Chapter 1 Introduction - INFLIBNET Centreshodhganga.inflibnet.ac.in/bitstream/10603/25036/11/12...Chapter 1 Introduction Suresh Gyan Vihar University, Jaipur 4 Where, S 0 is the solubility

Chapter 1 Introduction

Suresh Gyan Vihar University, Jaipur 1

01. INTRODUCTION

Analysis is considered to determine identity, strength, quality & purity of the drug

samples, synthetic intermediates and the final drug product, in pharmaceutical

industry. Hence analysis plays an important role right from the testing of raw

material, the in process control of every step to the final analysis of each batch of

finished drug product 1-2

.Analytical chemistry is always concerned with solubility

of drug. For analysis i.e identification of substances, the elucidation of its

structure and quantitative analysis of its composition for poorly soluble drug is a

very much challenging. Solubilisation of poorly soluble drugs is a frequently

encountered challenge in screening studies of new chemical entities as well as in

formulation design and development3, 4

. A number of methodologies can be

adapted to improve solubilisation of poor water soluble drug and further to

improve its bioavailability. Orally administered drugs completely absorb only

when they show fair solubility in gastric medium and such drugs shows good

bioavailability. Bioavailability depends on several factors, drug solubility in an

aqueous environment and drug permeability through lipophilic membranes being

the important ones5.

Solubilized drug molecules only can be absorbed by the cellular membranes to

subsequently reach the site of drug action (vascular system for instance). Any

drug to be absorbed must be present in the form of an aqueous solution at the site

of absorption6, 7

.

Therefore, the improvement of drug solubility thereby its oral bio-availability

remains one of most challenging aspects of drug development process especially

for oral drug delivery system. These in vivo and in vitro characteristics and the

difficulties in achieving predictable and reproducible in vivo/in vitro correlations

are often sufficiently difficult to develop formulation on many newly synthesized

compounds due to solubility issues8, 9

.



Pharmaceutical companies have been able to overcome difficulties with very

slightly soluble drugs, those with aqueous solubility of less than 0.1 mg/ml by

using solubility enhancement techniques. There are numerous approaches

available and reported in literature to enhance the solubility of poorly-water

soluble drug. The techniques are chosen on the basis of certain aspects such as

properties of drug under consideration, nature of excipients to be selected and

nature of intended dosage form. The techniques generally employed for

solubilisation of drug include, chemical modification, pH adjustment, solid

dispersion, complexation, co-solvency, micellar solubilisation, hydrotropy etc.

Pharmaceutical analysis utilized hydrotropy technique to increase the water

solubility of poorly water soluble drug molecule to preclude the use of organic

and costlier solvent.

1. SOLUBILITY

The term „solubility‟ is defined as maximum amount of solute that can be

dissolved in a given amount of solvent. Quantitatively it is defined as the

concentration of the solute in a saturated solution at a certain temperature. In

qualitative terms, solubility may be defined as the spontaneous interaction of two

or more substances to form a homogenous molecular dispersion. A saturated

solution is one in which the solute is in equilibrium with the solvent10-12

. The

solubility of a drug may express as Parts, Percentage, Molarity, Molality, Volume

fraction and Mole fraction13

.

As per IP solubility has been expressed as14

:

Table 1.1: Expression of Solubility

Definition Parts of Solvent Required

for One Part of Solute

Very soluble < 1

Freely soluble 1 - 10

Soluble 10 - 30

Sparingly soluble 30 - 100

Slightly soluble 100 - 1000

Very slightly soluble 1000 - 10,000

Insoluble > 10,000



1.1 Process of Solubilisation

The process of solubilisation involves the breaking of inter-ionic or

intermolecular bonds in the solute, the separation of the molecules of the solvent

which provide space in the solvent for the solute and then interaction between the

solvent and the solute molecule or ion occours10

.

Figure 1.1 Process of Solubilisation

1.2 Factors Affecting Solubility:-

The solubility depends on the physical form of the solid, the nature and

composition of solvent medium as well as temperature and pressure of system15

.

A. Particle size

The size of the solid particle influences the solubility because as a particle

becomes smaller, the surface area to volume ratio increases. The larger surface

area allows a greater interaction with the solvent. The effect of particle size on

solubility can be described by



Where,

S0 is the solubility of infinitely large particles

S is the solubility of fine particles

V is molar volume

g is the surface tension of the solid

r is the radius of the fine particle

B. Temperature

Temperature will affect solubility. If the solution process absorbs energy then the

solubility will be increased as the temperature is increased. If the solution process

releases energy then the solubility will decrease with increasing temperature.

Generally, an increase in the temperature of the solution increases the solubility of

a solid solute. A few solid solutes are less soluble in warm solutions. For all gases,

solubility decreases as the temperature of the solution increases16

.

C. Pressure

For gaseous solutes, an increase in pressure increases solubility and a decrease in

pressure decrease the solubility. For solids and liquid solutes, changes in pressure

have practically no effect on solubility 16

.

D. Nature of the solute and solvent

1 gram of lead (II) chloride can be dissolved in 100 grams of water at room

temperature where 200 grams of zinc chloride can be dissolved in same condition.

The great difference in the solubility‟s of these two substances is the result of

differences in their natures16

.

E. Molecular size

Molecular size will affect the solubility. The larger the molecule or the higher its

molecular weight the less soluble the substance. Larger molecules are more

difficult to surround with solvent molecules in order to solvate the substance. In

the case of organic compounds the amount of carbon branching will increase the

solubility since more branching will reduce the size (or volume) of the molecule

and make it easier to solvate the molecules with solvent17

.



F. Polarity

Polarity of the solute and solvent molecules will affect the solubility. Generally

non-polar solute molecules will dissolve in non-polar solvents and polar solute

molecules will dissolve in polar solvents. The polar solute molecules have a

positive and a negative end to the molecule. If the solvent molecule is also polar

then positive ends of solvent molecules will attract negative ends of solute

molecules. This is a type of intermolecular force known as dipole-dipole

interaction. All molecules also have a type of intermolecular force much weaker

than the other forces called london dispersion forces where the positive nuclei of

the atoms of the solute molecule will attract the negative electrons of the atoms of

a solvent molecule. This gives the non-polar solvent a chance to solvate the solute

molecules17

.

