pharmaceutical technology, apr 2, volume 36, issue 4, pp. 76-86

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Evaluating Impurities in Drugs (Part III of III)In Part III of a three-part article, the authors examine various degradation routes of APIs, impuritiesarising 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. 76-86Submitted: 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, [email protected]

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

API – excipient 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 (3 – 6). 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 (7 – 9).

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Degradation-related impurities

Degradation products are compounds

produced by decomposition of the

material of interest or active ingredient.

Several impurities may result because

of API degradation or other interaction

on storage, so stability studies need to 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.

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).

Formulation-relatedimpurities.

Several impurities in a drug

product or API can arise from

interactions with excipients used

to formulate the drug product. In

the process of formulation, a

drug substance is subjected to

various conditions that can lead

to its degradation or other

deleterious reactions. For

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.

NS

N

H

H

Cl

O2S

NH2 O O

NH2

S

Cl

O2S

NH2 O O

-(CH2O)n

hydrochlorothiazide Disulfonamide degradation product Figure 1: Degradation of hydrochlorothiazide. (ALL FIGURES ARE COURTESY OF

THE AUTHORS)

NH

N O N Cl

O

O

NN

N

O

DMF, EtOAc

KF

NaBH4

Ethanol, water

H2SO

4

NN

N

O

O

O

NN

N

O

OH

acetone, EtOAc

+

Mirtazapine impurity C (Ph. Eur

)

Figure 2: Reaction scheme for mirtazapine impurity. Ph. Eur is the European Pharmacopoeia.

DMF is dimethylformamide. EtOAc is ethyl acetate.



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 1 – 9

in buffered media; with peroxide and/or free-radical initiator; and light irradiation.

Figure 3 shows the

degradation pathway of

ketorolac in the solid andsolution states (12 – 14).

Dosage form-related

impurities. Impurities related

to the dosage form are

significant because many

times precipitation of the main

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 hydrochloridesolution in the presence of 5% dextrose in saline.

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.

L ight (ultr aviolet li ght) . 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 (17 – 19).

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.

O

N NHC(CH2OH)

3

O

O

NOH

O

NO

O

N

decarboxy analog

1-keto analog

- CO2

O2

O2

NH2-C-(CH

2OH)

3

solid state only

amide

O

N OH

O

1-hydroxy analogketorolac

Figure 3: Degradation pathway of ketorolac.



Table I. Effect of interactions among ingredients

Active

ingredient

Pharmaceutical aid Effect

Kanamycin Honey; sugar syrup Loss of activity at room

temperature

Cholecalciferol 2% polyoxy ethylene

ester surfactant;

polysorbate

Change in pH, degradation

of active ingredient.

Tetracyclines Calcium or magnesium

or metal ions

Complexation

Thiomersal Bromine; Chloride-

based compounds;

Iodide-based

compounds

Formed different soluble

halides of cationic

mercury compounds

Adrenaline Boric acid;povidone Stabilization

Table I: Effect of interactions among ingredients

I nteraction among ingredients . Vitamins are highly prone to instability after aging. Forexample, 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 acid – base hydrolysis (e.g., aspirin, atropine, and chloramphenical).

Oxidation . In pharmaceuticals, the most common form of oxidative decomposition is auto-

oxidation 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 materi als . Impurities may result from packaging materials (i.e., containers and

closures (23).



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 oxidationof 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 (26 – 34). 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.

NH

N

S

N

N

NH

N

S

N

N

OH

NH

N

S

N

N

OO

Acetoxymethylidine thione Hydroxymethylidine thioneOlanzapine

air

heating+

NH

N

OO

NN

O

N

NHS

NN

Olanzapine Thiolactam Impurity.Olanzapine Lactam Impurity.

1 2 Figure 4: Olanzapine impurities due to air, heating, and formulation.



Examples are

asenapine N-oxide,

asenapine desmethyl,

and ciprofoxacin ethyl

diamino impurity,

which are formed as

process impurities, but

are also metabolites ofthe same process (see

Figure 5). It put forth a

question whether

limiting such a

metabolite impurity in

the final API is still

required.

Select analytical

methodologies

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 (35 – 39). 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 sharesmany 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: GC – MS; liquid chromatography (LC) – MS; LC – diode-array detection (DAD) – MS; LC –

NMR; LC – DAD – NMR – MS; and LC – MS – MS.

.

O

N

Cl

HHCOOH

COOH

Asenapine maleate

O

Cl

N

HH

Asenapine N Oxide

O

N

ClO

N

Cl

O O

O

N

Cl

HH

O

* Thermal decomposition impurities

* CAS No 129385-60-0

* CAS No 129385-61-1 * CAS No 129385-59-7

O

CAS No 128949-51-9

O

Cl

NH

HH

Desmethyl Asenapine

O

N

HH

Deschloro Asenapine

O

Cl

N

HH

Cis AsenapineCAS No 128915-56-0

O

Cl

N

HH

O

Trans Lactam

O

Cl

N

HH

O

Cis lactam

Metabolite

Metabolite

Process impurity Process impurity

Process impurity

Process impurity

Process impurity

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

Chemical Abstracts Service number.



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, liquid – liquid 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 appropriatecontainer-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 impuritiesare 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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pharmaceutical technology, apr 2, volume 36, issue 4, pp. 76-86

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