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  1. 1. B.Lakshmi et al., IJSIT, 2012, 1(1), 08-16 A NOVEL RP-HPLC METHOD FOR THE QUANTIFICATION OF ICATIBANT IN FORMULATIONS B.Lakshmi, Kallam Haranadhreddy Institute of Technology, Guntur Prof T.V.Reddy , Prof Malla reddy College of Engineering, Secunderabad. ABSTRACTA simple, precise and accurate RP-HPLC method was developed and validated for rapid assay of Icatibanttablet dosage form. Isocratic elution at a flow rate of 1ml/min was employed on a symmetry Chromosil C18(250x4.6mm, 5µm in particle size) at ambient temperature. The mobile phase consisted of Methanol:Acetonitrile: water 57:13:30 v/v,. The UV detection wavelength was 224nm and 20µl ss was injected. Theretention time for Icatibant was 9.89min. The percentage RSD for precision and accuracy of the method wasfound to be less than 2%. The method was validated as per the ICH guidelines. The method was successfullyapplied for routine analysis of Icatibant tablet dosage form and bulk drug.Key Words: Icatibant, RP-HPLC, UV detection, recovery, precise, 224nm. 8 IJST (www.ijsit.com), Volume 1, Issue 1, September-October 2012
  2. 2. B.Lakshmi et al., IJSIT, 2012, 1(1), 08-16 INTRODUCTIONIcatibant (trade name Firazyr) is a peptidomimetic drug consisting of ten amino acids, which is selective andspecific antagonist ofbradykinin B2 receptors. It has been approved by the European Commission for thesymptomatic treatment of acute attacks,[1][2] of hereditary angioedema (HAE) in adults (with C1-esterase-inhibitor deficiency).Bradykinin is a peptide-based hormone that is formed locally in tissues, very often in response to a trauma. Itincreases vessel permeability, dilates blood vessels and causes smooth muscle cells to contract. Bradykininplays an important role as the mediator of pain. Surplus bradykinin is responsible for the typical symptoms ofinflammation, such as swelling, redness, overheating and pain. These symptoms are mediated by activation ofbradykinin B2 receptors. Icatibant acts as a bradykinin inhibitor by blocking the binding of native bradykininto the bradykinin B2 receptor. EXPERIMENTALMaterials: Working standard of Icatibant was obtained from well reputed research laboratories. HPLC gradewater, Methanol was purchased from E. Merck (Mumbai, India).Apparatus: A Series HPLC [6-11] system PEAK LC 7000 isocratic HPLC with PEAK 7000 delivery system. Rheodynemanual sample injector with switch (77251), Analytical column Chromosil C18. 250×4.6mm, Electronicbalance-DENVER (SI234), manual Rheodyne injector with a 20 μl loop was used for the injection of sample.PEAK LC software was used. UV 2301 Spectrophotometer was used to determine the wavelength of maximumabsorbance.Determination of wavelength of maximum absorbance: The standard solutions of Icatibant were scanned in the range of 200 -400 nm against mobile phaseas a blank. Icatibant showed maximum absorbance at 224nm. So the wavelength selected for thedetermination of Icatibant was 224nm. 9 IJST (www.ijsit.com), Volume 1, Issue 1, September-October 2012
  3. 3. B.Lakshmi et al., IJSIT, 2012, 1(1), 08-16Chromatographic equipment and conditions: To develop a High Pressure Liquid Chromatographic method for quantitative estimation ofICATIBANT an isocratic PEAK HPLC instrument with Zodiac C18 column (250 mm x 4.6 mm, 5μ) was used.The instrument is equipped with a LC 20AT pump for solvent delivery and variable wavelengthprogrammable LC – 7000 UV-detector. A 20μL Rheodyne inject port was used for injecting the samples. Datawas analyzed by using PEAK software. The mobile phase consisted of Methanol: Acetonitrile: water 57:30:13 v/v,. Injections were carriedout using a 20 μl loop at room temperature (20 + 2 °C) and the flow rate was 1 ml/min. Detection wasperformed at 224nm with 10min runtime.Standard and sample solutions: A 10 mg amount of Icatibant reference substance was accurately weighed and dissolved in 10 mlmobile phase in a 10 ml volumetric flask to obtain 1000 ppm concentrated solution. Required concentrationswere prepared by serial dilution of this solution. A composite of 20 (INTELENCE) tablets was prepared by grinding them to a fine, uniform sizepowder. 