10 chapter 1shodhganga.inflibnet.ac.in/bitstream/10603/13919/10/10_chapter 1.pdf · the growth of...

Chapter 1

Introduction &

Review of literature

1.1 Introduction:

The growth of pharmaceutical industry in last three decades has been a pointer towards the

requirement of quality medicines to alleviate diseases, ill health and suffering to animals and

mankind. The simplest therapy of known organic molecules like aspirin, sulphadiazine and

naturally occurring medicines from plant origin have been replaced now by complex organic

molecules, which are analogues, derived from multiple synthetic routes. In the event of usage of

such molecules the establishment of analytical profile of these molecules together with the

desired medicinal activity like safety and efficacy has been the focus of attention of world-

renowned scientists and pharmacist.

Drug substances are seldom administered alone, but rather as part of a formulation. Each

particular pharmaceutical product is a formulation unique into itself. In addition to the active

therapeutic ingredients, a pharmaceutical formulation also contains a number of non-therapeutic

agents. These agents are generally referred to as pharmaceutical adjuncts, excipients or

necessities, and it is through their use that a formulation achieves its unique composition and

characteristic physical appearance.

There are many different forms into which a medicinal agent used for treatment of a disease.

Most commonly known dosage forms are capsules, tablets, injections, suppositories, ointments,

aerosols and many more including modern drug delivery systems like use of prodrug.

An Analytical chemist used in the many studies and used in this documentation. Drug analysis

and assay play important role in the method development, actual manufacture of drug and use of

drugs in routine analysis. Drug analysis shows the quality and its specifications which is used for

full scale production.

1.2 Analytical Chemistry:

It is a stream in which the information is gain on the composition and nature of matter in space

and time by the mean of method development and analysis performed onn the instruments [1].

Analytical chemistry differs from the other branches of chemistry in both its scope and approach.

While the other disciples are aimed at acquiring knowledge and creating theories in their

respective fields, analytical chemistry develops method and tools necessary to acquire

information about the chemical composition, its changes over time, its spatial arrangement and

the structure of molecules and crystals. Scientists of chemical, physical, biological and engineers

have collect the qualitative and quantitative information about the sample constitute and

structure. The correct choice and efficient use of modern analytical instruments requires an

understanding of the fundamental principles of operation of these measuring devices [2]. The

student –analyst when attains such an understanding that, reasonable choices be made among the

several alternative means of solving an analytical problem; then student- analyst will be aware of

the pitfalls that accompany physical measurements, and only then will the student be sufficiently

attuned to the limitations in sensitivity, precision and accuracy of an instrumental

measurement[3].

Analytical chemistry is the science, which deals with methods for detection of active and

exipients, identification of them and quantification of chemical, biological and microbiological

species in matrices of chemical, biological and environmental importance. A analytical method

used for identifying the no of molecules atoms in sample. [4]

Thus analytical chemistry is a science of chemical characterization and measurement.

An analytical method is the way in which an analytical chemist obtains the required

information. Pharma analysis is also defined as analytical techniques in chemistry through which

the analysis was done on drugs both as API as well as finish product i.e. formulation. In

pharmaceutical industry generally a bulk drug is referred to as Drug Substance or API (active

pharmaceutical ingredient) where as pharmaceutical product is referred to as Drug Product or

Finished product.

However in academic and pharmaceutical industry many other ways of analysis done like as bio-

analytical chemistry, drug metabolism studies and biotechnology.

Drug analysis means identification, characterization and determination of drugs. Drug assay

refers to determination of drugs in mixtures such as dosage forms and biologic fluids.

Drugs may be gases, liquids or solids. Prior to the formulation and manufacture of dosage

forms, bulk drugs must be properly identified (qualitative analysis) and analyzed for drug content

and the related impurities (quantitative analysis). Qualitative and quantitative determination of

sample is also necessary once drugs are used in animals and humans during experiment

development and treatment of patients.

1.3 Analytical Tests included for finish product Analysis:

Analysis of bulk drugs and pharmaceutical products generally carried out for following tests[5]:

� Description

� Identification

� Dissolution

� Content Uniformity

� Assay

� Chromatographic Purity or Related impurities

� Residual solvent analysis

1.3.1 Description Test:

It is used to describe of active and there related impurities in drug product is what it is stated.

1.3.2 Identification Test:

It is used to identify the active as well as impurities in the formulation. It includes tests such as

melting point, color reactions, ultraviolet spectra, infra-red spectra and relative retention time by

TLC or HPLC.

1.3.3 Dissolution test:

It is done for active dissolved in disso medium and no of percentage of active calculated. The

major properties are also calculated by vivo tests on blood samples by bio-analytical methods.

But in routine it is done by on in vitro models, which obviously have to be correlated with the

in vivo data.

1.3.4 Content Uniformity:

Consistency of dosage units is important. All dosage should have uniformly percentage

distributed contents, i.e. Analysis of particular batch shows similar percentage of the active

component. This involves individual dosage form assay. This wanted in small amount active

contents i.e. bewtwen a few µg per dose to 50 mg per dose.

1.3.5 Assay Test:

Assay is the estimation of potency of an active principle in a unit quantity of preparation. Ideally

assays should not only be specific for chemical entity under examination, but also be stability

determining.

1.3.6 Chromatographic Purity or Related impurities:

It indicates the homologues, analogues and byproducts from the analysis of synthesis or

degradation. Chromatography has checking analyte purity and is currently giving the fingerprint

of a synthesis. Impurities and degraded products are checked from detection level to 150% of

working concentration. That is active components should be checked up to 1000 folds excess of

major compound. Because of this sometime minute amount of related substance gets overlap on

the active. Which effects the in peak purity test and which will be solved by using photodiode

array detection in liquid chromatography. The general specifications for related known

impurities are based on its toxicology, where as limit for unknown impurities are set in

accordance to maximum daily dose.

1.4 Development of new analytical methods:

In the last century’s second half, the technological developments of instrumental analysis were

so wide and rapid that today the field of analytical chemistry has expanded towards “Computer

Based Analytical Chemistry.” Mainly focus on control of impurities and content of drug

throughout the shelf life of the product, as it is in small amounts effected at drug safety and

efficacy. So the new Analytical methods for consistent quality are developed for checking the

shelf life of the product .

It is also necessary to remember the choice of the method, as a wrong choice can lead not only to

erroneous results but also a loss of valuable time and money. In order to arrive at the correct

decision, he must be familiar with the practical details of the various techniques and of

theoretical principle upon which they are based. The techniques have differing degrees of

sophistication, sensitivity, selectivity, cost and also of time requirements. During the

developmental stage of method it is very important for the analyst to know the final goal of the

method. Before initiating analysis, the analyst should consider the following points are as

follows:

� What kind of sample matrix is to be analyzed?

� What are the analytes to be determined and what are their expected concentration

levels?

� Are there likely to be any interfering substances in the sample?

� Are there any specific regulatory requirements, such as action level or reporting limit

to be met by the analysis?

� What analytical instrumentation and analytical skills are available?

� Is it to be screening procedure capable of detecting and identifying a number of

compounds with similar physical and chemical properties?

� The accuracy and precision required

� What level of detection is required?

� How robust should the method be? Is it intended for the use by skilled analyst or by

technical assistant?

� How expensive the method is?

� The time required for completing the analysis; this will be particularly relevant when

the analytical results are required quickly for the control of manufacturing processes.

After answering the above questions any of the analytical technique like classical or instrumental

techniques can be applied. Before developing any chromatographic method, one has to review

the nature of sample, and goals of the separation defined. The sample related information that

needs to be known prior to HPLC method development is given below [6]

�� Number of components present.

�� Chemical structures (functionality of components)

�� Molecular weight of compounds.

�� pKa values, melting point and boiling points of the compounds.

�� UV spectra of compounds

�� Concentration of compounds in sample

�� Sample solubility

�� Nature of sample – regular or special.

After the analysis of sample by suitable method, the data generated has to be processed and

logical conclusions should be prepared.

