Stability Indicating Bioanalytical Method Development and Validation for Estimation of Remogliflozin Etabonate by RP-HPLC in Human Plasma

Diabetes mellitus is a collection of metabolic disorders distinguished by high blood glucose levels that arise from deficiencies in the secretion of insulin, its action, or both. The primary cause of Type 1 diabetes is an autoimmune response that destroys the pancreatic β cells through humoral (B cell) and T-cell-mediated inflammation (insulitis).¹ 90-95% of cases of diabetes are Diabetes Mellitus Type 2 (T2DM), often called “non-insulin-dependent diabetes” or “adult-onset diabetes.” This category includes those with peripheral insulin resistance and relative insulin insufficiency. The actual etiologies remain unknown; however, patients do not have an autoimmune breakdown of β cells.² T2DM is marked by elevated blood sugar levels (hyperglycemia) and impaired glucose tolerance. The condition arises from the body’s insufficient responsiveness to insulin, resulting in an upsurge in insulin secretion and, subsequently, an insulin deficit.³ Glycemic control gradually deteriorates in people with T2DM. Sodium-dependent glucose co-transporter inhibitors are anti-diabetic drugs.⁴ The glucose reuptake in the proximal tubule of the kidneys is facilitated by the Sodium-Glucose co-transporter-2 (SGLT2), which exhibits a low-affinity mechanism.⁵ SGLT2 is specifically expressed in the renal system.^4,6

Remogliflozin Etabonate (REM) has the chemical composition represented by the formula C₂₆H₃₈N₂O₉.⁶ This prodrug is inactive, but when it is administered and absorbed, it transforms into its active state, remogliflozin.⁷ This active compound specifically targets SGLT2 in the renal proximal tubule and is employed in the management of type 2 diabetes.⁶ It Top of Formstops the kidneys from reabsorbing glucose, which lowers the blood glucose level in people with T2DM.⁸ Remogliflozin etabonate is not included in any pharmacopoeia.

A literature review reveals different analytical methodologies documented for the quantification of remogliflozin, either independently^6,7 or in conjunction with other medications.^8–10 The determination of remogliflozin in rat plasma has been documented using the LC-MS method.¹¹ Additionally, methods involving RP-HPLC and HPTLC have been documented for assessing REM levels in both bulk and tablet formulations.^12,13 However, at present, there is no method available for assessing REM in human plasma using RP-HPLC. The goal of this current study is to establish a new, precise and robust RP-HPLC method for quantifying REM in human plasma.

A gift sample of Pure REM was received from Swapnroop Drugs and Pharmaceuticals, Chha. Sambhajinagar, India. Methanol and acetic acid of HPLC grade were obtained from Omkar Traders Pvt. Ltd., Mumbai. The drug-free EDTA human plasma was obtained from the PMT Blood Bank, Pravaranagar.

The chromatographic analysis was performed using an HPLC binary gradient system (AGILENT 1100 series) with a reciprocating pump (1100). In the chromatographic study, the stationary phase was THERMO C₁₈ (250×4.6 mm, 5 μm) and a combination of methanol and 0.1% acetic acid was used as the mobile phase. The data was processed using CHEMSTATION software. A diode array detector was used for the detection of wavelength, which occurred at 224 nm.

The C₁₈ column was employed for chromatographic separation. Different compositions of the mobile phase were studied to obtain the best REM detection. The components of the mobile phase were degassed for duration of 15 min and were filtered prior to application. The standard and sample solutions were diluted using a diluent (mobile phase) in an 80:20%v/v ratio. The column was kept at a temperature of 25°C and 20 μL was the volume of injection with a flow rate of 1 mL/min.

Into a volumetric flask (10 mL), 10 mg REM was accurately weighed and transferred. Methanol (5 mL) was added to it and sonication was performed for 10 min to ensure the REM dissolves completely. The volume was adjusted with methanol to achieve a stock solution of 1000 μg/mL. 1 mL was drawn from the solution and placed into a volumetric flask (10 mL). The volume was adjusted with methanol to achieve the 100 μg/mL working standard solution.

