DRUG STABILITY STUDIES

Contents

1. Introduction
2. Chemical Kinetics
a.Zero order process
b.First order process
c.Second order process
d.Determination of order
3 Routes of degradation
a a.Hydrolysis
b b.Oxidation-Reduction
c.Photolysis
c d.Racemization

1. Accelerated stability analysis

2. Addition of overages
3. Factors affecting the stability
4. Preformulation studies
5. Storage considerations
a. ICH and WHO guidelines
 DRUG STABILITY STUDIES

INTRODUCTION

The term stability with respect to drug dosage form refers to chemical and physical
integrity of the dosage form unit and its ability to offer protection against microbial
contamination. The degradation occurs mainly because of the chemical reaction of the active
ingredients or additives.

Stability can be defined as the capacity of drug product to remain within specifications
established to ensure its identity, strength, quality, and purity. Precisely, it is the ability of the
product to resist deterioration. The stability of the product is expressed as the expiry period or
shelf life.

Application of the principles of chemical kinetics helps in determining the various factors
affecting stability. The results of such kinetic studies aids in selection of appropriate formulation
and predicting the shelf-life of the product. The shelf-life of the dosage form is the time lapse
from the initial preparation and packaging to the last day of specified expiration date i.e., when
the potency reaches 90% of the labelled value. It is defined as the time required for the
concentration of the reactant to reduce to 90% of its initial concentration. Shelf life is represented
as t 90 and has the units of time/concentration.

During drug development, several bulk lots are produced. The first lot used for stability
studied may not be the representative of the whole bulk, but it acts as a baseline for the
consequent lots to be analyzed. The application of certain physicochemical principles in the
performance of stability studies has proved to be considerable advantage in the development of
stable dosage forms.

Most of the drugs are susceptible to some form of decomposition. As a result, the physical
properties, chemical composition may change on ageing. Aged drug may contain significantly
less amount of the active medicament and hence may show poor bioavailability. So it is necessary
to mention the shelf life on the label. A manufacturer is obliged to indicate the shelf life of a drug
on the label unless it is greater than 3 years. No drug may be sold after 5 years.

Stability studies are important for the following reasons.

1. This is an assurance given by the manufacturer that the patient would receive a uniform dose
throughout the shelf life.
2. The drug control administration insists on manufacturers on conducting the stability studies,
identity, strength, purity and quality of the drug for an extended period of time in the
conditions of normal storage.
3. Stability testing prevents the possibility of marketing an unstable product.

Both physical and chemical degradation of drug can result in unstable product.
Physical degradation may be due to-

1. Loss of volatile constituents-Example: Nitroglycerine tablets.

2. Loss of water- Example:An effervescent substance loose water
3. Absorption of water-Example: Calcium chloride-A deliquescent substance absorbs water.
4. Crystal growth-Example:10% w/v Calcium gluconate injection gives precipitation and
undergo crystallization due to supersaturated solution.
5. Colour change-Example: Aspirin tablets become pink and Ascorbic tablets turns yellowish
brown when exposed to air.

Chemical degradation may be due to the functional group present. Some of them are:

1. Hydrolysis-Example: Aspirin and Procaine

2. Oxidation-Example: Ascorbic acid and vegetable oil.
3. Isomerisation- Example: (-) Adrenaline is more active and (+) Adrenaline is less potent.
Trans vitamin A palmitate is more active.
4. Absorption of carbondioxide-Example: Sodium hexobarbitone IV injection
5. Decarboxylation-Example: Procaine gives a dark colored liquid due to loss of carbon dioxide

Purpose of stability studies

Stability studeisis done to understand how to design a product and its packaging such tha
the product has appropriate physical .chemical and microbiological properties during a defined
shelf life when stored and used.

CHEMICAL KINETICS

Most of the degradative reactions in pharmaceutical formulations take place at definite

rates and chemical in nature. An effective and efficient study of these reactions requires the
applications of chemical kinetic principles. Most of the rate limiting phenomena is describable by
some equation systems. The reaction rate depends upon the concentration of reactants and this
describes the order of reaction. The degradation of most of the pharmaceuticals follows zero
order, first order and pseudo first order.

Reaction Kinetics is the study of rate of chemical change and the way in which this rate is
influenced by the conditions of the concentrations of reactants, products and other chemical
species which may be present and by factors such as solvent, pressure and temperature. In case of
pharmaceuticals, such information permits a rational approach to the stabilization of drug
products and prediction of shelf life and decisions regarding optimum storage conditions.

Zero order process

When the rate of disappearance of a reactant ‘A’ is constant and independent of its
concentration is said to be zero order process.
Mathematically it can be expressed as;

-dA = K or K= A0-At Where K is the rate constant

dt t At is the amount of A remaining
at time ‘t’
A0 is the initial concentration.
t90%=0.1 At
K

Example:
1.Photochemical reactions in which the rate determining factor is the light intensity rather
than concentrations of reactant are zero order reactions, like loss of colour in multisulfa product.

2.Pharmaceuticals like Riboflavin, Nifedipine are common drugs which are extremely
light sensitive and follow zero order process.

First order process

When the rate of disappearance of reactant ‘A’ is proportional to the concentration of ‘A’
at time ‘t’ is said to be first order process.

Mathematically it can be expressed as;

-dA/dt = KA or In At/Ao = kt

log a = Kt t½= 0.693

(a - x) 2.303 K

t90%= 0.105/K

Where, the rate constant K is in almost all cases a function of the temperature T. For most
pharmaceutical products, as t is increased, the rate constant and therefore the rate of degradation
increases.A0 is the initial concentration and at is the concentration at time t.

