A novel validated rphplc method for the estimation of liraglutide in bulk and parenteral dosage form
- 1. RESEARCH ARTICLE Am. J. PharmTech Res. 2013; 3(3) ISSN: 2249-3387 Please cite this article in press as: Susena S. et al., A Novel validated RP-HPLC method for the estimation of Liraglutide in bulk and Parenteral Dosage form. American Journal of PharmTech Research 2013. A Novel validated RP-HPLC method for the estimation of Liraglutide in bulk and Parenteral Dosage form S. Susena*1 , K. Vanitha Prakash1 , T.Radika1 , R.Tejaswini1 ,E.Manasa1 1. SSJ College of Pharmacy, V.N. Pally, Gandipet, Hyderabad-500075(India). Department: Pharmaceutical Analysis & Quality assurances) ABSTRACT A simple, accurate, precise and rapid reversed-phase high performance liquid chromatographic (RP-HPLC) method has been developed and subsequently validated for the estimation of Liraglutide in bulk and Parenteral Dosage form. The proposed method is based on the separation of drugs in reversed-phase mode using Waters HPLC 22695 model, Inertsil ODS column (250 x 4.6 mm, 5µm particle size).The optimum mobile phase consisted of methanol: phosphate buffer in the ratio of 85:15 v/v(Phosphate buffer pH 5.5) was selected as a mobile phase, flow rate of 1.0 ml/min and UV detection was set at 246 nm. The retention time was 3.25.The method was validated according to ICH guidelines. It was found to be accurate and reproducible. Linearity was obtained in the concentration range of 20-80 μg/ml . The percentage RSD for precision and accuracy of the method was found to be less than 2%. The proposed method can be successfully applied in the quality control of bulk and pharmaceutical dosage forms. Keywords:, Liraglutide ,RP-HPLC, Validation *Corresponding Author Email: susenaswrk@gmail.com Received 10 April 2013, Accepted 14 May 2013 Journal home page: http://www.ajptr.com/
- 2. Susena et. al., Am. J. PharmTech Res. 2013; 3(3) ISSN: 2249-3387 www.ajptr.com 2 INTRODUCTION Liraglutide is a long-acting glucagon-like peptide-1 (GLP-1) analog that has been developed by Novo Nordisk for the treatment of type2 diabetes. The product was approved by the European Medicines Agency (EMA) on July 3, 2009, and by the U.S. Food and Drug Administration (FDA) on January 25, 2010. Liraglutide intended to help lower blood sugar levels along with diet, exercise, and selected other diabetes medicines. It is not recommended as initial therapy in patients who have not achieved adequate diabetes control on diet and exercise alone. Liraglutide is an incretin mimetic. This means that it mimics the actions of incretin hormones in the body. As an incretin mimetic, liraglutide increases insulin production in response to meals and decreases the amount of glucose (sugar) that the liver produces. The medicine also slows the emptying of food from the stomach and decreases the amount of food that people eat. Figure 1: Structure of Liraglutide MATERIALS AND METHOD Apparatus: Waters HPLC 22695 series consisting 4 pump, Auto sampler, equipped with PDA detector, thermostat column connected with waters (alliance) Empower software ,Inertsil ODS C18 column (250x4.6mm, 5μm in particle size), Ohaus weighing balance and bath sonicator, borosil glass apparatus were used for experimental purpose. Chemicals and reagents: Liraglutide pure drug was obtained as a gift sample from HETERO LABS LIMITED. Hyderabad. Methanol and phosphate Buffer (PH-5.5) were purchased from S.D. Fine (P) Ltd. Mumbai. All chemicals and reagents used were of analytical HPLC grade. Preparation of mobile phase: Isocratic elution with Methanol: phosphate Buffer (PH-5.5) 85:15(V/V) was used at a flow rate of 1.0ml/min. The mobile phase was prepared freshly and degassed by sonicating for 5 min before use.
