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CHAPTER-1

INTRODUCTION

Introduction 2012

Ph. D. Thesis Page 1

1. INTRODUCTION

1.1. Herbal medicines

In man‟s quest for food during the early nomadic period of his existence he would

most certainly have encountered some plants that were poisonous, others that would

serve as adequate foods, and still others that produced bizarre and unusual effects by

altering his consciousness. Among this latter group were those that would

simultaneously relieve pain and counteract disease. These experiences, passed on

from generation to generation, are today recognized for their vital role in global health

care and the application of scientific method to ancient philosophical systems has led

to natural medicine (Emboden, 1997). Herbal medicines have been used in medical

practice for thousands of years and recognized especially as a valuable and readily

available resource for healthcare in East Asian nations. As per World Health

Organization (WHO) definitions herbal medicines are plant derived materials and

preparations with therapeutic or other human health benefits, which contain either raw

or processed ingredients from one or more plants, inorganic materials or animal origin

(Choi et al., 2002).One of the characteristics of oriental herbal medicine preparations

is that all the herbal medicines, either presenting as single herbs or as collections of

herbs in composite formulae, are extracted with boiling water during the decoction

process. This may be the main reason why quality control of oriental herbal drugs is

more difficult than that of western drugs (Liang et al., 2004). However, most herbal

medicines still need to be studied scientifically, although the experience obtained

from their traditional use over the years should not be ignored (Lewis, 2001).

Monographs of medicinal plants can be found in the United States Pharmacopeia,

Chinese Pharmacopeia, WHO monographs for medicinal plants, Japanese

Pharmacopeia and Herbal Medicine (Expanded Commission E Monographs). The

major compound types in herbal medicines include alkaloids, saponins, flavonoids,

anthraquinones, terpenoids, coumarins, lignans, polysaccharides, polypeptides and

proteins (Ong, 2004).

A list of commercially available products, containing medicinal plants, their common

medicinal uses and side effects are tabulated in Table 1.

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Table 1: Some of the common herbal products, their uses and side effects

Botanicals Common medicinal uses Side effects

Aloe vera Short-term treatment of occasional

constipation

Abdominal spasms and pain may

occur after even single dose.

Overdose can lead to colicky

abdominal spasms and pain, as

well as the formation of thin,

watery stools. Overdose will result

in electrolyte imbalance.

Echinacea To support and promote the

natural powers of resistance of the

body, especially in infectious

conditions, such as influenza and

cold in the nose and throat.

Chills, short-term fever reaction

and nausea and vomiting may

occur.

St. John‟s

Wort

For psychovegetative

disturbances, depressive moods,

anxiety and nervous unrest

Photosensitization is possible,

especially in fair skin individuals.

Gingko

biloba

For symptomatic treatment of

disturbed performance in organic

brain syndrome within the

regimen of a therapeutic concepts,

with the following principal

symptoms: memory deficients,

disturbances in concentration,

depressive emotional conditions,

dizziness, tinnitus and headache.

Very seldom, cases of stomach or

intestinal upset, headaches or skin

allergic skin reaction.

Garlic As a support to dietary measures

at elevated levels of lipids in the

blood and as a measure for age

dependant vascular changes.

In rare instances, there may be

gastrointestinal symptoms,

changes to the flora of the

intestine, or allergic reaction.

Ginseng A tonic for invigoration and

fortification times of fatigue and

debility or declining capacity for

work and concentration.

On prolong use and higher doses,

sodium and water retention and

potassium loss may occur,

accompanied by hypertension,

edema, hypokalemia and in rare

cases, myoglobinuria.

The WHO database has over sixteen thousand suspected herbal case reports. The most

commonly reported adverse reactions are hypertension, hepatitis, face oedema,

angiodema, convulsions, thrombocytopenia, dermatitis and death. In 1992, a list of

about 33 herbal drugs with serious risks prepared by the Committee for Proprietary

Medicinal Products (CPMP) was published by the European Commission. This list

included some plants such as Aconitum (all species), Aristolochia (all species),

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Claviceps purpurea, Convovulus scamonia L., Ocimum basilicum L., Strychnus nux-

vomica L., Vinca minor L. etc. (European Medicines Agency, 2005)

1.2. Traditional Herbal Medicines

Traditional medical systems (TMS), of which the most acknowledged and

investigated are Traditional Chinese medicine (TCM) and Indian Traditional

Medicine (Ayurveda, Sidha, Unani), are used worldwide (Cardini et al., 2006). The

World Health Organization (WHO) estimates that about 80% of the population living

in developing countries rely almost exclusively on traditional medicine for their

primary health care needs. In almost all the traditional medicine, the medicinal plants

play a major role and constitute backbone of the traditional medicine. Indian Materia

Medica includes about 2000 drugs of natural origin almost all of which are derived

from different traditional system and folklore practices. Out of these drugs derived

from traditional system, 400 are of mineral and animal origin while rest are of

vegetable origin. India has a rich heritage traditional medicine and the traditional

health care system have been flourishing for many years. Lot of efforts have been

taken by the government and private sectors for the development of traditional system

based on these methods.

Western medicine continues to show the influence of ancient practices. Current

examples are the use of cardiac glycosides from the purple foxglove Digitalis

purpurea and related plants, opiates from the opium poppy Papaver somniferum,

reserpine from Rauwolfia species, and quinine from Cinchona species. More recently,

there has been interest in other product from traditional systems of medicine.

Artemisin is an active active anti malarial compound isolated from Artemisia annua, a

constituent of the traditional Chinese antimalarial preparation Qinghaosu and

forskolin was isolated from Coleus forskohlii, a species used in Ayurvedic and Unani

preparations for cardiac disorders (Mukherjee, 2002).

Traditional medicine came into existence long before Western medicine was

developed in Europe. Traditional medicine is a scientific discipline, which develops

the related theories from the long-term clinical practices. Different from the modern

biomedical science, traditional medicine has no general experimental practice in

laboratory. In contrast, clinical practice or clinical experiment is the fundamental

research activity of traditional medicines (Zhou et al., 2010). The World Health

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Organization defined traditional medicine as the sum total of all knowledge and

practices, whether explicable or not, used in diagnosing, preventing, and eliminating

physical, mental, or societal imbalances. It relies exclusively on practical experience

and observation handed down from generation to generation, whether verbally or in

writing (WHO, 1978). The World Health Organization has estimated that 60–80% of

the population of non-industrialized countries rely on traditional healthcare for their

basic health care needs, either on its own or in conjunction with modern medical care.

