ANALYTICAL METHOD DEVELOPMENT AND VALIDATION OF TENELIGLIPTIN AND DAPAGLIFLOZIN API IN MARKETED FORMULATION

ANALYTICAL METHOD DEVELOPMENT AND VALIDATION OF TENELIGLIPTIN AND DAPAGLIFLOZIN API IN MARKETED FORMULATION

Aishwarya Kunchamwar and Vandana Jain *

Department of Quality Assurance, Oriental College of Pharmacy, Sanpada, Navi Mumbai, Maharashtra, India.

ABSTRACT: A simple, novel, precise, rapid, accurate, specific reverse phase high performance liquid chromatography (RP-HPLC) method for estimation of Teneligliptin hydrobromide hydrate and Dapagliflozin propanediol monohydrate API in marketed formulation was developed and validated. The separation was carried out on a Prontosil C18 (250 x 4.6 mm, 5 m) column using an acetonitrile: 3.5 pH Potassium dihydrogen phosphate buffer (60:40 v/v) mobile phase. The flow rate was 1 mL/min, and the UV detector was set to detect at 227 nm. Teneligliptin Hydrobromide Hydrate retained 2.37 min, while Dapagliflozin Propanediol Monohydrate retained 3.61 min. Teneligliptin hydrobromide hydrate revealed a linear response in the concentration range of 10-30ppm. In the concentration range of 5-15ppm, dapagliflozin propanediol monohydrate demonstrated a linear response. Teneligliptin hydrobromide hydrate and Dapagliflozin propanediol monohydrate had correlation coefficients ('R²' value) of 0.999 and 0.992, respectively. Various validation parameters were used to validate the analysis results. Teneligliptin hydrobromide hydrate and Dapagliflozin propanediol monohydrate percentage recoveries vary from 98% to 101%. Both precision and robustness study found to have % RSD values of less than 2. Thus, the suggested method may be successfully used in routine analysis for the method development and validation of Teneligliptin Hydrobromide Hydrate and dapagliflozin propanediol monohydrate.

Keywords: RP-HPLC, Teneligliptin hydrobromide hydrate, Dapagliflozin propanediol monohydrate, Method Development, Validation

INTRODUCTION: Teneligliptin hydrobromide hydrate (TEN) has the following chemical name (2S, 4S) pyrrolidin-2-yl) (1,3-thiazolidin-3-yl) (4-[4-(3-methyl-1phenyl-1Hpyrazol-5-yl) piperazin-1-yl] pyrrolidine-2-yl hemi methadone Fig. 1 ^{1, 2}. A dipeptidyl peptidase is Penta hydrobromide hydrateinhibitor ¹. Teneligliptin tends to slow the inactivation of incretin hormones in type 2 diabetes patients, enhancing bloodstream concentrations while decreasing fasting and postprandial glucose concentration levels in a glucose-dependent manner ³.

FIG. 1: CHEMICAL STRUCTURE OF TENELIGLIPTIN

Dapagliflozin is also known as (1s) -1, 5-anhydro-1-C-[4 – chloro – 3 - [(4 - ethoxyphenyl) methyl] phenyl] - 1, 5 – anhydro – 1 – C - [4 – chloro – 3 -[(4 – ethoxypheny – D - glucitol Fig. 2 ^1-2, Dapagliflozin inhibits renal glucose reabsorption via the solid- glucose cotransporter (SGLT), allowing for an insulin-independent technique of Controlling blood glucose levels in type 2 diabetes patients is essential ³.

Dapagliflozin is a first-generation selective SGLT inhibitor that is anticipated to be 100-fold more selective for SGLT2 than SGLT1 ³. Teneligliptin and Dapagliflozin are drugs that are used to improve glycemic control in people with type 2 diabetes ^{1, 3}.

FIG. 2: CHEMICAL STRUCTURE OF DAPAGLIFLOZIN

Several spectroscopic RP-HPLC techniques for estimating Teneligliptin and Dapagliflozin alone and in combination with other drugs have been published ^{3, 4, 7, 8}. As a result, it was thought desirable todevelop an accurate, precise, and cost-effective fast RP-HPLC method for the simultaneous measurement of Teneligliptin and Dapagliflozin in tablet dose form.

MATERIALS AND METHOD:

Instrumentation: Chromatography was carried out on a Shimadzu prominence - i 2030 system with data processing software from lab solution. The separation and quantification were performed on a Prontosil C 18 column (250×4.6 nm,5m).

