10

Chapter-2

2.1. Stages during quality control testing

Quality control is of crucial importance to the pharmaceutical industry

and for this reason numerous checks are made at every stage of production

to ensure that the quality is not compromised and that the code of Good

Manufacturing Process is adhered to quality control procedure include,

Sampling of raw materials.

All incoming raw materials are initially quarantined, and samples are

taken and tested to ensure that the material meets strict purity guidelines.

This testing involves both microbiological and chemical testing, as is laid out

in the relevant Pharmacopeia [1].

In process checks:

The manufacturing staff carries out checks on such things as, purity

of intermediates, Uniformity at intermediate levels and the parameters which

will effect on the production. At hourly intervals the quality control staff

takes samples to check for contamination and to ensure that the

composition is as expected.

Final product checking:

Checking the similar parameters to those measured during

production. Validated methods are used for final product checking.

Monitoring cleaning:

When a batch of certain drugs has been made, all equipment that has

been used must be cleaned. When the next pharmaceutical to be made on

11

that line is going to different, this cleaning must be particularly thorough to

prevent contamination. In this instance, after cleaning the quality control

staff take swabs off each piece of equipment and test them to see if they can

detect the presence active previously used.

Only when the equipments so clean that the previous active is

undetectable or below certain level which will not cause any effect on the

next production can the production of the next pharmaceutical commence.

The results of these tests are recorded on the batch records for the

pharmaceutical, as well as the name and batch number of the

pharmaceutical made immediately prior on the same production line.

2.2. Active Pharmaceutical Ingredient (API) (or Drug Substance)

Any substance or mixture of substances intended to be used in the

manufacture of a drug (medicinal) product and that, when used in the

production of a drug, becomes an active ingredient of the drug product.

Such substances are intended to furnish pharmacological activity or other

direct effect in the diagnosis, cure, mitigation, treatment, or prevention of

disease or to affect the structure and function of the body.

Modern medicines for human use are required to meet exacting

standards that relate to their quality, safety and efficacy. The evaluation of

the above factors in practice depends on the existence of adequate methods

for quality control of the drug substance and drug product. Thus great

demands are placed on the analytical methods that are used for the

determination of active drug and its related impurities in bulk drug

substance.

12

Drug substances are generally not administered in their native form.

The medicine is the whole pharmaceutical formulation (also called as dosage

form) in which the drug, that is active substance, combined with other

ingredients (also called as excipients) to form a convenient form of

administration, such as tablet, capsule, injection, ointment etc.

During manufacturing of the drugs by products, unreacted raw

materials, degradation products are the major impurities in drug

substances. These impurities often possess unwanted pharmacological or

toxicological effects like genotoxicity, carcinogenity due to which any benefit

from the administration of the drug is outweighed. In addition to above, the

impurities can be formed during the storage of drug substances due to

degradation. So it is very important to monitor the level of impurities present

in the drug substance during manufacturing, batch release and during its

storage.

It is very important for the analyst to develop a suitable analytical

method for the drug substance, which can monitor the level of impurities

and content during its manufacturing and release. The target of the analyst

is not only to test and release the batch and must ensure that the same

method shall also be used during the stability testing of drug substance

(stability-indicating).

An “expiry date” will be mentioned on the prescription or over-the-

counter (OTC) pharmaceutical products. Before this date, the product should

remain fully effective under normal storage conditions. The efficacy and

safety of pharmaceuticals cannot be ensured unless the quality of the

pharmaceuticals is maintained during their specified shelf lives. The analysis

13

of stability samples must be carried out with the use of stability-indicating

analytical methods (SIAMs).

A SIAM is a quantitative analytical procedure used to detect a

decrease in the amount of active pharmaceutical ingredient (API) present due

to degradation. During the Quality Control testing, batch release and

stability studies of a drug substance the HPLC technique is routinely used to

separate and quantitate the analytes of interest.

2.3. Drugs

Drug is a chemical substance that is used in the diagnosis, cure,

relief, treatment, or prevention of a disease. In medicine, a drug refers to any

substance with a potential to prevent or cure disease or enhance physical or

mental welfare and in pharmacology, to any chemical agent that alters the

biochemical and physiological processes of tissues or organisms [2]. The

medicinal value of plants has been recognized by almost every society on this

planet. In the earlier days, plant material (leaves or bark) extracts,

particularly provided the main source of folk medicines.

The idea that the effect of a drug in human body is mediated by

specific interactions of the drug molecule with biological macromolecules,

(proteins or nucleic acids in most cases), led scientists to consider that

individual chemicals may function well for interacting with biological

macromolecules and results in controlling their malfunctions. This made the

beginning of the modern era in pharmacology that pure chemicals, instead of

crude extracts, can be used as better drugs.

2.3.1. Drug development

14

In the latter part of the nineteenth century, biologically-active organic

molecules began to be isolated in relatively pure form for medicinal use. For

example, salicylic acid, the precursor of Aspirin, was isolated in 1874 from

willow bark. Various more potent painkillers, such as Morphine and

Codeine, were isolated from the opium poppy. The anti-malarial agent

Quinine was separated from cinchona (china bark). The leaves of the purple

foxglove plant provided an excellent source of digitalis that was purified for

use against heart disease. The synthesis of the first synthetic

pharmaceutical drug, aspirin, occurred in the latter half of the nineteenth

century,

Alexander Fleming [3] noticed that a fungus growing on an agar plate

had inhibited the growth of bacteria in the year 1928. Ten years later,

Howard Florey and Ernst Chain [3] isolated the chemical penicillin that was

affecting the bacteria. Penicillin is still most widely used antibiotic for the

treatment of bacterial diseases. The discovery of the antibiotics

Streptomycin, Chloramphenicol, and chlortetracycline followed the discovery

of penicillin.