G. Polymorphs

A solid has a rigid form and a definite shape. The shape or habit of a crystal of a

given substance may vary but the angles between the faces are always constant. A

crystal is made up of atoms, ions, or molecules in a regular geometric

arrangement or lattice constantly repeated in three dimensions. This repeating

pattern is known as the unit cell. The capacity for a substance to crystallize in

more than one crystalline form is polymorphism. It is possible that all crystals can

crystallize in different forms or polymorphs. If the change from one polymorph to

another is reversible, the process is called enantiotropic. If the system is

monotropic, there is a transition point above the melting points of both

polymorphs. The two polymorphs cannot be converted from one another without

undergoing a phase transition. Polymorphs can vary in melting point. Since the

melting point of the solid is related to solubility, so polymorphs will have

different solubilities. Generally the range of solubility differences between

different polymorphs is only 2-3 folds due to relatively small differences in free

energy18

.



1. 3 Techniques of Solubility Enhancement

There are various techniques available to improve the solubility of poorly soluble

drugs. Some of the approaches to improve the solubility are19

:

I. Physical Modifications

A. Particle size reduction

a. Micronization

b. Nanosuspension

c. Other techniques

B. Modification of the crystal habit

a. Polymorphs

b. Pseudopolymorphs

C. Drug dispersion in carriers

a. Eutectic mixtures

b. Solid dispersions

c. Solid solutions

D. Complexation

a. Use of complexing agents

E. Solubilization by surfactants:

a. Microemulsions

b. Self micro emulsifying drug delivery systems

II. Chemical Modifications

III. Other Techniques

A. Co-crystallization

B. Co-solvency

C. Hydrotropy

D. Solubilizing agents

E. Nanotechnology approaches

The approaches mentioned have been used widely in fields of pharmacy.

However, applications of hydrotropic solubilization have not been explored to

appreciable extent in various fields of pharmacy.



2. HYDROTROPY

The term "hydrotropy" has been used to designate the increase in solubility of

various substances due to the presence of large amounts of additives. Hydrotropy

is a solubilization process whereby addition of large amounts of a second solute

results in an increase in the aqueous solubility of another solute.

Hydrotropic agents are ionic organic salts. Additives or salts that increase

solubility in given solvent are said to “salt in” the solute and those salts that

decrease solubility “salt out” the solute. Several salts with large anions or cations

that are themselves very soluble in water result in “salting in” of non-electrolytes

called “hydrotropic salts” a phenomenon known as “Hydrotropism”13

. Increasing

the aqueous solubility of insoluble and slightly soluble drugs is of major

importance. Various techniques have been employed to enhance the aqueous

solubility of poorly water soluble drugs. Hydrotropic solubilization is one of

them.

In the hydrotropic solubilization phenomenon, addition of large amount of second

solute results in an increase in the aqueous solubility of another solute.

Concentrated aqueous hydrotropic solutions of urea, nicotinamide, sodium

benzoate, sodium salicylate, sodium acetate and sodium citrate have been

observed to enhance the aqueous solubility of poorly water soluble drugs. The

class of compounds that normally increase the aqueous solubility of sparingly-

soluble solutes is called hydrotropes.

A hydrotrope is a compound that solubilises hydrophobic compounds in aqueous

solutions. Typically, hydrotropes consist of a hydrophilic part and a hydrophobic

part (like surfactants) but the hydrophobic part is generally too small to cause

spontaneous self-aggregation. The hydrotropes are a special class of compounds

that exhibit distinct solution properties. They may self associate in aqueous

medium, comparable to amphiphile self-association or micellization. They are

efficient solubilizers and can influence the formation of micelle and micro

emulsion20

.



2.1. History of Hydrotropy and Basic Structure of Hydrotropic Agent

Winsor et al. speculated that hydrotropy is simply another type of solubilization

with the solute dissolved in oriented clusters of the hydrotropic agents. Some

workers proposed that this phenomenon is more closely related to complexation

with a weak interaction existing between the hydrotropic agent and the solute.

The characteristic that hydrotropic agents share is the ability of self association in

the aqueous solution, particularly at hydrotropic concentration more than 1 M21

.

Hydrotropy is the term originally put forward by Neuberg to describe the increase

in the solubility of a solute by the addition of fairly high concentrations of alkali

metal salts of various organic acids. However, the term has been used in the

literature to designate non-micelle-forming substances, either liquids or solids,

organic or inorganic, capable of solubilizing insoluble compounds. Hydrotropic

solubilization process involves cooperative intermolecular interaction with several

balancing molecular forces, rather than either a specific complexation event or a

process dominated by a medium effect, such as co-solvency or salting-in.

Neuberg’s postulated the chemical structure of the conventional hydrotropic salts

(proto-type, sodium benzoate) consists generally of two essential parts, an anionic

group and a hydrophobic aromatic ring or ring system. The anionic group is

obviously involved in bringing about high aqueous solubility, which is a

prerequisite for a hydrotropic substance. The type of anion or metal ion appeared

to have a minor effect on the phenomenon 22

.

Saleh and El-Khordagui suggested that the phenomenon of hydrotropy is not

confined to the metal salts of organic acids, certain cationic salts and neutral

molecules may be equally involved. They used procaine HCl, PABA HCl and

cinchocaine HCl as cationic salts and resorcinol and pyrogallol as neutral

molecules in their studies 23

.

Rasool et al showed that the aromaticity (Л-system) of the pyridine ring which

might promote the stacking of molecules through its planarity was an important

factor in complexation because the aromatic amide ligands enhanced the aqueous

solubility of the test drug to a greater extent than the aliphatic amide ligands24

.



On the other hand, planarity of the hydrophobic part has been emphasized as an

important factor in the mechanism of hydrotropic solubilization. This should

imply that hydrotropic agents are molecules having a planar hydrophobic

structure brought into solution by a polar group. Hence, it seems rational to

propose that molecules with a planar hydrophobic part and a polar group, which is

not necessarily anionic, can act as hydrotropic agents.

Gaikar et al investigated whether a drug with an amphiphillic structure can

exhibit hydrotropic properties. They sought to establish sodium ibuprofen as an

effective hydrotrope 25

.

Suzuki et al measured the aqueous solubility of nifedipine in presence of

nicotinamide, urea, and their analogues and concluded that the significant

contributor to the hydrotropic solubilization of nifedipine with nicotinamide was

therefore the ligand hydrophobicity rather than the aromaticity of the pyridine

ring 26

.

2.2 Commonly Used Hydrotropes

The hydrotropes are known to self-assemble in solution. The classification of

hydrotropes on the basis of molecular structure is difficult, since a wide variety of

compounds have been reported to exhibit hydrotropic behaviour. Specific

examples may include ethanol, aromatic alcohols like resorcinol, pyrogallol,

catechol, a- and b-naphthols and salicylates, alkaloids like caffeine and nicotine,

ionic surfactants like diacids, SDS (sodium dodecyl sulphate) and dodecylated

oxidibenzene. The aromatic hydrotropes with anionic head groups are mostly

studied compounds. They are large in number because of isomerism and their

effective hydrotrope action may be due to the availability of interactive pi-

orbitals. Hydrotropes with cationic hydrophilic group are rare, e.g. salts of

aromatic amines, such as procaine hydrochloride. Besides enhancing the

solubilization of compounds in water, they are known to exhibit influences on

surfactant aggregation leading to micelle formation, phase manifestation of

multicomponent systems with reference to nanodispersions and conductance

percolation, clouding of surfactants and polymers, etc27, 28

.