The demand for traditional medicine is increasing in many countries. The traditional

approach to medicine aims to preserve good health, to totally cure an illness and

believes in the balance of the „positive energy‟ in the body. Against this background,

Asian traditional medicine predominates in the Asian countries, and it is used for the

treatment of various physical and mental illnesses (Low and Tan, 2007).

1.3. Unani System of Medicine

The Unani System of Medicine, one of the oldest systems of medicine, had its origin

in Greece. The great Greek Philosopher and Physician, Hippocrates (460-377 B.C) is

the founder of Unani medicine later Galen and Avicenna enriched the system. Unani

system of medicine was introduced in India by Arabs in 13th

century. Due to its

efficacy and scientific base, it was accepted by masses and the system took firm roots

in India (Anonymous, 2007; Anonymous, 2006).

Today Unani system of treatment practised, taught and researched under its local

names in over 20 countries including Afghanistan, China, Canada, Denmark,

Germany, Finland, Netherlands, Norway, Poland, Korea, Japan, Saudi Arabia,

Sweden, Switzerland, Turkey, UK and USA. Unani system of medicine is unmatched

in treating chronic diseases like arthritis, asthma, mental, cardiac and digestive

disorders, urinary infections, and sexual diseases. Unani medicine established that

disease was a natural process and that symptoms were the reactions of the body to the

disease. Unani medicine plays a vital role when the individual experiences the

humoral imbalance. The correct diet and digestion can bring back the humoral

imbalance. Diet therapy aims at treating certain ailments by administration of specific

diets.

The Unani physician believes that the healthy state of the human body is maintained

by a power known as „Tabiyat‟ or „Quwwat-e-Mudabbira‟ (medicatrix naturae), gifted

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to it from its creator. The concept of „Tabiyat‟ is much vaster than the concept of

immunity system of body. It controls, regulates and restores the physiological

mechanisms of the body and helps in potentiating the immunity of the body and its

resistance against various ailments. Suppression of this gifted power leads to disease.

Therefore, the duty of the physician is to use such methods/treatments that encourage

the body‟s own innate healing response (Tabiyat). This can be achieved by

stimulating the „Hararat-e-Ghariziya‟ (Vital force of body), which is decreased in a

diseased person making him vulnerable to environmental and pathological challenges.

1.3.1. History of Unani system of medicine

Unani medicine originated from ancient Greece. The Unani system of medicine owes,

as its name suggests, its origin to Greece. The term "Unani" is derived from the work

"Unan" which means Greece in Arabic. In 460 BC the Greek Philosopher and father

of modern medicine, Hippocrates (Bukharath) who freed medicine from the clutches

of superstition and laid the foundation of Unani medicine. Another great scholar of

Unani medicine was Galen (131-210 AD) who stabilised the foundation of this

system. As Greek and then Roman civilisation declined, Greek medical texts survived

in the Islamic courts of the medieval Near East. In the eighth and ninth centuries AD,

many Greek texts were translated into Arab forming the basis of Unani medicine.

Some Islamic physicians like Al-Razi (Rhazes) (850-925 AD) and Ibn Sina

(Avicenna) (980-1037 AD) Al Zahravi (Albucasis) the surgeon and Ibn-Nafis etc.

contributed immensely to the system. In India, Unani system of medicine was

introduced by Arabs, and soon it took firm roots in the soil. When Mongols ravaged

Persian and Central Asian cities like Shiraz, Tabrez and Geelan, scholars and

physicians of Unani medicine fled to India. The physicians who came to India from

foreign countries also took advantage and derived benefits from indigenous or local

system of medicine i.e. Ayurveda and this tradition has been continuing ever since

that time right upto the times of Hakeem Ajmal Khan (Physician of the Nation) and

Hakeem Abdul Hameed.

1.3.2. Concepts of Unani system of medicine

In Unani System of medicine the human body is considered as a single unit, made of

seven components known as „Umoor-e-Tabiya‟. These seven components are Arkan

(Elements), Mizaj (Temperament), Akhlaat (Humours), Arwaah (Life force), Aaza

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(Organs), Quwa (Faculties), Afa‟al (Functions). According to Unani philosophy, the

body is made up of the four basic elements i.e. Earth, Air, Water and Fire which have

different temperaments i.e. Cold, Hot, Wet and Dry respectively. After mixing and

interaction of four elements a new compound having new Mizaj (temperament) comes

into existence i.e. Hot Wet, Hot Dry, Cold Wet, Cold Dry. The body has the simple

and compound organs, which receive their nourishment through four Akhlaat

(Humours) i.e. Dam (Blood), Balgham (Phlegm), Safra (Yellow Bile) and Sauda

(Black Bile). Each humour has its own temperament. Blood is hot and moist, phlegm

is cold and moist, yellow bile is hot and dry and black bile is cold and dry. Every

person attains a temperament according to the preponderance of the humours in

his/her body and it represents the person's healthy state. The temperament of a person

may be sanguine, phlegmatic, choleric or melancholic. Unani physician believes that

health is a state of body in which there is equilibrium in the humours and functions of

the body. To maintain the correct humoral balance there is a power of self-

preservation or adjustment called „Quwwat-e-Mudabbira‟ (medicatrix naturae) in the

body. When this power weakens, the equilibrium of the humours is disturbed

quantitatively or qualitatively or both and physiological functions of the body are

deranged due to the abnormal temperament of affected organ or system resulting in a

disease. Therefore, the aim of Unani physician is to find out the cause of the

underlying disruption of humours, so that it can be corrected and disease can be cured.

Imbalance of humours may be due to external factors such as an injury, incorrect diet,

environmental factors etc. or internal factors such as improper digestion or both. Signs

of humoral diseases are as follows:

a. Ghalba-e-Khoon (Sanguis Humour)

When there is excess of Dam (Blood) in the body the colour of skin appears red, veins

appear more prominent, pulse seems to be full and urine becomes high coloured.

