Development And Validation of Stability Indicating HPLC Method For Simultaneous Estimation of Dapagliflozin, Linagliptin and Metformine Hydrochloride in Its Tablet Dosage Form

Type 2 Diabetes Mellitus (T2DM) is a prevalent metabolic disorder worldwide, characterized by chronic hyperglycemia resulting from impaired insulin action and/or secretion. T2DM affects multiple organ systems and presents with various symptoms including excessive urination (polyuria), compensatory thirst (polydipsia), increased fluid intake, blurred vision, unexplained weight loss, lethargy, and altered energy metabolism. These manifestations result from the body’s inability to maintain proper glucose homeostasis [1].

Anti-Diabetic Agents :

Dapagliflozin :

Dapagliflozin (DAPA) is a member of a novel class of oral anti-diabetic agents known as Sodium-Glucose Co-Transporter 2 (SGLT2) inhibitors. It is indicated for the management of T2DM in conjunction with diet and exercise to improve glycemic control. Dapagliflozin acts by inhibiting SGLT2 in the renal proximal tubules, thereby preventing glucose reabsorption and promoting its excretion via urine [2].

• Chemical Name: (2S,3R,4R,5S,6R)–2-{4–chloro-3-[(4-ethoxyphenyl)methyl]phenyl}–6 (hydroxymethyl)oxane-3,4,5-triol
• Molecular Formula: C??H??ClO?
• Molecular Weight: 408.9 g/mol [3]

Linagliptin :

Linagliptin is an oral anti-diabetic drug belonging to the class of Dipeptidyl Peptidase-4 (DPP-4) inhibitors. It enhances glycemic control by inhibiting DPP-4, an enzyme that degrades incretin hormones. This inhibition increases insulin secretion and suppresses glucagon release in a glucose-dependent manner, providing an anti-hyperglycemic effect [4,5].

• Chemical Name: 8-[(3R)-3-aminopiperidin-1-yl]-7-(but-2-yn-1-yl)-3-methyl-1-[(4-methylquinazolin-2-yl)methyl]-1H-purine-2,6-dione
• Molecular Formula: C??H??N?O?
• Molecular Weight: 472.5 g/mol [6]

Metformin Hydrochloride :

Metformin Hydrochloride is a biguanide-class oral antihyperglycemic agent, commonly used in the treatment of non-insulin-dependent diabetes mellitus (NIDDM). It exerts its effects by decreasing hepatic glucose production, reducing intestinal glucose absorption, and enhancing insulin-mediated peripheral glucose uptake. Notably, metformin is associated with weight neutrality or modest weight loss and does not induce hypoglycemia when used alone. It is also indicated in the management of polycystic ovary syndrome and other insulin resistance-related disorders [7].

• Chemical Name: 1-carbamimidamido-N,N-dimethylmethanimidamide hydrochloride
• Molecular Formula: C?H??N?·HCl
• Molecular Weight: 165.62 g/mol [8]

Purpose of the Study :

The objective of this study is to develop a simple, precise, and rapid stability-indicating High-Performance Liquid Chromatography (HPLC) method for the simultaneous estimation of Dapagliflozin, Linagliptin, and Metformin Hydrochloride in a combined tablet dosage form. This method aims to provide robust analytical performance and will be validated according to the International Council for Harmonisation (ICH) guidelines. To date, no stability-indicating HPLC methods have been reported for the simultaneous estimation of these three drugs in a single tablet formulation.