To prepare a blank plasma, acetonitrile (4 mL) was introduced to 1 mL of collected plasma as an extracting solvent. The mixture was thoroughly mixed and left at room temperature for 5 min.

Then, centrifugation was performed at 10,000 rpm for 12 min. Finally, a clear fluid from the upper layer was separated, filtered and used further for the HPLC analysis.

To create calibration standards of REM, an appropriate amount of diluted standard solution was added to the blank plasma to achieve drug concentrations ranging from 5-13 μg/mL.

The validation of the developed method followed ICH guidelines for various parameters such as linearity, accuracy, precision, LOD, LOQ and robustness.

The developed method’s linearity was assessed by plotting a calibration curve by spiking plasma with a known quantity of REM ranging from 5-13 μg/mL. The regression equation was obtained from the calibration curve of peak area versus REM concentration.

The precision and accuracy of the developed method were assessed by spiking REM in human plasma. To assess intra-day precision, sample solutions of REM (7, 9, 11 μg/mL) were examined within the same day at low, medium and high concentration levels. The same concentrations were used to analyze inter-day precision.

The method’s accuracy was assessed through recovery studies conducted at three different levels of concentration (80%, 100% and 120%). To perform the recovery studies, a specific concentration of the standard drug (80%, 100% and 120%) was added to previously analyzed sample solutions. %RSD and %recovery were calculated.

The minimum detectable quantity in a sample from background noise but not quantitated is the LOD (Limit of Detection). The minimum quantity of substance in a sample that can be precisely and accurately quantified is the LOQ (Limit of Quantitation). Three times the noise level was used to determine LOD and ten times the noise level was used to calculate LOQ.

Robustness was examined by implementing slight modifications to the chromatographic conditions. Minor adjustments were made to the mobile phase ratio (79:21 and 81:19), flow rate (1 mL/min±0.1 mL/min) and detection wavelength (224 nm±1 nm). The %RSD was calculated.

Twenty tablets were weighed and then ground into a powdered form. An amount of powder equal to 10 mg of REM was mixed with methanol in a volumetric flask (100 mL). An appropriate quantity of plasma was introduced to a flask, the mixture was thoroughly mixed and centrifugation was performed for 15 min. After filtration, the volume was adjusted with methanol. A volume of 1mL from the solution was added to the volumetric flask (10 mL) and diluted with the mobile phase to achieve a 10 μg/mL REM solution. After sonication, the solutions were introduced into the system. The determination of peak areas was conducted and quantification was performed using a regression equation.

The studies of forced degradation were conducted to assess the intrinsic stability of REM and to find out the degradation pathways. During forced degradation studies, REM was subjected to diverse stress conditions such as acid (0.1 N HCl) and base hydrolysis (0.1N NaOH), peroxide degradation (3% H₂O₂) and photolytic degradation.

The different chromatography parameters such as column temperature, mobile phase concentration, flow rate and other conditions were optimized during the method development.

Temperatures of the column oven were examined in the 25-30°C range. The optimum column temperature was found to be 25°C. Different mobile phase ratios were tried in order to achieve better separation. A mixture of methanol and 0.1% acetic acid at 80:20%v/v ratios was used for the HPLC analysis, as it gives effective resolution and better separation. Various flow rates (ranging from 0.8-1 mL/min) were tested and it was discovered that 1 mL/min was the best for effective separation. The UV spectrophotometer was used to scan the solution in order to select the wavelength and the maximum absorbance was confirmed at 224 nm (Figure 1). Results for optimized conditions are given in Table 1.

The test for system suitability was conducted by injecting a standard solution of REM six times into the system. Different parameters, including resolution, tailing factor, retention time and theoretical plates were assessed to ensure the reproducibility of the chromatographic system. All the results were found to be within specifications proving the suitability of the method. Table 2 shows results for system suitability.

