stability testing of natural products_2003

DR. HARISH KAKRANI,PURVI KAKRANI

STABILITYStability can be defined as the extent to

which the drug product retains its original properties & characteristics during its storage period.

Expiry date is a date after which the potency of the activities would be lost or reduced to sub-potent levels.

Stability testing is necessary to ensure the product is of acceptable quality throughout quality throughout its entire storage period

2Dr. Harish Kakrani, Purvi Kakrani

SIGNIFICANCE OF STABILITY TESTING

Chemical degradation may lead to under-medication due to lowering of the concentration of drug in dosage form. Patient has to receive the right & standard quality drug.

During decomposition of drug substance or drug product may lead to toxic productSay in case of p-amino salicylic acid, it is converted to p-

amino phenol (toxic).In polyherbal formulations, degradation of any one

constituent may lead to its decreased concentration in blood. The patient has to receive a uniform dose of drug

throughout shelf life.Determination of shelf life of a product.Determination of degradation product and the

possibility degradation pathway.3Dr. Harish Kakrani, Purvi Kakrani

TYPES OF STABILITY

Dr. Harish Kakrani, Purvi Kakrani 4

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

PHYSICAL DEGRADATION1. Loss of volatile constituents:

Many constituents are volatile at ambient temperatures Due to this, they may be lost from pharmaceutical

preparations. Example: Dill water, camphor water.

2. Loss of water: Evaporation of water from liquid, semi-solid formulations

& o/w emulsions cracking of these systems. Loss of water from aqueous solution

crystallization of solute3. Absorption of water/ Moisture:

Higher humidity may lead to moisture absorption generally observed with solid pharmaceuticals.

This absorbed moisture may participate in drug degradation itself as a reactant leading to hydrolysis, hydration, isomerization or other biomolecular chemical reactions.


PHYSICAL DEGRADATIONRemedies for stabilization against moisture absorption:

Use of moisture proof packaging, dessicants are often used to eliminate moisture in packaging.

Moisture proof film coatings.

4. Crystal growth:Common in syrupsFluctuations in the ambient temperature i.e. in day &

night crystal growth occurs.Usually this problem occurs when the vehicle becomes

supersaturated with solute precipitation of solute occurs seeding occurscrystal growth.

Remedies: Select suitable storage conditions. Increase the product viscosity, so that diffusion of

solute towards crystal seed Incorporation of surface active agents.


PHYSICAL DEGRADATION5. Color changes:

Indicative of chemical or photochemical changes. Remedies for stabilization against

photodegradation: Use of photoprotective films Film coatings on the formulations, say capsules.

6. Polymorphic changes: Many substances exist in two or more polymeric

forms . Upon storage in the dry state or in suspension

inter-conversion of these forms alterations in solubilty.


CHEMICAL DEGRADATION1. HYDROLYSIS:

Considered to be major cause of deterioration of drugs, esp. in aqueous solutions

Defined as the reaction of a compound with water. 2 forms of hydrolysis:

Ionic hydrolysis: Occurs when the salts of weak acids (e.g. potassium acetate) & bases (e.g. codiene phosphate) interact with water to give alkaline & acidic solutions respectively

Molecular hydrolysis: Includes cleavage of drug molecule. It is much slower process & an irreversible too.

Hydrolysis may be of 3 types: Ester hydrolysis Amide hydrolysis Ring alterationDr. Harish Kakrani, Purvi Kakrani 9

CHEMICAL DEGRADATIONa) Ester hydrolysis:

Prevalent in fatty acids. Involves rupture of a covalent bond between C- atom

& O- atom. Alkaline hydrolysis of an ester: Irreversible &

quantitative because the resultant acid is at once neutralized.

Acid hydrolysis of an ester: Reversible process; may be essentially completed in either direction by an excess of water or alcohol.

Remedies: Lowering buffer concentration Partial or complete replacement of water with non-aqueous

solvent, say propylene glycol. Adding complexing agent, say caffiene. By formulating in form of dry powder.


CHEMICAL DEGRADATIONb) Amide hydrolysis:

Hydrolytic cleavage of an amide results in formation of an acid & an amine.

c) Ring alteration: A hydrolytic reaction can proceed as a result of

ring cleavage with subsequent attack by H+ or OH- ion.

The hydrolytic reaction was reported to proceed by ring opening for an amine attack by water or OH- ion to give a carbinolamine intermediate decomposition to formaldehyde & 4-amino-6 chloro- m- benzene disulfonamide.