Fig : 1 Structure of Dapagliflozin

Fig : 2 Structure of Linagliptin

Fig : 3 Structure of Metformine Hydrochloride

MATERIALS AND METHODS:  

Instrumentation:

• HPLC instrument with UV-visible detector – LC100
• Column - Kromasil C18 column (150mm x 4.6 mm, 5 μm)
• UV-visible Spectrophotometer – SHIMADZU-1800 Software-UV probe ,version-2.34
• Digital Analytical Balance - SCALE TEC, SAB 224 CL
• Ultra Sonicator - PHOENIX DSA – 50 SK2
• Digital pH meter - Chemiline ATC CL 120
• Controlled temperature water bath
• Hot air Oven - Lab Tech DST, Ahmadabad
• FTIR – SHIMADZU
• Volumetric flask - 10, 25, 50 and 100ml
• Pipette - 1, 2, 5 and 10ml.
• Measuring cylinder - 10, 100 and 500ml.
• Beakers - 250, 500 and 1000 ml

Chemicals and Reagents:

• Metformin Hydrochloride (API) –certified supplier
• Dapagliflozin –(API) – certified supplier
• Linagliptin (API) –certified supplier
• Potassium dihydrogen phosphate – Thermo Fischer Scientific Pvt. Ltd.
• Triethylamine –  SD Fine–Chem. Ltd., Mumbai, India
• Ortho Phosphoric acid HPLC Grade – Finar
• Acetonitril HPLC Grade – Finar
• Hydrochloric Acid – Rankem
• Methanol HPLC Grade – Life science Pvt Ltd
• Ethanol Analytical reagent – Fine Chemical Co.Ltd
• Sulphuric Acid HPLC Grade – Rankem
• Hydrogen Peroxide HPLC Grade – Rankem
• Sodium Hydroxide pellet –  Rankem
• 1-Pantanesulphonic acid Sodium salt HPLC Grade – Finar
• Potassium bromide (KBr) –  Rankem
• Water: Distill water, HPLC Grade water, - Rankem

IDENTIFICATION AND CHARACTERIZATION :

Solubility Stability Study :

For stability studies, the solubility of dapagliflozin, linagliptin and metformine hydrochloride were practically determinedby adding 100mg of dapagliflozin, linagliptin and metformine hydrochloride ti 100 ml volumetric flasks, then adding quantity of solvent (e.g., water or ethanol) at room temperature and shaking for a few minutes. The solubility was then classified based on the amount of solvent required to dissolve the solute.

Table : 1 Solubility table as per IP’2022 specification

                            Table : 2 Solubility Data for Dapagliflozin, Linagliptin and Metformin

Identification By Melting Point Determination

The melting point of Metformin, Dapagliflozin and Linagliptin were determined by using the open capillary method, a standard technique for this purpose. A small sample of dapagliflozin, Linagliptin and Metformine Hydrochloride are placed in an open capillary tube and heated gradually until it melts. The temperature at which the drug starts to melt is recorded as its melting point. Melting point for both the drug was observed and recorded in following table 3.

Table : 3 Melting Point of Drugs

IR Spectra:

The IR Spectra of Metformin, Dapagliflozin and Linagliptin along with its functional group identification, were shown in the following graph.

Fig : 4  IR Spectra of Dapagliflozin Sample

Table : 4  IR Spectra Interpretation for Dapagliflozin

Fig : 5  IR Spectra of Linagliptin Sample

Table : 5  IR Spectra Interpretation for Linagliptin

Fig : 6  IR Spectra of Metformin Sample.

Table :6 IR Spectra Interpretation for Metformin

METHOD DEVELOPMENT :

Preparation Of Buffer and Mobile Phase :

An amount of 1 mL of Methanol(85%) solution was taken in a 1000-mL volumetric flask; about 100 mL of milli-Q water was added and mixed well; then the final volume was made up to 1000 mL with milli-Q water, and the pH was adjusted to 3.0 with diluted Methanol(10 % v/v); 600 mL (0.1%) of p hosphate buffer (pH 3.0) and 400 mL of acetonitrile were mixed in the ratio of 60:40 (% v/v) and degassed in an ultrasonic water bath for 15 min and then filtered through a 0.45-μm membrane filter under vacuum.

