Evaluating Impurities in Drugs (Part III of III)

In Part III of a three-part article, the authors examine various degradation routes of APIs, impurities
arising from interactions in formulations, metabolite impurities, various analytical methods to
measure impurities, and ways to control impurities.
Apr 2, 2012
By: Kashyap R. Wadekar, Ponnaiah Ravi, Mitali Bhalme, S. Srinivasa Rao, K. Vigneshwar Reddy, L. Sampath
Kumar, E. Balasubrahmanyam
Pharmaceutical Technology Volume 36, Issue 4, pp. 76-86
Submitted: Sept. 19, 2011; Accepted Nov. 28, 2011.

Kashyap R. Wadekar , PhD ,* is a research scientist (II), Ponnaiah Ravi , PhD , is senior vice-president of R&D,
Mitali Bhalme , PhD , is an associate research scientist, S. Srinivasa Rao is a research associate,
K. Vigneshwar Reddy is a research associate, L. Sampath Kumar is a research chemist, and
E. Balasubrahmanyam is a research chemist, all with Neuland Laboratories, 204 Meridian Plaza, 6-3-854/1,
Ameerpet, Hyderabad, India, tel. 91 40 30211600, kashyapwadekar@neulandlabs.com

Controlling and monitoring impurities in APIs

and finished drug products is a crucial issue in drug
development and manufacturing. Part I of this article,
published in the February 2012 issue of Pharmaceutical
Technology, discussed the various types of and sources of
impurities with specific case studies (1). Part II, published
in the March 2012 issue, examined chiral, polymorphic, and
genotoxic impurities (2). In Part III, the authors examine
various degradation routes of APIs, impurities arising from
APIexcipient interaction during formulation, metabolite impurities, various analytical
methodologies to measure impurity levels, and ways to control impurities in pharmaceuticals.

Definition of impurity
The term impurity reflects unwanted chemicals that are present in APIs or that develop during
formulation or upon aging of the API in the formulated drug product. The presence of such unwanted
material, even in small amounts, could affect the efficacy and safety of pharmaceutical products.
Several guidelines from the International Conference on Harmonization (ICH) address impurities in
new drug substances, drug products, and residual solvents (36). As per the ICH guidelines on
impurities in new drug products, impurities present below a 0.1% level do not need to be qualified
unless the potential impurities are expected to be unusually potent or toxic (5). In all other cases,
impurities should be qualified. If the impurities exceed the threshold limits and data are not available
to qualify the proposed specification level, studies to obtain such data may be required. Several recent
articles describe a designed approach and guidelines for isolation and identification of process-related
impurities and degradation products using mass spectrometry, nuclear magnetic resonance (NMR)
spectroscopy, high-performance liquid chromatography (HPLC), and Fourier transform infrared
(FTIR) spectroscopy for pharmaceutical substances (79).

Degradation-related impurities
NH2

Degradation products are compounds

produced by decomposition of the
N
O 2S
S
-(CH2O)n
NH2
material of interest or active ingredient.
Cl
N
Cl
Several impurities may result because
H
hydrochlorothiazide
Disulfonamide degradation product of API degradation or other interaction
Figure 1: Degradation of hydrochlorothiazide. (ALL FIGURES ARE COURTESY OF
on storage, so stability studies need to
THE AUTHORS)
be conducted to ensure drug product
safety (10). Hydrochlorothiazide (see Figure 1) is a classical example of a degradation impurity. It
has a known degradation pathway through which it degrades to the starting material as disulfonamide
in its synthesis.
O 2S

NH2

Degradation products could result from the synthesis itself, storage, formulation of the dosage form,
and aging (11). These degradation pathways are further discussed.
Synthesis-related impurities. Impurities in a drug substance or a new chemical entity originate
mainly during the synthetic process from raw materials, solvents, intermediates, and byproducts. The
raw materials are generally manufactured to much lower purity requirements than a drug substance,
and thus, it is easy to understand why they can contain a number of components that can in turn affect
the purity of the drug substance.
1-Methyl-3-phenyl
piperazine
(see Figure 2) is present as an
unreacted starting material that
competes in all the stages
eventually leading to the
impurity
keto-piperazine
derivative of mirtazapine (see
Impurity C, Figure 2).