10 mg of Icatibant was accurately weighed and quantitatively transferred into a 100 ml volumetricflask. Approximately 25 ml mobile phase were added and the solution was sonicated for 15 min. The flaskwas filled to volume with mobile phase, and mixed. After filtration, an amount of the solution was dilutedwith mobile phase to a concentration of 10ppm.Method validation: Method validation was performed following ICH specifications for specificity, range of linearity,accuracy, precision and robustness. RESULTS AND DISCUSSIONSystem Suitability: Having optimized the efficiency of a chromatographic separation, the quality of the chromatographwas monitored by applying the following system suitability tests: capacity factor, tailing factor and theoreticalplates. The system suitability method acceptance criteria set in each validation run were: capacity factor >2.0,tailing factor ≤2.0 and theoretical plates >2500. In all cases, the relative standard deviation (R.S.D) for theanalytic peak area for two consecutive injections was < 2.0%. A chromatogram obtained from referencesubstance solution is presented. System suitability parameters were shown in Table.1. Standardchromatogram was given in Figure.2 10 IJST (www.ijsit.com), Volume 1, Issue 1, September-October 2012
  4. 4. B.Lakshmi et al., IJSIT, 2012, 1(1), 08-16 Api Concentration 10ppm Mobile Phase MeoH:ACN:H2O 57:30:13 Wavelength 224nm Column C18 Column PH 5.4 Concentration 10ppm Retention Time 9.89 Run Time 10min Area 115233 Th. Plates 4453 Tailing Factor 0.91 Pump Pressure 8.2 MPa Table1: System suitability parameters of ICATIBANTFigure 2: Standard chromatogram of IcatibantRange of linearity:Standard curves were constructed daily, for three consecutive days, using seven standard concentrations in arange of 10, 20, 30, 40, 50, and 60ppm for Icatibant. The linearity of peak area responses versusconcentrations was demonstrated by linear least square regression analysis. The linear regression equationwas y = 9304+ 3500x (r= 0.999). Linearity values can show in Table 2. S.No Concentration (µg/ml) Area 1 10 115233 2 20 300426 3 30 458762 4 40 621482 5 50 799879 6 60 953210 Slope 16249.92 Intercept 23355.9 CC 0.999127 Table 2: Linearity results of Icatibant 11 IJST (www.ijsit.com), Volume 1, Issue 1, September-October 2012
  5. 5. B.Lakshmi et al., IJSIT, 2012, 1(1), 08-16 1200000 1000000 800000 600000 400000 200000 0 0 10 20 30 40 50 60 70 -200000 Figure 3: Calibration curve of IcatibantPrecision: To study precision, six replicate standard solutions of Icatibant (10ppm) were prepared andanalyzed using the proposed method. The percent relative standard deviation (% RSD) for peak responseswas calculated and it was found to be which is well within the acceptance criteria of not more than 2.0%.Results of system precision studies are shown in Table.3 and Table.4. Sample (µg/ml) Area 1 115568 2 115798 3 116029 4 115867 5 114972 6 116003 RSD 0.342387 Table 3: Intraday Precision Results for Icatibant 12 IJST (www.ijsit.com), Volume 1, Issue 1, September-October 2012
