23

1.0 Introduction and importance of analytical method development

in pharmaceutical research and development

1.1 Impurity evaluation in pharmaceutical industry:

Over past two decades the pharmaceutical companies are investing in

Research and Development to develop and introduce new chemical

entities into the market. The control of pharmaceutical impurities is

currently a critical issue to the pharmaceutical industry. The impurity

may develop either during formulation, or upon aging of API’s and due to

the interaction of the excipients and the API’s in medicines. The presence

of these unwanted chemicals even in trace amount may influence the

efficacy and safety of pharmaceutical products. The composition of

impurities allows one to draw conclusions regarding the manufacturing

of the products and its adulteration, which is becoming widespread in all

countries of the world therefore, it is necessary to strictly control the

quality of pharmaceutical products and to determine the content of

impurities at all stages of production from raw materials to finished

medicinal forms [1-3]. The quality and safety of a drug is generally

assured by monitoring and controlling the impurities effectively. Thus,

the analytical activities concerning impurities in drug substances are

among the most important issues in modern pharmaceutical analysis.

Safety and efficacy of pharmaceuticals are two issues of fundamental

importance in drug therapy. The safety of a drug is determined by its

pharmacological and toxicological profile as well as the adverse effects

24

caused by the impurities in bulk and dosage forms. The impurities in

drugs often possess unwanted pharmacological or toxicological effects by

which any benefit from their administration may be outweighed [4].

Therefore, it is quite obvious that the products intended for human

consumption must be characterized upto the complete extent required.

The quality and safety of a drug is generally assured by monitoring and

controlling the impurities effectively. Thus, the analytical activities

concerning impurities in drugs are among the most important issues in

modern pharmaceutical analysis [5-8].

The best way to characterize the quality of API is to determine its

purity. There are two possible approaches to reach this goal, the

determination of the active ingredient content with a highly accurate and

precise specific method or the determination of its related impurities. In

the early years of drug analysis, when chromatographic techniques were

not available the characterization of the purity of drugs was based on the

determination of the active ingredient content by non-specific titrimetric

and photometric methods supported by the determination of physical

constants and some limit tests for known impurities based mainly on

colour reactions. The deficiencies of this approach are well known. In

many cases even highly contaminated drug materials could meet the

requirements set in the early editions of different pharmacopoeias.

As a consequence of the enormous development of the analytical

technology in the last two decades entirely new possibilities have

25

been created for the determination of the purity of drug materials. In

principle, it is now possible to replace all non-specific assay methods

with highly specific and precise (mainly HPLC) methods thus greatly

improving the value of the determination of the active ingredient

content of bulk materials. Nearly all organic impurities are

determined by chromatographic or related methods of which HPLC

has been the most important for over a decade and half.

A thorough literature search has revealed that many methods

based on HPLC, are used for analysis of impurities in drugs. Most of

the HPLC methods are in the reversed-phase mode with UV

absorbance detection, because this provided the best available

reliability, repeatability and sensitivity. In fact, this technique has set

a bench mark for other analytical techniques.

In the present era, there is a tremendous upsurge for the impurity

profiling of pharmaceutical products. Pharmaceutical industry is

emerging day by day with the aim to develop new drugs extracted from

natural products or synthetically produced chemical substances. But

one thing always remains important that the product should be as pure

as possible. Therefore, purity has always been considered as an essential

factor in ensuring drug quality.

To purify a material and remove the excess impurities one should first

recognize that whether they are actually present and what their nature

is. In the past, this was not always done. But presently drug analysis

26

and pharmaceutical impurities are the subjects of constant review in the

public interest. The International Conference on Harmonization (ICH)

guidelines achieved a great deal in harmonizing the definitions of the

impurities in new drug substances [9,10]. Impurities can be classified

into the following categories.

1) Organic impurities

2) Inorganic impurities

3) Residual solvents

1.1.1 Organic impurities:

Organic impurities, often called related, ordinary or synthesis-related

impurities may arise during the manufacturing process or storage of the

drug substance. They are derived from drug substance synthetic

processes and degradation reactions in drug substances and drug

products [11-13]. Synthetic process related impurities can be derived

from starting materials, intermediates, reagents, ligands, and catalysts

used in the chemical synthesis, as well as by-products from the side-

reactions during the chemical synthesis. Degradation products are

derived from the chemical degradation of drug substances and drug

products under storage or stress conditions. They may be identified or

unidentified, volatile or non-volatile, and include the following.

1.1.1.1 Starting Materials and Intermediates:

Starting materials and intermediates are the chemical building blocks

27

used to construct the final form of a drug molecule. Unreacted starting

materials and intermediates, particularly those involved in the last of few

steps of the synthesis, can potentially survive the synthetic and

purification process and appear in the final product as impurities [14].

1.1.1.2 Impurities in the Starting Materials:

Impurities present in the staring materials could follow the same

reaction pathways as the starting material itself, and the reaction

products could carry over to the final product as process impurities.

Knowledge of the impurities in starting materials helps to identify related

impurities in the final product, and to understand the formation

mechanisms of these related process impurities.

1.1.1.3 Reagents and Catalysts:

These chemicals are less commonly found in APIs however; in some

cases they may pose a problem as impurities. Chemical reagents, and

catalysts used in the synthesis of a drug substance can be carried over to

the final products as trace level impurities.

Many chemical reactions are promoted by metal based catalysts. For

instance, a Ziegler-Natta catalyst contains titanium, Grubb’s catalyst

contains ruthenium, and Adam’s catalyst contains platinum. In some

cases, reagents or catalysts may react with intermediates or final

products to form by-products.

1.1.1.4 Products of side reactions:

Frequently occurring side reactions are the formation of isomers.

28

Diastereomers as impurities occurs mainly in peptide derivatives. In the

majority of cases side reactions are unavoidable beside the main

reactions in organic syntheses even if pure starting materials and

reagents are used.

1.1.1.5 Impurities originated from reaction solvents:

Some solvents which are the part of the reaction act as a source of

impurities. In some cases the solvents of a reaction or an impurity in the

solvent is also transformed during the synthesis leading to an impurity.

1.1.1.6 Impurities Originating from Degradation of the Drug

Substance:

Impurities can also be formed by degradation of the end product

during manufacturing of bulk drugs. However, degradation products

resulting from storage are common impurities in the pharmaceuticals.

The definition of degradation product in the ICH guideline is a molecule

resulting from a chemical change in the substance brought about by

overtime or the effect of light, temperature, acid, base and peroxide.

1.1.1.7 Importance of chiral separations in the evaluation of

enantiomeric impurities:

Chirality is a major concern in the modern pharmaceutical industry. A

large percentage of commercial and investigational pharmaceutical

compounds are chiral and their enantiomers show significant differences

in their pharmacokinetics and pharmacodynamics. The importance of

29

chirality of drugs has been increasingly recognized and the consequences

of using them as racemates or as enantiomers have been frequently

discussed in the pharmaceutical literature during recent years [15,16].

The biological activity of chiral substances often depends upon their

stereochemistry. The body being amazingly chiral selective, will interact

with each racemic drug differently and metabolize each enantiomer by a

separate path way to produce different pharmacological activity. For

example, the enantiomers of chiral drugs such as omeprazole, ibuprofen

and DOPA(3,4-dihydroxyphenylalanine) exhibit different pharmacological

and pharmacokinetic activities because they interact with enzymes and

receptors consisting of amino acids and other chiral biomolecules [17].

Thus, one isomer may produce the desired therapeutic activities, while

the other may be inactive or, in worst cases, produce unwanted effects.

Consider the tragic case of the racemic drug of n-pthalyl-glutamic acid

imide that was marketed in the year 1960’s as the sedative Thalidomide.

Its therapeutic activity resided exclusively in the R-(+)- enantiomer. It

was discovered only after several hundred births of malformed infants

that the S-(+) enantioimer was teratogenic.

Nowadays, the regulatory authorities want drugs that are submitted

for approval to be single enantiomers, if it is at all possible. The U.S.

Food and Drug Administration, in 1992, issued a guideline that for chiral

drugs only its therapeutically active isomer be brought to market, and

that each enantiomer of the drug should be studied separately for its

30

pharmacological and metabolic pathways. In addition, a rigorous

justification is required for market approval of a racemate of chiral drugs.

