Development And Validation of RP-HPLC Method For Simultaneous Estimation of Dapagliflozin Propanediol Monohydrate and Bisoprolol Fumarate in Synthetic Mixture

The chemical name of Dapagliflozin Propanediol Monohydrate is (2S)-propane-1,2-diol (2S,3R,4R,5S,6R)-2-{4-chloro-3-[(4-ethoxyphenyl)methyl]phenyl}-6-(hydroxymethyl) oxane-3,4,5-triol hydrate and Bisoprolol Fumarate chemical name is (2E)-but-2-enedioic acid; bis(1-[(propan-2-yl) amino]-3-(4- {[2-(propan-2 yloxy)ethoxy] methyl}phenoxy)propan-2-ol). Dapagliflozin Propanediol Monohydrate is the salt form of Dapagliflozin, which belongs to the  Anti-Diabetic drugs. Dapagliflozin Propanediol Monohydrate that inhibits sodium-glucose transporter 2. They lower blood sugar by preventing the reabsorption of glucose by the kidney and are used in the treatment of type 2 diabetes mellitus. It has a molecular formula C₂₄H₃₅ClO₉ with molecular weight 502.99 g/mol. Another drug is Bisoprolol Fumarate is the salt form of Fumaric acid, which belong to the Beta-1 blocker. Bisoprolol is a Cardio selective β1-adrenergic antagonist. It blocks the β1 receptors and has a greater affinity for β1 receptors than β2 receptors. β1 receptors are primarily located in the heart. And also present in the juxtaglomerular cells of the kidneys. By inhibiting these receptors, Bisoprolol reduces the release of renin, reduces cardiac workload by decreasing contractility and reduction in blood pressure causing negative chronotropic and inotropic effects. It has a molecular formula C₁₈H₃₁NO₄)₂. C₄H₄O₄[with molecular weight 766.9582 g/mol. The purpose of this study is to develop a simple, precise and accurate RP-HPLC method for the simultaneous estimation of Dapagliflozin Propanediol Monohydrate and Bisoprolol Fumarate in synthetic mixture and to validate the developed method with study of different parameters as per ICH guidelines as no reported RP-HPLC method for simultaneous estimation of Dapagliflozin Propanediol Monohydrate and Bisoprolol Fumarate in synthetic mixture.

MATERIALS AND METHOD:

MATERIALS

Table 1.0 Materials & Sources

Instrumentation

• HPLC instrument with UV-visible detector
• Software: LC Solution
• Column - HYPERSIL ODS C18, 250 mm*4.6 mm
• UV-visible Spectrophotometer - SHIMADZU 1800 Software - UV probe, version- 2.42
• Digital Analytical Balance - Mettler Toledo
• Ultra Sonicator -PS 21
• Controlled temperature water bath
• Hot air Oven – Kesar
• FTIR – SHIMADZU
• Melting Point Apparatus – Gallenkamp Model: 889339

Chemicals and Reagents: Dapagliflozin Propanediol Monohydrate API, Bisoprolol Fumarate API, Acetonitrile HPLC Grade, Methanol HPLC Grade, Water: Distil water (Milli-Q), HPLC Grade water.

Preparation of Mobile Phase:  RP-HPLC method was followed by isocratic elution technique. Mobile phase comprised of Acetonitrile: Water (75:25 v/v/v %) ratio because it elutes both drugs peak efficiently in short time with satisfactory resolution, tailing factor and theoretical plates.

Preparation of Standard Stock Solution A: (Dapagliflozin Stock Solution):

Accurately weighed quantity of Dapagliflozin 10 mg was transferred into 10 mL volumetric flask, dissolved in methanol and diluted up to mark with methanol. This will give a stock solution having strength of 1000 µg/ml.

Preparation of Standard Stock Solution B: (Bisoprolol stock solution):

Accurately weighed quantity of Bisoprolol 10 mg was transferred into 10 mL volumetric flask, dissolved in methanol and diluted up to mark with methanol. This will give a stock solution having strength of 1000 μg/mL.

Preparation of standard solution:

Further, dilute 1 mL of standard stock solution A and 0.5 mL of standard stock solution B in to 10 mL of volumetric flask and makeup the volume upto the mark with diluent and mixed well. (Dapagliflozin: 10 μg/mL and Bisoprolol :5 μg/mL.)

Chromatographic Conditions:

Table 2.0 Chromatographic Conditions

Identification And Characterization

The identification of taken standard API for experimental work had done for confirmation of its identity, standard quality and purity. The identification had done by taking IR and UV spectra, solubility study and melting point determination.