  6. 6. B.Lakshmi et al., IJSIT, 2012, 1(1), 08-16 Sample (µg/ml) Area 1 114213 2 115689 3 115982 4 119689 5 115867 6 116021 RSD 1.565656 Table 4: Inter day Precision results of IcatibantLimit of Detection and Limit of Quantification: To determine the Limit of Detection (LOD) sample was dissolved by using Mobile phase and injecteduntil peak was disappeared. After 0.83ppm dilution Peak was not clearly observed, based on which 0.83ppmis considered as Limit of Detection and Limit of Quantification is 2.5ppm. Parameter Measured Value Limit of Quantification 2.5ppm Limit of Detection 0.83ppm Table 5: LOD and LOQ results of IcatibantRobustness: Typical variations in liquid chromatography conditions were used to evaluate the robustness of theassay method. The robustness study was performed by slight modification in flow rate of the mobile phase,composition of the mobile phase and wavelength of the detector. Icatibant at standard concentration wasanalyzed under these changed experimental conditions. It was observed that there were no marked changesin chromatograms, which demonstrated that the developed method was robust in nature. The robustnessacceptance criteria set in the validation were the same established on system suitability test describe above.Results were shown in table 6. 13 IJST (www.ijsit.com), Volume 1, Issue 1, September-October 2012
  7. 7. B.Lakshmi et al., IJSIT, 2012, 1(1), 08-16 S.NO Parameter Change Area % of Change 1 Standard ……………… 115233 ………… 2 MP Methanol:ACN:water 57:30:13 47:40:13 115489 0.22 116201 0.84 3 PH 5.6 115620 0.33 5.2 115976 0.64 4 WL 222nm 116097 0.74 226nm 116382 0.99 Table 6: Robustness results of IcatibantRuggedness: Ruggedness was performed by using six replicate injections of standard and sample solutions ofconcentrations which were prepared and analyzed by different analyst on three different. Ruggedness alsoexpressed in terms of percentage relative standard deviation. CONC INJECTION NO PEAKS AREA R.S.D SAMPLE (PPM) (Acceptance criteria ≤ 2.0%) 1 116421 2 115986 10ppm 3 116106 Icatibant 0.167061 4 115938 5 116036 6 115874 Table 7: Ruggedness results of Icatibant 14 IJST (www.ijsit.com), Volume 1, Issue 1, September-October 2012
  8. 8. B.Lakshmi et al., IJSIT, 2012, 1(1), 08-16Recovery:The accuracy of the method was determined by standard addition method. A known amount of standard drugwas added to the fixed amount of pre-analyzed tablet solution. Percent recovery was calculated by comparingthe area before and after the addition of the standard drug. Recovery test was performed at 3 differentconcentrations i.e. 90ppm, 120ppm, 150ppm. The percent recovery was calculated and results are presentedin Table. Satisfactory recoveries ranging from 98.07 to 101.14 were obtained by the proposed method. Thisindicates that the proposed method was accurate. Results are given in table.8 Icatibant % Target Conc., Spiked conc, Final Conc, Conc., % of Recovery Recovery (ppm) (ppm) (ppm) Obtained 50% 10ppm 10ppm 30ppm 29.84 99.46 50% 10ppm 10ppm 30ppm 30.28 100.9 50% 10ppm 10ppm 30ppm 30.51 101.7 100% 20ppm 20ppm 40ppm 39.32 98.3 100% 20ppm 20ppm 40ppm 40.27 100.6 100% 20ppm 20ppm 40ppm 40.61 101.5 150% 40ppm 40ppm 60ppm 59.28 98.8 150% 40ppm 40ppm 60ppm 60.41 100.6 150% 40ppm 40ppm 60ppm 60.53 100.8 Table 8: Recovery results of Icatibant Formulation Dosage Concentration Amount found % Assay INTELENCE 100mg 120ppm 119.80 99.83 Table 9: Formulation Analysis CONCLUSION The proposed method for the assay of Icatibant in tablets or capsules is very simple and rapid. Itshould be emphasized it is isocratic and the mobile phase do not contain any buffer. The method wasvalidated for specificity, linearity, precision, accuracy and robustness. Although the method could effectivelyseparate the drug from its products, further studies should be performed in order to use it to evaluate thestability of pharmaceutical formulations. 15 IJST (www.ijsit.com), Volume 1, Issue 1, September-October 2012
