1

CHAPTER - 1

INTRODUCTION

Analytical methods development, identification [1], characterization

[2] of impurities [3] and method validation play key role in the

pharmaceuticals discovery, development, and manufacturing. The new

drugs introduced into the market are increasing every year in number.

These drugs may be totally new or partial structural modification in

existing molecules.

Based on reports of toxicities and continuous wider use of these drugs

are indicated. Introduction of its better replacement, these new drugs

may include later in official pharmacopoeia. Therefore, due to this lag

time, analytical procedures, and standards may not available in

concerned pharmacopia, we can not find these drugs and impurities even

the drug substances [4] and drug products introduced into the market

are increasing every year. These drug substances and drug products may

be either partial structural modification of the existing drug substances

or new entities. This happens because of continuous and wider usage of

these drug substances and drug products and possible uncertainties,

introduction of better drugs by competitors and reports of impurity

toxicities. Under these circumstances, analytical procedures and

standards for these drugs and their related substances [5] may not be

available in the pharmacopoeias eg. Indian, IP [6], United Kingdom, BP

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[7], United States, USP [8], European, EP [9], Japan, JP [10] and

Martindale Extra [11] and public domain.

Combination products referred as with more than one drug (Fixed

dose combinations), previously not available to the patients by combining

theraputic effects of two or more drugs in one product. In such cases,

development of new analytical methods plays an important role. Study

drugs including structure, chemical nature, and synthesis composition

need to make use of science in the aspect of bio-chemical actions are

part of pharmaceutical chemistry [14-21]. Influence on an organism and

studies the physical and chemical properties, the methods of quality

control and conditions of their storage. India is the richest source for

Pharmaceutical products. Developed countries like United States of

America, Australia and Britain etc. have much demand for these

medicines; hence India is utilizing this opportunity to promote the

products. Presently we are exporting formulations to many developed

countries, but these countries are now imposing their regulatory

guidelines to accept our products into their markets. Australia is now

regulating manufactures by TGA [22] ―Therapeutic goods

administration‖, America by USFDA [23] ―United States Food and Drug

Administration‖ and European regulatory agency by EMEA [24], Brazil by

ANVISA [25] and other countries by ICH [26] and WHO [27]. According to

these stipulations the therapeutic products must satisfy defined

specifications like product description, identification, and assay for active

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constituents, related substances or degradation products, microbial

purity, and stability data for the exported product through out its shelf

life.

Indian manufacturers require analytical techniques and tools to

define the quality of their products and to retain their qualification in

world markets. The analytical tools include chemical, physico-chemical,

instrumental, biological techniques and also the combination of both

with instrumental methods (hyphenated techniques [28]) for developing

qualitative and quantitative determinations. Analytical chemistry plays

an imperative role to success the progression.

1.1 An Introduction to Pharmaceutical Formulations:

A pharmaceutical drug can be broadly defined as any chemical

substance intended for use in the medical diagnosis, cure, treatment, or

prevention of disease, also referred to as medicine, medicament or

medication [29- 31]. Pharmaceutical formulation is a produce of final

medicinal product by process of different chemical substances, including

the active drug are combined [32]. Pre-formulation, study has to be done

on behavior of a given steroid and antibiotic under a variety of stress

conditions such as freeze/thaw, temperature, shear stress among others

to identify mechanisms of degradation and therefore its mitigation [33].

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1.2 Types of pharmaceutical Formulations:

The drugs used in various forms in prophylactic or in therapeutic [34-

37] use. They are formulated as tablets, capsules, dry syrups, liquid

orals, creams or ointments, parenterals (injection in dry or liquid form),

lotions, dusting powders, aerosols etc. In tablets one or more among the

diluents such as starch, lactose, cellulose derivatives, calcium

phosphate, mannitol, sorbitol, sucrose, aerosols, acacia, polyvinyl

pyrrollidine, alginic acid, tragacanth, stearic acid, talc, magnesium

stearate, waxes, methyl paraben, sodium benzoate, permitted flavours,

and colors may be added. In capsules one or more among the diluents,

certified dyes, gelatine, plasticizers, preservatives, starch, lactose, talc

may be added. In dry syrups and liquid orals, sucrose, sorbitols,

preservatives, certified colors, and flavours might be added. In creams

and ointments, waxes, carbopol, petroleum jelly, surfactants,

preservatives permitted colors, and perfumes might be added. In

parenterals, water, vegetable oils, mineral oils, simulated oils, propylene

glycol, dioxalamines, dimethyl acetamide may be used as vehicles. Any

one or more among stabilizers, anti-oxidants, buffering agents like

citrates, acetates, phosphates, co-solvents, wetting suspending and

emulsifying agents like tween-80, sorbitol, oleate and preservatives may

be added. In lotions, dusting powders and aerosols, talc, silica

derivatives, alcohol, preservatives may be added.