CHEMICAL DEGRADATION2. OXIDATION:

Instability in number of formulations is due to oxidation of active ingredient when exposed to atmospheric oxygen.

Mediated by free radicals or by molecular oxygen. Oxidation is:

Removal of an electropositive atom, radical or electron Addition of electronegative atom or radical

Prediction of susceptibility of the compound to oxidation from a standard oxidation- reduction potential:

Eh = E0 + o.o592 n log [oxidized form]/ [reduced form] Where Eh= reduction potential E0= standard reduction potential n= number of ions transferred per ion.


CHEMICAL DEGRADATIONAuto-oxidation is free radical chain reaction which proceeds

quite slowly under the influence of molecular oxygenFree radicals are produced by reactions involving hemolytic

bond fission of a covalent bond.Mainly occurs in oils & fats containing unsaturated linkages.Only small amount of oxygen is needed just to initiate the

reaction.

Rancidity is a term covering many typical off- flavors formed by auto oxidation of unsaturated fatty acids present in oil or fat.

3.ISOMERISATION:Isomerisation: Conversion of an active drug into a

less active or inactive isomer having same structural formula but differing in stereochemical configuration.


CHEMICAL DEGRADATIONIn stereoisomerism, the atoms making up the

isomers are joined up in the same order, but still manage to have a different spatial arrangement.

Optical isomerism is one form of stereoisomerismOptical isomers are molecules that differ three-

dimensionally by the placement of substituents around one or more atoms in a molecule.

Optical isomers were given their name because they were first able to be distinguished by how they rotated plane-polarized light.

Optical isomerisation can be further divided into:Racemization: This involves conversion of an optically

active form of a drug into its enantiomorph. The process continues until equal concenttration of 2

optically active from is obtained.


CHEMICAL DEGRADATIONEpimerisation: a compound having more than 1

asymmetric carbon atom in the molecule.Example: epimerisation frequently found in

carbohydrates, more specifically sucrose.Geometrical isomerisation: Loss of activity is

due to difference in potency exhibited by - cis & -trans isomers of some organic compounds

Example: Active form of vitamin A molecule has all trans configuration.

4. POLYMERIZATION:Involves combination of 2 or more identical molecules to

form a much larger & complex moleculeThis is a major cause of degradation of antiseptic from

aldehyde.Dr. Harish Kakrani, Purvi Kakrani 15

CHEMICAL DEGRADATION5. DECARBOXYLATION:

Loss of CO2 Mainly occurs when parenteral solutions of

sodium bicarbonate autoclaved. Remedy: CO2 is passed into the solution for 1

minute.

6. ABSORPTION OF CO2: Most frequent occurrence than the

decarboxylation.


INTRODUCTIONPatient taking a pharmaceutical product expects the

product to be safe and efficacious. Pharmaceutical regulatory agencies world wide demand

that the product remains its identity, quality, purity and potency for the time the product is commercially available.

Various stability guidelines describing the types of studies &types of data needed are:Food and Drug Administration (FDA)International Conference on Harmonization (ICH)European Union Guidelines (EU)Japanese Guidelines (MHW)World Health Organization (WHO) GuidelinesGulf Central Committee (GCC) – For Gulf CountriesAssociation of South East Asian Nations (ASEAN)Eastern Mediterranean Region etc.Therapeutic Good Administration (TGA) – For Australia


INTRODUCTIONCurrently ICH guidelines are most commonly

accepted which provides information on stability testing within the areas of European union(EU), Japan, and United States.


ICHThe international conference of harmonization of

technical requirement for registration of pharmaceutical for human use (ICH) is a unique project that brings together the regulatory authorities of Europe, Japan, and USA and experts from the pharmaceutical industry in three regions to discuss scientific and technical aspects of product registration.