Example:

1. Decomposition of Hydrogen peroxide catalyzed by Potassium iodide.

2. Absorption, distribution. Metabolism and excretion of the drug.
Second order process

When the rate of disappearance of reactants ‘A’ and ‘B’ is proportional to the
concentration of each of ‘A’ and ‘B’at time ‘t’ is said to be second order process.

Mathematically it can be expressed as;

-dA/dt = dB/dt = K(AB) or

dx/dt = K(a-x) . (b-x)

K = 2.303 log b(a-x)

t(a-b) a(b-x)
Where (a-x) and (b-x) are the concentrations of A and B remaining at time‘t’ and x is the
concentration of drug decomposed at time t.

Example:

1. Hydrolysis of Chlorobutanol in presence of sodium hydroxide.

2. Alkaline hydrolysis of esters such as methyl acetate.

DETERMINATIONS OF ORDER

The order of a reaction may be determined by several methods,

Subsituation method

The data accumulated in a kinetic study may be substituted in the integrated form of the equations
that describe the various orders. When the equation is found in which the calculated ‘K’ value
remains constant within the limits of experimental variations, the reaction is considered to be of
that order.

Graphic Method

If a straight line results when concentration is plotted against t, the reaction is zero order. The
reacton is first order, if log (a-x) versus t yields a straight line and it is second order if 1 / (a-x)
versus t gives a straight line.When a plot of 1 / (a-x)2 against ‘t’ produces a straight line, with
all reactants at the same initial concentrates the reaction is third order.

Half-life method:

Order Integrated rate Equation Half life equation

0 x = Kt t½ = a
2k
 1 log a = Kt t½= 0.693
(a - x) 2.303 K

2 x = Kt t½= 1
a( a - x) aK

3 2ax - x2 = 2 Kt t½= 3 1
a 2 (a-x) 2 2
a K

The relationship between these results shows that in general the half-life of a reaction in
which concentration of all reactants are identical is given by

t½ ∝ 1
n–1
a

n is the order of the reaction. Thus if two reactions are run at different initial concentrations, a1
and a2, the half lifes t ½ (1) and t ½ (2) are related as follows,

log t ½ (1) = (n – 1) log a2

t ½ (2) a1

= n = log t ½ (1) / t ½ (2) +1

log (a2/ a1)
ROUTES OF DEGRADATION

The major routes of drug degradation are summerized in the following table.

OXIDATION HYDROLYSIS PHOTO CHEMICAL

DEGRADATION

Stabilization: Stabilization: Stabilization:

- Addition of anti - Adjustment of pH - Storage in Amber

Oxidants - Use of non-aqeous coloured containers
Solvent - Use of opaque wrapper
- Addition of reducing - Use of complexing - Use of opacifiers
Agents agent incorporation of UV
Light absorbers.
- Adjustment of pH

- Providing inert
Environment.

ROUTES OF DEGRADATION

RECEMIZATION

DECARBOXYLATION
ACYLATION

POLYMERIZATION

POLYMORPHISM SOLUATE - FORMATION

DEGRADATIVE PATHWAY
HYDROLYSIS

Many pharmaceuticals contain ester or amide functional groups, which undergo hydrolysis
in solution. Examples of drugs that tend to degrade by hydrolytic cleavage of an ester or amide
linkage are anesthetics, antibiotics, vitamins and barbiturates.

Many pharmaceuticals contain ester or amide functional groups, which undergo hydrolysis
in solution. Examples of drugs that tend to degrade by hydrolytic cleavage of an ester or amide
linkage are anesthetics, antibiotics, vitamins and barbiturates.

Ester hydrolysis:

The hydrolysis of an ester into a mixture of an acid and alcohol essentially involves the
rupture of a covalent linkage between a carbon atom and an oxygen atom. Although some of this
hydrolysis can be affected in pure water, in the majority of cases, the presence of a catalyst is
needed to promote the reaction.

These catalysts are capable of supplying hydrogen or hydorxyl ion to the reaction mixture.
(Polar compounds)

a. Hydrolysis effected by pure H2 O;

o o
|| + - ||
R - C - OR + H + - OH R - C - OH + HOR
(Acid) (Alcohol)

b. In case of acid catalysed reactions:

o o o
|| 1 + || 1 || 1
R - C - OR + H R - C - OR - - R – C – OH + R OH
|
H

c. In case of alkaline catalysed reactions:

o o
|| 1 || 1 1
2R - C - OR + O H R - C - OR RCOOH + R OH
|
OH
 A number of reports in the literature deal with detailed kinetic studies of the hydrolysis of
pharmaceutical ingredients containing an ester group in the molecule. Degradation of aspirin in
various buffer solutions and treated the overall reaction as pseudo – first order.

Aspirin hydrolysis:

The pH of optimum stability is at 2.4. At a pH of 5 to 7 the degradation reaction was

essentially pH – independent and at a pH above 10, the stability of aspirin was found to decrease
rapidly with increase in pH.

Other pharmaceutical materials that have been reported to degrade through ester
hydrolysis are procaine, atropine, and methyl P – amino benzoate.

A number of drugs degrade through ester hydrolysis. To enhance the stability of

pharmaceuticals undergoing this type of degradation, the following factors are to be considered.

1. pH: If physiologically permissible, the solution of the drug should be formulated as close as
possible to its pH of optimum stability.

