Stability-indicating RP-UPLC Method for Determination of Vildagliptin in Drug Substance and Its Tablet Dosage Form

Aim: The foremost purpose of this research work is to diminish the analysis time and to establish cost effective method for estimation of Vildagliptin by RP-UPLC. Study Design: UPLC based Quantification studies. Methodology: A simple, responsive and precised RP-UPLC method with good robustness was developed and validated as per ICH for the analysis of Vildagliptin in drug substance and separation of degradants generated by different forced degradation conditions. Productive separation of Vildagliptin was attained by the use of Luna C18 column (100x2.6mm and 1.6µm) with a mobile phase composition of 0.1% v/v Trifluoroacetic acid and Acetonitrile in 80:20 v/v, which was pumped with 0.5 ml/min flow rate. The eluted substances were examined with PDA detector at 239nm. Stressed degradation studies were performed with proposed method to determine the percentage degradation of Vildagliptin. Results: The RT of Vildagliptin was observed at 1.56 min. The developed method was validated as per ICHQ2B and proved that the method was precise, sensitive, specific and accurate.The lowest concentration of limit of detection (0.05µg/ml) and limit of quantification(0.5µg/ml) of Vildagliptin make obvious about the sensitivity of the method. The correlation coefficient found to be 0.9997 for given range of linear concentrations. The calculated average percentage recoveries of Vildagliptin in spiked solutions were found to be in the range of 99.1-100.5. The calculated % RSD was determined to be less than 2. Determination of degradation of amount of Vildagliptin by forced degradation studies representing the stability indicating nature of the proposed method. Conclusion: The developed method said to be highly sensitive, accurate, specific and robust, therefore this method has high probability to adopt in pharmaceutical industry for regular analysis of Vildagliptin.

concentration of limit of detection (0.05µg/ml) and limit of quantification(0.5µg/ml) of Vildagliptin make obvious about the sensitivity of the method. The correlation coefficient found to be 0.9997 for given range of linear concentrations. The calculated average percentage recoveries of Vildagliptin in spiked solutions were found to be in the range of 99.1-100. 5. The calculated % RSD was determined to be less than 2. Determination of degradation of amount of Vildagliptin by forced degradation studies representing the stability indicating nature of the proposed method. Conclusion: The developed method said to be highly sensitive, accurate, specific and robust, therefore this method has high probability to adopt in pharmaceutical industry for regular analysis of Vildagliptin.

INTRODUCTION
Diabetes or Hyperglycemia is the most of the prevailed diseases across the globe. Elevated blood sugar levels are diagnosed in diabetes. As per WHO study, around 462 million populations were agonized with diabetes [1]. A progressive research is done on diabetes and developing new way of treatments and novel antihyperglycemic agents Vildagliptin is one of the novel and efficient oral anti-hyperglycemic agent works by inhibiting dipeptidyl peptidase-4 (DPP-4) results in subdued the actions of glucagon-like peptide-1 (GLP-1) [2][3][4][5]. Vildagliptin chemically identified as acetyl]pyrrolidine-2carbonitrile. The chemical structure of Vildagliptin was represented in Fig. 1. Till date, few RP-HPLC methods were offered in the literature for the assessment and evaluation of Vildagliptinas as single entity or in blend with other antihyperglycemic agents in bulk and formulation forms [6][7][8][9][10][11][12]. Along with those few UPLC procedure were existed for vildaglitin alone or in blend with other agents [13].
In the reported method it was observed that complex mobile phase system, longer retention time and high sensitivity for Vildagliptin. Formerly, no RP-UPLC method was described for assessment and evaluation of Vildagliptin as single entity with simple solvent system, lesser retention time (RT) and exploration of degradants produced by stress conditions. The degradants in the drug substance particularly affects the excellence and purity of the drug substance. Hence, a simple UPLC method was developed with simple solvent system, lesser retention time (RT) and exploration of possible degradants produced by stress conditions.

2.1Reagents and Chemicals
Acetonitrile and Trifluoroacetic acid (TFA) of HPLC grade were procured from local supplier of Merck India. Vildagliptin API was obtained as gift sample from Hetero pharma, Hyderabad.

Instrumentation
Chromatographic separation was accomplished by using Waters alliance HPLC (2695) with photodiode array detector (PDA) and auto sampler. Data processing and integration was done with the Empower 2 tool.

Chromatographic Conditions
Productive separation of Vildagliptin was attained by the aid of Luna C18 column (100x2.6mm and 1.6µm) with a solvent composition of 0.1% v/v TFA and acetonitrile in 80:20 v/v, which was pumped with 0.5 ml/min flow rate. The eluted substances are examined in PDA detector at 239nm. Same solvent system was used in the preparation of stock solutions and dilutions.

Preparation of Standard Solution
Weighed accurately and transfer 50mg of Vildagliptin into 100ml of volumetric flask. Make up the volume with diluent to provide a solution of 0.5g/ml of Vildagliptin. 5ml of the above solution was again diluted to 50ml to get a solution of 50µg/ml of Vildagliptin.

Preparation of Sample Solution
62mg of Vildagliptin tablet powder was accurately weighed and transferred to 100ml of volumetric flask. Make up the volume with diluent to provide a solution of 0.5g/ml of Vildagliptin. 5ml of the above solution was again diluted to 50ml to get a solution of 50µg/ml of Vildagliptin.

Method Validation
The adopted method has been validated with respective to Q2 guidelines of ICH.

