Synergistic Detection and Quantification of Gliclazide and Dapagliflozin Propanediole Monohydrate from Tablet Dosage Form by Derivative Spectroscopy Method.

Gliclazide (GLIC) is an antidiabetic agent belonging to the sulfonylurea class, which acts by stimulating insulin secretion from pancreatic β-cells. It achieves this by blocking ATP-sensitive potassium channels, leading to cell depolarization and subsequent insulin release. Gliclazide is primarily used in the management of type 2 diabetes mellitus, either as monotherapy or in combination with other oral hypoglycemic agents. Dapagliflozin (DAPA), on the other hand, is a sodium-glucose cotransporter-2 (SGLT2) inhibitor. It is used alongside diet and exercise to enhance glycemic control in adults with type 2 diabetes. SGLT2 is a key transporter responsible for renal glucose reabsorption; its inhibition promotes glucosuria and reduces blood glucose levels in diabetic patients.

According to the clinical trial study of an ongoing international real-world investigation (ADD2DIA) assessing the effectiveness of adding an SGLT2 inhibitor, such as Dapagliflozin, to a Gliclazide MR (modified-release) regimen in individuals with type 2 diabetes mellitus (T2DM). This combination leverages complementary mechanisms: Gliclazide stimulates insulin secretion, while Dapagliflozin promotes urinary glucose excretion. The study found that this combination improved glycemic control without a significant increase in hypoglycemia risk and also contributed to weight reduction.

Figure 1 Structure of Gliclazide

Figure 2 Structure of Dapaglifozin

The above drug combination is marketed as a tablet dosage form with Gliclazide 30 mg and Dapagliflozin 10 mg. Several analytical methods have been reported for the individual or combined analysis of GLICLA and DAPA with other drugs, literature reveals no validated method specifically for their simultaneous estimation from a synthetic mixture or formulation. UV spectrophotometric techniques offer greater operational simplicity compared to chromatographic methods. Moreover, derivative spectroscopy enhances method specificity by allowing quantification at the Zero Cross Over Point (ZCP). Therefore, derivative spectroscopic analysis was chosen as the preferred method for the simultaneous estimation of Gliclazide and Dapagliflozin from their combined marketed formulation.

MATERIALS AND METHODS

Material

DAPA (99.98% pure) and GLICLA (99.96% pure) were obtained as gift sample for research purpose from, Cadila Healthcare Ltd., Sanand. Methyl alcohol (AR grade) was purchase from Finar. Sample processing was performed using Shimadzu UV1900i double beam spectrophotometer having path length of 1 cm matched pair of quartz cells. Obtain spectra of GLICLA and DAPA were derivatized 1^st order using UV probe 2.72 as software at delta λ of 10 nm. equipment.

Preparation of Master Stock Solution:

For the method development purpose, 30 mg of GLICLA and 10 mg of DAPA were precisely weighed and then transferred to 100 mL volumetric flasks. The level was raised with methanol to produce a solution containing 300 µg/mL and 100 µg/mL GLICLA and DAPA, respectively.   

Selection of Analytical Wavelength:

The working standards of GLICLA (15-75 µg/mL) and DAPA (5-25 µg/mL) were prepared in 10 ml volumetric flask using methyl alcohol as a solvent. They were scanned in the UV range of 200 – 400 nm and D⁰ spectra is recorded by UV spectrophotometer. All the D⁰ spectra of GLICLA and DAPA were transformed into D¹ spectra with the help of UV probe 2.42 software. For confirmation of D¹ spectra of GLICLA and DAPA, D⁰ and D¹ spectra of the same were overlapped.

Preparation of solutions for analytical method validation

Preparation of solution for linearity and range

To check linearity of method, GLICLA was prepared in the concentration range of 15-75 µg/mL and DAPA was prepared in the range of 5-25 µg/mL from master stock solution in 10 ml volumetric flask. When D1 Absorbance was plotted against concentration, non-linearity

was observed above 100 µg/mL for GLICLA and above 35 µg/mL for DAPA, so final range for validation was selected at mixture containing 15-75 µg/mL for GLICLA and 5- 25 µg/mL for DAPA. All prepared solutions were scanned between 200-400 nm and all spectra were derivatized to 1^st order. D¹ absorbance were obtained at selected wavelength and mean D¹ absorbance was plotted against concentration (To get mean D¹ absorbance, procedure was repeated for five times)

Intermediate precision (Repeatability)

To adjudge the repeatability of analytical method, solution of linearity studies were analysed for five time with same conditions. Mean D¹ absorbance was recorded at all concentrations for GLICLA and DAPA and were observed for relative standard deviation.

Method precision

Method precision was determined by performing intraday and interday precision. Mixture that represents overall range (15+5, 45+15 and 75+25 µg/mL) were analyzed on same day at different time interval for intraday precision. Mixture that represents overall range (15+5, 45+15 and 75+25 µg/mL) were analyzed on different days for interday precision.

Accuracy study

Accuracy of analytical method was adjudged by spiking of blank with standard solution. Standard solution was spiked at 50, 100 and 150% of target concentration (30+10 µg/mL) (Table 1). Each spiked concentration was analyzed for three times and mean % recovery was observed at each spiked level.