O
O

H
N

+
N

DMF, EtOAc
KF

O
N
N

Cl

O
N
NaBH 4
Ethanol, water
acetone, EtOAc

Formulation-relatedimpurities.
Several impurities in a drug
OH
product or API can arise from
H2SO 4
N
interactions with excipients used
N
N
N
O
to formulate the drug product. In
O
N
N
the process of formulation, a
drug substance is subjected to
Mirtazapine impurity C (Ph. Eur)
various conditions that can lead
to its degradation or other Figure 2: Reaction scheme for mirtazapine impurity. Ph. Eur is the European Pharmacopoeia.
deleterious
reactions.
For DMF is dimethylformamide. EtOAc is ethyl acetate.
example, if heat is used for
drying or for other reasons, it can facilitate degradation of thermally labile drug substances. Solutions
and suspensions are potentially prone to degradation due to hydrolysis or solvolysis. These reactions
also can occur in the dosage form at solid state, such as in the case of capsules and tablets, when
water or another solvent has been used for granulation.

There are two typical conditions in solid- and solution-state degradation studies. Typical conditions
for the API in a solid state might be 80 C, 75% relative humidity (RH); 60 C at ambient RH; 40 C
at 75% RH; and light irradiation. Typical conditions for an API in the solution state might be: pH 19
in buffered media; with peroxide and/or free-radical initiator; and light irradiation.

- CO 2
N

O
decarboxy analog

O2
OH

ketorolac

NH 2-C-(CH2OH) 3

Figure
3
shows
the
degradation
pathway
of
OH
N
ketorolac in the solid and
O
solution states (1214).
1-hydroxy analog

O2

Dosage
form-related
impurities. Impurities related
O
N
NHC(CH
OH)
to the dosage form are
2
3
N
O
O
significant because many
1-keto analog
amide
times precipitation of the main
Figure 3: Degradation pathway of ketorolac.
ingredient requires various
factors, such as pH or leaching, to be altered (15). For example, the precipitation of imipramine
hydrochloride with sodium bisulfite requires a subsequent pH alteration of lidocaine hydrochloride
solution in the presence of 5% dextrose in saline.
solid state only
O

Method-related impurities. A known impurity,1-(2,6-dichlorophenyl)indolin-2-one is formed in the

diclofenac sodium ampuls. Formation of this impurity depends on the initial pH of the preparation
and the conditions of sterilization (i.e., autoclave method, 123 C 2 C) that enforces the
intermolecular cyclic reaction of diclofenac sodium, forming indolineone derivative and sodium
hydroxide (16).
Environmental-related impurities. Environmental-related impurities may result from the following:
Temperature. Many heat-labile compounds, when subjected to extreme temperature, lose
their stability. Keeping this in mind, extreme care should be exercised to prevent them from
degradation.
Light (ultraviolet light). Exposure to light results in a photolytic reaction. Several studies
reported that ergometrine and ergometrine injections are unstable under tropical condition
such as light and heat (1719).
Humidity. Humidity is one of the important factors when working with hygroscopic
compounds. Humidity can be deleterious to bulk powders and formulated solid dosage forms.
Well-know examples are ranitidine and aspirin (19).
Impurities on aging. Generally, a longer stay on the shelf increases the possibility that impurities
will occur. Such impurities can be caused by several interactions as further described.

Table I. Effect of interactions among ingredients

Active
Pharmaceutical aid
Effect
ingredient
Kanamycin
Honey; sugar syrup
Loss of activity at room
temperature
Cholecalciferol 2% polyoxy ethylene
Change in pH, degradation
ester surfactant;
of active ingredient.
polysorbate
Tetracyclines
Calcium or magnesium Complexation
or metal ions
Thiomersal
Bromine; ChlorideFormed different soluble
based compounds;
halides of cationic
Iodide-based
mercury compounds
compounds
Adrenaline
Boric acid;povidone
Stabilization
Table I: Effect of interactions among ingredients