Chemicals and Reagents:

Active Pharmaceuticaling Redients used: Teneligliptin Hydrobromide Hydrate Purity of API is (99%) and Dapagliflozin Propanediol Monohydrate purity of API (99%).

Pharmaceutical Formulation: Tablets of Teneligliptin Hydrobromide Hydrate and Dapagliflozin Propanediol Monohydrate with strength 10 mgand 20mg Brand name as ZITA-D manufactured by Glenmark were used. These tablets were purchased from local pharmacy.

Reagentsand Chemicals used: Acetonitrile (Merck) and Milli-Qwater (HPLC Grade), Potassium dihydrogen Phosphate, Orthophosphoric Acid were used for preparing mobile phase.

Selection of Wavelength: Each solution of selected API was scanned using a twin beam UV visible spectrophotometer from 200 nm to 400 nm, yielding overlapping spectra. The wavelength chosen was 227 nm, which corresponds to the isosbestic point. Fig. 3 depicts the superimposed spectra of Teneligliptin and Dapagliflozin.

FIG. 3: UV SPECTRUM OF DAPAGLIFLOZIN AND TENELIGLIPTIN

Chromatographic Condition: A Prontosil C18 (250 x 4.6 mm, 5 m) column was used to develop the procedure. Acetonitrile: 3.5 pH potassium dihydrogen phosphate buffer (60:40) was used as the mobile phase. To identify the detection wavelength of 227 nm, a typical drug solution was scanned with a spectrometer over a wide range of wavelengths 200-400 nm. The pump's flow rate was set to 1 mL/min, and the capacity was set to 10µL. The temperature in the column was set to 30°C.

Preparation of Mobile Phase: 60 volumes of HPLC grade acetonitrile and 40 volumes of Buffer was used as mobile phase.

Preparation of Buffer pH 3.5: Dissolve 2.7 g of Potassium Dihydrogen Phosphate in 900ml water. Adjust the Ph 3.5 with Phosphoric Acid. Diluted to 1000 mL with water ⁹.

Preparation of Diluent: Water: Acetonitrile (60:40) was chosen as a diluent based on the solubility of the medication.

Preparation of Standard Solution: 10mg Teneligliptin and 10mg Dapagliflozin working standard were accurately weighedtransferredinto10 mL volumetric flask respectively. About 4ml diluent was added, sonicated to dissolve and diluted to 10mL using diluent. Accurately 2mL of Teneligliptin and 1mL of dapagliflozin both the solutions were transferred in to 10mL volume tricflask, Volume was make up with diluents to get 20ppm of Teneligliptin hydrobromidehydrate and 10ppm of Dapagliflozin propanediol monohydrate.

Preparation of Sample Solution: Ten tablets were crushed to make a fine powder. The tablet powder equivalent to 20 mg of Teneligliptin and 10 mg of Dapagliflozin was transferred to a 100 mL volumetric flask and dissolved in diluent for 30 minutes while the flask was ultrasonicated. Finally, diluent was used to get the volume up to the required level. This solution was further diluted by taking 1 mL from the above solution and diluting it to 10 mL with diluent.

Method Development: Chromatographic separation was accomplished using a Prontosil C18 (250 x 4.6 mm, 5 m) column with acetonitrile and buffer in the ratio of (60:40) as the mobile phase at a flow rate of 1 mL/min and column temperature of 30°C, with detection at 227 nm. The optimized approach resulted in an elution time of 2.37 min for Teneligliptin and 3.61 min for Dapagliflozin. The overall running time was 8 min. Fig. 2 and 3 exhibit chromatograms of Teneligliptin and Dapagliflozin standard and sample, respectively.

TABLE 1: OPTIMIZED CHROMATOGRAPHIC CONDITIONS FOR TENELIGLIPTIN AND DAPAGLIFLOZIN

RESULT AND DISCUSSION: The developed RP-HPLC method for Teneligliptin and Dapagliflozin was validated as per ICH guidelines ⁵.

Specificity: Specificity is defined as the capacity to evaluate an analyte definitively in the presence of predicted components ⁵. The specificity of the approach was determined by watching and comparing the test result obtained for the sample solution with the standard result obtained for a pure drug, as shown in Fig. 4, 5, 6.

