Abstract
The prevalence of diabetes is increasing day by day as per a report by the year 2045, 1 out of every 8th individuals may suffer from diabetes. This research article focuses on developing and validating Metformin and Dapagliflozin in combination by using high-pressure liquid Chromatography (RP-HPLC). The validation of the method was followed as per the guidelines provided by the International Conference on Harmonization (ICH) and United States Pharmacopeia (USP). Separation of both drugs takes place in less than 4 min. This separation takes place using Phosphate buffer (pH 6.8) and acetonitrile in a 45:55 (v/v) ratio at a 1.0 mL min−1 flow rate. Furthermore, studies on both drugs were conducted by using the bulk and pharmaceutical dosage forms (Tablets). The developed method was accurate as drug recoveries in both cases of Metformin, and Dapagliflozin ranged between (100.8, 99.6, 98.8%) to (100.8, 99.3, and 101.5%) respectively having a concentration range of solutions between 70, 100 and 130 μg mL−1 dilution. The recommended method for simultaneous quantification of Metformin and Dapagliflozin was established and validated and no excipient interactions were found.
Introduction
The use of medicines was started in ancient times. With human evolution, the process of taking medicines was also revolutionized. Over the last few decades, more work has been done in the field of the Pharmaceutical industry with special emphasis on increasing effectiveness, safety, and reducing side effects. The work was done to facilitate patients and improve their compliance with taking medication. The importance of combining active pharmaceutical ingredients is increasing day by day. So, such products aimed to reduce side effects, patient tolerance towards the use of medicines, and increase drug effectiveness. As per the report of the International Diabetes Federation about 382 million people throughout the world were affected by Diabetes Mellitus (DM). and still, the count is not going to slow down by the end of 2035 it is estimated that the number would be raised to 592 million.
DM is an endocrinological disorder that results due to irregularity in the insulin action, secretion of insulin, or both. This reduced insulin causes a permanent abnormality in glucose tolerance and high blood sugar levels. DM is considered the oldest human disease. From the 14th century, it was titled black death. It is mainly categorized into two categories when the pancreas is not able to produce enough insulin to meet the needs of the body and control sugar levels, termed Insulin-dependent diabetes mellitus. In this type of diabetes, the patient is completely dependent upon the parenteral form of insulin. Another type of DM is Non-insulin-dependent diabetes, In such type of diabetes, the body cells become resistant to insulin which is further treated with Biguanides, Sulfonylurea, and carbohydrate analogs the oral anti-diabetic drugs [1].
It is believed that type 2 DM is provoked by excessive nutrient diet consumption because more than 90% of the patients in Western countries are overweight or obese [2]. DM is the foremost reason for many serious problems such as renal and cardiovascular diseases. Microvascular (neuropathy, nephropathy, and retinopathy), hypertension, chronic hyperglycemia, and macrovascular (stroke, coronary artery disease) were regarded as distinctive properties of DM [3].
Non-adherence to medications is also a foremost reason for poor glycemic control in patients suffering from type 2 DM. That non-adherence leads to the progression of the disease, patient hospitalization, and even death. The cost of the medicines was so high that sometimes it became impossible for the patient to adhere to the dosage regimen. Benner et al. assessed a study of 6,000 patients who were admitted to hospitals and found an inverse relationship between the number of medicines taken and medication adherence [4].
The oral route of drug administration is still considered the most favorable route of administration [5]. Where a majority of the medications were administered in the form of capsules or tablets [6]. These dosage forms were prepared in a sequence of processes in which excipients were mixed with active pharmaceutical ingredients (APIs) to enhance the ease of their administration and to improve their manufacturing process [6]. A lot of quality control attributes were designed to ensure control, monitoring, and detection of any variations in the manufacturing processes [6].
So, the American Diabetic Federation (FDA) and the International Diabetic Federation (IDF) suggest Fixed dose combination therapy (FDC) in which two or more two APIs are added in fixed ratios. The advantages of FDC are to achieve desired therapeutic effects, ease of administration, synergistic effect, convenience, improved glycemic control, and decreased side effects [1].
