Type-2 diabetes mellitus is caused by either decreased insulin production from β-pancreatic cells or insulin resistance, where cells do not response to generate insulin. The pancreatic β cells must secrete a considerable amount of insulin. However, functional defects in secretion develops over time resulting in type-2 diabetes mellitus (Magdyet al., 2023). Diabetes develops as a result of risk factors such as obesity, a secondary lifestyle and genetic vulnerability (AlThikrallahet al., 2023).

Metformin (MET) (1,1-dimethylbiguanide) as Shown in Figure 1(a), an oral antidiabetic medication of the biguanide family, (Al-Rimawi F, 2009; Satheesh Kumaret al., 2014) used to manage the type-2 diabetes mellitus (Sha’at Met al., 2022). It stimulates the activity of the 5-Adenosine Monophosphate activated Protein Kinase enzyme (AMPK). AMPK activation enhances insulin sensitivity in body cells, increases glucose consumption and uptake peripherally, decreases glucose absorption from the gastrointestinal tract (Abou-Omaret al., 2021; Gedawyet al., 2019) with an absolute bioavailability of 50-60% reported for a single 500 mg dose. After intake, plasma protein binding is minor and it is eliminated as an unchanged form in the urine (Zarghi et al., 2023).

Figure 1:
Structure of (a) Metformin and (b) Furosemide.

Loop diuretics are a type of medicine that is commonly used in clinical practice to manage excessive fluid loads and maintain fluid balance. The pharmacologic effect of loop diuretics is that prevents the Na+, K+ and 2Cl- cotransporter, which transfuses from the tubular lumen to tubular cells. They block Na+ and Cl- reabsorption in the ascending limb of the loop of Henle, resulting in increased secretion of water, K+, Na+ and Cl- (Chenet al., 2021).

Furosemide (FU) chemically known as 4-chloro-N- furfuryl-5-sulfamoyl-anthranilic acid (Figure 1(b)) is a strong loop diuretic that is widely used to treat edema caused by chronic failure, hypertension or liver cirrhosis (Farthinget al., 1992; Chenet al., 2021). Fu is rapidly but incompletely absorbed after oral treatment and is strongly bound to plasma proteins (>90%) with up to 50% of the medication is eliminated in the urine, primarily as an unchanged drug (AL‐Hashimiet al., 2022).

Several studies have been conducted to detect Metformin in either its single form (Sha’at Met al., 2022; Zarghi et al., 2023) or in combination with other antidiabetic medicines such as Telmisartan (Borse et al., 2022), Pioglitazone (Mohamed AMet al., 2015), Empagliflozin (Abou-Omaret al., 2021), Vildagliptin (Satheeshkumaret al., 2014) and Gliclazide (Gedawyet al., 2019). Similarly, other approaches were used to detect Furosemide in either alone or in combination with other medications such as Spironolactone (Soraet al., 2010), Canrenone (Naguibet al., 2018) and Carbamazepine (AL‐Hashimiet al., 2022).

Metformin is used to treat type-diabetes and Furosemide is also used to treat the hypertension associated with edema state. To the best of our knowledge, no analytical methods have been reported for simultaneously determining Metformin and Furosemide using RP-UFLC method. Therefore, our purpose is to develop and validated a simple and sensitive RP-UFLC analytical method for the simultaneous quantification of Metformin and Furosemide and the developed method was validated in compliance with ICH guidelines.

Metformin (>98.0%) and Furosemide (>99.0%) were provided by Yarrow Chem Products (Mumbai). Methanol (HPLC grade) were supplied by Thermo Fisher Scientific India Pvt. Ltd., (Mumbai) whereas orthophosphoric acid (85w%, HPLC grade) was supplied by LOBA Chemie Pvt. Ltd., and Millipore water (HPLC grade) was provided by in house (B.G Nagar). All the chemicals and solvents were of analytical grade.

For HPLC analysis, ultra-fast liquid chromatography with UV detector, Shimadzu (Japan) was used. The separation was achieved by using C-18 column (5 µm, 4.6×250 mm); mobile phase was consisting of methanol and water containing 0.1% orthophosphoric acid in a 50:50 V/V ratio. The mobile phase was pumped at a flow rate of 1 mLmin^-1 and wavelength was kept at 240 nm. The detection was carried out at 25ºC; overall run time was 10 min with an injection volume was set to 20 µL. The mobile phase was prepared daily and degassed by ultra-sonicator before use. Peak integration and quantification were performed by using Lab-solution software.

To prepare stock and working standard solution, a standard solution of Metformin and Furosemide was prepared by weighing 0.01 g of each drug, diluting with mobile phase and sonicated for 5 min. The volume was then adjusted to achieve a concentration of 1000 µgmL^-1 of each drug. To prepare working solutions, the standard stock solution was serially diluted with mobile phase to achieve a final concentration of 1 µgmL^-1 of both drugs.

After optimization of chromatographic condition, the optimized method should be validated according to ICH guidelines that include linearity, system suitability, LOD and LOQ, accuracy, precision and robustness.

System suitability parameters such as retention time, number of theoretical plates, peak area and tailing factor were evaluated for Metformin and Furosemide by injecting black followed by six replicates of a 2 µgmL^-1 mixture containing two drugs.

Linearity parameter was performed by serial dilution of stock solution by using mobile phase to achieve a concentration ranging from 400-2400 ngmL^-1 for both drugs. Slope, standard deviation of Y-intercept and correlation coefficient of the calibration curve were calculated to ensure that the analytical procedure was linear.

