STABILITY TESTING OF
NATURAL PRODUCTS
Dr. Kirankumar Hullatti
Professor
KLE College of Pharmacy
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STABILITY
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SIGNIFICANCE OF STABILITY TESTING
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TYPES OF STABILITY
Type of Stability Condition Maintained throughout the shelf life of
drug product
Chemical Each active ingredient retains its chemical integrity
and labeled potency within the specified limit
Physical The original Physical properties including
appearance palatability, uniformity, dissolution and
suspendability
Microbiological Resistance to microbial growth is retained according
to specified requirement.
Therapeutic Therapeutic effect remains unchanged
Toxicological No significant increase in toxicity occurs
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FACTORS AFFECTING STABILITY OF HERBAL
FORMULATIONS
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PHYSICAL DEGRADATION
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PHYSICAL DEGRADATION
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PHYSICAL DEGRADATION
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PHYSICAL DEGRADATION
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CHEMICAL DEGRADATION
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Ester hydrolysis
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Amide hydrolysis
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Ring alteration
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OXIDATION
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OXIDATION
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ISOMERISATION
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ISOMERISATION
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Optical isomerization can be further divided
into
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POLYMERIZATION
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DECARBOXYLATION
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ICH GUIDELINES
FOR
STABILITY STUDIES
International Conference on Harmonization
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INTRODUCTION
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INTRODUCTION
Currently ICH guidelines are most commonly accepted which provides
information on stability testing within the areas of European union(EU), Japan,
and United States
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ICH
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TITLES UNDER ICH GUIDELINES
PARAMETER SUB- SUB-SECTION TITLE
SECTIONS
Stability Q 1A (R2) Stability testing in New drugs & products
Q 1B Photo- stability testing
Q 1C Stability testing: New dosage forms
Q 1D Bracketing & Matrixing Designs for stability
Testing of Drug Substances & Drug Products
Q 1E Evaluation of Stability data
Q 1F Stability Data Package for Registration in Climatic
zones ІІІ & ІV
Analytical Q 2A Definitions & terminology
validation
Q 2B Methodology
Impurities Q 3A Impurity testing in New drug
Q 3B Impurities in dosage forms: Addendum to the
guideline on impurities in New drug Substances
Q 3C Impurities: Residual solvents
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Q1A (R2): STABILITY TESTING NEW
DRUG AND PRODUCT
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TYPES OF STABILITY STUDIES
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TYPES OF STABILITY STUDIES- STABILITY PROFILES
• Accelerated stability study
Storage condition Testing condition
Controlled room temperature: 20-250C 400C and 75% RH for 6 months
Refrigerated condition: 2-80C 250C and 60% RH for 6 months
Freezer condition: -20 to -100C 50C for 6 months
• Intermediate stability study
Storage condition Testing condition
Controlled room temperature: 20-250C 300C and 60% RH for 6 months
Refrigerated condition: 2-80C 100C and 50% RH for 6 months
Freezer condition: -20 to -100C 50C for 6 months
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TYPES OF STABILITY STUDIES- STABILITY PROFILES
• Long term (real time) stability study:
Storage condition Testing condition
Controlled room temperature: 20-250C 250 C and 60% RH for 12 months
Refrigerated condition: 2-80C 50 C for 12 months
Freezer condition: -20 to -100C -200 C for 12 months
• Stress testing / short term stability study
Storage condition Testing condition
Controlled room temperature: 20-250C 600 C and 75% RH for 2 days
Refrigerated condition: 2-80C 400 C and 75% RH for 2 days
Freezer condition: -20 to -100C 250 C and 60% RH for 2 days
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WHO GUIDELINES ON
STABILITY
OF PHARMACEUTICAL
PRODUCTS
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WHO guidelines on stability studies