Various clinical trials support that the patients who were prescribed FDC had higher drug adherence rates. Extended-release medications were preferred over immediate or multiple-dose administration. Lastly, dosage forms that contain more than one API improve patient adherence and satisfaction. In that regard, the use of metformin and dapagliflozin improves patient compliance and adherence to medication [4].
Dapagliflozin (DAPA) is chemically defined as (1s)-1, 5-anhydro-1-C-[4-chloro-3-[(4-ethoxy phenyl) methyl] phenyl]-D-glucitol as represented in Fig. 1 [7].
Chemical structure of Dapagliflozin [8]
Citation: Acta Chromatographica 2025; 10.1556/1326.2024.01226
Dapagliflozin is an oral anti-diabetic agent, used for the management of type 2 DM due to its prolonged effects on reducing glycated and glycemia hemoglobin. Baker et al. in their systematic review described that dapagliflozin also causes a decrease in systolic blood pressure by 4–5 mmHg. Jayawardene et al. described that dapagliflozin decreases triglyceride concentrations and increases high-density lipoprotein cholesterol [9].
Metformin hydrochloride is chemically identified as (3-(diamino methylidene)-1, 1-dimethylguanidine; hydrochloride is represented in Fig. 2. Metformin has a molecular weight of 165.62 g mol−1 and has the molecular formula of C4H11N5 [11]. Metformin belongs to biguanide a class of anti-hyperglycemic agents and is used for the management of type 2 DM [12]. It is useful in treating type 2 DM, as it decreases the intestinal absorption of glucose, and hepatic glucose production, and improves insulin sensitivity by increasing utilization and uptake of peripheral glucose. All of these properties were initiated by the primary stimulation of AMP-activated protein kinase, which is a liver enzyme and plays an important part in insulin signaling, metabolism of fats and glucose, and whole body energy balance [11].
Chemical structure of metformin [10]
Citation: Acta Chromatographica 2025; 10.1556/1326.2024.01226
Combination therapy with dapagliflozin and metformin has the advantage that both APIs have a distinct mechanism of action in the kidney and liver and so lower the levels of glucose. This combination appeared to be helpful in patients with type 2 DM because they appeared to have a low risk of getting hypoglycemia as compared to other agents. Dapagliflozin is rapidly absorbed after oral administration and reaches systemic circulation rapidly. Such exposure to the drug is not affected by food or other factors [13].
Materials and method
Metformin (100.35% purity) was gifted by (Wanbury, Maharashtra, India), while Dapagliflozin (100.13% purity) was gifted by (Fuxin Long Rui Pharmaceutical C0., Ltd, China). Bulk tablets (Xiga-Met, CCL Pharmaceuticals) were purchased from the market. Potassium dihydrogen phosphate was obtained from Merck KGaA, Acetonitrile was purchased from Fisher Chemical, Potassium hydroxide was obtained from (KOH) Merck KGaA, and Methanol was purchased from Fisher Chemical. All of the used chemicals were of analytical grade.
Instrumentation
Chromatographic conditions
The analysis was performed by using the 1,260 infinity II LC system, (Agilent Technologies, USA), having software open lab CDS version 2.4. C 18 column of (Agela Technologies, USA) having dimensions (250 mm × 4.6 mm; 5 μm) was used for analytical purposes. The mobile phase was formulated by using buffer (Phosphate buffer pH 6.8) and acetonitrile in a 45:55 (v/v) ratio respectively. The mobile phase was injected at a flow rate of 1.0 mL min−1 having an injection volume of 10 µL. While the wavelength was set at 220 nm. The temperature of the HPLC was set at 25 °C.
HPLC analysis
An attempt has to be made to prepare a method that was precise, simple, and accurate for the simultaneous determination of Dapagliflozin and Metformin by using reverse-phase high-pressure Liquid Chromatography (RP-HPLC).
