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Development and Validation of Q-Absorbance Ratio by UV- Spectrophotometric Method for Simultaneous Estimation of Metformin and Empagliflozin in Bulk and Combined Dosage Form

Advantages of simultaneous stability studies are the identification of new degradation products, to understand mutual induction and/or inhibition of rates of degradation and to analyze the degradation products of both drugs. Various ultraviolet spectroscopic and high performance liquid chromatographic assay methods were reported for the estimation of metformin, sitagliptin, pioglitazone, glimepiride and simvastatin individually and in combination with other drugs. All the above reported methods were based on the estimation of metformin, sitagliptin, pioglitazone, glimepiride and simvastatin alone or in combination with other drugs. The degradation products were generated and successfully separated by the developed and validated high performance liquid chromatographic methods for the estimation of the selected anti-diabetic drug combinations. The aim of the study was to develop and validate of Q-Absorbance Ratio UV-Spectrophotometric Method for Simultaneous Estimation of Metformin and Empagliflozin in Bulk and Combined Dosage Form.


INTRODUCTION:
Type 2 Diabetes mellitus (T 2 DM) is the most prevalent metabolic disease worldwide. Inadequate management and control of hyperglycemia in patients with T 2 DM may lead to the risk of developing complications over the long term due to chronic and progressive nature of the disease arising from pathophysiology of beta-cell dysfunction, insulin resistance and increased hepatic glucose output [1][2][3] . Patients with T 2 DM often require a combination of therapeutic agents in order to achieve glycemic control over the long term. Fixed-dose combination (FDC) therapies have been shown to improve adherence by reducing costs, pill burden, and the complexity of treatment regimen. A treatment approach with a FDC that includes combination of anti-diabetic medications could be used to obtain adequate glycemic control in patients with type 2 diabetes. A combined formulation consisting of metformin, sitagliptin and glimepiride in a single tablet would potentially offer increased patient convenience and subsequent potential for increased therapeutic compliance and can be studied for the treatment of adults with inadequately controlled T 2 DM to improve glycemic control [4][5][6][7] . A clinical trial was conducted for evaluation of sitagliptin in combination with metformin and sulfonylurea. The aim of that clinical trial protocol was to determine the noninferiority of the effectiveness of sitagliptin compared to a control group of patients treated with thiazolidinedione as addon therapy; in low-income ethnic minority type 2 diabetic patients who are failing to maintain adequate control with maximal doses of metformin and a sulfonylurea agent [8][9][10][11][12]  Determination of Iso-absorptive Point and Wavelength of Maximum Absorbance ( max): Solutions of 10µg/mL of Metformin and Empagliflozin were scanned in the range of 200 nm to 400 nm against methanol as blank. The method involved Q-absorption ratio analysis using two wavelengths obtained by the overlay spectrum of the Metformin and Empagliflozin; with one being the maximum absorbance wavelength of Empagliflozin (225 nm, 2) and the other being the isoabsorptive point of both drugs (250 nm, 1).

Preparation of Sample Solutions from Standard Stock Solution:
The sample solutions of various concentrations were prepared from the standard stock solution by diluting aliquots of working stock solutions appropriately.

Calibration Curve (Linearity):
The linearity response was determined by analyzing six independent levels of calibration curve in the range of 5-17.5 μg/mL for each of Metformin and Empagliflozin respectively (n=3). Accurately measured stock solutions of Metformin and Empagliflozin (5.0, 7.5, 10.0, 12.5, 15.0 and 17.5 mL) were transferred to two separate series of 100 mL volumetric flask and diluted up to the mark with methanol. The absorbance of both solutions was taken at their respective max and at iso-absorptive point. The calibration curves were constructed by plotting concentration against absorbance where each data point was an average of three determinations.

Estimation of Standard Laboratory Mixture using proposed method:
The absorptivity coefficient of both the drugs was determined. The individual concentration Method Validation of Metformin and Empagliflozin was determined using the following equations: where, = 2/ 1, = 2/ 1, and = 2/ 1; 1 and 2 are the absorbance of the mixture at 250 nm and 240 nm respectively; 1 and 1 are absorptivities at 250 nm ( 1); 2 and 2 are absorptivities at 225 nm ( 2).