The introduction of Salvarsan for the treatment of syphilis by the

German hematologist Paul Ehrlich in 1911 [4] paved the way for the

introduction modern drugs like sulphonamide in 1930. Recent advances in

X-ray crystallography, NMR spectroscopy, mass spectrometry, developments

in electrophoresis, ultracentrifugation, chromatography etc. helped the

discovery of additional chemical entities with therapeutic activities.

15

The development of a new drug may take a minimum of 10 years. In

this long process, the substances that were identified in basic research need

to pass through pre-clinical & clinical tests. Pharmaceutical companies quite

often research and test around 30,000 different substances before one could

be successfully introduced into the market. Typical process flow chart for a

new drug is depicted in Figure 2.1.

Many aspects of drug development are focused on the following

regulatory requirements of the drug licensing authorities. They generally

constitute a number of tests designed to determine the major toxicities of a

novel compound prior to its use in human. It is a legal requirement that an

assessment of major organ toxicity be performed like effects on heart, lungs,

brain, kidney, liver and digestive system, as well as effects on other parts of

the body that might be affected by the drug.

For example, skin, if the new drug is to be delivered through the skin.

While these tests can be made using in vitro methods i.e. with isolated cells,

many tests are being done only using experimental animals, since it is in an

intact organism that the complex interplay of metabolism and drug exposure

on toxicity can be performed.

16

Figure 2.1: Typical process flow chart for the development of a new

drug

It is reported that average cost may be around $800 million to bring a

new drug in to the market for the beneficial use of people [5]. By law, drugs

are divided into two categories: prescription drugs and nonprescription

drugs [6]. Prescription drugs are those considered safe for use only under

medical supervision and may be dispensed only with a prescription from a

licensed professional Doctor with governmental privileges to prescribe.

Nonprescription drugs, those considered safe for use without medical

supervision and are sold over the counter. In the United States, the Food

and Drug Administration (FDA) is the government agency that decides which

drugs require a prescription and which may be sold over the counter.

2.4. Impurities and their origin

Impurities in drugs are the unwanted chemicals that remain with the

drug or active pharmaceutical ingredients (APIs), or develop during

formulation, or developed upon aging of both API and formulated products

[7]. The presence of these unwanted chemicals even in small amounts may

influence the efficacy and safety of the pharmaceutical products. Impurities

can have a strong adverse effect on human being due to undesirable

pharmacological and toxicological action, which can prevail over the positive

effect of the medicine, and can hinder the positive effect of the main

medicinal substance.

2.4.1. Nature of impurities

17

Medicines are the formulated forms of active pharmaceutical

ingredients. There are two types of impurities in medicines: (a) Impurities

associated with the active pharmaceutical ingredients and (b) Impurities that

are created during formulation and or with aging or that are related to the

formulated forms. Impurities associated with the active pharmaceutical

ingredients are classified into three categories as specified in the

International Conference of Harmonization (ICH) guidelines [8, 9 ].

Organic impurities that are either process and/or drug-related

Inorganic impurities

Residual solvents

2.4.2. Organic impurities

Organic impurities may come up in the manufacturing process and/or

during storage of the drug substance. They may generate during the

synthetic processes of a drug substance and due to degradation reactions of

the drug substances by the environmental parameters like temperature,

humidity, sunlight. The impurities can be originated from the following. (a)

Starting materials, (b) Intermediates, (c) By-products, (d) Enontiomers, (e)

Degradation products, (f) Reagents, ligands and catalysts

Starting materials & Intermediates: These are the most common

impurities found in every API unless a proper care is taken in every step

involved throughout the multi-step synthesis. Although, at the end of each

process step the products are always washed with solvents, there is every

chance that small concentration of these solvents remains with the main

drug. The non reacting impurities in the starting materials may also remain

18

with the drug unless the manufacturers are very careful about the

impurities.

In Paracetamol bulk drug substance; there is a limit test for p-

aminophenol, which is the starting material. This is clear from the synthesis

of Paracetamol shown in Figure 2.2. Impurities present in the staring

materials could also follow the same reaction pathways similar to the

starting material, and the reaction products could carry over to the final

product as process impurities. Knowledge of the impurities in the starting

materials helps to identify related impurities in the final product, and to

understand the formation mechanisms of these related process impurities.

By-products: In synthetic organic chemistry, getting a single end

product with 100% pure is very rare; there is always a chance of having

some by-products. In the case of Paracetamol bulk, shown in Figure 2.2

diacetylated paracetamol is the by-product which will remain as process

related impurity in the drug.

Figure 2.2: Synthesis route of Paracetamol showing the origin of

impurities.

Enantiomeric impurities: The single enantiomeric form of a chiral

drug is now considered as an improved chemical entity that may offer a

better pharmacological profile and an increased therapeutic index. For the

19

manufacturers of single enantiomeric drug, the undesirable stereo isomers

in the drug are considered in the same manner as the other organic

impurities. The prominent single isomer drugs, which are being marketed,

includes levocetrizine , esomeprazole, Eszopiclone etc. [10,11,12].

Degradation products: Impurities can also be formed due to

degradation of the drug during manufacturing of bulk drugs. However,

degradation of drug resulting from storage or formulation to different dosage

forms or aging are common impurities in the medicines. Also degradation

occurs by few pathways but the most often seen mechanism is hydrolysis.

Many drugs will show tendency of decomposition by hydrolysis.

In case of aspirin [13] the degradation of aspirin in various buffer

solutions was studied and the degradants are identified as salicylic acid and

acetic acid Figure 2.3. Oxidation and photolysis are the next two

mechanisms that degrade the main drug. From Figure 2.4, it can be seen

that exposure of Ciprofloxacin to light will lead to photolysis and formation

of Ethylene diamine analog of Ciprofloxacin [14].