PREPARATION OF SOLUTIONS :

Preparation Of Standard Solution :

Preparation Of Stock Solution of Metformin :

Accurately weighed Metformin (250 mg) was transferred into a 50-ml clean dry volumetric flask, 10 ml of a diluent was added, sonicated for 10 min, and made up to the final volume with diluents to give a stock solution of 5000 µg/ml of Metformin.

Preparation Of Working Standard Solution of Metformin :

From above stock solution pipette out 1 ml of aliquot and diluted up to 10 ml to give a solution having strength of 500 µg/ml of Metformin.

Preparation Of Stock Solution of Dapagliflozin

Accurately weighed Dapagliflozin (5 mg) was transferred into a 50-mL clean dry volumetric flask, 10 ml of a diluent was added, sonicated for 10 min, and made up to the final volume with diluents to give a stock solution of 100 µg/ml of Dapagliflozin.

Preparation of Working Standard Solution of Dapagliflozin:

From above stock solution pipette out 1 ml of aliquot and diluted up to 10 ml to give a solution having strength of 10 µg/ml of Dapagliflozin.

Preparation Of Stock Solution Of Linagliptin:

Accurately weighed Linagliptin (2.5 mg) was transferred into a 50-mL clean dry volumetric flask, 10 ml of a diluent was added, sonicated for 10 min, and made up to the final volume with diluents to give a stock solution of 50 µg/ml of Linagliptin.

Preparation of Working Standard Solution of Linagliptin:

From above stock solution pipette out 1 ml of aliquot and diluted up to 10 ml to give a solution having strength of 5 µg/ml of Linagliptin.

Preparation of Sample solution:

Preparation of Sample Stock Solution:

An accurate equivalent weight of the combination powder sample was transferred into a 100-ml volumetric flask; 50 ml of the diluent was added and sonicated for 25 min; further, the volume was made up with the diluent and filtered using HPLC filters (5000 μg/ml of Metformin, 100 μg/ml of Dapagliflozin, and 50 μg/ml of Linagliptin).

Preparation of Working Sample Solution:

Take 1 ml of the filtered sample stock solution was transferred into a 10-mL volumetric flask and made up with the diluent. The solutions prepared comprised 500 μg/ml of Metformin, 10 μg/ml of Dapagliflozin, and 5 μg/ml of Linagliptin.

VALIDATION OF PROPOSED METHOD :

The proposed method was validated according to ICH guidelines (2005) for system suitability, specificity, recovery, precision, linearity, and robustness.

System Suitability Test:

System suitability test is an integral part of LC methods. This test is used to verify that the chromatographic system is adequate for the intended analysis. HPLC system suitability was optimized per United States of Pharmacopeia (USP) general chapter on chromatography <621>. About 10 μl of the standard solution of drugs was injected six replicate injections into the chromatographic system. To determine the system suitability of the proposed method, the parameters such as retention time, theoretical plates, and tailing factor were calculated.

Specificity:

The specificity of the method was carried out to check whether there is any interference of any impurities in the retention time of the analyte peaks. The specificity was performed by injecting blank, placebo, and standard solutions of drugs.

Linearity:

The standard stock solutions of Metformin, Dapagliflozin, and Linagliptin were suitably diluted with the mobile phase to obtain a series of solutions containing  250, 375, 500, 625, and 750 μg/ml of Metformin; 5, 7.5, 10, 12.5, and 15 μg/ml of Dapagliflozin, and 2.5, 3.75, 5, 6.25, and 7.5 μg/mL of Linagliptin. The linearity was determined by calculating a regression line from the plot of the peak area to the concentration of the drug. The method was evaluated by the determination of correlation coefficient and intercept values according to ICH guidelines.

Precision:

Precision is expressed as the closeness of agreement between a series of measurements obtained from multiple sampling of the same homogeneous sample. Six replicate injections of a known concentration of Metformin (500 μg/ml), Dapagliflozin (10 μg/ml), and Linagliptin (5 μg/ml) were analyzed by injecting into a HPLC column on the same day. The intermediate precision was estimated by injecting samples prepared at the same concentrations on different days by different operators. The peak area of all injections was taken, and standard deviation and % relative standard deviation (RSD) were calculated.