2. Type of Solvent: Partial or full replacement of water with a solvent of lower dielectric
constant generally causes a considerable decrease in the velocity of ester hydrolysis.
Examples of these nonaqueous solvents are ethenol, glycols, glucose and mannital solutions
and substituted amides.

3. Complexation: The hydrolytic rates may be influenced in two ways by complex formation,
namely, by either stearic or polar effects.

Example: Caffine complexes with local anesthetics, such as Benzocaine, Procaine and
Tetracaine cause a reduction of the velocity of their hydrolytic degradation. The complexed
fraction of the ester undergoes essentialy no degradation.
The velocity of the base catalysed decomposition of riboflavin was decreased by the
Presence of caffeine in solution. It was found that the vitamin in its complexed form with
Caffeine possessed negligible reactivity towards alkaline hydrolysis.

4. Surfactants:Non ionic, catonic and anionic surfactans stabilize the drug against base
catalysis. Example: A 5% Sodium lauryl sulfate Solution (anionic) caused an 18 – fold
increase in the half – life of bernzocaine. When 2.46% cetyl trimethyl ammonium bromide
in solution (Cationic) is used, a ten fold increase in the half – life of benzocaine is seen.

5. Modification of chemical structure: By increasing the length of or by branching the acyl

or alkyl chain, the rate of hydrolysis of the ester usually decreases, owing to steric hindrance.
However, if an electrophilic or nucleophilic group is introduced into the acyl or alkyl side
 chain of aromatic esters, the rate of hydrolysis can be increased or decreased by the
electronic effect of these groups.

For example, alkaline hydrolysis of aromatic esters is promoted by the presence of

electrophilic groups on the benzene ring (halogen or NO2), which attract electrons away from
the reacton site (ester groups). The hydrolysis is retarded, on the other hand, by nucleophilic
groups (CH3, OCH3 and NH2 which cause electrons to move toward the point of reacton,
The reverse effect would be found in the case of hydrogen ion catalysed hydrolysis of
aromatic esters.

6. Salts and esters: Another technique that is sometimes employed to increase the stability of
pharmaceuticals undergoing degradation through ester hydrolysis is to reduce their
solubility by forming less soluble esters of the drug.

Amide hydrolysis

Pharmaceutical compounds containing an amide group can undergo hydrolysis resulting

in the formation of an acid and an amine.

o H o
|| | ||
R - C - N - R 1 + H2O R - C - OH + H2N - R 1
Amide Acid Amine

Pharmaceuticals such as niacinamide, phenethicillin, barbiturates and chloamphenical

degrade by amide hydrolysis.
The basic hydrolysis proceeded as follows:

o o o
|| slow | fast ||
R - C - NH R + OH
1
R - C - NH R 1
R - C - OH + R 1 NH

| FAST
OH
o
||
RC - O+ R 1 NH2

The rate determining step in the hydroxide ion – catalysed reaction is the nucleophilic attack by
the hydroxide ion.

The acid hydrolysis was as follows:

O O O
|| 1 fast || 1 slow |
RC - NHR + H3O RC – NH2R + H2O R - C - NH2R
|
O
|
H H
O O
|| 1 fast || 1
RC – OH + R NH3 RC – OH2 + R NH2
fast

The mechanism for acid hydrolysis of amides requires that subsituent should exert only
weak polar effects, but that when suitably situated, they should exert strong stearic effects.

The methods available for retarding deterioration through amide hydrolysis are similar to
those presented under ester hydrolysis.

Ring alteration

A hydrolytic reaction can proceed as a result of ring cleavage with subsequent attack by
hydrogen or hydroxyl ion. Examples of drugs that have been reported to undergo hydrolysis by
this mechanism include hydrochlorothiazide, pilocarpine and reserpine.

Example: The hydrolysis of pilocarpine in aqueous solution has been reported to involve a cyclic
equilibrium process, which is catalysed by hydrogen ion and hydroxyl ion.
 The concentration of pilocarpate and pilocarpic acid are influenced by pH. Pilocarpine is
relatively stable in solutions of acidic pH. As the pH increases, pilocarpine progressively becomes
unstable.

OXIDATION REDUCTION

The oxidative decomposition of pharmaceutical compounds is responsible for the

instability of a considerable number of pharmaceutical preparations. For example, steroids,
vitamins, antibiotics and epirephrine undergo oxidative degration. These reactions are medicated
either by free radicals or by molecular oxygen.

A substance is said to be oxidized if electrons are removed from it. Oxidation oftern
involves the addition of oxygen or the removal of hydrogen.

Ex: Ferrous ion is oxidised to ferric ion.

++ +++ -
Fe Fe + e

The most common form of oxidative decomposition is auto oxidation; it may

be defined as the reaction of any material with molecular oxygen.

The auto oxidation of an organic substance RH by a free radical chain

process can be simply described as follows’

Activation .
Initiation: RH R . + (H)
Light, heat

.
Propagation: R . + O2 RO2
.
RO2 + RH ROOH + R.

Hydroperoxide Decomposition:

ROOH RO. + . OH

Termination: RO2 . + X inactive products

RO2 + RO2 inactive products

Heavy metals, particularly those possessing two or more valency states, with a suitable
oxidation-reduction potential between them (copper, iron, cobalt and nickel) generally catalyze
oxidative deteriorations. These metals reduce the length of the induction period (the time in which
no measurable oxidation occurs) and increase the maximum rate of oxidation. They can affect the
rates of chain initiation, propagation and termination as well as the rate of hydroperoxide
decomposition.