The response of the calibration curve of REM was observed to be linear across 5-13 μg/mL concentration. The linearity equation was obtained as y=37.0414x-7.695 and the correlation coefficient was found to be 0.9992 (Figure 2). As a result, good linearity was shown by the developed method. Table 3 represents the linearity results.

The precision and accuracy of the developed method were calculated by using %RSD and percentage recovery. Three different concentrations were used to calculate precision. The method’s accuracy was assessed by adding specific concentrations of the standard drug (80%, 100% and 120%).

The %RSD values were found to be in the range of 0.08-0.73 for the intra-day precision and 0.04-0.30 for the inter-day precision. The mean recovery values for REM ranged from 98.63-100.81%, showing the method’s accuracy. %RSD for both precision and accuracy was within the limits which shows good precision and repeatability of the method. Results for precision and accuracy are given in Table 4A and Table 4B.

The detection limit for REM was determined to be 0.1390 μg/mL, while the quantification limit was 0.4213 μg/mL.

The study showed that after introducing minor changes in the ratio of mobile phase, flow rate and detection wavelength, no significant variations were found. Thus, the method demonstrated robustness, with a %RSD below 2.0%. Table 5 shows the results of robustness.

The analysis was conducted on a tablet formulation containing 100 mg of REM. The % recovery of REM was found to be 101.85±0.31 %w/w in plasma.

Stability investigation was carried out under several stress conditions, including the hydrolysis with acids (Figure 3A) and alkalis (Figure 3B), peroxide degradation (Figure 3C), photolytic degradation (Figure 3D) and neutral degradation (Figure 3E). The study on degradation revealed that REM exhibited high susceptibility to acid and base hydrolysis as well as peroxide degradation. The drug remained stable under neutral and photolytic degradation conditions. Table 6 represents the results of degradation studies.

Figure 1:
Representative chromatogram of Remogliflozin in human plasma.

Figure 2:
Calibration curve of REM spiked in human plasma.

Figure 3A:
Chromatogram of acidic degradation after 1 hr.

Figure 3B:
Chromatogram of basic degradation after 1 hr.

An RP-HPLC method was developed and validated to analyze Remogliflozin etabonate, a pure drug, using a mobile phase consisting of methanol and 0.1% acetic acid (80:20 v/v). Validation encompassed several parameters including specificity, linearity, accuracy, precision, LOD, LOQ, recovery and stability. When applied to a marketed formulation, the method effectively assessed pharmacokinetic parameters. The method demonstrated linearity from 5-13 μg/mL, indicating a strong linear relationship within the tested concentration range. Accuracy studies revealed results close to the expected value (100%), confirming the method’s accuracy, while precision assessment showed % RSD for intermediate precision below 2%, indicating its precision. A low LOQ emphasized the method’s sensitivity, enabling precise estimation of nanogram drug quantities. Robustness testing confirmed the method’s reliability against deliberate alterations.

Forced degradation studies exposed Remogliflozin etabonate’s susceptibility to acid and base hydrolysis, resulting in significant ester hydrolysis and conversion into the active drug, Remogliflozin. The method was found to be stable in the case of neutral conditions. Hence the suggested method can be successfully used for identification and quantification of Remogliflozin in various biological matrices.

A simple, accurate and precise RP-HPLC bioanalytical method was developed and validated for REM quantification in human plasma, following the ICH guidelines. The % RSD for validation parameters was found to be less than 2, which suggests that the method is appropriate for the identification and quantitative determination of REM. The developed method has better separation, and linearity and is highly accurate for the determination of drugs in human plasma. Forced degradation investigations were conducted, indicating the drug’s susceptibility to acid-base hydrolysis and peroxide degradation, which resulted in its conversion to the active form, Remogliflozin. The method is indicative of stability and is appropriate for the routine analysis of REM in biological fluids.

The authors are grateful to the Principal of Pravara Rural College of Pharmacy, Loni for providing the facility to carry out this study.