Advanced Drug Delivery Reviews 59 (2007) 3 – 11
`
`www.elsevier.com/locate/addr
`
`Assuring quality of drugs by monitoring impurities☆
`Satinder (Sut) Ahuja ⁎
`
`Ahuja Consulting, 1061 Rutledge Court, Calabash, NC 28467, USA
`
`Received 14 January 2006; accepted 25 October 2006
`Available online 16 November 2006
`
`Abstract
`
`To assure the quality of drugs, impurities must be monitored carefully. It is important to understand what constitutes an impurity and to identify
`potential sources of such impurities. Selective analytical methods need to be developed to monitor them. It is generally desirable to profile
`impurities to provide a yardstick for comparative purposes. New impurities may be observed as changes are made in the synthesis, formulation, or
`production procedures, albeit for improving them. At times it is necessary to isolate and characterize an impurity when hyphenated methods do not
`yield the structure or when confirmation is necessary with an authentic material. Availability of an authentic material can also allow toxicological
`studies and provide a standard for routine monitoring of the drug product.
`© 2007 Published by Elsevier B.V.
`
`Keywords: Characterization; Chiral impurity; Degradation product; Drug product; Drug substance; Isolation; Profiling; Selective analytical methodologies;
`Terbutaline
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`Contents
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`1.
`2.
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`Introduction .
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`Terminology .
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`2.1.
`Commonly used terms.
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`2.1.1.
`Starting material(s) .
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`2.1.2.
`Intermediates .
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`2.1.3.
`Penultimate intermediate .
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`2.1.4.
`By-products .
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`2.1.5.
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`2.1.6.
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`2.1.7.
`Related products .
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`2.1.8. Degradation products .
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`Compendial terminology .
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`2.2.1.
`Foreign substances .
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`2.2.2.
`Toxic impurities .
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`2.2.3.
`Concomitant components.
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`2.2.4.
`Signal impurities .
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`2.2.5. Ordinary impurities.
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`2.2.6. Organic volatile impurities (OVIs) .
`ICH terminology .
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`2.3.1. Organic impurities .
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`2.3.2.
`Inorganic impurities .
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`2.3.3. Other materials .
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`2.2.
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`2.3.
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`☆ This review is part of the Advanced Drug Delivery Reviews theme issue on “Pharmaceutical Impurities: Analytical, Toxicological and Regulatory Perspectives".
`⁎ Corresponding author. Tel.: +1 910 287 7565.
`E-mail address: sutahuja@atmc.net.
`
`0169-409X/$ - see front matter © 2007 Published by Elsevier B.V.
`doi:10.1016/j.addr.2006.10.003
`
`IPR2020-00770
`United Therapeutics EX2022
`Page 1 of 9
`
`

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`4
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`S. Ahuja / Advanced Drug Delivery Reviews 59 (2007) 3–11
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`2.4.
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`3.
`4.
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`Residual solvents .
`2.3.4.
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`Comments on various terminologies .
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`2.4.1.
`Chiral impurities .
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`Identification and qualification thresholds of impurities .
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`Sources of impurities .
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`4.1.
`Synthesis-related impurities .
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`4.2.
`Formulation-related impurities .
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`4.3. Degradation-related impurities .
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`4.3.1. Kinetic studies .
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`Selective analytical methodologies .
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`5.1.
`Spectroscopic methods .
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`Infrared spectrophotometry .
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`5.1.3. Mass spectrometry .
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`5.3. Hyphenated methods .
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`Impurity profiling .
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`6.1.
`Samples to be profiled .
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`6.2.
`Components seen in a profile .
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`Isolating impurities .
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`7.
`8. Characterization of impurities .
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`9. A case study.
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`9.1. HPLC methods .
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`9.1.1. Achiral impurities .
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`9.1.2.
`Chiral impurities .
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`10. Conclusions .
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`References .
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`6.
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`1. Introduction
`
`Webster's dictionary defines impurity as something that is
`impure or makes something else impure. An impure substance
`may be defined as follows: a substance of interest mixed or
`impregnated with an extraneous or usually inferior substance.