Indeed, to avoid the possible undesirable effects of a chiral drug, it is

imperative that only the pure, therapeutically active form be prepared

and marketed. High performance liquid chromatography (HPLC) for

separation of enantiomers plays an important role at the present time

[18]. Chiral HPLC has proven to be one of the best methods for the direct

separation and analysis of enantiomers.

Below are some examples of the chiral compounds where the

enantioseparation was achieved. E.g.: vildagliptin [Fig. 1.1.F1] and 2-

azido-3-methylbutanoic acid [Fig. 1.1.F2]

Fig. 1.1.F1: Enantiomers of vildagliptin

HO

HN

N

ONC

HO

HN

N

ONC

S-vildagliptin R-vildagliptin

31

Fig. 1.1.F2: Enantiomers of 2-azido-3-methylbutanoic acid

S-2-azido-3-methylbutanoic acid R-2-azido-3-methylbutanoic acid

According to ICH guidelines on impurities in new drug products,

identification of impurities below 0.10 % level is not considered to be

necessary, unless potential impurities are expected to be unusually

potent or toxic. According to ICH, limit for known and unknown impurity

is fixed as below.

Table 1.1.T1: Impurity limits

MaximumDaily Dose

ReportingThreshold

IdentificationThreshold

QualificationThreshold

Case-1 ≤ 2g/day 0.05 % 0.10 % or 1.0 mg

per day intake

(whichever is lower)

0.15 % or 1.0 mg

per day intake

(whichever is lower)

Case-2 ≥ 2g/day 0.03 % 0.05 % 0.05 %

1.1.2 Inorganic impurities:

32

Inorganic impurities are source from the manufacturing process.

They are normally known and identified. There are various possible

sources for inorganic impurities in drugs.

a) The starting materials, reagents and solvents of the synthetic

manufacturing process can be sources for salts of inorganic acids

(chlorides, sulphates and phosphates).

b) Heavy metals can originate also from the reaction vessels and

tubings used in the manufacturing process.

c) Filters, filter aids and adsorbents used mainly decolorizing the

solutions of bulk drug materials during their crystallization.

d) Traces of some inorganic reagents themselves or their

transformation products are also possible impurities.

e) The degradation of the drug material can also be a reason for the

presence of inorganic impurities (e.g. phosphate salts from the

hydrolysis of phosphate esters).

These impurities are normally detected and quantitated using

pharmacopeias or other appropriate procedures. Any carryover of

catalysts to the new drug substance should be evaluated during

development. The need for inclusion or exclusion of inorganic impurities

in the new drug substance specifications should be discussed. Limits are

based on pharmacopeial standards of known safety data.

1.1.3 Residual solvents:

33

Solvents are inorganic or organic liquids used during the preparation

of solutions or suspensions in the synthesis of a new drug substance.

Residual solvents are therefore usually present at least at trace level in

bulk drugs. Since these are generally of known toxicity, the selection of

appropriate controls is easily accomplished. Specified concentrations of

such solvents should not be exceeded in the end product. The potential

toxicity of the solvent and the technical feasibility of removing it from the

API have to be considered as part of the specification process. A number

of solvents that are used for the synthesis of API or formulation of drug

product can be present in the drug product. The content of these

solvents, which are commonly called organic volatile impurities (OVI) or

residual solvents, is generally determined by the OVI methods specified

in the compendia. Some solvents that are known to cause toxicity should

be avoided in the production of bulk drugs. Depending on the possible

risk to human health, residual solvents are classified in to four classes

[19].

Class1 solvents: Solvents to be avoided in the manufacture of

pharmaceutical products.

Examples: Benzene (2 ppm), Carbontetracloride (4 ppm), 1,2-

Dichloroethane (5 ppm), 1,1-Dichloroethane (8 ppm) etc.

Class2 solvents: Solvents to be limited

Examples: acetonitrile (410 ppm), Chloroform (60 ppm), Dichloromethane

(600 ppm), methanol (3000 ppm) etc.

34

Class3 solvents: Solvents with low toxic potential.

Examples: Acetone (5000 ppm), ethanol (5000 ppm), ethylacetate (5000

ppm), Isopropyl alcohol (5000 ppm) etc.

Class 4 solvents: Solvents for which no adequate toxicological data were

found are listed in below Table-1.5, where the limits were considered

based on daily dosage calculations.

Table 1.1.T2: Solvents with no adequate toxicological data

1,1-Diethoxypropane

1,1-Dimethoxymethane

2,2-Dimethoxypropane

Iso-octane

Isopropyl ether

Methyl isopropyl ketone

Methyltetrahydrofuran

Petroleum ether

Trichloroacetic acid

Trifluoroacetic acid

1.2 The role of chromatography in impurity evaluation:

Chromatography is considered as one of the most dynamic and

versatile analytical methods, it has drastically advanced since its

beginning in the twentieth century. The applications of chromatography

have grown explosively in the last fifty years, owing not only to the

development of several new types of chromatographic techniques but

also to the growing need by scientists for better methods for

35

characterizing complex mixtures. Chromatography is a procedure by

which solutes are separated by a dynamic differential migration process

in a system containing two migration phases, one of which moves

continuously in a given direction, other is stationary and in which the

individual substances exhibit different mobilities due to difference in

adsorption, partition, solubility, vapour pressure, molecular size and

ionic charge density [20-21] etc.

In the beginning of 19th century, a Russian scientist Tswett while

working on plant extracts encountered colour bands that moved down

the column. He coined the name chromatography for the technique. As

Tswett had coined the name, which is till date popular and prevalent, he

is considered as the father of chromatography. Chromatography as a

word stems from the Greek origin chroma, which means color, and

graphien, which means to write. Indeed the greatest advantage of the

chromatographic method over any other analytical procedure is the

ability of separating specific analytes, a feature that appeals to all

branches of science, which enables to discover and analyze unknown

elements and chemical compounds. Chromatography includes a group of

different methods that allow the separation of complex chemical

mixtures. All chromatographic techniques consist of two phases: mobile

phase and immiscible stationary phase. The mobile phase is the phase

which moves in a definite direction. The stationary phase is the

substance which is fixed in place for the chromatography procedure.

36

Chromatographic separation occurs due to the differential migration

of analytes along the column. The average rate of migration of an analyte

through the column depends on the fraction of time spent in the

stationary phase and the affinity of the analyte to the stationary phase.

Components that tend to reside in the mobile phase will move more

quickly than those that prefer the stationary phase.

1.2.1 Separation Mechanisms:

A useful classification of the various LC techniques is based on the

type of distribution that is responsible for the separation. The common

interaction mechanisms encountered in LC are classified as adsorption,

partition, ion-exchange, gel permeation or size exclusion, and chiral

interaction. In practice, most LC separations are the result of mixed

mechanisms. A brief description of the separation mechanisms is

presented below.

1.2.1.1 Adsorption Chromatography:

When the stationary phase in HPLC is a solid, the type of equilibrium

between this phase and the liquid mobile phase is termed ‘adsorption’.

All of the pioneering work in chromatography was based upon adsorption

methods, in which the stationary phase is a finely divided polar solid that

contains surface sites for retention of analytes. The composition of the

mobile phase is the main variable that affects the partitioning of

analytes. Silica and alumina are the only stationary phases used, the

37

former being preferred for most applications. Applications of adsorption

chromatography include the separation of relatively non-polar water-

insoluble organic compounds. Because of the polar nature of the

stationary phase and the impact of subtle variations in mobile phase

composition on the retention time, adsorption chromatography is very

useful for the separation of isomers in a mixture. The schematic diagram

of adsorption chromatography is shown in Fig. 1.2.F1.

Fig. 1.2.F1: The schematic diagram of adsorption chromatography.

1.2.1.2 Partition Chromatography:

The equilibrium between the mobile phase and a stationary phase

comprising of either a liquid adsorbed on a solid or an organic species

bonded to a solid is described as ‘partition’. The predominant type of

separation in HPLC today is based on partition using bonded stationary

phases. Bonded stationary phases are prepared by reaction of organo

chloro silane with the reactive hydroxyl groups on silica. The organic

functional group is often a straight chain octyl (C-8) or octyldecyl (C-18);

38

in some cases a polar functional group such as cyano, diol, or amino

may be part of the siloxane structure.

