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zidovudine hplc method

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development and validation of zidovudine by RP-HPLC mehtod

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zidovudine hplc method

  1. 1. DDEEVVEELLOOPPMMEENNTT AANNDD VVAALLIIDDAATTIIOONN OOFF ZZIIDDOOVVUUDDIINNEE BBYY RRPP--HHPPLLCC MMEETTHHOODD BY U. Aparna Rajeevi B.Pharm., 8thsem. GGuuiiddeedd bbyy PPrrooff.. TT.. SSaattyyaannaarraayyaannaa DD.. NNaarreennddrraa PPrriinncciippaall VViiccee pprriinncciippaall LLyyddiiaa ccoolllleeggee ooff pphhaarrmmaaccyy
  2. 2. 2 contents  Introduction  Principle and HPLC System  Drug profile  Review of literature  Method development  Validation  conclusion  References
  3. 3. 3 INTRODUCTION TO HPLC HPLC is a form of liquid chromatography used to separate compounds that are dissolved in solution. HPLC instruments consist of a reservoir of mobile phase, a pump, an injector, a separation column, and a detector.
  4. 4. 4 PRINCIPLE PRINCIPLE Compounds are separated by injecting a sample mixture onto the column. The different component in the mixture pass through the column at differentiates due to differences in their partition behavior between the mobile phase and the stationary phase. The mobile phase must be degassed to eliminate the formation of air bubbles.
  5. 5. 5 INTRODUCTION TO DRUG • Zidovudine or azidothymidine (AZT) is a nucleoside analog reverse-transcriptase inhibitor. • A type of antiretroviral drug used for the treatment of HIV/AIDS. • It is an analog of thymidine.AZT was the first approved treatment for HIV, sold under the names Retrovir and Retrovis. •
  6. 6. 6 ABSTRACT A new simple, rapid, selective, precise and accurate isocratic reverse phase high performance liquid chromatography assay has been developed and validated for the estimation of ZIDOVUDINE in tablet formulation. The separation was achieved by using C-18 column (250x4.6mm, 5μm in particle size) at ambient temperature coupled with a guard column of silica in mobile phase Acetonitrile: Water with the pH value adjusted to 4.8 .The flow rate was 1ml/min and the drug was detected by using UV detector at the wavelength 240nm and the run time was 10min. The retention time was found 4.7 minutes. The percentage of RSD for precision and accuracy of the method was found to be less than 2%. The method was validated as per ICH guidelines.The proposed method was found to be accurate, repeatability and consistent. It can be successfully applied for the analysis of the drug in marketed formulation and could be effectively used for the routine analysis of the same drug without any alteration in the chromatographic conditions
  7. 7. PLAN OF WORK
  8. 8. Materials and methods  In this analytical method, the development and validation of zidovudine was done by using reverse phase HPLC method.  The assay was performed on a Series HPLC system PEAK LC7000 isocratic HPLC with PEAK 7000 delivery system.  Rheodyne manual sample injector with switch (77251), Analytical column zodiac C18. 250×4.6mm, manual injector Rheodyne valve with 20μL fixed loop, PEAK LC software was used.  UV 2301 SPECTROPHOTOMETER was used to determine the λmax.
  9. 9. Chromatographic Conditions S.NO Parameter Result 1 Standard concentration 40μg/ml 2 Mobile phase Acetonitrile: water{90:10} 3 Wave length 240nm 4 pH 4.8 5 Flow rate 1.0 ml/min. 6 Retention time 4.752 min. 7 Run time 10 min. 8 Peak area 164899.2 9 Theoretical plates 7063.64 10 Pump pressure 5.2psi
  10. 10. Standard chromatogram 10
  11. 11. 11 RANGE OF LINEARITY Standard curves were constructed daily, for three consecutive days, using nine standard concentrations in a range of 20, 30, 40, 50, 60, 70, 80, 90,100 μg/ml for zidovudine. The linearity of peak area responses versus concentrations was demonstrated by linear least square regression analysis.. Linearity values are shown as Conc. ( PPM) Area 20μg/ml 99424.4 30μg/ml 135483.3 40μg/ml 199623.0 50μg/ml 243651.0 6oμg/ml 297651.0 70μg/ml 359899.0 80μg/ml 435861.9 90μg/ml 360732.4 100 μg/ml 528325.5
  12. 12. 12 CALIBERATION CURVE The linear regression equation was y = -12183.9+ 5130 X (r= 0.990)
  13. 13. 13 PRECISION To study precision, six replicate standard solutions of zidovudine (100ppm) were prepared and analyzed using the proposed method and was given as following CONCENTRATION AREA (%)RELATIVE STANDARD DEVIATION 100μg/ml 246756.1 100μg/ml 240286.5 100μg/ml 255178.7 1.96% 100μg/ml 244876.8 100μg/ml 246177.6 100μg/ml 247922.5