  9. 9. B.Lakshmi et al., IJSIT, 2012, 1(1), 08-16 REFERENCES1. Rooprai, H. K. et al. Evaluation of the effects of swainsonine, captopril, tangeretin and nobiletin on the biological behaviour of brain tumour cells in vitro. Neuropathol Appl Neurobiol 27, 29-39.2. Nakagawa, T. et al. Captopril inhibits glioma cell invasion in vitro: involvement of matrix metalloproteinases. Anticancer Res 15, 1985-1989.3. Geppetti, P. et al. Acute oral captopril inhibits angiotensin converting enzyme activity in human cerebrospinal fluid. J Hypertens 5, 151-154.4. Inoue, N. et al. Effect of angiotensin-converting enzyme inhibitor on matrix metalloproteinase-9 activity in patients with Kawasaki disease. Clin Chim Acta 411, 267-269.5. Yamamoto D, et al. Inhibitory profiles of captopril on matrix metalloproteinase-9 activity. Eur J Pharmacol. 588, 277-9. PubMed PMID: 18501888.6. Reinhardt, D. et al. Cardiac remodelling in end stage heart failure: upregulation of matrix metalloproteinase (MMP) irrespective of the underlying disease, and evidence for a direct inhibitory effect of ACE inhibitors on MMP. Heart 88, 525-530.7. Kichuk MR, et al. Angiotensin-converting nzyme inhibitors promote nitric oxide production in coronary microvessels from failing explanted human hearts. Am J Cardiol. 80, 137A-142A. PubMed PMID: 9293967.8. Efsen, E. et al. Ramiprilate inhibits functional matrix metalloproteinase activity in Crohns disease fistulas. Basic Clin Pharmacol Toxicol 109, 208-216.9. Tang, Q. et al. CD4+Foxp3+ regulatory T cell therapy in transplantation. J Mol Cell Biol 4, 11-21.10. Knutson KL, et al. Immunologic principles and immunotherapeutic approaches in ovarian cancer. Hematol Oncol Clin North Am. 17, 1051-7311. Curiel TJ, et al. Specific recruitment of regulatory T cells in ovarian carcinoma fosters immune privilege and predicts reduced survival. Nat Med. 10, 942-912. Preston, C. C. et al. Immunity and immune suppression in human ovarian cancer. Immunotherapy 3, 539-556.13. Gavalas NG, et al. Immune response in ovarian cancer: how is the immune system involved in prognosis and therapy: potential for treatment utilization. Clin Dev Immunol. 2010, 791603.14. Barnett, B. et al. Regulatory T cells in ovarian cancer: biology and therapeutic potential. Am J Reprod Immunol 54, 369-377.15. Wolf D, et al. The expression of the regulatory T cell-specific forkhead box transcription factor FoxP3 is associated with poor prognosis in ovarian cancer. Clin Cancer Res. 11, 8326-31.16. Sato, E. et al. Intraepithelial CD8+ tumor-infiltrating lymphocytes and a high CD8+/regulatory T cell ratio are associated with favorable prognosis in ovarian cancer. Proc Natl Acad Sci U S A 102, 18538- 18543.17. Kryczek I, et al. Relationship between B7-H4, regulatory T cells, and patient outcome in human ovarian carcinoma. Cancer Res. 67, 8900-5.18. Leveque L, et al. Interleukin 2-mediated conversion of ovarian cancer-associated CD4+ regulatory T cells into proinflammatory interleukin 17-producing helper T cells. J Immunother. 32, 101-8.19. Sehouli, J. et al. Epigenetic quantification of tumor-infiltrating T-lymphocytes. Epigenetics 6, 236-246.20. Söderlund J, et al. Acyclovir inhibition of IDO to decrease Tregs as a glioblastoma treatment adjunct. J Neuroinflammation. 7, 44. PubMed PMID: 20691089; PubMed Central PMCID: PMC2925358.21. Cannon MJ, et al. Dendritic cell vaccination against ovarian cancer--tipping the Treg/TH17 balance to therapeutic advantage? Expert Opin Biol Ther. 11, 441-5.22. Munn, D. H. et al. Indoleamine 2,3-dioxygenase and tumor-induced tolerance. J Clin Invest 117, 1147- 1154. 16 IJST (www.ijsit.com), Volume 1, Issue 1, September-October 2012