Solubility Study:

The solubility of Dapagliflozin & Bisoprolol practically determined separately by taking 100 mg of both the drugs in 100 ml volumetric flasks, adding required quantity of solvent at room temperature and shaken for few minutes. Solubility data for each study was observed and recorded in Table 4.0.

Table 3.0 Solubility Table

Table 4.0 Solubility Data for Dapagliflozin & Bisoprolol

Identification by Melting Point Determination:

Melting point of Dapagliflozin & Bisoprolol hydrochloride has been determined. The melting points of the compounds were taken by open capillary method.

Table 5.0 Melting Point of Drugs

IR Spectra:

The IR Spectra of Dapagliflozin with its functional group identification, were shown in the following graph. IR Spectra scanning of sample: Dapagliflozin.

Table 6.0 IR Spectra Interpretation for Dapagliflozin

The IR Spectra of Dapagliflozin with its functional group identification, were shown in the following graph. IR Spectra scanning of sample: Bisoprolol.

Table 7.0 IR Spectra Interpretation for Bisoprolol

Method Development

Selection Of Wavelength:

To determine wavelength for measurement, standard spectra of Dapagliflozin & Bisoprolol were scanned between 200-400 nm against diluents. Absorbance maxima of Dapagliflozin & Bisoprolol have detected at 270. Chromatogram was taken at 270 nm, both drugs give good peak height and shape. So, 270 nm was selected for Simultaneous estimation of Dapagliflozin & Bisoprolol in their formulation.

Selection Of Column:

For RP-HPLC Method, various columns are available but based on literature survey C-18 (id 4.6 x 250 mm, 5 µm) was selected over the other columns.

Selection of Mobile phase:

Table 8.0 Trial 1: Selection of Mobile Phase

Table 9.0 Trial 2: selection of mobile phase

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            <img alt="Fig 5.0 Trial 2.png" height="150" src="https://www.ijpsjournal.com/uploads/createUrl/createUrl-20250609191229-17.png" width="150">
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Fig 5.0 Trial 2: Chromatogram of Dapagliflozin & Bisoprolol Acetonitrile: Phosphate Buffer (50:50v/v)

Table 10.0 Trial 3: Selection of Mobile Phase

Table 11.0 Trial 4: Selection of Mobile Phase

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            <img alt="Fig 7.0 Trial 4.png" height="150" src="https://www.ijpsjournal.com/uploads/createUrl/createUrl-20250609191229-15.png" width="150">
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Fig 7.0 Trial 4: Chromatogram of Dapagliflozin & Bisoprolol Ethanol: Phosphate Buffer(60:40v/v)

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            <img alt="Fig 8.0 Trial 5.png" height="150" src="https://www.ijpsjournal.com/uploads/createUrl/createUrl-20250609191229-14.png" width="150">
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Fig 8.0 Trial 5: Chromatogram of Dapagliflozin & Bisoprolol Ethanol: Phosphate Buffer(70:30v/v)

Table 14.0 Trial 7: Selection of Mobile Phase

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            <img alt="Fig 10.0 Trial 7.png" height="150" src="https://www.ijpsjournal.com/uploads/createUrl/createUrl-20250609191229-12.png" width="150">
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Fig 10.0 Trial 7: Chromatogram of Dapagliflozin & Bisoprolol Chloroform: Acetonitrile (70:30 v/v)

Table 15.0 Trial 8: Selection of Mobile Phase

Table 16.0 Trial 9: selection of mobile phase

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            <img alt="Fig 12.0 Trial 9.png" height="150" src="https://www.ijpsjournal.com/uploads/createUrl/createUrl-20250609191229-10.png" width="150">
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Fig 12.0 Trial 9: Chromatogram of Dapagliflozin & Bisoprolol Chloroform: Acetonitrile (60:40 v/v)

Table 17.0 Trial 10: Selection of Mobile Phase

Table 18.0 Trial 11: Selection of Mobile Phase

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            <img alt="Fig 14.0 Trial 11.png" height="150" src="https://www.ijpsjournal.com/uploads/createUrl/createUrl-20250609191229-8.png" width="150">
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Fig 14.0 Trial 11: Chromatogram of Dapagliflozin & Bisoprolol Acetonitrile: Water(50:50v/v)

Table 19.0 Trial 12: Selection of Mobile Phase

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            <img alt="Fig 15.0 Trial 12.png" height="150" src="https://www.ijpsjournal.com/uploads/createUrl/createUrl-20250609191229-7.png" width="150">
        </a>
Fig 15.0 Trial 12: Chromatogram of Dapagliflozin & Bisoprolol Acetonitrile: Water (80:20v/v)