Figure 2.3: Degradation of Aspirin by hydrolysis

20

N

O

OH

O

F

N

NH

N

O

OH

O

F

NH

NH2

Sun Ligjt

Figure 2.4: Degradation of Ciprofloxacin by photolysis

Reagents, Ligands and Catalysts: These chemicals are less commonly found

in pharmaceutical compounds; however, in some cases they may pose a

problem as impurities .Chemical reagents, ligands, and catalysts used in the

synthesis of a drug substance can be carried over to the final products as

trace level impurities. For example, carbonic acid chloromethyl tetrahydro-

pyran-4-yl ester (CCMTHP), which is used as an alkylation agent in the

synthesis of ß lactam drug substance, is observed as an impurity in the final

product.

Metal-based catalysts promote many chemical reactions. For example,

use of Ziegler-Natta catalyst may result in Ti or TiO2 as impurity in the

main drug, Grubb‟s catalyst ruthenium, and Adam‟s catalyst platinum. In

some cases, reagents or catalysts may react with intermediates or final

products to form by-products. Pyridine, a catalyst used in the course of

synthesis of mazipredone, reacts with an intermediate to form a pyridinium

impurity [15].

2.4.3. Impurities that are formed during formulation

Apart from bulk drug-related impurities, the formulated form of API

may contain impurities that originated from various ways. Impurities arising

21

from excipients present in the drug product or extracted or leached from the

container system, due to environmental factor such as temperature, light

particularly UV light, humidity and storage conditions of the drug called

aging. A known impurity, 1-(2,6-diclorophenyl)indolin-2-one is formed in the

production of a parenteral dosage form of diclofenac sodium, if it is

terminally sterilized by autoclave. It is the condition of the autoclave method

(i.e., 123 + 2°C) that enforced the intramolecular cyclic reaction of diclofenac

sodium forming the indolinone derivative and sodium hydroxide.

In general, liquid dosage forms are very much susceptible to both

degradation and microbiological contamination. In this regard, water

content, pH of the solution/suspension, compatibility of anions and cations,

mutual interactions of ingredients, and the primary container are critical

factors. Ciprofloxacin (0.3% preparation) is available on the market for

topical ophthalmic use. If the ophthalmic solutions are exposed to sun light,

they may lead to formation of a degradation product, i.e. ethylenediamine

derivative of ciprofloxacin.

2.4.4. Inorganic impurities

Inorganic impurities arise from the manufacturing process, from

reagent, ligands, and catalysts. Inorganic salts, heavy metals, filter aids and

charcoal falls under this category. Metal residues also fall under this

category. Metal residues in pharmaceutical substances or the drug products

may originate from several sources like metal catalysts and metal reagents

used during the synthesis of the active pharmaceutical substance and the

excipients, manufacturing equipment and piping, bulk packaging and the

22

environment. As per European medicine agency recommendation [16], limits

for metal residues in the pharmaceutical substance are presented in Table

2.1.

Table 2.1: Specification limits for metal residues in pharmaceutical

products

Classification Oral Exposure Parenteral Exposure

Permitted

Daily

Exposure

(μg/day)

Concentration

(ppm)

Permitted

Daily

Exposure

(μg/day)

Concentratio

n

(ppm)

Class 1A: Pt, Pd

Class 1B: Ir, Rh, Ru,

Os

Class 1C: Mo, Ni, Cr, V

Metals of significant

safety concern

100

100

250

10

10

25

10

10

25

1

1

2.5

Class 2: Cu, Mn

Metals with low safety

concern

2500 250 250

25

Class 3: Fe, Zn

Metals with minimal

safety concern

13000 1300 1300 130

2.4.5. Residual solvents

Residual solvents may be present in pharmaceutical substance as they

are used or produced in the manufacture of the drug substances or

excipients, or in the preparation of drug products. Appropriate selection of

the solvent for the synthesis of the drug substance may enhance the yield, or

determine characteristics such as crystal form, purity, and solubility.

23

Therefore, the solvent may sometimes be a critical parameter in the

synthetic process. Since there is no therapeutic benefit from residual

solvents, all residual solvents should be removed to the extent possible to

meet product specifications. The solvents are not completely removed by

practical manufacturing techniques.

International conference on Harmonization (ICH) classified the solvents

based on the toxicity of solvents in to three classes, Class-1, Class-2, and

Class-3 [17]. Solvents which are known human carcinogens, strongly

suspected human carcinogens, and environmental hazards are classified

into Class 1, solvents that are to be avoided. The details of solvents,

concentration limits in the main drug and the effects they cause if in excess

are presented in the Table 2.2.

Table 2.2: Class 1 solvents and their specification limits in

pharmaceutical products

Solvent Concentration limit (ppm) Concern

Benzene 2 Carcinogen

Carbon tetrachloride 4 Toxic and environmental

hazard

1,2-Dichloroethane 5 Toxic

1,1-Dichloroethene 8 Toxic

1,1,1-

Trichloroethane 1500 Environmental hazard

Class 2 solvents: Solvents to be limited, Non-genotoxic animal carcinogens or

possible causative agents of other irreversible toxicity such as neurotoxicity

24

or teratogenicity. These are the solvents suspected of other significant but

reversible toxicities. The details of these solvents are presented in Table 2.3.

Class 3 solvents: Solvents with low toxic potential to human; no health-based

exposure limit are classified as Class 3. An exposure of 50 mg or more per

day and concentration limit is 5000 ppm is permitted for these solvents. The

list of such solvents is presented in Table 2.4.