There are two types of partition chromatography namely normal-

phase and reverse phase chromatography. Normal-phase

chromatography makes use of highly polar stationary phase and a

relatively less polar mobile phase. Here the least polar component is

eluted first where an increase in the polarity of the mobile phase will

decrease the retention times. The retention of analytes in reverse-phase

chromatography is fundamentally determined by their distribution

between a polar mobile phase (mixtures of water and organic modifiers)

and the less polar stationary phase. The schematic diagram of Partition

chromatography is shown in Fig. 1.2.F2.

Fig. 1.2.F2: The schematic diagram of Partition chromatography.

1.2.1.3 Ion Exchange Chromatography:

39

Ion exchange chromatography employs a stationary phase consisting

most often of a porous polymeric material to which acidic or basic

functional groups have been bonded. The acidic or basic groups give the

stationary phase an affinity for ions. An ion exchange reaction can thus

take place in which analyte ions displace the acidic or basic groups

comprising the stationary phase. The magnitude of the equilibrium

constant of the exchange reaction dictates the affinity of the analyte ions

for the stationary phase. The mobile phase used is typically an aqueous

solution containing an ionic species. Organic solvents such as Methanol

may also be present. The mobile phase ions compete with the analyte for

exchange sites on the stationary phase. The schematic diagram of Ion

Exchange Chromatography is shown in Fig. 1.2.F3.

Fig. 1.2.F3: The schematic diagram of Ion Exchangechromatography.

1.2.1.4 Molecular Exclusion Chromatography:

This is also known as gel permeation or gel filtration

chromatography. This technique separates analytes according to their

40

molecular size and shape. Resins for exclusion chromatography include

silica or polymer Particles, which contain a network of uniform pores into

which the solute and solvent molecules diffuse. As a sample moves

through the column the analytes are separated as the lower molecular

weight species are held back due to permeation of the particle pore

whereas the higher molecular weight species are larger than the average

size of the pore and are excluded. Thus the larger species move through

the column faster. Exclusion chromatography differs from conventional

chromatography, as there are no chemical or physical interactions between

the analytes and the stationary phase. The schematic diagram of

Molecular Exclusion Chromatography is shown in Fig. 1.2.F4.

Fig. 1.2.F4: The schematic diagram of Molecular exclusion

chromatography.

The following table depicts the typical classification of

chromatographic methods (Table 1.2.T1). Of all the types of

41

chromatographic techniques the most widely used technique in the bulk

drug and pharmaceutical applications for impurity evaluation is High

performance liquid chromatography, which was discussed further

elaborately [22-25].

Table 1.2.T1: Classification of chromatographic methods

Type of stationeryphase

Type ofmobilephase

Apparatus forstationaryphase

Type ofchromatography

Adsorptionchromatography

Competitionbetween a solidadsorbent and themobile phase

Gas Column Gas solidchromatography (GSC orGC)

LiqidColumn

Liquid columnchromatography (LC),High performance liquidchromatography (HPLC)

Planar layer Thin layerchromatography (TLC),paper chromatography(PC)

Partitionchromatography

Competitionbetween a liquidstationery phaseand the mobilephase

Gas Column Gas liquidchromatography (GLCor GC )

Liquid ColumnLiquid Liquidchromatography (LLC),High performance liquidchromatography (HPLC)

Ion exchangechromatography

Competition

42

between an ionexchange resinstationary phaseand liquid mobilephase

Liquid Column Ion exchangechromatography (IEC)

MolecularExclusionchromatography

Competitionbetween a polymermatrix and liquidmobile phase

Liquid Column Molecular Exclusionchromatography

1.2.2 High performance liquid chromatography (HPLC):

HPLC is defined as High Performance Liquid Chromatography or High

Pressure Liquid Chromatography. The utmost feature that is

characteristic of the development of the methodology for pharmaceutical

and biomedical analysis during the past 25 years is the use of HPLC,

which is undoubtedly the most important analytical tool for identification

and quantification of drugs either in the active pharmaceutical ingredient

or in the formulations, during the process of their discovery, development

and manufacturing. The schematic diagram of HPLC is shown in Fig.

1.2.F5.

Fig. 1.2.F5: The schematic diagram of HPLC

43

The various components that are present in HPLC equipment are:

1) Pump

2) The Injector

3) The column

4) The detector

5) Data handling device and microprocessor control.

1.2.2.1 Pump: The pump is one of the most important components in

HPLC, since it performance directly affects retention time,

reproducibility. A mobile phase is pumped under pressure from one or

several reservoir and flows through the column at a constant rate. A

degasser is needed to remove dissolved air and other gases from the

solvent. Various types of pumps are used in HPLC to propel the liquid

mobile phase through the system. They are:

1.2.2.1.1 Reciprocating Piston Pumps: Consist of a small motor

driven piston, which moves rapidly back and forth in a hydraulic

chamber that may vary from 35-400 l in volume. On the backstroke, the

44

separation column valve is closed, and the piston pulls in solvent from

the mobile phase reservoir.

On the forward stroke, the pump pushes solvent out to the column

from the reservoir. This type of pump system is significantly smoother

because one pump is filling while the other is in the delivery cycle.

1.2.2.1.2 Syringe Type Pumps: They are most suitable for small-bore

columns because this pump delivers only a finite volume of mobile phase

before it has to be refilled. These pumps have a volume between 250 to

500 ml. The pump operates by a motorized lead screw that delivers

mobile phase to the column at a constant rate. The rate of solvent

delivery is controlled by, changing the voltage on the motor.

1.2.2.1.3 Constant Pressure Pumps: The mobile phase is driven

through the column with the use of low pressure from a gas cylinder to

generate high liquid pressures. The valve arrangement allows the rapid

refill of the solvent chamber whose capacity is about 70ml. This provides

continuous mobile phase flow rates.

1.2.2.2 Injector: Samples are injected into the HPLC via an injection

port. The injection port of an HPLC commonly consists of an injection

valve and the sample loop. The sample is typically dissolved in the mobile

phase before injection into the sample loop. The sample is then drawn

into a syringe and injected into the loop via the injection valve. A rotation

of the valve rotor closes the valve and opens the loop in order to inject

the sample into the stream of the mobile phase. Loop volumes can range

45

between 10 µl to over 500 µl. In modern HPLC systems, the sample

injection is typically automated [26].

1.2.2.3 Column: High-performance liquid chromatography columns are

stainless steel tubes, typically of 10-30 cm in length and 3-5 mm inner

diameter. Short, fast analytical columns, and guard columns, which are

placed before an analytical column to trap junk and extend the lifetime of

the analytical column, are 3-10 cm long. The stationary phase or packing

is retained at each end by thin stainless steel frits with a mesh of 2µm or

less. The most popular material is octadecyl-silica, which contains C18

chains, but materials with C2, C6, C8 and C22 chains are also available

[27].

1.2.2.4 Detectors: Detector for HPLC is the component that emits a

response due to eluting sample compound and subsequently signals a

peak on the chromatogram. It is positioned immediately posterior to the

stationary phase in order to detect the compounds as they elute from the

column. The bandwidth and height of the peaks may usually be adjusted

using the coarse and fine-tuning controls and the detection and

sensitivity parameters may also be controlled. Regardless of the principle

of operation, an ideal LC detector should have the following properties:

a) Low drift and noise level (particularly crucial in trace analysis).

b) High sensitivity and fast response.

c) Wide linear dynamic range (this simplifies quantitation).

d) Low dead volume (minimal peak broadening).

46

e) Cell design, which eliminates remixing of the separated bands.

f) Insensitivity to changes in type of solvent, flow rate, and

temperature.

g) Operational simplicity and reliability.

h) It should be tunable so that detection can be optimized for

different compounds.

i) It should be non-destructive.

Many types of detectors can be used with HPLC.

1.2.2.4.1 Refractive index detectors: They measure the ability of

sample molecules to bend or refract light. This property is called

refractive index. Detection occurs when light is bent due to samples

eluting from the column and this is read as a disparity between the two

channels.

