Development And Validation of RP-HPLC Method for Simultaneous Estimation of Dapagliflozin Propanediol Monohydrate and Telmisartan in Synthetic Mixture

Dapagliflozin Propanediol Monohydrate

Dapagliflozin propanediol monohydrate is an oral antidiabetic agent that belongs to the class of SGLT2 (sodium-glucose co-transporter-2) inhibitors. It works by inhibiting SGLT2 in the renal proximal tubules, reducing glucose reabsorption and increasing urinary glucose excretion, thereby lowering blood glucose levels. It is primarily used in the management of type 2 diabetes mellitus, and also has beneficial effects in patients with heart failure and chronic kidney disease. The propanediol monohydrate form enhances its solubility and stability. Dapagliflozin is generally well-tolerated, with common side effects including urinary tract infections, genital infections, and increased urination. It may also cause volume depletion and, rarely, euglycemic diabetic ketoacidosis.

Dapagliflozin Propanediol Monohydrate

Telmisartan

Telmisartan is used alone or in combination with other classes of antihypertensive agents in the management of hypertension. Angiotensin II receptor antagonists, such as telmisartan, are considered one of several preferred antihypertensive drugs for the initial management of hypertension according to current evidence-based hypertension guidelines; other preferred options include angiotensin-converting enzyme (ACE) inhibitors, calcium-channel blockers, and thiazide diuretics.

Telmisartan

MATERIALS AND METHODS:

Materials

1. Name of API: Dapagliflozin Source: Glenmark Pharmaceuticals LTD.
2. Name of API: Telmisartan Source: CTX Life science

Instrumentation:

1. High presuure liquid chromatography Make and model: Shimadzu (LC 100)
2. Weighing balance  

Make and Model: Mettler Toledo

3. Metlting point apparatus

Make and model: Gallenkamp (889339)

4. UV double beam spectrophotometer

Make and model: Shimadzu (UV 1800)

Chemicals and Reagents: Dapagliflozin API, Telmisartan API Acetonitrile HPLC Grade, Phosphate Buffer, Ortho phosphoric acid, Hydrochloric Acid, Methanol HPLC Grade, Water: Distill water(Milli-Q), HPLC grade water.

Preparation of Mobile Phase: RP-HPLC method was followed by isocratic elution technique. Mobile phase comprised of Acetonitrile: Methanol (75:25 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 100 mL volumetric flask, dissolved in methanol and diluted up to mark with methanol. This will give a stock solution having strength of 100 μg/mL.Withdraw 0.5 ml from Stock Solution and make up to 10 ml with to get 5 μg/mL.

Preparation of Standard Stock Solution B: (Telmisartan stock solution): Accurately weighed quantity of Telmisartan 10 mg was transferred into 100 mL volumetric flask, dissolved in methanol and diluted up to mark with methanol. This will give a stock solution having strength of 100 μg/mL. Withdraw 2 ml from Stock Solution and make up to 10 ml with to get 20 μg/mL. Preparation of system suitability solution: Take standard solution for system suitability.

Chromatographic Conditions:

The chromatographic separation of Dapagliflozin and Telmisartan were achieved on C-18 (id 4.6 x 250 mm, 5 µm) by using mobile phase composed of Acetonitrile: Methanol (75:25 v/v/v %), at flow rate 1.0 ml/min with run time of 10 minutes. Detection of both drugs was carried out at 272 nm by using diluent as mobile phase.

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 & Telmisartan 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 2.0

Table 1.0 Solubility Table

Table 2.0 Solubility Data for Dapagliflozin and Telmisartan

Identification by Melting Point Determination

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

Table 3.0 Melting Point of Drugs

IR Spectra:

Dapagliflozin

Fig. 1 Structure of Dapagliflozin

Fig 2: IR Spectra of Dapagliflozin

Table 4.0 IR Interpretation Spectra for Dapagliflozin

Telmisartan

Fig 3 Structure of Telmisartan

Fig 4: IR Spectra of Telmisartan

Table 5 IR Interpretation Spectra for Telmisartan

METHOD DEVELOPMENT

SELECTION OF WAVELENGTH:

To determine wavelength for measurement, standard spectra of Dapagliflozin & Telmisartan were scanned between 200-400 nm against diluents. Absorbance maxima of Dapagliflozin & Telmisartan detected at 272 nm. Chromatogram was taken at 272 nm, both drugs give good peak height and shape. So, 272 nm was selected for Simultaneous estimation of Dapagliflozin & Telmisartan their formulation.