The objectives of such harmonization is: -A more economical use of human, animal, and

material resources.The elimination of unnecessary delay in the global

development and availability of new medicines.Maintaining safeguards on Quality, safety and

efficacy and regulatory obligations to protect public health


TITLES UNDER ICH GUIDELINESPARAMETE

RSUB-

SECTIONSSUB-SECTION TITLE

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 validation

Q 2A Definitions & terminology

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 solventsDr. Harish Kakrani, Purvi Kakrani 21

Q1A (R2): STABILITY TESTING NEW DRUG AND PRODUCTBasic Concepts For Stability Testing :-

There are two parts : 1. For Active Substance

Stress Testing Selection of Batches Container Closure System Specification Testing Frequency Storage Conditions Stability Commitment Evaluation Statements/Labelling


Q1A (R2): STABILITY TESTING NEW DRUG AND PRODUCT2. For pharmaceutical product:

Selection of BatchesContainer Closure SystemSpecificationTesting FrequencyStorage Conditions Stability CommitmentEvaluationStatements/Labelling In-use stabilityVariations

On-going Stability Studies


I. STABILITY TESTING FOR ACTIVE SUBSTANCE

1.1 GENERAL:For active substances not described in an official

pharmacopoeial monograph, stability studies are required.For active substances described in an official

pharmacopoeial monograph, which covers the degradation products and for which suitable limits have been set but a re-test period is not defined, two options are acceptable:The manufacturer confirms that the active substance complies

with the pharmacopoeial monograph immediately prior to the manufacture of the pharmaceutical product. In this case no stability studies on the active substance are required

The manufacturer establishes a re-test period based on the results of long term testing stability studies conducted on the active substance.


I. STABILITY TESTING FOR ACTIVE SUBSTANCE1.2 Stress Testing:Stress testing of the active substance can help

identify the likely degradation products, which can in turn help establish the degradation pathways and the intrinsic stability of the molecule.

For an active substance the following approaches may be used: No data are required on the degradation products,

if an active substance is described in an official pharmacopoeial monograph.

For active substances not described in an official pharmacopoeial monograph, there are two options:



When available, it is acceptable to provide the relevant data published in the literature to support the proposed degradation pathways;

When no data are available in the scientific literature, including official pharmacopoeias, stress testing should be performed.

Stress testing is likely to be carried out on a single batch of the active substance.It should include the effect of temperatures (in

10°C increments (e.g., 50°C, 60°C, etc.) above that for accelerated testing),

humidity (e.g., 75% RH or greater) where appropriate, oxidation and photolysis on

the active substance.Photostability testing should be an integral part of

stress testing.Dr. Harish Kakrani, Purvi Kakrani 26

1.3 Selection of BatchesData from formal stability studies provided on at

least 3 primary batches of the active substance.The primary batches should be manufactured to a

minimum of pilot scale.Pilot batches manufactured by the same synthetic

route and method of manufacture that simulates the final process to be used for production batches.

1.4 Container Closure SystemThe stability studies should be conducted on the

active substance packaged in a container closure system that is the same as or simulates the packaging proposed for storage and distribution.




1.5 Specification Stability studies should

Should cover, as appropriate, the physical, chemical, biological, and microbiological attributes.

Include testing of those attributes of the active substance that Are susceptible to change during storage and Are likely to influence quality, safety, and/or

efficacy



1.6 Testing FrequencyFor active substances with a proposed re-test period

of at least 12 months, the frequency of testing at the long term storage condition should normally be Every three months over the first year, Every six months over the second year, and Annually thereafter through the proposed re-test period.

1.7 Storage ConditionsThe storage conditions and the lengths of studies

chosen should be sufficient to cover storage, shipment, and subsequent use with due regard to the climatic zone(s) in which the active substance is intended to be stored.



The long-term testing Should cover a minimum of 12 months’ duration on

at least three primary batches at the time of submission and

Should be continued for a period of time sufficient to cover the proposed re-test period.

1.7.1 General Case:


Term Duration

Testing condition

Long term 12 months

•25°C ± 2°C/60% RH ± 5% RH or •30°C ± 2°C/65%RH ± 5% RH

Intermediate 6 months 30°C± 2°C/65% RH ± 5% RH

Accelerated 6 months 40°C± 2°C/75% RH ± 5% RH


If “significant change” occurs at any time during six months’ testing at the accelerated storage condition, additional testing at the intermediate storage condition should be conducted and evaluated against significant change criteria.

1.7.2 Active substances intended for storage in a refrigerator


Term Duration Testing conditions

Long term 12 months 5°C ± 3°C

Accelerated 6 months •25°C ± 2°C/60% RH ± 5% RH or•30°C ± 2°C/65% RH ± 5% RH


If significant change occurs between three and six months’ testing at the accelerated storage condition:The proposed re-test period should be based on the

real time data available at the long term storage condition.