1.2.2.4.2 Ultra violet (UV) detectors: The UV-visible absorbance

detector is the most common HPLC detector in use today since, many

compounds of interest absorb in the UV (or visible) region (from 190–600

nm).They measure the ability of samples to absorb light. This can be

established at one or several wavelengths. The schematic diagram of

UV detector is shown in Fig.1.2.F6.

Fig. 1.2.F6: The schematic diagram of UV detector.

47

Fixed wavelength: Measures at one wavelength, usually 254 nm.

Variable wavelength: Measures one wavelength at a time, but can detect

over a wide range of wavelengths.

Diode array: Measures a spectrum of wavelengths simultaneously. It

passes the total light through the flow cell and disperses it with a

diffraction grating. The dispersed light is measured by an array of

photosensitive diodes. The array of diodes is scanned by the

microprocessor. The reading for each diode is summed, and the total is

averaged.

The schematic diagram of Diode array detector is shown in Fig.1.2.F7.

Fig. 1.2.F7: The schematic diagram of Diode array detector.

48

1.2.2.4.3 Fluorescent detectors: They measure the ability of a

compound to absorb then re-emit light at given wavelengths. Each

compound has a characteristic fluorescence. The excitation source

passes through the flow-cell to a photodetector while a monochromator

measures the emission wavelengths. Fluorescence detection is usually

more sensitive than absorption detection.

1.2.2.4.4 Radio chemical detectors: Involves use of radio labeled

material usually tritium (3H) or carbon-14 (14C). It operates by detection

of fluorescence along with beta-particle ionization.

1.2.2.4.5 Electrochemical detectors: Used in analysis of compounds

that undergoes oxidation or reduction reactions. They measure the

difference in electrical potential when the sample passes between the

electrodes.

1.2.2.5 Data handling device and microprocessor control:

The last part of HPLC apparatus is data system. The visualization of

the detector signals helps to clarify the separation conditions. Pen

recorders were originally used but now the integrator is popular due to

49

the automatic reporting of both the retention time and the peak area or

the height. The use of the integrator makes quantitative analyses easier.

Computer based integrators are powerful for the storage and further

arrangement of data and can also be used for the column evaluation and

as a system controller [28].

1.2.3 Applications of HPLC [29]:

1.2.3.1 Chemical separation: This can be accomplished using HPLC by

utilizing the fact that, certain compounds have different migration rates

given a particular column and mobile phase. The extent or degree of

separation is mostly determined by the choice of stationary phase and

mobile phase.

1.2.3.2 Identification: For this purpose a clean peak of known sample

assay has to be observed from the chromatogram. Selection of column

mobile phase and flow rate matter to certain level in this process by

comparing with reference compound does identification and it can be

assured by combining two or more detection methods.

1.2.3.3 Quantification: It is the analyte confirmation by using the

known reference standards. Quantification of known and unknown areas

with respect to the principal peak by various methods like- area

normalization method, internal standard method and external standard

method.

1.2.3.4 Preparative HPLC: It refers to the process of isolation and

purification of compounds. Important is the degree of solute purity and

50

the throughput, which is the amount of compound, produced per unit

time. Typically this type of separations requires larger columns and the

chromatograms generally display broader peaks, due to the increased

column load. The separation is optimized for higher throughput

(weight/time), then sample load is maximized on small analytical

columns, and finally quantities are scaled up to the preparative column

according to the desired purity and recovery.

1.3 Ultra performance liquid chromatography:

High performance liquid chromatography (HPLC) has proven to be

the predominant technology used in laboratories worldwide during the

past 30-plus years. One of the primary drivers for the growth of this

technique has been the evolution of the packing materials used to impact

the separation. The underlying principles of this evolution are governed

by the van Deemter equation. The van Deemter equation is an empirical

formula that describes the relationship between linear velocity (flow rate)

and plate height (HETP or column efficiency). Since particle size is one of

the variables, a van Deemter curve can be used to investigate

chromatographic performance. It considers particle size as one of the

variables and therefore, it can be used to characterize theoretical

performance across various particle sizes. From the information in figure

1.3.F1 it can be realized that once moving below 2 µm in particle size, a

realm of chromatography is entered where not only higher efficiencies are

gained, but also these efficiencies no longer diminish with flow rate.

51

Fig. 1.3.F1: Van Deemter plot illustrating the evolution of particlesizes over the last three decades.

This new category of analytical separation science retains the

practicality and principles of HPLC while increasing the overall

interrelated attributes of speed, sensitivity and resolution.Ultra-

performance liquid chromatography (UPLC), using a new 1.7μm

reversed-phase packing material, operating at high pressure (upto

15000psi ), makes it possible to perform very high-resolution separations

in short periods of time with little organic solvent consumption [30],

which has attracted wide attention of pharmaceutical and biochemical

analysts [31].

1.3.1 Theory of separations using smaller particles:

According to van Demeter equation, smaller particles provide not

only increased efficiency, but also the ability to do work at increased

linear velocity without the loss of efficiency, providing both resolution

52

and speed. Efficiency is the primary separation parameter behind UPLC

since it relies on the same selectivity and retentivity as HPLC. In the

fundamental resolution equation (Rs), resolution is proportional to the

square root of N [32].

Where N is number of theoretical plates, α is Selectivity factor and k is

mean retention factor. But since N is inversely proportional to particle

size (dp).

As the particle size is lowered by a factor of three i.e. from 5 µm

(HPLC scale) to 1.7 µm (UPLC scale), N is increased by three and the

resolution by square root of three or 1.7. N is also inversely proportional

to the square of the peak width.

This illustrates that the narrower the peaks are, the easier they are

to separate from each other. Also peak height is inversely proportional to

the peak width (w):

So as the particle size decreases to increase N and subsequently Rs,

an increase in sensitivity is obtained, since narrower peaks are taller

peaks. Narrower peaks also mean more peak capacity per unit time in

53

gradient separations, desirable for many applications. Also, by using

smaller particles, analysis time can be decreased without sacrificing

resolution, because as particle size decreases, column length can also be

reduced proportionally to keep column efficiency constant. By using the

same HPLC mobile phase and flow rate, UPLC reduces peak width and

produced taller peaks which in turn increase the S/N from 1.8 to 8 fold,

thus improving both sensitivity and resolution.

1.3.2 Instrumentation

Also according to the van Deemter plot, use of particles smaller than

2 μm produces no loss in column efficiency with increasing flow rates.

However, by increasing flow rates to decrease analysis time, there is a

corresponding increase in system pressure. As a result, a system capable

of withstanding the proper pressures while still maintaining efficiency is

required. As well, a mechanically stable column is needed.

A completely new system design with advanced technology in the

pump, auto sampler, detector, data system, and service diagnostics was

required. The advent of UPLC has demanded the development of a new

instrumental system for liquid chromatography, which can take

advantage of the separation performance (by reducing dead volumes) and

be consistent with the pressures (about 8000 to 15,000 PSI, compared

with 2500 to 5000 PSI in HPLC). The ACQUITY UPLC system has been

designed for low system and dwell volume to take full advantage of low

dispersion and small particle technology.

54

1.3.2.1 Pimping systems

Achieving small particle, high peak capacity separations requires a

greater pressure range than that achievable by today's HPLC

instrumentation. The calculated pressure drop at the optimum flow rate

for maximum efficiency across a 15 cm long column packed with 1.7 μm

particles is about 15,000 psi. Therefore, a pump capable of delivering

solvent smoothly and reproducibly at these pressures, which can

compensate for solvent compressibility and operate in both the gradient

and isocratic separation modes, is required. The binary solvent manager

uses two individual serial flow pumps to deliver a parallel binary

gradient. There are built-in solvent select valves to choose from up to

four solvents. There is a 15,000-psi pressure limit (about 1000 bar) to

take full advantage of the sub-2μm particles.

1.3.2.2 Sample injection

In UPLC, sample introduction is critical. Conventional injection

valves, either automated or manual, are not designed and hardened to

work at extreme pressure. To protect the column from extreme pressure

fluctuations, the injection process must be relatively pulse-free and the

swept volume of the device also needs to be minimal to reduce potential

band spreading. A fast injection cycle time is needed to fully capitalize on

the speed afforded by UPLC, which in turn requires a high sample

capacity. Low volume injections with minimal carryover are also required

to increase sensitivity.

