1. INTRODUCTION:

Diabetes mellitus, which is typified by persistently elevated blood sugar levels, continues to pose an unparalleled worldwide health threat. An estimated 537 million persons (aged 20 to 79) were predicted to have diabetes in 2021. Predictions suggest that the number of people with diabetes worldwide is anticipated to rise significantly, reaching 643 million by 2030 and 783 million by 2045. Diabetes has severe consequences that include cardiovascular disease, neuropathy, retinopathy, and nephropathy. It also places an increasing strain on healthcare systems and jeopardizes the eminence of life of its patients¹.

The combination of vildagliptin (VILG) and remogliflozin etabonate (REMO) in a fixed-dose formulation serves as an antidiabetic medication². VILG functions as an antidiabetic agent by promoting insulin release and reducing the levels of hormones that elevate blood glucose³.

Meanwhile, REMO acts as an antidiabetic medication by facilitating the excretion of excess sugar through urine⁴. The structures of VIL and RMG are illustrated in Figure 1.

(A)

(B)

Figure 1: (A) Structure of Remogliflozin etabonate (B) Structure of Vildagliptin.

Comparing the HPTLC approach to RP-HPLC, less organic solvent is used, and time-consuming procedures such solvent pretreatment and column washing before and after drug analysis are eliminated⁵. There has been effective analytical methods reported for simultaneous estimation of anti-diabetic drugs^6-8. Vildagliptin (VILG) and Remogliflozin (REMO) have been quantified alone and in fixed-dose combinations with other medications using numerous analytical procedures, for example spectrophotometry, RP-HPLC and HPTLC^9-16. UV spectrophotometric methods for simultaneous estimation of Remogliflozin etabonate and Vildagliptin have also been reported^17,18. Despite existing methods, the literature review reveals an absence of an HPTLC approach for simultaneous assessment of VILG and REMO in its FDC (Fixed dose combination). The concurrent quantification of VILG and REMO has thus led to the development of a reliable, accurate, and precise HPTLC method, whose validation was completed in compliance with ICH recommendations¹⁹.

2. EXPERIMENTAL WORK:

2.1 Chemicals and ingredients:

Glenmark Pharmaceutical (Mumbai, India) provided pharmaceutical-grade standards for REMO and VILG. Glacial acetic acid, methanol, ethyl acetate, and toluene of analytical quality has been bought from Merck India Pvt. Ltd. (Mumbai, India).

2.1.1 Tablet dosage form:

Formulation available in the market, with a labeled content of REMO 100mg and VILG 50mg, was procured from nearby pharmacy store.

2.2 Instrumentation:

Chromatographic analysis was conducted using a high-performance thin-layer chromatograph. The HPTLC chromatograph was equipped with a Linomat-V automatic spotter device and Scanner IV, manufactured by CAMAG. The 20 x 10cm glass chamber was used to develop the chromatogram, and the ultraviolet (UV) chamber was used to visualize the bands Application of bands onto the TLC plates was performed using a 100 µL Hamilton syringe.

2.3 Chromatographic condition:

Aluminum plates (10 x 10cm) layered with silica gel 60 F254 were utilized for the separation process. Sample spotting on the TLC plate was performed using the Linomat V semi-automated spotting instrument furnished with a 100µL syringe. The mobile phase consisted of toluene:ethylacetate:methanol:glacial acetic acid in the ratio 2.0:6.0:0.5:0.1 (v/v/v/v). TLC plate development was conducted with a 15-minute saturation duration, and the migration distance was standardized to 90 mm. Subsequently, the bands on the air-dried plate were scanned using Scanner IV at a wavelength of 250 nm.

2.4 Standard Stock Solutions:

The stock solutions were prepared by precisely weighing and dissolving 10mg of Remogliflozin etabonate (REMO) and Vildagliptin (VILG) distinctly in volumetric flasks. The volume was adjusted to 10mL with the respective solvents, consequential in a concentration of 1000µg/mL for each drug. From the standard stock solution of REMO (1000μg/mL), 4 mL were collected, and from the standard stock solution of VILG (1000μg/mL), 2mL were taken. These volumes were diluted with 10mL of methanol, yielding solutions containing 400μg/mL of REMO and 200μg/mL of VILG. Subsequently, 5µL of each diluted solution were utilized, providing 2000ng/band for VILG and 1000 ng/band for REMO.