1.4.1 Purpose of analysis and define problem :

In order to define the problem, the following questions need to be answered,

� Whether primary goal is quantitative analysis, the characterization of unknown sample

components or isolation of pure component.

� Is it necessary to separated all sample components, degradants, and impurities? Sometimes

only main component and impurities separation is sufficient or other time it is required that

main component should be separated from degradants or impurities from each other.

� If quantitative analysis is looked for then quantitation upto what level is required that should

be known.

� Method development should be known to be done on how many different types of sample

matrices that is raw material, formulations, environmental sample. Will one or more HPLC

procedures require? Or is a single or similar procedure for all samples desirable?

� Chromatographer should consider number of samples to be analysed at one time. Sometimes

for large number of samples to run, one has to opt for shorter run time by shortening the

column length or increasing the flow rate.

In short what is required of the method should be clearly understood before method development

begins. From the answer to the above questions the goal of the experiment will be cleared.

1.4.2 Information of sample:

More the gathered information about a sample, the better is the beginning for the method

development. The important information concerning sample composition and properties are as

follows:

� No. of compounds present in the sample?

� Concentration range of compounds in the sample preparation?

� The other properties like the chemical structure, molecular weight, pKa value, UV

spectra, solution stability and solubility.

Based on the composition of the sample, choice of the initial conditions for an HPLC separation

will be decided.

1.4.3 Sample preparation and detection:

Sample comes in various forms:

� Solutions ready for injections

� Solutions that require dilution, addition of internal standard or other volumetric

manipulation.

� Solids must be dissolved or extracted from formulation matrix.

� Sample that require pre-treatment to remove interferences and to protect the column

from damage

In HPLC analysis sample preparation is an important throughout the analysis as it provide a

reproducible and homogenous results by injecting the column through auto sampler. Best results

are obtained when the composition of the sample solvent is close to that of the mobile phase,

since this minimizes baseline disturbance and does not affect the sample retention and resolution.

Before the first sample is injected during HPLC method development, the detector is selected

based on the information of UV spectra.

1.4.4 Mode of separation:

In HPLC there are two types of samples that need separation; that is the regular and special

samples. Regular samples are usually the mixtures of small molecules (<2000Da), while special

samples are those, which require specialized methodology for separation. Regular samples are

those that do not require special sample treatment and can be separated using a regular method.

Special samples are usually large macromolecules, enantiomers and various inorganic ions that

require special sample pretreatment and specialized type of chromatography for their separation.

Regular samples can be further classified as ionic or neutral. Ionic solutes can be generally

defined as organic molecules that contain one or more functional groups capable of acidic or

basic behavior in the pH range of 2 to 8.

1.4.5 Selection of Stationary phase / Chromatographic Column:

In HPLC analysis column is very important for separation of analytes and impurities. The

requirement of stable, high performance column is good for rugged, reproducible HPLC method.

The appropriate choice of separation column includes three different approaches,

� Selection of separation system

� The particle size of silica present and the nature of the packing material of column

� The physical parameters of the columns i.e. the length and the diameter.

Peak shape is equally important in method development. Columns with symmetrical peaks are

good while peaks with poor asymmetry can result in

� Inproper plate number and resolution measurement

� Bad precision results

� Distorted peak shape at tailing

� Retention time not consistent

1.4.6 Selection of Mobile Phase:

The choice of mobile phase for a given separation constitutes a very important stage in

producing a good separation in HPLC. The requirements for solvents is as mobile phase in High

Performance Liquid Chromatography are

�� High purity to avoid introduction of peaks that may overlap with the analyte peaks.

�� Ready availability at reasonable cost

�� Low viscosity and reactivity to avoid chemical interaction with the analytes.

�� Immiscibility with the stationary phase.

�� Compatibility with the detector. Thus, for absorbance detection, the solvent should not

absorb at wavelengths to be used. For refractive index detectors, the solvent refractive

index must be significantly different from that of the solutes.

�� Limited flammability and toxicity.

Acetonitrile and Methanol are the most popularly used organic solvents in HPLC. Acetonitrile is

usually used for the mobile phase. Acetonitrile and water combinally used because it has lower

UV cut-off (185 to 210 nm) and low viscosity leading to higher plate numbers and lower column

backpressures. However methanol which has a relatively lower UV cut-off (205nm) is a

reasonable alternative.

1.4.7 Selection of Detector:

The detector is chosen depending upon some characteristic property of the analyte like UV

absorbance, fluorescence, conductance, oxidation, reduction etc. Characteristics that are to be

fulfilled by a detector to be used in HPLC determination are:

� High sensitivity, facilitating trace analysis

� Negligible baseline noise, to facilitate lower detection

� Large linear dynamic range

� Response independent of variations in operating parameters, such as, pressure, flow

rates, temperature, etc.

� Response independent of mobile phase composition

� Low dead volume

� Non destructive to the sample

� Stable over long periods of operation

� Convenient and reliable to operate

� Inexpensive to purchase and operate

� Capable of providing information on the identity of the solute.

Multi component pharmaceutical preparations contain more than one active ingredient with

variable concentrations. Detecting each component at their maximum absorbance, the purpose of

using HPLC for their simultaneous determination is lost. Hence in such cases one has to explore

the possibility of selecting the wavelength at which all the components are detected. This single

wavelength is referred to as “most suitable wavelength”. While selecting the wavelength, the

interest of minor component in the formulation or component with low extraction co-efficient

needs special consideration. Variable wavelength detector or a photo diode array detector is

used.

1.4.8 Optimization of selected parameters:

One has to optimize the selected parameters merely by changing each and every parameter at a

time keeping all other parameters constant. This will provide when, what and how much each

parameter affect.

1.4.9 Anticipating problems:

What can give wrong results? Or what precautions one should take during the method

application so that method can serve the intended purpose. Like if hardness of the tablets

increases during the stability and if the method is with whole tablets then extraction of the active

from the tablet may require more shaking or sonication time as time require to disintegrate the

tablets may increase. If solution turns hazy after standing for long time may lead to wrong results

so method design should take care of all the possible variables and manual errors.

1.4.10 Validation of the method:

Validation of method is an necessary for submission of developed product. The aim of validation

of method is to show that it is suitable for outine analysis. As per the ICH guideline Q2 R2 for

Method development and validation, the official methods need not to be validated for entire

validation parameters but merely to be verified for their suitability and feasibility.

� Mainly validation parameters are as listed below :

1. Specificity

2. Accuracy

3. Linearity and range

4. Limit of detection

5. Limit of quantitation

6. Precision

� Repeatability

� Intermediate Precision

7. Ruggedness

1.5 Method validation:

Validation is a basically required for quality and reliability of the results for analytical

applications. The inhouse procedures are used for developments and applications. The method is

used for samples analysed. The exipient other than analytes in pharma industry is constant results

of batch is based on the results of procedures and tests. Required limits of the method are limit

values, often based on stability, in the assay of an analyte and in impurity, which is take large

safety factors into account.

1.5.1 Necessity of Validation:

The main reason for method validation is that the law requires it. Title 21, Part 211 of the Code

of Federal Regulations on Current Good Manufacturing Practice for Finished Pharmaceuticals

states:

The accuracy, sensitivity, specificity, and reproducibility of test methods employed by the

firm shall be established and documented.

Within the FDA[7], the Center for Drug Evaluation and Research (CDER) is take care of safe

and effective drugs are available to the public.

The ICH guidances were developed to harmonize the registration requirements of method

validation between the United States, Japan, and the European Union, and they do not

necessarily cover all requirements that may be required in other parts of the world. The United

States Pharmacopoeia (USP) [8][9] is published by a non-government organization whose

publications of standards for the analysis of pharmaceuticals (US Pharmacopoeia / National

Formulary) are officially recognized by the Federal Food, Drug and Cosmetic Act. General

Chapter <1225> within the USP covers the requirements for validation of compendial methods

and is generally included in any discussion of guidance documents relating to method validation.

