INTRODUCTION

gntrod'uctton

In the rapidly changing scenario of infectious and non-infectious diseases; new

and old diseases existing together with drug resistant strains of certain microganisms

coming to the fore; the need for better drugs with identified and specific site of action

has stirred researchers all around. Researchers are working around the globe to find

better cures and means of prevention of diseases. Biologists are looking for target

sites to design blockers, which will prevent the expressions of certain disease causing

genes. New techniques are being introduced every now and then, which provide fast

and result oriented synthesis and screening of compounds for their activity. Similarly

in the field of analysis, due to rapid advancement in technology, better techniques

with improved precision and accuracy are regularly being used. Techniques like

HPLC, HPTLC, DSC and UV -spetrophotometry are the choice for analysis.

There are various approaches, which a chemist adopts to synthesize a new

compound. The most common is to generate a lead compound from the other known

drugs for certain disease and to synthesize its analogs by altering its substituents.

Other than this, chemists take certain active groups and generate a series of

compounds. Then screening of the compounds is carried out to ascertain their primary

activity.

When the compound is found active for certain disease, several studies are

needed prior to toxicological and other pharmacological studies and developing a

formulation of the drug that is to be administered. Various physicochemical

parameters are to be developed and ascertained for a candidate drug. Prior to this a

validated method of analysis is required. Guidelines are available from ICH to

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harmonize and set a uniform standard of method validation and quantitation. After the

method is developed other pre-formulation studies are carried out.

The pre-formulation studies include: -

1. Establishment of physical parameters

11. Development of validated estimation procedure including impurity profile.

111. Isolation and purification of degradation products (if possible).

1v. Stability studies such as

)> Effect of temperature,

)> Effect of moisture,

)> Effect of change of pH ,

)> Effect of UV light on the compound

v. Formulation suggestion of candidate drug.

1.1. Preformulation studies- Preformulation studies [1-1 0] are an integral part of

drug development process. Any active compound has to undergo certain stages

of rigorous experiments including purity, stability and toxicity before it could be

approved for animal studies. All these studies play a very important role in

designing the formulation and deciding the route of administration of drug.

Many workers like G Jr Regdon et.al.[1], TX Viegas et.al.[2], PE Luner et.al[3],

NK Ebube et.al.[4], S Li et.al[5], S Tenjarla et.al[6], A Hatem et.al.[7], C

Graffner et.al. [8] have conducted and published works on various

preformulation studies.

1.1.1. Purity: The purity of drug substance plays the most significant role in all

studies carried out on it. For every new compound, depending on its dose and

toxicity, the limit of impurity is defined. Until and unless the purity ofthe drug

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is assured other studies like stability, degradation and toxicity cannot be

performed. Various parameters, which are considered to find the purity of the

drug substance, are

};> Melting point- Pure organic substance show a characteristic melting point.

Slight impurities can cause depression in melting point. It is a primary

indication of purity. (Figure- I)

};> UV absorption- The measurement of light absorption in the Visible and

UV range is now widely used as a means of identification. Samples with

chromophores in them give absorption at characteristic wavelength 'A,, which

in presence of impurity may get altered. Hence this can serve as a tool to

establish purity of the drug substance.

};> IR spectra- Comparing the IR spectrum of sample with that of standard

can give valuable information for identification of the drug substance.

};> TLC- more rapid and more sensitive technique, TLC forms the basis of a

number of important identity tests for drug substances in dosage forms. This

technique is still widely used as the basis of various important limit tests.

};> Quantitative determinations- Quantitative determinations of impurities is

done to check each batch for limit of impurities etc. the various parameters

checked are

•!• Limit of insoluble matter

•!• Limit of soluble matter

•!• Limits of moisture, volatile matter and residual solvents.

•!• Limit of nonvolatile matter

•!• Limit of residue on ignition

•!• Loss on ignition

•!• Ash values

[ 4]

dh dt

Primary Standard (NBS)

Temperature ~

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Fig. 1: Effect of Purity on Melting Endotherm of Benzoic Acid

L1H

2

TG TA

Temperature ~

Fig. 2: Example of DSC. 1, Endotherm, 2. Exotherm, Shaded area of peak is proportional to L1H

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• Detection of impurities- Both TLC and HPLC are used for detection of

impurities. Now a days these are used for identification and quantification of

impurities which are often very closely related in structure to the main compound

of interest. [11]

1.1.2. Thermal analysis - When a compound is subjected to temperature change, it

undergoes several transitions like glassy transition, melting, polymerisation and/or

decomposition. The Digital scanning calorimeter is used for studying the thermal

changes occurring in the compound due its exposure to high temperature. The

enthalpy of fusion, which is characteristics for pure substances used either as drugs or

formulation additives, may be calculated from the corresponding first order transition

in DSC thermograms. (Figure-2) Other first-order transitions, such as those

corresponding to evaporation and/or condensation of solvents present in solvated

crystals, polymorphic changes (solid-solid transitions), enantiotropic transitions and

monotropic transitions give valuable information about the behaviors of the drug

under conditions of storage and use. Second order transitions include Glass transitions

for amorphous substances, Electrical and curie transitions, and Lambda transitions.

Also purity analyses by DSC can be done on the basis ofVan't Hoff's law of melting

point depression.

Tr= To- X2 R*T02/~Hr

where T r = equilibrium melting temperature during fusion, T 0 = fusion temperature of

pure substance, x2 = mole fraction of impurities, R = gas constant, ~Hr = heat of

fusion of pure substance.

The molecular state of a molecule in a binary solid mixture has in recent years

been eluCidated mostly by use of thermal methods, in particular DSC and DT A. In

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DT A the temperature difference is measured between a sample and a reference

substance being heated together by a thermocouple. As the materials are heated, a

signal is observed only when the substance shows any thermal change. For an

endothermic change the temperature of the sample will become lower than the

reference, thus !}. T is plotted vs time and a curve is obtained. In DSC, the apparatus is

such that it records the energy necessary to establish 0 temperature difference between

a substance and a reference sample and in this case the thermogram will be !}.H vs !}.T.