Many oxidations are catalysed by hydrogen and hydroxyl ions. Example quinone to
hydroquinone.Although the oxygen concentration is of importance in the auto oxidation process,
its significance is usually not adequately considered. For the most part, oxidative degradations of
pharmaceutical compounds follow first order or second order kinetic expressions.

The solutions not containing any chelating agent degraded more rapidly as the buffer
concentration increased, while the buffered solutions containing chelating agent showed that the
rate of degradation was independent of the concentration of the buffer.

Rancidity, which can affect nearly all oils and fats is a widely known term covering many
typical off – flavors formed by the auto - oxidation of unsaturated fatty acids present in an oil or
fat. These off – flavours have a more or less distinct odour and are due to the volatile compounds
that are formed upon oxidation of the oils and fats. These volatile compounds are generally short
chain monomers that are formed by cleavage of the non-volatile hydroperoxide primary oxidation
product.

Determination of iodine numbers can be employed as an indication of whether oxidation

takes place across the double bond.The stability of pharmaceutical compounds undergoing
oxidative degradation can be increased by several approaches.

Oxygen Content

Since, in many cases, oxidative degradation of a drug takes place in aqueous solution, it is
helpful to keep the oxygen content of these solutions at a minimum. Most oxidative degradations
of pharmaceutical compounds are probably autooxidative in nature and involve chain reactions
that require only a small amount of oxygen for initiating the reaction, so it is necessary to add
agents such as antioxidants and chelating agents to obtain acceptable protection against oxidative
degradations.

Anti Oxidants:

The effect of antioxidants is to break up the chains formed during the propagation process
by providing a hydrogen atom or an electron. Water soluble antioxidants act by preferentially
undergoing oxidation in place of the drug. Oil – soluble antioxidants serve as free radical
acceptors and inhibit the free radical chain process.

Antioxidants commonly used for aqueous systems are:

Sodium sulfite Sodium dioxide Thioglycolic acid

Sodium metabisulfite Ascorbic acid Sodium thiosulfite
Sodium bisulfite Thioglycerol Cysteine hydro chloride
Antioxidants commonly used for oil systems are:

Ascorbyl palmilate Butylated hydroxy

Hydroquinone toluene
Propyl gallate α - tocopherol
Lecithin.

The effectiveness of these antioxidants can depend on the concentration used, whether
they are used singularly or in combination, the solution pH and the package integrity and non
reactivity. The effectiveness of antioxidants can be enhanced through the use of synergists such as
chelating agents.

Chelating Agents

Chelating agents tend to form complexes with the trace amounts of heavy metal ions
inactivating their catalytic activity in the oxidation of medicaments. Examples of some chelating
agents are ethylenediamine, tetra acetic acids derivatives and salts, dihydroxy ehtyl glycerine,
citric acid and tartaric acid.

pH
It is also desirable to buffer solutions containing ingredients that are readily oxidizable to
a pH in the acid range. This causes an increase of the oxidation potential of the system with a
concurrent increase in stability when oxidations are catalysed by hydrogen or hydroxyl ion. The
pH of optimum stability in the acid range, however, must be determined experimentally for each
drug.

Solvents
Solvents other than water may have a catalysing effect on oxidation reactions when used
in combination with water or alone. For example, aldehyde, ethers and ketones may influence free
radical reactions significantly.

Ascorbic acid Oxidation

The scheme of oxidation of ascorbic acid by cupric ions is as follows.

Ascorbate ion in solution---------------- Semiquinone-------------- Dehydro ascorbic acid----

Slow oxidation Rapid oxidation
Oxalic acid + Threonic acid ------ Ketogulanic acid

When solutions are free from traces of copper, ascorbic acid is not oxidized by molecular
oxygen to a measurable extent, except in alkaline solution. When CO and KCN are added they
form complexes with the metal ions and therefore oxidation is limited.
Ascorbic acid can exist as a singly charged and doubly charged ion. Oxygen ion react with
divalent ions at about 105 times faster compared to its reaction with the the monovalent ion. In
alkaline medium auto oxidation proceeds more rapidly.
PHOTOLYSIS

Degradative reactions, such as oxidation, reduction, ring arrangement or modification and

polymerization can be brought about by exposure to light at particular wavelength. According to
the equation E = 2.859 x 10 5 / Kcal per mole, the shorter the wavelength of light, the more
energy is absorbed per mole.

In a large number of systems that are photolyzed, free radicals are products that undergo
subsequent reactions. If the molecules absorbing the radiation take part themselves in the main
reaction, these actions is said to be a photochemical one. Where the absorbing molecules do not
themselves participate directly in the reaction, but pass on their energy, to other molecules that do,
the absorbing substances is said to be a photo sensitizer.

Examples of pharmaceutical compounds that undergoes, the photo decomposition are

chlorpromazine hydrochloride, alcoholic solutions of hydrocortisone, prednisolone and methyl
prednisolone.

RACEMIZATION

A racemization is a reaction in which, an optically active substance loses its optical

activity without changing its chemical composition. This reaction is important to the stability of
pharmaceutical formulations, since the biologic effect of the dextro form can be considerably less
than that of the levo form. For example, levo adrenaline is 15 to 20 times more active than dextro
– adrenaline.

Racemization reactions, in general, undergo degradation in accordance with first – order

kinetic principles. The racemization of a compound appears to depend on the functional group
bound to the asymmetric carbon atom. Aromatic groups tend to accelerate the racemization
process.