If significant change occurs within the first three months’ testing at the accelerated storage condition:A discussion should be provided to address the effect of

short term excursions outside the label storage condition, e.g. during shipping or handling.


I. STABILITY TESTING FOR ACTIVE SUBSTANCE1.7.3 Active substances intended for storage

in a freezer

In the absence of an accelerated storage condition for active substances intended to be stored in a freezer, testing on a single batch at an elevated temperature (e.g. 5°C ± 3°C or 25°C ± 2°C or 30°C ± 2°C) for an appropriate time period should be conducted to address the effect of short term excursions outside the proposed label storage condition


Term Duration Testing Conditions

Long term 12 months - 20°C ± 5°C


1.8 Evaluation: Evaluating the stability information (including, as

appropriate, results of the physical, chemical, biological, and microbiological tests), a re-test period is established applicable to all future batches of the active substance manufactured under similar circumstances.

The degree of variability of individual batches affects the confidence that a future production batch will remain within specification throughout the assigned re-test period.


I. STABILITY TESTING FOR ACTIVE SUBSTANCE1.9 Statements/Labelling:

A storage statement should be established for the labeling based on the stability evaluation of the active substance.

Where applicable, specific instructions should be provided, Example: Particularly for active substances that

cannot tolerate freezing, terms such as “ambient conditions” or “room temperature” must be avoided.


II. STABILITY TESTING FOR PHARMACEUTICAL SUBSTANCE 2.1 General:The design of the formal stability studies for

the pharmaceutical product should be based on:Knowledge of the behavior and properties of

the active substanceFrom stability studies on the active substance From pre-formulation studies.


2.2 Selection of Batches Data from stability studies should be provided on at

least three primary batches of the pharmaceutical product.

The primary batches should be of the same formulation and packaged in the same container closure system as proposed for marketing.

Two of the three batches should be at least pilot scale batches and the third one can be smaller, if justified.

Where possible, batches of the pharmaceutical product should be manufactured by using different batches of the active substance.


II. STABILITY TESTING FOR PHARMACEUTICAL SUBSTANCE

2.3 Container Closure System Conducted on the dosage form packaged in the

container closure system proposed for marketing.

2.4 SpecificationShould cover, as appropriate, the physical,

chemical, biological, and microbiological attributes, preservative content (e.g. antioxidant, antimicrobial preservative), and functionality tests (e.g. for a dose delivery system).

Analytical procedures should be fully validated.



II. STABILITY TESTING FOR PHARMACEUTICAL SUBSTANCE 2.5 Testing Frequency

For products with a proposed shelf life of at least 12 months, the frequency of testing at the long term storage condition should normally be Every three months over the first year, Every six months over the second year, and Annually thereafter through the proposed shelf life.

At the accelerated storage condition, a minimum of three time points, including the initial and final time points (e.g. 0, 3, and 6 months) from a 6-month study is recommended.


When testing at the intermediate storage condition is called for as a result of significant change at the accelerated storage condition, a minimum of four time points, including the initial and final time points (e.g. 0, 6, 9, 12 months) from a 12-month study is recommended.



2.6 Storage:In general, a pharmaceutical product should be evaluated

under storage conditions (with appropriate tolerances) that test:Its thermal stabilityIf applicable, its sensitivity to moisture or potential for

solvent lossPhotostability testing conducted on at least one primary

batch of the pharmaceutical product.Stability studies conducted on one batch of the

pharmaceutical product for up to three months at 50°C/ambient humidity may be useful to identify the formulation and packaging material adequate for extremely hot and dry conditions.



The storage conditions and the lengths of studies chosen should be sufficient to cover storage, shipment, and subsequent use with due regard to the climatic zone(s) in which the product is intended to be marketed.

2.6.1 General case



Term Duration

Testing condition

Long term 12 months

•25°C ± 2°C/60% RH ± 5% RH or •30°C ± 2°C/65%RH ± 5% RH

Intermediate

6 months

30°C± 2°C/65% RH ± 5% RH

Accelerated 6 months

40°C ± 2°C/75% RH ± 5% RH

2.6.2 Pharmaceutical products packaged in impermeable containers:

Stability studies for products stored in impermeable containers can be conducted under any controlled or ambient humidity condition.Reason being, Sensitivity to moisture or potential

for solvent loss is not a concern for pharmaceutical products packaged in impermeable containers that provide a permanent barrier to passage of moisture or solvent.



