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“Novel Analytical Method Development And Validation For Quantitative
Estimation Of Antiretroviral Drugs In Pharmaceutical Dosage Form”
SYNOPSIS FOR
M. PHARM DISSERTATION
SUBMITTED TO
RAJIV GANDHI UNIVERSITY OF HEALTH SCIENCES
BANGALORE, KARNATAKA
BY
SAVITHA K.
M. PHARM Part-I
DEPARTMENT OF PHARMACEUTICAL ANALYSIS
PES COLLEGE OF PHARMACY
BANGALORE-560050
(2010-11)
RAJIV GANDHI UNIVERSITY OF HEALTH SCIENCES
BANGALORE, KARNATAKA.
ANNEXURE-II
PROFORMA FOR REGISTRATION OF SUBJECTS FOR DISSERTATION
1.
Name of the candidate SAVITHA K.
Department of Pharmaceutical Analysis,
and address:
P.E.S. College of Pharmacy,
50 Feet Road, Hanumanthanagar,
BSK I-Stage,
Bangalore-560050.
Permanent Address
D/O Kempaiah C.
#10,2nd G cross, 8th main,
Opp new Baldwin school,
Muneshwaranagar
Hegganahalli main road
Bangalore-560091.
2.
Name of the institution:
3.
Course of
subject:
4.
Date of the admission:
5.
Title of the topic:
study
P.E.S. College of Pharmacy
50 Feet Road, Hanumanthanagar,
BSK I- Stage,
Bangalore-560050
and MASTER OF PHARMACY IN
PHARMACEUTICAL ANALYSIS
15/06/2010
“Novel Analytical Method Development And Validation For Quantitative
Estimation Of Antiretroviral Drugs In Pharmaceutical Dosage Form”
2
6.
BRIEF RESUME OF THE INTENDED WORK:
6.1 Need for the study:
Viruses have been producing an enormous health hazards continuously for the mankind since
ages. These challenges were constantly met by the mankind by producing the effective drugs. There
are number of new drug molecules that have been developed for the effective treatment of HIV
infection or other viral infections. As recently the mankind has encountered some viral infections
which are caused by newer viral strains like H1N1, Ebola virus, Hanta virus, Marburg virus etc
which has caused havoc in both developed & developing countries posing newer threats &
challenges to the mankind. These newer virus strains which have been recently identified are the
main cause for the increased mortality rate in both humans and animals. All these infections require
some new drugs so that these infections can be treated & prevented. So there is a need for
development of newer antiretroviral drugs and formulations which in turn makes it obligatory for
their quantifications either in APIs or in the formulations.
Introduction:
A retrovirus is any virus belonging to the viral family Retroviridae. They are enveloped viruses
possessing a RNA genome, and replicate via a DNA intermediate. Retroviruses rely on the enzyme
reverse transcriptase to perform the reverse transcription of its genome from RNA into DNA, which
can then be integrated into the host's genome with an integrase enzyme. The virus then replicates as
part of the cells DNA. HIV or the human immunodeficiency virus causes HIV infection. Once a
person becomes infected with this virus is infected for life resulting in AIDS (The Acquired
immunodeficiency syndrome).
Antiretroviral drugs are medications for the treatment of infection by retroviruses, primarily HIV.
Different classes of antiretroviral drugs act at different stages of the HIV life cycle. Combination of
several (typically three or four) antiretroviral drugs is known as Highly Active Anti-Retroviral
Therapy (HAART). Antiretroviral treatment should be considered if an HIV infected adult has a
CD4 count less than 200 cells, or has developed a serious (stage 4) illness. They lower the level of
the virus in the blood. This allows the immune system to recover.
3
Influenza, commonly referred to as the flu, is an infectious disease caused by RNA viruses of the
family Orthomyxoviridae. There are different strains of influenza viruses which includes H1N1,
which caused Spanish Flu in 1918, and Swine Flu in 2009 H2N2, which caused Asian Flu in
1957 H3N2, which caused Hong Kong Flu in 1968 H5N1, which caused Bird Flu in 2004 H7N7,
which has unusual zoonotic potential H1N2, endemic in humans, pigs and birds
H9N2,H7N2,H7N3,H10N7.(1)
Ebola and Marburg viruses are members of the Filoviridae family of the order Mononegavirales.
Both viruses are associated with recurrent outbreaks of deadly hemorrhagic fevers with mortality
rates as high as 90%. Currently, there are no approved vaccines, nor treatments for Ebola virus
(EBOV) infection. A better understanding of the molecular aspects of EBOV replication will be
necessary for successful development of specific treatments for EBOV infection.(2)
Hantaviruses form a separate genus within the Bunyaviridae family. There are currently more than