Patient complains of breathlessness, headache and scenes of blood in his/her dream.

b. Ghalba-e-Balgham (Phlegm Humour)

In the case of excess of Balgham (Phlegm) in the body, skin becomes whitish and

cold, pulse becomes slow and deep, urine becomes thick and low coloured. Patient

complains of forgetfulness, loss of appetite, increased sleep, laziness and scenes of

water in his/her dream.

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c. Ghalba-e-Safra (Choler Humour)

Choler humour results in yellowness of the skin, swifter pulse than ordinary and high

coloured urine. Patient appears irritated without any apparent cause and complains of

headache, disturbed sleep, bitterness in throat and scenes of fire, lighting, anger,

fighting etc. in his/her dream.

d. Ghalba-e-Sauda (Melancholer Humour)

When there is excess of Sauda (Black bile) in the body the skin appears rough, pulse

becomes weak, urine becomes thin, patient complains of loss of appetite and sourness

in throat. Patient remains busy with foolish imaginations and appears fearful without

any cause (Ahmad and Akhtar, 2007).

1.4. Regulatory norms for herbal drugs

The safety problems emerging with herbal medicinal products are due to a largely

unregulated growing market where there is a lack of effective quality control. Lack of

strict guidelines on the assessment of safety and efficacy, quality control, safety

monitoring and knowledge on traditional medicine/complementary and alternative

medicine are the main aspects which are found in different regulatory systems. Under

some regulatory systems plant may be defined as a food, a functional food, a dietary

supplement or a herbal medicine. Some of the parameters that help in understanding

the development of herbal drug regulation in a given nation are general policy

structure, drug registration system, development of pharmacopoeia, national

monographs, inclusion in essential medicine list and drug type (OTC or prescription).

The first International recognition of the role of traditional medicine and use in

primary health care was in „The Declaration of Alma-Ata‟. It states, inter alia, that

“…Primary health care relies, at local and referral levels, on health workers, including

physicians, nurses, midwives, auxiliaries and community workers as applicable, as

well as traditional practitioners as needed…” (WHO, 1998).

Herbal drug regulation in South East Asian and some Western Pacific countries are

compared in Table 2.

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Table 2: Herbal drug regulation in selected countries

Country

Regulation on

herbal drug

(year)

Herbal drug

registration

system

National

monograph Pharmacopoeia

Inclusion in

essential

medicine list

Drug type

Bangladesh 1992 Yes No Bangladesh National

Formularies on Unani

and

Ayurvedic medicine

Not available Prescription

and OTC

Bhutan No No In development In development 103(till 1998) Prescription

Democratic

People's

Republic of

Korea

1999 Yes Korean Herbal

Medicine

Monographs (1986)

Pharmacopoeia of the

Democratic People's

Republic of Korea

(1996)


and OTC

India 1940 Yes Yes Ayurvedic

Pharmacopoeia of

India and the Unani

Pharmacopoeia of India

Ayurveda

(2001)–315,

Unani (2000)–

244,

Siddha

(2001)–98

Prescription

and OTC

Indonesia 1993 Yes Materia Medika

Indonesia

(246 monographs)

Farmakope Indonesia No OTC

Maldives No No No No No OTC

Myanmar 1996 Yes Monograph of

Myanmar

Medicinal Plants

(2000)

In development In

development

OTC

Nepal 1978 Yes In development In development Not available Prescription

and OTC

Sri Lanka No No Compendium of

Medicinal Plants

Ayurveda

Pharmacopoeia

No Prescription

and OTC

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( 100 monographs) (1979)

Thailand 1967 Yes Yes, 21

monographs

Thai hErbal

Pharmacopoeia,

Traditional Formularies

of

Herbal Medicines (5

volumes)

16 herbal

preparation

Prescription

and OTC

Australia 1989 Yes No British Pharmacopoeia

is

used

No OTC

China 1963 Yes Yes, 92

monographs

Chinese Pharmacopoeia

(1963)

1242 Prescription

and OTC

Japan 1960 Approval system No Japanese

Pharmacopoeia


and OTC

Malaysia 1984 Yes Malaysian Herbal

Monograph

(1999)

No No OTC

Philippines 1984 Yes In development In development 2000 OTC

Republic of

Korea

1986 Yes No Korea Pharmacopoeia 515 OTC

Singapore 1998 No No No No OTC

Vietnam 1989 Yes Vietnam Medicinal

Plants

Vietnam Pharmacopoeia 267 Prescription

and OTC

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The Committee on Herbal Medicinal Products (HMPC) established within the

European Medicines Agency (EMEA), has introduced a simplified registration

procedure for traditional herbal medicinal products in EU member states. In the US,

herbal medicines have been regulated under the Dietary Supplement Health and

Education Act of 1994. A botanical drug product may be marketed in the United

States as an OTC drug monograph or as an approved NDA or ANDA. In India,

Department of Ayurveda, Yoga & Naturopathy, Unani, Siddha and Homoeopathy

(AYUSH) established in 1995 under the Ministry of Health & Family Welfare is

responsible for the regulation of herbal medicines. The Drugs & Cosmetics Act of

1940 lays down the various rules for production and marketing of Ayurveda, Siddha

and Unani (ASU) drugs (Sahoo et al., 2010). The various requirements for

registration and marketing authorization of herbal drugs in the EU, US and India are

depicted in Table 3.

Table 3: Herbal medicine regulation in European Union, United States and India

Country Regulatory

authority

Description Regulation/Act

EU European

Medicines Agency

(EMEA): The

Committee on

Herbal Medicinal

Products (HMPC)

Establishment of

HMPC and regulation

of herbal medicine

Registration Procedure

for traditional herbal

medicinal products

Directive 2004/24/EC

(Traditional Herbal

Medicinal Products

Directive) and

Regulation (EC)

US

USFDA: Center for

Drug Evaluation

and

Research (CDER)

Center for Biologics

Evaluation and

Research

(CBER)

Botanical drug

definition

Regulation of herbal

product

Procedure for

marketing of Botanical

drug as OTC drug

Regulation of

Allergenic extracts and

vaccines that contain

botanical ingredients

201(g)(1)(B), Federal

Food, Drug, and

Cosmetic Act Dietary

Supplement Health and

Education Act of 1994

Section 351 of the Public

Health Service Act (42

U.S.C.