`These definitions can help generate a more concise definition of
`an impurity: any material that affects the purity of the material
`of interest, viz., an active pharmaceutical ingredient (API) or
`drug substance [1–4]. The purity of a drug product is in turn
`determined on the basis of the percentage of the labeled amount
`of API found in it by a suitable analytical method. Later
`discussion will also reveal
`that a drug product can have
`impurities that need to be monitored even though they do not
`affect the labeled content. The presence of some impurities may
`not deleteriously impact on drug quality if they have therapeutic
`efficacy that is similar to or greater than the drug substance
`itself. Nevertheless, a drug substance can be considered as
`compromised with respect to purity even if it contains an
`impurity with superior pharmacological or
`toxicological
`properties. Consequently, in order to ensure that an accurate
`amount of the drug substance is being administered to the
`patient, drug substance purity must be assessed independently
`from these undesirable extraneous materials (e.g., inert, toxic, or
`pharmacologically superior impurities).
`
`2. Terminology
`
`A large number of terms have been used to describe the
`materials that can affect purity of the API. For the purpose of
`
`this discussion, they are all considered impurities. To better
`acquaint the reader with advantages and limitations of the use of
`various terms, a brief description of these terms is given below,
`followed by some comments.
`
`2.1. Commonly used terms
`
`A number of terms have been commonly used in the
`pharmaceutical industry to describe organic impurities:
`
`• Starting material(s)
`• Intermediates
`• Penultimate intermediate (Final intermediate)
`• By-products
`• Transformation products
`• Interaction products
`• Related products
`• Degradation products
`
`Some of these terms denote potential sources of impurities,
`e.g., intermediates; others tend to de-emphasize the negativity,
`e.g., related products. Let us review them individually.
`
`2.1.1. Starting material(s)
`These are the materials that are used to begin the synthesis of
`an API.
`
`2.1.2. Intermediates
`The compounds produced during synthesis of the desired
`material are called intermediates, especially when they have
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`been isolated and characterized. The most important criterion
`is characterization,
`i.e.,
`they cannot be just
`theorized
`potential
`reaction products (see by-products below). The
`theorized compounds are best designated as potential
`intermediates.
`
`2.1.3. Penultimate intermediate
`As the name suggests, this is the last compound in the
`synthesis chain prior to the production of the final desired
`compound. It is more appropriate to call it Final Intermediate.
`Sometimes confusion arises when the desired material is a salt
`of a free base or a free acid. In the opinion of this author, it is
`inappropriate to label the free acid or base as the penultimate
`intermediate if the drug substance is a salt.
`
`2.1.4. By-products
`The unplanned compounds produced in the reaction are
`generally called by-products. It may or may not be possible to
`theorize all of them. Hence, they present a challenging problem
`to the analytical chemist in that a methodology cannot be
`optimally planned if it is not known what needs to be excluded
`from evaluations.
`
`2.1.5. Transformation products
`This is a relatively nondescript term that relates to theorized
`and non-theorized products that may be produced in the reaction,
`which can include synthetic derivatives of by-products.
`Transformation products are very similar to by-products, except
`this term tends to connote that more is known about the reaction
`products.
`
`2.1.6. Interaction products
`Interaction products is a slightly more comprehensive term
`than the two described above (by-products and transformation
`products); however, it is more difficult to evaluate in that it
`considers interactions that could occur between various
`involved chemicals — intentionally or unintentionally. Two
`types of interaction products that can be commonly encountered
`are drug substance–excipient interactions and drug substance–
`container/closure interactions.
`
`2.1.7. Related products
`As mentioned before, the term related products tends to
`suggest that the impurity is similar to the drug substance and
`thus tends to play down the negativity frequently attached to
`the term impurity. Clearly these products generally have
`similar chemical structures as the API and may exhibit
`potentially similar biological activity; however, as discussed
`later, this by itself does not provide any guarantee to that
`effect.
`
`2.1.8. Degradation products
`These are the compounds produced because of decomposi-
`tion of the material of interest or active ingredient. This term can
`also include those products produced from degradation of other
`compounds that may be present as impurities in the drug
`substance.