20 recognised sero/genotypes and many others are under investigation. Each hantavirus type
appears to be specific to a different rodent host. Virus phylogeny very closely reflects rodent
phylogeny. The different hantavirus types are associated with different types of disease both in
terms of target organs and disease severity. Two major diseases are recognised: haemorrhagic fever
with renal syndrome (HFRS) and hantavirus pulmonary syndrome (HPS). HFRS is primarily a
disease of the old world while HPS is only recognised in the America.(3)
Classification and mechanism of antiretroviral agents:
Antiretroviral (ARV) drugs are broadly classified by the phase of the retrovirus life-cycle that the
drug inhibits.
Nucleoside and nucleotide reverse transcriptase inhibitors (NRTI):Nucleoside and nucleotide
reverse transcriptase inhibitors (NRTI) inhibit reverse transcription by being incorporated into the
newly synthesized viral DNA and preventing its further elongation. Ex: lamivudine, abacavir,
zidovudine, stavudine, didanosine, emtricitabine, tenofovir.
Non-nucleoside reverse transcriptase inhibitors (NNRTI): Non-nucleoside reverse transcriptase
inhibitors (NNRTI) inhibit reverse transcriptase directly by binding to the enzyme and interfering
with its function.Ex: delavirdine, efavirenz, etravirine, nevirapine.
4
Protease inhibitors (PIs) :Protease inhibitors (PIs) target viral assembly by inhibiting the activity
of protease, an enzyme used by HIV to cleave nascent proteins for final assembly of new virons.Ex:
amprenavir, fosamprenavir, atazanavir, darunavir, indinavir, nelfinavir, ritonavir, saquinavir,
tipranavir.
Integrase inhibitors: Integrase inhibitors inhibit the enzyme integrase, which is responsible for
integration of viral DNA into the DNA of the infected cell. There are several integrase inhibitors
currently under clinical trial, and raltegravir became the first to receive FDA approval in October
2007. Ex: raltegravir.
Entry inhibitors (or fusion inhibitors): Entry inhibitors (or fusion inhibitors) interfere with
binding, fusion and entry of HIV-1 to the host cell by blocking one of several targets. Ex:
Maraviroc and enfuvirtide.
Maturation inhibitors: Maturation inhibitors inhibit the last step in gag processing in which the
viral capsid polyprotein is cleaved, thereby blocking the conversion of the polyprotein into the
mature capsid protein. Because these viral particles have a defective core, the virions released
consist mainly of non-infectious particles.Ex: Alpha interferon, bevirimat and Vivecony.
The concept of analytical chemistry lies in the precise and accurate measurements. This
determination requires highly sophisticated instruments and methods like HPLC, Gas
chromatography, spectrophotometry, HPTLC etc.
On the literature survey it was found that various above mentioned anti-retroviral drugs were
estimated by several analytical methods either alone or in combination by HPLC methods likes, the
combination of emtricitabine (EMT) and tenofovir (TEN). Emtricitabine is a nucleoside reverse
transcriptase inhibitor (NRTI) for the treatment of HIV infection in adults. Tenofovir is a nucleotide
analogue reverse transcriptase inhibitor (NRTI). Literature survey reveals that, so far
Spectrophotometric method, HPTLC and RP-HPLC
UV
method has been reported for the
simultaneous estimation of EMT and TEN in formulation and in bulk is developed.(4-7). A new
HPLC-UV method to quantify simultaneously maraviroc and raltegravir levels in human plasma is
reported. Remarkably, this is the first method for maraviroc determination in human plasma.(8).
Determination of sulfate and glutarate ions in Abacavir Hemisulfate using ion chromatography with
conductivity detection.(9). Drugs like Zidovudine, lamivudine,stavudine (10) is estimated and
studied by HPLC which may be coupled with radio immune assay or solid phase extraction.(11-17).
5
Finally many antiretroviral drugs are estimated by HPLC flourimetric method other
chromatographic techniques and even in tendam mass spectrophotometry.(18-20).
Since we are doing the project in collaboration with Microlabs pvt Limited,
Bangalore at Analytical service department, at this instance it is not possible to disclose the name of
the anti-retroviral drugs on which the analytical methods were going to develop. In view of the
need in the industry for routine analysis of anti-retroviral drugs in formulations attempts shall be
made to develop simple and accurate instrumental method for estimation of antiretroviral drugs and
extend it for their determination in formulation.
6.2 REVIEW OF THE LITERATURE:

M.N Hemlata et al reported the application UV spectrophotometric method for estimation of
Emtricitabine in bulk and capsule.(4).

J. Maithilee et al. developed the HPTLC Method for the Simultaneous Estimation of
Emtricitabine and Tenofovir in Tablet Dosage Form.(5).

Rajesh sharma et al.developed the method for a validated RP-HPLC method for
simultaneous estimation of emtricitabine and tenofovir disoproxil fumarate in a tablet
dosage forms.(6)

S. Sagar et al. reported the stability indicating ion-pair RP-HPLC methods for the estimation
of Tenofovir Disoproxil fumarate in tablet dosage form.(7).

S. Notari et al. developed the simultaneous determination of maraviroc and raltegravir in
human plasma by HPLC-UV. (8).

Girish R Deshpande et al. developed method for the determination of sulfate and glutrate
ions in abcavir hemisulfate using ion chromatography with conductivity detection.(9).

V.K. Suresh et al. developed the formulation and in vitro evaluation of sustained release
matrix tablet of Stavudine.(10).

Dung et al. developed the validated specific HPLC method for determination of zidovudine
during stability studies.(11)

Lefebvre et al. reported the quantification of zidovudine and its monophosphate in cell
extracts by on-line solid-phase extraction coupled to liquid chromatography.(12)
6

J. D. Moore et al. developed the simultaneous quantization of the 5'-triphosphate
metabolites of zidovudine, lamivudine, and stavudine in peripheral mononuclear blood
cells in HIV infected patients by high-performance liquid chromatography tandem mass
spectrometry.(13)

K. Peter et al. developed the quantification of zidovudine and individual zidovudine
phosphates in peripheral blood mononuclear cells by a combined isocratic high performance
liquid chromatography radioimmunoassay method.(14)

Rita de Cassia et al. developed the rapid and sensitive method for simultaneous
determination of lamivudine and zidovudine in human serum by on-line solid-phase
extraction coupled to liquid chromatography or tandem mass spectrometry detection.(15)

Slusher et al. developed the Intracellular zidovudine (ZDV) zidovudine and phosphates as
measured by a validated combined high-pressure liquid chromatography-radioimmunoassay
procedure. (16)

Teresa Nadal et al. published the rapid and sensitive determination of zidovudine and
zidovudine glucuronide in human plasma by ion-pair high-performance liquid
chromatography.(17)

D Avolio A et al. developed the method for an improved HPLC fluorimetric method for the
determination of enfuvirtide plasma levels in HIV-infected patients.(18)

E.L. Marino et al. developed and validated the laboratory methods for antiretroviral
quantitation using HPLC.(19)

Anne-Kathrin et al. developed the electrospray tandem mass spectroscopic characterisation
of 18 antiretroviral drugs and simultaneous quantification of 12 antiretrovirals in
plasma.(20).
7
6.3 OBJECTIVES OF THE STUDY:
In the proposed work attempts shall be made to:

To develop new analytical method for the estimation of latest antiretroviral drugs
preferably by spectrophotometric and chromatographic methods.

Validate the proposed method in accordance with BP, USP and ICH guidelines for
the intended analytical application.