262).

India Department of

Ayurveda, Yoga &

Naturopathy, Unani,

Siddha and

Homoeopathy

(AYUSH)

Production and

marketing of ASU

drugs

GMP for ASU drugs

Drugs & Cosmetics Act,

1940 Drugs & Cosmetics

Rules,1945

Schedule T, Drugs &

Cosmetics Act, 1940

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1.5. Significance of quality control for traditional Unani formulations

About 25% of the drugs prescribed worldwide come from plants, 121 such active

compounds being in current use. Of the 252 drugs considered as basic and essential

by the World Health Organisation (WHO), 11% are exclusively of plant origin and a

significant number are synthetic drugs obtained from natural precursors (Rates, 2001).

Generally it is believed that the risk associated with herbal drugs is very less, but

reports on serious reactions are indicating to the need for development of effective

marker systems for isolation and identification of the individual components.

Standardization, stability and quality control for herbal drugs are feasible, but difficult

to accomplish (Sahoo et al., 2010).

The last few decades have seen rapid worldwide growth in the demand for herbal

medicines and their proprietary products in the pharmaceutical industry and medicinal

markets, especially in China, Japan, and countries in Europe and North America. As

demand grows so does the demand for mass production and quality assurance that

each batch of medicine meets certain standards both at the time of production and

over its shelf life. Quality control for herbal preparations or proprietary products,

however, is much more difficult than for synthetic drugs because of the chemical

complexity of the ingredients. As herbal preparations comprise hundreds of mostly

unique, or species-specific, compounds, it is difficult to completely characterize all of

these compounds. It is also equally difficult to know precisely which one is

responsible for the herbs or herbal preparation‟s therapeutic action because these

compounds often work synergistically in delivering therapeutic effects. Thus,

maintaining consistent quality in herbal preparations, both from batch to batch and

over time, is as problematical as it is necessary and has drawn serious attention

recently as a challenging analytical task (Xie et al., 2007).

The purpose of quality control of medicinal plant products is to ensure therapeutic

efficacy and to check any adulteration or non deliberate mixing in commercial

batches. The quality control of plant products is a general requirement to be fulfilled.

Good quality assurance is necessary when dealing with the plant products, intended to

be released in market as drug constituents or as test substances in basic

pharmacological experiments. Therefore efforts should be made to obtain and

maintain the high quality of these plant products.

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Since the herbal formulations are of mainly plant origin, they are susceptible to

contamination from different sources, detoriation and variations of chemical

composition which may occur due climatic and geographical changes. Therefore the

development of standardisation procedure for the herbal formulations is must. The

standardization of herbal drugs may give acceptance by worldwide moreover it

improves the therapeutic efficacy and safety of the drugs. The standardization gives a

clear picture about the intrinsic value of the drug i.e. the amount of medicinal

principles and constituents present, presence or absence of adulterants etc.

Quality control is the process of delivering a product with a specified minimum level

of one or more phytoconstituent (s), where we can make sure about the quality of the

product; broadly it covers the qualitative and quantitative part of analysis. Qualitative

analysis mainly covers the identification of the component(s) present in a particular

compound, whereas the quantitative analysis is accomplished by measuring the level

of a chemical in a crude herbal extract which are, present in that particular product

and establishing a standard amount of that chemical for future production. The

concept of standardized extracts definitely provides a solid platform for scientific

validation of herbals.

Plant materials and herbal remedies derived from them represent a substantial

proportion of global drug market and internationally recognized guidelines for quality

assessment are necessary. For pharmaceutical purposes, the quality of the medicinal

plant material must be as high as that of other medicinal preparations. However, it is

impossible to assay for a specific chemical entity when the bioactive ingredient is not

known. In practice, assay procedures are not carried for those medicinal plant

materials where there are known active ingredients (Dubey et al., 2004).

Depending upon whether the active principle of the plant is known or not, different

concepts („normalization‟ vs. „standardization‟) have to be applied in order to

establish relevant criteria for uniformity. It is often claimed and is widely believed

that remedies of natural origins are harmless and carry no risk. Nothing could be

further true, particularly where there is a risk of toxic plant being used by mistake or

where herbal preparation are marketed with the addition of undeclared potent

synthetic substances, as a result phytopharmaceuticals require further scientific

validation and standardization and to achieve this, herbal medicine must go through

the more rigid scientific scrutiny to which allopathic drugs are subjected. Proof of

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safety, however, should take precedence over establishing efficacy, and accuracy in

labeling the constituents of medicinal plant remedies is of critical for safety evaluation

and drug control. Reproducible efficacy and safety of phytopharmaceuticals is based

on reproducible quality (Anonymous, 1996).

Comparing with conventional preparations, herbal products represent a number of

unique problems when quality aspects are considered. These are because

phytopharmaceuticals are complex mixture of different secondary metabolites that

can vary considerably depending on environmental, genetic factors, growth, harvest,

drying, and storage conditions, stage of ripeness and geographic area where the plant

is grown. These complex positions of quality aspects of herbal drugs are further

complicated by use of herbal ingredients as are being used in traditional practice.

To ensure reproducible quality of herbal remedy, proper control of starting material is

utmost essential. The aspects that need to be controlled include authentication and

reproducibility of herbal ingredients, inter and intra specific variation in plants,

environmental factors, plant part used, time of harvesting, post harvesting factors,

contaminants of herbal ingredients, pesticides, fumigants and other toxic metals.

The polarity of the solvent, the mode of extraction, and the instability of the

constituents may also influence the composition and quality of the extracts and must

therefore be kept constant. The quality criteria of herbal drugs are based on a clear

scientific definition of the raw material (Mukherji, 2002).

1.6. WHO guidelines for quality control of standardized herbal

formulations

WHO has recognized the problem associated with the safety and efficacy of herbal

medicines and published guidelines to ensure the reliability and repeatability of

research on herbal medicines. This concept should be followed not only in research,

but also in the production and therapeutic application of phytopharmaceuticals. This

includes –

A therapeutic classification of medicinal plants in different countries.

Scientific criteria and methods for assessing the safety of medicinal plant

products.

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International standards and specification for identity, purity, strength and

manufacturing practices (Anonymous, 1999).