System suitability
System suitability of the proposed method has been carried out by introducing six homogenous replicate injections of standard solution (50µg/ml). Parameters like theoretical plates, % and tailing factor were evaluated for the gained chromatograms.

Specificity (Selectivity)
The blank, sample solution (50µg/ml), forced degradation solution(50µg/ml and standard solution(50µg/ml) were introduced in to UPLC system one after other. The derived chromatograms were interpreted to assess the occurrence of any interference from the blank, degradants and other substances with Vildagliptin peak.

Linearity
Linear response of the current procedure was established by assessing the correlation coefficient (R2) for the given series of concentrations ranges from 5 to 750 µg/ml Vildagliptin working standard solution.

Sensitivity
Standard deviation method has been adopted to calculate the limit of detection (LOD) and limit of quantification (LOQ). The following formulae were used to determine LOD and LOQ. LOD = 3σ/S LOQ = 10σ/S Here, σ is standard deviation of the intercept S is slope of the calibration curve

Precision
Intra-day precision of the proposed procedure was carried out by injecting prepared standard solution (50µg/ml) for 6 times in a day and inter day or intermediate precision was carried out by injecting prepared standard solution (50µg/ml). for 2 times in a day for 3 days continuously. The %RSD of the attained peak areas of Vildagliptin in both types of precessions was determined. The %RSD should be not more than 2.

Accuracy
To make sure the accuracy of the proposed procedure, percentage recovery procedure was implemented. In which predetermined amount of sample solution was spiked to Vildagliptin standard solution at, 50%, 100%, and 150% specification levels. Each spiked levels were injected in triplicate and average percentage recovery of sample concentration at different specification levels were assessed.

Robustness
Robustness of the present UPLC method was confirmed by modifies the flow rate and mobile phase composition slightly and deliberately. Standard solution (50µg/ml) was injected in triplicate with each modified parameter. %RSD of the attained peak areas of Vildagliptin in all modified cases were determined. The %RSD should be not more than 2.

Forced Degradation Studies (Stress Studies)
To find out the stability representing nature of the current method, standard solution (50µg/ml) was stressed by exposing to 1N HCl, 1NaOH, 10% H2O2, UV light at 254nm and 80°C/75% RH for 24 hours to produce degradation products. Each kind of stressed solution was injected and assess the percentage degradation of Vildagliptin.

Method Optimization
Productive separation of Vildagliptin was attained by the aid of Luna C18 column (100x2.6mm and 1.6µm) with a solvent composition of 0.1%v/v TFA and acetonitrile in 80:20, which was pumped with 0.5 ml/min flow rate. The eluted substances are examined in PDA detector at 239nm. Same solvent system was used in the preparation of stock solutions and dilutions. The RT of Vildagliptin was observed at 1.56min (Fig. 2). The optimized method condition have been satisfied the all the system suitability parameters (Fig. 2).

Method Validation
No interference was found at the retention time of Vildagliptin from sample, blank and degradants which extensively illustrates the specificity of the procedure (Fig. 3). The R 2 value calculated to be 0.9997 for Vildagliptin, which indicates good linearity (Fig. 4). The % RSD values of both precisions were etermined to be not more than 2 ( Table 1). The obtained results were given in and representative chromatogram in Fig. 5. The percentage recovery of Vildagliptin in spiked sample were in the range of 99.1-100.5 (Table 2), which significantly gives out the accuracy of the concerned method as of ICH limits. The LOD and LOQ values of Vildagliptin were assessed to be 0.05µg/ml and 0.5 µg/ml respectively. Deliberate changes in the flow rate and mobile phase composition could not affect the working properties of the method (Table 3) illustrate the robustness of the method.   The stability indicating property of the proposed method was confirmed through the degradation of Vildagliptin and well resolution of generated degradant peaks. The forced degradation conditions with percentage degradation in each stressed conditions were mentioned in Table 4 and Fig. 5. As comparison with available LC methods, the proposed method has less retention time of 1.55min, simple mobile phase composition of 0.1% TFA and Acetonitrile with good sensitivity in terms of LOD and LOQ.
In most cases stability indicating RP-HPLC method has noteworthy role in the analysis of the drugs. Until recently, a single stability indicating RP-HPLC method with high sensitivity and lower RT was not reported for Vildagliptin. In available methods sensitivity, RT and linear concentration range was not good. Hence attempt was made to develop an efficient, responsive stability indicating RP-HPLC method. The RT in the present developed method was 1.5 min for Vildagliptin outlines the method with lower RT, can be said as economical method. The statistical outcomes of the validation parameters of the current method were in the acceptance range ICH guidelines.

CONCLUSIONS
Based on established experimental results the proposed RP-UPLC method was reliable, highly sensitive, and cost effective for evaluation of Vildagliptin in drug substance and tablet form. The optimized FD conditions were effectively satisfying the stability indicating property of the established UPLC method. Hence it can be implemented in the regular analysis of Vildagliptin in API and tablet form in the pharmaceutical manufacturing units.

DISCLAIMER
The products used for this research are commonly and predominantly use products in our area of research and country. There is absolutely no conflict of interest between the authors and producers of the products because we do not intend to use these products as an avenue for any litigation but for the advancement of knowledge. Also, the research was not funded by the producing company rather it was funded by personal efforts of the authors CONSENT It is not applicable.

ETHICAL APPROVAL
It is not applicable.