- 3. Nagariya et. al., Am. J. PharmTech Res. 2013; 3(3) ISSN: 2249-3387 3 www.ajptr.com Preparation of standard and test solutions Liraglutide standard stock solution: 10 mg Liraglutide was accurately weighed and dissolved in 10 ml methanol then transferred to a 10 ml volumetric flask sonicate it for 5 min, finally volume was made up to the mark with methanol to make 1000μg/ml stock solution. Working solution: 1ml of the above stock solution was pipetted into a 10ml volumetric flask and diluted up to the mark with HPLC grade Methanol. The contents were mixed well and filtered through 0.45μm nylon filter paper to get this stock solution (100 μg/ml). Preparation of sample solution: For the analysis of the dosage form, Liraglutide in injection form manufactured by novonordisk Ltd. was Selected. By using Liraglutide injection 1.7ml(i.e.10mg)of solution is transferred to 10ml volumetric flask finally volume was made up to the mark with methanol to make 1000μg/ml sample solution Method validation The method was validated in accordance with ICH guidelines17. The parameters assessed were linearity, accuracy, and limit of detection (LOD), limit of quantification (LOQ), precision, reproducibility and robustness. RESULTS AND DISCUSSIONS Linearity The calibration curve obtained by plotting peak area against concentration showed linearity in the concentration range of 20-80 μg/ml Linear regression data for the calibration curves are given in Figure 2. Figure 2: Linear regression data for the calibration curve 0 1000000 2000000 3000000 4000000 5000000 6000000 7000000 8000000 0 20 40 60 80 100
- 4. Susena et. al., Am. J. PharmTech Res. 2013; 3(3) ISSN: 2249-3387 www.ajptr.com 4 Accuracy The % mean recovery obtained for Liraglutide was 101% .The %RSD is less than 2, results were given in Table 1. Table 1: Accuracy data for proposed method Spiked level of drug (%) Amount of drug added (μg/ml) Amount of drug found (μg/ml) % Recovery 50 20 20.29 105.0 100 40 40.2 101.0 150 60 60.7 98.0 Detection limit and quantification limit LOD for Liraglutide was 0.24 μg/ml respectively, while LOQ was 0.72 μg/ml Precision Results for repeatability expressed as %RSD, results were given in Table 2. The low values of %RSD indicate that the method is precise. Reproducibility was checked by analyzing the samples by another analyst using same instrument and same laboratory. There was no significant difference between the %RSD values, which indicates that the proposed method was reproducible, results were showed in Table 2. Table 2: Precision of the proposed HPLC method Conc. of Liraglutide (40 μg/ml) Peak area of Liraglutide Intra-day Inter-day Injection-1 3218338 3318621 njection-2 3230606 3326451 Injection-3 3250591 3412131 Injection-4 3242511 3332153 Injection-5 3257321 3365143 Average 3239873.4 3350899.8 Standard Deviation 7802.8 14435.5 % RSD 0.2 0.4 Table 3: Results of robustness for proposed method Factor Level Retention time Asymmetry A: Flow rate (ml/min) 0.8 -0.2 3.90 1.34 1.0 0 3.25 1.32 1.2 +0.2 2.89 1.28 %RSD 0.3 0.7 B: % of methanol (ml) 84 -1 3.70 1.34 85 0 3.26 1.32 86 +1 3.10 1.28 %RSD 0.2 0.7
- 5. Nagariya et. al., Am. J. PharmTech Res. 2013; 3(3) ISSN: 2249-3387 5 www.ajptr.com Detection limit and quantification limit LOD for Liraglutide was 0.24 μg/ml respectively, while LOQ was 0.72 μg/ml Robustness There was no significant change in the peak areas and retention times of Liraglutide, when the composition of mobile phase ±1ml and flow rate was varied by ±0.2 ml. The results are showed in Table 3. Specificity No interference from any of the excipients was found at retention times of the examined drugs. In addition, the chromatogram of each drug in the sample solution was found identical to the hromatogram received by the standard solution at the wavelengths applied. These results demonstrate the absence of interference from other materials in the pharmaceutical formulations and therefore confirm the specificity of the proposed method. System suitability The acceptance criteria are % RSD of peak areas and retention time less than 2%, theoretical plates numbers (N) at least 4500 per each peak and tailing factors less than 1.5 for Liraglutide the results are shown in the Table 4. Table 4: System suitability parameters Parameters Liraglutide Linearity (μg/ml) 20.