INTRODUCTION
Pharmaceutical analysis is a measurement science consisting of a set of
powerful ideas and methods that are useful in all fields of science and medicine.
Both quantitative and qualitative information‟s are required in an analysis.
Qualitative analysis establishes the chemical identity of a species in a sample
and quantitative analysis establishes the chemical identity of a species in a
sample and quantitative analysis determines the relative amount of these species
or analytes, in numerical terms. Analytical methods are applied throughout
pharmaceutical industry for their daily working.
Pharmaceutical analysis determines the quality of drug product via analytical
chemistry that involves a series of process for identification, determination,
quantification, and purification of a substance, separation of the components of
a solution or mixture or determination of structure of chemical compounds.
These teqniques are based on the various phytochemical properties of the
compounds of interest
The quantitative analysis can be performed by various instrumental and
chemical methods of analysis. Chemical methods of analysis executes various
chemical reactions for quantitative analytical measurement include gravimetric
,titrimetric and volumetric method of analysis but accuracy speed and
reproducibility are limited Instrumental method involves measurement of some
or physical properties of the compound or substance. Instrumental to methods
are proffered due to their selectivity, high speed, accuracy and simplicity of
analysis. These methods are employed for determination of minor or trace
concentration of element in the sample any change in the properties of the
system are detected by measurement of absorbance, specific rotation, refractive
index, migration difference, charge to mass ratio etc…
The selection of the analytical methods is in turn determined by the purpose and
objective of the investigation. In general, analytical method is selected
according to the analyte and matrix in the determined sample in order to ensure
the accuracy and reliability of the analytical result.
Traditional medicine is a broad term encompassing health practices, approaches,
knowledge and beliefs which incorporate herbal, animal and mineral based
medicines, spiritual therapies, manual techniques and exercises, applied
singularly or in combination to treat, diagnose and prevent illnesses or maintain
well-being.
Natural medicines provide valuable resources to meet the requirements for
global health care at affordable prices. Therefore, safety and efficacy need to be
proven in a comparable manner to conventional drugs. Evidence-based natural
and western medicine may merge to a “one-world medicine” for the sake of all
patients in industrialized and developing countries. In the present review, we
discuss strategies for preservation of traditional knowledge on natural
medicines, sustainability of medicinal herbs and natural products, and
standardization and quality control.
The objective of this study was to examine the methods used in systematic
reviews of safety across a range of complementary therapies to assess the
variation in approach and the potential for developing guidance on conduct and
reporting. Herbal medicine, or phototherapy, is the science of using herbal
remedies to treat the sick. It therefore covers everything from medicinal plants
with powerful actions; the term phototherapy was introduced by the French
physician Henri Leclerc (1870-1955). Herbal medicine has come a long way
since the days of the ancient „herbalism‟. The study of the use of medicinal
plants is now a scientific subject, a field of medicine in the same way as
chemotherapy, hydrotherapy, electrotherapy and others. Knowledge of
medicinal plants and their uses has been recorded from antiquity.
IMPORTANCE OF HERBAL MEDICINE
The points of thought are the common people divert to use the Ayurvedic,
Chinese and other herbal medicines because it is used all over the world, in
India, its use is much more because of their easy accessibility, no expert
consultation required, are considered safe to use and also because primary
health care services fall short of peoples‟ need both in qualitative and
quantitative terms. We should make all these easily marketed ayurvedic, and
other herbal medicines FDA approved and increase public awareness about pros
and cons of their uses. The common belief that anything natural is safe is not
correct. Herbal Medicines are readily available in the market from health food
stores without prescriptions and are widely used in India, China, and USA and
all over the world. According to recent survey the majority of people who use
herbal medicines do not inform their physicians about their consumptions that
can cause abnormal test results and confusion in proper diagnosis. However,
natural medicines seem to be barely able to provide convincing alternatives to
conventional western medicine for global health-care. Some reasons are
1) The knowledge of shamans and traditional healers is getting lost, since their
oral traditions being handed down from generation to generation for thousands
of years seem to be extinguished in modern times.
2) The traditional use of medicinal plants needs to be systematically
investigated and standardized. People outside the field of phytotherapy that
phytotherapy is inactive, because infectious diseases would otherwise not kill
many millions of patients each year, which is striking and embarrassing at the
same time for scientists working on natural medicines.
3) The rainforests and other biological habitats are getting extinguished with a
breath-taking velocity. Half of the rainforest worldwide has already been
destroyed during the past three decades. This has irreversible impact on drug
development
4) Overharvesting of medicinal plants from the wild presents a severe problem
of preserving many plant species endangered to be extinguished.
5) Global climate change may affect both the growths of medicinal plants as
well as their constituents.
STANDARDISATION OF HERBAL FORMULATION
pharmacognostic evaluation
chemical
parameters
standerdisation of herbal
formulations
chromatographic
and spectroscopic
analysis
microbial parameters
physico chemical
parameters
Quality Control of Herbal Drugs
Quality control for efficacy and safety of herbal products is of paramount
importance. Quality can be defined as the status of a drug that is determined by
identity, purity, content, and other chemical, physical, or biological properties,
or by the manufacturing processes. Quality control is a term that refers to
processes involved in maintaining the quality and validity of a manufactured
product.