Accuracy study was determined by spiking the marketed drugs with standard solution to get 50%, 100% and 150% concentrations. These mixtures were analysed by the proposed method in triplicate. % recovery, mean, standard deviation and % RSD of spiked drugs were calculated.

Both repeatability and reproducibility were assessed for Metformin and Furosemide. Repeatability was tested by injecting three different concentrations of Metformin and Furosemide in triplicate on the same day under same operational condition. Inter-day precision was determined by comparing the results from three consecutive days.

The linear regression equation was used to calculate the LOD and LOQ for Metformin and Furosemide based on the standard deviation of the Y-intercept and slope.

The parameter has been studied by examining the influence of small but deliberate modification in chromatographic parameters such as mobile phase ratio (altered by ±2%), flow rate (altered by ±0.1 mLmin^-1), wavelength (modified by ±2 nm) and column oven temperature (changed by ±2ᵒC). These chromatographic modifications were tested for resolution between Metformin and Furosemide peak areas, theoretical plate count and tailing factor.

Various mobile phase composition such as acetonitrile: water, methanol: water with 0.1% orthophosphoric acid in different ratios like 50:50, 55:45, 60:40 and 70:30 V/V were used for sensitive and selective determination of Metformin and Furosemide by RP-UFLC method for simultaneous estimation of Metformin and Furosemide. The optimized chromatographic condition was obtain using a C-18 column (5 µm, 4.6×250 mm) and a mobile phase was composed of methanol and water with 0.1% orthophosphoric acid in a 50:50 V/V ratio. The compounds were detected by using a UV detector with a wavelength of 240 nm. The flow rate was kept at 1 mLmin^-1 with an injection volume of 20 µL and temperature was maintained at 25ºC. The entire run time was 10 min and the retention time for Metformin was 1.93 min and for Furosemide was 6.05 min as indicated in Figure 2.

Figure 2:
(A)-Blank chromatogram, (B)-Chromatogram containing only Metformin (1 µg/mL) (C)-Chromatogram containing only Furosemide (1 µgmL^-1), (D)-Chromatogram containing Metformin (1 µgmL^-1) and Furosemide (1 µgmL^-1) with a retention time of 1.93 min and 6.05 min respectively.

Six replicates of the Metformin (1 µgmL^-1) and Furosemide (1 µgmL^-1) mixture were injected to determine the system suitability parameters for peak area, retention time, number of theoretical plates and tailing factor. In order to ensure good efficacy, theoretical plate count for Metformin and Furosemide in all chromatographic run was >1000 for Metformin and >6000 for Furosemide. The tailing factors for the Metformin and Furosemide peak never exceeded 1.339 and 1.392 respectively in all peak indicating good symmetrical peak (acceptance limit <2) and presented in Table 1.

Metformin and Furosemide were shown to have linearity between 400-2400ngmL^-1. The results showed that the correlation coefficient for Metformin and Furosemide were found to be 0.9982 and 0.9989 respectively. From the Figure 3, it was determined that the regression line equations for Metformin and Furosemide y=50.777x-278.04 and y=35.633x-723.32 respectively.

LOD and LOQ were determined from the calibration curve of Metformin and Furosemide by using the formula.

The SD of Y-intercept and slope for Metformin were found to be 1381.2234 and 50.7770 respectively.

The SD of Y-intercept and slope for Furosemide were found to be 767.4846 and 35.633 respectively.

The accuracy of the analytical method was assessed by creating a placebo of the drug formulation in accordance with the formulation procedure. To get concentrations of 600, 800 and 1000 ngmL^-1 (50%, 100% and 150%), a known quantity of standard solution of Metformin and Furosemide was added to the necessary amount of placebo. The results were presented in terms of the % recovery of Metformin and Furosemide from the spiked matrix. The suggested method’s validity was demonstrated by how well the recorded analyte values matched the stated theoretical concentrations at various levels. From the Table 2, it was found that the % recovery for Metformin and Furosemide was 98.68-100.23% and 101.39-105.04% respectively.

The levels of the Metformin and Furosemide at three concentration levels (400, 600 and 800 ngmL^-1) in triplicates were assessed on the same day (repeatability) and in between the days (intermediate precision). According to the Table 3, results shows that the suggested method has good precision and repeatability for both intra and inter-day determination. All data are expressed as % RSD and were never exceeds the acceptable levels, which is <2.

The developed method robustness was examined by checking the effect of small changes in the parameters of the experiment on the chromatographic response. After examining, % RSD reading was not significantly affected by the mobile phase (50±2%), flow rate (1±0.1 mLmin^-1), temperature (25±2ºC) and wavelength (240±2 nm). The robustness of Metformin and Furosemide are shown in Table 4.

A robust and effective RP-UFLC method was successfully developed for the simultaneous determination of Metformin and Furosemide in raw material and pharmaceutical dosage form. The optimized chromatographic conditions provide excellent separation of drugs with a retention time of 1.90 min for Metformin and 6.01min for Furosemide. This developed method shows a good linearity for Metformin (R²=0.9982) and for Furosemide (R²=0.9989) with a good detection and quantification limits (LOD=89.765 ngmL^-1, LOQ=272.017 ngmL^-1) for Metformin and (LOD=71.077 ngmL^-1, LOQ=215.385 ngmL^-1) for Furosemide with a good accuracy and precision (% RSD is <2%). All the results are within the limit as per ICH guidelines. Therefore, the optimized method could be helpful for routine analysis of Metformin and Furosemide in raw materials as well as in pharmaceutical dosage form.

We would like to thank Faculty of Pharmacy, Sri Adichunchanagiri College of Pharmacy, Adichunchanagiri University for providing the necessary apparatus and instrument to perform this study.