detailed forced degradation study and analysis of impurity involving types of impurity and its isolation and identification

1. Degradation study and analysis of impurities
Prepared by
Yachita Rajwadwala

2. Impurity
What is Impurity?
• Impurities in pharmaceuticals are the unwanted
chemicals that remain with the active pharmaceutical
ingredients (APIs), or develop during formulation.
• The presence of these unwanted chemicals even in small
amounts may influence the efficacy and safety of the
pharmaceutical products.
Impurity Control –Why is it important.
• Necessary to ensure the safety and efficacy of
pharmaceutical products.
• Required to determine the purity of the drug product.
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3. Impurity types and their sources
3
Impurity type Impurity source
1.Process-related drug substance -Organic
- Starting material
- Intermediate
2.Process-related drug product -Organic or inorganic
- Reagents, catalysts, etc
3.Degradation drug substance -Organic product
- Degradation products
4.Degradation drug product -Organic
- Excipient interaction
Yachita Rajwadwala

4. Source of
impurities in
formulation
Impurities
associated
with API
Organic
impurities
Inorganic
impurities
Residual
solvents
Impurities created during
formulation
Process
related
Environmental
related
Dosage form
related
Functional
group related
Yachita Rajwadwala

5. • Categories of impurities
– Organic
– Inorganic
– residual solvents
• Arise from the manufacturing process and /or during storage.
Classification:
– Identified:
• those whose structure has been determined
• specifications of
 0.2%- If toxicological data is available
0.1%.- If no toxicological data is available
– Unidentified:
• those whose structure and toxicology are unknown
• specifications of 0.1%.
Organic Impurities
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6. Organic Impurities includes
• Starting materials
• By-products
• Intermediates
• Degradation
• Products
• Reagents
• Ligands
• Catalysts.
IDENTIFICATION OF IMPURITIES
 It is based on the chemical reactions involved in the synthesis any
raw materials used which could contribute impurities , any possible
degradation products.
 understanding of the chemical reactions, reaction conditions, and
any metabolic degradation pathways is essential for
identification.
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7. Hydrochlorothiazide
• Hydrochlorothiazide degrades to the disulfonamide.
• A specific impurity test is conducted to determine the amount
of disulfonamide present, to ensure that the quantity is
acceptable.
• Tablet form
– An external standard solution of known concentration of
disulfonamide is prepared and the peak responses recorded
are used to calculate the amount of disulfonamide present
in the sample.
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8. • Arise from the manufacturing process known and identified.
• Inorganic impurities are determined when the drug substance
is tested, not in the final dosage form.
• Include
– Reagents, Ligands ,Catalysts ,heavy metals ,inorganic salts
• Pharmacopeial method for testing for these types of impurities
is called residue on ignition.
• Used to determine amounts impurities of different metals.
2. Inorganic Impurities
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9. Residual Solvents:
• Residual solvents : solvents that are used during the
manufacturing process. May be detected after the
product is in its final form. E.g. benzene, Chloroform.
• Comes from many different stages in the
manufacturing process
– Active substance granulation
– Milling
– Drug product coating
• The most common technique for measuring
residual solvents is gas chromatography (GC)
– Because of the small size and volatile nature of solvent molecules.
– External standard solutions of the solvent are prepared and
compared to sample solutions.
– The amount of residual solvent can then be calculated using the
peak responses.
3. RESIDUAL SOLVENTS AND WATER
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10. 1.Loss on drying (LOD) determines the amount of volatile
components that are released from a sample under specific
temperature and/or vacume conditions.
2.Thermal gravimetric analysis (TGA) measures the loss of
volatile components from a sample over a temperature gradient.
• Advantage:
• relatively quickly.
• Disadvantage:
• Non specific.
• Cannot account for volatile components that are trapped in lattice
structure of the compound.
Nonspecific methods:
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11. Water:
• In some cases, a specific amount of water is desirable, i.e.,
hydrates.
• In other cases, it is advantageous to keep water levels to a
minimum because water itself can accelerate the
degradation of a drug substance.
• Some drug substances are hydrophilic, so knowing how
the compound picks up water during stability studies is
important.
 Techniques used to determine water content
 TGA, loss on drying, gas chromatography,
dimethyoxyproprane titration, near-infrared (Near-
IR),NMR, Karl Fischer titration
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12. 1.Dimethyoxyproprane titration:
• A thermometric end point.
• Very fast and instantly.
• Difficult to work with for some drug substance.
2.NMR spectroscopy
• Not be practical.
• Can be used in cross-validation studies to support the primary
method.
3.Near-IR spectroscopy
• Provide good selectivity and sensitivity.
• A quick and nondestructive technique.
• Secondary method of analysis for water.
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13. 4.Karl Fischer titrations
• First introduced in 1935
• Widely used to determine water content in drug substances.
• The reaction is broken down into two parts and is shown in
Eqs. (1) and (2).
ROH + SO2 + R-N →[R-NH]SO3R ……………………………..(1)
H2O + I2 + [R-NH]SO3R + 2R-N →[R-NH]SO4R + 2[R-
NH]I ………………………………. (2)
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14. When one performs a volumetric Karl
Fischer titration, there are two basic
classes of reagents:
• The one-component reagents contain all of the reactants
(iodine, sulfur dioxide, and base) in an alcohol solvent. These
reagents have a large water capacity, and they are economical
and easy to use. The sample is dissolved in an alcohol, but co-
solvents can be used to improve sample solubility.
• Two-component volumetric reagents require two
separate solutions. The solvent contains sulfur dioxide and
base in an alcohol. The reagent is a solution of iodine in
methanol with a known titer.
Advantages : faster titrations and greater accuracy for small
amounts of water. There are, however, more possibilities for
side reactions within a two-component system.
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15. • Impurities resulting from chemical change in drug
substance brought during manufacturing and/or storage
of the new drug product by effect of light ,temperature,
pH, water or reaction with excipient and immediate
container closer system.
DEGRADATION
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16. Forced degradation study
Drug substance
Solid
Photolytic
Thermal
Thermal/
humidity
Solution /
Suspension
Acid
base
hydroly
sis
oxidativ
e
Drug product
Solid
Photolytic
Thermal
Thermal/h
umidity
oxidative
Semi-
solid
Photolytic
Thermal
Thermal/h
umidity
Solution /
Suspension
Photolyti
c
Thermal
oxidative
Various stress conditions used for degradation of drug substance and
drug product. 16Yachita Rajwadwala

