Novel Stress Indicating RP-HPLC Method Development and Validation for the Simultaneous Estimation of Ertugliflozin and Sitagliptin in Bulk and its Formulation

A selective, sensitive RP-HPLC method was developed for the simultaneous estimation of the Ertugliflozin (ETR) and Sitagliptin (SGT) in bulk and its dosage form. The separation and determination was carried on water’s C18 column capacitate (250X4.6 mm, 5 μm particle size), retention times of Ertugliflozin and Sitagliptin were found to be 2.39 and 4.60 min. respectively. The wavelength was fixed at 215 nm with PDA detection. The mobile phase was consisted mixture of 0.5 mM potassium dihydrogen ortho phosphate buffer: Methanol in the ratio of 55:45 v/v, pH 5.3 was adjusted with HCl and flow of mobile phase was maintained 1mL/min. The calibration curve was linear and regression co-efficient (R2) value found to be 0.999 and concentration ranging from 37.5-112.5 and 250-750 μg/mL for Ertugliflozin and Sitagliptin respectively. The quantization limit and detection limit of the method were found 0.1 & 0.3 μg/ml and 0.4 and 1μg/ml for Ertugliflozin and Sitagliptin. keywords: Ertugliflozin, Sitagliptin, Reversed Phase High Performance Liquid Chromatography, Methanol.


Reagents & chemicals
All the chemicals and reagents in this experiment were of analytical grade.Water was double distilled and filtered with a membrane filter.Methanol -HPLC grade (Merck, India), hydrochloric acid and potassium di hydrogen ortho phosphate (SD fine chem, India) were used to prepare mobile phase.Pharmaceutical grade standard drugs viz., Ertugliflozin and Sitagliptin were kindly gifted by Ajanta Pharma Ltd, Mumbai, India.The combined tablet formulation contains 15 mg of Ertugliflozin and 100mg of Sitagliptin (Steglujan, Natco) purchased from local market of Kurnool.

Preparation of standard solution
Weigh accurately 10 mg of Ertugliflozin & Sitagliptin and transferred in to individual 10 ml volumetric flasks with small quantity of mobile phase.The solution was sonicated for 10 min.and volume made with mobile phase and concentration 1000 µg/ml.This solution further diluted for the preparation of working standard solutions to get final concentrations of 75 µg/mL of Ertugliflozin and 500 µg/mL of Sitagliptin working standard solutions.

Preparation of sample solution
Twenty tablets were weighed and finely powdered.The average weight of tablets was determined.The powder equivalent to 10 mg of ETR was weighed and transferred to a 10 mL volumetric flask.10 mL of diluent was added to disintegrate tablets completely by using ultra sonicated for 10 minute.The aliquot portion of the filtrate was further diluted to get final concentrations 75 µg/mL of ETR and 500 µg/mL of SGT.The solution was filtered through membrane filter.The 20 µL of this solution was injected in to HPLC system.

Chromatographic Settings
The mobile phase used for the development of method was 0.5 mM potassium dihydrogen ortho phosphate buffer: Methanol in the ratio of 55:45 v/v, pH 5.3 was attuned with HCl and flow of mobile phase was filtered through membrane filter and flow rate was kept 1mL/min.The effluents were supervised at 215 nm with PDA detector and injected 20 µl of solution through chromatographic column.

Method development
The method was developed with different buffers and organic solvents but the composition of potassium dihydrogen ortho phosphate and methanol was showed good resolution, symmetrical peaks, high theoretical plates, and low retention times of both Ertugliflozin and Sitagliptin.The optimized parameters were showed in Table 1.

Method validation
The different method validation parameters were performed as per ICH norms.The all parameters showed good results and they met ICH guidelines of acceptance. 26

System suitability constraints
The system suitability parameters were showed good theoretical plates 3985 and 6425 for ETR and SGT.The tailing factor was less than 2 for both drugs.They showed good resolution between peaks 11.27 and showed fine peak areas.The chromatograms were showed in Fig. 3,4,5 and results were tabulated in Table 2.