55

1.3.2.3 Sample manager

The sample manager also incorporates several technology

advancements. Using pressure assisted sample introduction, low

dispersion is maintained through the injection process, and a series of

pressures transducers facilitate self-monitoring and diagnostics. It uses

needle-in-needle sampling for improved ruggedness and needle

calibration sensor increases accuracy. Injection cycle time is 25 seconds

without a wash and 60 sec with a dual wash used to further decrease

carry over. A variety of micro titer plate formats (deep well, mid height, or

vials) can also be accommodated in a thermostatically controlled

environment. Using the optional sample organizer, the sample manager

can inject up to 22 micro titer plates. The sample manager also controls

the column heater. Column temperatures up to 65°C can be attained.

1.3.2.4 UPLC columns

The promises of the van Deemter equation cannot be fulfilled without

smaller particles than those traditionally used in HPLC. The design and

development of sub-2μm particles is a significant challenge, and

researchers have been very active in this area to capitalize on their

advantages [33,34]. Although high efficiency nonporous 1.5μm particles

are commercially available, they suffer from low surface area, leading to

poor loading capacity and retention. To maintain retention and capacity

similar to HPLC, UPLC must use a novel porous particle that can

withstand high pressures. Silica based particles have good mechanical

56

strength, but suffer from a number of disadvantages. These include

tailing of basic analytes and a limited pH range. Other alternative,

polymeric columns can overcome pH limitations, but they have their own

issues, including low efficiencies and limited capacities.

In 2000, Waters introduced a first generation hybrid chemistry,

called XTerra, which combines the advantageous properties of both silica

and polymeric columns , they are mechanically strong, with high

efficiency, and operate over an extended pH range. XTerra columns are

produced using a classical sol-gel synthesis that incorporates carbon in

the form of methyl groups. However, in order to provide the kind of

enhanced mechanical stability that UPLC requires, a second generation

hybrid technology was developed called ACQUITY UPLC.ACQUITY 1.7μm

particles bridge the methyl groups in the silica matrix as shown in Fig.

1.3.F2, which enhances their mechanical stability.

57

Fig. 1.3.F2: Synthesis and Chemistry of ACQUITY 1.7μm particles

for UPLC

Not only do BEH columns have enhanced mechanical and chemical

stability, but also reduce peak tailing significantly for basic analytes

compared to silica columns, due to the reduced acidity of the unreacted

surface silanol groups. Four bonded phases are available for UPLC

separations (Fig. 1.3.F3).

Fig. 1.3.F3: Bonded phases available for UPLC

58

Each column chemistry provides a different combination of hydropho-

bicity, silanol activity, hydrolytic stability and chemical interaction with

analytes.

1.3.2.5 Detectors

For UPLC detection, the tunable UV/Visible detector is used which

includes new electronics and firmware to support Ethernet

communications at the high data rates. Conventional absorbance-based

optical detectors are concentration sensitive detectors, and for UPLC use,

the flow cell volume would have to be reduced in standard UV/Visible

detectors to maintain concentration and signal. According to Beer’s Law,

smaller volume conventional flow cells would also reduce the path length

upon which the signal strength depends. A reduction in cross-section,

means the light path is reduced, and transmission drops with increasing

noise. Therefore, if a conventional HPLC flow cell were used, UPLC

sensitivity would be compromised. The ACQUITY Tunable UV/Visible

detector cell consists of a light guided flow cell equivalent to an optical

fibre. Light is efficiently transferred down the flow cell in an internal

reflectance mode that still maintains a 10mm flow cell path length with a

volume of only 500nL. Tubing and connections in the system are

efficiently routed to maintain low dispersion and to take advantage of

leak detectors that interact with the software to alert the user to potential

problems.

59

1.4 Analytical method development approaches:

Many parameters must be evaluated and optimized during the

method development process. Proper development of a method, as well as

optimization and troubleshooting, requires an understanding of the

influence that each of these parameters plays in the overall process

[35,36]. The following parameters are to be evaluated critically in

developing a robust analytical method.

1) Literature collection

2) Chemical structure

3) Diluent selection

4) Selection of column

5) Detector selection

6) Mobile phase selection

7) Flow rate and Column temperature

8) Force Degradation studies

1.4.1 Literature collection:Thorough literature search to be carried out like, USP, EP, JP, IP,

Chromatography Journals, patents, etc., before initiating the method

development activity for same or similar type of drug molecules. This

should be the first element whenever one takes up the project on

establishment of a stability indicating HPLC method development. Collect

information if available on solubility profile (solubility of drug in different

solvents and at different pH conditions), analytical profile (physico-

60

chemical properties, Eg: pKa, melting point, degradation pathways, etc)

and stability profile (sensitivity of the drug towards light, heat, moisture

etc.

1.4.2 Chemical structure:

Collect the structures of the molecule and the impurities likely to be

present, starting material, byproduct, and intermediates in the reaction

and degradation products. Identify the closely related structures and

method design is to be made to get good resolution between the closely

related structures. Compare the structures of impurities, starting

material, byproduct, intermediates and degradation products with the

structure of drug substances and arrive at the polarity whether they are

less polar or more polar than the compound of interest.

1.4.3 Diluent selection:

Select a diluent in which impurities, starting material, byproduct,

intermediates and degradation products and the analyte is soluble. It is

advisable to check first in mobile phase. All the analytes should be

completely soluble and solution should be clear. Diluent should be

compatible with the Mobile phase to obtain the good symmetrical peak

shape.

1.4.4 Selection of column:

Bonding phase can be choose based on the polarity of the

molecule. For Reverse phase chromatography, a wide variety of

columns are available covering a wide range of polarity by cross-

61

linking the Si-OH groups with alkyl chains like C8, C18 and nitrile

groups (CN), phenyl groups (-C6H6) and amino groups (-NH2) etc [37]

[Fig. 1.4.F1].

Fig. 1.4.F1: Different alkyl chains attached to Si-OH.

Silica based columns with different crosslinkings in the

increasing order of polarity are as follows:

---------Non-polar--------moderately polar------------Polar-----------

C18 < C8 < C6/C4 < Phenyl < Amino < Cyano < Silica

Selection of chiral stationery phases:

There is no single chiaral stationary phase (CSP) that can be

considered as universal, i.e., has the ability to separate all classes of

racemic compounds. Choosing the right CSP for the enantioseparation of

62

a chiral compound is difficult. The decision relies mostly on empirical

data. Most chiral separations achieved on CSPs, however, were obtained

based upon the accumulated trial and error knowledge of the analyst,

intuition, and often simply by chance. An alternative way of choosing a

CSP is by using predictive empirical rules that have been developed

based on empirical structures. Neither scheme of choosing a right CSP

offers a guarantee for a successful enantiomeric separation. Although

enanatioseparation is hoped to be achieved by knowing the chemistry of

the racemic analytes and the CSP sometimes, however, it does not work

because the interactions of the mobile phase with both the racemic

analyte and CSP have to be considered. All three components, analyte,

CSP and mobile phase, must be taken into consideration when

developing a chiral separation method. The key, therefore, to a successful

enantiosepration of a particular class of racemates on a given CSP is the

understanding of the possible chiral recognition mechanisms. Below are

the different CSPs, which were mostly used in achieving the

enantioseparations.

i) Protein – Based chiral stationary phases: e.g.:

a) Chiral –AGP (- glycoprotein)

b) Chiral CBH (cellobiohydrolase)

c) Chiral HAS (human serum albumin)

ii) Pirkle type chiral stationary phases: e.g.:

a) Whelk-O1

63

b) Whelk-O2

iii) Polysaccharide based chiral stationary phases: e.g.:

a) Chiralcel OD

b) Chiralpak AD

c) Chiralcel OJ

iv) Cyclodextrin based chiral stationary phases: e.g.:

a) Cyclobond

b) Chiradex

v) Macocyclic glycopeptide antibiotic chiral stationary phases: e.g.:

a) Chirobiotic-T

b) Chirobiotech-V

1.4.4.1 Particle shape:

Particles are either spherical or irregular in shape [Fig. 1.4.F2].