Accuracy:

Accuracy is estimated using the standard addition method at different levels: 50, 100, and 150%. A known amount of the standard drug was added to the blank sample at each level. The mean recovery of  Metformin, Dapagliflozin, and Linagliptin was calculated

Robustness:

HPLC conditions were slightly modified to evaluate the analytical method robustness. These changes included the flow rate, column temperature, and mobile phase.

Forced Degradation Study :

Alkaline, acidic, oxidation, thermal, water, and direct exposure to UV were carried out.

Alkali Hydrolysis :

To 10 ml of the stock solution of Metformin, Dapagliflozin, and Linagliptin, 4 ml of 1.0 N sodium hydroxide was added and refluxed for 30 min at 60 °C. The solution was cooled to room temperature and neutralized with 4 mL of 1.0 N HCl, and finally, the solution was made up to the target concentration with the diluent.

Acid Hydrolysis :

To 10 ml of the stock solution of Metformin, Dapagliflozin, and Linagliptin, 4 mL of 1.0 N hydrochloric acid was added and refluxed for 30 min at 60 °C. The solution was cooled to room temperature and neutralized with 4 ml of 1.0 N NaOH, and finally, the solution was made up to the target concentration with the diluent

Oxidative Stress :

To 10 ml of the stock solution of Metformin, Dapagliflozin, and Linagliptin, 1 ml of 20% hydrogen peroxide (H2O2) was added, and the solutions were kept for 30 min at 60 °C. The solution was cooled to room temperature and made up to the target concentration with the diluent.

Thermal Degradation :

An amount of 10 mL of the standard stock solution of drugs was transferred into a 100 ml volumetric flask and placed in an oven at 80°C for 6 h to study dry heat degradation, and then the solution was cooled and made up to the target concentration in a 100 ml volumetric flask with diluent.

Photolytic Degradation :

Photo stability of the drugs was also studied by exposing 10 ml of the standard stock solution of Metformin, Dapagliflozin, and Linagliptin, to UV light in a glass beaker for 7 days, and then the solution was made up to the tar- get concentration in a 100 ml volumetric flask with the diluent.

Hydrolytic Degradation :

About 10 ml of the standard stock solution of drugs was transferred into a 100-ml volumetric flask; 10 ml of deionized water was added and heated on a water bath for 1 h. Finally, the solution was cooled and made up to the target concentration with the diluent.

RESULT AND DISCUSSION:

Selection Of Wavelength :

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

Selection Of Column :

For HPLC Method various columns are available but our main aim is to resolve drug in the presence of excipients, so the Kromasil  C18 column was selected for the estimation of Dapagliflozin, Linagliptin and Metformin.C-18 (id 4.6mm x 150 mm, 5 µm) column was chosen to give good peak shape and high resolution, which also provides high peak symmetry, good retention to drug and facilitates the separation of the drug without the interference of excipients within short run time.

Fig : 8  Kromasil C₁₈  (id 4.6mm x 150 mm, 5 µm) column

Selection Of Mobile Phase :

Trail 1

• Column: C-18 (id 4.6 x 150 mm, 5 µm)
• Mobile Phase: Methanol: Phosphate buffer(30:70v/v)
• Detection: 272 nm
• Flow rate:1 ml/min
• Run Time: 30 minutes
• Observations: No peak detected.

Fig : 9 Trial 1: Chromatogram of Dapagliflozin, Linagliptin and Metformin Hydrochloride Methanol : Phosphate buffer(30:70v/v)

Fig : 10 Trial 2: Chromatogram of Dapagliflozin, Linagliptin and Metformin Hydrochloride Methanol: Phosphate buffer(50:50v/v)

 Trail 3

• Column: C-18 (id 4.6 x 150 mm, 5 µm)
• Mobile Phase: Methanol: Phosphate buffer(20:80v/v)
• Detection: 272 nm
• Flow rate:1 ml/min
• Run Time: 30minutes
• Observations: Peaks detected and separated, but broad peaks observe.