-80 Correlation coefficient 0.998 Theoretical plates 9248 Tailing factor 1.32 LOD (μg/ml) 0.24 LOQ (μg/ml) 0.72 Quantification of Liraglutide in parentral dosage form The proposed method was applied to the estimation of Liraglutide in parentral dosage form. The results of the assay 99.84±0.24%, of label claim of the injection. The assay results showed that the method was selective for the estimation of Liraglutide without interference from the excipients used in the injection form. The results are shown in the Table 5. Table 5: Results of sample analysis for proposed method Brand name Analyte Label claim per injection (mg) % Analyte estimated (mean±SD) %RSD VICTOZA Liraglutide 10 99.84±0.24 0.193 In order to achieve the estimation of the Liraglutide initial trials was performed with the objective of selecting adequate and optimum chromatographic conditions. Parameters, such as
- 6. Susena et. al., Am. J. PharmTech Res. 2013; 3(3) ISSN: 2249-3387 www.ajptr.com 6 ideal mobile phase and their proportions, detection wave length and concentrations of the standard solutions were carefully studied. Several solvents were tested in varying proportions. Finally, a mixture of methanol: Phosphate buffer (85:15 v/v) was selected as the optimum mobile phase. The optimized chromatographic conditions were selected based on sensitivity, retention times and peak shape. The method was validated in terms of linearity, accuracy, precision, LOD, LOQ, robustness and specificity as per ICH guidelines. The accuracy data shows that the method is accurate within desired range. The LOD and LOQ values were low which indicates that the method is sensitive. The method was robust as minor changes in the chromatographic parameters did not bring about any significant changes in peak area and retention times of Liraglutide Name Retention Time Area % Area Height USP Tailing Symmetry Factor USP Plate Count Liraglutide 3.259 3250591 100.00 374481 1.329972 1.339972 9283 Figure. 3: Typical chromatogram of Liraglutide standard CONCLUSION A validated RP-HPLC method has been developed for the determination of Liraglutide in injection form. The proposed method is simple, rapid, accurate, precise and specific. Its chromatographic run time of 10 min allows the analysis of a large number of samples in short period of time. Therefore, it is suitable for the routine analysis of Liraglutide in pharmaceutical dosage form. The limit of detection for Liraglutide was found to be 0.24 μg/ml and the limit of quantification was found to be 0.72 μg/ml. It proves the sensitivity of method. REFERENCES 1. http://www.novonordisk.com/science/about_rd/quarterly_rd_update.asp Oct 2008 Inc esults of LEAD 6 extension 2. http://money.cnn.com/news/newsfeeds/articles/marketwire/0580389.htm January 2009 Liraglutide-3.259 AU 0.00 0.02 0.04 0.06 0.08 0.10 0.12 0.14 0.16 0.18 0.20 Minutes 0.50 1.00 1.50 2.00 2.50 3.00 3.50 4.00 4.50 5.00 5.50 6.00 6.50 7.00
- 7. Nagariya et. al., Am. J. PharmTech Res. 2013; 3(3) ISSN: 2249-3387 7 www.ajptr.com 3. Hirschler, Ben (January 13, 2010). "UPDATE 1-Novo starts tests on pill version of Victoza drug".Reuters. 4. "Sector Snap: New Diabetes Drugs under FDA Review". Forbes. Retrieved March 27, 2009. 5. http://www.fda.gov/downloads/AdvisoryCommittees/CommitteesMeetingMaterials/Drug s/Endocr inologicandMetabolic Drugs Advisory Committee/UCM148659.pdf 6. http://www.drugs.com/fda/victoza-liraglutide-rdna-origin-rems-risk-thyroid-c-cell- tumors-acutepancreatitis- 12979.html 7. http://diabetes.webmd.com/news/20080924/new-diabetes-drug-liraglutide-works Sept 2008 8. http://care.diabetesjournals.org/cgi/content/abstract/32/1/84 "Efficacy and Safety Comparison of Liraglutide, Glimepiride, and Placebo, All in Combination with Metformin, in Type 2 Diabetes" Diabetes Care. Oct 2008 9. "Novo Nordisk Limited, Eli Lilly and Company Limited, Grünenthal Ltd and Napp Pharmaceuticals Limited named in advertisements". Prescription Medicines Code of Practice Authority (PMCPA). Retrieved 2011-02-07. 10. http://www.mmm-online.com/novo-reps-to-spotlight-weight-loss-for-victozalaunch/ Article/163167/ | Medical Media & Marketing; Novo reps to spotlight weight loss for Victoza launch; 2/4/2010; Ben Comer.