Method Development and Validation for Estimation of Teneligliptin in Tablet Dosage Form by RP-HPLC

 

Bhanu Biswas¹, Manish Kumar¹, Jai Bharti Sharma¹, Vipin Saini², Shailendra Bhatt^1*

¹M.M. College of Pharmacy, Maharishi Markandeshwar (Deemed to be University), Mullana, Ambala

²Maharishi Markandeshwar University, Solan, Himachal Pradesh

 

ABSTRACT:

A new and simple technique of RP-HPLC was developed and validated for the estimation of Teneligliptin in the tablet dosage form. The partition or separation was accomplished on Kromasil C18 analytical column (150 mm × 4.6 mm, 5.0 μm) utilizing acetonitrile, water and trifluoroacetic acid as mobile phase A and acetonitrile and trifluoroacetic acid as mobile phase B at a flow rate of 1.0 ml/min. The detector used in the present study was UV detector and detection was detected at 245nm. The total chromatographic analysis time per sample was about 55.0 min with teneligliptin eluting at the retention time of about 11.2 minutes. The standard curve was found to be linear in the concentration range of 50-150μg/mL with R² value of 0.999. The developed method was validated for different parameters like accuracy, precision, specificity and linearity. The developed RP-HPLC method was found to be simple, specific, rapid, reliable and reproducible.

 

 

 

INTRODUCTION:

Diabetes mellitus is a heterogeneous metabolic disorder characterized by altered carbohydrate, lipid and protein metabolism. Diabetes and it's most abnormalities constitute a major health problem in modern society^1-3. Diabetes is a chronic, metabolic disease characterized by an elevated level of blood glucose. The most common is type 2 diabetes, usually in adults, which occurs when the body became resistant to insulin or doesn’t make enough insulin^4-6.

 

Teneligliptin is a newly advanced oral dipeptidyl peptidase 4 inhibitors used for the treatment of type 2 diabetes mellitus (T2DM) in grown-ups alongside exercise and diet⁷. Teneligliptin is an anti-diabetic drug as it increases the insulin secretion from the pancreas. Teneligliptin is depicted as [(2S,4S)- 4-[4-(5-methyl-2-phenyl pyrazole-3-yl)piperazin-1-yl]pyrrolidin-2-yl]-(1,3-thiazolidine-3-yl)methanone].

 

The chemical formula of Teneligliptin is C22H30N6OS with a sub-atomic mass of 426.58 g/mol^8-9. The chemical structure of teneligliptin is given in Figure-1.

 

Figure 1- Chemical Structure of Teneligliptin

 

It is a highly potent improves postprandial hyperglycemia and dyslipidemia¹⁰. Teneligliptin is a dipeptidyl peptidase-4 (DPP-4) inhibitor. DPP-4 inactivates the incretin hormone which is responsible for the secretion of the insulin. Incretin hormones, to be specific glucagon-like peptide-1 (GLP-1) and glucose-subordinate insulinotropic polypeptide (GIP), are discharged from enteroendocrine cells and improve insulin secretion^11-14.

 

At present, teneligliptin is available in Japan (Teneria), Argentina (Teneglucon) and India (Tenepure; Teneza) and the endorsement status of Teneligliptin is given in Table-1^15-16.

 

Table 1- Endorsement status of Teneligliptin

Phase of Development	Indication	Country
Marketed	T2DM	Japan
Marketed	T2DM	Argentina
Marketed	T2DM	India

 

Teneligliptin is enrolled in South Korea and is in the pre-enlistment stage in Indonesia. Moreover, teneligliptin is in phase II clinical preliminaries in Europe, and phase I clinical preliminaries in the US.

 

Different methods were reported for the estimation of drug by RP-HPLC. [17-20]. In the present study, we have developed a simple, accurate and more précised method for the estimation of teneligliptin in tablet dosage form by RP-HPLC.