2.6.3 Pharmaceutical products packaged in semi-permeable containers

Aqueous-based products packaged in semi-permeable containers should be evaluated for potential water loss, in addition to physical, chemical, biological, and microbiological stability.

Other comparable approaches can be developed and reported for non-aqueous, solvent-based products.



A 5% loss in water from its initial value is considered a significant change for a product packaged in a semi-permeable container after an equivalent of three months’ storage at 40°C/NMT 25% RH.

However, for small containers (1 ml or less) or unit-dose products, a water loss of 5% or more after an equivalent of three months’ storage at 40°C/NMT 25% RH may be appropriate.


II. STABILITY TESTING FOR PHARMACEUTICAL SUBSTANCE Term Duratio

n Testing condition

Long term 12 months

•25°C ± 2°C/40% RH ± 5% RH or •30°C ± 2°C/35% RH ± 5% RH

Intermediate

6 months

30°C ± 2°C/35% RH ± 5% RH

Accelerated 6 months

40°C ± 2°C/not more than 25% RH

In general, “significant change” for a pharmaceutical product is defined as: A 5% change in assay from its initial value; or failure

to meet the acceptance criteria for potency when using biological or immunological procedures;

Any degradation product exceeding its acceptance criterion;

Failure to meet the acceptance criteria for appearance, physical attributes, and functionality test conditions; and, as appropriate for the dosage form:

Failure to meet the acceptance criterion for pH; orFailure to meet the acceptance criteria for

dissolution for 12 dosage units. Dr. Harish Kakrani, Purvi Kakrani 46


2.6.4 Pharmaceutical products intended for storage in a refrigerator

If significant change occurs between three and six months’ testing at the accelerated storage condition, the proposed shelf life should be based on the real time data available from the long term storage condition.


Term Duration Testing conditions

Long term 12 months 5°C ± 3°C

Accelerated 6 months •25°C ± 2°C/60% RH ± 5% RH or•30°C ± 2°C/65% RH ± 5% RH


2.6.5 Pharmaceutical products intended for storage in a freezer

For pharmaceutical products intended for storage in a freezer, the shelf life should be based on the real time data obtained at the long term storage condition.

In the absence of an accelerated storage condition, testing on a single batch at an elevated temperature (e.g., 5°C ± 3°C or 25°C ± 2°C or 30°C ± 2°C) for an appropriate time period conducted to address the effect of short term excursions outside the proposed label storage condition.



Term Duration Testing Conditions

Long term 12 months - 20°C ± 5°C

2.7 Evaluation If analysis shows that the batch-to-batch

variability is small, it is advantageous to combine the data into one overall estimate. This can be done by first applying appropriate

statistical tests.If it is inappropriate to combine data from

several batches, the overall shelf life should be based on the minimum time a batch can be expected to remain within acceptance criteria.



2.8 Statements/Labelling The following statements should be used if

applicable:



Testing condition where the stability of the pharmaceutical product has been shown

Recommended labelling condition

For countries in Climatic Zones I and II: 25°C/60% RH (long term) 30°C/65% RH (intermediate) 40°C/75% RH (accelerated)

Store below 30˚C

for countries in Climatic Zones III and IVA: 30°C/65% RH (long term) 40°C/75% RH (accelerated)

Store & transport below 30˚C

2.9 In-use stability The purpose of in-use stability testing is to establish

– where applicable – the period of time during which a multi-dose product can be used whilst retaining acceptable quality once the container is opened and the first dose is removed.

A minimum of two batches, at least pilot scale batches, should be subjected to the test. At least one of the batches should be chosen towards

the end of its shelf life. If such results are not available, one batch should be

tested at the final point of the submitted stability studies.



2.10 Variations Once the pharmaceutical product has been

registered, additional stability studies are required whenever major variations are made like the following:

1.Change in the manufacturing process; 2.Change in the composition of the

pharmaceutical product; 3.Change of the immediate packaging.



2.11 On-going Stability Studies After approval, the stability of the

pharmaceutical product should be monitored according to a continuous appropriate programme that will permit the detection of any stability issue.

The purpose of the on-going stability programme is:To monitor the product over its shelf life andTo determine that the product remains, and can be

expected to remain, within specifications under the labelled storage conditions.