ACCELERATED STABILITY ANALYSIS

In the past it was the practice in many pharmaceutical manufacturing companies to

evaluate the stability of pharmaceutical preparations by observing them for a year or more,
corresponding to the normal time that they would remain in stock and in use. Such a method was
time – consuming and uneconomical. Therefore accelerated stability studies were carried out. The
objective of this study is to predict the shelf life of a product by accelerating the rate of
decomposition, preferably by increasing the temperature.

The method of accelerated testing of pharmaceutical products is based on the principles of

chemical kinetics. According to this technique the K values for the decomposition of a drug in
solution at various elevated temperatures are obtained by plotting function of concentration
against time, as seen in figure –1 and 2

70c
60c
Concentration 50c
40 40c
30c
50 Log K
25c
70 60 20c

|
1/T
Time in hours
Figure 1 accelerated breakdown of a Figure 2 – arrhenius plot for
drug in aqueous solution at predicting drug stability
elevated temperature. at room temperature.

The logarithms of the specific rates of decomposition are then plotted against the
reciprocals of the absolute temperatures as shown in figure and the resulting line is extrapolated to
room temperature. The K25 degree is used to obtain a measure of the stability of the drug under
ordinary shelf conditions.

Another similar method in which the fractional life period is plotted against reciprocal
temperatures and the time in days required for the drug to decompose to some fraction of its
original potency at room temperature is obtained. The approach is illustrated in figures 3 and 4.
 100—
300__
90—
40 200__
80— 150__
100__
70— 50 80__

60— 60 60__

50— Days t90 40__

70 log scale
40— 20__
10__
30— 8__

90 6__
| | | | | | | | | | | | | | | | |
0 24 48 72 96 120 2.7 2.8 2.9 3.0 3.1 3.2 3.3

| | | | | | | |
90 80 70 60 50 40 30 25
Time (days) Temperature (Degree Celsius)

Figure: 3 Time in days required for drug potency Figure:4 A log plot of t90 (i.e., time
to fall to 90% of original value. These timer, to 90% potency) on the vertical axis
designated as t90, are then plotted on a log scale against reciprocal temperature (both
Kelvin and centigrade scales are
Shown) on the horizontal axis.

As observed in Figure 3 the log percent of drug remaining is plotted against time in days
and the time for the potency to fall to 90 degree of the original value (i.e., t 90 ) is read from the
graph. In figure 4 the log time to 90% is then plotted against 1/T and the time at 25 degree celsius
gives the shelf-life of the product in days. The decomposition data illustrated in figure.3 result in
a t90 value of 199 days. Shelf-life and expiration dates are estimated in this way.

By either of these methods, the overage, that is, the excess quantity of drug that must be
added to the preparation to maintain at least 100% of the labeled amount during the expected shelf
of the drug, can be easily calculated and added to the preparation at the time of manufacture.
Limitations

1. Valid only when the breakdown depends on temperature.

2. Valid only when the Ea is 10-30 K Cal / mole.
3. Shelf life for one set of condition cannot be applied to other preparation of the same drug.
4. Stability prediction are of little use if the degradation is due to diffusion, Microbial
contamination, Photochemical reaction or excessive agitation
5. They cannot be used if the product looses its physical integrity at higher temperature like
co-agulation, denaturation, breaking of emulsion, melting of ointments etc.
6. Predictions will become erroneous when the order changes at elevated temperature.
7. Predictions will become erroneous when the reaction changes its order during the period of
study.

ADDITON OF OVERAGE

Addition of overages is done to attain the desired shelf life to keep the content of the
active ingredient within limits compatible with therapeutic requirements, for a predetermined
period of time, so that at least 100% of the labeled amount during the expected shelf life period is
maintained. The International Pharmaceutical Federation has recommended that overages be
limited to a maximum of 30% over the labeled potency of an ingredient.
Figure-5

110

100
% label claim
90

80

0 100 200 300 400 500 600 700 800

Time in days

Even if the pharmaceutical dosage form were found to extremely stable, the packaged product
would become unsightly if stored for excessive number of years. For these reasons, outdates no
longer than 60 months are used.

FACTORS AFFECTING STABILITY

 EXTRINSIC INTRINSIC BOUNDARY
Temperature PH Containers composition
Light Complexation Porosity
Gases Microbial growth Dosage from interactions
Moisture

PREFORMULATION STUDIES

Effects of Temperature

Most of the factors affecting drug stability can be controlled by careful selection of
adjuvant or container / closure system. But the effects of temperature are not in control as
majority of the marketed drugs are stored at room temperature. The room temperature may vary
from place to place and from season to season and hence the effects of temperature on drug
stability deserves a special attention.

The quantitative relationship of the specific reaction rate and temperature is given by the
Arrhenius equation:

In k = In A - Ea . T
R

Where, R = Gas constant ( 1.987 cal / degree . mole)

T = Absolute temperature
A = Frequency factor, Ea = Activation energy
A plot of K vs 1/T gives arrhenius plot from which Ea and A can be calculated.

FIGURE : 6 ARRHENIUS PLOT

100 --
log A
 50 ---

10 –
.