The heat (enthalpy), !}.H cal, associated with the change is proportional to the area

under the curve. [12-19]

1.1.3. Solubility- Solubility is one of the primary physical properties to be studied for

any compound, as it also plays an important role in the development of analytical

method and its formulation. The solubility of a compound depends upon the physical

and chemical properties of the solute and the solvent as well as upon such factors as

temperature, pressure, pH of the solution and to lesser extent the state of subdivision

Table: Terms of approximate solubility

Term Parts of solvent required for 1 part of solute

Very soluble Less than 1 part

Freely soluble 1 to 10

Soluble 10 to 30

Sparingly soluble 30 to 100

Slightly soluble 100 to 1000

Very slightly soluble 1000 to 10,000

Practically insoluble More than 10,000

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of the solute. The solubility of a drug can be expressed in many ways, the official

way, solubility of drug is given as the number of milliliters of solvent in which 1 gm

of solute will dissolve. Solubility is also quantitatively expressed in terms of molality,

molarity and percentage. For substances whose solubility is not definitely known, the

values are described by the use of certain general terms (Pharmaceutical compendia,

Table 1).

So~vent-solute interaction- The solubility of the compound in a particular solvent

depends upon its interaction with solvent on molecular level. It is a well known fact

that like dissolves like that is polar compounds and ionic salts dissolve in water easily,

whereas nonpolar, nonionic compounds dissolve in non polar organic solvents. This

can be further explained as the solubility of a drug is due to polarity of the solvent

(dipole moment). Also, another important factor in solubilisation of polar substance is

the formation of hydrogen bonds in protonated solvents. The solvent action of

nonpolar liquids differs from that of polar solvents. Only nonpolar compounds get

dissolved in nonpolar solvents through induced dipole interaction. Weak Van der

waal: london type forces are responsible for holding solute and solvent together.

1.1.4. Dissociation Constant (pKa) - Most of the drugs are either weak acids or

weak bases and according to the pH-partition hypothesis, unionized form of a drug

will be absorbed preferentially in a passive manner through a membrane. Similarly

weakly acidic drugs will be absorbed from acidic environment, i.e. stomach and

duodenum; where as weakly basic drug will be preferentially absorbed from alkaline

environment i.e. intestine. The effect of the ionization of weakly acidic or basic drug

and their salt is studied since drugs dissociates in adverse conditions. Ka is referred to

as ionization, dissociation or acidity constant for weak acid.

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Ka = aH30+ * a A- I aHA

Assuming the activity coefficients approach unity in dilute solution, the activities may

be replaced by concentrations:

Ka = [H30+] [K] I [HA]

The negative log ofKa is known as pKa (pKb-for weak base).

pKa =-logKa Or pKb = -logKb

These values provide a convenient means of comparing the strength of weak acid and

bases. The lower the pKa the stronger the acid, the lower the pKb the stronger the

base. The pKa and pKb are related by the equation -

pKa + pKb = pKw

This parameter is very important to know if spectra of pure species is to be recorded

in solution; if effects of pH changes on physical properties are to be interpreted.

Ideally, the dosage should be designed in accordance with the variation in pH of

different segments of gastro-intestinal tract.

1.1.5. Partition Coefficient (log P)

If a liquid or solid is added to a mixture of two immiscible liquids, it gets

distributed between the two phases in a definite concentration ratio. If C 1 and C2 are

the equilibrium concentrations of the substance in solvent 1 and solvent 2, the

equilibrium expression becomes:

Cl/C2 =K

where, the constant K is known as the distribution ratio, distribution coefficient or

partition coefficient. The principle of partition is important for a drug compound as it

is involved in several of the applications. These include formation of emulsions of oil

and water, drug action at nonspecific sites, and the absorption and distribution of

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drugs throughout the body. The solute may exist as associated molecule in one of the

phases or it may dissociate into ions in either of the liquid phases. But the law of

distribution applies to the species common to both the phases that is the monomer or

the simple molecules of the solute e.g. when benzoic acid is added to oil and water, it

gets distributed between the two. In oil phase it associates to form dimer whereas in

water phase it ionises. In lab conditions, octanol approximates the physico-chemical

properties of biological lipids and it is also insoluble in water. Studies on the

partitioning of candidate drug between octanol and water help in predicting directly

the interaction of the compound with the cell membrane. These results help to decide

the route of administration and design the formulation. [20,21]

Applications of partition coefficient:

• Extraction- in order to determine the efficiency with which one solvent can extract

a compound from a solution of another solvent.

• Solubility- Aqueous solubility of non-electrolytes has been correlated to

partitioning (Hansch et.al). To determine the aqueous solubility of liquid or

crystalline organic compounds workers like Y alkowsky and Valvani have put

forward an equation[22].

LogS= -log K- 1.11 Sr(mp-25)11364 + 0.54

when experimental values are not available, group contribution methods are used

for estimating partition coefficient of the compounds.

• Preservative action of weak acids in oil/water systems: solutions of food, drugs

and cosmetics are susceptible to enzymatic degradation by microorganisms. Thus,

protection of these are important from commercial point of view.

[ 10]

• Drug action and partitioning- the hypothesis that narcotic action of nonspecific

drug is a function of distribution coefficient of the compound between lipid and

water, was given by Meyer and Overton. Later it was proved and was similar to

theory given by Ferguson. For eg: partition coefficient of ethanol, n-propanol, n-

butanol and thymol are compared and found to be of same order, thus expected to

show same action. However, drugs with extremely high or extremely low partition

coefficient are less effective or ineffective. The reason is that highly oil soluble

drugs though cross the membranes but are unable to proceed to target; whereas

highly water soluble drugs are unable to cross the lipid membrane. A parabolic

relation is obtained, when Log partition coefficient is plotted against Log activity.

(Fig. 3 & 4).

10

8

X ::J 6 LL

~ ~ 4

2

0-3 -2 -1 0 1 2 3 4

log P ----.

Fig. 3: Maximum Penetrant Flux as a function of Oct/Water Partition Coefficient

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u -0 E J

0

0> -1 0

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log P ---+

Fig. 4: Log Jm + Maximum Flux as a Function of log P C = Minimum cone. of Nicotinic acid required to cause

vasodilation by Transdermal Delivery from Aq. solution

The oil/water partition coefficient is an indication of lypophilic or hydrophilic

character of a drug molecule. Passage of drugs through lipid membrane and

interaction with macromolecules at receptor sites sometimes correlates well with the

octanol/water partition coefficient of the drug. [23]

1.1.6. Stability Studies: The stability studies are done to determine shelf-life, and co-

related specifications, and it must take into account the chemistry of active ingredient

and its likely vulnerability to degrade by oxidation, hydrolysis,· isomerisation,

polymerisation, decarboxylation, moisture, heat and light [24]. Properly conducted

stability study must also include an examination of specific decomposition products

by appropriate techniques to establish identity and relative toxicity of the

decomposition products and the concentrations in which they are formed.