Table 1: Regulatory Guidance Documents:

Agency Title Published

ICH ICH-Q2A “Text on Validation of Analytical Procedure” 1994

ICH-Q2B “Validation of Analytical Procedures: Methodology” 1995

CDER

Reviewer Guidance: Validation of Chromatographic Methods 1994

Submitting Samples and Analytical Data for Method Validations 1987

Analytical Procedures and Method Validation 2000

Bioanalytical Method Validation for Human Studies 1999

USP <1225> Validation of Compendial Methods 2003

1.5.2 Validation requirements for Method Type:

The class of drugs are required for analysis are listed below and defined, with an table how they

are determined. Availability of procedure are defines which test and methods are to be used and

for which material. It is depend on which purpose for the procedure is used. A summation in

table is applicable to various analytical procedures is given in Table 2.

Table 2: Summary of Characteristics Applicable to various Analytical Procedures:

Class of drugs for different types of methods:

Class A: It is used for identification in bulk stage or finish product stage form.

Class B: It is used for detection and quantificationin bulk stage or finish product stage form.

Class C: It is used for assay calculation in active or finish product form.

Class D: it is used to determined characteristics of finished product forms, like dissolution

profiles and content uniformity.

Characteristic

Class

A

Class B

Class C

Class D Quantitative

tests

Limit

tests

Accuracy � � �

Precision � � �

Robustness � � � � �

Linearity and range � � �

Selectivity � � � � �

Limit of detection � �

Limit of Quantification �

Validation of methods is done by four common types [10][11[12]

� Identification Tests – is used for identification analyte in a sample. It is done by

spectrum, Chromatographic behavior, chemical reactivity, etc. and with the comparisons

to the reference standard

� Quantitative test for impurities’ It is mainly used for the peak purity analysis in the

sample.

� LOD and limit of quantification test for impurities

� Assay determines the percentage of analyte in the sample.

The assay is to determine the quantitatively analyte in the finish product. For drug product,

same validation parameters are apply when same methods are used e.g. dissolution. The

objective of method validation is to give the complete compliance data.

Method validation parameters are as below [13 [14]:

1. Specificity

2. Accuracy

3. Linearity and range

4. Limit of detection

5. Limit of Quantitation

6. Precision

a. Repeatability

b. Intermediate Precision

7. Ruggedness

8. Robustness

The detailed description is as follows:

1.5.3 Specificity / Selectivity:

The selectivity or specificity of a procedure is to determine that the analyte is free from any

contamination or overlapping of any impurity. When the impurities are known and available in

market then it is injected and compared with the retention time.

1.5.4 Accuracy:

The accuracy of an analytical method is the determine the recovery value to the true value.

Accuracy calculated by the percent recovery by the assay of known, added amounts of analyte.

The percentage recovery is determination of active in the dosage form is determined by adding

known amount of active in placebo to cover both above and below the normal levels (80%,

100% and 120 %) expected in samples. The peak area responses and assay of active in the three

ranges were calculated and the accuracy of the results was compared to the actual percentage of

drug added and the percentage of drug recovered. This is to know how accurately the active is

retrievable from the sample matrix. It is carried out at three different levels. The amount

recovered is calculated. Percent recovery at each level is calculated and statistical calculation

gives you % recovery which should be within 98-102%. Which gives the clear indication that

method is accurate.

1.5.5 Linearity and range:

The linearity of an analytical method is its ability to elicit test results data directly proportional to

the concentration of the analyte in samples within a given range. This parameter checks the

detector performance for the selected method. Series of dilutions are prepared and analyzed as

per the method. The graph is plotted for Concentration vs. response. The detector response is

said to be linear if Correlation coefficient of the graph when calculated is in the range of 0.9999 -

1.0.

1.5.6 Precision:

The precision is defined as the degree of agreement between test results. It is calculated by mean

results and individual results by standard deviation or as coefficient of variation (relative

standard deviation) when the complete procedure is applied repeatedly to separate identical

samples drawn from the same homogenous batch of material.

1.5.7 Repeatability (within-laboratory variation):

This is same as precision but the same analysis is done by different analyst under the same set of

conditions (same reagents, equipments, settings and laboratory) and within a short interval of

time. The repeatability is done on same batch or same homogenious material of same batch and

thus provides a measure of the precision of the procedure under normal operating conditions.

1.5.8 Reproducibility:

It is the precision of analysis which is carried on different conditions – usually in different

laboratories, on separate, identical samples taken from the same homogenous batch of material.

Comparisons of the results obtained by different analysts, by the use of different equipments, or

by carrying out the analysis at different times can also be provided valuable information.

1.5.9 Sensitivity:

It is determined by small variations in concentration and the same will be calculated by slope of

the calibration curve. A more general use of the term to encompass limit of detection and/or limit

of quantification should be avoided.

1.5.10 Limit of detection:

Limit of detection is a parameter of the limit test. It is the lowest concentration of analyte in a

sample that can be detected, but not necessarily quantitated under the stated experimental

conditions. Thus limit test merely substantiate that the analyte concentration is above or below a

certain level. The limit of detection is usually expressed as the concentration of analyte (e.g.

percentage or µg/ml) in the sample. The limit of detection calculated on lowest concentration of

analyte by the same method which is validated.

The limit of detection of active and its related impurities is determined by serially diluting at

lower concentration range, extended fairly close to the expected LOD. The concentrations were

selected based on the logic that the method must be enough sensitive to at least quantify less

than 0.5 times LOQ.

Duplicate injections of linear concentration of impurities are injected. The detection responses

were calculated from the calibration curve using the formula i.e. DL = [3.3∗ SyX/Slope]. Based

on the above calculation minimum detection limit are established.

1.5.11 Limit of quantitation (LOQ):

Limit of quantitation is a parameter of quantitative assay for low levels of compound in sample

matrices. It is lowest concentration of analyte in a sample that can be determined with

acceptance precision and accuracy under stated experiment condition”.

Duplicate injections of each impurity /active in the concentration range of 1- 150% of the

specified concentrations (approx.) are made to establish limit of detection and limit of

quantitation. The detection responses were calculated from the calibration curve using the

formula i.e. DL = [10∗ SyX/Slope]. Based on the above calculation minimum quantitation limit

are established

1.5.12 Ruggedness:

This parameter means calculation of degree of reproducibility of the results observed on same

samples under a variety of normal test conditions i.e. different instrument, analysts, days, etc.

The ruggedness of Assay test method of the dosage form is carried out by two different

analytical persons on two different instruments on different dates to know the %RSD of mean

assay results between the same sample.

1.5.13 Robustness:

When method is deliberately deviated by small amount the robustness of method is measure of

its capacity to remain unaffected and gives the reliability during normal usage.

The robustness of analytical method was evaluated by checking following parameters:

1. Effect on changing column oven temperature by ± 2°C

2. Effect on changing wave length by ± 2nm

3. Effect of pH of the mobile phase by ± 0.2

4. Effect on changing mobile phase composition by 5.0 ml.

5. Mobile phase flow rate changed by ± 0.1ml/min.

Method validation is very tedious procedures but it is necessary for the new developed method

since it is shows the quality of data generated and it is directly linked to the quality of procedure.

Time constraints often do not allow for sufficient method validations.

1.6 Antiviral drugs:

1.6.1 Definition: Antiviral drugs are medicines that cure or control virus infections.

1.6.2 Purpose:

Antivirals are used to treat infections caused by viruses. Unlike antibacterial drugs, which may

cover a wide range of pathogens, antiviral agents tend to be narrow in spectrum, and have limited

efficacy.