Apply the proposed methods for analysis of antiretroviral drugs in their dosage form
and in raw materials.
7. Materials and methods:
7.1 Source of data:

Literature survey was done at P.E.S. College of Pharmacy using Internet facilities
(RGUHS HELINET) and at Library.
 Reference from library at RGUHS bangalore.
 Indian Institute of Sciences, Bangalore.
7.2 Method of collection of data:
Analytical method development on anti-retroviral drugs shall be carried out in collabration
with M/s MICRO LABS PVT LIMITED (Analytical service department). The analytical
methods will be developed by using instruments like UV visible spectrometer, HPLC, and
HPTLC. The data so obtained is treated statistically to determine the compliance of the
experimental result as per ICH and Pharmacopeial guidelines and for the routine use of the
developed analytical methods in industry.
8
JOURNALS:
1. Indian Journal of Pharmaceutical Sciences.
2. Journal of Chromatography A
3. Journal of Chromatography B.
4. Asian Journal of Pharmaceutical and Clinical Research.
5. Eurasian Journal of Pharma Chemistry.
6. International Journal of Pharma Research and Development.
7. Journal of American Society for Mass Spectrometry.
8. Journal of Pharmaceutical and Biomedical Analysis.
9. Journal of Medical Microbiology.
10. Virology Journal.
11. Journal of Young Pharmacist.
12. Rapid communication in Mass Spectrometry.
7.3. DOES THE STUDY REQUIRE ANY INVESTIGATION OR INTERVENTIONS TO BE
CONDUCTED ON PATIENTS OR OTHER HUMANS OR ANIMALS?
NO
7.4. HAS ETHICAL CLEARENCE BEEN OBTAINED FROM YOUR INSTITUTION
IN CASE OF 7.3?
NOT APPLICABLE.
9
8.
REFERENCES:
1.
Reed F, Johnson., Peter B, Ronald NH. Effect of Ebola virus proteins GP, NP and VP35 on
VP40 VLP morphology. Virology Journal. 2006;3(31).
2.
Neal N, Olen MK. From Emergence to Eradication: The Epidemiology of Poliomyelitis
Deconstructed American Journal of Epidemiology. 2010;172(11):1213-29.
3.
Mccaughey C, Hart CA. Hantaviruses Journal of Medical Microbiology.
2000;49(2000):587-99.
4.
Hemlata MN, Rajesh JO, Mahesh BF. Application UV spectrophotometric method for
estimation of Emtricitabine in bulk and capsule. International Journal of Pharma Research and
Development 2010;2(8).
5.
Maithilee J, Nikalje AP, Shahed M, Dehghan M. HPTLC Method for the Simultaneous
Estimation of Emtricitabine and Tenofovir in Tablet Dosage Form. Indian Journal of
Pharmaceutical Sciences. 2009;71(1):95–7.
6.
Rajesh S, Pooja G. A Validated RP- HPLC Method for Simulataneous Estimation of
Emtricitabine and Tenofovir Disoproxil Fumarate in a Tablet Dosage Form. Eurasian Journal of
Analytical Chemistry 2009 19 May
4(3):276-84.
7.
Sagar S, Panda., Pratik D, Annapurna M. Stability indicating ion-pair RP-HPLC methods
for the estimation of Tenofovir Disoproxil fumarate in tablet dosage form. International Journal of
Pharma Research and Development 2009;1(10).
8.
Notari S, Tommasi C, Nicastri E, Bellagamba R, Tempestilli M, Pucillo LP, et al.
Simultaneous determination of maraviroc and raltegravir in human plasma by HPLC-UV.
Antimicrobial Agents and Chemotherapy. 2009 61(4):470-5
9.
Girish RD, Mallikarjuna RB, Someswara RN, Pallavi V. Determination of sulfate and
glutarate ions in Abacavir Hemisulfate using ion chromatography with conductivity detection.
Journal of Chemical and Pharmaceutical Research. 2010;2(2):321-9.
10.
Suresh VK, Ranjit k, P., Shankar MS, Someshwara R, B., Ashok K, P., Ramesh B.
Formulation and in vitro evaluation of sustained release matrix tablet of Stavudine. Asian Journal