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In some instances drugs are applied in small doses and they are often

mixed with excipients as combinations. The assay of various dosage

forms raises several special and skilful sampling for the preparation of

sample solutions. Hence standard techniques must be employed to

ascertain the homogeneity of the sample before collecting for analysis.

1.3 Preparation of Sample Solution for Analytical Investigations:

Preparation of sample solution may not always a straightforward

situation. Preparation often includes processes such as quantitative

extraction frequently causes serious problem, extraction of drugs into the

most important solvents and their nature to be bound to excipients, each

case it can be solved seperately. The most difficult problems arise when

selective extraction is necessary. It is also observed often that the

specificity of the extraction is insufficient. In these instances separation

of the components by extractor and its further chromatography are

widely used for purification. The most exact method of extracting drugs

from formulation is to treat the drugs with a proper solvent, RS method

chosen such that the resulting extract can be used directly in the assay

without having the intereference of associating ingredients. When the

finely pulverized sample is agitated and/ or assisted with boiling the

solvent for a period from few minutes to several hours to get sufficient

extraction achieved. In general, a sample later used as is or concentrated

well, if the content is very less to improve the sensitivity of the analysis.

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Increasing the selectivity of the two-phase extraction is generally used

method for decreasing the absorption loses. Here one of the solvents is

always water. Water soluble components such as the generally used

method for decreasing the adsorption losses and increasing the

selectivity of the extraction are two-phase extraction. Here one of the

solvents is always water. Lactose, is generally main ingradient of the

excipients to get favorable conditions for extraction of the drugs and their

related substances. Starch is also critical from the point of view of

adsorption losses, which can be eliminated by dissolving treatment with

diastase and other solvents, which are immiscible with water. The

organic solvents genereally used dichloromethane, chloroform, ethyl

acetate, diethyl ether, iso-octane and few others have also been used.

1.4 Introduction to Analytical Chemistry [38]:

Analytical chemistry is a science deals with the identification,

characterization and estimation of the components of a sample. The

primary interest of an analytical chemist is to develop experimental

methods of measurement to obtain information about the qualitative and

quantitative tests for given composition of a sample. Analytical chemistry

involves a multisided approach to obtain information of every individual

chemical species present in any sample. A knowledge of analytical

chemistry helps to develop methods, select an appropriate instruments,

and strategies to obtain information on the composition and nature of

sample. The number of analytical techniques, their degree of

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sophistication and area of applications has increased tremendrously [39-

40]. Analytical chemistry [41] as a whole has evolved world wide

supporting pillar of human culture, industry and trade, providing

numerous goods of urgent daily need to the human kind, such as

pharmaceuticals, chemicals and also food ingredients. Analytical

chemistry stands in the development of science [42]. The most important

definition of analytical chemistry was proposed by the working committee

on analytical chemistry of the FECS [43] (Freedom of European Chemical

Societies). It reads all branches of chemistry, and techniques drawn on

the ideas of analytical chemistry [44-45]. Analytical chemists [46] meet

the demands for better chemical measurements reliability of existing

techniques to be improved. Analytical chemist efforts to develop new

methods those methods developed are kept purposely static; so that data

can be compared over long periods of time and this is true in industrial

quality assurance (QA). In Analytical chemistry the use of a tunable laser

to increase the sensitivity and specificity of a spectrometric method plays

an important role in the pharmaceutical industry, where aside from QA

[47], it is used in discovery of new drug candidates. Analytical chemistry

has been important because Modern analytical chemistry [48]

categorized in terms of the analytical target, in providing methods for

determining elements and chemicals are present in the world around us.

