INTRODUCTION:

Individuals with type 2 diabetes (T2D) experience elevated reabsorption of sugar in the kidneys and increased glucose production in the body (Gluconeogenesis). Additionally, there appears to be an upregulation of sodium-glucose transporter 2 (SGLT-2) proteins, which are in charge of about 90% of sugar reabsorbed in the kidneys^1,2. The inhibition of SGLT-2 reduces the absorption of glucose in the renal proximal tubules and lowers the blood sugar threshold in the kidneys. As a consequence, there is a rise in the elimination of glucose through urine^3,4. T2D can be treated with a variety of SGLT-2 inhibitors².

Bexagliflozin (Figure 1) is an SGLT-2 inhibitor (Brenzavvy™) that is taken orally and has been developed to treat the symptoms of type 2 diabetes (T2D) and high blood pressure^5,6. Its chemical nomenclature is (2S,3R,4R,5S,6R)-2-[4-chloro-3-[[4-(2-cyclopropyloxyethoxy) phenyl] methyl] phenyl]-6- (hydroxymethyl) oxane-3,4,5-triol. Bexagliflozin is a white/off-white to pale yellow powder ^{with the molecular formula C}₂₄^H₂₉^ClO₇ ^{and a molecular mass of 464.94g/mol4}. Bexagliflozin is a potent SGLT-2 inhibitor (through an inhibitory constant of 2 nmol/L) and has 2435 times greater selectivity than SGLT-1⁷. It is absorbed within 2-4 hours, but this process can be delayed by food or medications that slow down gastric emptying. It binds to proteins in the bloodstream. Bexagliflozin is metabolized by UGT1A9 and CYP3A enzymes and is eliminated through urine⁴. The literature survey revealed that no methods have been developed to analyze Bexagliflozin^8,9. For the first time, we have developed an innovative analytical approach that allows quantification of Bexagliflozin in Bexagliflozin tablets^10,11.

MATERIALS AND METHODS:

Chemicals:

Bexagliflozin (API) was obtained as a complimentary sample courtesy of Spectrum Research Private Limited located in Hyderabad, India, while bexagliflozin tablets were acquired from a nearby pharmacy. In this study, all reagents, solvents, and chemicals utilized had been of HPLC grade and purchased from Rankem Chemicals Pvt. Ltd.

Instrumentation:

HPLC experiment is conducted on a Waters HPLC 2695 System using quaternary pumps, a PDA, and an Autosampler. Empower 2 Software was used for data collection and analysis. The ^{analytical column
employed for the separation was Discovery C}₁₈ ^{(150mm x
4.6mm, I.D. 5}µm). An electronic balance (Denver), ultra-sonicator (BVK enterprises), and pH meter (BVK enterprises) were also used.

Wavelength selection:

Bexagliflozin (20µg/mL) was subjected to scanning within the 200-400nm range with the wavelength maxima of 220nm (Figure 2) chosen as the detecting wavelength.

Composition of mobile phase:

Developing a method for quantifying bexagliflozin had been initiated by experimenting with various solvent combinations at different ratios (50:50, 60:40, 65:35). Although, finally a combination of 0.01 N Sodium hydrogen phosphate: methanol (55:45, v/v) has shown satisfactory resolution for bexagliflozin.

Preparation of standard solution:

In a 50mL volumetric flask, 20mg of bexagliflozin powder was precisely weighed and combined with 70 mL of diluent, followed by 25 minutes of sonication (200μg mL^-1). Further 1mL dilution in a 10mL volumetric flask resulted in a final concentration (20μg mL^-1).

Preparation of sample solution:

The average weight of each tablet was determined after weighing ten of them. The weight equivalent of one tablet was then added to a 100mL volumetric flask along with 70mL of diluent. The mixture was filtered after being sonicated for 25 minutes, with the volume being adjusted with diluent (200μg mL^-1). Further 1mL dilution in a 10mL volumetric flask resulted in a final concentration (20μg mL^-1).

Chromatographic parameters:

Bexagliflozin separation methods were developed using a variety of solvents, but ultimately ^{the separation was
accomplished through a mobile phase of 0.01 N Na}₂^HPO₄^:
CH₂^OH(55:45, v/v), pumping at a flow rate of 0.9mL min^-1. At 220nm, the eluent had been identified by observing through the PDA detector¹². The mobile phase underwent filtering before its utilization.

Method validation:

The new technique was validated in compliance with ICH, United States Pharmacopeia (USP), about system suitability, specificity, range, solution stability, linearity, precision, accuracy, LOD, LOQ, Robustness and forced degradation tests^13-16.