Irregular particles have higher surface areas and higher carbon loads.

Spherical particles provide higher efficiency, better column stability and

lower back-pressures compared to irregularly shaped particles.

Fig. 1.4.F2: The shapes of spherical and irregular particles

1.4.4.2 Particle size:

64

Particle size for HPLC column packings refers to the average diameter

of the packing particles [Fig. 1.4.F3]. Particle size affects the back-

pressure of the column and the separation efficiency. Column back-

pressure and column efficiency are inversely proportional to the square

of the particle diameter. As the particle size decreases, the column back-

pressure and efficiency increase. The particle diameter range is about 2–

20 µm. Smaller particles offer higher efficiency. Fast, high-resolution

separations can be achieved with small particles packed in short (10-50

mm length) columns.

Fig. 1.4.F3: Different particle sizes of HPLC column packing

3 µM PARTICLE SIZE 5 µM PARTICLE SIZE

1.4.4.3 Surface Area:

The surface area is the sum of particle outer surface and interior pore

surface in square meters per gram [Fig. 1.4.F4]. High surface areas

generally provide greater retention, capacity, and resolution for

separating complex, multi component samples. The physical structure of

the particle substrate determines the surface area of the packing

material. Surface area is determined by pore size. Pore size and surface

area are inversely related. A packing material with a small pore size will

65

have a large surface area, and vice versa. High surface area materials

offer greater capacity and longer analyte retention times. Low surface

area packings offer faster equilibration time and are often used for large

molecular weight molecules.

Fig. 1.4.F4: The schematic diagram of surface area.

1.4.4.4 Pore size:

The pore size of a packing material represents the average size of the

pores within each particle [Fig. 1.4.F5]. They range in value from 60 Å to

10,000 Å. Larger pores allow larger solute molecules to be retained

through maximum exposure to the surface area of the particles.

Generally pore size of 150 Å or less is chosen for samples with molecular

weights less than 2000 and a pore size of 300 Å or greater for samples

with molecular weights greater than 2000.

Fig. 1.4.F5: A representative diagram of pore size

66

60A PORE SIZE 100A PORE SIZE

1.4.4.5 Carbon load:

The carbon load is the amount of bonded phase attached to the base

material, expressed as the percentage of carbon [Fig. 1.4.F6]. High

carbon loads generally offer greater resolution and longer run times for

hydrophobic samples. Low carbon loads shorten run times and often

show different selectivity.

Fig. 1.4.F6: A representative diagram for carbon load.

1.4.4.6 pH limitations of HPLC column:

In general, HPLC columns are stable within a pH range of 2 to 8.

Modern HPLC columns can be used outside that pH range. The new

67

bonding chemistries allow use down to pH 1 for some stationary phases.

However, best lifetimes are obtained between pH 2.0 and pH 6.8.

1.4.4.7 Effect of variables on column efficiency:

The performance of the chromatographic column is key in the

separation process. Van Deemter plots are commonly used to describe

column performance by plotting the height equivalent to a theoretical

plate (HETP or H) against the average linear velocity (u). The general form

of the van Deemter equation is given by,

HETP (H) = A + B / u + C u

A - Eddy diffusion : The first term, eddy diffusion accounts for the

geometry of the packing. This term describes the change in pathway and

velocity of solute molecules in reference to the zone center. Therefore,

when the sample migrates down the column, each molecule has different

paths and each path is of a different length. Some molecules take the

longer paths and other take the shorter paths. The various possible

pathways result in different retention times as the mobile phase carries

sample molecules through the packed stationary phase and this effect is

directly proportional to the diameter of the particles packing the column

[Fig. 1.4.F7].

68

Fig. 1.4.F7: Pictorial diagram for understanding the eddy

diffusion.

Initial Packed bed Final

B/u - Longitudinal diffusion : It describe the band broadening process

in which solutes diffuse from the concentrated centre of a zone to the

more dilute regions ahead of and behind the zone centre. The

contribution of the longitudinal diffusion is inversely proportional to the

mobile phase velocity. The B/u term in the van Deemter equation is

negligible due to the small solute diffusion coefficient at practical flow

rates in HPLC relative to gases [Fig. 1.4.F8].

Fig. 1.4.F8: Pictorial diagram for understanding the longitudinal

diffusion

69

Cu - Resistance to mass transfer : The analyte takes a certain amount

of time to equilibrate between the stationary and mobile phase. If the

velocity of the mobile phase is high, and the analyte has a strong affinity

for the stationary phase, then the analyte in the mobile phase will move

ahead of the analyte in the stationary phase. The band of analyte is

broadened. The mass-transfer broadening is related to both the size of

packing particles and the column flow rate. The faster the mobile phase

moves, the bigger the particle size, the less time there is for equilibrium

to be approached [Fig. 1.4.F9].

Fig. 1.4.F9: Pictorial diagram for understanding the resistance tomass transfer

Stationary phase mass transfer Mobile phase mass transfer

1.4.4.8 Detector selection:

PDA (photodiode array) detector is useful for initial method

development based on the chromophores present in the compounds to be

separated. Select the initial wavelength analyzing the UV spectra of the

compounds using UV-VIS Spectrophotometer. If the compounds are not

having chromophores, choose other detectors like RI, ELSD/CCAD [38].

Reverse phaseBonded

70

Modern Diode array detector technology is a powerful tool for

evaluating specificity. Diode array detectors can collect spectra across a

range of wavelengths at each data point collected across a peak, and

through software data processes involving multidimensional vector

algebra, they compare each of the spectra to determine peak purity. In

this manner, Diode array detectors today can distinguish minute spectral

and chromatographic differences not readily observed by simple overlay

comparisons. To be successful, three components are required:

A UV chromophore, or some absorbance in the wavelength range

selected.

Some degree of chromatographic resolution.

Some degree of spectral difference.

1.4.4.9 Mobile phase selection:

In reversed phase HPLC, the retention of analytes is related to their

hydrophobicity. The more hydrophobic the analyte, the longer it is

retained. When an analyte is ionized, it becomes less hydrophobic and,

therefore, its retention decreases. Acids lose a proton and become ionized

when pH increases and bases gain a proton and become ionized when pH

decreases is shown in the figure below. Therefore, when separating

mixtures containing acids and/or bases by reversed phase HPLC, it is

necessary to control the pH of the mobile phase using an appropriate

buffer in order to achieve reproducible results [Fig. 1.4.F10].

71

Fig. 1.4.F10: The Effect of pH on the Retention of Acids and Bases.

As acids lose a proton and become ionized (with increasingpH), their retention decreases. As bases gain a proton andbecome ionized (with decreasing pH) their retentiondecreases.

1.4.4.9.1 Fixing of the pH:The pH of the Buffer in the mobile phase is selected based on the pKa

of the analyte, which is based on the structure of the molecule [Table

1.4.T1]. If compound is acidic, use acidic mobile phase. For a basic

compound, use low pH or basic mobile phase. For a neutral compound,

neutral mobile phase is suitable. For most practical purposes and for

most robust methods, the pH of the mobile phase can be ± 1 units from

the pKa of analytes of interest. Acidic compounds are more retained at

low pH; while basic compounds are more retained at higher pH (neutral

compounds are of course unaffected). At pH values used traditionally (pH

4-8); a slight change in pH would result in a dramatic shift in retention

(up-slope or down-slope of curve).

72

Table 1.4.T1: pKa values for different functional groups.

When the pH of mobile phase is near the pKa value of the analytes,

even a small change in pH will have a major impact on the resolution.

Acidic compounds: Preferable to use acidic mobile phase as the

compound will be in unionised form and will retain more.

Basic compounds: In Acidic mobile phase compound will be ionised and

will elute early; peak shapes will be better. In basic mobile phase

compound will be unionised and will retain more but peaks may tail due

to active silanols at basic pH.

Neutral Compounds: Neutral mobile phase is suitable.