17. Degradation conditions
1. Hydrolytic condition
2. Oxidation condition
3. Photolytic condition
4. Thermal condition
HYDROLYTIC CONDITION
• Hydrolysis is one of the most common degradation chemical
reactions over a wide range of ph.
• Chemical process that includes decomposition of a
chemical compound by reaction with water.
• Under acidic and basic condition involves. Acid or base
stress testing involves forced degradation of a drug
substance by exposure to acidic or basic conditions which
generates primary degradants in desirable range.
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18. • The selection of the type and concentrations of acid or
base depends on the stability of the drug substance.
– For acid hydrolysis : Hydrochloric acid or sulphuric
acids(0.1–1 M)
– For base hydrolysis : Sodium hydroxide or potassium
hydroxide(0.1–1M)that are suitable reagent for
hydrolysis.
• If the compounds for stress testing are poorly soluble
in water, then co-solvents can be used to dissolve them
in HCl or NaOH.
• The selection of co-solvent is based on the drug
substance structure.
• Stress testing trial is normally started at room
temperature and if there is no degradation, then the
tem. Is increase.
• Stress testing should not exceed more than 7days. 18Yachita Rajwadwala

19. Oxidation conditions
• Hydrogen peroxide is widely used for oxidation of drug
substances in forced degradation studies but other oxidizing
agents such as metal ions, oxygen, and radical initiators can
also be used.
• It is reported that subjecting the solutions to 0.1–3% hydrogen
peroxide at neutral pH and room temperature for seven days
or upto a maximum 20% degradation could potentially
generate relevant degradation products .
• The oxidative degradation of drug substance involves an
electron transfer mechanism to form reactive anions and
cations.
• Amines, sulphides and phenols are susceptible to electron
transfer oxidation to give N-oxides, hydroxylamine, sulfones
and sulfoxide.
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20. • The functional group with labile hydrogen like
benzylic carbon, allylic carbon, and tertiary carbon
or α-positions with respect to hetro atom is
susceptible to oxidation to form hydro peroxides,
hydroxide or ketone.
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21. Photolytic conditions
• Photo stability studies are performed to generate
primary degradants of drug substance by exposure to
UV or fluorescent conditions.
• Samples of drug substance and solid/liquid drug
product should be exposed to a minimum of 1.2 million
lx hand 200Wh/m2 light. The most commonly
accepted wavelength of light is in the range of 300– 800
nm to cause the photolytic degradation .
• The maximum illumination recommended is 6 million
lx h . Light stress conditions can induce photo oxidation
by free radical mechanism.
• Functional groups like carbonyls, nitro aromatic, N-
oxide, alkenes, aryl chlorides, weak C–H and O–H bonds,
sulphides and polyenes are likely to introduce drug
photo sensitivity. 21Yachita Rajwadwala

22. Thermal conditions
• Thermal degradation (e.g. , dry heat and wet heat)
should be carried out at more strenuous conditions
than recommended ICHQ 1 A accelerated testing
conditions.
• Samples of solid-state drug substances and drug
products should be exposed to dry and wet heat,
while liquid drug products should be exposed to
dry heat.
• Studies may be conducted at higher temperatures
for a shorter period. Effect of temperature on
thermal degradation of a substance is studied
through the Arrhenius equation:
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23. k = AeEa/RT
where k is specific reaction rate,
A is frequency factor,
Ea is energy of activation,
R is gas constant(1.987 cal/deg mole) and
T is absolute temperature.
Thermal degradation study is carried out at 40–80 ℃.