`
`2.2. Compendial terminology
`
`The United States Pharmacopoeia (USP) deals with
`impurities in several sections:
`
`Impurities in official articles
`Ordinary impurities
`Organic volatile impurities
`
`The USP acknowledges that concepts about purity are
`susceptible to change with time, and purity is intimately related
`to the developments in analytical chemistry. What we consider
`pure today may be considered impure at some future date if
`methods are found that can resolve other components contained
`in a particular compound. Inorganic, organic, or polymeric
`components can all be considered impurities. The following
`terms have been used by the USP to describe impurities:
`
`▪ Foreign substances
`▪ Toxic impurities
`▪ Concomitant components
`▪ Signal impurities
`▪ Ordinary impurities
`▪ Organic volatile impurities (OVIs)
`
`2.2.1. Foreign substances
`The materials that are introduced by contamination or
`adulteration, not as a consequence of synthesis or preparation,
`are labeled foreign substances, e.g., pesticides in oral
`analgesics.
`
`2.2.2. Toxic impurities
`These impurities have significant undesirable biological
`activity, even as minor components; and they require individual
`identification and quantification by specific tests.
`
`2.2.3. Concomitant components
`Bulk pharmaceutical chemicals may contain concomitant
`components, e.g., antibiotics that are mixtures and are
`geometric and optical isomers (see Section 2.4.1).
`
`2.2.4. Signal impurities
`These are distinguished from ordinary impurities discussed
`below in that
`they require individual
`identification and
`quantification by specific tests. These impurities include some
`process-related impurities or degradation products that provide
`key information about the process.
`
`2.2.5. Ordinary impurities
`The species of impurities in bulk pharmaceutical chemicals
`that are innocuous by virtue of having no significant undesirable
`biological activity in the amounts present are called ordinary
`impurities.
`
`2.2.6. Organic volatile impurities (OVIs)
`This term relates to residual solvents that may be found in the
`drug substance. OVIs are generally solvents used in the
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`synthesis or during formulation of the drug product. The
`solvents have been classified as follows by ICH.
`
`Class I (to be avoided): benzene, carbon tetrachloride, 1,2-di-
`chloromethane, 1,1-dichloroethane, and 1,1,1-trichloroethane.
`Class II
`(should be limited): acetonitrile, chloroform,
`methylene chloride, 1,1,2-trichloroethane, 1,4-dioxane, and
`pyridine.
`Class III: low toxic potential and permitted daily exposure
`(PDE) of 50 mg or more.
`Class IV: solvents for which adequate toxic data are not
`available.
`
`2.3. ICH terminology
`
`2.3.1. Organic impurities
`Starting materials
`Process-related impurities
`Intermediates
`Degradation products.
`
`2.3.2. Inorganic impurities
`Salts
`Catalysts
`Ligands
`Heavy metals or other residual metals.
`
`2.3.3. Other materials
`Filter aids
`Charcoal.
`
`2.3.4. Residual solvents
`Organic and inorganic liquids used during production and/or
`crystallization.
`
`2.4.1. Chiral impurities
`Chiral impurities have the identical molecular formula and
`the same connectivity between various atoms, and they differ
`only in the arrangement of their atoms in three-dimensional
`space. The differences in pharmacological/toxicological pro-
`files have been observed with chiral impurities in vivo [4,5].
`This suggests that chiral
`impurities should be monitored
`carefully.
`
`3. Identification and qualification thresholds of impurities
`
`The International Conference on Harmonisation addresses
`questions relating to impurities as follows [6]:
`
`Q1A (R) stability testing of new drug substances and
`products
`Q3A (R) impurities in drug substances
`Q3B (R) impurities in drug products
`Q3C impurities: residual solvents
`Q6A specifications: test procedures and acceptance criteria
`for new drug substances and new drug products; chemical
`substances
`
`the identification and qualification
`ICH guidelines for
`threshold of impurities and degradation products are provided
`in Table 1.
`As can be seen from the data in Table 2, ICH treats the
`degradation products slightly differently than impurities even
`though for all intents and purposes the degradation products are
`impurities.
`
`4. Sources of impurities
`
`Discussed below are three important sources of impurities.