  14. 14. 14 Interday Precision CONCENTRATION AREA (%)RELATIVE STANDARD DEVIATION 100μg/ml 121221.4 2.45% 100μg/ml 199471.8 100μg/ml 212402.4 100μg/ml 232883.1 100μg/ml 236348.5 100μg/ml 277456.8
  15. 15. 15 SYSTEM SUITABILITY Having optimized the efficiency of a chromatographic separation the quality of the chromatography was monitored by applying the following system suitability tests: capacity factor, tailing factor and theoretical plates. The system suitability method acceptance criteria set in each validation run were: capacity factor >2.0, tailing factor ≤2.0 and theoretical plates >2000.13. In all cases, the relative standard deviation (R.S.D) for the analytic peak area for two consecutive injections was < 2.0%. A chromatogram obtained from reference substance solution is presented. System suitability parameters were shown in Table.1. Standard chromatogram was given in Figure
  16. 16. SYSTEM SUITABILITY CHROMATOGRAM 16 SPECIFICITY The specificity of the method was determined by comparing test results obtained from analysis of sample solution containing recipients with that of test results those obtained from standard drug
  17. 17. 17 LIMIT OF DETECTION AND QUANTIFICATION 1 LOD 5ppm 2 LOQ 16.5ppm
  18. 18. 18 ROBUSTNESS SAMPLE(100 μg/ml) RETENTION TIME (min.) AREA % OF RECOVERY % OF DIFFERENCE Standard Wave length 240nm Flow rate 1ml/min 4.76 528325.5 100 0.0 Changed parameters Wave length 240 nm Flow rate 0.8 ml/min 5.9 341790.8 64.6 25.4 Wave length 240 nm Flow rate 1.2 ml/min 3.9 210465.4 39.8 60.2 Wave length 237nm Flow rate 1.0 ml/min 4.78 215568.8 40.8 59.2 Wave length 243nm Flow rate 1.0 ml/min 4.78 286167.7 54.1 45.9
  19. 19. 19 RECOVERY CONCENTRATION OF SAMPLE DRUG RECOVERY % DRUG RECOVERY %Average recovery 50% 20 μg/ml 21 PPM 105 100% 40 μg/ml 39.6 PPM 99 101% 150% 60 μg/ml 60.5 PPM 100
  20. 20. 20 The drug zidovudine being non polar is preferably analysed by reverse phase columns and accordingly C18 column was selected. The concentration of acetonitrile and water were optimized to give symmetric peak with short run time based on asymmetric factor and peak area obtained
  21. 21. CONCLUSION • The proposed method for the assay of zidovudine in tablet or capsule is very simple and rapid. • It should be emphasized that,it is isocratic and the mobile phase do not contain any buffer. • This method was validated for linearity, precision, system suitability, specificity, LOD, LOQ and robustness. 21
  22. 22. 22 REFERENCES • WHO Model List of Essential Medicines" (PDF). World Health Organization. March 2005. Retrieved 2006-03-12. • Connor E, Sperling R, Gelber R, Kiselev P, Scott G, O'Sullivan M, VanDyke R, Bey M, Shearer W, Jacobson R (1994). "Reduction of maternal-infant transmission of human immunodeficiency virus type 1 with zidovudine treatment. Pediatric AIDS Clinical Trials Group Protocol 076 Study Group". N Engl J Med 331 (18): 1173–80. • Sun, R.; Eriksson, S.; Wang, L. (2010). "Identification and Characterization of Mitochondrial Factors Modulating Thymidine Kinase 2 Activity". Nucleosides, Nucleotides and Nucleic Acids 29 (4–6): 382–385. • Richman, D. (1990). "Susceptibility to nucleoside analogues of zidovudine-resistant isolates of human immunodeficiency virus". The American journal of medicine 88 (5B): 8S–10S. • Broder, S. (2009). "The development of antiretroviral therapy and its impact on the HIV-1/AIDS pandemic". Antiviral research 85 (1): 1–2. 10.1016/j.antiviral.2009.10.002. • Horwitz, JP; Chua J; Noel MJ (1964). "The monomesylates of 1-(2-deoxy-bd-lyxofuranosyl) thymines". Org. Chem. Ser. Monogr 29 (7): 2076–9. • Henry D (writers/directors) (2002). I am alive today (history of an AIDS drug) (Film). ADR Productions/Good & Bad News. • Development and Validation of RP-HPLC Method for the Estimation of Abacavir, Lamivudine and Zidovudine in Pharmaceutical Dosage Form, T. Raja and A. LakshmanaRao International Journal of PharmTech Research, Vol. 3, No.2, pp 852-857 • Validated specific HPLC method for determination of zidovudine during stability studies,  Ashenafi Dunge, Nishi Sharda, Baljinder Singh, Saranjit Singh, Journal of Pharmaceutical and Biomedical Analysis, Received 20 May 2004. Revised 14 September 2004. Accepted 14 September 2004, Available online 17 November 2004. • Reversed phase HPLC determination of zidovudine in rat plasma and its pharmacokinetics after a single intranasal dose administration, RUBIANA M. MAINARDES, and MARIA PALMIRA D. GREMIÃO, Biological Research, Biol. Res. v.42 n.3 Santiago 2009.
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  24. 24. 24 Pareto Diagram Example

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