The analytical methods and their research applications [49-54] are

Material analysis, Bio-analytical chemistry, Chemical analysis and

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Forensic chemistry. Analytical methods are differntiated as wet-chemical

and instrumental methods. Wet-chemical methods include gravimetric

and volumetric analysis while instrumental methods include mass

spectrometry [55], spectrophotometry [56-58] and colorimetry,

electrophoresis, crystallography, microscopy, electrochemistry, optical

methods such as emission and absorption spectroscopy etc., separation

methods such as chromatography, electro analytical methods like

potentiometry, voltammetry etc., radiochemical methods like

scintillation, tomography and biological methods like RIA and ELISA

tests [59]. Analytical techniques [60-63] are broadly classified as titration

techniques, spectrometric techniques and chromatographic separation

techniques. Different type of detectors [64] used in chromatographic

technique are Photo Diode Array detector, Light scatering Evaporative

detector, Refractive index detector, Ultraviolet detectors, Mass detectors,

Potentiometric detectors, Fluorascence detectors, Amperometric

electrochemical detectors, Flame ionization detectors, Thermal

conductivity detectors, Electro chemical detectors. Sophisticated

instrumentation given predominant status to Modern Analytical

chemistry, the basic roots of analytical chemistry and some of the

methods used in advanced instruments are from titration [65-66],

gravimetry [67], inorganic qualitative analysis [68-69], crystallography

[70-71], electrochemical analysis [72], thermal analysis [73], separation

techniques [74], hybrid techniques [75-77], microscopy [78], and

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traditional analytical techniques [79-81]. Chromatographic separation

techniques are multi-stage separation methods.

In two phases, we can find the components of the sample, one is

stationary and another one is mobile. The stationary phase prepared is a

solid or a liquid supported on a solid or a gel. The stationary phase is

packed in a column, spread as a layer, or distributed as a film, the

mobile phase may be gaseous or liquid or supercritical fluid. The

separation may be based on adsorption, mass distribution (partition), ion

exchange, etc., or may be based on differences in the physico-chemical

properties of the molecules such as size, mass, volume, etc.

chromatographic separation methods are used for simulltanious

estimation of combination drugs. These methods offer accuracy and

precision and good reproducibility. Chromatography classification [82-

85] is based on its interaction with the stationary phase.

1.5 High Performance Liquid Chromatography [86-110]:

High Performance Liquid Chromatography (HPLC) is a different

branch of column chromatography in which the mobile phase is forced

through the column at high speed. As a result the analysis time is

decreased by 1-2 orders of magnitude relative to classical column

chromatography and the use of much smaller particles of the adsorbent

or support becomes possible increasing the column efficiency

substantially.

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HPLC General Considerations [111-115] involving a polar stationary

phase and a non polar mobile phase are indicated as normal phase

systems and this combination of phases. Solute retention generally

increases with solute polarity. High performance liquid chromatography

comprises of a solvent delivery system or a pump for controlled flow of

mobile phase and injection system for precise sample introduction.

Variety of modified, stable chemically bonded stationary phases capable

of being operated at high pressures, leading to better efficiency of

separations. Sophisticated detector system capable of handling small

flow rates and detecting small concentrations. The sample related

information that needs to be known prior to HPLC method development.

High performance liquid chromatography column is selected

depending on the nature of the solute and the information about the

analyte. Reversed phase mode of chromatography facilitates a wide range

of columns like octadecaylsilane (C18), Octylsilane (C8), butyl silane (C4),

phenyl, nitro, amino, cyanopropyl (CN), dimethyl silane (C2) etc. were

chosen for this study since it is retentive, rugged and widely available.

C18 and C8 columns are applied to approximately 80% of the reverse

separation problems. The other available phases are often significantly

less retentive than the C18 and C8 phases, and finding an appropriately

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weak mobile phase that will accomplish the separation is not always

possible due to the polar to semi polar nature of the analytes.

Table 1.01

Column Particle Characteristics for HPLC

Features Utility

5 μm totally porous particles Most separations

3 μm totally porous particles Fast separations

1.5 μm pellicular particles Veryfast separations (especially macromolecules)

+50% from mean particle-size distribution

Stable, reproducible, more efficient columns with lower pressure drop

7 to 12 nm pores, 150 to 400 m2/g

narrow pore

Small molecule separations

15 to 100nm pore, 10 to 150 m2/g wide pore

Macromolecular separation.

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Figure 1.01 Steps in HPLC Method Development

Information on sample, define separation goal

Need for special HPLC procedure sample

pretreatment etc.?

Choose detectors and detector settings

Choose LC method; preliminary run; estimate

best separation conditions

Optimization separation conditions

Check for problems or requirement for special

procedure

Recover purified

material

Quantitative

Calibration

Qualitative

method

Validate method for release to routine analysis

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Figure 1.02

Method Development Conditions for the Initial Experiment.