K hours-1

2.68 2.84 2.92 3.0 3.1 3..2 3..3 3.4

1/T x 103
Arrhenius plot showing the method for determining activation energy and temperature dependency of degradation

During early phases of drug development, the greatest amount of information is sought in
the least amount of time. The Kinetic and Predictive studies are, therefore, generally carried out
under accelerated conditions. The objectives of accelerated tests are’

1. Aids in rapid detection of deterioration.

2. Aids in selection of the best formulation (Fig. 2)
3. Shelf life prediction (Fig. 3)
4. A rapid means of quality control.
 FIGURE - 7 FIGURE - 8

Formulation 1
(least stable) X High Stress
Amount of
Decomposition X
Obtained after the
Formulation 2 time t, is used to
Predict value of Y
after time t2
Formulation 3 Y
(Most stable) Low stress

Time Time t1 t2

There are number of situations when Arrhenius predictions become invalid or errneous
(e.g emulsions, viscous dispersions, drugs very sensitive to oxygen or water vapour etc.,)

Realistically, FDA Stability Guidelines stress on requiring that stability studies be

performed in intended marketed container under conditions of 37 - 40 degree Celsius and 75 -
85% RH for three months. This can simulate tropical conditions also. This gives a more realistic
shelf-life, though a longer period is required. Shelf-life (t90 %) at ambient conditions can be
determined from Arrhenius plot.

FDA states that a drug product which is stable for three months at 37 - 40 degree C and
75% RH can be given a tentative experiment period of two years, if it does not contain an
overage.

Influence of pH on degradation

The magnitude of the rate of hydrolytic reactions catalyzed by hydrogen and hydroxyl ion
can vary considerably with pH. Hydrogen ion catalysis predominates at the lower pH range
whereas hydroxyl ion catalysis operates at the higher pH range. For such a study, product samples
are kept at pH 2-12 at one selected temperature between 55-900 C for two weeks. The data can be
presented in the form of pH rate profile plots of log K or K against pH. The pH of maximum
stability can be read from the plot.
 To determine the influence of pH on the degradative reaction, the decomposition is
measured at several hydrogen ion concentrations. The pH of optimum stability can be determined
by plotting the logarithm of the rate constant versus pH.
Figure-9
The point of inflection of such a plot represents
the pH of optimum stability. Knowledge of this point
is extremely useful in the development of stable dosage
form, provided the pH is within safe physiologic limits. pH OF
pp
MAXIMUM
STABILITY
K-Hours

1 2 3 4 5 6 7 8

pH

General acid-base catalysis of degradation:

Buffer salts are commonly used in the formulation of pharmaceutical liquids to regulate
the pH of the solution. Although these salts tend to maintain the pH of the solution at a constant
level, they can also catalyze the degradation.

Commonly buffer salts such as acetate, phosphate and borate have been found to have
catalytic effects on the degradation rate of drugs in solution.

Influence of ionic strength on degradation:

The rate of reaction can be influenced by the strength of the solution in accordance with
the following equation,

Log K = log Ko + 1.02 ZAZB u

Where,ZA and ZB are the charges carried by the reacting species in solution.

U- the ionic strength

K- the rate constant of degradation
Ko- the rate constant of infinite dilution.

The ionic strength is defined as half the sum of the terms obtained by multiplying the
concentration of each of the ionic species present in the solution by the square of its valence.
 +
When the drug is positively charged and is ^
undergoing hydrogen ion catalysis, an increase in
ionic strength caused by the addition of a salt,
such as NaCl, cause an increase in the rate of degradation. 0

A decrease in the rate of degradation results Reaction

if the positively charged, drug is undergoing rates
hydroxyl ion catalysis and the ionic strength is
increased by addition of a salt. | >
u

Figure 10: Square root of ionic

strength dependence of reaction rates
on ionic strength.

The change in the pH of a drug solution during stability testing can be indicative of either
degradation of the active ingredient on interaction of one or more of the constituents of the
solution either the container-Plastic, glass or the rubber.

Photo Stability Studies

Exposure to sunlight can change the color of products, degrade packaging or even
lead to chemical decomposition of active ingredient. However to study the detrimental effects of
light on drug product, samples are placed in open petri dishes and packaged in both clear and
amber containers. Controls are placed in light – resistant container such as, amber, glass, foil-
wrapped or in a cardboard box. These are placed into a well-vented temperature monitored light
cabinet of specified lumens, exposed for at least four weeks and analyzed.

Effect of Humidity

In order to obtain relevant information, it is preferable to employ a range of

humidities. This is achieved by wing saturated salt solution. The test should be carried out on both
final packaged product and the unpacked material to get information regarding formulation
adjuvant, type of environment suitable for a drug and the type of package needed.

Effect of Oxygen

To study the effect of oxygen, the product samples are placed into containers and
stored at 75 degree celcius for one week. Prior to sealing, the head space is evacuated and purged
with an inert gas such as argon or nitrogen. Air head space samples are used as positive controls.
Oxidation may be evident by potency loss and /or color change.
Autoclaving Studies

For parenterals, determination of stability to autoclaving is necessary. Solutions at

an established pH range are exposed to autoclaving conditions of 121 degree celcius at 30 psig for
20, 30, 45 and 90 minutes. Assay data are recorded together with evaluation of change in color,
pH and particulate matter.

Studies of Microbial Quality

The presence of microbes in the product poses a threat to stability causing

degradation of drug resulting in dosage impotency or toxic products. Bacterial or mold growth is
undesirable from therapeutic and an aesthetic point of view .

For evaluating microbiological stability, it is necessary to monitor the preservative

content at intervals through out the projected expiration period. This can be done by microbial
challenge tests and by chemical assays of the preservatives. Products that do not contain
preservatives but require control of microbes are subjected to microbial limit tests.