FIG. 4: CHROMATOGRAM OF BLANK SOLUTION

FIG. 5: CHROMATOGRAM OF STANDARD SOLUTION OF TENELIGLIPTIN AND DAPAGLIFLOZIN

FIG. 6: CHROMATOGRAM OF SAMPLE SOLUTION OF TENELIGLIPTIN AND DAPAGLIFLOZIN

System Suitability: In order to confirm the optimal settings, the system suitability parameter was studied. A system appropriateness test was performed on the chromatograms in accordance with USP requirements ⁵. The following parameters were assessed: retention time, tailing factor, theoretical plate, and resolution. The results are summarized in Table 2.

TABLE 2: SYSTEM SUITABILITY PARAMETER RESULTS

Linearity: The concentrations of teneligliptin and dapagliflozin were utilized to produce the standard curve. The method's linearity was examined using linear regression analysis ⁵. The linearity graph was generated by displaying the drug concentration on the X-axis and the corresponding peak area on the Y-axis, as illustrated in Fig. 7 and 8. Table 3 summaries the linearity statistics.

TABLE 3: LINEARITY DATA OF TENELIGLIPTIN AND DAPAGLIFLOZIN

FIG. 7: CALIBRATION CURVE OF TENELIGLIPTIN HYDROBROMIDE HYDRATE   

FIG. 8: CALIBRATION CURVE OF DAPAGLIFLOZIN PROPANEDIOL MONOHYDRATE

Precision: An analytical procedure's precision defines the level of agreement between a number of measurements taken from multiple sampling if the same sample is used under identical conditions ⁵. The precision of an analytical technique expresses the degree of agreement between a set of measurements acquired from multiple samplings of the same sample under prescribed conditions. To achieve system precision (20 ppm), six replicate injections of Teneligliptin standard solution were employed. The average, standard deviation (SD), and percentage RSD of area were calculated and reported for six replicate injections. Furthermore, triplicate injections of Dapagliflozin (10 ppm) and Teneligliptin standard and sample solutions (20 ppm) were done. Its assay, average, standard deviation (SD), and percent RSD were computed and presented as follows. Table 4 highlights the findings of the system precision and technique precision tests.

TABLE 4: SYSTEM PRECISION & METHOD PRECISION RESULT

Accuracy /Recovery Studies: Recovery tests were carried out to investigate the suggested method's accuracy and reproducibility ⁵. The standard medication was added to a predetermined amount of pre-analyzed sample at 50%, 100%, and 150% concentrations. Each level was repeated three times. Table 5 displays the Teneligliptin and Dapagliflozin content discovered utilizing the proposed approach. Teneligliptin and Dapagliflozin had mean recoveries of 99.89% and 99.46%, respectively.

TABLE 5: ACCURACY DATA OF TENELIGLIPTIN AND DAPAGLIFLOZIN

Assay of Marketed Formulation: Ten tablets were weighed and finely pulverized. Transfer tablet powder equivalent to 20 mg of Teneligliptin and 10 mg of Dapagliflozin into a 100 mL volumetric flask, add 60 mL diluent, sonicate for 30 minutes, and make up the volume to the mark.

This solution was diluted further by placing 1 mL from above solution in a 10 mL volumetric flask and filling the remaining capacity with diluent to achieve 20 ppm of Teneligliptin acid and 10 ppm of Dapagliflozin.

TABLE 6: ANALYSIS OF THE FORMULATION

Robustness: Robustness is a measure of its ability to remain unaffected by minor changes in the chromatographic technique parameters and indicates its dependability. The ability to stay unaffected by tiny changes in the chromatographic technique parameters implies robustness, which indicates dependability. The retention time of Teneligliptin and Dapagliflozin was measured after making slight purposeful modifications in the chromatographic conditions at three different levels. Flow rate, column temperature and wavelength were chosen as parameters. There were no deliberate changes in the chromatogram, demonstrating the robustness of the developed RP- HPLC method, Table 7 describes the outcome.

TABLE 7: RESULT OF ROBUSTNESS STUDY

CONCLUSION: According to the findings, the RP-HPLC method for simultaneous estimates of Teneligliptin and Dapagliflozin pharmaceutical formulation was successfully established. Both medications have a high resolution with short analysis time of 8-min. The developed HPLC method was found to be simple, accurate, linear, precise, and robust. It is capable of determining the individual and concurrent concentrations of Teneligliptin and Dapagliflozin in pharmaceutical medication items and substances. It can be used to determine the assay of medicinal products, mix consistency, and content uniformity. The developed approach was validated in compliance with ICH recommendations, and the findings were equivalent.