Table 2.3: Class 2 solvents and their specification limits in

pharmaceutical products

Solvent Permitted Daily

Exposure (mg/day)

Concentration

limit (ppm)

Acetonitrile 4.1 410

Chlorobenzene 3.6 360

Chloroform 0.6 60

Cyclohexane 38.8 3880

1,2-Dichloroethene 18.7 1870

Dichloromethane 6.0 600

1,2-Dimethoxyethane 1.0 100

N,N-Dimethylacetamide 10.9 1090

N,N-Dimethylformamide 8.8 880

1,4-Dioxane 3.8 380

2-Ethoxyethanol 1.6 160

Ethylene glycol 6.2 620

Formamide 2.2 220

Hexane 2.9 290

Methanol 30.0 3000

2-Methoxyethanol 0.5 50

Methylbutyl ketone 0.5 50

Methylcyclohexane 11.8 1180

N-Methylpyrrolidone 5.3 530

Nitromethane 0.5 50

Pyridine 2.0 200

Sulfolane 1.6 160

Tetrahydrofuran 7.2 720

Tetralin 1.0 100

25

Toluene 8.9 890

1,1,2-Trichloroethene 0.8 80

Xylene 21.7 2170

Table 2.4: List of Class 3 solvents

Solvents

Acetic acid Ethanol 3-Methyl-1-butanol

Acetone Ethyl acetate Methyl ethyl ketone

Anisole Ethyl ether Methyl isobutyl ketone

1-Butanol Ethyl formate 2-Methyl-1-propanol

2-Butanol Formic acid Pentane

Butyl acetate Heptane 1-Pentanol

tert-Butylmethyl ether Isobutyl acetate 1-Propanol

Cumene Isopropyl acetate 2-Propanol

Dimethyl sulfoxide Methyl acetate Propyl acetate

2.5. Quality Control of drugs

Quality is the most important factor for the products and

manufacturing firms. Quality control is a process by which entities review

the quality of all factors involved in production. Quality control is a

mechanism for ensuring that the product conforms to predetermined

specifications. Quality is important in every product but it is vital in

medicine as it involves life of human beings. Unlike ordinary consumer‟s

goods, there is no second quality in drugs.

Synthetic bulk drugs are produced in the production plants in large

quantities using different manufacturing technologies. The purity of a drug

and the nature and quantity of any impurities present in that drug are very

26

important for the modern pharmaceutical analyst. Synthetic precursors, by-

products, un-reacted raw materials, intermediates and products of

degradation are the potential components of manufacturing processes likely

to be present as impurities in the bulk drugs and their formulations.

Impurities in reagents or starting materials may be carried out intact

through the synthesis or react to produce new impurities. These impurities

often pose unwanted pharmacological or toxicological effects due to which

any benefit from the administration of the drug is outweighed. Impurities in

reagents or starting materials may be carried out intact through the

synthesis or react to produce new impurities.

Threshold for impurities specified in ICH impurities guideline [7],

which is mentioned in the following Table 2.5. In most of the cases, the daily

dosage of drugs may be less than 2 grams per day; hence general limit for

any unknown impurities should be less than 0.10% and for any identified

impurity limit is not more than 0.15%. As per ICH recommendation, the limit

of quantification (LOQ) requirement should be at least 50% target

concentration, in case of unknown impurities, the LOQ requirement is less

than 0.05%. Hence, the sensitivity of preferred method for determination of

impurities is very critical.

Table 2.5: ICH guidelines for impurities present in a drug

Maximum

Daily Dose

Reporting

Threshold

Identification

Threshold

Qualification

Threshold

2g/day 0.05% 0.10% or 1.0 mg per day

intake (whichever is

lower)

0.15% or 1.0 mg per

dayintake (whichever is

lower)

27

> 2g/day 0.03% 0.05% 0.05%

2.5.1. Different measurement techniques for impurities determination

a. Organic impurities

Majorly organic impurities like related substances, raw materials used

in process intermediates, by products and degradants are being determined

frequently by using thin layer chromatography (TLC), high performance

liquid chromatography (HPLC), and in case of volatile, thermally stable

materials, Gas chromatography (GC) is used. Other chromatographic and

electrophoretic techniques such as supercritical fluid chromatography,

capillary electrophoresis and capillary electro chromatography are also being

used. Mass spectroscopy (MS) and Nuclear magnetic Resonance (NMR) can

also contribute in the identification and determination of the impurities [14].

b. Inorganic impurities

For determination or inorganic impurities like chlorides, sulfate,

sulfites etc conventional gravimetric and titremetric techniques may be

adopted. Ion chromatographic techniques are being employed as they are

very specific and sensitive. The heavy metal concentration is reported to be

estimated by the conventional sulfide precipitation techniques [18]. As the

above techniques lack the sensitivity, specificity, and poor recovery to

monitor properly low levels of these metals, for the determination of toxic

28

metal impurities United States Pharmacopeia [19] proposed inductively

coupled plasma–atomic emission spectrometer ICP-AES and inductively

coupled plasma mass spectrometer ICP-MS methods.

c. Residual solvents

For Class 3 solvents, non-specific method such as loss on drying may

be used. Class 1 and Class 2 solvents are to be determined using

chromatographic techniques such as gas chromatography. For making

injection, “Head space” sampling technique is being employed to avoid

interferences of the product and to achieve desired sensitivity [20].

2.6. ICH activity and quality guidelines

The ICH was established in 1990 as a joint regulatory and industry

project to improve, through harmonization, the efficiency of the process for

developing and registering new medicinal products in Europe, Japan and the

United States of America, to make these products available to patients with a

minimum of delay. The six parties involved in ICH process represent the

regulatory bodies and pharmaceutical industry in the three regions, Europe,

Japan and the USA.

The parties involved from these three regions are – European

Commission (EC), European Federation of Pharmaceutical Industries and

Associations (EFPIA); Ministry of Health, Labor and Welfare (MHLW), Japan

Pharmaceutical Manufacturers Association (JPMA); and US Food and Drug

Administration (FDA), and the Pharmaceutical Research and Manufacturers

of America (PhRMA).

The fourth International Conference on Harmonization in 1997

29

marked the completion of the first phase of activities. In the first phase the

exercise was directed towards elimination of redundant and duplicates

technical requirements for registration in individual countries, and lay down

of minimum standards applicable uniformly irrespective of where the

product is manufactured and/or marketed in the three regions. This was

done with development of over 40 guidelines covering Efficacy, Quality and

Safety aspects of drug development. The list of „Quality‟ guidelines is given in

Table 2.6 [21].