System suitability:

To ensure that a method is adequate for a given analysis, system suitability is frequently used. Theoretical plate count, tailing factor, area resolution and reproducibility, limit of detection, and limit of quantification were among the parameters that were confirmed in the current study.

Linearity, range and calibration curve:

To construct a calibration curve, the peak area was plotted against various concentrations (25-150% nominal), and a calibration curve was then used to assess the linearity and operating range of the method. Calculations were made for slope, intercept, regression coefficient (R²), and linear equation.

Precision:

The two commonly used levels of precision evaluation are intra-day (repeatability) and inter-day (intermediate precision). Three replicates at each concentration level were used in the current evaluation of the three concentration levels of intra-day precision. To assess intermediate precision, two HPLC systems were applied in two altered days. The %RSD was determined.

Accuracy:

The data is collected through a minimum of nine analyses, spanning at least three concentration levels encompassing the recommended range. Recovery in the current study was utilized to assess accuracy at three different concentrations.

Specificity:

To ensure that no excipients interfered with the drug peaks in the chromatogram, the specificity of the approach was evaluated. The specificity of the drugs was evaluated using peak purity indices in addition to visual inspection.

Solution stability:

The stability of the drug in the diluent was assessed by storing test solutions in sealed vials in the refrigerator at 5°C for 24h. The solutions at 0 and 24hours were analyzed using the established methodology. There was no obvious change in the area. The %RSD, which measures medication stability in the diluent, was found to be less than 2.0%.

Robustness:

By intentionally and deliberately changing the chromatograph's flow rate, mobile phase, and temperature, robustness was determined¹⁰.

Forced degradation study:

These investigations are carried out to intentionally induce degradation in a sample. These investigations are carried out to assess the sensitivity of an analytical technique. The development of multiple dosage forms and the rational formulation of the treatment are both aided by the vital information on the drug breakdown pathway provided by forced degradation research. When exposed to acid, base, oxidizing, thermal, ultraviolet (UV) radiation, and water, drug compounds or drug products deteriorate by 10–30%. The technique is then used to examine the degraded samples to determine if any interactions exist among the drug molecule and any associated substance(s)^16-18.

Assay of Bexagliflozin tablets:

The assay method used bexagliflozin tablets (Brenzavvy™) with a label claim of 20mg (API). The average weight of ten tablets was calculated using precise weight measurements. The weight of one tablet, which was determined after the tablets were ground up, was subsequently placed into a 100mL volumetric flask. Filter the powder after it has been dissolved in the diluent and sonicated for 30 minutes. With diluent, the filtered solution was diluted to the desired volume. The mobile phase was used to dilute aliquots of the sample solutions. 10µL of the solution had been introduced into the apparatus. The chromatogram was analyzed to record the peak area and retention time¹⁹.

RESULTS AND DISCUSSION:

Preliminary screening:

An initial screening and literature search were conducted to identify the significant factors as well as their effects and levels. Acetonitrile (50%) and 0.1% v/v acetic acid in water buffer (50%) with a flow rate of 1.0mL/min mixture separate bexagliflozin according to screening experiments, but the peak is not symmetrical, the resolution is outside of acceptable ranges, and the retention time is also much longer than anticipated. Whereas in the second trial, Methanol (35%) and 0.1N KH₂PO₄ buffer (65%) mixture was used as the mobile phase and flow rate 1.0mL/min, by maintaining all the chromatographic conditions constant, the peak is asymmetrical with tailing, and additional peaks were observed. In the third trial, 0.1N KH₂PO₄ buffer was replaced by 0.1% Sodium dihydrogen orthophosphate, and the mobile phase consisting of methanol (60%) and 0.1% Sodium dihydrogen orthophosphate (40%) with flow rate 1mL/min, was taken into consideration. In this trial, Bexagliflozin peak was eluted but the USP plate count was not in the acceptance criteria (>2000). So, further trials were carried out.

