Impurity Profile The Review Biology Essay

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1. Introduction

An impurity in a drug substance as defined by the International Conference on Harmonization (ICH) Guidelines [1] is any component of the drug substance that is not the chemical entity defined as the drug under the act and having impact on the purity of active pharmaceutical ingredient or drug substances.

Impurity is not very much liked word in pharmaceutical industries so there is no clear definition for impurity in the pharmaceutical world. Most frequently used terms for impurity are by-products, transformation products, degradation products, interaction products, and related drug products. The impurities related to residual solvents used in the preparation of pharmaceutical compounds or marketed drug products are frequently referred to as organic volatile impurities (OVI) [2] and the impurities relating to the inert ingredients (excipients) used in pharmaceutical formulation or pharmaceutical adjuvant used in the preparation of the marketed drug products are rarely mentioned.

Bulk pharmaceutical chemicals (BPCs), can be obtained or synthesized from multiple sources and, therefore, it is very important that impurities in them be carefully monitored and controlled. Therefore the different Pharmacopoeias, such as the British Pharmacopoeia (BP), United States Pharmacopeia (USP) and Indian Pharmacopoeia (IP) are slowly incorporating limits to allowable levels of impurities present in the API's or formulations [3-4].

Present article thoroughly review on different impurities found in the pharmaceuticals, methods for isolation, extraction and identifying possible impurities.

Impurity should be defined as Identified Impurity: An impurity for which a structural characterization has been achieved and Unidentified Impurity: An impurity for which a structural characterization has not been achieved and that is defined solely by Qualitative analytical properties (e.g. Chromatographic retention time)

Impurities present in new drug substances are covered under two aspects [5]:

Chemistry Aspects include classification and identification of impurities, generation of report for different impurities, list of impurities present in any substances, and a brief discussion of analytical procedures for impurity detection; and

Safety Aspects include those impurities that is present considerably at low amount or not present at all in a discovery of new drug substance used in clinical and safety trials.

2. Impurity term commonly used

A number of terms have been commonly used to describe an impurity or impurities [6, 9]:


Penultimate intermediate


Transformation products

Interaction product

Related products

Degradation products

Foreign substances

Toxic impurities

Concomitant components

Ordinary impurities

Organic volatile impurities (OVI)

2.1 Intermediates: The compounds produced during synthesis of the desired material are called intermediates or reaction intermediates. Product that has undergone a partial processing and is used as raw material in a successive productive step.

2.2 Penultimate Intermediate: As the name suggests, this is the compound found in the synthesis chain before the production of the desired compound. Sometimes confusion arises when the desired material is a salt of a free base or 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.

2.3 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 thorny problem to the analytical chemist. A by-product can be useful and marketable or it can be considered waste.

2.4 Transformation Products: This is a relatively nondescript term which relates to theorized and non-theorized products that may be produced in the reaction. Transformation products are very similar to by-products, except the term tends to connote that more is known about the reaction products.

2.5 Interaction Products: This term is slightly more comprehensive and more difficult to evaluate than the two described above, i.e. by-products and transformation products, in that it considers interactions that could occur between various involved chemicals-- intentionally or unintentionally.

2.6 Related Products: As mentioned, 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. These products can have similar chemical structure and potentially similar biological activity; however, as we shall discuss later, this by itself does not provide any guaranty that effect.

2.7 Degradation Products: The compounds produce due to decomposition of the material of interest or active ingredients are often referred to as degradation products.

2.8 Foreign Substances: The materials that are introduced by contamination or adulteration, and not as consequences of synthesis or preparation, are labeled foreign substances, e.g., pesticides in oral analgesics.

2.9 Toxic Impurities: These impurities have significant undesirable biological activity, even as minor components, and require individual identification and quantitation by specific tests.

2.10 Concomitant Components: Bulk pharmaceutical chemicals may contain concomitant components, e.g., geometric and optical isomers and antibiotics that are mixtures.

2.11 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.

2.12 Organic Volatile Impurities: This term relates to residual solvents that may be found in the drug substance.

3. Classification of Impurities

Organic Impurities (Process-and Drug Related)

Inorganic Impurities

Residual Solvents

Polymorphic Forms

Enantiomeric Impurities

3.1 Organic impurities come into existence during the synthesis of the active and inactive materials. It may come up while manufacturing and/or storage of the materials. These impurities can be deduced from degradation reactions and ongoing synthesis in active pharmaceutical entity and drug products. Impurities generated during the synthetic process are starting materials, intermediates, by-products, degradation products, reagents, ligands, and catalysts used in the chemical synthesis, as well as by-products from the side-reactions of the chemical synthesis [9, 10].