Table 2.6: List of topics, codes and corresponding quality guidelines

developed by ICH

Topics /

Code Quality guidelines

Q1A(R2) Stability Testing of New Drug Substances and Products

Q1B Stability Testing: Photo stability Testing of New Drug Substances and Products

Q1C Stability Testing for New Dosage Forms

Q1D

Bracketing and Matrixing Designs for Stability Testing of Drug Substances and

Drug Products

Q1E Evaluation of Stability Data

Q1F Stability Data Package for Registration in Climatic Zones III and IV

Q2(R1) Validation of Analytical Procedures: Text and Methodology

Q3A(R2) Impurities in New Drug Substances

Q3B(R2) Impurities in New Drug Products

Q3C(R4) Impurities: Guideline for Residual Solvents

Q4 Pharmacopeias

Q5A(R1)

Viral Safety Evaluation of Biotechnology Products Derived From Cell Lines of

Human or Animal Origin

Q5B

Quality of Biotechnological Products: Analysis of the Expression Construct in

Cells Used for Production of R-DNA Derived Protein Products

Q5C

Quality of Biotechnological Products: Stability Testing of Biotechnological/

Biological Products

Q5D

Derivation and Characterization of Cell Substrates Used for Production of

Biotechnological/Biological Products

Q5E Comparability of Biotechnological/Biological Products Subject to Changes in Their Manufacturing Process

Q6A

Specifications: Test Procedures and Acceptance Criteria for New Drug

Substances and New Drug Products: Chemical Substances

30

Q6B Specifications: Test Procedures and Acceptance Criteria for Biotechnological/Biological Products

Q7 Good Manufacturing Practice Guide for Active Pharmaceutical Ingredients

Q8(R2) Pharmaceutical Development

Q9 Quality Risk Management

Q10 Pharmaceutical Quality system

2.7. Techniques applied during quality control testing

In the field of drug analysis, the analytical investigation of bulk drug

materials, the intermediates in their synthesis, products of drug research,

drug formulations, impurities and degradation products, and biological

samples containing the drugs and their metabolites is a very important area

of research. From the point of view of public health, the safety, efficacy, and

economy of drug therapy are extremely important issues.

The importance of drug impurity and stability-related issues has also

been characterized by a number of books and articles devoted to this subject

[22].The determination of drugs and metabolites in biological samples [23],

with particular attention to toxicological and forensic analysis, requires

special techniques and a special manner of thinking [24, 25], as reflected by

many books and articles on these issues.

Drug discovery requires a solid analytical background, with a great

variety of methods to be used. Innumerable drug-related chapters have also

appeared in general analytical books and special issues of scientific journals.

During synthesis of drugs and during their stability studies lot of impurities

may form which maybe organic, inorganic and residual solvents.

2.7.1. Application of Chromatographic Techniques

31

Rapid development of analytical methodology in pharmaceutical and

biomedical analyses has led to various forms of high-performance liquid

chromatography (HPLC) becoming undoubtedly the most important

methods. The theoretical and practical foundations for this method were laid

down at the end of 1960s and the beginning of 1970s [26]. The latter decade

was the period that saw a rapid spread of this technique.

In pharmaceutical analysis, the HPLC method shares its importance

with various techniques. HPLC has been used to solve no less than 50% of

the problems, leaving the other 50% to about 15 other chromatographic,

spectroscopic, and other methods, about 10% to gas chromatography (GC),

5% to thin-layer chromatography (TLC), 10% to ultraviolet (UV)

spectrophotometry, and the rest to electro analytical methods.

The contribution of HPLC in drug analysis has further increased in

this long period. For example, in 1983, a UV detector was applied almost

exclusively, leaving a little share to refractive index, fluorimetric, and

electrochemical detection, but in 2005, a mass spectrometer was applied as

a detector in about one-third of the analysis.

HPLC coupled with mass spectrometry, HPLC/MS (MS) or LC/MS (MS)

due to high sensitivity and selectivity have become the predominant method

in bioassays and pharmacokinetic and metabolic studies, as well as in the

structure elucidation of drug impurities and degradation products. A new

development in the field of HPLC/MS has been the introduction of column

packing with ultrafine particles (< 2 um)[27,28,29], enabling short columns

(5 cm or less) to be used, and rapid analyses (e.g., 5 minutes or even less

32

than 1 minute) to be carried out by ultra performance liquid

chromatography(UPLC).

In the compendial analysis of small organic molecules, the breakthrough

of HPLC was also extremely rapid. In the twenty-ninth United States

Pharmacopoeia, HPLC was applied to the assay of bulk drug materials of this

type in about 45% of the monographs [30]. This share was somewhat higher

than that of the non-selective ones, but with titration methods that were less

time-consuming, leaving only about 10% to other methods, mainly the

similar, non-selective UV-Vis spectrophotometry. HPLC and TLC were used

almost exclusively, with almost equal shares for the purity control of bulk

drug materials and the related compounds test.

Even more spectacular was the propagation of HPLC in the assay of

pharmaceutical formulations, which needed specific methods indicating

stability. No other method had spread so rapidly in pharmaceutical analysis.

Among other chromatographic methods the important application field of

modern TLC is the separation of the components of complex mixtures, for

example, impurities and degradation products of drug materials and extracts

of medicinal plants.

The speed and the resolution could be greatly improved by the

introduction of special techniques, such as high-performance thin-layer

chromatography (HPTLC), using ultra thin layers and coatings with ultrafine

particles or over pressured-layer chromatography (OPLC). The development

of densitometers enables classical TLC and the latter techniques could be

successfully used as tools for the quantitative analysis of complex mixtures.

33

The introduction and rapid spread of HPLC and HPLC/MS decreased

the importance of GC and GC/MS in pharmaceutical analysis. Nevertheless,

these are still important techniques in many fields of drug analysis [31],

where the analytes are volatile and thermally stable. In the past 15 to 20

years a new field application is being used for the determination of residual

solvents in drugs. Almost always the headspace technique is used to fulfill

the demanding requirements, that is, determination of solvents at the 10-

ppm level; and down to the ppm level in the case of carcinogenic or genotoxic

solvents.