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WHO guidelines on stability studies
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WHO guidelines on stability studies
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WHO guidelines on stability studies
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WHO guidelines on stability studies
Factors affecting stability of herbal
drugs
Storage
Moisture conditions Herb-
content Excipients
interaction
STABILITY
Microorganisms Environmental
Containers & factors
closures used
Challenges in stability testing of Herbal products
Active substances present in HMPs consist of complex
mixtures of constituents and in most cases the constituents
responsible for the therapeutic effects are unknown
In case pf polyherbal formulation, they have similar
constituents and this gives rise to even more analytical
challenges
Herbal substances/herbal preparations consist of
specific active principles; and many of them are known
to be unstable
Some herbal substance tend to undergo degradation and
microbial contamination. Also Enzymatic activities of
various enzymes present in herbs influences the shelf life
Stability Testing as per ICH Q1A(R2) & WHO
guidelines
Intended Study Test temperature & Test temperature &
storage humidity as per ICH humidity as per WHO
conditions
25 °C ± 2 °C/60% RH ± 5% RH or
25°C ± 2°C/60% RH ± 5% RH or
Long term 30 °C ± 2 °C/65% RH ± 5% RH or
30°C ± 2°C/65% RH ± 5% RH
30 °C ± 2 °C/75% RH ± 5% RH
General case
Intermediate 30°C ± 2°C/65% RH ± 5% RH 30 °C ± 2 °C/65% RH ± 5% RH
Accelerated 40°C ± 2°C/75% RH ± 5% RH 40 °C ± 2 °C/75% RH ± 5% RH
Drug Long term 5°C ± 3°C 5 °C ± 3 °C
substances
intended for 25 °C ± 2 °C/60% RH ± 5% RH or
storage in a Accelerated 25°C ± 2°C/60% RH ± 5% RH 30 °C ± 2 °C/65% RH ± 5% RH or
refrigerator 30 °C ± 2 °C/75% RH ± 5% RH
Drug
substances
intended for Long term - 20°C ± 5°C -20 °C ± 5 °C
storage in a
freezer
Testing frequency
Study Testing frequency
Long term 0, 3, 6, 9, 18, 24 and then
annually
Accelerated 0, 3 and 6
Parameters to be studied
Organoleptic
Physico-
parameters
chemical
parameters
• Color
• Odour chemical
• Taste • Determination parameters Microbiological
of PH parameters
• Phytochemical
• Moisture
analysis
content
• Specific • Total
• Extractive
biomarker microbial
value
• Fingerprinting count
• Ash value
profile
Conclusion
Conclusion
Reference
• Stability testing of new drug substance and products
Q1A(R2). ICH harmonised tripartite guideline, February
2003.
• Stability testing of active pharmaceutical ingredients
and finished pharmaceutical products. World Health
Organisation. 2009.
• Bansal G, Suthar N, Kaur J, Jain A. Stability Testing of
Herbal Drugs: Challenges, Regulatory Compliance and
Perspectives. Phytother. Res. 2016.
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STABILITY
TESTING
OF
HERBAL DRUGS
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Indicative substance for stability test
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INDICATIVE SUBSTANCE FOR
STABILITY TEST
• Factors considered in selection of indicative substances:
• Multiplicity of constituents, their qualitative & quantitative differences
through the growing period & numerous other influences.
• So the most labile compound must be chosen as
indicative substance.
• Each type of preparation requires its own type of indicative substance,
as its lability differs greatly under various environmental conditions.
• A hygroscopic substance would not be a suitable marker for
a dry preparation
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INDICATIVE SUBSTANCE FOR
STABILITY TEST
• Similarly in case of substances sensitive to oxidation or light,
evaluation done in following ways:
• Organoleptic: Taste, smell & appearance.
• Chemical & physical.
• For any alterations in the formulation during storage, evaluation done
by:
• Determination of density, refractive index, alcohol content, in cases of
liquid formulations
• TLC comparison of fresh material with that on completion of manufacture.
• Care should be taken not to interpret every little alteration as a
serious instability
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STABILIZATION & STABILITY
• Stabilization of a pharmaceutical preparation consists of
measures that guarantee its keeping quality or stability.
• METHODS OF STABILIZATION OF SOLIDS:
1. Drying:
• Physical, chemical or microbial contaminations take place more
easily in liquid preparations, esp. in aqueous medium, than in dry
products
• So drying of preparations & esp. keeping such preparations dry is the
simplest & best method of protection
• Residual moisture content in the dried extracts is limited to a
maximum of 5%; as extracts are dried more than this limit, they
readily attract water from surrounding atmosphere.