Each hydrotropic agent is effective in increasing the water solubility of selected

hydrophobic drugs. No universal hydrotropic agent has been found effective to

solubilize all hydrophobic drugs. Thus finding the right hydrotropic agent for a

poorly water-soluble drug requires screening of large number of candidate

hydrotropes. However, once the effective hydrotropic agent is identified for a

series of structurally different drugs, the structure activity relationship can be

established.

Various investigated done to check the effect of various hydrotropes such as

sodium benzoate, sodium salicylate and piperzine on the solubility of nimesulide.

The solubility enhancement of nimesulide by the hydrotropes observed in

decreasing order as piperazine>sodium ascorbate> sodium salicylate> sodium

benzoate> nicotinamide. Parenteral formulations using piperazine as a hydrotrope

were developed and studied for physical and chemical stability29

.

Jain et al. investigated the effect of various hydrotropes such as urea,

nicotinamide, resorcinol, sodium benzoate, sodium p-hydroxy benzoate on the

solubility of indomethacin. The solubility enhancement of indomethacin by the

hydrotropes was observed in decreasing order as sodium p-hydroxyl benzoate>

sodium benzoate> nicotinamide> resorcinol> urea. Aqueous injectable

formulations using sodium p-hydroxyl benzoate, sodium benzoate and

nicotinamide as hydrotropes were developed and studied for physical and

chemical stability30

.

2.3 Mechanism of Hydrotrope Action

A hydrotrope is a compound that solubilises hydrophobic compounds in aqueous

solutions. Typically, hydrotropes consist of a hydrophilic part and a hydrophobic

part (like surfactants) but the hydrophobic part is generally too small to cause

spontaneous self-aggregation. Hydrotropes do not have a critical concentration

above which self-aggregation, 'suddenly' starts to occur (as found in micelle and

vesicle-forming surfactants, which have a critical micelle concentration and a

critical vesicle concentration or cvc, respectively). Instead, some hydrotropes

aggregate in a step-wise self-aggregation process, gradually increasing

aggregation size.



However, many hydrotropes do not seem to self-aggregate at all, unless a

solubilisate has been added. Badwan et al studied the solubility of benzodiazepine

in sodium salicylate solution. It was suggested that molecular aggregation takes

place and benzodiazepine molecule were induced in these aggregates. A donor

acceptor type of interaction between sodium salicylate and benzodiazepine

molecules is assumed to stabilize such inclusions and determined the degree of

solubility31

.

Hydrotropes are used in detergent formulations to allow more concentrated

formulations of surfactants. Examples of hydrotropes include sodium p-

toluenesulfonate and sodium xylene sulfonate.

Poochikian et al studied the solubilization of chartreusin by hydroxybenzoate.

Plane to plane orientation of ligand molecules and chartreusin brought together by

electrostatic and hydrophobic interactions was suggested as possible

mechanism32

.

Jain et al investigated the solubilization of ketoprofen, by means of various

physiologically active hydrotropic agents. In order to gain an insight into probable

mechanism of solubilization, solubility, spectral, typical properties of

hydrotropes, solution properties, gel formation, paste formation, TLC and IR

spectral studies were carried out with structural variation in hydrotropes. The

results indicated that the enhanced solubility of ketoprofen in presence of

hydrotropes in low concentration is due to weak ionic interaction. At higher

concentration, the formation of molecular aggregates seemed to be the possible

mechanism of hydrotropic solubilization33

.

Rawat et al studied the effects of various hydrotropes such as nicotinamide,

sodium benzoate, sodium salicylate in the solubility of rofecoxib, celecoxib and

meloxicam, and were investigated to gain an insight into the mechanism of

solubilization. The results indicated that the enhanced solubility of these drugs in

the presence of hydrotropes in low concentration is due to weak ionic interaction.

At high hydrotropic concentration the formation of molecular aggregation seems

to be the possible mechanism of solubilization34

.



Coffman et al studied the effect of nicotinamide and urea on the solubility of

riboflavin in various solvents. Their study examined the mechanism of

hydrotropic solubilization. The most commonly proposed mechanism for

hydrotropic solubilization is complexation35

.

2.4 Mixed Hydrotropy

Mixed hydrotropy is a synergistic effect on enhancement in solubility of a poorly

water-soluble drug by mixing two hydrotropic agents. Maheshwari et al and

Jain et al was employed urea, sodium citrate and other mixed hydrotropic blend

to solubilize a poorly- water soluble drug, to carryout spectrophotometric analysis

precluding the use of organic solvents36

.

2.5 Advantages of Hydrotropic Solubilization Technique

1. It precludes the use of organic solvents and thus avoids the problem of

residual toxicity, error due to volatility, pollution, cost etc.

2. It is new, simple, cost-effective, safe, accurate, precise and environmental

friendly method for the analysis (titrimetric and spectrophotometric) of poorly

water-soluble drugs by titrimetric and spectrophotometrically precluding the

use of organic solvents.

3. It only requires mixing the drug with the hydrotrope in water.

4. Hydrotropy is suggested to be superior to other solubilization method, such as

miscibility, micellar solubilization, cosolvency and salting in, because the

solvent character is independent of pH, has high selectivity and does not

require emulsification.

5. It does not require chemical modification of hydrophobic drugs, use of organic

solvents, or preparation of emulsion system.

6. Mixed hydrotropy reduce the large total concentration of hydrotropic agents

necessary to produce modest increase in solubility by employing combination

of agents in lower concentration.

2.6 Pharmaceutical Applications of Hydrotropic Agents

1. Quantitative estimations of poorly water-soluble drugs by uv-visible

spectrophotometric analysis precluding the use of organic solvents37

.



2. Quantitative estimations of poorly water-soluble drugs by titrimetric

analysis.

3. Preparation of hydrotropic solid dispersions of poorly water-soluble drugs

precluding the use of organic solvents.

4. Preparation of ready to use syrups of poorly water-soluble drugs.

5. Preparation of dry syrups (for reconstitution) of poorly water-soluble drugs.

6. Preparation of topical solutions of poorly water-soluble drugs, precluding

the use of organic solvents.

7. Prepration of injection of poorly water soluble drugs38, 39

.