2.7.2. Application of Capillary Electrophoresis

Since the introduction of the commercially available instruments

capillary electrophoresis (CE), related methods such as micellar electro

kinetic chromatography (MEKC), micro emulsion electro kinetic

chromatography (MEEKC), and capillary electrochromatography (CEC), have

attracted great interest in pharmaceutical analysis as possible alternatives

or amendments to HPLC [32].

This share is an underestimation, as there are researchers specializing

in CE analysis. It is an overestimation because, despite CE having several

advantages such as a flat flow profile that results in an extremely high

column efficiency, due to its limitations with regard to its general

applicability, it does not yet seem to be a real rival to HPLC in the practice of

compendial-industrial pharmaceutical analysis.

The separation and quantification of enantiomeric mixtures are among

the greatest challenges of the past years in pharmaceutical and biomedical

34

analysis. The main problems to be solved are, to determine the enantiomeric

purity of drugs being used in therapy as pure enantiomers and the

simultaneous determination of the components of race-mates in the

biological samples [33]. The latter type of enantiomeric separation has been

successfully adapted to CE. The present situation can be characterized by

the spread of this technique and the continuous development and

commercialization of new types of chiral HPLC columns [34].

2.7.3. Application of Spectroscopy

In pharmaceutical and biomedical analysis, the development of

nuclear magnetic resonance (NMR) and mass spectrometry (MS), along a

road paved with Nobel Prizes, has also been successfully exploited. The

dramatic decrease in the demanding requirements for sample size and the

solution of the difficult problems of interfacing these techniques with

chromatographic (and electrophoretic) separation methods have greatly

expanded their field of application.

In addition to the offline applications that are still widely used, online

HPLC/MS, HPLC/ NMR, HPLC/NMR/MS, and other hyphenated methods,

are becoming leading methods, for example, the structure elucidation of

drug impurities, degradation products, metabolites, and bioactive

components in natural products. Due to its high sensitivity and selectivity,

HPLC/MS/MS has become the predominant method, even in the

quantitation of these minor components (e.g., in pharmacokinetic and

bioequivalence studies) [35].

35

UV spectroscopy [36] is observable due to the availability of diode-

array detectors attached to HPLC and TLC densitometers, both suitable for

obtaining good-quality spectra, which are often useful; in identifying

impurities for example. As for the quantitative analytical application of this

technique, approximately 10% of the share in pharmacopoeias for the assay

of bulk drug materials and pharmaceutical formulations is very slowly

decreasing.

The most important field of application of infrared (IR) [37] and near-

infrared (NIR) spectroscopy [38] is the identification of drugs. IR has greatly

decreased (almost completely eliminated) the importance of the classical

color tests, while NIR is a method of increasing importance in the in-process

control of manufacturing pharmaceutical formulations. IR and Raman

spectroscopy, together with solid-phase NMR, X-ray diffraction, and thermal

methods are the up-to-date methods in solid-phase characterization, which

is of great importance in developing pharmaceutical formulations, with

optimal bioavailability [39].

In recent times FTIR spectroscopy has been coupled with ATR

(attenuated Total Reflection).FTIR spectroscopic imaging in ATR mode is a

powerful tool for studying biomedical samples and dissolution of

pharmaceutical formulations and drug release. One of the key advantages of

ATR-FTIR imaging is that is requires minimal or no sample preparation prior

to spectral measurements. Consequently, this approach is particularly

suitable to measure substances with strong infrared absorption such as

water.

36

The application of ATR-FTIR imaging also allows for the

characterization of biomedical materials in tissue engineering. Also the

quantitative information about the spatial distribution of chemical

components on pharmaceutical tablets in contact with water, as a function

of time, provides an important basis for building new mathematical models

for the optimization of controlled drug delivery. ATR-FTIR imaging is suitable

for imaging of realistic tablets in contact with aqueous solutions because of

the shallow penetration of the evanescent wave into the sample.

In pharmacopoeias, for the study of toxic metal impurities, the

classical sulfide and other limit tests are still widely used. At present, the

rapidly increasing importance of the much more selective and sensitive

atomic spectroscopic methods can be observed, such as, graphite furnace

atomic absorption spectrometry (GF-AAS), inductively coupled plasma

atomic emission spectrometry (ICP/AES), and mass spectrometry (1CP/ MS).

2.7.4. Other Analytical Techniques

The classical titrations non-selective method is still widely used in

compendial analysis for the assay of bulk drug materials. Even in the USP,

where the breakthrough of HPLC has been much faster, more than 40% of

the low molecular weight organic compounds are determined by aqueous or

non-aqueous titration. Other electro analytical methods have always been

only modestly important in pharmaceutical analysis. Classical polar graphic

methods using toxic mercury electrodes are being driven out from practice

and replaced by new electrodes, for example, glassy carbon electrodes

modified with carbon nanotubes, which provide highly sensitive analyses.

37

Another field where remarkable results have been obtained is the

development of ion-specific and molecule-specific sensors. Flow-injection

analysis with various detectors such as spectroscopic, electro analytical

chemiluminescence is often used in the analysis of drug formulations. All

antibiotics, 30 years ago, were determined using microbiological methods.

In modern pharmacopoeias [40], in the majority of cases, these are

replaced by much more selective and informative methods [41], mainly

HPLC. Although the importance of immunoassays has decreased in the

recent years, they are still often used in the determination of some bioactive

compounds in the biological samples. Radioimmunoassay has been greatly

superseded by various enzyme-immunoassay methods.