Table 20.0 Trial 13: Selection of Mobile Phase

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            <img alt="Fig 16.0 Trial 13.png" height="150" src="https://www.ijpsjournal.com/uploads/createUrl/createUrl-20250609191229-6.png" width="150">
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Fig 16.0 Trial 13: Chromatogram of Dapagliflozin & Bisoprolol Acetonitrile: Water (75:25 v/v)

Chromatographic conditions for optimized mobile phase trial (Bisoprolol):

Table 21.0: optimized mobile phase trial of Bisoprolol

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            <img alt="Fig 17.0.png" height="150" src="https://www.ijpsjournal.com/uploads/createUrl/createUrl-20250609191229-5.png" width="150">
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Fig 17.0: Optimized mobile phase trial for optimized chromatogram of Std. Bisoprolol: 6.115 min

Table 22.0: optimized mobile phase trial of Dapagliflozin

Table 23.0: optimized mobile phase trial of Dapagliflozin & Bisoprolol

Introduction Of Method Validation Parameter:

Method validation is the process of documenting or proving that an analytical method provides analytical data acceptable for the intended use. The need to validate a method and the procedure to be followed are matters of professional judgement, although well-prescribed procedures and guidelines are now available that aid in decision making. According to that the various validation parameters to validate each and every above stated method are:

• Accuracy and Recovery
• Precision (Repeatability and Reproducibility)
• Linearity
• Range
• Robustness/ Ruggedness
• Limit of Detection (LOD)/ Limit of Quantification (LOQ)

Accuracy and Recovery:

The capability of a procedure to generate outcomes close to the actual or standard value (Standard value may be reference value given in official compendia).  The chosen concentration for precision investigations must encompass the complete concentration range (i.e. one may the lowest concentration, one may the middle and one may be the last of range).

Accuracy is performed by performing recovery studies by spiking in 2 ways:

1. Spiking of sample with standard (in case placebo are not available).
2. Spiking of placebo with standard.

It can be performed at different level like 50, 100 and 150 % of test concentration or 80, 100, 120% of test concentration.

Reproducibility and Precision:

It is defined as the degree of similarity between test findings obtained from many samplings of the same sample. C.V. or relative standard deviation is capable of expressing it is further classified as reproducibility, repeatability, and intermediate precision.  Repeatability is the capability of an analytical procedure to produce identical test findings when performed in a similar setting by the same operator over a brief period of time. It is stated as R.S.D. when a minimum of three concentrations is analysed at least six times each, and R.S.D. is determined.   At least 6 repetitions encompassing 100% of the target concentration or 9 repetitions covering the complete linear range must be assessed. (i.e., three replications for every concentration). Reproducibility is the capacity of an analytical procedure to yield same test findings when applied to identical samples under various conditions and by different analysts. Conditions of operation vary, but the variance is still within acceptable parameters. It is a crucial validation parameter if the procedure must be executed under diverse situations.

Range and Linearity:

It is the capability of the method to generate findings that are directly proportional to the concentration of a specific component in the samples. It is shown via certain mathematical modifications.

1. It may be conducted directly on the material being examined or using a suitable standard and dilution using the suggested approach.
2. It can be accomplished by measuring at least five injections in a series of standards. Eighty to one hundred twenty percent of the recommended concentration range is permissible.
3. A mathematical equation must illustrate the reaction of the sample to the concentration.
4. The linear regression approach requires a substantial zero intercept, and if it is not reached, one must demonstrate that there is no significant influence on accuracy. 
5.  Another method that may be applied is the response factor, which is generated by dividing the response by its corresponding concentration to obtain the relative response. On a logarithmic scale, a graph showing relative reaction against concentration is presented. Therefore, the resultant horizontal line must be linear over the full range. Negative deviation is often observed at greater doses. On the graph, a horizontal line is drawn parallel to the x axis to represent 95 to 105 percent of the horizontal line. The reaction is linear with respect to concentration up until the intersection of the horizontal line and the 95 percent line.

It is the concentration interval between which quantitative analysis may be done with appropriate precision and linearity. The analytical method's range is stated in the same units (such as PPM or %).

Robustness:

A technique has the ability to be unaffected by tiny, purposeful changes to operating settings, but these changes are still within the method's range. The effects of these adjustments on the method's output are evaluated. By conducting robustness, one may assess if revalidation is necessary or not. Variable technique parameters include flow rate, mobile phase composition, mobile phase pH, and detection wavelength. This modification can be within the permissible range (i.e., 2% to 5% of the original value). The essential method parameter that identifies the susceptibility of the technique to

change must be reported following ICH rules. However, it is not a registration requirement.