In the fourth trial, Acetonitrile (60%) and 0.1% Sodium dihydrogen orthophosphate (40%) were taken as mobile phase with a flow rate of 1.0mL/min. By changing buffer composition both peaks were eluted but retention times were longer. The research was done using various pH levels and buffer systems. A general guideline suggests maintaining the pH of the mobile phase buffer either 2 units higher or lower than the drug's pKa values. The pKa value of Bexagliflozin is 3.23 which remains undissociated at a pH of 2 units lower than 3 (i.e., 1.23), which causes its retention for a longer period and necessitates the use of a higher percentage of methanol to reduce its retention. For this reason, we chose pH 5.32, which is roughly two units over the pKa value of Bexagliflozin; at this pH, the drug should persist undissociated. Potassium dihydrogen ortho Phosphate buffer and buffer with 0.1% orthophosphoric acid were the two choices for the mobile phase. In the current situation, a 0.1% Sodium dihydrogen orthophosphate buffer was employed because it forms a less harmful salt with a larger molecular structure when the pH of the mobile phase approaches or exceeds 8.0. Final separation was carried out on a Discovery C₁₈ column (150mm x 4.6 mm, I.D. 5 µm) with a flow rate of 0.9 mL min^-1 and 55% 0.01N Na₂HPO₄: 45% Methanol as mobile phase. To achieve the optimized separation, the eluents were analyzed at a wavelength of 220 nm. Table 1 displays the optimal chromatographic conditions, and Figures 3 and 4 show typical HPLC chromatograms of the blank, standard, and samples, respectively along with trails.

Method validation^20-21:

System suitability:

The system suitability parameters, including peak area, USP plate count, and tailing factor, all met the desired criteria, and the results have been summarized in Table 2.

Solution stability:

Vials containing working standards at the nominal concentration were subjected to stability tests in this solvent, with one set stored under ambient conditions and another set placed in a cold storage environment. Tests were conducted at 0 and 24 hours to assess stability. The peak areas have been then contrasted with those at 0 h. No appreciable alterations in the area were seen. The drugs appeared to be stable in the diluting solvent as the RSD was discovered to be less than 2.0%.

Linearity, working range, and accuracy:

In the current validation process, the designated standard concentration was 20μg/mL for Bexagliflozin. To create the standard solutions for calibration, we generated samples at concentrations of 25%, 50%, 75%, 100%, 125%, and 150% relative to the nominal concentration, and then plotted the peak area against their respective concentrations. The drug had a determination coefficient (R²) of 0.9996, indicating a satisfactory fit of the data by the regression line. The linearity was found to be 5-30 μg/mL for Bexagliflozin (Figure 5). The regression equation was as follows [Eq (1)]:

Bexagliflozin:

Y= 12845x+988.82 (R²=0.9996) …………………… (1)

The percentage of recoveries fell comfortably within the acceptable limit (100±2%), showing how accurate the method was at determining the amount of drug. Tables 3, and 4 present the findings.

Precision:

In the current study, triplicate injections of three different solutions with known quantities of added drugs were conducted. Regression equations were used to calculate percent recoveries (concentrations). Table 5 displays the findings of the precision study. The low RSD of the total variation demonstrates the precision of the technique within the necessary recovery range.

Specificity:

Upon visual examination of the standard chromatogram, the chromatogram from the assay sample, and the chromatogram from the forced degradation sample, there were no indications of any interference from other impurities or excipient peaks. Additionally, peak purity in each was reviewed and was found to be greater than 0.9998, demonstrating the technique specificity for the drug molecules. The standard and sample chromatograms are shown in Figure 4 (b) and 4 (c). The chromatogram of a blank obtained by a PDA detector is shown in Figure 4 (a).

Robustness:

The robustness was investigated by making a small but intentional change to the intrinsic method parameters. The flow rate, mobile phase concentration, and column temperature were examined in our robustness investigation. The RSDs for peak areas were found to be less than 2%. Table 6 displays the outcomes.

Forced degradation study:

These experiments were conducted in 0.1M HCl, 0.1M NaOH, 10% H₂O₂ solution, 105°C for 6 h, H₂O and a UV chamber for seven days or 200 W∙h/m². After being kept in the dark for 24h, samples were examined using the recently created technique. Their peak areas were compared with those of a freshly made standard solution. Bexagliflozin was found to be significantly stable to all environmental factors (Figure 6), degrading the most in acid (4.11%) and oxidative media (4.84%). As the percentage of degradation is within the limits (5-20%), it has been proven that bexagliflozin was stable at pH 4. The data can be used for the analytical method development for the determination of bexagliflozin in tablet preparations. To maintain a pH of about 4.0, a buffering agent can be added, because bexagliflozin demonstrated a significant propensity for degradation in acidic media but was practically stable in the diluting solvent (pH 4.0). The results are given in Table 7.

Assay of Bexagliflozin tablets:

Bexagliflozin tablets with a 20mg (API) label claim were used for the assay method. The amount of Bexagliflozin was found to be 98.55 - 100.35% (Table 8) and there was no interference of excipients¹⁹.