2.5 Preparation of Sample Solutions:

First, the average weight of twenty tablets, each containing 100mg of Remogliflozin (REMO) and 50mg of Vildagliptin (VILG), was determined collectively. Then, the tablet powder equivalent to 40mg of REMO was accurately measured and shifted into a 10ml volumetric flask. Subsequently, 5ml of methanol was added to the flask, after that 15minutes of sonication. The volume of the solution was attuned to 10ml, resulting in a concentrated solution with a concentration of 4000μg/ml for REMO and 2000μg/ml for VILG. This concentrated solution underwent further dilution, where 10ml was taken and diluted to 100ml by methanol, yielding solutions with strength of 400μg/ml for REMO and 200μg/ml for VILG.

3. METHOD VALIDATION:

The validation of the developed methods was conducted in agreement with the validation parameters outlined by the ICH Q2(R1) guidelines^19,20.

3.1 Linearity:

Calibration curves were produced over a concentration range of 200 to 1400ng/band for Remogliflozin (REMO) and 100 to 700ng/band for Vildagliptin (VILG). Standard working solutions of REMO and VILG were applied to the plate in predetermined aliquots. The relationship between peak area and concentration was established by plotting the data using winCATS software, thereby constructing the calibration curves.

3.2 Accuracy (recovery):

Accuracy assessment has been performed by standard addition technique by determining the % recoveries of REMO and VILG. Known quantities of a mixed standard solution containing REMO (400, 800, 1200 ng/band) and VILG (200, 400, 600ng/band) were added to sample solutions of the test substances. The determination of REMO and VILG amounts was accomplished by applying the peak area values to the regression equations derived from the calibration curve.

3.3 Precision:

In these evaluations, mixed standard solutions containing three different concentrations of Remogliflozin (REMO) and Vildagliptin (VILG) were injected onto a plate using the same syringe. The concentrations used were 400, 800, and 1200ng/band for REMO and 200, 400, and 600ng/band for VILG. Subsequently, peak areas were noted, and the percentage relative standard deviation (%RSD) was calculated for each concentration level to assess precision.

3.4 Limit of detection and limit of quantification:

The following formulas were used to calculate Limit of Quantification (LOQ) and Limit of Detection (LOD) for both drugs:

LOD = 3.3 × σ/S

LOQ = 10 × σ/S

3.5 Specificity:

To evaluate specificity blank, standard solution containing REMO and VILG and sample solution were injected. Any interference from blank, sample excipients and mobile phase to the peak of interest was checked.

3.6 Robustness:

By systematically changing the saturation period (13 min and 17min), wavelength (248nm and 252nm), and distance travelled from solvent front (8.5cm and 9.5 cm), the robustness was evaluated. Three duplicates of a single standard concentration (400ng/band of VILG and 800ng/band of REMO) were used in the evaluation. Mean area values and the percentage %RSD were calculated, and the observed effects of parameter changes were reported.

3.7 Assay of REMO and VILG in dosage form:

Sample solution containing 400 and 200μg/ml solutions for REMO and VILG respectively was applied. The quantification of REMO and VILG was achieved by determining their respective amounts through the application of peak area values to the regression equations derived from the calibration curve.

4 RESULTS AND DISCUSSION:

4.1 HPTLC chromatographic conditions:

To achieve high-resolution and consistent peaks in HPTLC tests, various mobile phase proportions were tested. Optimal results were attained using a mobile phase consisting of Toluene: Ethyl Acetate: Methanol: Glacial Acetic Acid in the ratio of 2.0: 6.0: 0.5: 0.1 (v/v/v/v). These experiments were conducted at room temperature (25±2°C), with a solvent migration distance of 90mm and a chamber saturation period of 15 minutes. This approach yielded symmetrical and well-defined peaks, with Rf (Retention factor) of 0.36 and 0.71 for Vildagliptin (VILG) and Remogliflozin (REMO), correspondingly.

4.2 Linearity:

Calibration curves yielded linear regression data that showed a high linear association within concentration range of 200‒1200ng/band for REM and 100‒700 ng/band for VILG, as shown in Figure 2. As shown in Table 1, the regression equations and their coefficient of determination (r²) values showed good linearity, with r² values of 0.998 for VILG (Figure 4) and 0.999 for REM (Figure 3).

Figure 2: 3D chromatogram of linearity for REMO and VILG.

Figure 3: Calibration curve for linearity study of REMO

Figure 4: Calibration curve for linearity study of VILG

Table 1: Linearity, Rage, LOD and LOQ for REMO and VILG.