Fig :11  Trial 3: Chromatogram of Dapagliflozin, Linagliptin and Metformin Hydrochloride Methanol: Phosphate buffer(20:80v/v)

 Trail 4

• Column: C-18 (id 4.6 x 150 mm, 5 µm)
• Mobile Phase: Methanol: Water(30:70v/v)
• Detection: 272 nm
• Flow rate:1 ml/min
• Run Time: 30 minutes
• Observations: No peak detected.

Fig :12  Trial 4: Chromatogram of Dapagliflozin, Linagliptin and Metformin Hydrochloride Methanol: Water(30:70v/v)

Trail 5

• Column: C-18 (id 4.6 x 150 mm, 5 µm)
• Mobile Phase: Methanol: Water (50:50v/v)
• Detection: 272 nm
• Flow rate:1 ml/min
• Run Time: 30minutes
• Observations: only two peak detected with broad spectrum.

Fig : 13 Trial 5: Chromatogram of Dapagliflozin, Linagliptin and Metformin Hydrochloride Methanol: Water (50:50v/v)

Trail 6

• Column: C-18 (id 4.6 x 150 mm, 5 µm)
• Mobile Phase: Methanol: Water (20:80v/v)
• Detection: 272 nm
• Flow rate:1 ml/min
• Run Time: 30minutes
• Observations: Two Peaks detected and separated, but broad peaks observe.

Fig : 14 Trial 6: Chromatogram of Dapagliflozin, Linagliptin and Metformin Hydrochloride Methanol: Water (20:80v/v)

Trail 7

• Column: C-18 (id 4.6 x 150 mm, 5 µm)
• Mobile Phase: Acetonitrile: Phosphate buffer(30:70v/v)
• Detection: 272 nm
• Flow rate:1 ml/min
• Run Time: 30 minutes
• Observations: No peak detected.

Trail 8

• Column: C-18 (id 4.6 x 150 mm, 5 µm)
• Mobile Phase: Acetonitrile: Phosphate buffer(50:50v/v)
• Detection: 272 nm
• Flow rate:1 ml/min
• Run Time: 30minutes
• Observations: only one peak detected.

Trail 9

• Column: C-18 (id 4.6 x 150 mm, 5 µm)
• Mobile Phase: Acetonitrile: Phosphate buffer(20:80v/v)
• Detection: 272 nm
• Flow rate:1 ml/min
• Run Time: 30minutes
• Observations: Peaks detected and separated, but broad peaks observe.

Fig : 17 Trial 9: Chromatogram of Dapagliflozin, Linagliptin and Metformin Hydrochloride Ortho-phosphoric acid: Phosphate buffer(20:80v/v)

Trail 10

• Colum: C-18 (id 4.6 x 150 mm, 5 µm)
• Mobile Phase: Acetonitrile: Phosphate buffer(60:40v/v)
• Detection: 272 nm
• Flow rate:1 ml/min
• Run Time: 30minutes
• Observations: Good peaks with Adequate solution were observed.

Fig : 18 Trial 10: Chromatogram of Dapagliflozin, Linagliptin and Metformin Hydrochloride Acetonitrile: Phosphate buffer(60:40v/v)

Chromatographic conditions for optimized mobile phase trial :

• Stationary phase: C-18 (id 4.6 x 150 mm, 5 µm)
• Mobile Phase: Acetonitrile: Phosphate buffer (60:40v/v)
• Detection: 272 nm
• Flow rate:1 ml/min
• Run Time: 30 minutes
• Detector: UV detector
• Injection volume: 20 μl
• Column Temperature: 40ºC
• Mode: Isocretic

Fig : 19 Optimized mobile phase trial for optimized chromatogram of Std Dapagliflozoin:25.515 min, Linagliptin: 15.115 min, Metformin Hydrochloride: 3.113 min

