.
The data of stability of sotagliflozin at different storage conditions
| Condition | Recovery level | Fortified concentrations (ng/mL) | Recovered concentrations (ng/mL) | Accuracy (%) | Precision (%CV) |
|---|---|---|---|---|---|
|
Room Temperature (5 h) |
LQC | 30.08 | 31.10 | 103.39 | 3.14 |
| HQC | 949.390 | 999.90 | 105.32 | 0.64 | |
|
Autosampler (24 h) |
LQC | 30.08 | 30.43 | 101.91 | 3.11 |
| HQC | 949.390 | 969.34 | 102.72 | 2.62 | |
|
Freeze-thaw (5 cycle) |
LQC | 30.08 | 31.12 | 103.49 | 4.48 |
| HQC | 949.390 | 987.36 | 104.00 | 2.62 | |
|
Long-term stability at -70 °C (for 20 days) |
LQC | 30.08 | 30.01 | 99.78 | 1.49 |
| HQC | 949.390 | 947.94 | 99.87 | 1.66 |
Potential application in non-clinical studies
The developed LC–MS/MS method demonstrates high sensitivity, accuracy and reproducibility making it suitable for application in actual pharmacokinetic and toxicokinetic studies of sotagliflozin. The lower limit of quantification (10.20 ng/mL) allows reliable monitoring of plasma concentrations across different dosing regimens in rabbit models. Since rabbits are widely used as a non-rodent species in regulatory toxicology and safety pharmacology studies, this method can support dose-proportionality assessment, bioavailability studies, drug–drug interaction evaluation, and preclinical safety investigations. Furthermore, the short runtime (4 min) enables high-throughput analysis, which is advantageous in large-scale non-clinical studies.
Conclusion
A sensitive, precise and reproducible LC-MS/MS method with simple protein precipitation extraction was developed and validated for the estimation of sotagliflozin in rabbit plasma. Significantly acceptable accuracy and precision were obtained over the linearity range. The method emphasizes simplicity, rapidity and provides information about control solution’s stabilities. The proposed method has advantages with respect to simple extraction, procedure, processing method, sensitivity and reproducibility. The validation data showed that the method is precise, free from matrix effects and can be used for pharmacokinetics estimation.
Acknowledgements
The authors gratefully acknowledge Jai Research Foundation, India for providing facility and necessary guidance to do work.
Abbreviations
- QC
Quality control
- MQC
Middle quality control
- HQC
High quality control
- LLOQC
Lower limit of quantification quality control
- LLOQ
Lower limit of quantification
- IS
Internal standard
- MRM
Multiple reaction monitoring
- ULOQ
Upper limit of quantification
- LQC
Low quality control
Author contributions
SJP: Methodology, Design of Experiment, Analysis and Data Compilation; HM: Guidance, Data Review and Writing; BAS: Methodology, Analysis and Data Compilation; NAK: Design of Experiment, Guidance, Data and Review.
Funding
This research did not receive funding.
Data availability
Data are available on reasonable request from the corresponding author.
Declarations
Ethics approval and consent to participate
Not applicable.
Consent for publication
Not applicable.
Competing interests
The authors declare no competing interests.
Footnotes
Publisher’s Note
Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
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Associated Data
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Data Availability Statement
Data are available on reasonable request from the corresponding author.