1.6.3 History:

In the mid- to late-20th century, medical science and practice included an array of effective tools,

ranging from antiseptics to vaccines and antibiotics, but no drugs to treat viral infections. While

vaccines were effective in preventing many viral diseases, they could not help once a viral

infection set in. Prior to the development of antivirals, when someone contracted a virus, there

was little that could be done other than treating the symptoms and waiting for the disease to run

its course. It was not until the 1980s, when the full genetic sequences of viruses began to be

unraveled, that researchers began to learn how viruses worked in detail, and exactly what

chemicals were needed to thwart their reproductive cycle. Dozens of antiviral treatments are now

available, and medical research is rapidly exploiting new knowledge and technology to develop

more. [15]

1.6.4 Introduction of antiviral drugs:

Inspite of the antiretroviral drugs other than used in HIV (AIDS) therapy, only 11 antiviral drugs

is available in market which covers four types of virus. Acyclovir (Zovirax), famciclovir

(Famvir), and valacyclovir (Valtrex) are used in herpesvirus, including herpes zoster and herpes

genitalis. They are also used for viruses caused by herpes, such as chickenpox and shingles. The

disease is not cure by these drugs but reduce the pain of a herpes outbreak and they shorten the

time of viral shedding.

Amantadine (Symmetrel), oseltamivir (Tamiflu), rimantidine (Flumadine), and zanamivir

(Relenza) are is inn treatment of influenza virus. Amantadine, rimantadine, and oseltamivir drugs

are taken orally for the flu season for cure the patients who not takes the influenza virus vaccine.

Antiviral drugs are often nucleoside analogues, (fake DNA building-blocks), which viruses

mistakenly incorporate into their genomes during replication. After taking the antiviral drugs the

life cycle was stopped because of new synthesised DNA become inactive. Due to which these

analogues are lack the hydroxyl groups, which, along with phosphorus atoms, link together to

form the strong "backbone" of the DNA molecule. It is known as DNA chain termination.

Examples of nucleoside analogues are aciclovir for Herpes simplex virus infections and

lamivudine for HIV and Hepatitis B virus infections. Aciclovir is antiviral drugs which is oldest

and commonaly used as antiviral drugs.

1.6.5 Antiviral agents:

Antiviral drugs are used to stop the production of viruses which cause disease. When virus is

replicating in that case most antiviral agents are effective.

It is not possible to find the medicines which is specific for virus because of viruses share the

metabolic processes of the host cells. Some enzymes are mainly present in viruses and which is

the main targets for antiviral drugs. A drug classified by the chemical type of the active

ingredient or by the way it is used to treat a particular condition. Every drug classified into one

or more drug classes.

Antiviral drugs which inhibit the transcription of the viral genome are DNA polymerase

inhibitors and reverse transcriptase inhibitors. Antiviral drugs injet the virus from attaching to or

penetrating the host cell. Immunomodulators induce production of host cell enzymes, which stop

viral reproduction. Integrase strand transfer inhibitors prevent integration of the viral DNA into

the host DNA by inhibiting the viral enzyme integrase. Neuraminidase inhibitors block viral

enzymes and inhibit reproduction of the viruses.

1.7 Acyclovir: Aciclovir or acyclovir, chemical name acyclo guanosine, abbreviated as ACV , is a guanosine

analogue antiviral drug, is available in market by the name of Zovirax and Xovir. It is most

commonly used antiviral drugs. It is basically treated against the treatment of herpes simplex

virus infections and also the treatment of varicella zoster (chickenpox) and herpes zoster

(shingles).

1.7.1 Properties of Acyclovir:

Chemical name:

2-amino-1, 9-dihydro-9-[(2-hydroxyethoxy)methyl]-6H-purin-6-one

Empirical formula C8H11N5O3

Chemical structure:

1.7.2 Physical properties:

Molecular weight: 225

Appearance and color: white, crystalline powder

Solubility: Acyclovir is freely soluble in dimethylsulfoxide, slightly soluble in water, and very

slightly soluble in ethanol. Acyclovir dissolves in dilute hydrochloric acid.

Therapeutic category: Antiviral Agent

Dosage: - Tablet

1.7.3 Pharmacology: A investigation of antiviral drugs are initiated by Burroughs Wellcome in the 1960s and it is

resulted to find the acyclovir in 1974. In 1977 Bioanalytical investigation was broug

drug to clinical trials and the first form of the drug (topical) was available to physicians in 1982.

This drugs are mainly used against herpes 1 and herpes 2, and in less against varicella zoster, still

less against Epstein-Barr, and very little a

when it is phosphorylated in infected cells by a viral

monophosphate is phosphorylated to diphosphate and triphosphate forms by cellular enzymes in

the infected host cell where the drug is concentrated. Acyclovir triphosphate inactivates viral

deoxyribonucleic acid polymerase. Acyclovir incorporation into the growing viral

deoxyribonucleic acid chain causes its termination. The antiviral process has relatively little

effect on normal, uninfected cells. An important toxic effect of acyclovir is its potential to cause

obstructive nephropathy. The drug is excreted primarily by the kidney, which may require

smaller doses in patients with decreased kidney function. Oral do

recommended for herpes simplex are probably not adequate for varicella zoster infections.

The related impurity of Acyclovir is as follows:

white, crystalline powder

Acyclovir is freely soluble in dimethylsulfoxide, slightly soluble in water, and very

slightly soluble in ethanol. Acyclovir dissolves in dilute hydrochloric acid.

Antiviral Agent

A investigation of antiviral drugs are initiated by Burroughs Wellcome in the 1960s and it is

acyclovir in 1974. In 1977 Bioanalytical investigation was broug



Barr, and very little against cytomegalovirus. Acyclovir is an antiviral agent

when it is phosphorylated in infected cells by a viral-induced thymidine kinase. Acyclovir


t cell where the drug is concentrated. Acyclovir triphosphate inactivates viral





smaller doses in patients with decreased kidney function. Oral dosages of acyclovir as


The related impurity of Acyclovir is as follows:

Acyclovir is freely soluble in dimethylsulfoxide, slightly soluble in water, and very

A investigation of antiviral drugs are initiated by Burroughs Wellcome in the 1960s and it is

acyclovir in 1974. In 1977 Bioanalytical investigation was brought this



gainst cytomegalovirus. Acyclovir is an antiviral agent

induced thymidine kinase. Acyclovir


t cell where the drug is concentrated. Acyclovir triphosphate inactivates viral





sages of acyclovir as


1.7.4 Acyclovir related impurity A:

Chemical name: 2-[(2-amino-6

Chemical structure:

Empirical formula: C12H15N5O

Molecular weight: 309.28

1.7.5 History of Acyclovir:

Aciclovir is termed as the new start in antiviral therapy,

specific as well as less cytotoxic. Aciclovir is synthesis from nucleosides isolated from a

Caribbean sponge, Cryptotethya crypta

acyclovir is Howard Schaffer and further it was cariied by with Robert Vince,

Gurwara on the adenosine analog acycloadenosine which showed promising antiviral activity.

After Schaffer joined Burroghs

Pharmacologist Gertrude B. Elion

for HIV patients.[23] Elion was awarded the 1988 Nobel Prize in Medicine, partly for the

development of aciclovir. Dr. Richard Whitley, a University of Alabama at Birmingham

researcher and pioneer in antiviral therapy, was the first to successfully use the drug in humans.

1.7.6 Pharmacology:

Mechanism of action:

Acyclovir is contain the sugar ring

previous nucleoside analogues in containing only a partial nucleoside structure. By the mean of

viral thymidine kinase acyclovir converted into acyc

GMP). It is far more effective in

the monophosphate form is further phosphorylated into the active

guanosine triphosphate (acyclo-GTP), by cellular

viral than cellular polymerase.

Acyclovir related impurity A:

6-oxo-1, 6-dihydro-9H-purin-9-yl)methoxy]ethyl acetate

O5

Aciclovir is termed as the new start in antiviral therapy, [17] as Acyclovir is very useful and

ic as well as less cytotoxic. Aciclovir is synthesis from nucleosides isolated from a

Cryptotethya crypta.[18][19][20] The Scientist is involve in discovery of

acyclovir is Howard Schaffer and further it was cariied by with Robert Vince,

Gurwara on the adenosine analog acycloadenosine which showed promising antiviral activity.

