Development and Validation of RP-HPLC Method for the Estimation of Gemigliptin
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1 Human Journals Research Article September 2018 Vol.:13, Issue:2 All rights are reserved by Hajera Khan et al. Development and Validation of RP-HPLC Method for the Estimation of Gemigliptin Keywords: Gemigliptin, RP-HPLC, Validation. ABSTRACT Hajera Khan*, Vaishali Shelke P. Department of Quality Assurance SSS Indira College of Pharmacy, Vishnupuri, Nanded Maharashtra India. Submission: 20 August 2018 Accepted: 27 August 2018 Published: 30 September 2018 The RP-HPLC method has been developed for the estimation of Gemigliptin. The quantification was carried out C 18 bonded phase i.e. Zorbax Eclipse XDB-C 18 ( mm 5μ) with particle size 5 μm in an isocratic mode with a mobile phase consisting of Methanol: Water (20:80 % v/v). The detection was carried out using a UV detector at 233 nm. The solutions of Gemigliptin was chromatographed at a constant flow rate of 1 ml/min & the retention time of the drug was found to be 2.3 min. The linearity range of Gemigliptin was found to be from 1-35 µg/ml. linear regression coefficient was As per ICH guideline, the method was validated for recovery, Precision, ruggedness and linearity.
2 INTRODUCTION Gemigliptin is Dipeptidyl Peptidase 4 inhibitor class of anti-diabetic drug 1. Its chemical formula is C 18 H 19 F 8 N 5 O 2 2. Gemigliptin adjunct to diet and exercise to improve glycemic control in adults with type 2 diabetes mellitus and can be taken with or without food. It is DPP-4 inhibitors which block the cleavage of the gliptins and thus lead to an increase insulin level and a reduced glucagon level in a glucose-dependent way 3. The structure of Gemigliptin is as shown in figure 1. Literature survey revealed that very few analytical methods have been reported for estimation of Gemigliptin. Rapid and sensitive RP-HPLC method for analysis of Gemigliptin and Metformin Hydrochloride was available 4, 5. Figure 1. Gemigliptin MATERIALS AND METHODS Material Gemigliptin was received as a gift Sample from Manus Akkteva Biopharma LLP, Ellisbridge, Ahmedabad. Methanol (HPLC grade) and Water were procured from Merk India. INSTRUMENT 6 The instrument used was Agilent 1220 LC series HPLC instrument. The instrument is consist of Agilent 1220 LC pump and variable wavelength programmable UV detector and 20 µl inject port. 225
3 Chromatographic conditions C 18 Column Zorbax Eclipse XDB- C 18 ( mm 5μ) was used for separation. The mobile phase containing Methanol: Water in the ratio 20:80 v/v. was delivered at flow rate 1 ml/ min and elution was monitored at 233 nm. Injection volume was 20 µl and analysis was performed at ambient temperature. Figure 2. Gemigliptin Chromatogram Preparation of mobile phase: A mobile phase consisting of Methanol (HPLC grade), water in the ratio of 20:80 v/v was prepared and then filtered through a 0.45 µ membrane filter. Preparation of standard stock solution: Accurately, about 10 mg of standard Gemigliptin was weighed and transferred to separate 10 ml volumetric flasks. The drugs was dissolved in methanol then volume made up to the mark with the same solvent to obtain a standard stock solution of drug of concentration 1000 μg/ml. 226
4 Preparation of working solution: Appropriate volume 0.01 ml of a standard stock solution of Gemigliptin Hydrochloride was transferred into 10 ml volumetric flask, diluted to mark with Distilled Water to give a desired concentration of drug. The resulting solution was scanned at 233 nm. Construction of calibration curve Appropriate aliquots of the standard stock solutions of Gemigliptin were pipetted out and transferred to a series of 10 ml volumetric flasks respectively. The volume was made up to the mark with water to obtain working standard solutions of Gemigliptin. The concentrations 1-35 µg/ml of Gemigliptin and. From these solutions, 20 µl injections of each concentration of the drug were injected into the HPLC system three times separately. Evaluation of the drug was performed with the UV detector set at 233 nm and the peak areas were recorded. The standard calibration curve for Gemigliptin was plotted as peak area Vs the respective concentration of Gemigliptin. Good linearity was obtained in the concentration range of 1-35 μg/ml for Gemigliptin. Figure No. 3: Calibration Curve of Gemigliptin 227