Stability studies should not only take account of the physical state in which the

compound is likely to be used, but also the immediate biological environment likely

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to be met on administration. Thus, substance for tabletting, encapsulation and

preparation of suspension, should be examined primarily in solid state. Substances for

injection, which must be subjected to some form of sterilization procedure, must be

examined particularly for stability at elevated temperature for possible hydrolysis or

rearrangement in aq. Media and effects of exposure to C02 and light. Similarly all

substances intended for oral administration must be chemically stable to the pH and

enzymatic conditions likely to be met in the gastro-intestinal tract.

Hence, stability studies must be conducted on the drug substance in the solid

state over a range of temperature, at varying degrees of humidity, and in both light

and dark. Also, if a product is to be used in multiple dose form in the tropics with

fluctuation in temperature, which should be stored ideally in cool or refrigerated

conditions, then stability tests should include a study of the effects of fluctuating

temperature. [25-43]

Rate Kinetics: rate process are of fundamental concern to everyone connected with

pharmaceutics, from manufacturer to patient. At the manufacture stage, it must be

clearly shown that the drug (dosage) produced is sufficiently stable for storage for a

reasonable time without degradation. The rate of reaction is given by

Rate= dC/dt

Where C is concentration and t is time.

1.1.6.1. Effect of temperature on the compound (temperature stability) - It is

carried out during storage testing and is of particular importance for products destined

to be used in tropical countries or which are subjected to heat sterilization. This tells

about the shelf life of the compound and its half-life at the required temperature.

These can be calculated from Arrhenius equation which is-

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Log k = log A - Ea I 2.303 R T

"There- A= frequency factor; Ea =activation energy; k =stability constant; R =gas

constant; T = temperature (in kelvin scale). According to this equation plot of log k as

a function of reciprocal temperature should be linear with a slope of(- Ea I 2.303 R)

from which activation energy can be calculated.(Figure5&6)

~~0 40°C (!)

80 so0c 70 ::::> 60 e::: 0 50 _J 40 <( 60°C ::::> 30 0 (f) w e::: 20 ~ 0

10 0 7 14 21 28 35

TIME (Days) • Fig. 5: Residual Doxycycline Hyc. at Different Temperatures and time intervals

1000

100 '<;!"

0 --><

I 40°C (f)

~ \ 0

~ 10 . \ \

\ \

\ 25°C

2.9 3.0 3.1 3.2 3.3 3.4

1/TX 103

Fig. 6: Arrhenius Plot for Doxycycline Hy. to Find kat 25°C

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The accelerated stability studies are conducted-

+ To determine the shelflife/halflife ofthe product.

+ To find the most suitable conditions for formulation development.

+ To suggest the storage and transportation conditions.

+ To give the rate of reaction and stability constants.

Although shelf lives can be estimated in accelerated studies, it is noted that

expiration dates cannot be estimated from accelerated data, it can only be determined

on the basis of noting down the real time degradation pattern. From the analytical

chemist's point of view as well as of product formulator, there are a few more

advantages to accelerated stability studies. It obviously can serve for screening

purposes in preformulation studies, [ 44] as a guide in formulation selection in initial

development, and for supporting evidence of physical equivalence of new/ revised

formula/ procedure changes that would be needed for new drug application.

Further reasons for accelerated studies are:

a) They serve as early warnings of unsuspected decomposition paths in early and

middle development phases.

b) They present the analytical chemist with stressed systems having products that

may interfere with a stability-indicating assay.[45-50]

1.1.6.2. Effect of moisture (humidity stability) - During manufacture, storage and

transport of the compound (drug), its protection from moisture is necessary as it may

lead to decomposition of the drug and the impurity may be harmful or toxic; if taken

as such. The single most important cause of loss of potency of a drug substance is the

presence of moisture. It can be present as surface moisture, which dissolves the drug

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to the extent of its solubility S, if the moisture is abundant (water is in excess) then

rate of decomposition can be pseudo first order. Some workers demonstrated that

extra moisture decreased the lag time and increased the zero order rate constant for

the decomposition of aminosalicylic acid. Others showed a linear relationship

between log k and the relative humidity. When moisture content is very high,

decomposition of solid dosage form may be treated as kinetics of saturated solution.

In such cases it follows zero order kinetics. It is seen that for 'C= Co- ko nt', when

weight is plotted vs time, a straight line results with time. The slope is independent of

C0, as opposed to the reactions of l51 order, in such cases it follows zero order kinetics.

But in other cases the effect of humidity follows first order kinetics. Hence it becomes

necessary to study the effect of moisture on the compound (Figure7&8) [51-53].

Drugs in solid dosage forms decompose much more slowly than the same in

suspensions or in solution form. The reason for this is that hydrolysis is a predominant

decomposition route, and thus solution dosage forms constitute a system much more

prone to hydrolysis because of abundance of water. Secondly the molecules in solids

are fairly fixed in space positionwise whereas in solution and suspension they are

subject to random movement, so that the interaction possibilities are greater in

solution than in solids. Moreover, dissolved oxygen in solution is readily available for

reaction, whereas in solid phase, only surface oxidation with gaseous oxygen occurs

and thus internal molecules are protected.

Assays of drugs in solid dosage form are less precise than in solutions because

of the heterogeneity of the dosage form. The problem also exists that the

decomposition is not exactly same in each unit, so that the highly precise

decomposition kinetics often encountered in solution system can never be found in

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tf ' 0%:

--- A:t

r fl"" .. 4t

~ a J :.2. d. Q)

t: l 0

00 <2.o 4.o c:o !O too Rfi:t.A i"t v Cii HllMt~.T~ "·

Fig. 7: Effect of Temperature on Water Content of Ranitidine HCI in 2 hrs

100 50%

so

oJ1 '0 4>. :2: 85"/o z < 4o

~ 2.0·

~

() 0 .2.00 4c)O (nO t.oo IOOO

liMti!.,""" -+

Fig. 8: Effect of RH on Degradation of Ranitidine at 45°C

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case of solid dosage forms. Whereas the kinetics in solution might at times be carried

out through many half-lives and assigned fractional reaction orders, a solid dosage

form is rarely studied beyond one half-life, and can only be distinguished between

first and zero order decomposition patterns. Frequently the decomposition is a

hydrolysis, where water is used up as the drug degrades for eg: thiamine contains an

ethylene bridge (R-CH2-R') and hydrolyses to RCH20H + R'H, such that one mole of

water is consumed per 1 mole of thiamine decomposition. In most cases if hydrolysis

alone is occurring and free moisture is not abundant, the potency drops down to a

level corresponding to that of water, and then stop. But often the hydrolysis is

accompanied by other parallel decomposition reaction.[54-69]

1.1.6.3. Effect of UV light on the compound -The prediction of decomposition rate

from experiments in which the effect of light is exaggerated; is conducted to find the

relation between the flux of light and the degradation rate. This has gained importance

in recent years because of the complex chemical structure of many new drugs.