After Schaffer joined Burroghs-Wellcome and continued the development of aciclovir with

Gertrude B. Elion.[22] Vince later went on to invent abacavir, the

Elion was awarded the 1988 Nobel Prize in Medicine, partly for the


iviral therapy, was the first to successfully use the drug in humans.

sugar ring is replaced with an open-chain structure whic

in containing only a partial nucleoside structure. By the mean of

viral thymidine kinase acyclovir converted into acyclo-guanosine monophosphate

GMP). It is far more effective in phosphorylation than cellular thymidine kinase. Subsequently,

form is further phosphorylated into the active triphosphate

GTP), by cellular kinases. This is 100 times greater effected than

yl)methoxy]ethyl acetate

as Acyclovir is very useful and

ic as well as less cytotoxic. Aciclovir is synthesis from nucleosides isolated from a

The Scientist is involve in discovery of

acyclovir is Howard Schaffer and further it was cariied by with Robert Vince, S. Bittner and S.

Gurwara on the adenosine analog acycloadenosine which showed promising antiviral activity.[21]

Wellcome and continued the development of aciclovir with

Vince later went on to invent abacavir, the NNRTI drug

Elion was awarded the 1988 Nobel Prize in Medicine, partly for the


iviral therapy, was the first to successfully use the drug in humans.

chain structure which is differs from

in containing only a partial nucleoside structure. By the mean of

guanosine monophosphate (acyclo-

than cellular thymidine kinase. Subsequently,

triphosphate form, acyclo-

. This is 100 times greater effected than

Acyclovir is considered a prodrug. Mainly administered in less active form and is metabolised

into a more active species after administration.

It is useful to treat herpesvirus family:

• Herpes simplex virus type I (HSV-1)

• Herpes simplex virus type II (HSV-2)

• Varicella zoster virus (VZV)

• Epstein-Barr virus (EBV)

• Cytomegalovirus (CMV) least activity

Activity is used to treat mostly to HSV, and in less effective to VZV. Acyclovir is having very

limited effect on EBV and CMV. This drugg is almost inactive on latent viruses in nerve

ganglia.

1.7.7 Resistance:

Acyclovir is having low power to resistance , but in patients on chronic antiviral prophylaxis

which means transplant recipients, people with HIV infection. Process of resistance in HSV

include deficient viral thymidine kinase and mutations to viral thymidine kinase and/or DNA

polymerase, altering substrate sensitivity. [24] he has shown cross-resistance with valacyclovir and

famcyclovir.

1.7.8 Pharmacokinetics:

Since it is less water soluable and poor oral bioavailability (15–30%), hence for good effect it is

injected direct in intravenous and high concentrations are used. When it is taken orally effect of

this drugs observed after 1–2 hours. It is having high distribution rate for protein binding and it

is range from 9 to 33%. Acyclovir is having half-life is approximately 3 hours for elimination. It

is renally excreted, partly by glomerular filtration and partly by tubular secretion.

The poor oral bioavailability of acyclovir improved by administering Valaciclovir which is

having more oral bioavailability is of 55%. It is i.e. Valaciclovir then converted to acyclovir by

esterification via hepatic first-pass metabolism.

1.7.9 Indications:

It is used for the treatment of HSV and VZV infections such as:

• Genital herpes simplex (treatment and prophylaxis)

• Herpes simplex labialis (cold sores)

• Herpes zoster (shingles)

• Chickenpox

• Herpes simplex encephalitis

• Mucocutaneous HSV infections

• Herpes simplex keratitis (ocular herpes)

• Herpes simplex blepharitis

• Prophylaxis against Herpesviruses as patients undergoing cancer chemotherapy[25]

Dr Jairam Lingappa shown that the progress of HIV-1 can be e delayed by using aciclovir .

overall 16% it is effective in cases, which is delayed to 1–2 years with the HAART . In the

University of Washington, Seattle. in a trial period of 2 year , they observed the 284 people with

the progress of the HIV-1, and 324 who had not been treated with aciclovir. According to there

study they shows that the evidence for the effectiveness of topically applied cream for recurrent

labial outbreaks is weak. Before scientific literature showed that if aciclovir is applied at an early

stage of an outbreak , there is some effect in reducing the number and duration of lesions. But in

oral therapy for non immuno compromised patients based on costs and benefits, In those

countries where aciclovir is drug which is only available on prescription.

1.7.10 Dosage forms:

Aciclovir is available in market in tablets (200 mg, 400 mg, 800 mg and 1 gram), topical cream

(5%), intravenous injection (25 mg/ml) and ophthalmic ointment (3%). Cream preparations are

used primarily for labial herpes simplex. For high concentration is needed it is directly given in

intravenous as a form of injection. The ophthalmic form as ointment is used for herpes simplex

keratitis.

1.8 Valacyclovir:

Valaciclovir is an antiviral drug which is used for herpes simplex, shingles, and herpes B. It is a

prodrug of acyclovir so it gets converted into vivo to aciclovir. GlaxoSmithKline marketig this

drugs under the trade names Valtrex and Zelitrex . From Nov 2009 it is available in amrket as

generic form of valaciclovir in the U.S. [26]

1.8.1 Properties of Valacyclovir:

Chemical name:

(S)-2-[(2-amino-6-oxo-6, 9-dihydro

Empirical formula: C13H20N6O

Chemical structure:

Physical properties:


Description and color: It is a white, crystalline powder.

Solubility: It is having maximum

Therapeutic category: Antiviral Agent

Dosage: Tablet

1.8.2 Pharmacology:

It is prodrug of acyclovir, is taken in orally.

tract and liver, converting more than 95% to acyclovir, to provide significantly greater

bioavailability than oral acyclovir.

1.8.3 The related impurity of Valacyclovir is as follows:

Valacyclovir related compound C

2-[(2-amino-6-oxo-1, 6-dihydro-

hydrochloride

Chemical structure:

NH

NH2

dihydro-3H-purin-9-l) methoxy] ethyl-2-amino-3- methylbutanoate

O4. HCl

white, crystalline powder.

maximum solubility in water at 25°C is 174 mg/ml

Antiviral Agent

It is prodrug of acyclovir, is taken in orally. It is hydrolyzed by esterases in the gastrointestinal


bioavailability than oral acyclovir.

The related impurity of Valacyclovir is as follows:

related compound C:

-9H-purin-9-yl) methoxy] ethyl N-methyl-Lvalinate

N

N

N

O

ONH

O

CH3

CH3CH3

O

.HCl

methylbutanoate

It is hydrolyzed by esterases in the gastrointestinal


Lvalinate

Molecular formula: C15H21N3O3S.HCl


Chemistry:

Mainly it is synthesized from the natural proteinogenic amino acid L-valine.

1.8.4 Pharmacology:

Mechanism of action:

It is a prodrug, having more oral bioavailability (about 55%) than aciclovir (10–20%) can be

produce by esterified version of aciclovir. Valacyclovir is converted to esterases in the form of

aciclovir and same as the amino acid valine. Aciclovir is selectively converted into a

monophosphate form by viral thymidine kinase, which is far more effective (3000 times) in

phosphorylation than cellular thymidine kinase. Valacyclovir is 100 times more affinity to viral

than the cellular polymerase. Valacyclovir monophosphate form is more effective against viral

DNA, forms the chain termination. From study it is observed that the viral enzymes cannot

remove aciclo-GMP from the chain. By the mean of cellular phosphatases Aciclo-GTP is fairly

rapidly metabolised within the cell.

Valacyclovir is mainly used in against HSV, and in low effective or in small amount to VZV.

This drug is very limited efficacy against EBV and CMV; however, valacyclovir this drug

currently shown to lower or eliminate the presence of the Epstein–Barr virus in subjects afflicted

with acute mononucleosis for reduction of severity of symptoms.[27][28][29] This drug is

completely inactive against latent viruses in nerve ganglia.

1.8.5 Ingredients and dosage:

Valtrex is available in market in 500 mg and 1 gram tablets forms, the analyte is used as

valacyclovir hydrochloride and the exipients is used as carnauba wax, colloidal silicon dioxide,

cross povidone, FD&C Blue No. 2 Lake, hypromellose, magnesium stearate, microcrystalline

cellulose, polyethylene glycol, polysorbate 80, povidone, and titanium dioxide.