Specificity
The stress degradation studies were implies the specificity of the method.Different parameters were evaluated depend upon separation between degradants and active moiety, as well as method showed ability to analyze analyte in the presence of other products.

Common Suggested procedure for Linearity
The calibration curve linear over concentration range and R 2 values were found to be 0.999 for both Ertugliflozin and Sitagliptin.The standard solution was showed linearity concentration range from 37.5-112.5µg/mLfor Ertugliflozin & 250-750 µg/mL for Sitagliptin.The data of graphs were showed in Figures 6 & 7.

Precision
The precision was assessed through system precision and method precision.The method precision was estimated through assay.The optimized concentrations of standard and sample solutions were injected in to chromatographic system for the system precision and method precision.The %RSD values varied from 0.55-0.66%.The results of the method showed good precision of the values.The results were tabulated in Table 3 and 4.

Limit of detection & Limit of quantification
The LOD and LOQ were estimated 12.71µg/ml-42.37µg/mlfor Ertugliflozin and 8.59µg/ml-28.65µg/mlfor Sitagliptin.The limit of detection and quantitation limits performed based on the slope and standard deviation.The method showed ability to detect Ertugliflozin & Sitagliptin at low level of concentrations.The chromatograms were showed in Figures 8, 9.

Robustness
The robustness of the method was performed with deliberate change of flow rate, temperature and mobile phase composition.The changed parameters were showed good percentage assay values.The percentage assay values were in between 99.24% -101.47% for Ertugliflozin and 99.51-101.08 for Sitagliptin respectively.They met acceptance criteria according to ICH guidelines.The results were tabulated in Table 6.

Assay of Ertugliflozin and Sitagliptin in commercial dosage form
The assay of the method was performed for tablet formulation.Powdered 20 tablets from that accurately weighed powder equivalent to 161.56 mg of Ertugliflozin.The final concentration was prepared as 75 µg/mL of Ertugliflozin and 500 µg/ mL of Sitagliptin.The % assay values were 99.02% & 99.40% for Ertugliflozin and Sitagliptin.The method was used for routine analysis of Ertugliflozin and Sitagliptin estimation in combined dosage form.The results were showed in Table 7.

Force degradation studies
The stability studies were implemented on the Ertugliflozin and Sitagliptin.The method showed, there was no interference of degradants and blank.The developed RP-HPLC method verifies the proficiency of stability indicating method for the analysis of Ertugliflozin and Sitagliptin.Different stress indicating studies were conducted with 0. nm for 5 days).The % degradation in all the stress conditions were observed up to 9%.Proposed method was found to be resolved the degraded products from the analytes peak.The average assay results in all the conditions were approximately 90%.The results were tabulated in Table 8 and chromatograms of degradation studies were showed from Figure 10 to 14.

CONCLUSION
The developed and validated simultaneous estimation of Ertugliflozin and Sitagliptin by RP-HPLC method was showed low tailing factor and high theoretical plates.The method was exposed good precision, accuracy and robustness, met the all values with in the limit according to ICH guidelines.The linearity graphs showed good linearity between different concentrations solutions of ETR and SGT, the R 2 value were found to be 0.999 for both ETR and SGT.The LOD and LOQ values were found to be 0.1 and 0.4 µg/ml for ETR and 0.3 and 1 µg/ml for SGT.The results of LOD and LOQ specified sensitivity of the method and detected ETR and SGT at low concentration.The forced degradation studies were accomplished with acid, alkaline, peroxide, hydrolytic, UV-light conditions.The results of the method were showed high stability and method was used for the routine analysis bulk and its pharmaceutical dosage forms.

Table 4 : Precision results of ETF & SGT
Sitagliptin respectively.The concentration of 150% solution showed % mean recovery 100.84 and 99.86 for Ertugliflozin & Sitagliptin respectively.The results were tabulated in Table5.