 

MATERIAL AND METHOD:

Chemicals and Reagents:

Teneligliptin HBr bulk drug was procured as a gift sample from Lupin Pvt Ltd. Pune, India, as a gift sample. Acetonitrile (HPLC grade) was purchased from Central Drug House (P) Ltd., India. The 0.45μm nylon filters were purchased from Advanced Micro Devices Pvt. Ltd. Chandigarh, India. The marketed tablets of Teneligliptin HBr were obtained from a local market (Glytrin tablets). All the other reagents and samples used were of analytical grade.

 

Table-2 Gradient programme

Time (minutes)	Module	Mobile Phase A	Mobile Phase B
0.01	Pumps	88	12
5.00	Pumps	88	12
20.00	Pumps	62	38
30.00	Pumps	25	75
40.00	Pumps	25	75
40.10	Pumps	88	12
55.00	Pumps	88	12
55.01	Controller	Stop	Stop

 

HPLC Instrumentation and conditions:

The analysis was carried out on an HPLC System (CyberlabLC-UV100) consist of a stainless-steel column (Kromasil C18 150 cm X 4.6 mm, 5µ), which was maintained at 40°C. The analytical wavelength was set at 245 nm and samples of 10 µl were injected to the HPLC system. It was a gradient system with a flow rate of 1.0 ml/min. The column temperature was 40°C while sampler temperature was 15ºC. The run time was 55 minutes. The gradient programme is given in Table 2.

 

Preparation of Mobile Phase A & B:

The mobile phase A consists of Acetonitrile: Water: Trifluoroacetic Acid (60: 1940: 2) v/v and the mobile phase B consists of Acetonitrile: Trifluoroacetic Acid (2000: 2) v/v. Both were sonicated for 10 minutes to remove air bubbles.

 

Preparation of Diluent:

The diluent contains Acetonitrile: Water (300: 700) v/v. They were properly mixed and sonicated for 10 minutes to remove air bubbles.

 

System Suitability:

For the system suitability, the following requirements were checked,

·       The tailing factor for the teneligliptin peak should not be more than 2.0

·       The relative standard deviation of the areas from each peak of teneligliptin of first 6 injections must not be greater than 5.0 %.

·       The relative standard deviation of the areas from each peak of teneligliptin for all reference solution injections must not be greater than 5.0 %.

 

The HPLC system needs to be checked if the above requirements are not met.

 

Preparation of Standard Solution:

The standard stock solution was prepared by accurately weighing (31g) Teneligliptin HBr hydrate and transferred to a 100ml clean and dry volumetric flask. To this 70ml of diluent was added and sonicated for 10 minutes. It was then cooled and volume was made up with diluent. The standard solution was loaded in HPLC and six injections were taken from the same vial. The chromatogram of the standard solution is given in Figure 1.

 

Preparation of Test Solution:

Ten intact tablets were accurately weighed and it was properly crushed into a fine powder. The powder, equivalent to 20mg of Teneligliptin was properly weighed and transferred to a 100ml clean and dry volumetric flask. It was then sonicated for 15 minutes and vigorously shaken in a mechanical shaker for another 15 minutes to complete dissolution of the drug. The test solution was then cooled and volume was made up with diluent. The test solution was centrifuged at 4000rpm for 10 minutes. The test solution was then filtered with a 0.45µ nylon filter and injected and chromatographed in duplicates. The peak area of Teneligliptin was estimated at 245nm and chromatograms were recorded. The drug content (mg/tablet) was evaluated by comparing the mean peak area of the standard with that of the sample and per cent labelled claim was calculated. The chromatogram of the test solution is given in Figure 2.

 

Method Validation:

The developed method was validated as per ICH guidelines.

 

Specificity:

The specificity of the method was determined to identify the presence of any interfering compound. For specificity study, a fixed concentration of standard, test and blank solution was injected to the HPLC system and the chromatograms were measured at 245nm.

 

Precision:

The method precision was determined by making six replicate injections on the same day for intraday precision. The % RSD was calculated.