Method development and validation in the mobile phase
Preparation of phosphate buffer (pH 6.8) for mobile phase
Accurately weigh 6.8 g of potassium dihydrogen phosphate by using an analytical balance (Mettler Toledo ME204 ME Series Balance, Ohio, USA) then transfer it to a 1,000 mL beaker and dissolve it in 1,000 mL of HPLC grade water. Adjust the pH of the solution to 6.8 with KOH.
Preparation of standard solution
Solution A
Accurately weigh 40 mg of Dapagliflozin on an analytical balance and shift it to a volumetric flask (capacity 200 mL) up to the final volume with Acetonitrile, and sonicate for 5 min. This constitutes solution A.
Solution B
Weigh accurately 40 mg of Metformin HCL on an analytical balance and transfer it to a 200 mL volumetric flask, add 20 mL of methanol, and sonicate for 5 min and this constitutes solution B.
Preparation of sample solution
First of all, take 10 tablets and weigh them with the help of an analytical balance. The tablets were crushed by using mortar and pestle. The powder obtained was again weighed by using analytical balance and sufficient powder which is equivalent to 250 mg metformin and 2.5 mg Dapagliflozin was transferred to a 100 mL volumetric flask and finally diluted with 50 mL methanol. The mixture was sonicated for 15 min. It is again diluted with methanol and again sonicated it for another 30 min. Take 2 mL from this solution into a 25 mL volumetric flask dilute up to the mark with buffer and shake well.
Procedure
Separate chromatograms of the sample and standard were obtained by injecting equal volumes of 10 µL. The areas of the major peaks were recorded and the percentage of Dapagliflozin and Metformin HCL were calculated.
System suitability
Two replicates of the sample and six replicates of standards were injected to verify the precision and accuracy of the chromatographic system. Different parameters of system suitability which include retention time, resolution, column theoretical plates, and tailing factor were studied.
Linearity
The linearity of both drugs was determined by plotting a graph between peak area and drug concentrations. Different concentrations of Metformin and Dapagliflozin were injected into the HPLC system to construct a linearity curve and determine the coefficient of correlation (R2). The column of HPLC was equilibrated with the mobile phase for 30 min before the start of the experiment [14].
Precision
The precision of the method was established by injecting 6 samples at a level of 100% concentration. The value of % RSD for 6 samples must be below 2.0%.
Robustness
Robustness was established by changing the composition of the mobile phase, pH of the mobile phase, and flow rate. Each of the varied conditions was studied thoroughly to ensure the system's suitability. The developed method would be proven suitable only if all the varied conditions complied with the criteria of the system's suitability [8].
Accuracy
Different concentrations (80, 100, and 120%) of Metformin and dapagliflozin were constructed to study the recovery studies [15].
Limit of detection and quantitation (LOD/LOQ)
Specificity
Specificity studies were conducted to estimate the actual separation of analyte from placebo, and all other related peaks. The placebo solution usually consists of all excipients employed for dosage form manufacturing [7].
Results
Controlling pH is considered an important factor because it directly reflects the symmetry, splitting, and broadening of the chromatogram peaks. The pH directly impacts the retention time as it manages both protonated and non-protonated species. The most suitable and readily employed pH ranges between 2 and 7. The selection of buffer is also an important factor so highly compatible, readily available, and cheap buffer is selected. Most commonly phosphate buffers were used in HPLC analysis [17].
Another factor that determines how fast a mobile phase travels in a column is the flow rate. Flow rate is responsible for the utilization of the mobile phase. Retention time is defined as the time of elution of the sample after it is injected. It depends upon the column used, rate of flow, and mobile phase used [18] (Fig. 3).
Chromatogram of metformin HCL and Dapagliflozin
Citation: Acta Chromatographica 2025; 10.1556/1326.2024.01226
Different trials using buffer, water, and organic solvents (methanol, acetonitrile) were conducted. Their flow rate varied ranging from 0.5 to 1.5 mL min−1 as represented in Table 1. With an increase in flow rate, there appears a decrease in retention time [19]. It is observed from Table no 1 that, a decrease in retention time was observed as the flow rate increases in both the drugs. For better elution, the wavelength was adjusted to 220 nm.