ACKNOWLEDGEMENTS: Authors are thankful to Oriental College of Pharmacy, Sanpada, Navi Mumbai for providing necessary facilities.

CONFLICTS OF INTEREST: Nil

REFERENCES:

1. Patel, Anokhi, Preeti Jadeja and Rajashree Mashru: Analytical Method Development and Validation for simultaneous estimation of Dapagliflozin and Teneligliptin hydrobromide hydrate from synthetic mixture by three different UV spectrophotometric methods." World Journal of Pharmaceutical Research 2022; 11(7): 770-783.
2. Rao Modalavalasa R, Pharma KBC, Sura DS and Kothapalli CB: Validation of a Newly Developed Stability Indicating RP-Liquid Chromatographic Method for the Quantitative Deter. Related papers Development and Validation of St ability Indicating RP-Liquid Chromatographic Method for the 2018; ISSN 0975-413XCODEN (USA): PCHHAX Validation of a Newly Developed Stability Indicating RPLiquid Chromatographic Method for the Quantitative Determination of Dapagliflozin.
3. Jani BR, Shah KV and Kapupara PP: Development and validation of UV spectroscopic method for simultaneous estimation of dapagliflozin and metformin hydrochloride in synthetic mixture. International Journal of Research and Development in Pharmacy & Life Sciences 2015; 4(3): 1569-76.
4. Deepan T and Dhanaraju MD: Stability indicating HPLC method for the simultaneous determination of dapagliflozin and saxagliptin in bulk and tablet dosage form. Current Issues in Pharmacy and Medical Sciences 2018; 31(1): 39-43.
5. ICH Harmonised Tripartite Guideline Validation of Analytical Procedures: Text and Methodology Q2 (R1). International Conference on Harmonisation of Technical Requirements for Registration of Pharmaceuticals for Human use, Geneva 2005; 1-13.
6. National Center for Biotechnology Information (2023). PubChem Compound Summary for CID 9887712, Dapagliflozin. Retrieved May 26, 2023 from https://pubchem.ncbi.nlm.nih.gov/compound/Dapagliflozin.
7. Bhavyasri K, Surekha: bioanalytical method development and validation of dapagliflozin and metformin hydrochloride in combined dosage form using uv spectroscopy. Gedrag & Organisatie Review 2020; 33.
8. Sharma A, Jain V and Prabhu V: Stability–indicating RP_HPLC method development and validation for simultaneous estimation of teneligliptin hydrobromide hydrate and metformin hydrochloride in combination tablet dosage form. European Journal Biomedical Pharm 2018; 5(5): 453-62.
9. Government of India, Ministry of Health. Pharmacopoeia of India: (the Indian Pharmacopoeia). Delhi: Manager of Publications 1955.
10. Bichala, Prem Kumar, K. Jeevan Kumar, R. Suthakaran, and Ch Shankar: "Development and Validation of an Analytical Method for the Estimation of Metformin and Teneligliptin in its Bulk and Tablet Dosage Form by using RP-HPLC." Asian J of Pharma Analy 2020; 10(1): 11-14.
11. Kant R, Bodlarb, Kapoor G and Bhutani R: optimization of a single HPLC-PDA method for quantifying metformin, gliclazide, pioglitazone, dapagliflozin, empagliflozin, saxagliptin, linagliptin and teneligliptin using central composite design. Bioorga Chem 2019; 12(91): 103111.
12. Asmita M, Rao CS, Akkamma HG, Arpitha S and Krishna PT: Analytical method development and statistical validation of dapagliflozin in the tablet dosage form and bulk drug. Indian Research Journal of Pharmacy and Science 2018; 16: 1344-56.
13. Patel V, Pandya C, Patel Z, Patel D and Pandya A: Isocratic RP-UHPLC method development and validation of stability-indicating for simultaneous determination of teneligliptin and metformin in fixed-dose combination. Current Chemistry Letters 2021; 10(4): 503-16.
    

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    How to cite this article:

    Kunchamwar A and Jain V: Analytical method development and validation of teneligliptin and dapagliflozin API in marketed formulation. Int J Pharm Sci & Res 2024; 15(1): 170-76. doi: 10.13040/IJPSR.0975-8232.15(1).170-76.

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