Fig : 20 Chromatogram of blank Acetonitrile: Phosphate buffer (60:40v/v)

Linearity :

For the purpose of linearity, accurately weighed amount of Dapagliflozin (10 mg), and Linagliptin (5 mg) and Metformin Hydrochloride (500 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 Ranitidine, and 100 µg/ml of Ondansetron. 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+250, 7.5+3.75+375, 10+5+500, 12.5+6.25+625 and 15+7.5+750 µg/ml of Dapagliflozin, Linagliptin and Metformin Hydrochloride respectively.

Table : 7  Linearity data for Dapagliflozin, Linagliptin and Metformin Hydrochloride

Fig : 21 Calibration curve of Dapagliflozin ( 5-15 μg/mL) R2 = 0.9999

Fig : 22 Calibration curve of Linagliptin (2.5-7.5 μg/mL) R² = 0.9999

Fig : 23 Calibration curve of Metformin HCl (250-750 μg/mL) R² = 0.9993

Fig : 24 Overlain Linearity Spectra of Dapagliflozin, Linagliptin and Metformin Hydrochloride

Table : 8  Linearity results for Dapagliflozin, Linagliptin and Metformin Hydrochloride

Precision :

Repeatability :  

The data for repeatability for Dapagliflozin, Linagliptin and Metformin Hydrochloride is shown in table 8.13. The % R.S.D For Repeatability data was found to be 0.50 % for Dapagliflozin , 0.38% for Linagliptin and 0.86% for Metformine Hydrochloride.

Table : 9 Repeatability data for Dapagliflozin, Linagliptin and Metformin Hydrochloride

Inter-day precision  :

The data for interday precision for Dapagliflozin, Linagliptin and Metformin is shown in table 8.14. The % R.S.D for intraday precision was found to be 0.35-0.66 % for Dapagliflozin and 0.68-0.86 Linagliptin and 0.07-0.40 % for Metformin Hydrochloride.

Table : 10 Inter-day precision data for estimation of Dapagliflozin, Linagliptin and Metformin Hydrochloride

Intra -day precision :           

The data for intra-day precision for Dapagliflozin, Linagliptin and Metformin Hydrochloride is shown in table 11. The % R.S.D for intraday precision was found to be 0.35-0.66 % for Dapagliflozin, 0.68-0.86% for Linagliptin and 0.32 -0.93 % for Metformin Hydrochloride.

Table : 11 Intra-day precision data for estimation of Dapagliflozin, Linagliptin and Metformin Hydrochloride

Accuracy :

The difference between theoretically added amount and the practically achieved amount is called the accuracy of the analytical method. Accuracy of the method was performed by spiking API to sample at specified levels. Accuracy was determined at three different levels 80%, 100%, and 120% of the target concentration in triplicate. The data show that the proposed method is accurate. Accuracy data is shown in the table. 12 :

Table : 12 Data of recovery study of Dapagliflozin, Linagliptin and Metformin Hydrochloride

LOD and LOQ :

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

Table: 13 LOD and LOQ Limit for Dapagliflozin, Linagliptin and Metformin Hydrochloride

Limit of Detection (LOD) is the lowest concentration of a substance that can be reliably distinguished from the absence of that substance (i.e., background noise).

Equation for LOD:

LOD=3×σ? /s

Where:

• σ = Standard deviation of the blank (background noise)
• S = Slope of the calibration curve

Limit of Quantification (LOQ) is the lowest concentration of a substance that can be quantitatively measured with acceptable precision and accuracy.

Equation for LOQ:

LOQ=10× σ/s?