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24. • Drug substance used as pharmaceutical have different
molecular structure and therefore susceptible to many and
variable degradation pathway.
• Predicting the drug stability would be very helpful in design of
stability study in earliest stage of drug development and
identifying the way to minimize the chemical degradation.
1. Hydrolysis
2. Dehydration
3. Isomerization and Racemization
4. Decarboxylation and Elimination
5. Oxidation
6. Photolysis
CHEMICAL STABILITY OF DRUG
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25. HYDROLYSIS
• It is major cause of deterioration of drug , especially for those in
aqueous solution.
DEHYDRATION
• Removal or loss of water.
• Sugars such as glucose and fructose are known to undergo
dehydration to form 5-(hydroxymethyl)furfural.
• Erythromycin is susceptible to acid catalyzed dehydration.
ISOMERIZATION AND RACEMIZATION
Isomerization:
• Conversion of active drug into less active or inactive drug.
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26. Racemization:
• Conversion of optically active drug into its enantiomer.
• The best known racemization reaction of drugs are
epinapherine, pilocarpine , ergotamine and tetracycline.
DECARBOXYLATION AND ELIMINATION
• Drug substance have carboxylic acid groups undergo
decarboxylation reaction. 4-amino salicylic acid is good
example.
• In elimination reaction, two substituents are removed
from molecule in either a one or two step mechanism.
OXIDATION
• Drug can be affected by the availability of oxygen.
• Some photo degradation reactions involve photo oxidative
mechanisms that are dependent on conc. Of oxygen.
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27. • Oxygen participates as reactant and also alters the degradation
rate.
PHOTOLYSIS
• Reaction such as oxidation-reduction, rig alteration and
polymerization can be catalyzed or accelerated by exposure to
sun or artificial light.
• Photolytic degradation can be very complex , the product such
degradation being numerous and difficult to identify.
• Exposure to light can cause discolouration of both drugs and
excipients even when degradation is modest and non even
detectable analytically. This can lead to “off colour “ product ,
perceived by the patient as a quality deficiency.
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28. • Drug substance exist in various microscopic physical state
for eg. Amorphus, crystalline, hydrated and solvated state.
• With time drug or excipients change from one state to more
thermodynamically stable state.
• Rate of conversion depend on the free energy difference
between the state and energy barrier that must be overcome
for the conversion to take place.
1. Crystallization of amorphous drug
2. Transitions in crystalline state
3. Formation and growth of crystal
4. Vapour phase transition including sublimation
5. Moisture adsorption
PHYSICAL STABILITY OF DRUG SUBSTANCE
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29. CRYSTALLIZATION OF AMORPHOUS DRUG
• Poorly water soluble drug formulate in amorphous form
because the solubility of amorphous form is generally
higher than of same substance in crystalline form.
• But, crystalline state has lower free energy, so the
amorphous substance tend to change their more
thermodynamically stable crystalline phase with time.
• Therefore crystallization of amorphous drug substance
may occur during the long term storage and may lead to
change release characteristics of drug substance and
hence, change in its toxicological and clinical behavior.
• Amorphous nifidipine prepared by spray dried method
also exhibit time dependent crystallization result into
altered solubility and dissolution behavior. This
crystallization can be inhibited by addition of B-
cyclodextrine. 29Yachita Rajwadwala