Degradative reactions, such as oxidation-reduction, ring rearrangement, or

modification and polymerization can occur within the molecule by exposure to light

especially by UV light as they have short wavelength and higher energy. This

phenomenon is very common in the drugs, which have labile or photosensitive groups

in them. For such compounds it becomes mandatory to protect them from direct or

indirect exposure of light. [70-83]

1.1.6.4. Effect of pH (stability of compound at different pH)- the compound tends to

degrade by various reaction mechanisms when it comes in contact with buffers of

different pH. The stability of a drug with respect to different pH is important in

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. 2 pH ~ ,.o ~ ,.qq 7·0

i \·q8 S'"-0 t.·o

A \· q "l !·O ~ ,.q, 2.·0 ~ \·~S' e-o ~ C\·0 s \·~~

lo•O \·ql

0 l TtME

Fig. 9: Effect of Different pH on Butaperazine Dimaleate

Fig. 10: Plot of log k vs pH to find pH of maximum stability

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designing the formulation and route of administration. This is very important as

different parts in the body have different pH environments and the compound has to

be stable in that pH to be effective. For oral dosage forms, the drug comes in contact

with highly acidic pH in stomach to neutral to basic pH in the intestine. The

degradation of compound may vary with different H+ ion concentration in the

solution. Also due to different mechanism of degradation the final products could be

totally different. This study also becomes important to find the pH of maximum

stability, which can be very useful in designing delivery system and route of

administration (Figure9& 1 0). [84-93]

1.1.6.5. Dosage form: in the formulation, all constituents, active ingredients and

excipients must conform to a fully stated specification, pharmacopoeial or otherwise

appropriate to their use in human medicine. Few exemptions from licensing of

medicines are allowed in respect of herbal remedies. The choice of excipients should

be carefully considered and be capable of rationale. Ideally, these should be limited to

the minimum to ensure uniformity of dosage and stability throughout the period of

proposed shelf-life of the product.

There are various works reported on interactions between active medicaments

and excipients in formulations [94-103]. The rationale approaches to the excipients

choice as well as to their interactions with medicaments have been shown as a basis

for modem modeling of pharmaceutical formulations. The importance of

complexation, hydrogen bonding, ion-dipole, dipole-dipole and van der Waals

attractions as the tools, which can modify the physicochemical, pharmacological or

pharmacokinetical behavior of medicaments, has been emphasized. In vivo and in

vitro studies as well as studies of chemical stability and bioavailability also serve as a

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proof of these interactions. Therefore excipients are important components of

pharmaceutical formulations and they can take an active part in the improvement in

the characteristics of formulations. Several studies have investigated the mechanism

of dissolution from basic, acidic drug -excipient combination [ 104]. The early

prediction of drug-excipient incompatibility is vital in the pharmaceutical industry to

avoid costly material wastage and time delays. In particular, the low availability of

drug and the time constraints associated with the early stages of formulation

development have made such predictions desirable. Thus choice of technique 1s ,!((~\~~-; r.-.;

mainly DSC for conducting interaction studies [ 105,1 06]. (('~~ . ~i

TH- I o 7- fJ-2_ \:~ . . ·:;/ There is also increasing interest in optimizing the efficacy of drug activity through the \::~~:>~, .···.-~ .. -

·· .. ,::.."~:::·/ ~.:· ;: --._...:__

use of rationally designed drug carrier materials. Cyclodextrins { CDs} are strong

candidates for a role, modifying physical, chemical and biological properties of drug

molecules through the formation of inclusion complexes. The hydrophilic CDs are

used to enhance the solubility and dissolution rate of poorly water-soluble drugs,

whereas hydrophobic CDs are used to slow the dissolution rate of water-soluble

drugs. Advanced controlled release can be achieved by a combination of the two.

When CDs are used to solubilize water insoluble drugs, it is assumed that inclusion

complexes are formed. That is the lypophilic water-insoluble drug molecules, or some

lypophilic moieties of the drug molecule, are taken into the hydrophobic central

cavity of the water-soluble CDs. Linear phase-solubility diagrams {solubility vs CDs

concentration} are usually assumed to indicate formation of 1:1 Drug/CDs inclusion

complexes or first order with respect to CDs. Positive deviation from linearity is

thought to indicate higher-order inclusion complexes such as 1 :2 drug/CyDs

complexes.[107-111]

r? 1 1

The choice of container and pack should reflect the stability characteristics of

the product. Light sensitive materials should be packed to exclude light; for eg: tablets

in metal foil strip packs; amber glass is no longer considered effective. Thus

injectibles or other liquid preparations should be packed in clear glass vessels, if

necessary wrapped individually in foil, but otherwise in packages capable of

excluding light. The type of container and its nature (material of container) should be

carefully selected according to the drug or active ingredient.

Considering the important role of physicochemical parameters mentioned

above, the present investigations were undertaken to develop the same for selected

CDRI compounds. The model candidate drugs chosen for this purpose were CDRI

compound no. 93/478, 97/78, Sapindus saponin and Aabulaquine- antimalarial kit

developed by CDRI.

The present studies have been described under-

(i) Sapindus saponin (Spermicidal agent)- isolation of marker,

fingerprinting of the extract and development of validated method of

estimation.

(ii) CDRI no. 93/478 (Anti ischemic & Anti hypertensive agent)­

physicochemical parameters, stability studies and methods of estimation.

(iii) Aabulaquine [Anti-malarial combination kit containing Chloroquine

and Bulaquine (CDRI no. 80/53)]- development of validated HPLC and

HPTLC methods for estimation.

(iv) CDRI no. 97/63 & 97178 (Anti malarial trioxane derivative)­

physicochemical parameters, stability studies and method of estimation.

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These studies and their results will play a determining role in the formulation

development, quality control assessment and specification of storage conditions of the

compounds and their formulations in the long run. Before giving an account of the

work done, it may be of interest to review the guidelines given by ICH for method

development and validation, and also to introduce various analytical techniques used

for analysis.