HPLC CE GC SFC

TLC

Nature of Sample

Regular Special

Neutral Ionic

Exploratory run

Reverse-phase

Isocratic

Gradient

Ion-pair

NARP

Normal-phase

Inorganic ions

Isomers

Enantiomers

Biological samples

Peptides

Carbohydrates

Nucleotides

Macromolecules

Proteins

Nucleic acids

Carbohydrates

Synthetic polymers

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Table 1.02

Column Selection for HPLC Method Development

Reversed-phase and ion-pair method

C18 (Octadecyl or

ODS)

Rugged, highly retentive, widely available.

C8 (Octyl) Similar to C18 but slightly less retentive.

C3, C4 Less retentive and mostly used for peptide and proteins

C1[trimethylsilyl (TMS)]

Least retentive and least stable

Phenyl, phenethyl Moderately retentive and some selectivity

differences

CN (cyano) Moderately retentive and used for both reversed

and normal phases

NH2 Amino Weak retention, used for carbohydrates and less stable

Polystyrene Stable with 1<pH<13 mobile phase and better peak shape and longer column life for some

separations.

Normal-phase method

CN Rugged and fairly polar general utility

OH More polar than CN

NH2 Highly polar and less stable.

Silica Very rugged and cheap, less convenient to operate

, used in prep LC

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Table 1.02 (Continued)

Column Selection for HPLC Method Development

Size-exclusion method

Silica Very rugged and adsorptive

Silanized silica Less adsorptive, wide solvent compatibility, used

with organic solvents

OH Less stable, used in aqueous SEC gel filtration

Polystyrene Used widely for organic SEC (GPC), generally incompatible with water and highly polar organic solvents.

Ion-exchange methods

Bonded phase Less stable and reproducible

Polystyrene Less efficient, stable, more reproducible.

Mobile phase:

Mobile phases in reversephase chromatography contains mixture of

aqueous phase and organic phase, buffer is not required for neutral

samples. Whenever acidic or basic samples are separated, it is strongly

advisable to control mobile-phase pH by adding a buffer. It is strongly

recommended that the pH of the buffer be adjusted before adding

organic. In selecting buffer several considerations should be kept in

mind. Buffer capacity is determined by pH, buffer pKa, and buffer

concentration. As for the case of sample compound, buffer ionization

occurs over a range in pH given by pKa + 1.5 and buffer can be effective

only in this pH range. Buffers selected for a particular separation should

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be used to control pH over a range pKa +1 and concentration of 10 to 50

mM is usually adequate for reversed phase separation. A mobile phase

with marginal buffer capacity will give less reproducible separation for

compounds that are partially ionized at the pH of the mobile phase. In

this case, retention may change from run to run, and distorted peaks

may result.

Table 1.03

Buffer Selection for HPLC

Buffer pKa Buffer Range UV Cutoff

Trifluoracetic acid 2.0 1.5 to 2.5 210 nm

Phosphoric acid/mono or di

potassium phosphate

2.1,

7.2, 12.3

Less than 3.1

6.2 to 8.2 11.3 to 13.3

200 nm

Citric acid/tri potassium citrate

3.1,4.7,5.4 2.1 to 6.4 230 nm

Formic acid 3.8 2.8 to 4.8 210 nm

Acetic acid 4.8 3.8 to 5.8 210 nm

Mono /dibasic potassium carbonate

6.4 5.4 to 7.4 Less than 200 nm

Bis-tris propane HCl/ Bis-

tris propane

10.3 9.3 to 11.3 Less than

200 nm

Tris HCl / Tris 8.3 7.3 to 8.3 205 nm

Ammonium chloride/

Ammonia

9.2 8.2 to 10.2 200 nm

1-Methyl piperidine HCl/1-Methyl piperidine

10.1 9.1 to 11.1 215 nm

Triethylamine HCl/ Triethylamine

11.0 10 to 12 200 nm

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Buffer capacity is determined by pH, buffer pKa, and buffer

concentration. In case of sample compound, buffer ionization occurs over

a range in pH given by pKa + 1.5 and buffer can be effective only in this

pH range. Buffers selected for a particular separation should be used to

control pH over a range pKa +1 and concentration of 10 to 50 mM is

usually adequate for reversed phase separation. A mobile phase with

marginal buffer capacity will give less reproducible separation for

compounds that are partially ionized at the pH of the mobile phase. In

this case, retention may change from run to run, and distorted peaks

may result.

