Analytical Method Development and Validation of Teneligliptin by using RP-HPLC with ICH Guidelines

Copyright © 2019 by author(s) and International Journal of Trend in Scientific Research and Development Journal. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (CC BY 4.0) (http://creativecommons.org/licenses/ by/4.0) ABSTRACT Teneligliptin is drug used against type 2 diabetes mellitus and it is also a member of class of anti-diabetic drugs known as dipeptidyl peptidase-4 inhibitors or "gliptins". A simple, sensitive and accurate RP-HPLC method has been developed for the determination of Teneligliptin in bulk formulation. The λmax of the Teneligliptin was found to be 246 nm in Methanol: Phosphate buffer pH:3 [70:30 (v/v)]. The method shows high sensitivity with linearity 10 to 50 μg/ml (regression equation: y = 54647x 74133; r2 = 0.9968). The various parameters according to ICH guidelines are followed for validating and testing of this method. The Detection limit and quantitation limit were found to be 0.109 μg ml –1 and 0.3305 μg ml–1 in Methanol: Phosphate buffer pH: 3 [70:30 (v/v)] respectively. The % purity of tablet formulation was found to be 99.57%. The results demonstrated that the procedure is accurate, specific and reproducible (RSD < 2%), and also being simple, cheap and less time consuming and appropriate for the determination of Teneligliptin in bulk and pharmaceutical formulation.


INTRODUCTION
A new class of anti-diabetic drugs, Dipeptidyl peptidase-4 (DPP-4) inhibitors have recently introduced, that show enthusiastic results in the treatment of glycemic control with a minimal risk of hypoglycemia and weight gain. Teneligliptin, a novel Dipeptidyl peptidase-4 inhibitor, has a unique structure characterized by five consecutive rings, which produce a potent and long-lasting effect. Teneligliptin is now used as treatment in cases of insufficient improvement in glycemic control even after diet control and exercise and also a combination of diet control, exercise, and sulfonylurea-or thiazolidine-class drugs. Teneligliptin is administered orally at a dosage of 20 mg once daily in adults, which can be increased up to 40 mg per day. Because of the excretion of the metabolites of this drug are via renal and hepatic excretion, no special dose adjustment is necessary in patients who having renal impairment. Mitsubishi Tanabe Pharma Corporation (Osaka, Japan) are doing original synthesis of Teneligliptin and was the first drug of its kind to be synthesized in Japan. The drug under the brand name TENERIA® is sold jointly by Mitsubishi Tanabe Pharma Corporation and Daiichi Sankyo Co, Ltd, (Tokyo, Japan).
Teneligliptin is 1-(3-methyl-1-phenyl-1H-pyrazol-5-yl)-4-[(3S,5S)-5-(1,3-thiazolidine-3-carbonyl)pyrrolidin-3yl]piperazine (C22H30N6OS) and its structure is shown in Figure: 1 Analytical method validation provides that various HPLC analytical techniques shall give repeatable reliable and results; As it is providing information about accuracy, linearity, precision, detection, and quantitation limits and hence it is crucial step in developing new dosage forms. ICH guideline says that, "the objective of validation of an analytical procedure is to demonstrate that it is suitable for its intended purpose." It is now mandatory in the process of drug development to provide the validation data for the responsible authorities. The Guidelines for analysis method validation include ICH guidelines. By the literature survey a very few methods reported for determination of Teneligliptin in bulk drug as well as pharmaceutical preparation. This research is tries to develop a new sensitive and rapid HPLC method for the determination of Teneligliptin in Bulk preparation, and this method was also validated according to ICH guidelines.

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Preparation of Mobile phase
Preparation of Phosphate buffer pH 3: Dissolve 1.36g of Potassium dihydrogen orthophosphate & 2 ml of triethylamine in 800ml of HPLC water, adjust the pH to 3 with orthophosphoric acid and add sufficient HPLC water to produce 1000ml.The mobile phase was sonicated for 15 min and filtered through a 0.45 µm membrane filter paper.