Interaction among ingredients. Vitamins are highly prone to instability after aging. For
example, the presence of nicotinamide containing four vitamins (nicotinamide, pyridoxine,
riboflavin, and thiamin) caused the degradation of thiamin to a substandard level during a
one-year shelf life (20). Table I lists some examples of interactions among ingredients.
Hydrolysis. Many drugs are derivatives of carboxylic acids or contain functional groups
susceptible to acidbase hydrolysis (e.g., aspirin, atropine, and chloramphenical).
Oxidation. In pharmaceuticals, the most common form of oxidative decomposition is autooxidation through a free-radical chain process. Drugs that are prone to oxidation include
methotrexate, adinazolam, catecholamine, conjugated dienes (i.e., vitamin A), and nitroso and
nitrite derivatives. Olanzapine is especially prone to oxidative degradation in the presence of
oxygen (see Figure 4) (21).
Photolysis. Photolytic cleavage on aging products occurs with APIs or drug products that are
prone to degradation on exposure to UV light. For example, the ophthalmic formulation of
ciprofloxacin drops 0.3%, when exposed to UV light and undergoes photolysis to form
ethylene diamine, an analog of ciprofloxacin (22).
Decarboxylation. Carboxylic acid (COOH) tends to lose carbon dioxide from carboxyl
groups when heated. For instance, a photoreaction of a rufloxacin enteric tablet coated with
cellulose acetate phthalate and subcoated with calcium carbonate causes hydrolysis of
cellulose acetate phthalate. This reaction liberates acetic acid, which on reacting with calcium
carbonate, produces carbon dioxide as a byproduct.
pH. It is well understood that pH, particularly extreme levels of pH, can encourage hydrolysis
of the API when ionized in an aqueous solution. This situation necessitates buffer control if
such a dosage form is required.
Packaging materials. Impurities may result from packaging materials (i.e., containers and
closures (23).

N
N

air

heating
N
H

N
H

N
O

N
N

N
H

S
NH

Olanzapine Lactam Impurity.

1

OH

NH
N

Hydroxymethylidine thione

Acetoxymethylidine thione

Olanzapine

N
N

Olanzapine Thiolactam Impurity.

2

Figure 4: Olanzapine impurities due to air, heating, and formulation.

Two
impurities
in
olanzapine have been
identified as 1 and 2 (see
Figure 4) (24). The
structures indicate that
the two impurities are
degradation
products
resulting from oxidation
of the thiophene ring of
olanzapine.
From
a
regulatory
perspective,
forced
degradation
provides
data to support the

following (25):
Forced degradation is typically carried out using one batch of material.
Forced-degradation conditions are more severe than accelerated stability testing, such as 50
C; 75% RH; light conditions exceeding ICH standards; high and low pH; and oxidation.
Photostability should be an integral part of forced-degradation study design (10).
Degradation products that do not form in accelerated or long-term stability may not have to be
isolated or have their structure determined.
Mass balance should be considered.
Various issues are not addressed in regulatory guidance (3-6). Some key issues not addressed are:
Exact experimental conditions (temperatures, duration, and extent of degradation)
Experimental design (left to the applicant's discretion).
Metabolite impurities
Metabolite impurities are byproducts formed in the body after a drug substance is ingested. During
metabolism, the API and drug product in the body are exposed to various enzymes, from which
metabolite impurities can be formed (2634). Drug metabolism is traditionally divided into two
phases: metabolic (i.e., hepatic) clearance and the Phase I and Phase II process. The division is based
on the observation that a drug substance first undergoes oxidative attack (e.g., benzene to phenol),
and the newly introduced hydroxyl function will undergo glucouronidation (e.g., phenol to phenyl
glucouronic acid). Some metabolites are formed as impurities during the development of a process.
Control of these process-related metabolite impurities in the final API may not be necessary if control
of other metabolites has already occurred and taken into consideration. Tightening the limits,
therefore, may not be needed.

Examples
are
asenapine
N-oxide,
asenapine desmethyl,
and ciprofoxacin ethyl
diamino
impurity,
which are formed as
process impurities, but
are also metabolites of
the same process (see
Figure 5). It put forth a
question
whether
limiting
such
a
metabolite impurity in
the final API is still
required.

O
Cl
H

Cl

N
H

H
N

Desmethyl Asenapine
CAS No 128915-56-0
Metabolite
Process impurity

H
N

Deschloro Asenapine
Process impurity

Cis Asenapine
Process impurity

Cl
H
O

O
O

Cl
H
O

COOH

Cl

Trans Lactam
Process impurity

COOH
O
Cl

Asenapine maleate

H
N

O
Cl

Cl

* CAS No 129385-60-0

Cis lactam
Process impurity

Cl

Select analytical
methodologies

H
O

* CAS No 129385-61-1

Asenapine N Oxide
CAS No 128949-51-9
Metabolite

* CAS No 129385-59-7

* Thermal decomposition impurities

Figure 5: Process impurities, thermal decomposition impurities, and metabolites of asenapine. CAS No. is
Chemical Abstracts Service number.