UV absorbance of the selected buffer should exhibit as low as UV

absorbance for proper detection, and to get good sensitive method.

Retention in reversed phase chromatography affected by mobile phase

composition such as choice of % B (organic), mobile-phase strength,

column and temperature effects. Selectivity in reversed-phase

chromatography influence factors such as solvent-strength selectivity,

solvent-type selectivity, column type selectivity, temperature selectivity.

An effective approach to method development begins with a very

strong mobile phase. (e.g., 100% Acetonitrile) The initial use of strong

mobile phase makes it likely that the run time of the first experiment will

be conveniently short, and strongly retained compounds will all be

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eluted. If no peaks observed after 30 to 40 min with 100% acetonitrile,

weaker mobile phase is required, strength of acetonitrile gradually

reduced to 80%, 60% which ever the amount until required separation

and optimum run time attained.

During HPLC method optimization stage, the initial sets of conditions

that have evolved from the first stages of development are improved or

maximized in terms of resolution, peak shape, plate counts, asymmetry,

capacity, elution time, detection limits, limit of quantization, and overall

ability to quantify the specific analyte of interest. When optimizing any

method, an attempt should be made to provide analytical figures of merit

which are needed to meet the assay requirements defined at the initial

stages of method development. In other words, the required detection

limits, limits of quantization, accuracy and precision of quantification,

and specificity must be defined. Without adequate and definitive

requirements, it is difficult to optimize any analytical method [116].

Two- columns strategy method used to enable rapid early method

development along with a four-column method for commercial method

development of the analytical methods utilized to evident the quality of

drug substance or drug product. Mobile phases contain methanol or

acetonitrile with aqueous trifluoroacetic acid for low pH screening and

ammonium hydroxide for high pH screening [117].

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To determine the success of the method developed, HPLC column

stability is one of the important factors. A systematic approach for the

detection of HPLC column stability has been improved with emphasis on

the usage of the application to pharmaceutical analysis. The specifics of

the design, evaluation criteria mentioned and result obtained for some of

the mostly used analytical columns from reputable column

manufacturers. A stability profile over the pH range was identified that

may serve as a reference for column scouting during method

development [118]

A brief study on the development of validated stability- indicating

assay methods (SIAMs) for drug substances and products focused on

critical issues related to method development of SIAMs. Frequent

problems encoutered were in-sufficient impurity profiling and non-

suitability of pharmapoeial methods for the testing the stability samples

[119].

The chromolith monolithic column indicates an efficiency that is

comparable to that of columns compacted with spherical particles of 3

μm and 4 μm. We can compare the theoretical analysis speed of different

seperation media by a kinetic plot analysis. At 200 bar, the monilith

column indicated the highest performance when the requisit plate

number was higher than 5000, while lower range, identified with

compacted columns. If the possibility of optimum performance was used

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the monolith column would provide the least efficiency while the column

compacted with 1. 5 µm particles offered the shortest impedance time

[120].

1.6 Identification and Characterization of Impurities [121-122]:

The United States Food and Drug Administration (FDA) have

endorsed the guidance prepared under the auspices of the ICH. The

guidance developed with the joint efforts of regulatory agencies and

industry. Professionals from the European Union, Japan and the United

State to ensure, that the different regions have consistent requirement

for the data that should be submitted in the drug substance and drug

products, in regards guidelines are not only aid the sponsors of latest

drug applications (NDA) or abbreviated new drug applications (ANDA)

with the type of information that should be submitted with their

applications, but also assist the FDA reviewers of field investigators in

their consist interpretation and implementation of regulations. FDA

guidance on the impurities [123] for IND requires and maintain

appropriate standards of identity, strength, quality and purity as needed

for safety and give significance to clinical investigation made with the

drug statement on the method, facilities and controls used for

manufacturing, processing, and packing of new drug to establish NDA

demands, more specific and explicit information, including stability

studies to guarantee that all predetermined attributes of the drug

product are maintained until its expiration date. The FDA may initiate

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action under the F&D act to bring about removal of a product from the

market, or as it granted in the law, a manufacturer may voluntarily

withdraw from the market place any batches that do not meet the

approved specifications. In each country regulatory authorities use their

own standards for conducting clinical studies on a new drug product

[124] and drug substance that has been formulated or the product that

is approved for commerce. All efforts taken to characterize the source of

actual impurities present in the new drug substance at a level greater

than 0.1 per cent should be described. ICH guidelines also emphasize the

characterizing the impurities present in the new drug products appearing

as degradation products, identified in stability studies conducted on

storage conditions at a level greater than the identification threshold (1 %

for maximum daily dose of less than 1.0 mg and 0.1 % for a maximum

daily dose of greater than 2 grams [125].