2.7.5. Application of Microscopy Techniques

A useful instrument for the structural and morphological study of

novel films, nanoparticles, hydrogels, matrices, and porous scaffolds, is

offered by modern microscopy tools, such as, atomic force microscopy (AFM),

scanning electron microscopy (SEM), and confocal Raman microscopy

(CRM).The imaging tools are important for analyzing the surface of soft

organic materials, as understanding these surfaces may help to shed light

on the interactions that occur between crystals and their surrounding

environment[42].

Drug release is the result of a complex interplay between the drug, its

carrier, and the release environment. Study of the surface structure and

morphology of pharmaceutical substances contributes to an understanding

of surface activity and is of critical importance to the pharmaceutical

38

industry. Modern microscopes are tools that are applicable for the collection

of topographic data and morphological investigation of different applicable

pharmaceutical materials.

2.7.6. Future Trends

Globalization of the drug market and the sharpening concurrence

among the drug companies has caused pharmaceutical analysis to become

one of the battlefields in the struggle. The importance of issues related to

drug safety has greatly increased and this has led to the continual increase

of demands with regard to securing the quality of drugs, and often over

securing the safety of drug therapy. It became necessary to harmonize the

demands and analytical strategies [43].

The first step was the establishment in the European Pharmacopoeia,

of which the Sixth Edition is now official. This became the basis of the

national pharmacopoeias of the member states of the European Union. The

next step was the formation of ICH, which was done with the aim of

harmonizing the efforts of registration agencies, principal pharmacopoeias

(Ph. Eur., USP, and Japanese Pharmacopoeia), and pharmaceutical

manufacturers' organizations, to improve the quality of drugs and the safety

and efficacy of drug therapy.

The guidelines issued by ICH are authoritative worldwide with respect

to drug quality issues. It has to be noted that requirements with regard to

the quality of drugs and drug formulations in the drug market are, in

practice, much greater than those prescribed in the pharmacopoeias and

ICH guidelines.

39

The greater change in the pharmacopoeias in the past years has been the

increasing importance of purity tests. At the beginning only a very limited

number of monographs contained tests related to impurities.

Thanks to the development of TLC and HPLC, at present, an

overwhelming majority of the monographs on bulk drugs, and in a fairly high

proportion of those on formulations, contain these tests. The impurity profile

has become the most informative indicator of the quality of bulk drug

materials. At the same time, the importance of assaying bulk drugs has

decreased considerably; moreover, there are opinions that even this

importance is questionable.

The tendencies toward globalization and harmonization mentioned earlier

and the necessity of increasing the safety of drug therapy, have prompted

the validation of analytical methods to the forefront; moreover, it has become

one of the most important issues in contemporary drug analysis. However,

some negative tendencies are also apparent:

A fully validated method meeting the requirements of various guidelines

needs much more analytical data than would be strictly necessary

Many drug analysts, especially among the young generation, feel that the

essence of pharmaceutical analysis is the mass production and handling

of data, rather than problem solving

The way of thinking is changing, with many people, mainly outside the

circles of drug analysts, believing that possession of up-to-date,

automated computerized instruments and validated methods

automatically give good

40

and reliable results. Pharmaceutical analysis is an important field of

activity in the interest of suffering mankind, through increasing the safety

of drug therapy.

2.8. Importance of HPLC in the quality control of Drugs

Thin layer Liquid Chromatography (TLC), Gas Chromatography (GC)

and High Performance Liquid chromatography or Liquid Chromatography

(HPLC or LC) are the important chromatographic techniques, widely used in

the quality control of drugs in the pharmaceutical industry. HPLC is the

dominant method in pharmaceutical analysis in quality control, because it is

most versatile technique, having high resolving power, sensitivity, advanced

column, detector technologies, and commercial availability, flexible

separation modes and capability of computerized optimization of separation.

In the past two decades, LC has replaced GC in most of the QC tests

due to its simplicity and applicability to polar and thermally labile species.

Most synthetic drugs are in this category. HPLC is a powerful separation

method, suited to resolving a large number of similar substances in a short

time. HPLC is especially significant for the pharmaceutical industries

because it allows for both qualitative and quantitative analysis. HPLC has

been used to solve no less than 50% of the problems, leaving the other 50%

to 15 other chromatographic, spectroscopic, and other methods.

A new development in the field of HPLC has been the introduction of

column packing with ultrafine particles (< 2 um), enabling short columns (5

cm or less) to be used, and rapid analyses (e.g., 5 minutes or even less than

41

1 minute) to be carried out by Ultra Fast Liquid Chromatography (UFLC) or

Ultra Performance Liquid Chromatography (UPLC) .

2.9. Importance of Stability- indicating methods

a. Introduction:

Stability studies need to be performed to provide evidence of how the

quality of a drug substance or drug product varies with time under the

influence of variety of environmental factors such as temperature, humidity

and light [44]. As a result of stability testing, a re-test period for the drug

substance or a shelf-life for the drug product can be established and storage

conditions can be fixed for that drug. The analytical test method used for

testing samples which are under stability evolution shall be capable enough

to detect the changes happening in the product [45].

As per US FDA guideline, a stability-indicating method can be defined

as “validated quantitative analytical methods that can detect the changes

with time in the chemical, physical, properties of the drug substance and

drug product, that are specific so that the contents of the active ingredient,

degradation products and other components of interest can be accurately

measured without interference [46].

The purpose of establishing the stability-indicating power of the

method is to ensure that while analyzing the test samples all the related

substances, process impurities and degradation products etc. are separated

from each other and from the peak of the main drug of interest. The

requirement of stability-indicating methods is specified in various regulatory

guidelines. World Health Organization (WHO) stability guidelines specify that

42

analytical procedures used for stability monitoring should be fully validated

and stability-indicating.

The ICH guidelines on Good Manufacturing Practices state that the

test procedures used in stability testing should be validated and stability-

indicating [47]. European guidelines state that the testing should cover all

the appropriate, physical, chemical, biological, and microbiological

attributes. Validated stability-indicating analytical procedures should be

applied [48].