1.2. Method development and validation

1.2.1. Strategy for developing an analytical procedure by HPLC

The first step of developing chromatographic analysis is to define the problem and

state the purpose of analysis. In order to define the problem, the following questions

should be asked,

~ Is the analysis going to be used routinely for a large number of samples?

~ Is a qualitative or quantitative analysis required?

~ Is it necessary to separate all the constituents in the sample or a small group?

~ Are the constituents similar in structure or widely diverse?

~ Are the constituents present in similar concentration or is one constituent present

in a large amount and other in trace amount?

1.2.1.1. Conditions for high resolution

1. Stationary phase- There are four important parameters involved in the

stationary phase that can be varied; Column length and internal diameter,

Particle size, Type of bonded phase and Surface coverage. These are adjusted

and best suitable column is used.

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2. Mobile phase- Before considering any solvent as eluent, sufficient solubility

of solute in that solvent must be ensured. If the sample to be analyzed contains

very complex or a mixture of compounds of diverse structure & retention

behavior, then gradient elution is preferred. In some cased buffer solution can

be used at different pH, ionic strength. Methanol and acetonitrile are the

widely chosen organic modifier.

3. Mode of elution and flow rate- Whenever possible, isocratic elution should be

used because it eliminates turn around time on the column and shortens the

analysis time. Retention reproducibility is more predictable with isocratic

elution because equilibrium of the column is unchanged. But when adequate

resolution cannot be achieved within a reasonable length of time then gradient

elution is advisable, which can be stepwise or continuous. Flow rate may be

increased within specified limit.

4. Optimization of an analysis- In solving a general elution problem compromise

may be necessary among the goals of optimization of analysis time, resolution,

detection sensitivity. Some times optimization of one of these parameters is

made at expense of one or more of the other parameters.

At this stage validation of the method must be done to ensure the reproducibility

and accuracy ofthe method [108-111].

1.2.2. Validation of analytical procedure [112-114]-

1. Specificity- An investigation of specificity should be conducted as a primary

step during the validation of identification tests, the determination of

impurities and the assay. Suitable identification tests should be able to

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discriminate between compounds of closely related structures, which are likely

to be present. It may be confirmed by comparing a known reference material

with samples containing the analyte, coupled with samples, which do not

contain the analyte.

For critical separations, specificity can be demonstrated by the

resolution of the two components, which elute closest to each other, in the

presence of impurities and/or excipients. The approach is similar for both

assay and impurity tests. This can be done by spiking pure substances with

appropriate levels of impurities and/or excipients and demonstrating that the

result is unaffected by the presence of these materials by comparing with the

result obtained on unspiked samples.

2. Detection Limit- Several approaches for determining the detection limit are

defined, depending on whether the procedure is a non-instrumental or

instrumental.

Based on Signal-to-Noise: This approach can only be applied to analytical

procedures, which exhibit baseline noise. Determination of ratio is performed

by comparing measured signals of samples with known low concentrations of

analyte with those of blank samples and establishing the minimum

concentration at which the analyte can be detected. A signal-to-noise ratio

between 3: 1 is generally considered acceptable for estimating the detection

limit.

Based on the Calibration Curve: A specific calibration curve should be studied

using samples containing an analyte in the range of DL. In cases where an

estimated value for the detection limit is obtained by calculation or

[ 25]

qntroduction

extrapolation, this estimate may subsequently be validated by the independent

analysis of a suitable number of samples known to be near or prepared at the

detection limit.

3. Quantitation Limit- Similar approach is used for determining the quantitation

limit, depending on whether the procedure is a non-instrumental or

instrumental.

Based on Signal-to-Noise Approach: By comparing measured signals from

samples with known low concentrations of analyte with those of blank

samples and by establishing the minimum concentration at which the analyte

can be quantified with acceptable accuracy and precision. A typical signal-to­

noise ratio is 10:1.

Based on the Calibration Curve: A specific calibration curve should be studied

using samples, containing an analyte in the range of QL. The residual standard

deviation of a regression line or the standard deviation of y-intercepts of

regression lines may be used as the standard deviation.

4. Linearity- A linear relationship should be evaluated across the range of the

analytical procedure. It may be done directly on the active substance by

dilution of a standard stock solution and/or by separate weighing of synthetic

mixtures of the product, using the proposed procedure. Linearity should be

evaluated by appropriate statistical methods of a plot of signals as a function

of analyte concentration, for example, by calculating a regression line by the

method of least squares. A plot of the data, correlation coefficient, y-intercept,

slope of the regression line and residual sum of squares should be given. For

linearity, a minimum of 5 concentrations is recommended.

[ 26]

qntrod'uction

5. Range- The specified range is normally derived from linearity studies and

depends on the intended application of the procedure. It is established by

confirming that the analytical procedure provides an acceptable degree of

linearity, accuracy and precision when applied to samples containing amounts

of analyte within or at the extremes of the specified range of the analytical

procedure.

6. Accuracy- Accuracy should be established across the specified range of the

analytical procedure. By applying an analytical procedure to reference

material; and or comparing results of the proposed analytical procedure with

another procedure. Accuracy may be inferred once precision, linearity and

specificity have been established. In cases where it is impossible to obtain

samples of certain impurities and/or degradation products, it is acceptable to

compare results of an independent procedure, but determination of impurities

e.g., weight/weight or area percent, with respect to the major analyte should be

clear. Accuracy should be assessed using a minimum of 9 determinations over

a minimum of 3 concentration levels covering the specified range (e.g. 3

concentrations/ 3 replicates each of the total analytical procedure).

7. Precision- Validation of tests for assay and for quantitative determination of

impurities includes an investigation of precision. Repeatability should be

assessed using a minimum of 9 determinations covering the specified range

for the procedure (e.g. 3 concentrations/ 3 replicates each) or a minimum of 6

determinations at 1 00% of the test concentration. Reproducibility is assessed

by means of an inter-laboratory trial, especially in case of the standardization

of an analytical procedure for inclusion of procedures in pharmacopoeias. The

[ 27]

qntroduction

SD, RSD (coefficient of variation) should be reported for each type of

precision investigated.

8. Robustness- The evaluation of robustness can be done during the

development phase, such that to show the reliability of an analysis with

deliberate variations in method parameters. If measurements are susceptible to

variations in analytical conditions, the analytical conditions should be suitably

controlled or a precautionary statement should be included in the procedure.

Typical variations which can be done are: stability of analytical solutions,

extraction time, influence of variations of pH in a mobile phase, influence of

variations in mobile phase composition, different columns (different lots

and/or suppliers), temperature, flow rate.