1.8.6 Indications:

Valaciclovir is used against the treatment of HSV and VZV infections, including: [29]

• Oral and genital herpes simplex (treatment and prophylaxis)

• Reduction of HSV transmission to uninfected individuals

• Herpes zoster (shingles): the typical dosage for treatment of herpes is 1,000mg orally

three times a day for seven consecutive days.

• Before organ transplantation the prevention of CMV disease done.

• Prophylaxis against Herpesviruses in patients undergoing cancer chemotherapy

• It has done treatment for infectious mononucleosis, and it is preventively taken in

suspected cases of herpes B virus.

1.9 REVIEW OF LITERATURE:

General Literature review from Journals for determination of Antiviral

drugs:

Various analytical methods reported in literature studies show for the estimation of

individual drug substances of anti-viral drugs.

Acyclovir:

The literature review of Acyclovir antiviral drugs showed that limited

work was done on this antiviral drugs and most of the work was on

biological fluids.

Bahrami G. et al. (2005) [30] reported the HPLC method for determination of acyclovir in

human serum. The sample preparation method is based on liquid–liquid extraction of acyclovir

and internal standard (vanillin) is used in dichloromethane-isopropyl alcohol (1:1 v/v) as an

diluents. Chromatographic condition used as Column: ODS column using Mobile phase as

methanol-phosphate buffer (0.05 M) containing sodium dodecyl sulfate (200 mg/l) and

triethylamine (2 ml/l, v/v) and pH adjusted to 2.3. the mobile phase ration used as 5:95 and the

flow rate used as 2 ml/min. This method is selective and linear in the concentration between

10–2560 ng/ml. The limit of quantitation is shown 10ng/ml. It is used in bioequivalence study

of two separate preparations which is used on 12 healthy volunteers administration of 400 mg.

Basavaiah K. et al. (2003) [31] reported that an assay method for the determination of acyclovir

in pharmaceutical preparations is developed for the determination of product quality using

HPLC. The chromatographic conditions used as 1) column: reversed-phase C18 column (250

x/4.6 mm i.d.) 2) Mobile phase: Acetonitrile: 20 mmol aqueous ammonium acetate buffer of

pH 4.5 (40:60). 3) The flow rate used as 0.8 ml/min and at wavelength used at 250 nm. The

method is linear in the range 1.98-59.4 mg/ ml. and in accuracy and recoveries values from

between 96.64 to 99.53%.

Hung C. V. et al. (2002) [32] reported that a method developed and validation done on sensitive

plasma assay on acyclovir. Acyclovir is make separate using Oasis HLB columns from plasma

components. Separation was obtained with no plasma interference using micellar electro

kinetic chromatography (175 mM SDS) and hydroxypropyl-b-cyclodextrin (100 mM) in 90

mM borate buffer (pH 8.8) containing 0.2% NaCl. High sensitivity was achieved by large

volume sample introduction and stacking. The coefficient for linearity found for concentration

from 20 to 10000 ng/ml which shows the limit of quantitation is 20 ng/ml.

Emami J. et al. (2009) [33] reported that quantitate levels of acyclovir in human plasma by

rapid reverse phase HPLC method which is a sensitive and accurate. For sample preparation

done by using internal standard (metronidazole) and phosphate buffer (0.05 M) were added to

serum samples. Serum sample vortex for 30 sec. the solvent mixtures of isopropyl alcohol:

dichloromethane (60:40) was then added into it and the same is vortexed for 3 min. Samples

centrifuged and the upper layer was taken for analysis, the layer was dryed under N2 gas,

reconstituted in mobile phase and sample was analysed is 50 µl on the column used a µ-

bondapack C18 (250 × 3.9 mm) column with 3% acetonitrile in deionised water and 0.5%

orthophosphoric acid (pH 2.5) at wavelength 254 nm. The linearity of standard ranges between

100-1500 ng/ml concentration range and found linear, relative errors were within 0.79 to

17.4% and the CV% ranged from 3.81 to 18.2. The LOQ and LOD of the method was

100ng/ml and 25ng/ml respectively.

Pradeep B. et al. (2010) [34] reported that acyclovir is used safely and effectively against

inhibitor of herpes virus as it is specific and selective. Since it is having poor bioavailability

which results in poor absorption of drug. Valacyclovir is the prodrug of Acyclovir and which is

used against of Herpes simplex virus and Varicella zoster virus. It is effective after oral

administration as it is converted in acyclovir in the Gastro intestinal tract and liver more

rapidly.

Smith J. et al. (2010) [35] reported that a Pharmacokinetics of Acyclovir and Its Metabolites in

Cerebrospinal Fluid and Systemic Circulation after Administration of High-Dose Valacyclovir

in Subjects with Normal and Impaired Renal Function. Valacyclovir, the L-valyl ester prodrug

of acyclovir (ACV) is widely prescribed to treat infections caused by varicella zoster virus or

herpes simplex virus. They characterized the steady state pharmacokinetics of ACV and its

metabolites 9-[(carboxy methoxy) methyl] – guanine (CMMG) and 8 – hydroxyl – acyclovir (8-

OHACV) in cerebrospinal fluid (CSF) and the system are circulation. We administered multiple

doses of high dose valacyclovir to 6 subjects with normal renal function and 3 subjects with

chronic renal impairment (creatinine clearance [CrCl] 15 to 30 ml/min). Dosages were 2,000

mg every 6h and 1,500 mg every 12h respectively. The CSF penetration of analyte is reflected

by the CSF-to-plasma area under the concentration-time curve over the 6 or 12 h dosing interval

(AUC) ratio, did not differ based on renal function.

Loregiana, A. et al. (2001) [36] reported that a method for Separation of acyclovir and related

antiviral compounds. In this method Carbon Silica columns from7.5 to 1830 cm is used for

analysis and for separation at 25°C in less than 10 min is required for analysis at 1.0 to 1.5

ml/min off low rate. Its required absorbance at 250-254nm and at 260-285nm, 375-380nm,

respectively. The LOD is 0.310ng/ml when body fluids are under examination. The calibration

linearity found linear in the range of 0.2-20.0 ng/ml, LOD is 0.15 g/ml.

Fernandeza M. et al. (2003) [37] reported that a technique for validation by liquid

chromatography for the analysis of acyclovir in plasma samples. The plasma samples prepared

with acyclovir and in presence of 59 – N – methyl carboxy-amido adenosine (MECA) is used

as internal standard and sample preparation done by solid phase extraction technique with

Waters Oasis HLB columns. The column used as LiChrospher 100 RP-18 column and

wavelength used between of 250 – 260nm. The mobile phase prepared as 18 % acetonitrile:

sodium dodecyl sulphate 5mM and phosphate buffer at pH 2.6. the run time is 13 min/sample.

The RT for acyclovir was of 5.0 min and retention time for internal standard is about 11 min.

The linearity curve was linear between 0.05 and 1.80 mg/ml. The LOD was 0.006 mg/ml with a

LOQ of 0.020 mg/ml observed in the method.

Ibrahim A. et al. (2005) [38] reported that a simple and sensitive fluorimetric method for

determination of antiviral drugs-Acyclovir ,ribavirin and amantadine hydrochloride has been

developed. This is done by oxidation of drugs by cerium (IV) in presence of perchloric acid and

same will be monitored under fluorescence of the induced cerium (III) at k excitation 255 and k

emission 355 nm. Under the defined conditions, calibration curve for linear relationships was

observed is 0.9978–0.9996 which is within the limit and the concentration found in the range of

50–1400 ng/ml. The assay limits of LOD and LOQ were 20–49 and 62–160 ng/ml, respectively.

The precision found within the limit for the method . The values of relative standard deviations

did not exceed more than 2.0%. The recovery found from 99.2 to 101.2 ± 0.48–1.30%.

The literature review of Acyclovir antiviral drugs showed that some

analytical methods are published by authors on work done on UV

Spectroscopy.