With increasing pH acids losses a proton and become ionized. When

acids are ionized, it becomes less hydrobhobic and more hydrophilic

pKa

Aliph a Aromatic b Aliph a Aromatic b

Sulfonic acid ----SO3H 1 1 ---- ----Amino acid, ---C(NH2)---COOH 2 - 4 ---- 9 - 12 ----Carboxylic acid, ---COOH 4 - 5 4 - 5 ---- ----Thiol,--SH 10 - 11 6 - 7 ---- ----Purine ---- 2 - 4 ---- 9Phenol, ---OH ---- 10 - 12 ---- ----Pyrazine ---- ---- 1 ----Sulfoxide, --SO ---- ---- 1 - 2 ----Thiazole, ---- ---- 1 - 3 ----Amine, ---NH2, ---Nr2, Pyridine ---- ---- 8 - 11 5Imidazole ---- ---- 7Piperazine ---- ---- 10

Note a Aliph , Aliphatic Substituent(e.g., acetic acid for ---COOH)

Noteb

Arom , Aromatic Substituent(e.g.,benzoic acid for ---COOH)

Acid BaseFunctional Group

73

resulting in decreased retention. With decreasing pH bases gain a proton

and become ionized. When bases are ionized, it becomes less

hydrophobic and more hydrophilic resulting in decreased retention [Fig.

1.4.F11].

Fig. 1.4.F11: Representative chromatogram of retention factorversus pH.

Mobile phase pH should be selected so that it is atleast ± 1.5 pH units

from the analyte’s pKa. This assures that the analytes are either 100%

ionized or 100% non-ionized and helps in controlling the run-run

reproducibility.

1.4.4.9.2 Fixing of the Buffer:

Buffer imparts constant ionic strength to the mobile phase. Therefore,

it is always better to use buffer in aqueous portion of the mobile phase

for reverse phase chromatography. Buffering increases the ruggedness of

the method. Most commonly used buffers were tabulated in Table 1.4.T2.

74

Table1.4.T2: Commonly used Buffers for Reversed Phase HPLC

Buffer concentration: The concentration of the mobile phase buffer

usually has little effect on retention in reversed phase HPLC, just as long

as the buffer concentration is high enough to control pH. A buffer

concentration in the range of 25 to 50 mM is adequate for most reversed

phase applications. This concentration is also low enough to avoid

problems with precipitation when significant amounts of organic

modifiers are used in the mobile phase and, in the case of phosphate

buffers, low enough to minimize the abrasive effect on pump seals. It is

seldom advisable to use a buffer concentration less than 10 mM.

For ionic compounds which behave as highly polar and difficult to

retain by reverse phase, ion pairing chromatography can be helpful.

Negatively charged reagent such as alkyl sulfonic acid will be used to

retain positively charged ionic bases. Positively charged reagent such as

75

tetrabutyl ammonium salts will be used to retain negatively charged ionic

acids. Alkyl sulphonates are good first choice for basic compounds and

Quaternary amines are useful for acidic compounds [Fig. 1.4.F12].

Fig. 1.4.F12: The chemical structures of ion pair reagents.

1.4.4.9.3 Fixing of organic modifier:Acetonitrile and Methanol are the first choice for organic modifier.

Acetonitrile is best among the two due to low UV cutoff and Low

viscosity. Methanol is a proton donar and acetonitrile is proton acceptor

and so selectivity will be significantly different IPA & THF are other

alternates, but the THF mobile phases are not stable. For acetonitrile

mobile phases, to avoid pumping problems associated with 100%

acetonitrile use always with about 5-10% aqueous portion. An example

for the solvent strength presented in Fig. 1.4.F13.

76

Fig. 1.4.F13: Representative chromatogram on different solventstrengths.

1.4.4.10 Flow rate and column temperature:

Initial flow rate of 1.0 mL min-1 or 1.5 mL min-1 and column

temperature as ambient (25-30°C) is preferable.

1.4.4.11 Degradation studies:

Stressing the API in both solutions and in solid-state form generate

the sample that contains the products most likely to form under most

realistic storage conditions, which is in turn used to develop the stability

indicating methods. In simplest terms, the goal of the stability indicating

method is to obtain baseline resolution of all the resulting products (the

API and all the degradation products) with no co-elutions.

The degradation products generated in the stressed samples are

termed as “potential” degradation products that may or may not be

77

formed under relevant storage conditions. Below are the major forced

degradation studies:

1. Acid degradtion

2. Base degradation

3. Oxidative degradation

4. Thermolytic degradation,

5. Photolytic degradation,

Some general conditions used in conducting forced degradation studies

for drug substances are mentioned in the below table 1.4.

Table 1.4.T3: General conditions used in conducting forceddegradation

Sample condition Time of stressingSolid / 60 – 70 ºC 7 – 10 days

Solid / 60 – 70 ºC / 75% RH 7 days

Solid / simulated sunlight 1-2 weeks x ICHphoto exposure

0.1 to 2 N HCl solutions either at RT or at 60 – 70ºC 1 – 2 days

0.1 to 1 N NaOH solutions either at RT or at 60– 70ºC 1 – 2 days

Hydrogen peroxide (0.3 to 6%) at RT or at 60 – 70ºC 1 – 2 days

Solution in water 1 – 2 days

1.5 Analytical method validation process:

Validation is the process of providing documented evidence that

something does what it is intended to do. Validation is a constant,

evolving process that starts before an instrument is placed on-line and

78

continues long after method development and transfer. A well-defined

and documented validation process provided regulatory agencies with

evidence that the system and method is suitable for its intended use. By

approaching method development, optimization, and validation in a

logical, stepwise fashion, laboratory resources can be used in a more

efficient and productive manner [39-44]. The biggest advantage of

method validation is that it builds a degree of confidence, not only for the

developer but also to the user.

The USP has published specific guidelines for method validation for

compound evaluation. USP defines eight analytical performance

parameters for validation.

1) Precision

2) Accuracy

3) Limit of Detection

4) Limit of Quantitation

5) Specificity

6) Linearity and Range

7) Ruggedness

8) Robustness

1.5.1 Precision:

Precision is the measure of the degree of repeatability of an analytical

method under normal operation and is normally expressed as the

79

percent relative standard deviation for a statistically significant number

of samples.

According to the ICH, precision should be performed at three different

levels: repeatability, intermediate precision, and reproducibility.

Repeatability refers to the results of the method operating over a short

time interval under the same conditions (inter-assay precision). It should

be determined from a minimum of nine determinations covering the

specified range of the procedure (for example, three levels with three

repetitions each), or from a minimum of six determinations at 100% of

the test or target concentration. Intermediate precision refers to the

results from within-lab variations due to random events such as

differences in experimental periods, analysts, equipment, and so forth.

In determining intermediate precision, experimental design should be

analyzed so that the effects (if any) of the individual variables can be

monitored. Reproducibility refers to the results of collaborative studies

among laboratories. Documentation in support of precision studies

should include the standard deviation, relative standard deviation,

coefficient of variation, and confidence interval.

1.5.2 Accuracy:

Accuracy is the measure of exactness of an analytical method, or the

closeness of agreement between the measured value and the value that is

accepted either as a conventional, true value or an accepted reference

value. Accuracy is measured as the percentage of analyte recovered by

80

assay, by spiking samples in a blind study. For the assay of a drug

substance, accuracy measurements are obtained by comparison of the

results with those of a standard reference material, or by comparison to

a second, well-characterized method. For the assay of a drug product,

accuracy is evaluated by analyzing synthetic mixtures spiked with known

quantities of components. For the quantitation of impurities, accuracy is

determined by analyzing samples (drug substance or drug product)

spiked with known amounts of impurities.

1.5.3 Limit of Detection:The limit of detection (LOD) is defined as the lowest concentration of

an analyte in a sample that can be detected, though not necessarily

quantitated. It is a limit test that specifies whether or not an analyte is

above or below a certain value. It is expressed as a concentration at a

specified signal-to-noise ratio, usually about 2 or 3 [Fig. 1.5.F1].

1.5.4 Limit of Quantitation:

The limit of quantitation (LOQ) is defined as the lowest concentration

of an analyte in a sample that can be determined with acceptable

precision and accuracy under the stated operational conditions of the

method. Like LOD, LOQ is expressed as a concentration, with the

precision and accuracy of the measurement also reported. The signal-

to-noise ratio about 10 is considered for the determine LOQ [Fig. 1.5.F1].