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STABILIZATION & STABILITY
• Rate at which the extracts absorbs moisture depends on its
specific surface area.
• This means that spray dried & freeze dried extracts
are more sensitive than roller or oven dried ones.
• This fact kept in mind while grinding extracts:
• Not done in rooms where there is high atmospheric
humidity.
• Also there should be no noticeable downward
temperature difference from the air in the room to the
material being milled, as otherwise water vapor will
condense on the cold extract.
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STABILIZATION & STABILITY
• Large quantities of extracts cannot be stored over a drying
agent, as uneconomically large quantities of the drying agent
will be required to ensure an adequate absorption capacity.
• Physical changes rarely occur at all under the above
mentioned conditions.
• Chemical changes, such as enzymatic reactions, hydrolysis,
oxidations , proceed extremely slowly when storage in a cool
place protected from light, as well as adequate drying, are
guaranteed.
• Entry of oxygen must be restricted by the choice of suitable
packing materials.
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STABILIZATION & STABILITY
• Impermeability of packing material to oxygen should be
checked if this packing material is a synthetic (plastic)
substance.
• In extreme cases:
• Material must be vacuum packed
• Sealed under an inert gas.
• Microbial alterations, i.e. generally multiplication of the
bacteria present in the product, is dependent on residual
moisture content in the product.
• METHODS OF STABILIZATION OF LIQUID
PREPARATIONS:
• Alternative processes proceed more rapidly in liquid
phytopharmaceutical preparations than in dry ones.
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STABILIZATION & STABILITY
• The following alterations are relatively easy to recognize:
• Physical alterations such as formation of sediments, color changes, etc.
• Alterations due to microbial growth, recognizable by the formation of
pellicle of mould, cloudiness or a sediment which can be easily
disturbed.
• Chemical alterations, when these can be detected organoleptically by
smell, taste or appearance.
• Chemical alterations such as hydrolytic decomposition, racemization,
oxidation, etc., detected with difficulty i.e. with analytical apparatus &
agents.
• Prevention of microbial growth in liquid formulations by
diethylpyrocarbonic ester prohibited, as this substance may
possibly form carcinogenic urethanes with free amino acids or
amines in the substrate.
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PARAMETERS FOR EVALUATION
OF STABILITY
• Appearance, Colour, Odour, Taste
• Particle size, Flowability, Viscosity, Clarity, pH
• Moisture content
• Sedimentation, Flocculation
• Emulsion breakage
• Friability, Hardness
• Extractive Values ( in selected solvents)
• Volatile matter contents, Free fatty acids/ acidity, Peroxide
values
• Microbial parameters
• Total viable count (TPC)
• Yeast and mould count ( YMC)
• Coil form count and other pathogens
• Specific parameters applicable to formulation / dosage form
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VALIDATION OF ANALYTICAL
PROCEDURES
• Various parameters considered in validation of analytical
procedures can be explained through foll. parameters:
• Purity of reagents, reference substances, solvents, etc.
• Reagents, reference substance & solvents are required for identity
testing & later quantitative analysis. Their purity must be specified or
guaranteed by 2 independent analysis methods e.g. GC-MS & NMR.
VALIDATION PARAMETERS:
1. Identity testing: In order to be certain in, for example, TLC,
that a certain band corresponds to a defined reference
substance, the distances migrated on chromatography are
compared with each other.
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VALIDATION OF ANALYTICAL
PROCEDURES
2. Specificity: A chromatographic method can only determine
an active substance specifically if the chromatographic
system selectively separates the active substance from
impurities, degradation products & excipients.
3. Linearity: The basis of any analytical method is a
functional relationship between the concentration of a
substance & the measured value. Ideally there should be a
linear calibration curve which passes through the origin
when tested graphically.
4. Sensitivity: The sensitivity describes the ability of an
analysis method to react to changes in the substances (e.g.
concentration).
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VALIDATION OF ANALYTICAL
PROCEDURES
5. Precision: The precision of an analytical method is given
by the coefficient of variation of the method. This is a
measure of the repeatibility of a determination method for
which exact methodological instructions are available.
6. Trueness: The trueness of an analysis is determined by
the systematic errors involved. The trueness can be
defined by the agreement of the measured analysis value
with the ‘actual value’.
