Analytical Method Development And Validation of Dapagliflozin and Saxagliptin by HPLC in Bulk Drug and pharmaceutical Dosage Form

In patients with type 2 diabetes, Dapagliflozin is used to  lower blood sugar levels and reduces the risk of kidney damage, blindness, and limb loss. Additionally  prevented are nerve problems and sexual function. This medication is also prescribed to patients with type 2. Chemically speaking, Dapagliflozin is known as (1s)-1,  5-anhydro1-C-[4-chloro-3-[(4-ethoxyphenyl) methyl] phenyl]-Dglucitol. Its molecular weight is 408.98 and its  molecular formula is C24H33ClO8. (16), Its Solubility in Methanol, Water, and Acetonitrile [1-4]

Fig 1: Strcture of Dapagliflozin

Dapagliflozin is a newly developed sodium-glucose cotransporter 2 (SGLT2) inhibitor, is a promising agent to treat type 2 diabetes mellitus (T2DM) and cardiometabolic comorbidities

Fig 2:Structure of Saxagliptin

Saxagliptin

Chemically speaking (1S,3S,5S-2-[(2S-2-amino-2-(3-hydroxy-1-adamantyl)acetyl]-2-azabicyclo hexane-3-carbonitrile.,its molecular formula is  C₁₈H₂₅N₃O₂ molecular weight is 314.5 g/mol [5] _.It is a Selective dipeptidyl peptidase-4 (DPP-4) inhibitors recently  emerged as the forefront of intervention in pathophysiology of type 2 diabetes mellitus  (T2DM) to reverse the progressive rising of type 2 diabetes mellitus due to the β-cells  insulin resistance.This medication leads to  lengthening of incretin actings, in increasing the insulin released by pancreatic 1-cells in a situation reliant on glucose and in inhibiting the glucagon released by 2-cells,  primarily in postprandial state.[6-9]

MATERIALS AND METHODS

Chemicals

The pure drug samples of Dapagliflozin and Saxagliptin were received as a gift sample  from Reliable’s Shree Industrial Training Centre and Laboratory, Jalgaon. The  marketed tablet formulation Dapaglyn L was purchased from local medical. HPLC grade  methanol, acetic acid, and purified water were purchased from Merck India Ltd. All  solvents and reagents were filtered through a 0.45 µm membrane filter and then  degassed prior of using for removal of any particulate matter and dissolved gases. The  chemicals and reagents used in the study were of analytical reagent grade or HPLC grade for ensuring accuracy, reliability, and reproducibility of the analytical method.

Instrumentation and analytical conditions: The chromatographic analysis was carried out using an Agilent 1100 HPLC system equipped with a diode array detector (DAD, G1314B)and ChemStation software for data acquisition and processing. The system was capable of operating at a maximum pressure of 400 bar with a discharge flow rate range of 0.001 to 5 mL/min. The pressure display accuracy was maintained at ±5%, and the system could accommodate up to four mobile phases with a mixing ratio range of 0 to 100%. The pump unit was a high-performance reciprocating pump (HP-1100), which ensured precise and consistent solvent delivery during analysis.

Wavelength selection:

The prepared working solutions of Dapagliflozin and Saxagliptin in methanol were separately scanned in the wavelength range of 200–400 nm using methanol as blank.  Dapagliflozin demonstrated a sharp absorption maximum at approximately 225 nm,  while Saxagliptin showed an absorption maximum at approximately 213 nm. The overlay spectrum also revealed an isosbestic point at 220 nm, that indicated a common  analytical wavelength which could be used for simultaneous estimation of both drugs.  The final chromatographic detection was carried out at 230 nm under the optimized RP HPLC condition.

Preparation of standard solution

Accurate quantities of 10 mg of Dapagliflozin and 5 mg of Saxagliptin were weighed and transferred separately into two clean, dry 10 mL volumetric flasks. Approximately 20 to 30 mL of methanol was added to each of the flask, and the contents were sonicated for 10 minutes, ensuring complete dissolution. made up to the mark with methanol to obtain stock solutions with concentrations of 1000 μg/mL of Dapagliflozin and 500 μg/mL of Saxagliptin respectively

Preparation of Sample solution: From the above stock solutions, suitable aliquots were withdrawn and diluted with methanol in separate volumetric flasks to obtain working solutions of lower concentration for analytical studies. The prepared working solutions were mixed thoroughly before use and were used for the determination of the absorption maxima and for method validation studies.

Result and Discussion:

Method optimization:

RP-HPLC method development for simultaneous estimation of Dapagliflozin and Saxagliptin was performed with the evaluation of different compositions of methanol  and 0.1% acetic acid at different flow rates. The aim of the optimization was to achieve  adequate separation, good peak shape, and suitable run time for both analytes. Several  trial conditions were analysed, and the chromatographic performance was evaluated on the basis of retention time, symmetry, theoretical plate count, and  resolution. The optimized condition was selected on the basis of the best overall  chromatographic behaviour and was used for all subsequent validation experiments.

Final chromatographic condition: The chromatographic conditions were optimized using trial-and-error method and were kept constant throughout the experimental study. The analysis was performed with an Agilent 1100 HPLC system equipped with an autosampler and ChemStation software. Separation was performed using a C18 column (4.6 mm × 250 mm, 5 μm). The mobile phase consisted of methanol and 0.1% acetic acid in the ratio of 70.7:29.3 (v/v), that was selected on the basis of optimum peak resolution and system suitability. The flow rate was constant at 0.9 mL/min, detection was performed at 230 nm, the column temperature was maintained at 33°C, and the injection volume was fixed at 20 μL. Under these optimized conditions, both analytes provided well-defined peaks with satisfactory separation and acceptable retention times.

Fig. 3:  UV absorption spectrum of Dapagliflozin (10 μg/mL) in methanol

Fig 4 :UV absorption spectrum of Saxagliptin (10 μg/mL) in methanol

Fig 5:  Isosbestic point of Dapagliflozin and Saxagliptin at 220 nm

Linearity: Linearity of the method was determined in the concentrations range of 10-50 μg/mL for Dapagliflozin and 5-25 μg/mL for Saxagliptin. These findings revealed that there was a proportional and definitive enhancement in the peak area with the rise in concentration of both drugs which verifies that the detector follows a linear response  at the chosen range. For Dapagliflozin, the mean areas were 1907.76, 3360.37,  4903.09, 6476.53, and 8041.46 at 10, 20, 30, 40, and 50 μg/mL, respectively. Saxagliptin, the corresponding mean areas were 485.93, 871.82, 1289.56, 1706.26, and 2129.07 at 5, 10, 15, 20, and 25 μg/mL.

A regression equation was obtained of y=153.8356x+322.774 with the correlation coefficient of R2=0.999761896 as part of the calibration curve of Dapagliflozin. Saxagliptin showed the regression equation y=82.4144x+60.312 with R2=0.999724629. These values show the existence of great linearity in both analytes within the analysed range.

 

Table no 2: Linearity Parameters of Dapagliflozin and Saxagliptin

The developed method was validated as per the International Conference on Harmonization (ICH) 14, 15 guidelines with respect to linearity and range, specificity, precision, accuracy, robustness, limit of detection and limit of quantification.

CONCLUSION

The developed RP-HPLC method provides a reliable analytical tool for simultaneous estimation of Dapagliflozin and Saxagliptin in bulk and dosage forms. Its validation as per ICH guidelines confirms suitability for pharmaceutical quality assurance and regulatory applications. The method’s simplicity, reproducibility, and robustness make it ideal for routine use in industry and academia.

REFERENCES

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