9. System Suitability Testing- The tests are based on the concept that the

equipment, electronics, analytical operations and samples to be analysed

constitute an integral system that can be evaluated as such. System suitability

test parameters to be established for a particular procedure depend on the type

of procedure being validated.

1.2.3. Quantitation of analysis-

Quantitation means different things to different analyst. At times, quantitation

has been defined as relative or percent area of individual peaks in a chromatogram, all

summing to 100%. Quantitation also has been defined as showing that a substance is

present at or above a certain threshold level. In reality, true quantitation relates to an

ability to demonstrate exactly how much of a particular analyte is present in a

particular sample. It means determining the concentration present in ppm, mcg/mL,

M, or other terms that de-note the amount of material (mass) in a given amount of the

[ 28]

qntroauction

original sample (or volume of solution). Many chromatographic methods for

pharmaceutical analysis must demonstrate the quantity of the analytes present per

gram of original solid formulation or per volume of the original aqueous solution.

Concentration is not area percent or relative area; it is the absolute level or mass per

unit volume of an analyte present in a sample matrix [115-120].

A method, which depends on the accuracy and precision of measurements, can

not be validated without first being able to accurately and precisely quantitate the

level of analytes present in the original samples. Quantitation in samples requires a

well-characterized standard of the analyte to be used. The very essence of analytical

chemistry relies on accurately and precisely quantitating the analytes of interest in the

samples. The U.S. Pharmacopoeia (USP) and the International Conference on

Harmonization of Technical Requirements for Registration of Pharmaceuticals for

Human Use (ICH) both recognize the unique significance of quantitation in deriving a

validated high performance liquid chromatography (HPLC or HPTLC) method. [121]

Unfortunately, neither the USP nor ICH guidelines direct analysts to the best possible

method of quantitation. Therefore, chemists must review books and search the

literature available that deal with these topic. Also FDA will never dictate nor even

suggest particular quantitation methods for particular samples or HPLC analysis

modes. These decisions are to be taken by the individual analysts. As a start, analysts

must use a trial-and-error approach to determine the best quantitation method for a

particular analyte in a particular sample matrix.[122-130]

Quantitation has no hard and fast rules or guidelines, except that the final

method selected must provide the best accuracy and precision possible, the best

[ 29]

qntrod'uctlon

repeatability, and a high degree of intermediate precision or reproducibility

(ruggedness) from analyst to analyst, day to day, and laboratory to laboratory. The

method so chosen for quantitation should accomplish above-mentioned objectives in

the shortest time possible with a minimum amount of operator involvement and the

smallest amount of sample, resources, and instrument time.

1.2.3.1. Specific goals of an ideal quantitative method

The ideal quantitative method should possess attributes and advantages such as

+ Rapid sample throughput with minimum cost, manpower, and instrument

requirements.

+ High accuracy and precision.

+ Lack of interference and contamination from matrix and analytical

reagents.

+ An accounting for percent recovery of analyte.

+ An accounting for loss of sample and analyte during sample work-up or

analysis.

+ Easy routine operation and application by suitably trained technicians.

1.2.3.2. The real question remains: Which of the numerous quantitation methods

should one select for a particular sample? It often depends on the nature of the

sample, whether it is simple with a few peaks or very complex with numerous peaks.

Usually, simpler samples with few analytes, simple matrix can use simpler

quantitation methods and external standard calibration plots or even single-point

calibrations. The use of single-point, external standard calibration for samples reduces

cost, time, and manpower requirements and leads to higher sample throughput, but

[ 30]

qntrod'uction

this can be done only with standard concentrations that are close to the actual

concentration of the unknown samples within the linear range of the method. But first

the HPLC peak purity and chromatographic selectivity should be proved. This

requires photodiode-array or mass spectrometry (MS) detection after HPLC

separation.

No single method of quantitation works best for any and all samples. The best method

for any given situation is derived by experimentation. [ 131-13 8]

1.3. Methodologies and techniques

1.3.1. High Performance Liquid Chromatography

1.3.1.1. Introduction

The Russian botanist Tswett originally developed adsorption chromatography in

1906. The separation of compounds occurs due to adsorption at solid-liquid interface

with different degree of affinity and the interaction between adsorbent and component

that must be reversible.

The High Performance Liquid Chromatograph (HPLC) instruments as we see it today

is due the fundamental work of Howard and Martin. As compared to classical column

liquid chromatography, which is dependent on gravity takes long separation time. In

HPLC, small diameter columns with fine material like silica or alumina with llm

particle size are used and the solvent is pumped through the column under pressure

with a constant flow rate, which requires less time for separation. Due to the versatile

nature of bonding phase available with a choice of solvent systems HPLC has gained

importance and is being used by researchers around the world for various

applications. [139-157]

[ 31 ]

gntrocfuction

There are two types ofHPLC:

• Analytical HPLC: In analytical HPLC columns of small internal diameter are

used. These columns use very small amount of the analyte, usually in ng or !J.g

quantity.

• Preparative HPLC: In this type of HPLC, columns of big internal diameter that

have large amount of packing material are used. This allows loading in larger

quantity mg or g and can be used for separation and collection of different

components. The flow rate range can be from 1 to 800 mL/min or more. This is

mainly used to purify mixture of compounds and preparation of reference

standards.

1.3.1.2. Basic Theory: The basic principle of separation for HPLC is dependent on

the fact that substances to be separated have different relative affinities for the

stationary and mobile phase. Thus a substance with relatively higher affinity for the

stationary phase moves slower than others, through the column and mixtures are

resolved by differential migration of their constituents during passage through a

chromatographic column. The separation procedure is governed by the distribution of

substances between two phases- mobile phase (carrier phase) and stationary phase.

The forces acting on solute can be polarity, ion pairing or van der Waal forces. After

separation, each component reaches the detector, which generates a signal due to

change in the constitution of mobile phase. These signals are traced as a

chromatogram on a recorder. The integrator enables qualitative and quantitative

analysis of each component through specification of the individual retention time and

concentration value.

[ 32]

"lmroaucnon

The separation in HPLC can be achieved by:

a) Adsorption b) Partition

c) Ion-exchange d) Size Exclusion.

or by a combination of above methods.

1.3.1.3. Terminologies Used

1. Retention Time: The degree of retention of a particular compound of a mixture

on the column is expressed as its retention time (RT). It is defined as the time

that elapses from the moment of sample is introduced to the point of

maximum concentration of the eluted peak. Retention time is affected by:

change in temperature, column, mobile phase and flow rate.