Basavaiah K. et al. (2002) [39] reported a method for determination of acyclovir in bulk drug

and in formulations which is simple and cost effective spectrophotometric. The coloured

analytes and exicipients having absorption maximum at 760 nm and follows Beer’s law in the

concentration between 50–450 g/ml. when the concentration of acyclovir increases the

absorbance of the same will increases, corroborated by the calculated correlation coefficient

value of 0.9998 (n=9). The molar absorptivity and Sand ell sensitivity was found 1.65×102

l/mol/cm and 1.36 g/cm2 respectively. The coefficient of correlation and slope of the equation

of the regression line are 8.33×103 and 6.87×104, respectively. The LOD was 5.68 g/ml and the

LOQ was 18.95g/ml.

Gandhi P. et al. (2006) [40] reported that a Spectrophotometric method for determination of

Acyclovir in Analytical Dosage Forms. A spectrophotometric method was developed for

analysis of acyclovir in bulk and pharmaceutical dosage. Acyclovir had absorbance at 253 nm

and molar absorptivity of 1.3733 ×104 l/mol × cm followed the Beer’s law in the concentration

range of 2-20 µg/ml. which results that the method used for analysis were validated statistically

and by recovery studies.

Ayad M. et al. (2007) [41] reported the method for analysis on acyclovir and acebutolol

hydrochloride by Spectrophotometric and spectrofluorimetric. The methods are mainly done by

oxidation of the selected drugs with cerium(IV) ion in acidic medium and at absorbance at

320nm or the fluorescence intensity of the produced cerous (III) ion at 361–363nm (excitation

at 250nm). This method follows Beer’s law in 2 to 8, 0.25 to 2.51g/cm acyclovir, 1 to 7 and

0.25 to 2.51g/ml acebutolol hydrochloride when used the spectrophotometric and

spectrofluorimetric method respectively. This method is used for determination of the selected

drugs in their pharmaceutical preparations with good recoveries.

The literature review of Acyclovir antiviral drugs showed that some

analytical methods are published by authors on work done on IR

Spectroscopy.

Liyan Y. et al. (2008) [42] reported that a method by near infrared spectroscopy for Quantitative

determination of acyclovir in plasma. The aim of this study to analysis of acyclovir in plasma

was to assess the feasibility of near infrared spectroscopy (NIRS). In the plasma samples

concentration of acyclovir was calculated employing a 6-factors PLS calibration using the

spectral in formation in the range of 6102–5450 per cm. The RSDof prediction (RMSEP) were

observed is 1.21 for acyclovir. The method of developed PLS-NIRS procedure analysed 120

samples/h not require any sample pretreatment and avoids waste generation.

Valacyclovir:

The literature review of Valacyclovir antiviral drugs showed that some

analytical methods are published by authors on work done on HPLC:

Jadhav A.S. et al. (2007) [43] reported a validated method for the enantiomeric resolution of

Valacyclovir using chiral high performance liquid chromatographic method. Columns used for

resolved the enantiomers of Valacyclovir on a Chiralpak AD (250mm×4.6 mm, 10µ) column

and mobile phase system containing n-hexane: ethanol: diethylamine (30:70:0.1, v/v/v). The

resolution found more than four between the enantiomers. The LOD and LOQ of (d)-

enantiomer were found to be 300 and 900 ng/ml, respectively, when injected the 20 µL injection

volume. The Linearity showed excellent linearity on the concentration between 900 ng/ml

(LOQ) to 6000 ng/ml for (d)-enantiomer. The recovery of (d)-enantiomer was found between

97.50 to 102.18 satisfactory. Stability of sample solution and mobile phase was stable for 48 h.

Chuong P. et al. (1999) [44] reported that a rapid high-performance liquid chromatographic

assay with isocratic elution is developed for the simultaneous quantification of valaciclovir

(VACV) prodrug and converted in acyclovir (ACV). For serum, the samples are deproteinized

with perchloric acid in presence of 1-methylguanosine as the internal standard (IS). For urine

and dialysis liquid, IS is used in sample prepared and diluted with the help of mobile phase and

filtered. Mobile phase prepared by acetonitrile–ammonium phosphate buffer for separation of

VACV, ACV and the IS . the column used as Symmetry Shield ERP-8 column and wavelength

254 nm. Therun time is about 12 min. The relative standard deviations (RSD) of VACV and

ACV standards are between 0.5 and 3.5%. With the help of this technique interference of most

endogenous nucleosides and their metabolites.

Huidobro A. et al. (2005) [45] reported that a LC methods for acyclovir and related impurities

determination. Acyclovir, guanine, and impurity A were baseline separated with isocratic

conditions at pH to 3.0 and run time maintained 15 min by using the columna SB CN from

Agilent (150 mm × 4.6 mm and 3.5m). Moreover, when run time was increased to 40 min six

impurities (guanine, impurities A,F,G, Vir ¾ and N7 ) plus acyclovir were separated. The mobile

phase used as buffer preparation prepared by 25mM H3PO4 and pH maintained at 3.0 with the

help of KOHH and Acetonitrile in composition 96:4(v/v).

Sinha V. et al. (2007) [46] reported that a method on Stress Studies of Acyclovir. The drug is

stable for solid stage degradation. It is stable for light exposure in a solid state degradation.

However in photolytic degradation the drug is stable is exposed as a solution in water. The drug

is degraded majorly in acidic hydrolysis and photolysis as it comparison with the standard. The

column used as C-18 column for separation of analytes and impurities under various conditions

with the help of water –methanol in the ratio of 90:10used as mobile phase. Analysis is done at

252 nm and flow is maintained 1 ml/min. The validation parameters are checked for parameters

like linearity, precision, accuracy, selectivity, specificity, and robustness. The slope and

correlation coefficient are 39.307 and 0.9998 observes and RSD values less than 2%. The values

observed for accuracy and recovery is found in the range of 97.34 % to 102.35 %.

Patil G. et al. (2009) [47] reported that a Method for the Determination of Valacyclovir in Bulk

Drug and in Tablet Dosage Form by HPLC. The study was performed as per ICH guideline.

Column used for analysis was C8 and in mobile phase of Acetonitrile: Phosphate buffer pH 3 (25

mM) in the ratio of 10:90 v/v. Analysis performed on flow rate was maintained at 1 ml/min and

wavelength used was 254 nm. The Analyte found highly sensitive for alkaline, acidic and

oxidative conditions, and also in the presence of light (in alkaline environment). Less

degradation was observed in neutral but it is stable for thermal, humidity stress.

Srinivas K. et al. (2011) [48] reported a method for estimation of Valacyclovir in bulk and tablet

dosage forms by HPLCand the method development and validation done for the same. Column

used for this is oyster (250 x 4.6, 5µm particle size) column. Buffer prepared as 0.025M

potassium dihydrogen phosphate and pH adjusted to 2.0: acetonitrile: methanol (40: 55: 5 %

v/v/v). The pH of buffer was adjusted with hydrochloric acid or sodium hydroxide solutions

between 1.84–7.94 and floe rate maintained at l/min and at wavelength 254 nm.


analytical methods are published by authors on work done on UV

Spectroscopy:

Ganesh M. et al. (2009) [49] reported a method for determination of valacyclovir in bulk and

tablet dosage form by UV spectrophotometric. Valacyclovir shows maximum absorbance at 255

nm in 0.1N HCl. By using Beer’s law on concentration of 5-25 mcg/ml with 1.0910 x 104

mol/cm. this method is validated on the parameters like : slope, intercept, correlation

coefficient, detection and quantitation limits . The percentages assay of valacyclovir HCl in

tablet was 99.82%. The method complies the validation parameters like sensitivity, accuracy

and precision which determines suitability of the developed method for the routine estimation

of valacyclovir in bulk and solid dosage form.