`
`2.4. Comments on various terminologies
`
`4.1. Synthesis-related impurities
`
`The impurities that may be present in the starting material(s)
`can potentially be carried into the active ingredient of
`interest. And the impurities that relate to the solvents used
`during synthesis and the inert ingredients (excipients) used
`for formulation must also be considered potential impurities that
`may be found in API or drug product. Inorganic impurities
`may also be found in compendial articles. These impurities may
`be as simple as common salt or other compounds that are
`controlled, such as heavy metals, arsenic, etc., which can
`be introduced during various synthetic steps. Potential reaction
`by-products, degradation products, and drug substance–excip-
`ient
`interactions must also be evaluated. All of these im-
`purities have the potential of being present in the final drug
`product.
`Of the various terminologies described above, the Interna-
`tional Conference on Harmonisation (ICH) provides a simple
`classification to adequately address various impurities that may
`be present in pharmaceutical products. However, all of these
`terminologies fail to adequately highlight that enantiomeric
`(chiral) impurities might warrant additional considerations.
`
`Impurities in a drug substance or a new chemical entity
`(NCE) originate mainly during the synthetic process from raw
`materials, solvents, intermediates, and by-products. The raw
`materials are generally manufactured to much lower purity
`requirements than a drug substance. Hence,
`it
`is easy to
`understand why they can contain a number of components that
`can in turn affect the purity of the drug substance.
`Similarly, solvents used in the synthesis are likely to contain
`a number of impurities that may range from trace levels to
`significant amounts that can react with various chemicals used
`
`Table 1
`Thresholds for reporting impurities
`
`Maximum
`daily dose
`
`Reporting
`threshold
`
`Identification
`threshold
`
`Qualification
`threshold
`
`Less or equal
`to 2 g/day
`>2 g/day
`
`0.05%
`
`0.03%
`
`0.10% or 1.0 mg/day
`(whichever is lower)
`0.05%
`
`0.15% or 1.0 mg/day
`(whichever is lower)
`0.05%
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`Table 2
`Threshold for reporting degradation products in a new drug product
`
`4.3. Degradation-related impurities
`
`Maximum daily dose
`
`1 g
`>1 g
`
`Threshold
`
`0.1%
`0.05%
`
`in the synthesis to produce other impurities. Intermediates are
`also not generally held to the purity level of
`the drug
`substance—hence the remarks made for the raw materials
`apply. It is not reasonably possible to theorize all by-products;
`as a result, any such products that may be produced in the
`synthesis would be hard to monitor. The “pot reactions,” i.e.,
`when the intermediates are not
`isolated, are convenient,
`economical, and timesaving; however,
`they raise havoc in
`terms of the generation of impurities because a number of
`reactions can occur simultaneously. Incidentally, this problem
`of numerous reactions occurring simultaneously can be also
`encountered in single reactions where intermediate is isolated.
`The final
`intermediate is generally controlled in the
`pharmaceutical synthesis by conducting regulatory impurity
`testing. This typically entails residual solvents (that are not used
`in further downstream processing) or process impurities (in
`cases where they conclusively demonstrate that these moieties
`are not also degradation products). It is important to remember
`that this step is the last major source of potential impurities,
`therefore, it is very desirable that the methods used for analysis
`at this stage be rigorous. It should be remembered that base-to-
`salt or acid-to-salt conversions could also generate new
`impurities. Furthermore,
`thermally labile compounds can
`undergo decomposition if any further processing involves
`heating.
`
`4.2. Formulation-related impurities
`
`A number of impurities in a drug product can arise out of
`interactions with excipients used to formulate a drug product.
`Furthermore, in the process of formulation, a drug substance is
`subjected to a variety of 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 degrada-
`tion that is due to hydrolysis or solvolysis (see kinetic studies
`discussed below). These reactions can also occur in the dosage
`form in a solid state, such as in the case of capsules and tablets,
`when water or another solvent has been used for granulation.
`Not only can the water used in the formulation contribute its
`own impurities,
`it can also provide a ripe situation for
`hydrolysis and metal catalysis. Similar reactions are possible
`in other solvents that may be used.
`Oxidation is possible for easily oxidized materials if no
`precautions are taken. Similarly,
`light-sensitive materials
`can undergo photochemical reactions. Details are provided
`in Chapter 6 of reference [1] regarding how various ex-
`cipients can contribute to degradation and the resulting
`impurities.
