CHAPTER-1 INTRODUCTION - Information and Library...
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CHAPTER-1
INTRODUCTION
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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)
Not available Prescription
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
Not available Prescription
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
Introduction 2012
Ph. D. Thesis Page 17
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
Ph. D. Thesis Page 18
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
Ph. D. Thesis Page 19
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
Ph. D. Thesis Page 20
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
Ph. D. Thesis Page 21
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
Ph. D. Thesis Page 22
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
Ph. D. Thesis Page 23
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
Ph. D. Thesis Page 24
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
Ph. D. Thesis Page 25
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
Ph. D. Thesis Page 26
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
Ph. D. Thesis Page 27
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.