SELECTION OF CHROMATOGRAPHIC CONDITIONS

Proper selection of the HPLC method depends upon the nature of the sample (ionic or ionisable or neutral molecule), its molecular weight, pKa and solubility. RP-HPLC was selected for the initial separation based on literature survey and its simplicity and suitability. To optimize the chromatographic conditions the effect of chromatographic variables such as mobile phase, flow rate and solvent ratio were studied. Finally, the chromatographic condition was chosen that give the best resolution, symmetry and capacity factor for estimation of both drugs.

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.

METHOD VALIDATION

As per ICH guideline (Q2R1), the method validation parameters studied were specificity, linearity, accuracy, precision, limit of detection, limit of quantitation and robustness.

Specificity

The analytical method for specificity was evaluated by injecting the following solutions. Diluent was prepared and inject into the HPLC system in triplicate. Sample solution was prepared with appropriate levels of excipients as a placebo sample and inject into the HPLC system in triplicate for all the dosage strengths. Placebo was prepared by mixing all excipients without active ingredients. Standard and sample solutions were prepared for assay (100% Conc.) and inject into the HPLC system in triplicate.

Linearity and Range

Preparation of Solution for linearity studies: For the purpose of linearity, accurately weighed amount of Dapagliflozin (10 mg), and Telmisartan (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 Telmisartan. 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 containing 5+20, 7.5+25, 10+30, 12.5+35 and 15+40 µg/ml of Dapagliflozin and Telmisartan respectively.

Precision

Prepared standard working solution of mixtures having concentration of Dapagliflozin (5 μg/ml) and Telmisartan (20 μg/ml) were injected at volume of 20 μL into column by employing optimized chromatographic conditions. Each standard mixture was injected 5 time and peak area was monitored. Each concentration was monitored for repeatability by RSD.

Intra-day and Inter-day Precision

Method precision was determined by performing intraday and inter day precision.

Mixture that represents overall range (Dapagliflozin +Telmisartan = 5+20, 10+30 and 15+40 µg/ml) were analyzed on same day at different time interval for intraday precision.

Mixture that represents overall range (Dapagliflozin +Telmisartan = 5+20, 10+30 and 15+40 µg/ml) were analyzed on different days for inter-day precision.

System Suitability Parameters

Solution of Dapagliflozin + Telmisartan (5+20 μg.ml-1) was injected 3 times for determination of System suitability parameters which includes Retention time (Rt), Tailing factor (Tf), Resolution (Rs) and number of theoretical plates. System suitability parameters   for selected concentration were determined by C.V.

Accuracy

Accuracy of the analytical method has been performed by spiking of sample with the standard. Spiking of the placebo was performed at 50,100 and 150 % of the target concentration

Limit of detection and Limit of Quantification

The limit of detection (LOD) and the limit of quantification (LOQ) were calculated using the standard deviation of y-intercept of calibration curve. The limit of detection (LOD) and the limit of quantification (LOQ):

LOQ = 10 σ/s and LOD = 3.3 σ/s

          Where, σ = the standard deviation of the response.

          S = the slope of the calibration curve

Robustness

Following parameters were altered one by one for determination of robustness of the method and their effect was observed by comparing with the standard preparation. Mobile phase flowrate (± 0.1 mL/min), optimized flowrate was 1.0 mL/min. Mobile phase composition (± 2 mL), in optimized ratio 2 determinations of Dapagliflozin + Telmisartan = 5+20 µg/mL for each alteration were carried out and RSD was measured.