of Pharmaceutical and Clinical Research. 2009;3(3):216.
11.
Dunge A, Sharda N, Singh B, Singh S. Validated specific HPLC method for determination
of zidovudine during stability studies. Journal of Pharm Biomedical Analysis. 2005;37(5):1109-14.l
10
12.
Lefebvre I, J.-Y. Puy CP, Perigaud CQozaimicebo-ls-pectlcJoCB-. Quantification of
zidovudine and its monophosphate in cell extracts by on-line solid-phase extraction coupled to
liquid chromatography. Journal of Chromatography B. 2007;858(1-2):2-7.
13.
Moore DJ, Gilles V, Albert D, Xiao-Jian Z, Jean-Pierremmadossi. Simultaneous
quantitation of the 5′-triphosphate metabolites of zidovudine, lamivudine, and stavudine in
peripheral mononuclear blood cells of HIV infected patients by high-performance liquid
chromatography tandem mass spectrometry. . Journal of American Society for Mass Spectrometry.
2000;11(12):1134-43.
14.
Peter K, Lalezari JP, Gambertoglio JG. Quantification of zidovudine and individual
zidovudine phosphates in peripheral blood mononuclear cells by a combined isocratic high
performance liquid chromatography radioimmunoassay method. journal of Pharm Biomedical
Analysis. 1996;14(4):491-9.
15.
Rita-de CE, Estrela., Myriam C, Salvadori., Guilherme S. A rapid and sensitive method for
simultaneous determination of lamivudine and zidovudine in human serum by on-line solid-phase
extraction coupled to liquid chromatography/tandem mass spectrometry detection. . Rapid
Communication in Mass Spectrometry. 2004;18(10):1147-55.
16.
Slusher JT, Kuwahara SK, Hamzeh FM, Lewis LD, Kornhauser DM, Lietman PS.
Intracellular zidovudine (ZDV) and ZDV phosphates as measured by a validated combined highpressure liquid chromatography-radioimmunoassay procedure. Antimicrobial Agents And
Chemotherapy. 1992;36(11):2473-7.
17.
Teresa N, Jordi O, Jose AP. Rapid and sensitive determination of zidovudine and
zidovudine glucuronide in human plasma by ion-pair high-performance liquid chromatography.
Journal of Chromatography A. 1996;721(1):127-37.
18.
D' Avolio A, de Requena, Ibanez A, Bonora S, Di Perri G. An improved HPLC fluorimetric
method for the determination of enfuvirtide plasma levels in HIV-infected patients. Therapeutic
Drug Monitoring. 2006;28(1):110-5.
19.
Marino EL, Albert V, Ferrer MS, Modamio P, Lastra CF. Development and validation of
laboratory methods for antiretroviral quantitation using HPLC. Journal of Young Pharmacists 2009
20 April;52(2):534–8.
20.
Anne-Kathrin G, Gerd M, Walter EH, Jurgen B. Electrospray tandem mass spectroscopic
characterisation of 18 antiretroviral drugs and simultaneous quantification of 12 antiretrovirals in
plasma. Rapid Communications in Mass Spectrometry. 2007 30 August 21(16):2704–16.
21.
FDA Food And Drug Administration
11
22.
Websites:
www.rguhs.ac.in
www.pubmed.com
www.medline.com
www.chemindustry.com
www.google.com
www.sciencedirect.com
www.ingentaconnect.com
12
9.
Signature of the candidate
(Savitha K)
10. Remarks of the guide
11. Name and Designation of
11.1 Guide
Mr. VENKATESH PRASAD
Asst.Professor
Department of Pharmaceutical Analysis,
P.E.S. College of Pharmacy,
Hanumanthanagar,
Bangalore-560050
11.2 Signature
11.5 Head of the department
Dr. Nagaraj
Professor & Head
P.E.S College of Pharmacy,
Hanumanthanagar,
Bangalore-560050
11.6 Signature
12. Remarks of the Chairman and
Principal
Prof. Dr. S. Mohan
Principal & Director,
P.E.S College of Pharmacy,
Hanumanthanagar,
Bangalore-560050
12.1 Signature
13