The term “herbal drugs” denotes plants or plant parts that have been converted
into phytopharmaceuticals by means of simple processes involving harvesting,
drying, and storage [3]. Hence they are capable of variation. This variability is
also caused by differences in growth, geographical location, and time of
harvesting. A practical addition to the definition is also to include other crude
products derived from plants, which no longer show any organic structure, such
as essential oils, fatty oils, resins, and gums. Derived or isolated compounds
(e.g. strychnine from strychnous nux-vomica) or mixtures of compounds (e.g.
abrin from Abrus precatorius).
In general, quality control is based on three important pharmacopeial definitions
- it should have one herb
– it should not have any contaminant other than herb
-the active constituents should be within the defined limits.
It is obvious that the content is the most difficult one to assess, since in most
herbal drugs the active constituents are unknown. Sometimes markers can be
used which are, by definition, chemically defined constituents that are of
interest for control purposes, independent of whether they have any therapeutic
activity or not .
Identity can be achieved by macro and microscopical examinations. Voucher
specimens are reliable reference sources. Outbreaks of diseases among plants
may result in changes to the physical appearance of the plant and lead to
incorrect identification [5,6]. At times an incorrect botanical quality with
respect to the labeling can be a problem.
Purity is closely linked with safe use of drugs and deals with factors such as ash
values, contaminants (e.g. foreign matter in the form of other herbs), and heavy
metals. However, due to the application of improved analytical methods,
modern purity evaluation also includes microbial contamination, aflatoxins,
radioactivity, and pesticide residues. Analytical methods such as photometric
analysis, Thin layer chromatography (TLC), High performance liquid
chromatography (HPLC), High performance thin layer chromatography
(HPTLC), and Gas chromatography (GC) can be employed in order to establish
the constant composition of herbal preparations.
Content or assay is the most difficult area of quality control to perform, since in
most herbal drugs the active constituents are unknown. Sometimes markers can
be used. In all other cases, where no active constituents or marker can be
defined for the herbal drug, the percentage extractable matter with a solvent
may be used as a form of assay, an approach often seen in pharmacopeia.
A special form of assay is the determination of essential oils by steam
distillation. When active constituents (e.g. sennosides in senna) or markers (e.g.
alkydamides in Echinacea) are known, a vast array of modern chemical
analytical methods such as ultraviolet/visible spectroscopy(UV/VIS),
TLC,HPLC, HPTLC, GC, mass spectrometry, or a combination of GC and
MS(GC/MS), can be employed.
Techniques in Herbal Drug Identification and Characterization
1. HPLC
Preparative and analytical HPLC are widely used in pharmaceutical industry for
isolating and purification of herbal compounds. There are basically two types of
preparative HPLC: low pressure HPLC (typically under 5 bar) and high
pressure HPLC (pressure >20 bar). The important parameters to be considered
are resolution, sensitivity and fast analysis time in analytical HPLC whereas
both the degree of solute purity as well as the amount of compound that can be
produced per unit time i.e. throughput or recovery in preparative HPLC.
Vasicine, the major bioactive alkaloid of Adhatoda vusica, was estimated by
HPLC in two polyherbal drug formulations - Shereeshadi Kashaya and
Yastyadivati, Standardization of the Triphala (an antioxidant-rich herbal
formulation) mixture of Emblica officinalis, Terminalia chebula and T. belerica
in equal proportions has been reported by the HPLC method by using the RP18
column with an acidic mobile phase.
2. Liquid chromatography- massspectroscopy (LCMS)
LC-MS has become method of choice in many stages of drug development.
Chemical standardization of an aqueous extract of the mixture of the 20 herbs
provided 20 chemical compounds serving as reference markers using LC-MS
Further, LC-MS analysis of aminoglycosides showed that these drugs are highly
soluble in water, exhibited low plasma protein binding, and were more than
90% excreted through the kidney. Further this technique helps in analysis of
aminoglycosides in plasma samples with ion pairing chromatography. Two
HPLC methods, one combined with a photodiode array detector (LC/UV) and
another with mass spectrometry (LC/MS), were reported for the analysis of
aristolochic acid I and II in herbal medicines.
3. Gas chromatography (GC) and gas chromatography-mass spectroscopy
(GC-MS)
GC-MS instruments have been used for identification of large number of
components present in natural and biological systems. The identification and
quantification of chemical constituents present in polyherbal oil formulation
(Megni) consisting of nine ingredients, mainly Myristica fragrans, Eucalyptus
globulus, Gaultheria procumbens and Mentha piperita was analyzed by GCMS
method.
4. Supercritical fluid chromatography (SFC)
Supercritical fluid chromatography is a hybrid of gas and liquid
chromatography. This technique is an important third kind of column
chromatography that is beginning to find use in many industrial, regulatory, and
academic laboratories. supercritical fluid chromatography is of importance
because it permits the separation and determination of a group of compounds
that are not conveniently handled by either gas or little chromatography.