 

Accuracy:

For the accuracy study, teneligliptin standard concentration recovery was performed at 50, 100 and 150%. Three individual samples were prepared and percentage recovery, as well as %RSD, were calculated.

 

 

Linearity:

The linearity study was performed by analysing the standard stock solution. The stock solution was prepared by dissolving 50mg Teneligliptin HBr in 100ml volumetric flask with diluent.

 

Dilutions were made to get concentrations of 50µg/ml, 80µg/ml, 100µg/ml, 120µg/ml and 150µg/ml from the above stock solution. Two dilutions of 50µg/ml and 150 µg/ml were injected thrice were as remaining concentrations were injected twice. The calibration curve was constructed by plotting the peak areas of analyte versus the corresponding drug concentration.

 

RESULT AND DISCUSSION:

Assay method:

The % drug content of teneligliptin was calculated and it was found to be 98.65 % and it was found that the assay method has passed the system suitability criteria. The results for the assay method are shown in Table 3 and Table 4.

 

Table 3: Result of Standard solution for the assay method

 

Table 4: Result of the Assay method

 

Method Validation:

Specificity:

From the values and calculation, it was also found that the specificity parameter has passed and the chromatographic peaks are well separated and there is no interfering peaks were observed. The peak purity is also more than 990 at the scale of 1000 for the principal peak.

The final result is given in Table 5.

Table 5: Result of Specificity parameter

 

Table 6: Result of Method Precision

 

Method Precision:

The %RSD was found to be 0.42%. As the value of %RSD is less than 2% the method precision parameter has passed. The result of the method precision parameter is given in Table-6

 

Accuracy:

The accuracy parameter was performed at three different levels of teneligliptin. The highest %RSD was found to be 1.43 for teneligliptin. The accuracy parameter has passed as the individual recovery is between 97% - 103% and the mean recovery is between 98% - 102%. The accuracy result was found to be 100%. The final result is given in Table 7.

 

Table 7: Result of Accuracy parameter

 

Linearity:

The peak obtained have a perfect relationship with the concentration. The linearity for Teneligliptin HBr in the concentration range from 50-150 µg/ml. The correlation coefficient was found to be (R² ) = 0.9999 respectively. The linearity plot is given in Figure 3 and the result of linearity is given in Table 8. The % RSD was found to be less than 1%.

 

Table 8: Result of Linearity

 

CONCLUSION:

The RP-HPLC method was successfully developed and validated the estimation of Teneliglptin HBr in the tablet dosage form. The suggested method was found to be sensitive, specific, precise, accurate and linear as compared to other methods. The drug was well separated from the analyte peaks, indicates the stability nature of the developed method. The developed RP-HPLC method could be employed for the routine analysis of raw materials and dissolution study of a tablet dosage form containing Teneligliptin HBr.

 

REFERENCES:

1.      Nachiket D, Ganesh S and Jyoti V. Simultaneous Estimation, Validation  and Force  Degradation Study of Metformin Hydrochloride and Empagliflozin by RP HPLC Method. Research Journal of Science and Technology. 2019: 11(2): 135-147.

2.      Rahul G, Ganesh S. and Shraddha J. Simultaneous Estimation and Validation of Dapagliflozin and Saxagliptin in Bulk Drug and Dosage Form by RP-HPLC. Research Journal of Science and Technology. 2019: 11(1): 59-63.

3.      Gandla S, Lalitha R, Mounika C, Soumya B and Sudheer K. A Validated RP-HPLC Method for Simultaneous Determination of Metformin and Canagliflozin in Pharmaceutical Formulation. Asian Journal of Pharmaceutical Analysis. 2018: 8(2): 73-77.

4.      Santosh J, Sanjay P, Virendra Y and Ashpak T. Zero Order and Area Under Curve Spectrophotometric methods for determination of Atenolol in Pharmaceutical Formulation. Research Journal of Pharmaceutical Dosage Forms and Technology. 2015: 7(3): 185-189.