8. The use of hydrotropic solubilizers as permeation enhancers.

9. The use of hydrotropy to give fast release of poorly water-soluble drugs

from the suppositories.

10. Application of mixed-hydrotropy to develop injection dosage forms of

poorly water-soluble.

11. Application of hydrotropic solubilization in nanotechnology (by controlled

precipitation

12. Application of hydrotropic solubilization in extraction of active

constituents from crude drugs.

Table 1.2: Hydrotropic Solubilization Studies of Various Poorly Water-

Soluble Drugs

Sr.

No.

Drugs Hydrotropic Agent with Working λ Ref.

No.

1 Cefixime 8 M Urea, 4 M Sodium acetate and 1.25 M

Sodium citrate) 40

2 Hydrochlorothiazide 10 M Urea 41

3 Hydrochlorothiazide

and Indomethacin

2 M Sodium benzoate 42

4 Ketoprofen 4 M Sodium aetate 43

5 Tinidazole 8 M Urea, 4 M Sodium acetate and 1.25 M

Sodium citrate 44

6 Ofloxacin 2 M Sodium benzoate 45



7 Norfloxacin & Tinidazole 8 M Urea 46

8 Cephalexin 8 M Urea 47

9 Metronidazolem, Nalidixic

acid, Norfloxacin,

Tinidazole

2 M Sodium benzoate and 2 M

Niacinamide 48

10 Amoxycillin 10 M Urea 49

11 Paracetamol 10 M Urea 50

12 Piroxicam 2 M Sodium benzoate 51

13 Frusemide 2 M Sodium benzoate 52

14 Norfloxacin 8 M Urea 53

15 Diclofenac sodium 8 M Urea 54

16 Gatifloxacin 2 M Sod. Benz. and 1.5 M Metformin 55

17 Amoxicillin 5 M Potassium acetate 56

18 Cefixime 8 M Pot. acetate & 6 M Amm.acetate 57

19 Cefixime 0.5 M Potassium citrate 58

20 Aceclofenac 22.5%w/v Urea and 22.5% w/v Sod. citrate 59

21 Famotidine 1.5 M Metformin HCl 60

22 Hydrochlorothiazide 2 M Niacinamide 61

23 Cefixime trihydrate 0.5 M Metformin Hydrochloride 62

24 Hydrochlorothiazide 20 % Chlorpheniramine Maleate 63

25 Hydrochlorothiazide 1M Lignocaine hydrochloride 64

26 Metronidazole &

Norfloxacin

8 M Urea solution 65

27 Naproxen 2 M Sodium benzoate 66

28 Naproxen 0.5 M Ibuprofen solution 67

30 Chartreusin Sodium benzoate, Sodium p- hydroxyl

benzoate, Sodium m-hydroxybenzoate,

Sodium o-hydroxy benzoate, Sodium 2,4-

dihydroxybenzoate, Sodium 2,5 dihydroxy

benzoate.

32



31 Theophylline,

Hydrocortisone,

Prednisolone, Phenacetin

Sodium benzoate, Sodium o-hydroxy

benzoate, Sodium m-hydroxybenzoate,

Sodium p-hydroxybenzoate, Sodium 2,4-

dihydroxy benzoate, Sodium 2,5-

dihydroxybenzoate, Sodium 2,6-



dihydroxybenzoate, Sodium 3,4,5 –

trihydroxybenzoate

68

32 Nimuselide Sodium salicylate 69

33 Valsartan effect of ethyl alcohol,

propylene glycol and pH 70

34 Riboflavin Nicotinamide 71

35 Ketoprofen Sodium benzoate, Sodium o-hydroxy

benzoate, Nicotinamide, Sodium m-

hydroxy benzoate, Sodium ascorbate,

Sodium 2,5-dihydroxybenzoate

33,

72

36 Piroxicam Nicotinamide, Sodium ascorbate, Sodium

benzoate, Sodium o-hydroxybenzoate,

Sodium m-hydroxybenzoate, Sodium 2,5-

dihydroxybenzoate

73,

74

37 Etoposide Sodium benzoate, Sodium salicylate,

Sodium gentisate, Sodium m-hydroxy

benzoate, Sodium p-hydroxybenzoate,

Sodium 2,4-dihydroxybenzoate, Sodium

2,6-dihydroxy benzoate, Sodium 2,4,6-

trihydroxybenzoate

75

38 Saquinavir Nicotinamide, Ascorbic acid, Dimethyl

Urea, 76

39 Glimeperide Benzoic acid, Ascorbic acid, Citric acid 23



40 Progesterone,

Testosterone,

17- Estradiol,

Diazepam and

Griseofulvin

Nicotinamide, Isonicotinamide,

Nipecotamide, N-methylnicotinamide,

N, N-dimethylnicotinamide 24

41 Salicylamide,

Acetaminophen

Pheniramine maleate,

Chlorpheniramine maleate,

Brompheniramine maleate

77

42 Carbamazepine Sodium salicylate, Sodium benzoate 78

43 Nimesulide Sodium ascorbate,

Sodium salicylate,

Sodium benzoate,

Nicotinamide

29

44 Norfloxacin Ascrobic acid 79

45 Albendazole Sodium salicylate,

Sodium benzoate,

Nicotinamide,

Sodium ascorbate

80

46 Rofecoxib,

Celecoxib,

Meloxicam

Sodium salicylate,

Sodium benzoate,

Nicotinamide

81

3. ANALYTICAL METHOD

There is a need of a sensitive, accurate, precise analytical method for

determination of concentration of drug in the bulk drug as well as in the dosage

formulation. With the rapid development of pharmaceuticals and higher

challenges of quality, the volume of analytical work is increasing day by day. This

force to development of analytical methods, that are rapid, accurate, precise and

reproducible82, 83

.



3.1 Type of Analytical Methods82

I. Qualitative Methods

II. Quantitative methods

I. Qualitative Methods : It refers identify of the product i.e., it yields useful

clues from which the molecular or aromatic species, the structural feature, or

the functional groups in the samples can be deduced.

II. Quantitative: It refers purity of the product, i.e. the results are in the form of

numerical data corresponding to the concentration of analytes. In the

analysis, the required information is obtained by measuring a physical

property that is characteristically related to the component of interest (the

analyte). In the present age, the physical, chemical and biological analysis,

involve computerized techniques to facilitate better results.