The development of a
new drug mandates
that meaningful and
reliable analytical data be generated at various steps of drug development. The drug also should
exhibit excellent stability throughout its shelf-life. To meet these requirements, methodologies need
to be developed that are sensitive enough to measure low levels of impurities. This need has led to
analytical methods that are suitable for determining trace and ultra-trace levels (i.e., submicrogram)
quantities of various chemical entities (3539). Various methods are available for monitoring
impurities.
Spectroscopic methods. Various spectroscopic methods can be used for characterization of
impurities, such as UV-visible spectroscopy, FTIR spectroscopy, NMR spectroscopy, and mass
spectrometry (MS).
Separation methods. Various separation methods can be used, including thin-layer chromatography
(TLC), gas chromatography (GC), HPLC, capillary electrophoresis (CE), and supercritical fluid
chromatography (SFC). A review of these methods is provided in the literature (39). CE is an
electrophoretic method that is frequently lumped with chromatographic methods because it shares
many of the common requirements of chromatography. A broad range of compounds can be resolved
using TLC by using different plates and mobile phases. GC is a useful technique for quantification. It
can provide the desired resolution, selectivity, and ease of quantification. This technique is useful for
organic volatile impurities. SFC offers some of the advantages of GC in terms of detection and HPLC
in terms of separation.
Hyphenated methods. The following hyphenated methods can be used effectively to monitor
impurities: GCMS; liquid chromatography (LC)MS; LCdiode-array detection (DAD)MS; LC
NMR; LCDADNMRMS; and LCMSMS.

Isolating impurities. It is often necessary to isolate impurities because the instrumental methods are
not available or further confirmation is needed. The following methods have been used for isolation
of impurities: solid-phase extraction, liquidliquid extraction, accelerated solvent extraction,
supercritical fluid extraction, column chromatography, flash chromatography, TLC, HPLC, CE, and
SFC.
Impurity profiling
Ideally, an impurity profile should show all impurities in a single format to allow monitoring of any
variation in the profile. The driving forces for studying an impurity profile are quality considerations
and regulatory requirements.
Samples to be profiled. Impurity profiling should be done for APIs, process check of the synthesis
or formulation, and final drug product.
Components in an impurity profile. Ideally, an impurity profile should show synthesis-related
impurities, formulation-related impurities, degradation products, and interaction products.
Crucial factors for controlling impurities in APIs. Several factors are important in controlling
impurities in APIs as further outlined.
Crystallization. The size of crystals not only determines the quality, but also the stability of the drug.
During crystallization, fine crystals should be formed to prevent entrapment of minute amounts of
chemicals from the mother liquor, which in turn causes degradation of the drug.
Wet-cake washing. Many unwanted chemicals, including residual solvents, could be removed by
thorough washing of the wet cake, which if not done correctly, could lead to retention of solvents and
impurities in the cake.
Drying. Use of vacuum dryer or a fluid-bed dryer is always advisable in comparison to a tray dryer.
Use of the former reduces drying time and brings about uniform drying, which is helpful in drying
sensitive drug substances.
Appropriate packaging. The packing of bulk drugs should be based upon their nature and
sensitivity. Light-sensitive products should be packed in light protective packing. Use of opaque
containers for ciprofloxacin eye-drops preparations protects the active ingredients from
photodegradation (22). Use of ampuls with either black carbon paper or aluminum foil for
ergometrine produced negligible degradation (40). It is important to determine the most appropriate
container-closure system.
Production methods based on stability studies. A manufacturer of a bulk drug should perform a
detailed investigation of the process, including stability studies while finalizing the method of
preparation. For example, for producing diclofenac sodium injections, the aseptic filtration process is
better than the autoclave method that produces the impurity (16).

Measures by pharmacopoeias. Pharmacopoeias should take steps to incorporate impurity limits for
drug substances made from a raw material in which that particular impurity is controlled. It becomes
convenient for the users if the impurity limit is mentioned in the dosage forms.
Conclusion
Parts I, II, and III of this article discussed the types, origin, causes, chemistry, and impact of
impurities in APIs and drug products (1, 2). Parts I and II explained how, when, and why impurities
are formed. This article, Part III, highlighted the degradation-related, formulation-related, and
metabolite impurities, the various analytical techniques available for their identification and
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