5.      Anand C and Murli K. Evaluation and marker quantification of antidiabetic herbal tablets: Steve Tab and Andro Tab by HPLC method. Research Journal of Pharmaceutical Dosage Forms and Technology. 2013: 5(1): 17-21.’

6.      Ashwini P, Sandeep S, Santosh C and Sanjay K. Development and validation of RP-HPLC method for simultaneous estimation of ezetimibe and glimepiride in a tablet dosage form. Asian Journal of Pharmaceutical Analysis. 2017: 7(3): 185-188.

7.      Surendra K, Girish P, Pradeep G and Onkar S. Diabetes, Metabolic Syndrome and Obesity. Targets and Therapy. 2016; 9: 251–260.

8.      Manjusha K and Barhate V. Spectrophotometric Determination of an Anti-Diabetic Drug Teneligliptin Bulk And Pharmaceutical Formulations. World Journal of Pharmaceutical Research. 2016; 5(5): 1625-1632.

9.      Chandana M, Prasad R, Samrajyam B and Naga P. Analytical Method Development and Validation of Teneligliptin In Pharmaceutical Dosage Form By RP-HPLC Method. Journal of Health Sciences and Nursing. 2017; 1(12): 1-12.

10.   Atul T, Arjun R and Suresh G. HPLC and UV-Spectrophotometric Estimation of Teneligliptin from Tablet Dosage Form. Asian Journal of Pharmaceutical Analysis and Medicinal Chemistry. 2016; 4(3): 148- 156.

11.   Raja H, Babu C And Gowri D. Development And Validation Of LC-MS/MS Method For Quantification Of Teneligliptin In Human Plasma And Its Application To A Pharmacokinetic Study. World Journal of Pharmacy and Pharmaceutical Sciences. 2017; 5(5): 838-850.

12.   Vishnu C, Kiran B, Sanjay C. Development And Validation Of UV Spectrophotometric Method And High-Performance Thin-Layer Chromatographic (HPTLC) Method For Estimation Of Teneligliptin Hydrobromide In Pharmaceutical Preparation. Der Pharmacia Lettre. 2017; 8(8): 291-301.

13.   Amit M, Sonawane K, Dhokale, V. A Simple UV- Spectrophotometric Method Development and Validation of Teneligliptin In Tablet Dosage Form; Indo American Journal Of Pharmaceutical Research. 2017; 6(4): 206-215.

14.   Maladkar M, Sankar S and Kamat, K. Teneligliptin: Heralding Change In Type 2 Diabetes; Journal Of Diabetes Mellitus. 2016; 6: 113-131.

15.   Kishimoto M. Teneligliptin: A DPP-4 Inhibitor for The Treatment Of Type 2 Diabetes. Diabetes Metabolism Syndrome Obvs. 2013; 6: 187–195.

16.   Scott L. Teneligliptin: A Review In Type 2 Diabetes; Clini Drug Invest. 2015; 35(11): 765–772.

17.   Anil M and Nandini P. Simultaneous Determination of Eight Phyoconstituents in Triphala churna by HPLC-DAD. Research Journal of Pharmaceutical Dosage Forms and Technology. 2011: 3(2): 62-66.

18.   Sunitha N, Sujitha Y and Manohar S. Development and validation of RP-HPLC Method for the Simultaneous Estimation of Ketorolac Tromethamine and Olopatadine Hydrochloride in pure and Pharmaceutical Formulation. Research Journal of Pharmaceutical Dosage Forms and Technology. 2014: 6(1): 37-43.

19.   Santanu G and BB Barik. Quantization of Aceclofenac in Pharmaceutical Formulations by RP-HPLC. Research Journal of Pharmaceutical Dosage Forms and Technology. 2010: 2(1): 52-55.

20.   Sushil P, Sunil and C.D. Upasani, Development and Validation of Stability Indicating RP-HPLC Method for Empagliflozin. Asian Journal of Pharmaceutical Analysis. 2016: 6(4): 201-206.

 

 

 

Received on 04.07.2019         Modified on 11.07.2019

Accepted on 04.08.2019         © RJPT All right reserved