2. Resolution and efficiency: the aim of any analysis is to separate the

components of a mixture such that each compound can be easily identified

under convenient conditions to obtain a satisfactory analysis. For a given

mixture of compounds, the main requirement is to achieve a minimum degree

of resolution between them. In peak resolution, it is assumed that all band

profiles have Gaussian shape. Kirkland has shown that if the peak asymmetry

is moderate, conventional equation is used for Guassian peaks. The resolution

between two Guassian peaks is given by:

Where trl and wl are the retention time and base width of peak 1.

3. Number oftheoretical plates: The number of theoretical plates (n) is expressed by

the following mathematical expression,

[ 33 ]

qntroauction

n = 16 (Tr I W) 2 or

n = 5.54 ( Tr I W 112 ) 2

Where, W= Peak width; W 112= Peak width at half height of the peak;

Tr= Retention time of peak.

4. Height equivalent to theoretical plate: The thickness of the layer of the column in

which one "Partition" is considered to occur is called the height equivalent to a

theoretical plate (HETP) and is given by the expression,

HETP = Length of column I n

Note: The smaller the HETP, the greater is the number of theoretical plates in the

column and the greater is the separating ability of the column.

1.3.1.4. Apparatus For HPLC:

There are four basic components of a modem HPLC device

1. A solvent delivery system or pump.

2. A means of introducing the sample i.e. sample injection system.

3. A chromatographic column.

4. Detector and recording system.

1.3.1.5. Criteria for selecting mobile phase: - The choice of mobile phase depends

upon the kind of separation desired. The mobile phase should be- of desired polarity/

of proper elution power, economical; non-toxic; easily available. Its polarity, the

nature of stationary phase and that of sample components determine the elution power

of the mobile phase. Optimum separating conditions can often be achieved by making

use of a mixture of two solvents, gradient elution is also used where mobile phase

r 34 1

Cintrod"uction

components vary widely in polarity, effecting the affinity of the sample to stationary

phase. Solvent delivery in HPLC can be done in two different ways:

1. !socratic: one particular solvent or ratio of different solvents is maintained

constant through out the analysis period; and

2. Gradient system: In this type of delivery the ratio of the solvents is changed

during the analysis period.

1.3.1.6. Columns: Different columns may be used for analytical, semi-analytical and

preparative works. The most widely used column is C-18 in which octadecyl

derivative of silica is used.

Efficiency of column: the efficiency of column is measured by its number of

theoretical plates. The column is considered as being made up of a large number of

parallel layers of "Theoretical plates" and when the mobile phase passes down the

column, the components of a mixture on the column distribute themselves between

the stationary and mobile phases in accordance with their affinities. The rate of

movement of the mobile phase is assumed to be such that a dynamic equilibrium is

established between each theoretical plate and the components move down the

column at a definite rate depending on the rate of movement of mobile phase.

1.3.1.7. Detector: The function of detector in HPLC is to monitor the eluent as it

emerges from the column. The detectors for HPLC consists of photometric detector

fitted with a low volume flow cell (about 10 J..ll). The detector response is usually

presented on a time scale as a peak displaying the components. Various types of

detectors used are:-

1. UV detector.

[ 35]

2. Fluorescence detector.

3. EC detector.

4. Mass detector.

5. NMR detector.

6. Refractive index detectors etc.

1.3.2. High Performance Thin Layer Chromatography

Thin layer chromatography (TLC) is a method of analysis in which the

stationary phase, a finely devided silica, is spread as a thin layer on a rigid supporting

plate and the mobile phase is allowed to migrate across the surface of the plate. It

differs from other techniques in that the separation does not take place in a close

column, but rather on a plane surface and the mobile phase does not flow under the

influence of gravity or high pressure but is driven on the plate by capillary action. The

stationary phase is arranged in the form of a planar bed on a glass plate or aluminum

sheet or plastic sheet. [ 15 8-161]

The TLC process unlike HPLC is an offline process. A number of samples are

chromatographed simultaneously side-by-side. Since the stationary phase is used only

once, lifetime considerations have no bearing unlike a column. Sample preparation is

very simple and necessary to ensure that extraneous material in sample applied to the

plate do not retain any of the analyte. In the open TLC process, all fractions are stored

on the plate. Their optical properties are measured by densitometry (TLC Scanner).

This process of scanning can be repeated without doing all the work again and also

combining with post-chromatographic derivatisation.

[ 36]

gntrod'uction

Although separation efficiencies equivalent to those obtained with GLC or HPLC

can't be obtained by this method. Separation efficiency is expressed as the number of

theoretical plates (N) in the chromatographic system. HPTLC yields a maximum of

5000 plates as compared to HPLC which has 10,000 to 15,000 plates. The reason for

the lower performance of TLC is due to its capillary flow behavior and length

limitation. But, it has the advantage of speed, versatility and simplicity. It is used for

such diverse purposes as trial runs to test stationary and mobile phases for liquid

chromatography, monitoring the progress of synthetic reactions, clinical diagnosis and

monitoring the drug abuse.[162-167]

Recent developments in automation of thin layer chromatography have resulted in a

breakthrough in performance which has led to the expression 'high performance thin

layer chromatography. These developments have not been the result of any specified

advance in the instrumentation (as per HPLC), but rather the culmination of

improvements in the various operations involved in TLC. The modem HPTLC system

available nowadays consists of

1. Automated TLC plate spotter;

2. Automated TLC plate developer

3. TLC plate Scanner with different lamps e.g. Tungsten, Mercury & Deuterium.

Thus, allowing handling of more samples in one day than in fully automated

HPLC system. [168-173]

[ 37]

qntroduction

1.3.2.1. The absorbent material-

The substances most often used as stationary phase are silica gel, alumina and

cellulose to give stable layers, containing binders such as calcium phosphate

(gypsum) or starch. It may also contain an inorganic fluorescent indicator (e.g. zinc

silicate), which fluoresces when irradiated at suitable wavelength.

Layers of the HPTLC are prepared using smaller particle size (6J...L) and less

thickness (1 00 or 200J...Lm) and particularly whose particle size distribution is similar.

Due to these reasons, the resolution achieved in HPTLC is much more. The

uniformity of layer enables separations to be achieved in much shorter times.

Although reverse phase layers are also available, but maximum TLC separations are

carried out on normal phase silica gel.

1.3.2.2. Methods of sample application.

Due to more sample capacity of HPTLC layer, the amount of sample applied

to the layer is reduced. A further advantage is that the compact starting spots allow an

increase in the number of samples, which may be applied to the HPTLC plates.