3.2 Types of Chemical Analysis

1. Classical Method

A. Titrimetric methods

(a). Acidimetry and Alkalimetry

(b). Redox titration

(c). Precipitation titration

B. Gravimetric method

2. Instrumental Methods

A. Spectroscopic Methods

(a). Ultraviolet and visible spectroscopy

(b). Infra-red spectroscopy

(c). Raman spectroscopy

(d). Atomic absorption spectroscopy

(e). X-ray diffraction

(f). X-ray fluorescence

(g). Fluorometry and phosphorimetry

(h). Nephelometry and Turbidimetry

(i). Mass spectroscopy

(j). Nuclear magnetic resonance spectroscopy



B. Electrochemical method

(a). Potentiometry

(b). Polarography

(c). Amperometric methods

(e). Coulometry method

(f). Conductance techniques

C. Chromatographic method

(a). Gas chromatography

(b). Liquid chromatography (HPLC)

(c). High-performance thin layer chromatography (HPTLC)

(d). Paper chromatography

D. Miscellaneous Method

(a). Thermal analysis

(b). Refractrometry method

(c). Polarimetry method.

3.3 Advantages of Instrumental Methods

Highly sensitive

Measurements are reliable

Determination is very fast

Easy to handle complex samples

Small quantity of samples can be used

3.4 Limitations of Instrumental Methods

Initial and continuous calibration is required

Sensitivity and accuracy depends upon the instrument or the chemical

methods.

High cost of equipment

Limited concentration range



4. SPECTROSCOPY 82-84

Spectroscopy is the branch of science dealing with the study of interaction of

electromagnetic radiation with matter. The most important consequence of such

interaction is that energy is absorbed or emitted by the matter in discrete amounts

called quanta. A number of analytical methods both qualitative and quantitative

involve the interaction of radiant energy with matter. Two important experimental

parameters are

The energy of radiation absorbed or emitted by the system

Intensity of spectral lines

4.1 Theory of Spectroscopy

1. Intensity of emergent light (It) = Intensity of incident light (Io), therefore no

absorption of energy takes place, i.e. I t = Io, no change in energy takes place

and hence no information about the molecule can be derived.

2. Reflection. Refraction or scattering. (Scattering of light by particles) where

some studies like nephlometry or turbidimetry are being made.

3. Intensity of emergent light < Intensity of incident light, where there is

absorption of energy takes place and some information about the molecule can

be derived.

4.2 Principle of Ultraviolet Spectroscopy

The wavelength range of UV radiation starts at the blue end of visible light 400

nm to 200 nm. The UV region is subdivided into two spectral regions.

The region between 200 nm-400 nm is known as near UV region

The region below 200 nm is called the far or vacuum UV region.

The UV radiation has sufficient energy to excite valence electrons in many atoms

or molecules consequently UV is involved with electronic excitation. The UV

absorption spectra arise from transition of electron or electrons within a molecule

or an ion from a lower to a higher electronic level and UV emission spectra arise

from the reverse type of transition. For radiation to cause electronic excitation, it

must be in the UV region of the electromagnetic spectrum.



4.3 Laws of Absorption

a. Lambert’s law:

When a beam of monochromatic radiation passes through a homogeneous

absorbing medium, the rate of dicrease of intensity of radiation with thickness of

absorbing medium is proportional to the intensity of the incident radiation.

Mathematically, the law is expressed as

di

- = KI ---------------------------------------1

dx

Where I = intensity of radiation after passing through as thickness of the

medium.

di = infinitesimally small decrease in the intensity of radiation on passing through

infinitesimally small thickness dx of the medium di / dx = rate of decrease of

radiation with thickness of the absorbing medium k = proportionality constant or

absorption coefficient. Its value depends upon the nature of the absorbing

medium.

Let Io be the intensity of radiation before entering the absorbing medium (x= 0).

Then I, the intensity of radiation after passing through any thickness, say x of the

medium can be calculated as

I x = x

dI

I ---------------------2

Io x =o

Or In I ---------------------3

Io

Or I ---------------------4

Io

I = I0e-Kx

---------------------5

The intensity of the radiation absorbed, Iabs is given by

Iabs = Io – I = Io (1-e-kx

) ---------------------6

The above Lambert‟s law equation can also be written by changing the natural

logarithm to the base 10

I = Io 10-ax

---------------------7

Where a = excitation coefficient of the absorbing medium. a = K/ 2.303

= kdx

= -Kx

= e

-Kx



b. Beers’ Law

This law stats that when a beam of monochromatic radiation is passes through a

solution of an absorbing substance, the rate of decrease of intensity of radiation

with thickness of the absorbing solution is proportional to the intensity of the

incident radiation as well as concentration of the solution.

Mathematically this law stated as –

dI

dx ----------------------1

Where C = conc. of the solution in moles litre-1

k1 = molar absorption coefficient and its value depends the nature of the substance

suppose Io be the intensity of the radiation before entering the absorbing solution.

(When x=a), I after passing through the thickness x, of the medium can be

calculated:

I x = x

dI -------------------2

Io

x=o

Or I = Ioe-k1cx ---------------------- 3

The above equation can written by changing the natural logarithm to the base 10

Here K1 = a

1

2.303

Where a1 = molecular extinction coefficient of the absorbing medium

5. QUANTITATIVE SPECTROPHOTOMETRIC ASSAY OF MEDICINAL

SUBSTANCE

The assay of an absorbing substance may be quickly carried out by preparing a

wavelength. The wavelength normally selected is a wavelength of maximum

absorption (max) where small errors in setting the wavelength scale have little

effect on the measured absorbance. Ideally, the concentration should be adjusted

to give an absorbance of approximately from 0.9, around which the accuracy and

precision of the measurement are optimal. The preferred method is to read the

absorbance from the instrument display under non-scanning condition, i.e., with

the monochromator set at the analytical wavelength.

- = K

1IC

= - k\cdx



5.1 Use of a Standard Absorptivities Value

The procedure is adopted by official compendia, e.g. British Pharmacopoeia, for

stable substance such as methyl testosterone that have reasonably broad

absorption bands and which are practically unaffected by variation of instrumental

parameters, e.g. slit width, scan speed .The use of standard A (1%, 1cm) or

value avoids the need to prepare a standard solution of the reference substance in

order to determine its absorptivity, and is of advantage in situations where it is

difficult or expensive to obtain a sample of the reference substance.

5.2 Use of a Calibration Graph

In this procedure the absorbance of a number (typically 4-6) of standard solutions

of the reference substance at concentration encompassing the sample

concentration are measured and a calibration graph is constructed, The

concentration of the analyte in sample solution is read from the graph as the

concentration corresponding to the absorbance of the solution.

5.3 Single-or Double Point Standardization

The single-point procedure involves the measurement of the absorbance of a

sample solution and standard solution of the reference substance, the standard and

sample solution is prepared in a similar manner; ideally, the concentration of the

standard solution should be close to that of the sample solution. The concentration

of the substance in the sample is calculated from the proportional relationship that

exists between absorbance and concentration.