CONCLUSION:

The present approach was observed to be simple, specific, linear, precise, reliable, robust, economical and validated according to the regulations of ICH Q2B. From the stability studies of the present analytical method, the degradation behavior of Bexagliflozin was known and it has been demonstrated that this method can successfully distinguish the degradation peak from the analytical peak. Symmetrical peaks have been observed within less analysis time and the percent RSD values for all parameters were as per the limits for the developed method. This shows that the results and assay achieved by this approach are in good accordance. Hence the present analytical method developed can be used for the estimation of Bexagliflozin in pure and pharmaceutical formulations and also for regular analysis in quality control purposes.

Table 1: Optimized chromatographic conditions

Parameters	Conditions
Stationary phase	^{Discovery C}18 ^{column (150 mm x 4.6 mm I.D, 5 µm)}
Mobile phase	^{0.01 N Na}₂^HPO₄^{: CH}₂^{OH (55:45 v/v)}
Detector	PDA
Flow rate	0.9 mL min^-1
Run time	6 min
Column temperature	30°C
Injection volume	10 μL
λmax	220 nm
Retention time	2.724 min

Table 2: Analysis of system suitability

S. No	Parameters	Bexagliflozin	Limit
1	RSD of Peak area, %	0.8	RSD ≤ 2%
2	RSD of Retention, %	0.3	RSD ≤ 2%
3	Tailing factor	1.25	USP Tf ≤2.0
4	LOD µg/mL	0.004	S/N = 3:1
5	LOQ µg/mL	0.011	S/N = 10:1

Table 3: Linearity data

S. No	Concentration (μg mL^-1)	Peak area
1	5	64992
2	10	129947
3	15	193270
4	20	262934
5	25	318131
6	30	386323

Table 4: Accuracy data of bexagliflozin

% Level	Amount Spiked (μg mL-1)	Amount recovered (μg mL-1)	% Recovery	Mean Percent Recovery
50%	10	9.91	99.15	99.97%
	10	10.10	100.99
	10	9.96	99.64
100%	20	19.99	99.97
	20	20.19	100.93
	20	20.00	99.99
150%	30	30.01	100.02
	30	29.87	99.56
	30	29.86	99.52

Table 5: Intra-day & inter-day precision of bexagliflozin

S. No	Intra-day Precision		Inter-day precision
S. No	RT	Peak Area	RT	Peak Area
1	2.707	173940	2.753	169426
2	2.712	172391	2.754	169713
3	2.725	175074	2.764	169763
4	2.727	175081	2.764	170357
5	2.728	173643	2.764	169496
6	2.728	171797	2.765	167169
Mean		173654		169320
SD		1354.8		1104.09
%RSD		0.8		0.6

Table 6: Robustness study

S. No	Variation of parameter	Peak area	RSD, %
1	Flow rate minus (0.8 mL/min)	164490	1.5
2	Flow rate plus (1 mL/min)	173020	1.4
3	Mobile phase minus (Na₂HPO₄:CH₂OH (40:60 v/v))	168455	0.8
4	Mobile phase plus (Na₂HPO₄: CH₂OH (50:50 v/v))	166447	0.7
5	Temperature minus (-3°C)	183800	0.6
6	Temperature plus (+3°C)	186161	1.1

Table 7: Forced degradation study

S. No	Degradation Condition	% Drug degraded	% Drug undegraded	Retention Time (RT)	Acceptance Limit
1	Acid Degradation (0.1 N HCl/60°C/30 min)	4.11	95.89	2.770	Less than 20%
2	Alkali Degradation (0.1 N NaOH/60°C/30 min)	2.38	97.62	2.770
3	Oxidative Degradation (20% (w/v) H₂O₂/60°C/30 min)	4.84	95.16	2.770
4	Thermal Degradation (105°C/1 day)	1.97	98.03	2.758
5	Photolytic Degradation (UV radiation at 200 W h/m²)	0.93	99.07	2.755
6	Nuetral Degradation (water/60°C/6 h)	0.15	99.85	2.755

Table 8: Assay of Bexagliflozin tablets

S. No	Standard Area	Sample area	% Assay	Acceptance Criteria
1	173940	174436	100.35	%Assay must be 90-110%. %RSD must be less than 2
2	172391	173405	99.76
3	175074	173408	99.76
4	175081	173764	99.96
5	173643	171316	98.55
6	171797	173784	99.97
Avg	173654	173352	99.73
S. D	1354.9	1066.1	0.6133
%RSD	0.8	0.6	0.61