It is measured by analyzing samples containing known quantities of the

81

analyte and determining the lowest level at which acceptable degrees of

accuracy and precision are attainable.

Linear regression method also can be used for the estimation of LOD

and LOQ value. For a linear calibration curve, it is assumed that the

instrument response say y is linearly related to the standard

concentration say x for a limited range of concentration. It can be

expressed as:

Y = a + b x

Where X is the explanatory variable and Y is the dependent variable.

The slope of the line is b, and a is the intercept .

This model is used to compute the sensitivity b and the LOD and

LOQ. Therefore the LOD and LOQ can be expressed as:

LOD = 3Sa

b

LOQ = 10Sa

b

Where Sa is the standard deviation of the response and b is the slope of

the calibration curve.

This method can be applied in all cases, and it is most applicable

when the analysis method does not involve background noise.

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Fig. 1.5.F1: Pictorial diagram of limit of detection and limit ofquantitation via signal to noise.

1.5.5 Specificity:

The terms selectivity and specificity are often used interchangeably.

The term specific generally refers to a method that produces a response

for a single analyte only, while the term selective refers to a method that

provides responses for a number of chemical entities that may or may

not be distinguished from each other. If the response is distinguished

from all other responses, the method is said to be selective. Since there

are very few methods that respond to only one analyte; the term

selectivity is usually more appropriate.

Specificity is the ability to measure accurately and specifically the

analyte of interest in the presence of other components that may be

expected to be present in the sample matrix. It is a measure of the

degree of interference from such things as other active ingredients,

excipients, impurities, and degradation products, ensuring that a peak

response is due only to a single component; that is, that no co-elutions

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exist. Specificity is measured and documented in a separation by the

resolution, plate count (efficiency), and tailing factor. Specificity can also

be evaluated with modern photodiode array detectors that compare

spectra collected across a peak mathematically as an indication of peak

homogeneity. Sample to be diluted and to be used for the accurate

estimation of peak purity, if concentration over ranges.

1.5.6 Linearity and Range:

Linearity is the ability of the method to elicit test results that are

directly proportional to analyte concentration within a given range.

Linearity is generally reported as the variance of the slope of the

regression line. Range is the interval between the upper and lower levels

of analyte that have been demonstrated to be determined with precision,

accuracy, and linearity using the method. The range is normally

expressed in the same units as the test results obtained by the method.

The ICH guidelines specify a minimum of five concentration levels,

along with certain minimum specified ranges. For assay tests, the

minimum specified range is 80-120% of the target concentration. For

impurity tests, the minimum range is from the reporting level of each

impurity to 120% of the specification. (For toxic or potent impurities, the

range should be commensurate with the controlled level.)

1.5.7 Ruggedness:

Ruggedness, according to the USP, is the degree of reproducibility of

the results obtained under a variety of conditions, expressed as %

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relative standard deviation (RSD). These conditions include differences

in laboratories, analysts, instruments, reagents, and experimental

periods. In the guideline on definitions and terminology, the ICH does

not address ruggedness specifically. This apparent omission is really a

matter of semantics, however, as ICH chooses instead to cover the topic

of ruggedness as part of precision.

1.5.8 Robustness:

Robustness is the capacity of a method to remain unaffected by small

deliberate variations in method parameters. The robustness of a method

is evaluated by varying method parameters such as percent organic

solvent, pH, ionic strength, or temperature, and determining the effect (if

any) on the results of the method. As documented in the ICH guidelines,

robustness should be considered early in the development of a method.

The variations such as stability of analytical solutions, different

equipment and different analysts should be studied. In addition, if the

results of a method or other measurements are susceptible to variations

in method parameters, these parameters should be adequately controlled

and a precautionary statement included in the method documentation.

1.6 Role of Mass Balance during stability indicating analytical

method (SIAM) validation:

Mass balance correlates the measured loss of a parent drug to the

measured increase in the amount of degradation products. It is a good

quality control check on analytical methods to show that all degradation

85

products are adequately detected and do not interfere with quantitation

of the parent drug (i.e Stability–indicating methods). In mass balance

calculations, the loss of parent drug or the amount of drug remaining is

determined from a sample assay, and the measured increase in

degradation products is determined by a related substances method. The

fundamental approach for determining mass balance is to quantitate the

decomposition peaks using degradation methods and then reconcile the

measured loss in the parent drug with the amount of degradation

products. If the loss in potency can be reasonably accounted for by the

amount of degradation products measured, then mass balance is

achieved.

The assessment of degradation in pharmaceutical products involves

two aspects of analytical measurement, firstly, a specific or selective

analytical method must be available for accurate assay of parent drug

compound, in-order to measure any loss, second and methodology

should be in place for the quantification of the degradation products

formed. Ideally, when degradation occurs, the measured amount of

parent drug lost should correlate well with the measured increase in

degradation products. This correlation is referred to as “Mass balance”

More recently, the ICH has provided definition of “mass balance”;

material balance” as follows.

The process of adding together the assay value and levels of

degradation products to see how closely these add up to 100% of initial

86

value, with the consideration of the margin of analytical precision. The

concept is useful scientific guide for evaluating data, but it is not

achievable in all circumstances. The focus may instead be on assuring

the specificity of the assay, the completeness of the investigation of route

of degradation, and the use, if necessary, of identified degradants as

indicators of the extent of degradation via particular mechanism.

Mass balance is also useful in method validation. In order to

demonstrate that analytical methods are stability-indicating, unstressed

and stressed materials are often compared. Any increase in degradation

a product that correlates well with loss of parent drug, aids in

demonstrating that the methods can accurately assess degradation.

Stability studies are used to establish the re-test period for the active

ingredient-that is the length of time it can be stored and used without

analyzing immediately before use and the shelf life of the finished

product. The release and shelf life specifications for the product may

differ to accommodate degradation of active ingredient or other

acceptable changes, which may occur on storage. The ICH drug stability

test guideline Q1A (R2) requires that analysis of stability samples should

be done through the use of validated stability-indicating analytical

methods (SIAMs). Additional guidance is given only for photo stability

testing. It also recommends carrying out the stress testing on drug

substance to establish its inherent stability characteristics and to

support the suitability of proposed analytical procedure.

87

1.7 Scope and Objective of research work:

The significance of drug evaluation and impurity profiling in the

pharmaceutical world has been the driving force for the current research

work and this work unravels the usage of the chromatographic

techniques for effective impurity estimation and thus developing unique,

sensitive, rapid and efficient methods with a special focus on the method

validation. The present research work thus focuses on the development

of new stability-indicating analytical methods for some of the active

pharmaceutical ingredients and the development of new chiral hplc

methods for API and key starting raw material (KSM). The work also

includes the validation of the developed methods as per ICH

requirements and demonstrates the suitability of developed methods to

assess the stability of API (Table1.7.T1).

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Table 1.7.T1: The list of Active pharmaceutical compounds (API) andits key starting raw material (KSM) taken for research study.

S.No. API/KSMchemical name

Structure Therapeutic activity

1.0 (S)-1-[N-(3-hydroxy-1-adamantyl)glycyl]pyrrolidine-2-carbonitrile

Anti-diabetic drug.

2.0 7-[(3R)-3-amino-1-oxo-4-(2,4,5-trifluorophenyl)butyl]-5,6,7,8-tetrahydro-3-(trifluoromethyl)-1,2,4-triazolo[4,3-a]pyrazine phosphatemonohydrate

An oral anti-diabetic, for theimprovement ofglycemic control inpatients with type IIdiabetes mellitus.

3.0 N,N-dimethylimido-dicarbonimidicdiamidehydrochloride

Anti-diabetic agent.

4.0 (S)-2-azido-3-methylbutanoic acid

Key starting rawmaterial (KSM) ofvalganciclovir HCl,an antiviral drug.

5.0 2'-deoxy-2',2'-difluorocytidinemonohydrochloride

Chemotherapy drug

New chiral HPLC and UPLC stability–indicating analytical methods

were developed for above API and key starting raw material. The

developed methods can be successfully implemented during the quality

89

monitoring and also well employed for the assessment of quality during

its storage and stability.

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