Optimized flow rate and pH
| Parameter | Used | Retention time of metformin | Retention time of dapagliflozin |
| Flow Rate | 0.5 mL min−1 | 2.567 | 4.26 |
| 1.0 mL min−1 | 1.955 | 3.602 | |
| 1.5 mL min−1 | 1.56 | 3.251 | |
| pH | 2 | 1.103 | 2.96 |
| 3 | 1.43 | 2.45 | |
| 4 | 1.52 | 2.432 | |
| 4.6 | 1.631 | 2.523 | |
| 5 | 1.85 | 3.5 | |
| 6.8 | 1.957 | 3.662 | |
| 7 | 2.01 | 3.81 |
Method validation
Linearity and range
Sample solution preparation
An accurate amount of both the drugs i.e. Metformin and Dapagliflozin was weighed by using an analytical balance after that dilution of both the drugs was prepared in such a way that for Metformin the concentration was 40–200% considering 100% is equal to 40 mg of the drug and from that stock solution separate dilution of 80–400 ppm was prepared for Metformin and 0.8–4 ppm for Dapagliflozin. The prepared dilutions were injected into the HPLC for further analysis the peak area of each dilution was determined and finally the determined concentrations were plotted against the peak area. Metformin and Dapagliflozin both of the drugs were separately identified quite satisfactorily and successfully. Metformin represents a retention time of 1.957 while, Dapagliflozin at 3.662 min respectively. The linearity of both drugs was evaluated between 50 and 250 μg mL−1 concentration. Both Metformin and Dapagliflozin showed significant linearity values of R2 0.9934 and 0.9996 respectively.
Precision
Precision was estimated as both precision and repeatability. Samples were analyzed in a replicate manner on the same day and their RSD was estimated to analyze repeatability. Regarding the calculation of precision same sample was investigated on two different days, and columns, equipment, and differences were calculated (Tables 2 and 3).
Precision studies of Metformin HCl and Dapagliflozin regarding the developed method
| Injections | Metformin HCl average standard AUC | Dapagliflozin average standard AUC | Metformin HCl average sample AUC | Dapagliflozin Average sample AUC |
| 1 | 11274.7 | 114.1 | 11862.2 | 128.1 |
| 2 | 11210.5 | 114.4 | 11853.6 | 128.2 |
| 3 | 11246.7 | 114.2 | 11847.8 | 128.2 |
| 4 | 11266.1 | 114.3 | 11825.7 | 128.0 |
| 5 | 11271.9 | 114.5 | 11852.6 | 128.2 |
| 6 | 11276.6 | 114.5 | 11845.9 | 127.9 |
| Mean | 11257.8 | 114.3 | 11848.0 | 128.1 |
| SD | 25.56 | 0.26 | 12.31 | 0.13 |
| % RSD | 0.06 ± | 0.03 | 0.12 | 0.07 |
Recovery studies of metformin HCl and Dapagliflozin after replicated injection (n = 6)
| Drugs | % Solution | Amount recovered (μg mL−1) | % Recovery | SD | % RSD |
| Metformin HCl | 70 | 143.57 | 100.8 | 16.605 | 0.21 |
| 100 | 200.63 | 99.6 | 11.053 | 0.1 | |
| 130 | 256.28 | 98.8 | 32.481 | 0.23 | |
| Dapagliflozin | 70 | 1.76 | 100.8 | 0.1513 | 0.19 |
| 100 | 2.48 | 99.3 | 0.1683 | 0.15 | |
| 130 | 3.33 | 101.5 | 0.4078 | 0.27 |
Limit of Quantitation (LOQ) and Limit of Detection (LOD)
Robustness
Method robustness was estimated to evaluate the impact of deliberate variations within the chromatographic conditions on the determination of Metformin HCl and Dapagliflozin Propanediol. Robustness was determined by varying the rate of flow by ±15%, Change in Column Temperature by ±5 °C, Mobile Phase Composition Variation by ±3%, and the pH of buffer in the mobile phase by ±0.2.
Accuracy
Each sample recovery should lie within the range of 98.0%–102.0%. Acceptance criteria for accuracy are met, hence the method is accurate.