Developments in the field of magnetic resonance have been evidant

strides in increasing sensitivity levels. This is most important in the

structural characterization of drug impurities and degradation of

products, which frequently are available only in extremely less

quantities. The non-invasive nature of NMR spectroscopy makes it a

valuable tool for the characterization of low level impurities and

degradation products. In addition NMR can be considered close to a

universal detector for hydrogen and carbon, as well as for other

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magnetically active nuclei. This is both good and bad because all signals

are detected, those arising from the compound of interest and all other

compounds in the sample, such as solvents and starting materials. The

popularity of LC-MS-MS systems for complex mixtures analysis of

thermally liable biologically relevant molecules is largely attributed to the

soft nature of atmospheric pressure ionization techniques such as electro

spray ionization(ESI), atmospheric pressure photo ionization(APPI),

attributes of various mass analyzers and scan modes used for collision-

induced dissociation experiments and issues. The next step generally is

to obtain molecular mass and fragmentation data via HPLC_MS. It is

essential to determine the molecular mass of the unknown, as it helps in

tracking the correct peak by HPLC when isolation becomes necessary. To

run LC-MS a mass spectrometry, a compatible HPLC method is

necessary. If such a method is not available, it has to be developed which

adds considerable time to the identification process. Relevance of

Impurities characterization: The characterization effort serves as the

basis of understanding for chemical and physiological process upon

which successful development of a medicine depends. Traditionally, the

product development process for pharmaceuticals has relied on

separation techniques such as HPLC employing non specific detectors

(refractive index detectors, UV/Vis absorption, electrochemical,

fluorescence, etc.) These detectors provide sufficient information in many

instances and are inexpensive, reproducible, rugged, and simple to

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operate sample in the absence of authentic standards, they do not

indicate any information that might lead to the recognition of

compounds. In contrast, the use of a specific mode of detection, such as

mass spectrometry alleviates the dependency on standards because

compounds can be identified directly, provided they respond under the

conditions of the analysis and that the molecular mass can be associated

with the identification of particular compound. The most prevalent use of

qualitative mass spectrometry during the course of pre clinical

pharmaceutical development is structural elucidation of related

substances (metabolites, synthetic impurities, and degradants), often at

trace levels. Insight into chemical structure is a key factor in referring

pharmaceutically desirable attributes of molecule such as potency, safety

and bioavailability, thus elucidation of structure is critically important

for accelerating the pace of the development process. The number of

synthetic impurities and their levels are indicative of the overall process

quality [126].

1.7 Validation of Analytical Method [127-130]:

Method validation constitutes the important part of any analytical

methods. A Success in these areas is identified to several important

factors, we can find in the development of pharmaceuticals and all of

them have great contribution to regulatory compliance. Validation proves

under standardised set of conditions that any procedure, process,

equipment, material, activity or system performs; it assures that a

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method works reproducibly, when proved by a same or different person,

in same or different laboratories, using different reagents, different

equipments, etc. Validation is important one to understand the

parameters or characteristics involved in the validation process.

Method validation is explained as the process of explained an

analytical method acceptance in scientific method for its intended use.

Guidelines for methods improvement and validation for noncompendial

and compendial test methods are standerdised by the FDA and ICH

documents, ―Analytical Procedures and Methods Validation: Chemistry,

Manufacturing and Controls Documentation [104] and ICH topic Q2B.

This latest document indicates to the method development and validation

process for products included in investigational new drug (IND), new

drug application (NDA) and briefed new drug application submissions

[59]. In recent years, trails have been developed to the harmonization of

pharmaceutical regulatory requirements in the United States, Europe,

and Japan. The FDA methods validation draft guidance and USP refer to

ICH guidelines [131].

1.7.1 Accuracy:

The accuracy of an analytical method indicates the similarity of

agreement between the value, which is accepted either as a conventional

25

value or a proved reference value and the value identified. Accuracy is

calculated as percentage recovery of the analyte by the assay of known

added amount of the analyte in the sample. Accuracy was studied at

four concentration level at LOQ level, 50% level, 100% level and 150%

level of working concentration for related substances and 80% level,

100% level and 120% level for assay in triplicate. Each preparation was

prepared independently by spiking impurities in to sample for related

substances and analyte in the placebo for Assay. Percent of recovery was

calculated by comparing values got in spiked sample with those obtained

in standard and got results were recorded.