Ctest = A test x Cstd (single point calculation)

A std

Where Ctest and Cstd are the concentration of the sample and standard solution

respectively, and Atest and Astd are the absorbance of the sample and standard

solution respectively.

Ctest = (Atest – Astd1) (Cstd1 – Cstd2) + Cstd1 (Astd1 – Astd2) (Double point)

Astd1 - Astd2

Where, the subscripts std.1 and std. 2 refers to the more concentrated standard and

less concentrated standard respectively.



5.4 Assay of Substance in Multicomponent Sample

The spectrophotometric assay of drugs rarely involves the measurement of

absorbance of samples containing only one absorbing component. The

pharmaceutical analyst frequently encounters the situation where the

concentration of one or more substance is required in samples known to contain

other absorbing substances, which potentially interfere in the assay. If the recipe

of the sample formulation is available to the analyst, the identity and

concentration of the interferents are known, the extent of interference in the assay

may be determined. Alternatively interference, which is difficult to quantify, may

arise in the analysis of formulation from manufacturing impurities, decomposition

products and formulation excipients. Unwanted absorption from these sources is

termed irrelevant absorption and if not removed, imparts a systematic error to the

assay of the drug in the sample. A number of modifications to the simple

spectrophotometric procedure described above for single-component samples is

available to the analyst, which may eliminate certain sources of interference and

permit the accurate determination of one of the absorbing component. Each

modification of the basic procedure may be applied if certain criteria are satisfied.

5.5 Assays as Single-Component Sample

The concentration of a component in a sample which contains other absorbing

substance may be determined by a simple spectrophotometric measurement of

absorbance as described above, provided that the other component have a

sufficiently small absorbance at the wavelength of measurement. This condition

is satisfied if the concentration of the interfering substances and their absorptivity

or the path length of the solution is sufficiently small that their product (i.e. the

absorbance) can be ignored. A systematic error of less than 1% would normally

be considered to be acceptable. For example, if the contribution to a total

absorbance of 1.00 from the interferents is less than 0.01 and if there is no

chemical interaction between the components. The sample may be analysed for

its principal absorbing component by a simple direct measurement of absorbance

at its max.



5.6 Assay Using Absorbance Corrected for Interference

If the interference from other absorbing substance is large or if its contribution to

the total absorbance cannot be calculated, it may be possible to separate the

absorbing interferents from the analyte by solvent extraction procedures. The

judicious choice of pH of the aqueous medium and of immiscible solvent may be

affect the complete separation of the interferents from the analyte, the

concentration of which may be obtained by a simple measurement of absorbance

of extract containing the analyte.

5.7. Dual Wavelength Spectroscopy

The principle for Dual wavelength method is “the absorbance difference between

two points on the mixture spectra is directly proportional to the concentration of

the component of interest”. The two-wavelength data processing programme is

based on the above principle and can be utilized to a great extent without much

complication to calculate the concentration (unknown) of particular of interest in

a mixture. The two-wavelength calculation is expressed as:

The following mathematical discussion will show that the absorbance difference

between two wavelengths (1&2) on the mixture spectra is directly proportional

to the component of interest, independent of the individual interfering component.

Since, AA1 = AB1 + Ac2

AA2 = AB2 + Ac2

then, AA1 = AA2 (AB1=Ac1)-AB1+Ac2)

= (AB1=AB2) + (Ac1+Ac2)

Since, 2 is chosen such that (Ac1=Ac2)

Then, AA1-AA2+AB1+AB2.

Let, b= Concentration of the pure component of interest B.

D= Concentration of the pure interfering component C.

Then from beer‟s law we get,

AB1=KB1 b, and AB2=KB2. b

Where, KB1= absorption coefficient of the component of interest at1.

KB2 = absorption coefficient of the component of interest at 2.

Therefore,



AA1 = AA2 = (KB1.b) – (KB2.b)

(KB1=AB2). B

Since the expression (KB1 = AB2) is simply a constant, then

AA1 = AA2 = Constant x b.

Thus, it clearly can be seen that absorbance difference (AA1 – AA2) between two

points (1 and 2) on the mixture curve is directly proportional to the

concentration (Cx) of the component of interest (B) independent of the interfering

component (C).

5.8. Simultaneous Equation Method

If a sample contains two absorbing drugs X and Y, each of which absorbs at the

wavelength maximum of the other, it may possible to determine the

concentrations of both drugs by the techniques of simultaneous equations

(Vierordt‟s method) provided that certain criteria may apply. The information

required is:-

1) The absorbtivities of X at 1 and 2 are ax1 and ax2 respectively.

2) The absorbtivities of Y at 1 and 2 are ay1 and ay2 respectively.

3) The absorbance of the diluted sample at 1 and 2 are A1 and A2 respectively.

Let Cx and Cy be the concentrations of X and Y respectively in the dilute sample.

Two equations are constructed based upon the face i.e.at at 1 and 2 the

absorbance of the mixture in the sum of individual absorbance of X and Y.

At 1, A1 = ax1bcx + ay1bcy………eqa-1

At 2, A2 = ax2bcx + ay2bcy………..eqa-2

If b=1 cm, equation (2) becomes,

Substitution of Cy in equation (1) gives,

5.9. Graphical Absorbance Ratio Method

The method depends upon the property that, for a substance, which obeys Beer‟s

law at all wavelengths, the ratio of absorbance at any two wavelengths, are a

constant value independent of concentration of path length. In the USP, this ratio

Cx= (A2ay1 – A1ay2)

Ax2ay1 – ax1a2

Cy= (A1ay2 – A2ax1)

Ax2ay1 – ax1ay2



is referred to as Q value, the wavelength of maximum absorption of the two

components. A simple straight- line graphs can be drawn to show the relationship

between the absorbance ratio and the fraction or relative concentration of the two

components. The concentration of individual component may be calculated by

mathematical treatment of the simultaneous equation.

Cx and Cy be the concentration of X and Y respectively, in the sample.

CX = Qm-Qy/Qx-Qy)×A1 /ax1

CY = Qm-Qx/Qy-Qx)×A1 /ay1

Where,

Qm = A2/A1

A1 = absorbance of sample at isoabsorptive point.

A2 = absorbance of sample at max of one of the two components.

Qx = ax2 / ax1

Qy = ay2 / ay1

Where, ax1 and ay1 is absorbtivity of X and Y at isoabsorptive points.

ax2 and ay2 - absorptivity of X and Y at max of one of the two components

5.10. Derivative Spectrophotometry

In derivative spectrophotometry, the first to fourth even higher order derivative of

absorbance with respect to wavelength is employed as the ordinate in plotting

spectral data. Such plots often reveal spectral details that are less apparent in

normal absorption spectral plot and hence, can be utilized for analysis of a

substance in the presence of other substance too.