1.7. General protocol for the quality control of herbal drugs

In order to assure a consistent and acceptable quality herbal product, care should be

taken right from the identification and authentication of herbal raw materials to the

verification process of final product. The following parameters are recommended.

Table 4: General protocol for the quality control of herbal drugs

1 Authentication The first stage is the identification of the plant species or

botanical verification by the currently accepted Latin

binomial name and synonym. The steps involved in the

authentication are taxonomic, macroscopic and

microscopic studies. records of collection, parts of the

plant collected, regional status, botanical identity such as

phytomorphology, microscopial and histlogical analysis,

taxonomical identity etc.

2 Physical parameters Physical tests include organoleptic evaluation (sensory

characters such as taste, appearance, odour feel of the drug

etc.), viscocity, moisture content, pH, disintegration time,

friability, hardness, flowability, sedimentation and ash

value.

3 Chromatographic

and spectroscopic

evaluation

Sophisticated modern techniques of standardization such

as spectroscopic UV-vis spectrophotometry, TLC, HPTLC,

HPLC, NMR, near infra red spectroscopy provide

quantitative and semi quantitative information about main

active constituents or marker compound present in the

crude drug and herbal products. Markerd play an important

role in fingerprinting of herbs. Quality of the drug can also

be assessed by chromatographic fingerprint.

5 Pesticide residue

analysis

Standard limits of pesticide have been set by WHO and

FAO (Food and Agricultural Organization). Some

common pesticide that can cause harm to human beings

such as DDT, BHC, toxaphene and aldrin should be

analyzed.

6 Aflatoxin analysis Aflatoxins are group of toxic compounds produced by

certain molds, especially Aspergillus flavus. Aflatoxin is

the strongest known naturally occurring carcinogen.

Among 18 different types of aflatoxins identified, major

members are aflatoxin B1, B2, G1 and G2.

7 Heavy metal

analysis

Toxic metals such as Cu, Zn, Fe and particularly Cd, As,

Pb and Hg should be analyzed. In the analysis of metals

their speciation is to be taken into consideration

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Figure 1: General protocol for standardizing herbal drugs

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1.8. Conventional methods for quality control and limitations

Most of the regulatory guidelines and pharmacopoeias suggest macroscopic and

microscopic evaluation and chemical profiling of the botanical materials for quality

control and standardization (Anonymous, 2002; Philipsom, 1996; WHO, 1998). With

respect to this, Department of AYUSH Govt. of India gave some parameters for drug

development, standardization & quality of Ayurveda, Siddha and Unani drugs, which

include five protocols as follows Protocol-I (Standardization of single plant material),

Protocol-II (SOP of preparation of extracts), Protocol-III (Standardization of plant

extract), Protocol-IV (SOP of finished product) and protocol-V (Standardization of

formulations). These protocols based on most common parameters such as

morphological evaluation, microscopical evaluation, physico-chemical evaluation,

particle size , bulk density and tap density in case of powder crude drugs or powder

formulations, assay for constituents (marker %, major compounds like alkaloids,

flavonoids/saponin compounds). With respect to above parameters test for

heavy/toxic metals (Lead, Cadmium, Mercury and Arsenic), microbial contamination

(total viable aerobic count, total Enterobacteriacea and total fungal count), test for

specific pathogen (E. coli, S. spp., S. aureus, P. aeruginosa), pesticide residue (DDT,

HCH, Endosufan, Alderin, Malathion and Parathion), test for aflatoxine (B1, B2, G1,

G2) and chelating agent (for Bhasma, Lepa, Aswarista etc.). However, these

parameters are judged subjectively and substitutes or adulterants may closely

resemble the genuine material (Joshi et al., 2004). So chemical profiling is an

essential parameter for standardization, which establishes a characteristic chemical

pattern for a plant material, its fractions or extracts.

1.9. Emerging techniques in quality control of traditional Unani

formulations

In general, one or two markers or pharmacologically active components in herbs and

or herbal mixtures were currently employed for evaluating the quality and authenticity

of herbal medicines, in the identification of the single herb or herbal formulation

preparations, and in assessing the quantitative herbal composition of an herbal product

this kind of determination, however, does not give a complete picture of a herbal

product, because multiple constituents are usually responsible for its therapeutic

effects. These multiple constituents maywork „synergistically‟ and could hardly be

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separated into active parts. Moreover, the chemical constituents in component herbs

in the herbal medicinal products may vary depending on harvest seasons, plant

origins, drying processes and other factors. Thus, it seems to be necessary to

determine most of the phytochemical constituents of herbal products in order to

ensure the reliability and repeatability of pharmacological and clinical research, to

understand their bioactivities and possible side effects of active compounds and to

enhance product quality control (Yan et al., 1999). Thus, several chromatographic

techniques, such as high performance liquid chromatography (HPLC), gas

chromatography (GC), capillary electrophoresis (CE) and thin layer chromatography

(TLC), can be applied for this kind of documentation. According to this concept, a

chemical profile, such as a chromatographic fingerprint, for a herbal product should

be constructed and compared with the profile of a clinically proven reference product.

Chemical fingerprints obtained by chromatographic and electrophoretic techniques,

especially by hyphenated chromatographies, are strongly recommended for the

purpose of quality control of herbal medicines, since they might represent

appropriately the “chemical integrities” of the herbal medicines and therefore be used

for authentication and identification of the herbal products.

1.9.1. Chromatography and chemical fingerprints of Unani medicines

A single herbal medicine may contain many natural constituents, and a combination

of several herbs might give rise to interactions with hundreds of natural constituents

during the preparation of extracts, the fingerprints produced by the chromatographic

instruments, which may present a relatively good integral representation of various

chemical components of herbal medicines. Chromatographic technique such as

HPLC, TLC, GC and capillary electrophoresis, spectroscopic methods such as IR,

NMR, and UV-may also be used for fingerprinting.

1.9.1.1. Thin layer chromatography

Thin-layer chromatography is, at present, the most popular method in the

authentication of traditional herbal medicines. TLC is used as an easier method of

initial screening with a semi quantitative evaluation together with other

chromatographic techniques (Wagner et al., 1996; Nyiredy, 2003). It provides visible,

UV images or fluorescent ones. Compared with the column chromatography, it has an

additional colour parameter. This method identifies several samples at the same time.