1.7.2 Precision:

Precision can be defined as ―the degree of agreement among the

individual test results when the procedure is applied repeatedly to

multiple sampling of a homogeneous sample‖ a more comprehensive

definition proposed by International conference on Harmonization (ICH).

Precision defined in to three types (1) repeatability (2) Intermediate

precision and (3) reproducibility.

To compare the system precision (repeatability) for peak response

obtained with six replicates of dilute standard at limiting concentration.

To check repeatability (method precision) of method for independent six

different sample preparations were injected and % RSD with six sample

26

preparation found to be within 2.0%. To demonstrate Intermediate

precision, the method be compared on two days on two different systems.

1.7.3 Linearity and Range:

The linearity of the method is a measure of how well a calibration plot

of response versus concentration approximates a straight line. To

establish linear relationship between concentration and response with

correlation coefficient will be more than 0.999. To propose best fitting

equation of least square line (y = mx +c).

1.7.4 Limit of Detection:

The lowest concentration of analyte that can be identified, but not

able to determine in a quantitative method by using a specific method

under the required experimental states. The instrumental method limit of

detection determination is carried out by detecting the signal-to-noise

ratio by comparing test values from the samples with known

concentration of analyte with those of blank samples and the minimum

level at which the analyte can be truly identified. A signal-to-noise ratio

of 2:1 or 3:1 is standardised, the IUPAC approach gives the standard

seperation of the intercept (Sa) which may be related to LOD and the

slant of the calibration curve, b, by LOD = 3 Sa / b.

1.7.5 Limit of Quantitation:

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Impurities in bulk drugs and degradation products in finished

pharmaceuticals identified as per Limit of quantitation is a quantitative

assay parameter for low levels of compounds in sample matrices. The

limit of quantitation is the lowest determination concentration of analyte

in a sample with standardised precision and accuracy when the standard

procedure is applied. The standard deviation multiplied by a factor gives

finalisation of the limit of quantitation. In many instances, approximately

the limit of quantitation is double the limit of detection.

1.7.6 Specificity and Selectivity:

The selectivity of an analytical method is its ability to measure

accurately and especially the analyte of interest in the present of

components that may be expected to be identify in the sample matrix.

Chosen analytical method is capable to resolve and differentiate various

items of a mixture and identify the analyte qualitatively. A method is

said to be exact when it measures or proves quantitatively the

component of interest in the sample matrix without separation. Hence

primary deviation in the selectivity and speciality is that, while the

previous one is restricted to qualitative detection of the components of a

sample while the latter quantitative measurement of one or more analyte.

Selectivity may be defined with respect to the bias of the assay results

got. When the process is applied to the analyte in the presence of

assumed levels of different components, compared the results got. When

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the process is applied to the analyte in the presence of assumed levels of

other components, compared to the results got on the same analyte

without added substances.

1.7.7 Robustness and Ruggedness:

The robustness is the analytical capability, when measured on

deliberate differences in method parameters, final values remains

unaffected by these variations in parameters; it is reliability shown

during normal usage. Robustness of an analytical method was identified

by changing the pH of the buffer and found to acceptable for organic

addition, the 5 % v/v change in organic was influencing the retention

time of peaks, hence method validation advice to be cautious while

adding organic phase.

The ruggedness is degree of redevelopment of results, when measured

under a various of Laboratory conditions, other analyst, column and

environmental situations, but followed specified analytical parameters

given in method, final values remains unaffected by these variety of

conditions; it is reliability indication during normal usage, this is

recommended when method is to be changed to be used in more than

one lab.

1.7.8 Stability of Solutions:

Stability of a standard solution, sample solution and reagents used

for colour development reaction is necessary for a reasonable time to

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generate reproducible and accurate results. For example, 24 h stability

is sufficient for all solutions and reagents that necessary to be prepared

for each analysis.