Table 1 Preparation of solutions for accuracy studies

Average weight of 10 tablet was calculated, 325.75 mg powder (equivalent to 30 mg of GLICLA + 10 mg of DAPA) transferred to 10 ml volumetric flask. Volume make up was done with methanol (GLICLA+DAPA = 300+100 µg/mL) Sonicated for 10 minutes and filter through 0.45 micron Whatman filter paper) Withdraw 1.0 mL of master stock solution in 10 ml volumetric flask; make up the volume with methanol, which contain 30 µg/mL of GLICLA and 10 µg/mL of DAPA. This mixture was scanned between 200-400 nm and was derivatized to 1^st order. D¹ absorbance was measured at selected wavelengths and were transformed to concentration with help of linear regression equation. This mixture was analyzed for three times and mean % assay was drawn.

Result and Discussion

Selection of analytical wavelength

Four different ZCP at 212 nm, 228 nm, 260 nm and 287 nm were observed in overlain D¹spectra of GLICLA (Figure 3). Five different ZCP at 217 nm, 222 nm, 255 nm, 275 nm and 300 nm different ZCP at 256 nm, 275 nm and 305 nm were observed in overlain D¹ spectra of DAPA (Figure 4). For determination of analytical wavelength D¹ spectra of GLICLA and DAPA were overlapped (Figure 5). But there is very less difference between absorbance values of DAPA at 260 nm and hence difficulty in quantifying the same. Discussed problem can be eliminated at 222 nm, where the D¹ absorbance values of DAPA are linear with significant difference (Figure 6). In similar way at ZCP of DAPA, linearity was observed only at 228 nm for GLICLA (Figure 7). So 222 nm and 228 nm were selected as analytical wavelength for quantitative determination of DAPA and GLICLA respectively.

Figure 3 Zero cross over point of GLICLA

Figure 4 Zero cross over point of DAPA

Figure 5 Overlain D1 spectra of GLICLA and DAPA

Figure 6 Determination of DAPA (25 µg/mL) at ZCP of GLICLA

Figure 7 Overlain of D1 spectra of GLICLA [15-75µg/mL] and DAPA [5-25 µg /mL]

Analytical method validation

All validation parameters were studied as per ICH guidelines.25,26

Linearity and range

When D⁰ spectra of GLICLA was taken between 15 – 75 µg/mL, non-linearity was observed over 100 µg/mL. So, linearity for GLICLA was observed between 15 – 75 µg/mL. for method development purpose range was selected between 15 - 75 µg/mL (based on beer — lambert’s law). In similar way D⁰ spectra of DAPA was taken between 5 - 25 µg/mL, but non-linearity was observed over 35 µg/mL. So, linearity for DAPA was observed between 5 - 25 µg/mL. and for method development purpose range was selected between 5 - 25 µg/mL (based on beer — lambert’s law). So final range for validation was selected at mixture containing 15 – 75 µg/mL for GLICLA and 5 - 25 µg/mL for DAPA. When calibration curve was plotted for given concentration range (Figures 7 and 8), value of linear regression coefficient was found to be 0.9971 for GLICLA and 0.9961 for DAPA. Regression equation was found to be y = 0.0007x - 0.0016 for GLICLA and y = -0.0008x + 0.0005 for DAPA. Linearity data for both drugs is shown in Tables 2 and 3.

Table 2 Linearity data of GLICLA

y= 0.0007x - 0.0016, Correlation Coefficient R² = 0.9971

Figure 8 Calibration curve of GLICLA (15-75 µg/mL)

Table 3 Linearity data of DAPA

y= -0.0008x + 0.0005 Correlation Coefficient R²= 0.9961

Figure 9 Calibration curve of DAPA (5-25 µg/mL)

Repeatability

When all mixtures were analyzed at all concentration, calculated relative standard deviation at each level was found to be less than 2 so that method was found to be repeatable over the range of 15 - 75 µg/mL for GLICLA and 5 - 25 µg/mL for DAPA. Repeatability data are shown in table 4 and 5 for GLICLA and DAPA respectively.

Table 4 Repeatability data of GLICLA

n= 5 determinations

Table 5 Repeatability data of DAPA

n= 5 determinations

For determining inter day and intraday precision, % RSD was monitored at selected concentration level which was found to be less than 2 so method was found to be precise for estimation of GLICLA and DAPA. Data for intermediate precision are given in Tables 6 and 7 for GLICLA and DAPA, respectively.

Table 6 Intraday and Inter-data of GLICLA

n = 3 determinations

Table 7 Intraday and Inter-data of DAPA

n= 3 determinations

Accuracy study

Spiked placebo with standard solution at 50, 100 and 150% level was analyzed for % recovery which was found within 98 to 102, so method was found to be accurate (Table 8 & 9).

Table 8 Accuracy data of GLICLA

n= 3 determinations for each set

Table 9 Accuracy data of DAPA

n= 3 determinations for each set

When prepared synthetic mixture was analyzed by developed and validated method, % assay was found to be 97.89 ± 3.56 for GLICLA and for 99.23 ± 1.57 DAPA (Table 10)

Table 10 Determination of GLICLA and DAPA from mixture