Introduction 2012


Combined with the scanning technology, digital imaging, and data treatment, it may

promptly form outlines and show the integration value of the peaks. Because of the

increased information and the enhanced complex analysis function, TLC is more

suitable for the fingerprint analysis of the traditional herbal medicines. TLC has the

advantages of many-fold possibilities of detection in analyzing herbal medicines. In

addition, TLC is rather simple and can be employed for multiple sample analysis. For

each plate, more than 30 spots of samples can be studied simultaneously in one time

(Jianliang et al., 2008). The advantages of HPTLC over HPLC are that several

samples can be run simultaneously by use of a smaller quantity of mobile phase and

mobile phases of pH 8 and above can be used for HPTLC. Another advantage of

HPTLC is the repeated detection (scanning) of the chromatogram with the same or

different conditions (Verma et al., 2007). For the chemical fingerprint of traditional

herbal medicine TLC is one of the commonly used method because of simplicity,

versatility, high velocity, specific sensitivity and simple sample preparation. Thus,

TLC is a convenient method of determining the quality and possible adulteration of

herbal products.

Table 5: Thin-layer chromatographic analysis of herbal products

S.

N

o

Title Analyte TLC System References

1. Application of

HPTLC to

alternative

medicines –

qualitative and

quantitative

evaluation of

the

Ayurvedic

formulation

„Triphala

Churna‟

„Amla‟ (Emblica

officinalis), „Beheda‟

(Terminalia belerica), and

„Harhra‟ (Terminalia

chebula) and Gallic acid)

Stationary

phase: Silica gel

Mobile phase

Lalla et al.,

2000

2. Herbal products

a new

approach for

diabetic

patients

Azadirachta indica,

Catharanthus roseus and

Momordica charntia

Stationary

phase: Silica gel

Mobile phase :

Dichloro

methane –

methanol, 2:8

Habib et

al., 2000

3. Screening of

Chinese

Myristica fragrans,

Plantago asiatica, Rubia

Stationary

phase: Silica gel

Tezuka et

al., 2001

Introduction 2012


herbal drug

extract for

inhibitory

activity on

nitric

oxide

production and

identification of

an active

compound of

Zanthoxylum

bungeanum

cordifolia, and

Zanthoxylum

bungeanum

Mobile phase:

Methanolwater,

3:7

4. Reactions of p-

coumaric

acid with

nitrite: product

isolation and

mechanism

studies

Coumaric acid, 4-

hydroxybenzaldehyde, 1,4-

dihydroxybenzeneacetalde

h

yde, 4-

hydroxybenzenepropanoic

acid, 4-

hydroxy-3-

nitrobenzenepropanoic acid

Stationary

phase: silica gel

Mobile phase:

Dichloromethan

e – methanol –

formic acid

190:10:1;

188:12:1 and

dichloromethane

– methanol 47:3,

19:1

Torres et

al., 2001

5. Occurrence and

activity of

natural

antioxidants in

herbal spirits

Alcoholic or

hydroalcoholic solutions of

volatile substances with

flavoring or medicinal

properties

Stationary

phase: Silica gel

Mobile phase:

Toluene – ethyl

formate – formic

acid, 79:20:1

Imark et

al., 2001

6. Identification,

isolation,

and

determination

of

flavones in

Origanum

vulgare from

Macedonian

flora

Apigenin, Luteolin,

Chrysoeriol, and Diosmetin

Stationary

phase: Silica gel

Mobile phase

Toluene – ethyl

acetate – formic

acid 58:33:9,

Chloroform –

methanol, 97:3,

Chloroform –

nhexanemethanol

,

40:40:3 and

Toluene –

methyl ethyl

ketone – acetic

acid, 18:5:1

Kulevanov

a et al.,

2001

7. Fractionation

and

antioxidants

screening of

Quercus cortex

Quercus Cortex, Caffeic

acid, p-Cumaric acid,

Ellagic acid, (+)-

epicatechin, (+)-catechin,

Quercetin, Rutin,

Stationary

phase: Silica gel

Mobile phase

Ethyl acetate –

formic acid –

Rajani et

al., 2001

Introduction 2012


extract Protocatechu acid, Quinic

acid, Synapic

water 17:2:3

8. Recent

investigations

on St. John‟s

Wort by

HPTLC

Hyperoside, Quercetin,

hyperforin, Quercitrin and

Biapigenin

Stationary phase:

Silica gel

Mobile phase

Ethyl acetate –

dichloromethane

– acetic

acidformic

acidwater

100:25:10:10:11

Blatter et

al., 2001

9. Antioxidant

Activities and

Phenolic

Composition of

Extracts from

Greek Oregano,

Greek Sage, and

Summer Savory

Plant extracts and Catechin

and Epicatechin

Stationary

phase:

Cellulose,,

Silica gel, and

Cyano-, amino-,

and RP-18

modified silica

Mobile phase

Acetone – acetic

acid 93:7 and

Water –

methanol –

formic acid ,

84:15:1 or

69:30:1

Exarchou et

al., 2002

10. A new and

convenient

method for

quantitative

estimation of

chrysophanol,

an antioxidant

in the rhizomes

of Rheum emodi

(Roxb)

Chrysophanol Stationary

phase: Silica gel

Mobile phase

Hexane – ethyl

acetate 9:1

Kumar et

al., 2002

11. Characterizatio

n of tannins

from rhubarb by

TLC/HPTLC

Rhubarb extract Stationary

phase: Silica gel

Mobile phase:

Acetone – water

– formic acid

18:1:1 or

toluene- acetone

– formic acid

3:6:1 over 75

mm after partial

chamber

saturation

Sievers et

al., 2002

12. HPTLC-aided

phytochemical

Ushaq (ammoniacum gum) Stationary

phase: Silica gel

Reich et

al., 2003

Introduction 2012


fingerprinting

analysis as a

tool for

evaluation of

herbal drugs

Mobile phase:

n-hexane and

ethyl acetate,

5:5

13. New amides

and

gastroprotective

constituents

from the fruit of

Piper chaba.