Aswani Kumar C. et al. (2010) [50] reported that a spectrophotometric method for the

determination of valacyclovir and cefotaxime after developed. The 1, 2- napthaquinone-4-

sulfonic acid sodium (NQS) in alkaline medium was condensed of valacyclovir and cefotaxime

to yield orange colored products respectively. Both the analytes shows valacyclovir and

cefotaxime showed maxima at 495nm and 475nm with linearity was observed in the

concentration range of 20-120 µg/ml and 20-140 µg/ml respectively. The RSD of 0.363% for

valacyclovir and 0.66% for cefotaxime were obtained. The recoveries found for valacyclovir

and cefotaxime injections were in the range 96.01 ± 0.52 and 98.12 ± 0.96 respectively.


analytical methods are published by authors on work done on Biological

fluid.

Wang L. et al. (1996) [51] reported that a method for Valaciclovir in Geriatric Volunteers with

and without Concomitant Diuretic Therapy. A study was done to evaluate the safety and

pharmacokinetics of acyclovir of valaciclovir (three times a day for 8 days) in geriatric

volunteers (65 to 83 years of age). This study was done on three groups: normotensive subjects

given 500-mg doses of valaciclovir (n11), normotensive subjects given 1,000-mg doses of

valaciclovir (n9), and thiazide diuretic-treated hypertensive subjects given 500 mg doses of

valaciclovir (n9). Valaciclovir converted and it gets absorbed rapidly to acyclovir. The plasma

concentrations generally undetectable or <0.4 g/ml for valacyclovir. The peak concentration of

drug in plasma (Cmax) for acyclovir occurred at 1 to 2h, and the half life of acyclovir was 3 to

4h in all three l derly groups. The Cmax and area under the concentration time curve from 0h to

infinity (AUC0) values of acyclovir obtained on days 1 and 8 indicated no unexpected

accumulation at steady state. The steady-state acyclovir Cmax (4.30 and 5.98 g/ml) and daily

AUC0 (44 and 74 hg/ml) following dosing of valaciclovir (500 and 1,000 mg) three times a day

were two to three times greater than those expected after high dose oral acyclovir treatment (800

mg, five times daily). There were no valaciclovir – related changes or abnormal it is in safety

parameters and no reports of serious adverse experiences in the seelderly volunteers.

Kasiari M. et al. (2008) [52] reported that a method for the quantification of valacyclovir and its

metabolite in human plasma. Filtration done on Sample after it was prepared by protein

precipitation with acetonitrile. Valacyclovir, acyclovir and ganciclovir (internal standard) were

separated on column (2.1 mm × 125.0 mm i.d., particle size 5), when used a mobile phase of

acetonitrile/water with 0.05% (v/v) diethylamine (50:50,v/v). the mobile phase flows at rate of

0.15 ml/min in 4.0 min. Detection was performed by negative electrospray ionization using the

selected ion monitoring mode of the deprotonated molecularionsat m/z 323.0 for valacyclovir,

224.0 for acyclovir and 254.0 for ganciclovir. The linearity of assay was checked after plotting

calibration curves over the range 0.020–0.800 g/ml for valacyclovir and 0.100–20.00 g/ml for

acyclovir. For the above method accuracy and precision were within the acceptance limit of

15%.

Yadav M. et al. (2009) [53] reported that a method for valacyclovir and acyclovir in human

plasma for stability evaluation by mass spectrometry . The analysis done with the spiking of IS

into the analyte. The method involved solid phase extraction of the analytes and Internal standard

from 0.5 ml human plasma with no reconstitution and drying steps (direct injection of eluate).

The column used for analysis is Gemini C18 on isocratic mobile phase, prepared by 0.1 %

formic acid and methanol (30:70v/v), analysis done on the flow of 0.8 ml/min. The

precursor→production transition for valacyclovir (m/z 325.2→152.2), acyclovir (m/z

226.2→152.2) and IS (m/z 307.1→220.3) were monitored on a mass spectrometer, operating in

the MRM mode. The method was validated over the concentration range between 5.0 –1075

ng/ml and 47.6–10225 ng/ml for valacyclovir and acyclovir respectively. The mean recovery of

valacyclovir (92.2%), acyclovir (84.2%) and IS (103.7%) from spiked plasma samples was

consistent and reproducible.

Granero G. et al. (2006) [54] reported that the method on absolute bioavailability of

valacyclovir when it is taken by oral administration is 54.5%. The pH of the analyte shows the

stability of the drug hence it is found to be dependent on pH. This drug is stable in acidic

medium (under 4), while this drug degrades in alkaline medium rapidally. In intestinal fluid

degradation of the drug valacyclovir increases rapidally than in phosphate buffer at the same

pH. In human and dog stomach there was no appreciable release of Valacyclovir in phosphate

buffers at pHs fewer than 4, though the drug degrade faster in the human and dog stomach.

The literature review showed that some combined analytical methods are

published by authors on Acyclovir and Valacyclovir:

Anil Kumar T. et al. (2011) [55] reported a methods for assay determination of acyclovir and

valacyclovir in tablets. For the method auther used oxidimetric reagent is N-bromosuccinimide

(NBS) and for dye methyl orange is used. The method determination is done as in acidic

medium addition of excess NBS is done on acyclovir and valacyclovir. Then it is followed by

determination of residual NBS by reacting with a methyl orange and same will be measured at

508 nm. The analytes are showed maximum absorbance at 508 nm with the concentration of

linearity was ranged of 1-5 µg/ml and 5-10 µg/ml respectively. The rsd of 0.024 % for acyclovir

and 0.018 % valacyclovir were obtained. The recovery tests via standard-addition method was

done for checking of accuracy and reliability of methods. The values shows the good recoveries

of acyclovir and valacyclovir tablets i.e. 99.26 ±0.52, 99.47±0.96 respectively.

Bomgaars L. et al. (2008) [56] reported that a method on Pharmacokinetics in Immune

compromised Children of Valacyclovir and Acyclovir drugs. This is orally administered pro-

drug of acyclovir i.e. Valacyclovir and it is mainly utilized din against therapy of herpes simplex

and herpes zoster infections. This study is conducted on total of 37 immune compromised

children which were enrolled on one of two studies. The study data of pharmacokinetic data were

of 32 patients who is taken the valacyclovir (15 mg/kg) administration, out of which 11 was also

had pharmacokinetic sampling following IV acyclovir administration. The treatment for

valacyclovir is given to three patients who is received for herpes zoster infections results. Results

for the same is Mean (SD) Cmax values for acyclovir where valacyclovir is taken by orally were

18.87 mM with a total exposure of 4, 1061, 519mM min. The mean values for bioavailability of

acyclovir from valacyclovir was 64%. valacyclovir related toxicity like Grade1nausea and

emesis is occurred in five patients . Treatment for herpes zoster had complete scabbing of lesions

by day 9 for two of the three patients .

1.10 Objectives:

The prime importance of drug analysis to gather the analytical information in the aspects of the

qualitative and quantitative composition of analytes present in the compound, from which we can

know that what a substance is added in the drug product and exactly in how much. This

information guides development of the manufacturing operation and therapeutic action of drugs.

India, a developing country, with fast industrialization and rapid progress on all fronts, is making

big strides towards global recognition. The flip side is that the economic prosperity and modern

way of life is translating into an increase in lifestyle related diseases. Home to nearly millions of

viral diseases today, India is fast becoming the viralatic capital of the world.

• The aim to develop and validate the “Analytical methods for Antiviral drugs” with better

selectivity and this method applies in industry to assure the good quality of drugs.

• The developed method shall be linear, precise, accurate and robust and will help

scientist for regular analysis in pharmaceutical organization by Quality control

department.

• The validated method will be used for analysis of stability testing in pharma

industry.

• Assay test is important part in determining the potency of analytes in active

drug. Assay value determines the purity of product and error in measurement

of assay will be creating the impact on human being. hence accuracy of results

will be determine by HPLC. Method will be help to industry to estimate the

potency of drugs accurately.

• The method to be develops for help in “Analytical development lab” to provide

the support for experimental study of process development and formulation

development study.

• The method to be develops for analytical support in pre-formulation study in

pharmaceuticals industry.