zidovudine hplc method

DEVELOPMENT AND DEVELOPMENT AND VALIDATION OF VALIDATION OF

ZIDOVUDINE ZIDOVUDINE BY RP-HPLC METHODBY RP-HPLC METHOD

Guided byGuided by

Prof. T. Satyanarayana D. NarendraProf. T. Satyanarayana D. Narendra

Principal Vice principal Principal Vice principal

Lydia college of pharmacyLydia college of pharmacy

BY U. Aparna Rajeevi B.Pharm., 8thsem. Regd. no - 29131647

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contents

Introduction Principle and HPLC System Drug profile Review of literature Method development Validation conclusion

References

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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.

INTRODUCTION TO HPLC

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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.

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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.

•

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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

PLAN OF WORK

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.

Chromatographic Conditions S.NO Parameter Result

1 Standard concentration

40µg/ml

2 Mobile phase Acetonitrile: water{90:10}

3 Wave length 240nm4 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

Standard chromatogram

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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) Area20µg/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

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CALIBERATION CURVE

The linear regression equation was y = -12183.9+ 5130 X (r= 0.990)

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To study precision, six replicate standard solutions of zidovudine (100ppm) were prepared and analyzed using the proposed method and was given as following

PRECISION

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

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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

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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

SYSTEM SUITABILITY CHROMATOGRAM

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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

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LIMIT OF DETECTION AND QUANTIFICATION

1 LOD 5ppm

2 LOQ 16.5ppm

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ROBUSTNESS

SAMPLE(100 µg/ml)

RETENTION TIME (min.)

AREA% OF

RECOVERY% OF

DIFFERENCE

StandardWave length 240nmFlow rate 1ml/min 4.76 528325.5 100 0.0

Changed parametersWave length 240 nm

Flow rate 0.8 ml/min 5.9 341790.8 64.6 25.4

Wave length 240 nmFlow rate 1.2 ml/min

3.9 210465.4 39.8 60.2

Wave length 237nmFlow rate 1.0 ml/min 4.78 215568.8 40.8 59.2

Wave length 243nmFlow rate 1.0 ml/min 4.78 286167.7 54.1 45.9

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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

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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

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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.

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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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Pareto Diagram Example