1.7.9 System Suitability:

Test assurance of system suitability, accuracy and precision of results

on a specific occasion, system suitability tests performed every time

while method is used either before or during analysis. The results of

system suitability test are compared with standard values criteria, the

method are satisfactory on that occasion if values are within limit of

creiteria. A

1.8 Stastistics-treatment of Analytical Data [132-134]:

Collecting, organising, summarising and analysing data from

experiments are important elements in the presentation of all scientific

data. Laboratories produce data that are associated with some degree of

variability that affects the evaluation and interpretation of the data,

therefore knowledge on degree of uncertainty is essential. Statistics

forms the basis of this knowledge and is the key to drawing valid

conclusions and making reasonable decisions. Statistics is defined as a

science of data collection, interpretation of numerical data, presentation

and analysis.

Statistics is also the key to condensing data into a form that makes

main features of a data clearer. The term error is defined as the

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departure of the computed value from its real value. Thus, most of the

difference between the two values, greater will be the magnitude of error

involved. A large proportion of observations in a group of observations on

some variables have a tendency to cluster around some central value.

This is known as central tendency. Mean is calculated by adding all data

values and dividing added value by the number of measurements, X=∑

X/n.

Median is one of the measures of central tendency. It divides the

ordered sequences of data into two equal groups, half of the values will

be less than the median and half will have values greater than the

median. If n (number of observations) is odd there will be one and only

one middle value or median i.e. ½ (n+1) th observation from both ends of

the order of sequence. If ‗n‘ is even there is no middle observation but the

median is declared by convention as the mean of two middle

identifications, i.e. the (n/ 2) th and ((n+1)/2) th observation from one

end in the ordered sequence [70].

The difference between the observed value and arithmetic mean of the

set of observations in known as deviation, di = Xi – X. Mean deviation is

the arithmetic mean of all deviations irrespective of all the sign of the

deviation d = n

dnddd 321

The standard deviation of an infinite set of data is theoretically the

mean of the squares of the difference between the individual value xi and

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the mean of the infinite observation µ. Scientific experiments often have a

small number of observations. Standard deviation is calculated using (n-

1) observations because it represents better estimate of the standard

deviation of a small population from which the sample is taken,

S =

(x )2

n 1

i

For large number of observations (n-1) = n.

Therefore, S =

(x )2

n

i

The variance of a set of data is a square of the standard deviation.

Variance (standard deviation) measures the extent to which the data vary

amongst themselves. The relative measures of dispersion is expressed by

the term coefficient of variance for a set to measurements having X

(Mean) and S (standard deviation), the coefficient of variance can be

computed as

S 100

X

The statistical interval set about the mean ‗X‘, so that, for a given

number of replicate measurements and for a given probability level the

population mean ‗µ‘ lies within the specified range. These statistical

limits are known as confidence limits and the interval so generated is

called as a ‗confidence interval‘.

Confidence interval = X N

DS ..

X z

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Where, X is the observed mean, SD is standard deviation for the

experiment containing N number of observations. ‗z‘ is the student ‗t‘

value obtained from the 12 student ‗t‘ table for the given level of

confidence and for a given degree of freedom. Confidence intervals are

usually calculated at 95 % and 99 % level of confidence. The smaller the

confidence interval at 95 % and 99% levels of confidence, the more

precise are the results.

The statistical tool to expect the unknown values of one differenciative

from known values of another differnciative is called as regression. In

regression analysis the average relationship between the two variables is

revealed and thus it is possible to make a prediction of the dependent

variable. The variable whose value is influenced is called as the

dependent variable ‗Y‘; the variable, which exerts the influence, is called

as the independent variable ‗X‘. In case of linear regression, it will be

seen that a unit change in the value of the independent variable (X) will

produce a constant and absolute change in the dependent variable (Y).

When the two variables have a linear relationship the regression line can

be used to determine the value of the dependent variable. The correlation

coefficient obtained during the analysis of a regression line plays an

important role in deciding the relationship between the independent and

the dependent variable.

Most cases analytical methods are based on a calibration curve in

which a calculated quantity y is plotted as a function of the known

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concentration x of a series of standards. A plot of obtained data could be

a straight line. However, the complete data points fall exactly on the line

because of the random errors in the calculation process. Thus, we try to

derive the ―best‖ straight line from the points. A statistical technique

called the method of least squares provides the means for objectively

obtaining an equation for such a line and also for specifying the

uncertainties associated with its subsequent use. In applying the method

of least squares, one has to assume, that there is a linear relationship

between the peak area (y) and the analyte concentration (x), by the

equation y = mx + b, in this, b is the intercept (the value of y when x is

zero) and m is the slant of the line. Residual value is the vertical

difference of each point from the straight line. Total of the squares of the

residuals minimizes by the line generated by the least-squares method.