The protocol for an on-going stability programme should extend to the end of the shelf life period the following parameters: Number of batch(es) per strength and different batch

sizes, if applicable; Relevant physical, chemical, microbiological and

biological test methods; Acceptance criteria; Reference to test methods; Description of the container closure system(s); Testing intervals (time points); Description of the conditions of storageOther applicable parameters specific to the

pharmaceutical product.Dr. Harish Kakrani, Purvi Kakrani 54


Accelerated stability studyIntermediate stability studyLong term (real time) stability studyStress testing / Short term stability studyForced degradation testing &Thermal cycling stability study


TYPES OF STABILITY STUDIES

Accelerated stability study

Intermediate stability study


TYPES OF STABILITY STUDIES- STABILITY PROFILES

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


Controlled room temperature20-250C


Refrigerated condition2-80C


Freezer condition-20 to -100C

50C for 6 months

Long term (real time) stability study:

Stress testing / short term stability study


TYPES OF STABILITY STUDIES- STABILITY PROFILES




Refrigerated condition: 2-80C 50C for 12 months

Freezer condition: -20 to -100C -200C for 12 months



600C and 75% RH for 2 days

Refrigerated condition: 2-80C 400C and 75% RH for 2 days

Freezer condition: -20 to -100C 250C and 60% RH for 2 days

WHO GUIDELINES ON STABILITY STUDIES

Accelerated stability testing on two batches should be conducted, one of which must be either commercial batch or a pilot-scale batch. A pilot batch should not be smaller than one eighth of

the commercial batch.Initially real time stability testing should be

conducted on two batches for the expected shelf life of the product, one of which should be commercial batch. One batch can be a pilot scale batch.

Stability testing should be conducted on the dosage form packaged in the container closure system proposed for marketing


Stability studies should include testing of those attributes of the pharmaceutical product that are susceptible to change during storage and are likely to influence quality, safety, and efficacy. The testing should cover, as appropriate, the physical,

chemical, biological and microbiological attributes, preservative content (e.g. antioxidant, antimicrobial preservative).

Analytical procedures should be fully validated and stability indicating

If significant change occurs between 3 and 6 months’ testing at the accelerated storage condition, the proposed shelf-life should be based on the real-time data available from the long- term storage condition.



Stability studies for products stored in impermeable containers can be conducted under any controlled or ambient humidity condition.

If no significant change occurs during 6 month's accelerated and real time stability testing, the product will be allowed to place in the market with a provisional shelf-life of up to 24 months.

However, real time stability testing should be continued up to the proposed shelf-life.



Once the pharmaceutical product has been registered, additional stability studies are required whenever variations that may affect the stability of the active pharmaceutical substance or pharmaceutical product are made, such as major variations like the following:Change in the manufacturing process.Change in the composition of the pharmaceutical

product.Change of the immediate packaging.

The ongoing stability programme should be described in a written protocol, and results formalized as a report.



Condition for real time stability testing: 30°C ± 2°C/65% RH ±5% RH; condition for accelerated stability testing: 40°C

± 2°C/75% RH ± 5% RH.



INDICATIVE SUBSTANCE FOR STABILITY TESTIndicative substance can be termed as

biomarker.Biomarker is a chemical entity in the plant

material that may or may not be chemically defined and serves as a characteristic fingerprint of that plant.

Utility of indicative substance:Active substances are necessary to be evaluated

for the shelf life of the formulation.So if the active constituent is unknown, use of

indicative substance to evaluate the stability limits.Dr. Harish Kakrani, Purvi Kakrani 65

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


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 formulationsTLC comparison of fresh material with that on completion

of manufacture.Care should be taken not to interpret every little

alteration as a serious instability


INDICATIVE SUBSTANCE FOR STABILITY TEST

STABILIZATION & STABILITYStabilization 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.


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.


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.



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 packedSealed 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.



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.



Appearance, Colour, Odour, TasteParticle size, Flowability, Viscosity, Clarity, pHMoisture contentSedimentation, FlocculationEmulsion breakageFriability, HardnessExtractive Values ( in selected solvents)Volatile matter contents, Free fatty acids/ acidity,

Peroxide valuesMicrobial parameters

Total viable count (TPC)Yeast and mould count ( YMC)Coil form count and other pathogens

Specific parameters applicable to formulation / dosage form


PARAMETERS FOR EVALUATION OF STABILITY

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.


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).



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’.



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Documents

y y y yy y y common

y y loss of water

yy y y hydrolysis

y y ionic hydrolysis

y patient

y prevalent

y acid hydrolysis

y alkaline hydrolysis