Analytical-grade reagents and chemicals were used for the study. The samples were obtained from a local distributor representing Lupin Pharma, specifically the brand Gibtulio Met in mixed dosage form. Methanol (HPLC grade) was sourced from Merck Life Sciences Pvt. Ltd.

A Shimadzu UV-1900i double-beam UV-Vis spectrophotometer with 1 cm path length quartz cells was used for the analysis. The materials were weighed using a Shimadzu electronic analytical balance, and a Professional Ultrasonic Cleaner was employed for sonication of the prepared solutions.

Gibtulio Met was dissolved in a 50 ml standard flask using a Methanol-Water mixture (50:50) to achieve a concentration of 1 mg/ml (Solution A) for each component. The standard stock solution was further diluted by taking 1 ml of Solution A and diluting it with the Methanol-Water mixture to 10 ml, yielding a concentration of 100 μg/ml (Solution B). From this, 5 ml was accurately pipette and diluted to 50 ml to obtain a 10 μg/ml solution (Solution C). For the calibration curve, 2, 4, 6, 8, and 10 ml of Solution C were precisely transferred into 10 ml standard flasks, and the volume was adjusted to the appropriate level using the Methanol-Water mixture.

A 10 μg/ml solution of Gibtulio Met was prepared, with a Methanol-Water mixture used as the blank. This solution was subjected to UV spectrophotometric scanning across the wavelength range of 200-400 nm to determine the λ_max of Gibtulio Met. The scanning spectra revealed two distinct peaks at 225 nm and 235 nm, respectively, indicating the absorption maxima. Additionally, the overlay spectra of the 10 μg/ml solution were recorded for further analysis, as shown in Figure 2 this data provides critical insight into the compound’s UV absorbance characteristics for accurate quantification.

The absorbance of the further diluted solutions of Solution C, corresponding to Gibtulio Met concentrations ranging from 2 to 10μg/ml, was measured at wavelengths of 225 nm and 235 nm, respectively shown in Figure 2.

Twenty tablets of Gibtulio Met (12.5/500 mg Empagliflozin and Metformin HCl) were weighed and triturated. A portion of the powder, equivalent to 1.25 mg of Empagliflozin and 50 mg of Metformin HCl, was transferred into a 10 ml volumetric flask with 5 ml of a Methanol-Water mixture. The flask was sonicated for 10 minutes, and after adjusting to the appropriate volume, the mixture was filtered using Whatman filter paper. 1 ml of the filtrate was diluted to 10 ml with the Methanol-Water mixture, yielding a solution with concentrations of 500μg/ml Metformin HCl and 12.5μg/ml Empagliflozin. Subsequently, 0.1 ml of this solution was further diluted in a 10 ml volumetric flask with the Methanol-Water mixture to achieve concentrations of 5μg/ml Metformin HCl and 0.125μg/ml Empagliflozin.

A multi-component system consisting of two components X and Y, each of which absorbs at the λmax of the other, λ1 (234 nm) being the wavelength of maximum absorbance of X (Metformin HCl) and λ2 (225 nm) being the wavelength of maximum absorbance of Y (Empagliflozin). Cx and Cy be the concentration of X and Y respectively in the diluted sample. Two equations are constructed based up on the fact that at λ1 and λ2 the absorbance of the mixture is the sum of the individual absorbances of X and Y.

At λ1

At λ2

For measurements in 1cm cell, b= 1, therefore, Rearrange Equation 2

CY= A2-ax2cx/ay2

Cx = (A2ay1-A1ay2) / (ax2ay1ax1ay2) (3)   

Cy = (A1ax2-A2ay2) / (ax2ay1ax1ay2) (4)

The absorptivities of X at λ1 and λ2 = ax1 and ax2 respectively.

The absorptivities of Y at λ1 and λ2, = ay1 and ay2 respectively.

The absorbance of the diluted sample at λ1 and λ2 = A1 and A2 respectively. Cx and Cy be the concentrations of X and Y respectively in the diluted sample.

Linearity (Calibration curve): Calibration curves were constructed by plotting absorbance vs. concentrations of MFN and EMPA, at their respective λ_max, and regression equations were computed. The calibration curves were graphed over five distinct concentrations for MFN ranging from 2 to 10 μg/mL and six distinct values for EMPA ranging from 0.1 to 5μg/mL (Figure 4).

Where, σ = Standard deviation, S= Slope of Calibration curve.

Authors don’t have any conflict of interest.