United States Pharmacopoeia (USP) has a requirement for adopting a

method to USP, which says that whenever possible, a stability-indicating

procedure should be used for the Assay [49]. Generally, chromatographic

procedures are stability-indicating and titration procedures are not. During

registration of any product with US or Europe agencies, when a non-stability

indicating assay is proposed, a separate stability-indicating impurity

procedure should be provided.

b. Evaluation of stability-indicating power

Stress testing of the active substance can help identify the likely

degradation products, which can in turn help establish the degradation

pathways and the intrinsic stability of the molecule and validate the

stability-indicating power of the analytical procedure used. Stress testing is

likely to be carried out on a single batch of the drug substance. The ICH

guidelines Q1A suggest the following conditions to be employed [50]:

(i) 10°C increments above the accelerated temperatures (e.g.50°C, 60

°C,etc.),

43

(ii) Humidity where appropriate (e.g. 75% or greater),

(iii) Hydrolysis across a wide range of pH values, Oxidation and Hydrolytic

degradation studies need to be performed using 0.1M HCl /NaOH,

oxidation using 3% H2O2. If reasonable degradation is seen study will be

stopped otherwise extended to further harsh condition along with heat

or reflux for longer duration. Alternatively, if total degradation is seen

after subjecting the drug to those conditions, acid/alkali strength can

be decreased along with decrease in the reaction temperature.

(iv) Photolysis, exposing the sample to light providing an overall

illumination of not less than 1.2 million lux hours and an integrated

near ultraviolet radiation with an energy of not less than 200 watt

hours/square meter [51].

The target decomposition of the product shall be about 5 to 10%. The

stressed samples are subjected to analysis under proposed method

conditions to study the separation of degradation products. Well separated

peaks with high resolution are expected for a stability-indicating method.

Also homogeneity of peaks need to be established that to ensure the purity of

peaks and simultaneously to prove no co elution of peaks. For peak

homogeneity, the most popular technique is the Photo Diode Array (PDA)

analysis, the principle of which is the comparison of the spectra of the

analyte peak, taken upslope, at the apex and on the down slope.

If these spectra do not match then the peak is non-homogeneous.

Homogeneity of the peak also can be established by liquid chromatography

mass spectrometry (LC-MS) technique by scanning entire peak for its mass

44

value. Therefore PDA or LC-MS results suggest that if the products are

different but are co-eluting, then suitable modification should be done in the

chromatographic method to achieve a satisfactory resolution.

2.10. Role of Mass Balance during HPLC development

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

measured increase in the amount of degradation products [52]. It is a good

quality control check on analytical methods to show that all degradation

products are adequately detected and do not interfere with quantitation of

the parent drug (i.e., stability-indicating methods). Regulatory agencies use

mass balance to assess the appropriateness of the analytical method as a

stability-indicating method and determine whether all degradants have been

accounted for [53].

In mass balance calculations, the loss of parent drug or the amount of

drug remaining is determined from a sample assay, and the measured

increase in degradation products is determined by a related substances

method. The fundamental approach for determining mass balance is to

quantitate the decomposition peaks using degradation methods and then

reconciles the measured loss in the parent drug with the amount of

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

by the amount of degradants measured, then mass balance is achieved.

The assessment of degradation in pharmaceutical products involves

two aspects of analytical measurement. Firstly, a specific or selective

analytical method must be available for accurate assay of parent drug

compound, in order to measure any loss. Second, methodology should be in

45

place for quantification of the degradation products formed. Ideally, when

degradation occurs, the measured amount of parent drug lost should

correlate well with the measured increase in degradation products. This

correlation is referred to as “mass balance”. More recently, the ICH has

provided definition of “mass balance; material balance” as follows [54]

The process of adding together the assay value and levels of

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

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

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

in all circumstances.

The focus may instead be on assuring the specificity of the assay, the

completeness of the investigation of route of degradation, and the use, if

necessary, of identified degradants as indicators of the extent of degradation

via particular mechanism [55]. The analyst must balance time and resource

demands to provide the information necessary to understand degradation

without going to extreme measures of quantify components of little interest.

Mass balance in pharmaceutical analysis is very important for several

reasons. By demonstrating the degradative losses of parent drug correlate

well with the measured increase in degradation products unaccounted for.

Conversely, if one observes, for example, a 20% loss of parent drug but only

measures a 5% increase in degradation products, it is likely that additional

degradation products formed are not accurately determined by the given

method(s). Because unknown degradation products could potentially be

toxic or otherwise compromise the safety of drug, it is important to have

46

methods that detect all major degradation products. Thus, safety is the

major reason for the study of mass balance.

Mass balance is also useful in method validation [56]. In order to

demonstrate that analytical methods are stability-indicating, unstressed and

stressed materials are often compared. Any increase in degradation a

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

that the methods can accurately assess degradation.

Mass balance is also important in understanding alternative

degradation pathways [57]. For example, consider a situation where both

acid-catalyzed and oxidative degradation produces a substantial loss of

parent compound in stress-testing studies. If good mass balance is achieved

for the acid-catalyzed degradation, but not for the oxidative degradation,

further work to better understand the oxidative degradation pathway(s) is

warranted.

It may be that the poor mass balance in the latter case results from

important oxidative products that are unaccounted for or from structures,

which need to be more fully elucidated to understand response factor

differences. Mass balance is an important consideration in assessing

degradation pathways of pharmaceutical products. Often, response factor

differences between degradation products and the parent compound are

responsible for mass balance problems. Relative Response Factor (RRFs)

should be incorporated, when possible, in the quantification of degraded

samples.

47

Mass balance is also an approach to establish the validity of stability-

indicating method. It is the process of adding together the assay value and

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

the initial value, with due consideration of the margin of analytical error.

Mass balance may not also be established due to formation of volatile

impurities during degradation, non availability of reference standards, some

of the products are strongly bound to stationary phase and do not elute at

all or elute after very long periods, variability of response or reduced

response in UV detector, drug content variation in drug products etc. [58].