Robustness:

A technique has the ability to be unaffected by tiny, purposeful changes to operating settings, but these changes are still within the method's range. The effects of these adjustments on the method's output are evaluated. By conducting robustness, one may assess if revalidation is necessary or not. Variable technique parameters include flow rate, mobile phase composition, mobile phase pH, and detection wavelength. This modification can be within the permissible range (i.e., 2% to 5% of the original value). The essential method parameter that identifies the susceptibility of the technique to change must be reported following ICH rules. However, it is not a registration requirement.

Limit of Quantification and Limit of Detection:

It refers to the analytical method's capacity to identify analyte in the presence of a matrix with sufficient accuracy and precision. The method may be able to detect the analyte but not quantify it. There may be confusion between technique sensitivity and LOD. Sensitivity can distinguish between minute differences in concentration. It may be described as the slope of the regression line.

Following methods are available other than signal to noise ratio that are as follows:

1. Visual determination: it is possible to determine by personally injecting the lowest known concentration that is detectable but cannot be measured by mathematical manipulations.
2. Standard deviation of response based on standard deviation of blank: it may be determined by examining the number of blank samples and determining the standard deviation of the blank answers. This approach suggests the capability of a method to provide a response other than analyte concentration.
3. Using slope of regression line to estimate standard deviation of response: one may measure the S.D. of regression line of a given calibration curve or use S.D. of intercept.
4. It is also calculable using the mathematical formulae shown below.

RESULT AND DISCUSSION:

Analytical method Validation

Linearity

For the purpose of linearity, accurately weighed amount of Dapagliflozin (10 mg), and Bisoprolol (10 mg) was taken into the volumetric flask (10 ml) and volume of the flask was raised to 10 ml with methyl alcohol to give stock solution containing 100 µg/ml of Dapagliflozin, and 100 µg/ml of Bisoprolol. Various aliquots from this stock solution were transferred to another 10 ml volumetric flask and volume was raised to the mark with mobile phase to give final solutions containing 5+2.5, 7.5+3.75, 10+5, 12.5+6.25 µg/ml and 15+7.5 µg/ml of Dapagliflozin and Bisoprolol respectively.

Table 25.0 Linearity Data for Bisoprolol

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            <img alt="Fig 20.0.png" height="150" src="https://www.ijpsjournal.com/uploads/createUrl/createUrl-20250609191229-2.png" width="150">
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 Fig 20.0: Overlain Linearity Spectra of Dapagliflozin and Dapagliflozin

Precision

Repeatability

The data for repeatability for Dapagliflozin and Bisoprolol is shown in table 6.13. The % R.S.D For Repeatability data was found to be 1.10 % for LID and 1.45 % for DIL.

Inter-day precision

The data for inter-day precision for Dapagliflozin and Bisoprolol is shown in table 6.14. The % R.S.D for intraday precision was found to be 0.18-0.34 % for Dapagliflozin and 0.06 -0.70 % for Bisoprolol.

Inter-Day Precision for Dapagliflozin:

Inter-Day Precision for Bisoprolol:

Intra-Day Precision for Bisoprolol:

Accuracy:

Accuracy of the method was confirmed by recovery study from synthetic mixture at three level standard additions. Percentage recovery for Dapagliflozin & Bisoprolol was found to be 99.48- 99.78% and 99.33-100.59 % respectively. The results are shown in table 32.0 – 33.0.

Recovery For Dapagliflozin:

Recovery For Bisoprolol:

LOD and LOQ:

The limit of detection (LOD) and Limit of Quantification (LOQ) was found to be as per below:

Robustness:

The method is found to be robust as the results were not significantly affected by slight variation in Mobile Phase Composition and flow rate of mobile phase. The results are shown in table 35. Variation seen was within the acceptable range respect to peak asymmetry and theoretical plates, so the method was found to be robust.

Analysis of marketed product:

The proposed method was successfully applied to analysis of the commercially available tablet formulation. The % drugs were found satisfactory, which is comparable with the corresponding label claim.

Summary Of Method Validation:

Summary of validation parameter are shown in below table. All the parameters for substance met the criteria of ICH guideline for the method validation and found to be suitable for routine quantitative analysis in pharmaceutical dosage forms. The result of linearity, accuracy, precision proved to be within limits with lower limits of detection and quantification. Robustness of method was confirmed as no significant in the were observed on analysis by subjecting the method to slight change in the method condition. Assay results obtained by proposed method are fair agreement.

Table 37.0 Summary of validation parameter of RP-HPLC method

Table 38.0 Validation Parameters