RESULT AND DISCUSSION

SELECTION OF WAVELENGTH

To determine wavelength for measurement, standard spectra of Dapagliflozin & Telmisartan were scanned between 200-400 nm against diluents. Absorbance maxima of Dapagliflozin & Telmisartan have detected at 272 nm. Chromatogram was taken at 272 nm, both drugs give good peak height and shape. So, 272 nm was selected for Simultaneous estimation of Dapagliflozin & Telmisartan in their formulation.

Fig 5.0: UV Graph for Dapagliflozin and Telmisartan

SELECTION OF MOBILE PHASE

Trail 1

Column: C-18 (id 4.6 x 250 mm, 5 µm)

Mobile Phase: Acetonitrile: Water (30:70v/v)

Detection: 272 nm

Flow rate:1 ml/min

Run Time: 10 minutes

Observations: No peak detected.

Fig 6 Trial 1: Chromatogram of Dapagliflozin & Telmisartan Acetonitrile: Water (30:70v/v)

Trail 2

Column: C-18 (id 4.6 x 250 mm, 5 µm)

Mobile Phase: Acetonitrile: Water (50:50v/v)

Detection: 272 nm

Flow rate:1 ml/min

Run Time: 10 minutes

Observations: Only one Peak detected but broad peaks observe.

Fig 7 Trial 2: Chromatogram of Dapagliflozin & Telmisartan Acetonitrile: Water (50:50v/v)

Trail 3

Column: C-18 (id 4.6 x 250 mm, 5 µm)

Mobile Phase: Acetonitrile:Water (80:20v/v)

Detection: 272 nm

Flow rate:1 ml/min

Run Time: 10 minutes

Observations: Only one Peak detected but broad peaks observe.

Fig 8 Trial 3: Chromatogram of Dapagliflozin & Telmisartan Acetonitrile: Water(80:20v/v)

Trail 4

Column: C-18 (id 4.6 x 250 mm, 5 µm)

Mobile Phase: Methanol: Phosphate Buffer(60:40v/v)

Detection: 272 nm

Flow rate:1 ml/min

Run Time: 10minutes

Observations: only one peak detected.

Fig 9 Trial 4: Chromatogram of Methanol : Phosphate Buffer(60:40v/v)

Trail 5

Column: C-18 (id 4.6 x 250 mm, 5 µm)

Mobile Phase: Methanol: Phosphate Buffer(70:30v/v)

Detection: 272 nm

Flow rate:1 ml/min

Run Time: 10minutes

Observations: only one peak detected

Fig 10 Trial 5: Chromatogram of Methanol: Phosphate Buffer(70:30v/v)

Trail 6

Column: C-18 (id 4.6 x 250 mm, 5 µm)

Mobile Phase: Methanol: Phosphate Buffer(50:50v/v)

Detection: 272 nm

Flow rate:1 ml/min

Run Time: 10minutes

Observations: Peaks detected and separated, but broad peaks observe.

Fig 11 Trial 6: Chromatogram of Methanol: Phosphate Buffer (50:50v/v)

Trail 7

Column: C-18 (id 4.6 x 250 mm, 5 µm)

Mobile Phase: Ortho phosphoric acid: acetonitrile (80:20 v/v)

Detection: 272 nm

Flow rate:1 ml/min

Run Time: 10 minutes

Observations: No peak detected.

Fig 12 Trial 7: Chromatogram of Dapagliflozin & Telmisartan Ortho phosphoric acid: acetonitrile (80:20 v/v)

Trail 8

Column: C-18 (id 4.6 x 250 mm, 5 µm)

Mobile Phase: Ortho phosphoric acid: acetonitrile (60:40 v/v)

Detection: 272 nm

Flow rate:1 ml/min

Run Time: 10 minutes

Observations: Peaks detected and separated, but broad peaks observe.

Fig 13 Trial 8: Chromatogram of Dapagliflozin & Telmisartan Ortho phosphoric acid: acetonitrile (60:40 v/v)

Trial 9

Column: C-18 (id 4.6 x 250 mm, 5 µm)

Mobile Phase: Acetonitrile:Methanol(50:50v/v)

Detection: 272 nm

Flow rate:1 ml/min

Run Time: 10minutes

Observations: Only one peak detected with broad spectrum.