Method Validation
Linearity and Range: Linearity, consisting of the basic elements input → converter → output, is the assumption that there is a straight-line relationship between the input ( ) and output ( ) variables that can be written mathematically by the expression = ( ), if the straight-line crosses through the origin or by the expression = ( ) + , if the straight line does not cross through the origin.
The linear range corresponds to the valid interval of functional dependence of the signal on concentration or mass that assumes homoscedasticity of the measurements over the linear range. The linear response of Metformin and Empagliflozin was determined by analyzing six independent levels of the calibration curve in the range of 5-17.5 g/mL.
Precision: ICH guidelines define the term precision as the closeness of agreement between quantity values obtained by replicate measurements of a quantity under specified conditions [11] . Assessing the precision implies expressing numerically the random error or the degree of dispersion of a set of individual measurements by means of the standard deviation, the variance, or the coefficient of variation.
Repeatability: It is the concordance of a series of measurements of the same quantity when the experiments are conducted under same conditions (analyst, apparatus, instrument, and day) in a rapid succession. Standard solution of Metformin and Empagliflozin (10 g/mL each) was prepared and analyzed six times as per the proposed method.
Intermediate Precision: It is the concordance of a series of measurements of the same quantity when the experiments are conducted within the same laboratory under different conditions (analyst, apparatus, instrument, and day). Standard solution of Metformin and Empagliflozin (10 g/mL each) was prepared and analyzed as per the proposed method.

Accuracy (%Recovery):
The accuracy of an analytical procedure expresses the closeness of agreement between the value that is accepted either as a conventional true value or an accepted reference value and the value found. The recovery experiments were carried out in triplicate by spiking previously analyzed samples with three different concentrations of standards.

Limit of Detection (LOD) and Limit of Quantification (LOQ):
The detection limit of an individual analytical procedure is the lowest amount of analyte in the sample that can be detected but not necessarily quantitated as an exact value. The quantitation limit of an individual analytical procedure is the lowest amount of analyte in the sample that can be quantitatively determined with suitable precision and accuracy. The LOD and LOQ of the proposed method were determined by using calibration curve: Sandell's Sensitivity: Sandell's sensitivity, the concentration of the analyte (in g/mL or g/cm 2 ) that will give an absorbance of 0.001 in a cell of path length 1cm, was calculated. It gives valuable information regarding sensitivity of the method.

RESULTS AND DISCUSSION
The solutions of 10 g/mL of both Metformin and Empagliflozin were analyzed and the max was found to be 225 nm and 240 nm respectively. Two iso-absorptive points: 230 nm and 250 nm were obtained by overlaying the spectra and the iso-absorptive point 250 nm was selected for further analysis.
The calibration curve of Metformin and Empagliflozin individually and the mixture of both drug at 225 nm ( 1) and 250 nm ( 2) were plotted (Figures 1 and 2). The relationship between the absorbance and the concentration of Metformin and Empagliflozin was linear in the range of 5-17.5 g/mL at both wavelengths 225nm and 250nm. The representative linear equations were calculated by the least squares method and the correlation coefficients were linear ( Table 1). Evaluation of repeatability and intermediate precision was done and coefficients of variation (CV) or percent relative standard deviation (%RSD) values were <2%, indicating good precision ( Table 2). Accuracy of the proposed method was calculated by percent recovery in standard addition method. Accuracy ranged between 100.28 and 104.24% for Metformin and 96.88 and 104.06% for Empagliflozin (Table 3). The limit of detection (LOD) of Metformin and Empagliflozin at iso-absorptive point (250 nm) was 0.106 g/mL and 0.078 g/mL; at 225 nm, LOD was 0.186 g/mL and 0.211 g/mL for Metformin and ISSN: 2250-1177 [16] CODEN (USA): JDDTAO Empagliflozin respectively. The limit of quantification (LOQ) of Metformin and Empagliflozin at iso-absorptive point (250 nm) was 0.321 g/mL and 0.238 g/mL; at 225 nm, LOQ was 0.563 g/mL and 0.639 g/mL for Metformin and Empagliflozin, respectively.
Sandell's sensitivity was 0.0347 and 0.0348 g/cm 2 at 250 nm and 0.022 and 0.035 g/cm 2 Simultaneous Estimation of Metformin and Empagliflozin in Bulk and Combined Dosage Form at 225 nm for Metformin and Empagliflozin, respectively. Various validation parameters are summarized in Table 4.

CONCLUSION:
The UV spectrophotometric Q-absorption ratio method was developed and validated for the simultaneous analysis of Metformin and Empagliflozin. The results together established that the method is simple, accurate, precise, reproducible, rapid, and sensitive. The method can be applied successfully and economically for the simultaneous estimation of Metformin and Empagliflozin in bulk and in the synthetic laboratory mixture and also for the quantitation of generic equivalents in pharmaceutical industries.