Where:

• σ = Standard deviation of the blank (background noise)
• S = Slope of the calibration curve

Selectivity :

There is no interference in the mixture

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

Table :14  Robustness data for Dapagliflozin, Linagliptin and Metformin Hydrochloride

Alkali hydrolysis  degradation : 

The densitogram showed peak  at Rf of 0.58 along with two degradation peaks at Rf 0.49 (2) and 0.83 (3) indicating 15 % degradation Peak 1 (standard peak; Rf: 0.58), peak 2 (degradant 2;Rf: 0.49), peak 3(deradant3; Rf: 0.83).

Fig: 25 Densitogram of Alkali hydrolysis degradation with two degradants

Peak 1 (standard peak; Rf: 0.58), peak 2 (degradant 2;Rf: 0.49), peak 3(deradant3; Rf: 0.83).

Acid Hydrolysis degradation :

The densitogram showed peak at Rf of 0.59 along with seven peaks of degradation products at Rf 0.01 (4), 0.03(5), 0.30 (6), 0.39 (7), 0.45(8), 0.50 (9) and 0.86 (10) resulting in 20.20 degradation (Figure 6.6 and Table 6.7).

Fig : 26 Densitogram of Acid Hydrolysis degradation with seven degradants

Peak 1  (standard peak; Rf : 0.59), peak 4 (degradant 4; Rf: 0.01),peak 5 (degradant 5; Rf: 0.03), Peak 6 (degradant 6; Rf: 0.30), peak 7 (degradant 7; Rf : 0.39), peak 8 (degradant 8; Rf: 0.45), peak 9 (degradant 9; Rf: 0.50), peak 10 (degradant 10; Rf: 0.86).

Hydrolytic  degradation :

The densitogram showed peak at Rf of 0.59 along with seven peaks of degradation products at Rf 0.01 (4), 0.03(5), 0.30 (6), 0.39 (7), 0.45(8), 0.50 (9) and 0.86 (10) resulting in 20.20 % degradation.

Fig : 27 Densitogram of Hydrolytic  degradation with seven degradants

Peak 1 (standard peak; Rf : 0.59), peak 4 (degradant 4; Rf : 0.01), peak 5 (degradant 5; Rf : 0.03), Peak 6 (degradant 6; Rf : 0.30), peak 7 (degradant 7; Rf : 0.39), peak 8 (degradant 8; Rf : 0.45), peak 9 (degradant 9; Rf : 0.50), peak 10 (degradant 10; Rf: 0.86).

Oxidative degradation :

The densitogram of subjected to oxidative stress showed a peak of UPA at Rf of 0.58 along with two degradation peaks at Rf 0.86 (10) and 0.04 (11) indicating17.25 % degradation.

Fig : 28 Densitogram of oxidative degradation with two degradants

Peak 1 (standard peak; Rf : 0.58), peak 10 (degradant 10; Rf: 0.86), peak 11; (degradant 11; Rf: 0.04)

Thermal degradation :

The densitogram showed peak of at Rf of 0.61 along with one degradation peak at Rf 0.86 (10) resulting in 10.02 % degradation.

Fig : 29 Densitogram of thermal degradation with one degradant

Peak 1 (standard peak; Rf : 0.61), peak 10 (degradant 10; Rf : 0.86)

Photodegradation :

In the photodegradation, the peak of drug was observed at Rf of 0.59 along with three degradation peaks at Rf of 0.01 (4), 0.86 (10) and 0.75 (12)

Fig : 30 Densitogram of photodegradation with three degradants

Peak 1standard peak; Rf: 0.59), peak 4 (degradant peak 4; Rf : 0.01), peak 10 (degradant 10; Rf: 0.86), peak 12 (degradant 12; Rf: 0.75)

Analysis of marketed product :

The proposed method was successfully applied Solid dosage form (Dapagliflozin 10mg, Linagliptin 5mg, Metformine hydrochloride 500mg) to analysis of the commercially available tablet formulation. The % drugs were found satisfactory, which is comparable with the corresponding label claim.

Table : 15 Analysis of marketed formulations

SUMMARY OF METHOD VALIDATION :

Table : 16 Summary of validation parameter of RP-HPLC method