`
`A number of impurities can be produced because of API
`degradation or other interactions on storage. Therefore, it is very
`important to conduct stability studies to predict, evaluate, and
`ensure drug product safety [7]. Stability studies include
`evaluation of stability of API, preformulation studies to evaluate
`compatibility of API with the excipients to determine its stability
`in the formulation matrix, accelerated stability evaluations of the
`test or final drug product, stability evaluation via kinetic studies
`and projection of expiration date, routine stability studies of drug
`products in marketed, sample or dispensed package under
`various conditions of temperature light, and humidity.
`The stability studies under various exaggerated conditions of
`temperature, humidity, and light can help us determine what
`potential impurities can be produced by degradation reactions
`(for details see Chapter 8 of reference [1]). It is important to
`establish a viable stability program to evaluate impurities. A
`good stability program integrates well the scientific considera-
`tions with regulatory requirements. The importance of kinetic
`studies in monitoring and evaluating impurities is discussed
`below.
`
`4.3.1. Kinetic studies
`Most of the degradation reactions of pharmaceuticals occur
`at finite rates and are chemical in nature. These reactions are
`affected by conditions such as solvent, concentration of
`reactants, temperature, pH of the medium, radiation energy,
`and the presence of catalysts. The order of the reaction is
`described by the manner in which the reaction rate depends on
`the concentration of
`reactant. The degradation of most
`pharmaceuticals can be classified as zero order, first order, or
`pseudo-first order, even though they may degrade by compli-
`cated mechanisms, and the true expression may be of higher
`order or be complex and noninteger.
`An understanding of the limitations of experimentally
`obtained heat of activation values is critical
`in stability
`predictions. For example, the apparent heat of activation of a
`pH value where two or more mechanisms of degradation are
`involved is not necessarily constant with temperature. Also, the
`ion product of water, pKw, is temperature-dependent, and −ΔHa
`is approximately 12 kcal, a frequently overlooked factor that must
`be considered when calculating hydroxide concentration. There-
`fore, it is necessary to obtain the heat of activation for all
`bimolecular rate constants involved in a rate–pH profile to predict
`degradation rates at all pH values for various temperatures.
`It is incumbent upon the chemist to perform some kinetic
`studies to predict stability of a drug substance and to evaluate
`degradation products. However, it is also important to recognize
`the limitations of such predictions. The importance of kinetic
`studies and the effect of various additives on the reaction rates
`are discussed at some length in Chapter 7 of reference [1].
`
`5. Selective analytical methodologies
`
`Development of a new drug mandates that meaningful and
`reliable analytical data be generated at various steps of the new
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`drug development [7]. Ensuring the safety of a new pharma-
`ceutical compound or drug requires that it meet the established
`purity standards as a chemical entity or when admixed with
`animal feeds for toxicity studies or pharmaceutical excipients for
`human use. Furthermore, it should exhibit excellent stability
`throughout its shelf life. These requirements demand that the
`analytical methodology that is used be sensitive enough to mea-
`sure low levels of impurities. This has led to analytical methods
`that are suitable for determination of trace/ultratrace levels, i.e.,
`sub-microgram quantities of various chemical entities [8–12].
`A variety of methods are available for monitoring impurities.
`The primary criterion is the ability to differentiate between the
`compounds of interest. This requirement reduces the availabil-
`ity of methods primarily to spectroscopic and separation
`methods or a combination thereof.
`
`5.1. Spectroscopic methods
`
`The following spectroscopic methods can be used:
`
`▪ Ultraviolet (UV)
`▪ Infrared (IR)
`▪ Nuclear magnetic resonance (NMR)
`▪ Mass spectrometry (MS)
`
`UV at a single wavelength provides minimal selectivity of
`analysis; however, with the availability of diode array detectors
`(DAD),
`it
`is now possible to get sufficient simultaneous
`information at various wavelengths to ensure greater selectivity.
`
`5.1.1. Infrared spectrophotometry
`Infrared spectrophotometry provides specific information on
`some functional groups that may allow quantification and
`selectivity. However,
`low-level detectability is frequently a
`problem that may require more involved approaches to
`circumvent the problem.