Dosage form factors:

The objective of preformulation studies is to identify compatible, potentially useful

pharmaceutical excipients, so that a stable formulation can be developed. Generally, a drug or a
mixture of drugs along with excipients like preservatives, buffering agents, antioxidants and
chelating agents are subjected to accelerated studies. This requires screening of a large number of
excipients under several storage conditions, thus analytical, chemical and physical data are
developed for the new drug substance. Incompatibility tests are also carried out.

Three types of dosage forms are there, if it is a

6. Solid state dosage form:

Crystallinity, UV, I.R, TLC, HPTLC analysis should be carried out.

7. Semi-solid:
X-ray crystallinity, particle size, shape and particle distribution should be found
out.

8. Liquids:
pH studies, cosolvant studies are to be carried out. If it is a suspension-particle
size distribution, sedimentation, rate should be carried out.

Effect of package on stability

The package has been described as an economical method of providing convenience,

identification, presentation and protection for a given product until such time that is consumed.
Protection is the main emphasis for packaging pharmaceutical products and should act to protect
the drug during product shelf-life.

The commonly used packaging materials include glass, metal, plastic and rubber .For solid
dosage form , strip and blister packaging are used. Containers are made up of plastic or bottle,
even silica bag is used to avoid the absorbency of moisture. Proper polymers should be selected.
In glass maybe containers leaching or sorption happen for which proper test ought to be carried
out.

For liquid dosage form,bottles are most commonly used. For air resistance, moisture
resistance, amber colored bottles are used. Blue colored bottles are used for milk of magnesia. For
parenterals ampoules and vials are used as containers. For this powdered glass tests, water attack
test, alkalinity test, thermal shock test, internal pressure test, whole ampoule test are carried out.
For plastic containers, test for plastic should be carried out. For rubber closures, test for rubber
should be carried out..

For semi-solid dosage form – Collapsible tubes are used as a container which is made up
of plastic, aluminum and rarely tin. Respective tests should be carried out.

STORAGE CONSIDERATIONS

The storage information can be found on the label of the immediate pack or subsequent
package. The pharmacist should be highly concerned about maintaining the integrity of the
product and necessity of storing it in the proper environment. In addition to this general storage
restrictions should be given to certain individual drug applicable to particular drug product. When
no specific compendial storage direction or limitation is provided, it is understood that the storage
conditions include protection from moisture freezing and excessive heat. Expiration date is
meaningless unless accompanied by labeled direction for storage under controlled condition.
Data is provided in the following tables for world climatic conditions and calculated storage
conditions for world wide stability testing.

Both ICH and WHO guidelines are framed on the concept of derived storage conditions of
temperature and % relative humidity in the various climatic zones around the world.

Compendial storage temperature definitions

I.P. USP/NF
Cold Temperaure not exceeding 80 C, Same
Usually between 20-80 C

Cool Temperature between 80- 250 C Temperature

between 80-150 C

Room temperature The temperature prevailing in a Same

Working area

Warm temperature between 300- 400 C Same

Excessive Heat Temperature above 400 C Same

World climate zones

Climatic Zone I Zone II Zone III Zone IV

Conditions (Temperare) (Sub tropical) (Hot Dry) (Hot Humid)

Mean Annual 20.50 C20.5 – 240 C 240 C 24 0C

Temperature

Kinetic Mean 21 0C 250 C 300 C 300 C

Temperature

Mean Annual 45% 60% 35% 70%

Relative Humidity

ZoneI: UK, North Europe, Canada, Russia.

Zone II: United states, Japan, Southeren Europe
ZoneIII: Iran, Iraq, Sudan,
Zone IV: Brazil, Iondonesia, Philippines.

Calculated storage conditions for World Wide Stability Testing

Climatic Zones Storage Conditions

Temperature RH

1. Temperature Climate 19 C 40 – 60%

2. Mediterranean and Sub Tropical Climate 26 C 60 – 65%
3. Hot and Dry Climate 31 C < 65%
4. Hot and Humid Climate 31 C > 65%

A pharmacist developing a product exclusively for export market may utilize this data.
 General Considerations of ICH and WHO Drug Stability Testing Guidelines

ICH WHO
1. Concept ICH guidelines have been developed to WHO guidelines similarly are meant to
harmonize drug stability data required to be harmonize drug stability requirements of
submitted to registration authorities in ICH registration authorities in WHO associated
countries. countries.
2. Agencies Drug regulatory authorities and experts from WHO expert committee involving regulatory
involved in the pharmaceutical industry. authorities, scientists, and the pharmaceutical
development industry.
3. Countries of 17 Countries in three regions viz. USA, Global, meant to cover 170 WHO member
application Japan and EC. states outside the ICH exercise.
4. Applicability New Chemical Entities and their finished Pharmaceutical products containing well
products. established drug substances in conventional
dosage forms
5. Stage of Final draft endorsed by ICH steering Appeared recently as Annexure 4 to the report
development committee on 27 October, 1993. of 34th Meeting of Expert Committee on
Specifications for Pharmaceutical
Preparations.
6. Date of 1 January, 1998 Not available
implementation
7. Contents • Preamble • General
• Objective • Definition of Terms
• Scope • Purpose of Stability Testing
• Drug Substance • In the development Phase for the
• General registration dossier in the post-registration
• Stress Testing period
• Formal Studies • Intended market
• Selection of batches • Design of Stability Studies
• Test Procedures • Test Samples
• Specifications • Test Conditions
• Storage Conditions • Accelerated Studies
• Testing Frequency • Real Time Studies
• Packaging Containers • Frequency of Testing and Evaluation of
• Evaluation Test Results
• Statements/Labelling • Analytical Methods
• Drug Product • Stability Report
• General • Shelf-Life and Recommended Storage
• Selection of Batches Conditions
• Specifications • References
• Test Procedures • Appendix 1. Survey of Stability of
Pharmaceutical Preparations included in
• Storage Conditions
the WHO model list of essential drugs
• Testing Frequency
• Packaging Containers • Appendix 2. Stability Testing - Summary
• Evaluation Sheet
• Statements/Labelling
• Annexure 1. Glossary and Information
 Technical Features of ICH and WHO Drug Stability Testing Guidelines