30. TRANSITIONS IN CRYSTALLINE STATE
• Polymorphs are different crystalline form of same drug.
Because this form have different free energy and chemical
potentials, depending upon temp condition, transition
between polymorph can occur.
• Polymorphic transition during storage can alter properties of
the drug because the solubility and dissolution rate of
substance generally vary with change in the crystalline form.
Temp and humidity affect polymorphic transition.
• Polymorphic transition observed between two crystalline form
of benoxaprofen and two form of pyridoxal phosphate.
• Transition between anhydrous and hydrate form have been
reported for many drug substance such as phenobarbital,
nitrofurantoin and theophylline. Significant difference in
solubility can exist between anhydrous and hydrate form of
same drug.
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31. FORMATION AND GROWTH OF CRYSTAL
• Molecules in crystal should not be considered static. Crystal
can grow or decrease in size provide that there is medium
across which molecule can travel.
• It may be liquid or gaseous phase into which molecule can
sublime.
• For eg. Drug substance in solid dosage form, such as tablet or
granule may recrystallize or sublime onto the surface of solid
dosage form during storage.
• So, called whisker crystallization was observed in tablet of
caffeine anhydride.
• As mention earlier, carbamazepine tablet containing stearic
acid from column shaped crystal on tablet surface during
storage at high temp.
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32. VAPOUR PHASE TRANSITION INCLUDING
SUBLIMATION
• Some pharmaceutical compounds that sublime easily may
undergo change in drug content because of the sublimation
of drug substance or excipient.
• In case of nitroglycerine, which is liquid with a significant
vapour pressure. So, the sublingual tablet of it show
significant variation in drug content during storage because
the inter tablet migration through vapour phase. This
transfer can be inhibited by adding water soluble nonvolatile
fixing agent such as polyethylene glycol.
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33. Moisture adsorption
• Drug degradation in heterogeneous system such as solid and
semisolid states is affected by moisture.
• Moisture plays important role in catalyzing chemical
degradation:
1) Water participates in the drug degradation process itself as a
reactant, leading to hydrolysis; hydration etc. here
degradation rate is directly affected by the concentration of
water , hydronium ion , hydroxide ion.
2) Water absorbs onto the drug surface and forms a moisture-
absorbed layer in which the drug is dissolved and degraded.
• eg: Sodium ampicillin , potassium propicillin.
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34. ISOLATION AND IDENTIFICATION OF
IMPURITIES/DEGRADANTS
• The identification of degradation products can provide an
understanding of impurity formation and define degradation
mechanisms.
• If the identification process is performed at an early stage of
drug development, there is adequate time for improvements in
the drug substance process and drug product formulation to
prevent these impurities and degradants long before the filing
stage.
• A number of methods can be used for isolating impurities
and/or degradants.
– TLC
– flash chromatography (column chromatography)
– preparative HPLC.
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35. Thank you
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