5. Capillary electrophoresis (CE)
Researchers evaluated the importance of CE for quality control of herbal
medicinal products. Several CE studies dealing with herbal medicines have
been reported and two kinds of medicinal compound i.e. alkaloids and
flavonoids have been studied extensively. The methodology of CE was
established to evaluate one herb drug in terms of specificity, sensitivity and
precision, and the results were in agreement with those obtained by the HPLC
method.
6. Thermal analysis of herbal drugs
Thermo gravimetric analysis (TGA), differential thermal analysis (DTA) and
differential scanning calorimetry (DSC) have been employed to study any
physical or chemical changes in various products including herbal drugs and
also used to study preformulation or drug excipient compatibility. TGA may be
operated under subambient conditions to analyse ethanol in herbal formulations
such as asavas and arista. TGA and DTA analysis of mercury based Indian
traditional metallic herbal drug Ras-sindoor indicated the presence of mercury
sulphide based on a sharp peak at 354o C which corresponded to melting
temperature of mercury sulphide.
7. X-ray powder diffractometry (X-RPD)
This technique is used to identify minerals, crystalline materials and metallic
based herbal formulations. The tin based herbal drug Vanga Parpam was
estimated by XRD and the intense sharp diffraction peaks clearly confirmed the
presence of high crystallinity in Vanga Parpam. XRD analysis of metallic based
Indian traditionally medicine Ras-sindoor indicated the presence of mercury
sulphide which is represented by sharp peak. X-ray powder diffractometry data
confirmed the formation of phospholipid complex with emodin, naringenin ,
quercetin, Gallic acid.
8. Infrared spectroscopy
FTIR along with the statistical method principal component analysis (PCA) was
applied to identify and discriminate herbal medicines for quality control in the
fingerprint region 400-2000 cm-1. The ratio of the areas of any two marked
characteristic peaks was found to be nearly consistent for the same plant from
different regions, thereby, an additional discrimination method for herbal
medicines. PCA clusters herbal medicines into different groups, clearly showing
that IR method can adequately discriminate different herbal medicines using
FTIR data. Near-infrared spectroscopy technique has been used for rapid
determination of active components, species, geographic origin, special
medicinal formula, on-line quality control, identification of counterfeit and
discrimination of geographical origins of Chinese herbal medicines.
9. High performance thin layer chromatography (HPTLC)
Among the modern Analytical tools HPTLC is a powerful analytical method
equally suitable for qualitative and quantitative analytical tasks. HPTLC is
playing an important role in today analytical world, not in competition to HPLC
but as a complementary method. One of the most obvious orthogonal features of
the two techniques is the primary use of reversed phases in HPLC versus
unmodified silica gel in HPTLC, resulting in partition chromatography and
adsorption chromatography respectively. Unlike other methods, HPTLC
produces visible chromatograms complex information about the entire sample is
available at a glance. Multiple samples are seen simultaneously, So that
reference and test samples can be compared for identification. Similarities and
differences are immediately apparent and with the help of the image
comparison. Several chromatograms can be compared directly, even from
different plates. In addition to the visible chromatograms, analog peak data are
also available from the chromatogram. They can be evaluated either by the
image based software Videoscan or by scanning densitometry with TLC
Scanner, measuring the absorption and/or fluorescence of the substances on the
plate. TLC is an offline technique: the subsequent steps are relatively
independent, allowing parallel treatment of multiple samples during
chromatography, derivatization and detection. Some of the steps can be repeated
independently of others, for example in post chromatographic derivatization,
some reagents can be applied in sequence allowing multiple derivatization and
thus multiple detection of the same sample. In view of the above article
describes the key features of traditional thin layer chromatography and modern
HPTLC advantages.
Key feature of HPTLC
1. Simultaneous processing of sample and standard - better analytical precision
and accuracy less need for Internal Standard.
2. Several analysts work simultaneously.
3. Lower analysis time and less cost per analysis.
4. Low maintenance cost.
5. Simple sample preparation - handle samples of divergent nature.
6. No prior treatment for solvents like filtration and degassing.
7. Low mobile phase consumption per sample
8. No interference from previous analysis – fresh stationary and mobile phases
for each analysis - no contamination.
9. Visual detection possible - open system.
HPTLC- High Performance Thin Layer Chromatography is a sophisticated
and automated form of TLC.Main Difference of HPTLC and TLC Particle and Pore size of Sorbents.
HPTLC
TLC
Layer of sorbent
Efficiency
Separations
Analysis Time
particle size
generated
- 5 cm
- 15 cm
distance and the
analysis time is greatly
reduced
Solid support
stationary phases like
Alumina &
silica gel for normal Kiesulguhr
phase and C8 ,
C18 for reversed phase
modes
Development chamber
less amount of
mobile phase
Sample spotting
Scanning
Fluorescence
scanner scans the entire
chromatogram
qualitatively
and
quantitatively and the
scanner is an advanced
type of
densitometer
Steps involved in HPTLC :
Selection of chromatographic layer
Sample and Standard Preparation
Activation of pre-coated plates
Application of sample and standard
Selection of mobile phase
Pre- conditioning (Chamber saturation)
Chromatographic development and drying
Detection and visualization
Quantification
Documentation
Multiple Detection
Scanning densitometry