ICH WHO Comments

1. Consideration Zone II Zone II and IV ICH countries mostly fall in
of climatic zone Zone II with very few in
Zone I. WHO guidelines,
being meant for global
marketing, cover both Zones
II and IV.
2. Test
Conditions

- Accelerated 400C ± 20C/75% RH± 5% for 400C ± 20C/75% RH± 5% The ICH and WHO
for 6 months for Zone IV guidelines are built upon the
6 months concept of derived
countries.
400C ± 20C/75% RH± 5% temperature (calculated
for 3 months for Zone II from mean kinetic
countries. temperatures) in four
different zones. The testing
conditions suggested in ICH
250C ± 20C/60% RH± 5% for 30 C ± 2 C/60% RH± 5%
0 0

- Real time for IV countries. guidelines are based on

12 months, assurance to be 250C ± 20C/60% RH± 5% derived conditions existing
for zone II countries. in Zone II countries while
provided for continuity of
Data for 6 months minimum WHO guidelines are based
the test upto the end of the shall be available at the time upon derived storage
of registration conditions in both zone II
expected shelf-life and IV countries. The use of
zone II conditions for zone
I, and zone IV conditions
for zone III means testing in
adverse conditions which is
reasonable as it provides an
element of safety and also is
rational since subsequent to
harmonisation the products
would move frequently
from one country to the
other.
3. Test samples/ Stability information on For test samples containing In WHO guidelines,
selection of long-term and accelerated fairly stable active classification of batches is
batches testing to be provided on ingredients -from two based on stable and
three batches of the same different production sensitive active ingredients,
formulation, with two of batches. For products which is not the case in ICH
three batches essentially containing easily degradable guidelines.
being of pilot scale. active ingredients - three
Information on three batches batches to be sampled.
also desired for drug Sampled batches out to be
substance representative of the
manufacturing process, pilot
plant or full production
scale.
4. Testing Testing suggested to be For accelerated studies, Testing frequencies vary in
frequency carried out every three 0,1,2,3 and when the two guidelines. The
months during the first year, appropriate, 6 months. For matrixing and bracketing
every six months during the real time studies, 0,6,12 designs allowed under ICH
second year and then months and beyond that guidelines are meant to
annually for drug substance once a year rationally reduce the testing
as well as the drug product. frequency without
ICH guidelines suggests use sacrificing on the end
of matrixing and bracketing results. This aspect is
designs, if application is adopted from US FDA
justified guidelines of 1987. Not
covered by WHO guideline.
5. Packaging Packaging containers same Studies to be done on final Conceptually, requirements
containers or shall simulate the actual dosage form in its final of the two guidelines are
proposed packaging. The container and packaging same with respect to testing
guideline also recommends of packaged samples. The
generation of data on the ICH directive of testing of
unprotected product under unpackaged drug substance
the accelerated conditions for need to be incorporated by
the purpose to study the other guidelines as the same
worse effects of storage on gives a lot of information on
product properties inherent stability of the
compound in the
formulation environment
6. Interpretation Required to be presented in a WHO guidelines permit This aspect or ICH
of stability data systematic form and assignment of a tentative guideline is also adopted
summarised. Shelf-life shelf-life of 24 months from US FDA guidelines of
estimates to be made through provided the active 1987. WHO guidelines,
statistical methods such as ingredient is known to be however, are straight and
regression analysis on stable, stability studies as suggest direct assignment of
transformed and suggested above have been shelf-life of 24 months if
untransformed data. To be performed without formulation shows no
 followed when significant significant changes, changes under the testing
degradation or change in the supporting data indicate that conditions
properties of the product similar formulations have
occur, meaning that the been assigned a shelf-life of
change is outside the 95% 24 months or more and the
confidence limits of the manufacturer continues with
analytical method real-time studies until the
proposed shelf-life is
covered.
7. Labelling Product to be labelled with Requires the product to be ICH guidelines suggest
storage temperature range PROMINENTLY labelled labelling based on
based on national/regional with the following storage national/regional
requirements. Use of conditions: requirements and don’t
`ambient conditions’or room -store under normal define the storage
temperature as terms for conditions conditions. WHO guidelines
storage conditions not -store between 2-80C are more specific and the
accepted. However allows (under refrigeration, no labelling conditons for
mention of specific storage freezing) storage are defined.
conditions. -store below 80C (under
refrigeration)
-store below -180C (in a
deep freezer)
The normal conditions have
been defined as `storage in
dry, well ventillated
premises at temperatures of
15-250C or depending on
climatic conditions,up to
300C
Extraneous
odours,contamination and
intense light have to be
excluded Where normal
conditions are not met,
WHO guidelines even
recommend determination
of storage conditions at
level of country of
designated use.