Repeatability
Six independent samples and standards were prepared and were performed using the analytical procedure and their percentage recovery was determined.
Acceptance criteria
The % RSD of the assay must not be more than 2.0%. % RSD for repeatability of Metformin HCl and Dapagliflozin is 0.26% and 0.40% respectively (i.e. less than 2.0%) hence repeatability is accepted.
Limit of Quantitation (LOQ) and Limit of Detection (LOD)
In the case of Metformin LOD was 23.4 ppm, and LOQ 70.9 ppm. While for Dapagliflozin LOD and LOQ were 0.05 and 0.17 ppm respectively.
Specificity
Drug samples were exposed to base, oxidizing agent, and acid. Peaks were then observed regarding any presence of degradation in the sample.
Acceptance criteria
The resolution should be more than 2.0.
The peak purity of the degraded spiked placebo sample, the principal peak is NLT 990. Resolution was found to be more than 2.0 (See chromatograph). The purity factor of Metformin HCl is 999.096 which is within the calculated threshold limit. The purity factor of Dapagliflozin Propanediol is 999.760 which is within the calculated threshold limit (Table 4).
Calculated theoretical plate, tailing factor, resolution, and retention time of both drugs
| Drugs | Mean | % RSD | Theoretical plated | Tailing factor | Resolution | Retention time | SD |
| Metformin HCl | 11,632 | 0.46% | 2,991 | 0.91 | 5.1 | 1.957 | 53.49 |
| Dapagliflozin | 166 | 0.36% | 10,443 | 0.98 | 9.63 | 3.662 | 0.59 |
| USP recommendation | <2 | >2,000 | 0.8–1.5 | >2 |
Stability of the mobile phase
The mobile phase and the prepared solutions showed better stability under the provided circumstances. The accuracy of the method accuracy was in the range of 99–101% for Metformin and Dapagliflozin respectively.
Discussion
It was observed from the obtained chromatogram that the highest peaks of Metformin were observed as compared to Dapagliflozin. Solutions of the same concentration were constituted for both drugs. The possible reason for the highest peak observed for Metformin was due to high absorption compared to Dapagliflozin at the selected wavelength for the quantification of both drugs. Alkaline pH is not recommended in HPLC as high pH reduces the column's life and may damage the HPLC silica support. As the concentration of acetonitrile increases a shorter retention time could be observed [20].
Meanwhile, two important factors in HPLC are retention time and flow rate. Retention time depends upon the mobile phase's polarity, flow rate, pH, and composition. Flow rate and retention time both have an inverse relation. Similar types of work were performed by Nasser, S., et, al [21], and Darshak Patel [22] separately, but in both these research papers a high retention time of 2–8 min, was observed. An increase in retention time leads to more consumption of analytical solvents. It also leads to more time-consuming. In this research, we address both issues and we can obtain results with minimum solvent usage and in less time. Peak symmetry, tail factor, and theoretical plate number were the important factors to be adjusted which are directly related to flow rate and retention time.
The developed method is specific and accurate as no extra peak was observed. The present method is stable, highly precise, and simple. So, it suggests that the method is validated and can be further used to quantify both drugs in a bulk form and in dosage form.
Conclusion
To enhance patient compliance and reduce dosing frequency bi-layer tablets of Metformin and Dapagliflozin were prepared. Combination therapies have always attractive therapies for patients as they reduce dosing frequency and increase patient compliance. A simultaneous method for quantification of both drugs was ensured by using HPLC. Acetonitrile was frequently used in RP-HPLC as a solvent for better elution determinations. Water was used as a polar and acetonitrile as a non-polar solvent on the mechanism of like dissolves like. The selection of the mobile phase was based on the most optimized chromatograms. Finally, the proposed method is possibly used for the determination of both drugs simultaneously. A validated method has been developed and validated for the simultaneous determination of both drugs in tablets furthermore the developed method was found to be precise, accurate, and robust and could be used in any pharmaceutical industry for commercial purposes.
Acknowledgment
The authors thank CCL and Riphah International University for providing research facilities and raw materials.
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