Principle and HPLC System
Review of literature
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
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
• Zidovudine or azidothymidine (AZT) is a nucleoside analog
• A type of antiretroviral drug used for the treatment of
• It is an analog of thymidine.AZT was the first approved
treatment for HIV, sold under the names Retrovir and
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
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
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.
S.NO Parameter Result
2 Mobile phase Acetonitrile:
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
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
100 μg/ml 528325.5
The linear regression equation was y
= -12183.9+ 5130 X
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
100μg/ml 255178.7 1.96%
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
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
LIMIT OF DETECTION AND QUANTIFICATION
1 LOD 5ppm
2 LOQ 16.5ppm
The drug zidovudine being non polar is
preferably analysed by reverse phase
columns and accordingly C18 column was
The concentration of acetonitrile and
water were optimized to give symmetric
peak with short run time based on
asymmetric factor and peak area
• 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
• 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
• 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,
• 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.