Fig 14 Trial 9: Chromatogram of Dapagliflozin & Telmisartan Acetonitrile: Methanol (50:50v/v)

Trail 10

Column: C-18 (id 4.6 x 250 mm, 5 µm)

Mobile Phase: Acetonitrile:Methanol(60:40v/v)

Detection: 272 nm

Flow rate:1 ml/min

Run Time: 10minutes

Observations: Peaks detected and separated, but broad peaks observe

Fig 15 Trial 10: Chromatogram of Dapagliflozin & Telmisartan Acetonitrile: Methanol (60:40v/v)

Trial 11 (Final Trial)

Column: C-18 (id 4.6 x 250 mm, 5 µm)

Mobile Phase: Acetonitrile: Methanol (75:25v/v)

Detection: 272 nm

Flow rate:1 ml/min

Run Time: 10minutes

Observations: Good peaks with Adequate solution was observed.

Fig 16 Trial 11: Chromatogram of Dapagliflozin & Telmisartan Acetonitrile: Methanol (75:25v/v)

7.3 Chromatographic conditions for optimized mobile phase trial

Stationary phase: C-18 (id 4.6 x 250 mm, 5 µm)

Mobile Phase: Acetonitrile: Methanol (75:25v/v)

Detection: 272 nm

Flow rate:1 ml/min

Run Time: 10 minutes

Detector: UV detector

Injection volume: 20 μl

Column Temperature: 40ºC

Mode: Isocretic

Fig 17: Optimized mobile phase trial for optimized chromatogram of Std.Dapagliflozin:5.115 min, Telmisartan: 2.225 min

Fig 18: Chromatogram of blank Dapagliflozin: Telmisartan (75:25v/v)

Method Validation

Linearity

For the purpose of linearity, accurately weighed amount of Dapagliflozin (10 mg), and Telmisartan (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 Telmisartan. 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+20, 7.5+25, 10+30, 12.5+35 and 15+40 µg/ml of Dapagliflozin and Telmisartan respectively.

Table 6 Linearity data for Dapagliflozin and Telmisartan

Fig 19: Overlain Linearity Spectra of Dapagliflozin and Telmisartan

Fig 20: Calibration curve of Dapagliflozin

Fig 21: Calibration curve of Telmisartan

Repeatability

The data for repeatability for Dapagliflozin and Telmisartan is shown in table 8. The % R.S.D For Repeatability data was found to be 1.10 % for LID and 1.45 % for DIL.

Table 8 Repeatability data for Dapagliflozin and Telmisartan

The data for interday precision for Dapagliflozin and Telmisartan is shown in table 9. The % R.S.D for intraday precision was found to be 0.37-1.71 % for Dapagliflozin and 0.36-1.22 % for Telmisartan.

Table 9 Inter-day precision data for estimation of Dapagliflozin and Telmisartan

Intra -day precision

The data for intra-day precision for Dapagliflozin and Telmisartan is shown in table 10. The % R.S.D for intraday precision was found to be 0.20-0.38 % for Dapagliflozin and 0.09-1.26 % for Telmisartan.

Table 10 Intra-day precision data for estimation of Dapagliflozin and Telmisartan

Accuracy of the method was confirmed by recovery study from synthetic mixture at three level standard additions. Percentage recovery for Dapagliflozin & Telmisartan was found to be 99.48- 99.78% and 99.33-100.59 % respectively. The results are shown in table.11-12.

Table 11 Recovery data for Dapagliflozin

Table 12 Recovery data for Telmisartan

7.4.4 LOD and LOQ

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

Table 14 LOD and LOQ Limit for Dapagliflozin & Telmisartan

7.4.5 Selectivity

There is no interference in the mixture.

7.4.6 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 15. Variation seen was within the acceptable range respect to peak asymmetry and theoretical plates, so the method was found to be robust.

Table 15 Robustness data for Dapagliflozin & Telmisartan

7.4.7 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.

Table 16 Analysis of marketed formulations

7.5 Summary of Method Validation

Table 7.17 Summary of validation parameter of RP-HPLC method