Preparation of Standard solutions
10mg Teneligliptin was accurately weighed and transferred into 10 ml volumetric flasks, dissolved using mobile phase and the volume was made up with the same solvent to obtain primary stock solution of concentration 1000µg/ml of the drug (Working stock solution).
Preparation of Sample Solution 20 tablets of Teneligliptin were initially weighed and powdered and an amount equivalent to 10mg was accurately weighed into a 10ml volumetric flask, mixed with 10ml of mobile phase and sonicated for 5 min after making final volume up to 10 ml with mobile phase. Then solution was filtered through 0.45µm membrane filter. The solution contains 1000µg/ml of Teneligliptin. From the above stock solution 0.1ml aliquot was transferred in to a 10 ml volumetric flask, volume was made up to the mark with mobile phase to obtain a final concentration of 10 µg/ml of metformin.

Optimization of RP-HPLC method
The HPLC method was optimized with an aim to develop a estimation of Teneligliptin. Different mobile phases were tried for the method optimization, but acceptable retention times, theoretical plates and good resolution were observed with Methanol, Phosphate buffer pH 3 (70:30 v/v) using C18 column [Cosmosil C18 (250mm x 4.6ID, Particle size: 5 micron)] Table:1 and a typical chromatograph of teneligliptin was shown in figure 3.

Parameter Condition
Column

Validation of the RP-HPLC method
Validation of the optimized method was performed according to the ICH Q2 (R) guidelines.

Linearity
For the determination of linearity, appropriate aliquots were pipetted out from 1000µg/ml (working stock solution). 0.1 -0.5 ml was pipetted out in to a series of 10ml volumetric flasks and volume was made up with the solvent to obtain concentration ranging from 10-50µg/ml of metformin. Each solution was injected in triplicate. Calibration curves were plotted with concentration against observed peak areas followed by the determination of regression equations and calculation of the correlation coefficients. The calibration curves for Teneligliptin were shown in figure 2 and their corresponding linearity parameters given in table 2.

Accuracy
To ensure the reliability and accuracy of the recovery studies were carried out by % recovery method (standard addition method). A known quantity of pure drug was added to preanalysed sample and contents were reanalysed by the proposed method and the percent recovery was reported.
The results were given in tables 3 and 4.

Precision
The repeatability of the method was verified by calculating the % RSD of three replicate injections of 100% concentration (30µg/ml of Teneligliptin) on the same day and for intraday precision % RSD was calculated from repeated studies. The results were given in table 5.

Limit of Quantitation (LOQ) and Limit of Detection (LOD)
The LOD and LOQ were calculated from the slope(s) of the calibration plot and the standard deviation (SD) of the peak areas using the formulae LOD = 3.3 s/s and LOQ = 10 s/s.

Robustness
Robustness was verified by altering the chromatographic conditions like mobile phase composition, flow rate, detection wave length, etc. and the % RSD should be reported. In the operational conditions Small changes were allowed and the extent to which the method was robust was determined. A deviation of ± 2 nm in the detection wave length and ± 0.1 ml/min in the flow rate, were tried individually. Solutions of 100% test concentration with the specified changes in the operational conditions were injected to the instrument in triplicate. % RSD was reported in the table 6. Page: 261 and the amount equivalent to 10 mg of pure teneligliptin was dissolved in 10 ml of solvent. From this stock solution 30 ppm dilution was prepared and injected. The % purity was calculated by comparing the result with result obtained from 30 ppm standard drug and are reported in table 7.

System suitability
It was ensuring that from the system suitability parameters, the method can generate results of acceptable accuracy and precision. System suitability was carried out with three injections of solution of 30 µl/ml of Teneligliptin in to the chromatographic system. Number of theoretical plates (N) obtained and calculated tailing factor (T) was reported in table 8.

RESULT AND DISCUSSION Linearity:
It was clarified from the analytical method linearity as the ability of the method to obtain test results that are directly proportional to the analyte concentration, within a specific range. The peak area obtained from the HPLC chromatograph was plotted against corresponding concentrations to obtain the calibration graph. The results of linearity study (Figure 1) gave linear relationship over the concentration range of 10 -50 µg/ml for metformin. From the regression analysis, a linear equation was obtained y = 54647 x -74133, and the goodness-of-fit (r 2 ) was found to be 0.9968, indicating a linear relationship between the concentration of analyte and area under the peak.

Precision
Precision is "the closeness of results obtained from multiple sampling of the same homogeneous sample under the prescribed conditions," and it is expressed in the form of relative standard deviation. The repeatability, intra-day and inter-day precision results are shown in the table 5. The RSD were calculated for all the results are within limits. Precision was not more than 2.0% RSD, as demonstrated in Table 5.