Parameter	REMO	VILG
Concentrationrange	200–1400ng/spot	100–700ng/spot
Slope	3.5	2.8
Intercept	205.66	291.33
Correlationcoefficient	0.998	0.999
LOD	2.37 ng	2.45 ng
LOQ	7.19 ng	7.43 ng

4.3 Accuracy:

The recovery findings were within the acceptable range as defined by the ICH guideline. The range of recovery percentages for VILG was 98.45% to 100.03%, and for REMO it was 99.82% to 100.05%. As shown in Table 2, these values were calculated for formulation samples as well as pure drug samples at 50%, 100%, and 150% levels.

Table 2: Accuracy study of REMO and VILG.

Drug	Level (%)	Amount of sample (ng/band)	Amount of std. spiked (ng/band)	Total amount (ng/band)	% Recovery (avg.)
REMO	50	200	400	600	99.82
	100		800	1000	99.84
	150		1200	1400	100.05
VILG	50	100	200	300	100.03
	100		400	500	99.86
	150		600	700	98.45

4.4 Precision:

Precision study of the process was assessed depending upon its repeatability. For VILG and REMO, intra- and inter-day fluctuations were measured at three distinct concentration levels: 200, 400, and 600ng/band for VILG, and 400, 800, and 1200ng/band for REMO. The %RSD was achieved to be less than 2% for each of these concentration levels. The particular outcomes are revealed in Table 3.

Table 3: Precision study of REMO and VILG

Drug	Conc. (ng/band)	Intraday %RSD (Area) (Day 1)	Intraday %RSD (Area) (Day 1)	Interday %RSD (Area) (Day 2)	Interday %RSD (Area) (Day 3)
REMO	400	0.0825	0.3735	0.0599	0.1815
	800	0.0492	0.0590	0.1216	0.1160
	1200	0.0153	0.0449	0.0287	0.0347
VILG	200	0.2561	0.1243	0.3818	0.2160
	400	0.1490	0.1622	0.0892	0.1624
	600	0.1664	0.0533	0.1138	0.1633

4.5 LOD and LOQ:

The evaluation of sensitivity was conducted by shaping the Limit of Detection (LOD) and Limit of Quantification (LOQ). The obtained values for REMO and VILG are 2.37ng and 7.19ng, and 2.45ng and 7.43 ng, respectively, as presented in Table 1.

4.6 Specificity:

The R_f values corresponding to the chromatographic peaks of REMO and VILG in the sample matched those in the standard chromatogram for REMO and VILG. As shown in Figure 4, these experimental results demonstrate the purity of the chromatographic peaks for VILG and REMO in both the sample and standard.

Figure 5: 3D Chromatogram for specificity study

4.7 Robustness:

The %RSD of method less than 2% was obtained by calculating the standard deviation of peak areas for each experimented parameters, as shown in Table 4.

Table 4: Robustness study for REMO and VILG

Parameter	Condition	%RSD
Parameter	Condition	REMO	VILG
Unaltered		0.0471	0.1146
Change in saturation time	13 min	0.0882	0.1088
Change in saturation time	17 min	0.0350	0.1553
Change in wavelength	248 nm	0.1086	0.1088
Change in wavelength	252 nm	0.0532	0.1553
Change in distance travelled	8.5 cm	0.0493	0.1761
Change in distance travelled	9.5 cm	0.0350	0.2530

4.8 Assay of REMO and VILG in dosage form:

Table 5 displays the results of the quantification of the marketed tablet. After analysis, the percentages of VILG and REMO were found to be 98.94% and 98.83%, respectively.

Table 5: Assay of REMO and VILG in the marketed dosage form

Drug	Label claim	Amount obtained	% Assay
REMO	100 mg	98.83 mg	98.83
VILG	50 mg	49.47 mg	98.94

5. CONCLUSION:

The HPTLC method developed facilitated rapid separation within a brief timeframe, enabling the simultaneous measurement of numerous samples with better accuracy, virtuous sensitivity, and meticulous precision. This characteristic is particularly vital in quality control processes. The method, noted for its simplicity, speed, sensitivity, accuracy, precision, specificity, and efficiency, underwent validation in accordance with ICH guidelines.

6. REFERENCES:

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Received on 08.03.2024 Revised on 20.06.2024

Accepted on 28.08.2024 Published on 24.12.2024

Available online from December 27, 2024

Research J. Pharmacy and Technology. 2024;17(12):5697-5701.

DOI: 10.52711/0974-360X.2024.00867