`
`5.1.2. Nuclear magnetic resonance spectroscopy
`Nuclear magnetic resonance spectroscopy provides fairly
`detailed structural information on a molecule and is a very
`useful method for characterization of impurities; however, it has
`limited use as a quantitative method because of cost and time
`considerations.
`
`5.1.3. Mass spectrometry
`Mass spectrometry provides excellent structural informa-
`tion, and, based on the resolution of the instrument, it may
`provide an effective tool for differentiating molecules with
`small differences in molecular weight. However, it has limited
`use as a quantitative technique because of cost and time
`considerations.
`In summary, IR, NMR, and MS are excellent techniques for
`characterization of impurities that have been isolated by any of
`the techniques discussed above. UV has been found to be
`especially useful for analyzing most samples with high-pressure
`liquid chromatography. This combination is commonly used in
`pharmaceutical analysis.
`
`5.2. Separation methods
`
`The following separation methods can be used:
`
`▪ Thin-layer chromatography (TLC)
`▪ Gas chromatography (GC)
`▪ High-pressure liquid chromatography (HPLC)
`▪ Capillary electrophoresis (CE)
`▪ Supercritical fluid chromatography (SFC)
`
`A brief account of the above-listed methods is given here to
`provide a quick review of their potential use [10].
`Except for CE, all these techniques are chromatographic
`methods. CE is an electrophoretic method that is frequently
`lumped with the chromatographic methods because it shares
`many of
`the common requirements of chromatography.
`However, it is not strictly a two-phase separation system — a
`primary requirement in chromatography. Hyphenated methods
`such as GC–MS, LC–MS, GC–LC–MS, LC–MS–MS, etc. are
`all discussed later in this chapter.
`A broad range of compounds can be resolved using TLC by
`utilizing a variety of different plates and mobile phases. The
`primary difficulties related to this method are limited resolution,
`detection, and ease of quantification. The greatest advantages
`are the ease of use and low cost.
`Gas chromatography is a very useful technique for quanti-
`fication. It can provide the desired resolution, selectivity, and
`ease of quantification. However, the primary limitation is that
`the sample must be volatile or has to be made volatile by
`derivatization. This technique is very useful for organic volatile
`impurities.
`High-pressure liquid chromatography is frequently casually
`referred to as high-performance liquid chromatography today.
`Both of these terms can be abbreviated as HPLC, and they are
`used interchangeably by chromatographers. This is a useful
`technique with applications that have been significantly
`extended for the pharmaceutical chemist by the use of a variety
`of detectors such as fluorescence, electrometric, MS, etc.
`Capillary electrophoresis is a useful technique when very
`low quantities of samples are available and high resolution is
`required. The primary difficulty is assuring reproducibility of
`the injected samples.
`the
`Supercritical
`fluid chromatography offers some of
`advantages of GC in terms of detection and HPLC in terms of
`separations, in that volatility of the sample is not of paramount
`importance. This technique is still evolving, and its greatest
`application has been found in the extraction of samples.
`
`5.3. Hyphenated methods
`
`The following hyphenated methods can be used effectively
`to monitor impurities [3]:
`
`▪ GC–MS
`▪ LC–MS
`▪ LC–DAD–MS
`▪ LC–NMR
`
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`▪ LC–DAD–NMR–MS
`▪ LC–MS–MS
`
`these methods are not always available or
`Of course,
`applicable — a detailed discussion is included in Chapters 4
`and 9 of reference [1] as to why it is not always possible to use
`these methods. In case it is necessary to procure authentic
`material for purposes of structure confirmation, synthesis or
`isolation methods should be utilized.
`
`6. Impurity profiling
`
`Ideally an impurity profile should show all impurities in a
`single format to allow monitoring of any variation in the profile
`because of planned or unplanned changes in synthesis,
`formulation, or stability, etc. The driving forces for studying
`an impurity profile are
`
`Quality considerations
`Regulatory (FDA) requirements
`
`▪ Accelerated solvent extraction
`▪ Supercritical fluid extraction
`▪ Column chromatography
`▪ Flash chromatography
`▪ Thin-layer chromatography
`▪ Gas chromatography
`▪ High-pressure liquid chromatography
`▪ Capillary electrophoresis
`▪ Supercritical fluid chromatography.
`
`Isolation should be ini