5 VALIDATION OF PROPOSED METHOD The proposed method was validated as per ICH guidelines 7,8 The solutions of the drugs were prepared as per the earlier adopted procedure is given in the experiment. a) Recovery study The accuracy of an analytical method is the closeness of the test results obtained by that of the true value. The accuracy of the proposed method has been carried out by recovery studies. It was performed by recovery study using standard addition method at 80, 100, and 120 % level; the known amount of standard Gemigliptin was added to the pre-analyzed sample (8, 10, 12 µg/ml) and subjected them to the proposed HPLC method. Results are shown in Table No. 2. b) Precision The precision of an analytical method is the degree of agreement among individual test results. The precision of the method was verified by using stock solutions containing 1 µg/ml Gemigliptin. System repeatability was done by repeating the assay three times of the same concentration after every two hours on the same day for intraday precision. Inter-day precision was carried out by performing the assay sample sets after 24 hours and 48 hours, results are reported in Table No. 3. c) Sensitivity The sensitivity of the proposed method was estimated in terms of Limit of Detection (LOD) and Limit of Quantitation (LOQ). LOD = 3.3 SD/S and LOQ = 10 SD/S, where SD is the residual standard deviation and S is the slope of the line. LOD was found to be 0.38 ng/ml & LOQ were found to be 1 ng/ml. d) Ruggedness From the stock solution, a sample solution of Gemigliptin (1 μg/ml) was prepared and analyzed by two different analysts using similar operational and environmental conditions. Peak area was measured for the same concentration solutions. The results are shown in Table No
6 e) Linearity and range It was performed using different test concentrations. The response was Linear in the range of 1 to 35 μg/ml for Gemigliptin (Figure no. 1). RESULT Developed HPLC for determination of Gemigliptin was sensitive, specific, precise, rugged and robust. For this method, the retention time was found to be min and mobile phase was Methanol: Water 20:80 v/v and the flow rate was 1.0 ml/ min. % label claim for Gemigliptin found to be indicative of the accuracy of the method. Statistical analysis proves developed method is rapid and economical & can be used for routine analysis of said drug. CONCLUSION The proposed method for the determination of Gemigliptin is accurate, precise, linear, robust, simple and rapid. Hence the present RP-HPLC method is suitable for ascertaining the quality control of Gemigliptin bulk drug. ACKNOWLEDGMENT The author is thankful to Manus Akkteva Biopharma LLP, Ellisbridge, Ahmedabad & Swapnroop Pharmaceuticals, Aurangabad for providing gift samples. The author is also thankful to the chairmen Mr. Santukrao Humberde, Indira College of Pharmacy, Nanded for providing the necessary facilities for the project work. REFERENCES 1. Luhar Shailesh V, Patel, Kajal R Dr. Jani GK, Dr. Narkhede; Narkhede; Sachin B Stability Study of Gemigliptin and Simultaneous Estimation of Gemigliptin and its Degradation Product by RP-HPLC Method, J Pharm Sci Bioscientific Res. 2016;6(3): Gemigliptin: migliptin 3. Wei Zeng, Donald Musson G, Alison Fisher L, Li Chen, Michael Schwartz S, Eric Woolf J, Amy Qiu Wang. Determination of sitagliptin in human urine and hemodialysate using turbulent flow online extraction and tandem mass spectrometry. J. Pharm. Biomed.Anal.2008; 46(3): Luhar Shailesh V, Patel, Kajal R Dr. Jani GK, Dr. Narkhede; Narkhede; Sachin B Stability Study of Gemigliptin and Simultaneous Estimation of Gemigliptin and its Degradation Product by RP-HPLC Method, J Pharm Sci Bioscientific Res. 2016;6(3):
7 5. Dr. Kumar GV, Dr. Nair D. Naresh, Ayana Ramesh; Analytical Method Development and Validation for the Simultaneous Estimation of Metformin and Gemigliptin by RP-HPLC Method, IJMPR, 2016;4(6): Instruction manual model HPLC-1220 Infinity LC, Agilent Technologies. 7. ICH, Q2A, Text on validation of analytical products, International Conference on Harmonization, Geneva, October 1994: ICH, Q2B, Text on validation of analytical products, International Conference on Harmonization, Geneva, November 1996: Table No. 1: Calibration Table for Gemigliptin Table No. 2: Result of Recovery Study Concentration in µg/ml Peak Area of GEM Level of % recovery Amount present (µg) 1 1 Amount of standard added (µg) The total amount recovered (µg) % Recovery Mean SD % RSD SE (n=3) *Average of three replicate at each level of recovery S.D Standard deviation, % RSD- Relative standard deviation. 230
8 Table No. 3: Result of Intraday Precision Sr. No. Conc. In µg/ml GEM Peak Area % Estimation Mean SD % RSD SE (n=3) *Average of three replicate at each level of recovery S.D Standard deviation, % RSD-Relative standard deviation. Table No. 4: Result of Ruggedness Sr. No. Conc. In µg/ml Peak Area GEM % Estimation GEM Analyst Analyst (n=3) *Average of three replicate at each level of recovery S.D Standard deviation, % RSD- Relative standard deviation. Table 5: Summary of validation parameter Parameters GEM Linearity range [µg/ml] 5-35 Regression equation y = 55103x [Y = mx +C] Recovery [% RSD] Precision [% RSD] Intra-day Inter-day Ruggedness [% RSD] Specificity Specific LOD 0.38 LOQ 1 231
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