piperine, piperanine,

pipernonaline,

dehydropipernonaline,

piperlonguminine,

retrofractamide B,

guineensine, N-isobutyl-(2

E,4 E)-octadecadienamide,

N-isobutyl-(2 E,4 E,14 Z)-

eicosatrienamide ( 13), and

methyl piperate

Stationary

phase: Silica gel

Mobile phase:

Chloroform

methanol 20:1

Morikawa

et al., 2004

14. Qualitative

identification

of herbal drugs

by

preparative

TLC

Different herbals Stationary

phase: Silica gel

Mobile phase:

Toulene-ethyl

acetate, 3:7

Reich et

al., 2006

1.9.1.2. Gas chromatographic fingerprint

It is well-known that many pharmacologically active components in herbal medicines

are volatile chemical compounds. Thus, the analysis of volatile compounds by gas

chromatography is very important in the analysis of herbal medicines. The GC

analysis of the volatile oils has a number of advantages. Firstly, the GC of the volatile

oil gives a reasonable “fingerprint” which can be used to identify the plant. The

composition and relative concentration of the organic compounds in the volatile oil

are characteristic of the particular plant and the presence of impurities in the volatile

oil can be readily detected. Secondly, the extraction of the volatile oil is relatively

straightforward and can be standardized and the components can be readily identified

using GC–MS analysis (Liang et al., 2004). The non-volatile substances can be also

analyzed by GC with precolumn derivative technology, by pyrolysis GC or by flash

GC. A number of detectors are used in gas chromatography. The most common are

the flame ionization detector (FID) and the thermal conductivity detector (TCD). Both

are sensitive to a wide range of components, and both work over a wide range of

concentrations. However, the most serious disadvantage of GC is that it is not

convenient for its analysis of the samples of polar and non-volatile compounds. For

this, it is necessary to use tedious sample work-up which may include derivatization.

Introduction 2012


Therefore, the liquid chromatography becomes another necessary tool for us to apply

the comprehensive analysis of the herbal medicines (Jianliang et al., 2008; Zuin et al.,

2003; Nahoko et al., 2009; Janete et al., 1994; Yan et al., 2000; Carmen et al., 2011;

Yue et al., 2009; Zhou et al., 1998; Nishiguchi et al., 2001; Li et al., 2010).

1.9.1.3. High-Performance liquid chromatography

HPLC is a popular method for the analysis of herbal medicines because it is easy to

learn and use. Moreover is not limited by the volatility or stability of the sample

compound. In general, HPLC can be used to analyze almost all the compounds in the

herbal medicines. The applicability of HPLC is much wider than that of GC.

Equipped with different mobile phases and detectors, HPLC can detect the majority of

organic compounds.

Popularity of the universal detectors such as evaporative light scattering detector

(ELSD), electrochemical detector, photo-diode detector (PDA) and MS detector has

led to the active fingerprint analysis of the compounds which have no UV absorption

or poor UV absorption. Reversed-phase (RP) columns may be the most popular

columns used in the analytical separation of herbal medicines (Karamanos et al.,

1997; Yan et al., 2006; Chang et al., 2008; Wang and Zhou, 2006; Pan et al., 2007;

Saravanan et al., 2007; Jianjun et al., 2005; Penissi et al 2003; Alev et al., 2007;

Kazuo et al., 2008; Yoshiyuki et al., 2007).

1.9.2. Hyphenation procedures

In the past two decades, combining a chromatographic separation system on-line with

a spectroscopic detector in order to obtain structural information on the analytes

present in a sample has become the most important approach for the identification

and/or confirmation of the identity of target and unknown chemical compounds. For

most (trace-level) analytical problems in the research field of herbal medicines, the

combination of column liquid chromatography or capillary gas chromatography with

a UV–Vis or a mass spectrometer (HPLC–DAD, CE-DAD, GC–MS and LC–MS,

respectively) becomes the preferred approach for the analysis of herbal medicines

(Rajani and Kanaki, 2008).

The data obtained from such hyphenated instruments are the so-called two-way data,

say one way for chromatogram and the other way for spectrum, which could provide

much more information than the classic one-way chromatography. Mass spectrometry

Introduction 2012


is the most sensitive and selective method for molecular analysis and can yield

information on the molecular weight as well as the structure of the molecule.

Combining chromatography with mass spectrometry provides the advantage of both

chromatography as a separation method and mass spectrometry as an identification

method.

1.9.2.1. Gas Chromatography–Mass Spectrophotometry

GC–MS was the first successful online combination of chromatography with mass

spectrometry, and is widely used in the analysis of essential oil in herbal medicines

(Guetens et al., 2002). GC–MS, can produce not only a chromatographic fingerprint

of the essential oil of the herbal medicine but also the information related to its most

qualitative and relative quantitative composition.

The significant advantages for GC–MS is: (1) with the capillary column, GC–MS has

in general very good separation ability, which can produce a chemical fingerprint of

high quality; (2) with the coupled mass spectroscopy and the corresponding mass

spectral database, the qualitative and relatively quantitative composition information

of the herb investigated could be provided by GC–MS, which will be extremely useful

for the further research for elucidating the relationship between chemical constituents

in herbal medicine and its pharmacology in further research (Li et al., 2003a; Gong et

al., 2001; Gong et al., 2003; Li et al., 2003b).

1.9.2.2. HPLC–DAD, HPLC–MS

HPLC–DAD has become a common technique in most analytical laboratories in the

world now. With the additional UV spectral information, the qualitative analysis of

complex samples in herbal medicines turns out to be much easier than before. For

instance, checking peak purity and comparing. With the introduction of electrospray

mass spectrometry, the coupling of liquid chromatography and mass spectrometry has

opened the new way to widely and routinely applied to the analysis of herbal

medicines with the available standard spectrum of the known compound to the one in

the investigated sample. Combined HPLC–DAD–MS techniques take advantage of

chromatography as a separation method and both DAD andMS as an identification

method. DAD and MS can provide on-line UV and MS information for each

individual peak in a chromatogram.With the help of this hyphenation, in most cases,

one could identify the chromatographic peaks directly on-line by comparison with

Introduction 2012


literature data or with standard compounds, which made the LC–DAD–MS becomes a

powerful approach for the rapid identification of phytochemical constituents in

botanical extracts, and it can be used to avoid the time-consuming isolation of all

compounds to be identified (He, 2000; Qian et al., 2001; Mo et al., 2009; Dan et al.,

2009).