5.11. Difference Spectrophotometry

The essential feature of difference spectrophotometric assay is that the measured

value is the difference in absorbance (dA) between two equimolar solutions of the

analyte in different chemical forms: which exhibit different spectral

characteristics. The criteria of applying difference spectrophotometry to the assay

of a substance in the presence of other absorbing species are that,

1. Reproducible changes may be induced in the spectrum of the analyte of one or

more reagent.

2. The absorbance of the interfering substance is not altered by the reagents.



5.12. Orthogonal Polynomial Method

This technique is another mathematical correction method, which involves

complex calculations. The basis of this method is that an absorption spectrum

may be represented in terms of orthogonal functions as follows:-

A() = Po Po (1) + P1 P1 (2) + ……. pn Pn (n)

Where, A denotes the absorbance of wavelengths belonging to a set of (n+1)

equally spaced wavelengths, at which orthogonal polynomials P0 (), P1 (1)… Pn

(n) are each defined.

6. ANALYTICAL METHOD VALIDATION84, 85

Method validation is the process of demonstrating that analytical procedures are

suitable for their intended use. The method validation process for analytical

procedures begins with the planned and systematic collection of data by the

analyst, which support the analytical procedures. The analyst evaluates the

analytical procedures and validation data is submitted in the NDA or ANDA.

Each BLA (Biologic license application) and PLA (product license application),

must include a full description of the manufacturing methods, analytical

procedures, that demonstrate, that the manufactured product meets prescribed

stands of safety, purity, potency, accuracy and reliability. Also validation method

for method development should be submitted, to prove that the method is suitable

for its intended use. All analytical procedure is of equal importance form a

validation perspective.

In CGMP guidelines of March 28, 1979, the need for validation was done into

sections.

1. Section 211.145, where the word validation was used.

2. Section 211.194, where the proof of suitability, accuracy and reliability

was made compulsory for regulatory submissions.

Subsequently, a guideline was issued on 1st Feb 1987, for submitting samples and

analytical data for method validation. The international conference on

harmonization (I.C.H.) has published a guideline for “validation of analytical

procedures”.



6.1 Validation Characteristics

For validation of analytical methods applicant should follow characteristics or

parameters needed for validation according to ICH Q2A and ICH Q2B.

Typical validation characteristics are:

1. Linearity

2. Range

3. Accuracy

4. Precision

Repeatability

Intermediate precision

Reproducibility

5. Limit of Detection (LOD)

6. Limit of Quantitation (LOQ)

7. Specificity

8. Robustness

9. Ruggedness

Table 1.3: Type of Validation Required for Analytical Methods

Type of analytical

procedure

characteristics

Identification Testing for

impurities

Assay, dissolution

(measurement

only) content /

potency

Accuracy - + - +

Precision :

Repeatability - + - +

Intermediate Precision - + (1) - + (1)

Specificity (2) + + +

Detection limit - - (3) + -

Quantitation limit - + - -

Linearity - + 1 +

Range - + - +

(-) Signifies that this characteristic is not normally evaluated

(+) Signifies that this characteristic is normally evaluated

(1) In cases where reproducibility has been performed, intermediate

precision is not needed

(2) Lack of specificity of one analytical procedure could be

compensated by other supporting analytical procedures

(3) May be needed in some cases



6.1.1. Linearity

The linearity is the ability of analytical procedure to produce test results which are

proportional to the concentration (amount) of analyte in samples within a given

concentration range, either directly or by means of a well-defined mathematical

transformation. Linearity should be determined by using a minimum of six

standards whose concentration span 80 –120% of the expected concentration

range.

The linearity of a method should be established by visual inspection of a plot of

analytical response as a function of analyte concentration. If there is a linear

relationship, test results should be evaluated by appropriate statistical methods,

for example, by calculation of the regression line by the method of least squares.

Reports submitted must includes, the slope of the line, intercept and correlation

coefficient data. The measured slope should demonstrate a clear correlation

between response and analyte concentrations. The results should not show a

significant deviation from linearity, which is taken to mean that the correlation

coefficient, r > 0.99, over the working range (80-120%).

6.1.2. 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.

6.1.3. Accuracy

The accuracy of an analytical method is defined as the degree to which the

determined value of analyte in a sample corresponds to the true value. Accuracy

may be measured in different ways and the method should be appropriate to the

matrix. Analysing a sample of known concentration and comparing the measured

value to the „true‟ value. However, a well characterised sample (e.g., reference

standard) must be used.

The accuracy of an analytical method may be determined by any of the following

ways:



Spiked-Placebo (Product Matrix) recovery method- In the spiked-placebo

recovery method, a known amount of pure active constituent is added to

formulation blank [sample that contains all other ingredients except the active(s)],

the resulting mixture is assayed, and the results obtained are compared with the

expected results.

Standard addition method- In the standard addition method, a sample is

assayed, a known amount of pure active constituent is added, and the sample is

again assayed. The difference between the results of the two assays is compared

with the expected answer.

Recommended data

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). Accuracy

should be reported as percent recovery by the assay of known added amount of

analyte in the sample or as the difference between the mean and the accepted true

value together with the confidence intervals.

6.1.4. Precision

The precision of an analytical procedure expresses the closeness of agreement

(degree of scatter) between a series of measurements obtained from multiple

sampling of the same homogeneous sample under the prescribed conditions.

Precision may be considered at three levels: repeatability, intermediate precision

and reproducibility.

For these guidelines, a simple assessment of repeatability will be acceptable. The

precision of an analytical procedure is usually expressed as the variance, standard

deviation or coefficient of variation of a series of measurements. A minimum of 5

replicate sample determinations should be made together with a simple statistical

assessment of the results, including the percent relative standard deviation. If

considered appropriate, a suitable test for outliers (Dixon‟s or Grubbs Test) may

be applied to the results. Where outliers have been discarded that fact must be

clearly indicated. An explanation as to the reason for the occurrence of individual

outliers must be attempted.



(a) Repeatability

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 100% of the test concentration.

(b) Intermediate precision

The extent to which intermediate precision should be established depends on the

circumstances under which the procedure is intended to be used. The applicant

should establish the effects of random events on the precision of the analytical

procedure. Typical variations to be studied include days, analysts, equipment, etc.

It is not considered necessary to study these effects individually. The use of an

experimental design (matrix) is encouraged.

(c) Reproducibility

Reproducibility is assessed by means of an inter-laboratory trial. Reproducibility

should be considered in case of the standardisation of an analytical procedure, for

instance, for inclusion of procedures in pharmacopoeias. This data is not part of

the marketing authorization dossier.