The best way of loading sample on a precoated TLC plate is to apply it

sequentially. This has been achieved with automatic TLC sampler, which applies

samples using a platinum-iridium capillary (0.05mm2) of fixed volume (100 or

200nL), sealed into a glass support capillary of large bore. The samples are dispersed

by contact or spray technique. By this way the position of spots is precise and sample

loading time is less with minimum damage to the layer surface.

[ 38]

qntroduction

1.3.2.3. Chromatogram Development:

Generally speaking, a planer chromatogram can be developed in three modes­

linear, circular and anti-circular, widely used is linear mode. The usual way to

develop a TLC is to immerse the plate with lower edge in the solvent system

contained in a twin trough chamber. Solvent ascends on the plate layer and with that

the samples move according to their affinity.

+ Automated chromatogram development (ACD): this is different as the instrument

controls it. Preconditioning, tank or sandwich configuration, solvent migration

distance and the drying conditions are selected. Development is automatic and

takes place under controlled conditions; a sensor monitors the development

progress. When it is complete the development is stopped and plate is dried with

filtered warm or cold air and stored there.

+ Automated multiple development (AMD): in this the plate is developed in the

same direction over different migration distances, with different ratio of solvents.

This way a stepwise gradient is obtained. Between each change of solvent the

plate is dried under vacuum. Unlike a gradient in column chromatography, an

AMD gradient starts with the solvent having the strongest elution power. In the

successive runs, the solvent is varied towards decreasing elution power.

1.3.2.4. Densitometers- chromatogram evaluation:

For densitometric measurements of thin layer chromatogram, its separated

tracks are scanned with a light beam in the form of a slit selectable in length and

width. The reflected light is measured by a photosensor. The difference between the

optical signal from the sample free background and that from a sample zone (fraction)

[ 39]

qntroduction

is correlated with the amount of the respective fraction of calibration standards loaded

on the same plate. Densitometric measurements can be made by absorbance or by

fluorescence. The majority of such measurements are carried out in absorbance mode.

TLC/HPTLC Scanner- The densitometer is designed specifically for HPTLC. The

stage is designed for linear and circular scanning and can accommodate all plate size. \•

Absorbance, fluorescence, and fluorescence quenching can be determined in

reflection mode. The geometry of the optical system is such that the incident beam is

projected onto the plate at an angle of 30°. The reflected light is measured with a

photo-multiplier at a 0° angle. A Tungsten, deuterium, or high-pressure mercury lamp

can be selected.

The evaluation of thin layer chromatogram takes place in a light scattering

medium, in contrast to the photometry of the solution. Thus, beer's law cannot be

strictly applied to these types of measurements. So far, there is no simple and

generally applicable mathematical equation, which can express the relationship

between response and sample concentration. At low sample concentrations, there is

linear relation between sample concentration and signal over a concentration range of

approximately one order of magnitude. Non -linear response can be easily overcome

by means of calibration curve using programmable calculators.

There are basically two different approaches to the photometric evaluation of thin -

layer chromatograms:

Transmittance measurement: - In a trasmittance measurement the light source and

detector are arranged on opposite sides of TLC plate. The range of usable

wavelengths is limited by absorption in the glass plate and the layer below 330nm.

[ 40]

qntroduction

This constitutes a substantial limitation of the method because approximately 70% of

all samples analyzed by TLC absorb in the range of 200to 360nrn. Also transmittance

measurement is very sensitive to differences in layer thickness, which results in a

relatively high signal-to-noise ratio.

Reflectance measurement: - In a reflectance measurement, the light source and the

detector are arranged on the same side of the TLC plate. The method allows the full

use of wavelength range between 200-800nrn. The signal-to-noise ratio is basically

determined by the surface quality of the layer and it is generally better than in

transmittance measurements. For this reason measurements in the reflectance mode

are used exclusively in modem TLC scanners.

Optical System of a TLC Scanner: Since there is no universal light source in the range

of 200-800 nrn, the instrument is equipped with three different lamps.

+ Deuterium lamp: has a usable spectral range of 200-400 nrn and 1s used

principally for absorption measurements.

+ Tungsten lamp: has a usable spectral range of 400-800 nrn.

+ Mercury high-pressure lamp: The very intense light of the various emission lines

is mainly used for fluorescence measurements. It can also be used for absorption

measurements. The intensity of the mercury high-pressure lamp is orders of

magnitude higher than the output of the deuterium or tungsten lamp.

The slit size can be adjusted according to the bandwidth. The light passes through

the slit and strikes the object at the track at right angles. The photo-multiplier for

reflectance scanning is aligned at an angle of 30 to normal. All functions of the

scanner are controlled from a personal computer that is linked via interface. The

[ 41 ]

CJntroduction

scanner transmits all measured data in digital form to the computer for processing

with TLC specific CATS software.

1.3.3. Digital Scanning Calorimeter-

In DSC, a sample and reference material is placed in separate crucibles and the

temperature of each crucible is increased or decreased at a predetermined rate. When

the sample reaches its melting point, it remains at this temperature until all the

material has passed into the liquid state, because of endothermic process of melting. A

temperature difference therefore exists between sample and a· reference, as the

temperature of the two materials is raised gradually. A second temperature circuit is

used in the DSC to provide a heat input to overcome this temperature difference. In

this way the temperature of the sample is maintained at the same value as the

reference. The difference is heat input to the sample, and the reference per unit time is

fed to a recorder and plotted as dH/dT versus the average temperature to which the

sample and reference are being raised. The differential heat input is recorded with a

sensitivity of ± O.lm Cal per second, and the temperature range over which the

instrument operates is -175° to 725° C.

Although DSC is used most widely in pharmacy to establish identity and

purity, it may be employed to obtain heat capacities and heats of fusion. It is also

useful for preparing phase diagrams to study the polymorphs and for carrying out the

kinetics of decomposition of solids. DSC, as well as other thermal analytical methods,

has a number of applications in biomedical research and food technology. Guillory

and associates have explored the applications of thermal analysis, particularly, DSC

and DTA, in conjugation with IR spectroscopy and X-ray diffraction. Using these

[ 42]

CJntroduction

techniques, they have characterized various solids forms of drugs, such as the

sulfonamides, and have correlated a number of physical properties of crystalline

materials with interactions between solids, dissolution rates and stability in the

crystalline and amorphous states. DSC has found increasing use in standardisation of

lipophilization process. Crystal changes and eutectic formation in the frozen state can

be detected by DSC when the instrument is operated below room temperature.[l74-

179]

qntroduction

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