1.9.3. Method development, optimization and validation

Method development is a systematic series of logical steps which should be optimized

before the determination of performance characteristics so that all the parameters are

final. After the individual components of method have been finalized, the

optimization of the various parameters is performed. Experimental design approach is

used to determine the experimental factors that have significant impact on the method.

This is very important in determining the conditions that are required to be

investigated in the robustness testing during the method validation.

Method validation is a process that demonstrates that the method will successfully

meet or exceed the minimum standards recommended in the Food and Drug

Administration (FDA) guidance for accuracy, precision, selectivity, sensitivity,

reproducibility, and stability. Validation involves documenting, through the use of

specific laboratory investigations, that the performance characteristics of the method

are suitable and reliable for the intended analytical applications (Hill, 2000; Parveen

et al., 2009; Ansari et al., 2005; Ahmad et al., 2008; Alam et al., 2009).

The International Conference on Harmonization (ICH) has produced guidelines on the

validation of analytical procedures for pharmaceutical product registration

applications. One of the key aims of harmonization is the mutual acceptance of data

from different regulatory authorities. Validation does not imply that the method is free

from errors. It only confirms that it is suitable for the purpose. Any modifications to a

method during its use require its revalidation (ICH, 1996).

1.9.3.1. Specificity and Selectivity

The term selectivity and specificity are often used interchangeably. International

Union of Pure and Applied Chemistry (IUPAC) and Western European Laboratory

Accreditation Conference (WELAC) have defined the difference between the

specificity and selectivity. Even inconsistent with ICH, the term specificity generally

refers to a method that produces a response for a single analyte only while the term

Introduction 2012


selective refers to a method which provides responses for a number of chemical

entities that may or may not be distinguished from each other.

Selectivity or specificity should be assessed to show that the intended analytes are

measured and that their quantitation is not affected by the presence of the biological

matrix, known metabolites, degradation products, or co-administered drugs. Specifi

city should be determined for each analyte in the assay.

1.9.3.2. Linearity and Calibration curves

The linearity of an analytical method is its ability to elicit test results that are directly

proportional to the concentration of analytes in samples within a given range.

Linearity is determined by a series of three to six injections of five or more standards

whose concentration ranges from 80-120% of the expected concentration range. For

impurity methods, linearity is determined by preparing standard solutions at five

concentration levels over a range from LOQ of the impurity to 120% of expected

concentration. The response should be directly or by means of a well-defined

mathematical calculation, proportional to the concentration of the analytes.

Frequently, the linearity is evaluated graphically or mathematically. The evaluation is

made by visual inspection of a plot of signal height or peak area as a function of

analyte concentration. Acceptability of linearity data is often judged by examining the

correlation coefficient and y-intercept of the linear regression line for the response

versus concentration plot. Correlation coefficient r2 of > 0.999 is generally considered

as evidence of acceptable fit of the data to the regression line.

1.9.3.3. Accuracy

Accuracy of an analytical method is the closeness of the measured value to the true

value for the sample. The true value for accuracy assessment can be obtained in

several ways. One alternative is to compare results of the method with results from an

established reference method. This approach assumes that the uncertainty of the

reference method is known. Secondly, analyzing a sample with known concentrations,

for example, a certified reference material and comparing the measured value with the

true value as supplied with the material can assess accuracy. If such certified

reference material is not available, a blank sample matrix of interest can be spiked

with a known concentration by weight or volume. After extraction of the analyte from

the matrix and injection into the analytical instrument, its recovery can be determined

Introduction 2012


by comparing the response of the extract with the response of the reference material

dissolved in a pure solvent. Because this accuracy assessment measures the

effectiveness of sample preparation, care should be taken to mimic the actual sample

preparation as closely as possible.

1.9.3.4. Precision

The precision of a method is the extent to which the individual test results of multiple

injections of a series of standards agree. The measured standard deviation can be

subdivided into three categories: repeatability, intermediate precision and

reproducibility. Repeatability is obtained when one operator using one piece of

equipment over a relatively short time span carries out the analysis in one laboratory.

At least 5 or 6 determinations of three different matrices at two or three different

concentrations should be done and the relative standard deviation is calculated. The

acceptance criterion for precision depends very much on the type of analysis. While

for compound analysis in pharmaceutical quality control, precision of better than 1%

RSD is easily achieved, for biological samples the precision is more likely to be 15%

at the concentration limits and 10% at other concentration levels. For environmental

and food samples, the precision is very much dependent on the sample matrix,

concentration of the analyte and on the analysis technique. It can vary between 2-

20%.

Intermediate precision is a term that has been defined by ICH as the long-term

variability of the measurement process and is determined by comparing the results of

a method run within a single laboratory over a number of weeks. Intermediate

precision may reflect discrepancies in results obtained by different operators, from

different instruments, with standards and reagents from different suppliers, with

columns from different batches or a combination of these. The objective of

intermediate precision validation is to verify that in the same laboratory the method

will provide the same results once the development phase is over.

1.9.3.5. Limit of Detection

The limit of detection is the point at which a measured value is larger than the

uncertainty associated with it. It is the lowest concentration of analyte in a sample that

can be detected but not necessarily quantified. In chromatography, the detection limit

is the injected amount that results in a peak with a height at least three times as high

Introduction 2012


as the baseline noise level. The detection limit needs to be determined only for

impurity methods in which chromatographic peaks near the detection limit are

observed.

1.9.3.6. Limit of Quantitation

The limit of quantitation is the lowest level of analyte that can be accurately and

precisely measured. This limit is required only for impurity methods and is

determined by reducing the analyte concentration until a level is reached where the

precision of the method is unacceptable. If not determined experimentally, the

quantitation limit is often calculated as the analyte concentration that gives signal to

noise ratio S/N = 10.

1.9.3.7. Robustness

Robustness tests examine the effect of operational parameters on the analysis results.

For the determination of a method‟s robustness a number of chromatographic

parameters, for e.g. flow rate, column temperature, injection volume, detection

wavelength and mobile phase composition are varied within a realistic range and the

quantitative influence of the variables is determined. If the influence of the parameter

is within a previously specified tolerance, the parameter is said to be within the

method‟s robustness range.

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