6.1.5. Limit of detection

The detection limit of an analytical procedure is the lowest amount of an analyte

in a sample that can be detected, but not necessarily quantitated as an exact value.

The LOD may be determined by the analysis of samples with known

concentrations of analyte and by establishing the minimum level (lowest

calibration standard) at which the analyte can be reliably detected. The lowest

calibration standard which produces a peak response corresponding to the analyte

should be measured n times (normally 6-10). The average response (X) and the

standard deviation (SD) calculated. The LOD is X + (3 x SD).

6.1.6. Limit of quantitation

The limit of quantitation is the lowest amount of the analyte in the sample that can

be quantitatively determined with defined precision under the stated experimental

conditions. The limit of quantitation is a parameter of quantitative assays for low

levels of compounds in sample matrices and is used particularly for the



determination of impurities and/or degradation products or low levels of active

constituent in product.

The LOQ may be determined by preparing standard solutions at estimated LOQ

concentration (based on preliminary studies). The solution should be injected and

analyzed n times (normally 6-10). The average response and the standard

deviation (SD) of the n results should be calculated and the SD should be less

than 20%. If the SD exceeds 20%, a new standard solution of higher

concentration should be prepared and the above procedure repeated. The LOQ is

X + (10 x SD).

Approach-1: Based on visual evaluation

Visual evaluation may be used for non-instrumental methods but may also be used

with instrumental methods. The detection limit is determined by the analysis of

samples with known concentrations of analyte and by establishing the minimum

level at which the analyte can be reliably detected.

Approach-2: Based on signal-to-noise ratio

This approach can only be applied to analytical procedures which exhibit baseline

noise. Determination of the signal-to-noise ratio is performed by comparing

measured signals from samples with known low concentrations of analyte with

those of blank samples and establishing the minimum concentration at which the

analyte can be reliably detected. A signal-to-noise ratio between 3 or 2:1 is

generally considered acceptable for estimating the detection limit.

Approach-3: Based on the standard deviation of the response and the slope

The detection limit (DL) expressed as:

DL = 3.3

s

= standard deviation of the response s = Slope of the calibration curve

The quantitation limit (QL) may be expressed as:

QL = 10

s

Several approaches for determining the detection limit (LOD) and the quantitation

limit (LOQ) are possible, depending on weather the procedure is a non-

instrumental or instrumental. The slope may be determined from the calibration



curve of the analyte. The estimation of s may be carried out in variety of ways, for

example.

(1) Based on the standard dividing of number of blank samples

(2) Based on Calibration curve.

A specific calibration curve should be studied using an analyte in the range

detection limit. 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.

6.1.7. Specificity

Selectivity of a method refers to the extent to which it can determine particular

analyte(s) in a complex mixture without interference from other components in

the mixture. The terms selectivity and specificity have often been 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 very few analytical methods

respond to only one analyte, the use of the term selectivity is more appropriate

than specificity. The International Union of Pure and Applied Chemistry (IUPAC)

have expressed the view that “Specificity is the ultimate of Selectivity‟.

The selectivity of the analytical method must be demonstrated by providing data

to show the absence of interference peaks with regard to degradation products,

synthetic impurities and the matrix (excipients present in the formulated product

at their expected levels).

6.1.8. Selectivity

It an analytical procedure is able to separate and resolve the various components

of mixtures and detects the analyte quantitatively, the method is called as

selectivity. It is an essential requirement for all types of methods used in

identification. Spectroscopic, chromatographic or chemical selectivity is restricted

to quantitative detection of the components of a sample matrix and it is applicable

to separate methods.



Identification

It insures the identity of an analyte. Suitable identification tests should be able to

discriminate between compounds of closely related structures, which are likely to

be present. The discrimination of a procedure may be confirmed by obtaining

positive results (by comparison with a known reference material) from samples

containing the analyte, coupled with negative results from samples which don‟t

contain the analyte. In addition identification test may be applied to materials

structurally similar to or closely related to the analyte, to conform that, the

positive response is not obtained.

Assay and impurity test

For chromatographic procedure, representative chromatograms should be used to

demonstrate specificity and individual components should be labeled

appropriately. Similar considerations should be given to other separation

techniques. Critical separation in chromatography should be investigated at an

appropriately level for critical separation. Specificity can be demonstrated by the

resolution of the two components, which eluted closest to each other. In case

where a non-specific assays is used to demonstrate overall specificity. The

approach is similar for both assay and impurity tests.

6.1.9. Robustness

The evaluation of robustness should be considered during the development phase.

It should show the reliability of an analysis with respect to 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. One consequence of

the evaluation of robustness should be that a series of system suitability

parameters (e.g., resolution test) is established to ensure that the validity of the

analytical procedure is maintained whenever used:-

Influence of variations of pH in a mobile phase,

Influence of variations in mobile phase composition,

Different columns (different lots and/or suppliers),

Temperature



6.1.10. Ruggedness

The ruggedness of an analytical method is the degree of reproducibility of test

result obtained by the analysis of the same, under a variety of normal test

conditions, such as different laboratories, reagents, analyst, instruments, days,

assay temperature, etc. It is normally expressed as the lack of influence on test

results of operational and environmental variables of the analytical method. To

determine the methods ruggedness, the results obtained after the changes have

been implemented with the assays, should be compared with the precision of the

assay under normal conditions.

7. STATISTICAL PARAMETER

There is some statistical parameter listed below and their formula for calculation

Table 1.4: Commonly Used Statistical Parameters and their Calculation

S. No. Statistical

Parameter

Formula Significance

1. Arithmetic mean X= x1 +x2 + ….+xn

n

Easy and ideal measures of central

tendency.

Very much affected by extreme

observation

2. Standard

deviation

(S)

S = (x –x)2 / n-1

Summarises the deviation of a

large distribution from mean in

one figure used as a unit of

variation.

For small observations, it does not

estimate the range within which

the true mean may be found

3. Relative standard

deviation (RSD)

RSD = s/x A measure of precision

4. Coefficient of

variance (CV)

CV = S x 100/x Percentage of RSD.

Compare the variability of two or

more than two series.

5. Standard error of

mean (Sex)

SEx = s/ n

Improved by increasing the

number of measurements

6. Standard error of

standard

deviation

SE = s/ 2n

Significance of standard deviation

7. Confidence of

Interval

= x + ts

n

Estimate the range within which

the true mean may be found

Where, x = value of particular value, n = number of observations. = true value,

t = a parameter that depends upon the number of degrees of freedom v and

confidence level required, V = (n=1) = degree of freedom

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