3.1.1 Starting materials and intermediates are the chemical compositions used to synthesize the desired constitute of a drug substance molecule. Starting materials and intermediates which are not reacted in the reaction especially when the synthesis is about to complete will remain in final product as impurities [11-13]. One such example is 4-aminophenol is starting material for synthesis of paracetamol bulk drug which might be present in final product as impurity having toxic effect on liver.

According to Dir. 2001/83/EC [14], for biological medicinal products, "starting materials shall mean any substance of biological origin such as micro-organisms, organs and tissues of either plant or animal origin, cells or fluids (including blood or plasma) of human or animal origin, and biotechnological cell constructs (cell substrates, whether they are recombinant or not, including primary cells)." GMP measures (e.g. contract between supplier and manufacturer of medicinal product, audit system) should be adequate to ensure an appropriate control while allowing sourcing of starting materials or early intermediates in different locations from third countries.

An intermediate is a molecular entity that is formed from the reactants (or preceding intermediates) and reacts further to give the directly observed products of a chemical reaction. An intermediate is material produced during steps of the synthesis of a new drug substance that undergoes further chemical transformation before it becomes a new drug substance.

3.1.2 By Product the term "by product" is generally used to denote one or more products of relatively small total value that are produced simultaneously with a product of greater total value. The product with the greater value, commonly called the "main product", is usually produced in greater quantities than the by products.

3.1.3 Degradation products are chemical breakdown compounds of the drug substance formed during storage. In rare cases, degradants are formed when the drug substance chemically interacts with other compounds or contaminants.

In addition, degradants may also be formed by physical degradation, for example, aggregates of proteinaceous material, dimers, trimers, and so forth, of synthetic compounds, polymorphs of synthetic compounds. Different pharmaceutical preparations against the common cold contain acetaminophen, phenylephrine hydrochloride, and chlorpheniramine maleate. A degradation product had been discovered in these preparations after short- and long-term stability studies. This degradation product was isolated and found to be adduct of phenylephrine and maleic acid [17]. The definition of degradation product in accordance with the ICH guideline is "a molecule come forth from a chemical change in the substance brought about by overtime and/or action of e.g. Light, temperature, pH or water or by reaction with excipient and/or the intermediate container closure system" [1, 16].

3.1.4 Reagents, Ligands and Catalysts are very rarely present in the final products; however, in some cases they may pose a problem as impurities [10, 15]. For the synthesis of the drug substance or any excipient various catalysts, chemical reagents and ligands are used that can be conveyed to the final products as trace level impurities. For example, carbonic acid chloromethyl tetrahydro-pyran-4-yl ester (CCMTHP) [16], which is used as an alkylating agent in the synthesis of a β lactam drug substance, was observed in the final product as an impurity.

3.1.5 Products of over-reaction formed when reactions for the synthesis are not selective as much as necessary, so reagents does not react with the intermediate at the desired site. For e.g. in the synthesis of nanodralone decanoate, the last step of the synthesis is the decanoylation of the 17 -OH group. In the course of overreaction the reagents also attracts the 4ene- 3 oxo group leading to an enol ester- type impurity (3, 17 β-dihydroxyestra-3, 5- diene disdecanoate) [11, 16].

3.1.6 Contamination organic impurities are not drug related but are inadvertently introduced during processing or storage, and are not part of the synthesis, extraction, or fermentation process. For drug substances from plants, examples of contaminant impurities could be herbicides, for example, diquat and glyphosate, or pesticides, and carbofuran and endrin, sprayed in the environment [21].

3.2 Inorganic Impurities

Inorganic impurities that may be derived from the manufacturing process of bulk drugs include reagents, catalysts, ligands, heavy metals, and other materials (e.g., filter aids, charcoal). For example, inorganic impurities may be present in the raw materials or may be derived from reagents, such as phosphate buffers, used during the production of the pharmaceutical. While the presence of many inorganic impurities at low concentrations have few toxicological consequences, significant variation in the impurity levels from batch-to-batch can indicate that the manufacturing process of the drug product is not adequately controlled [15, 18-20]. In most cases, these impurities should be removed or at least minimized in the final product. Therefore, the identification, quantification, and control of impurities are important during drug development in the pharmaceutical industry.

Inorganic impurities may derive from the manufacturing process. They are normally known and identified and include:

· Reagents, Ligands and Catalysts

· Heavy Metals

· Inorganic Salts

· Other Materials (e.g., Filter Aids, Charcoal, etc.)

3.2.1 Contamination inorganic impurities, Contamination impurities are unexpected adulterating compounds found in the drug substance. Current manufacturing technology has reduced many of the contaminant impurities observed in drugs prepared decades ago. For example, heavy metals like lead that leached from pipes or manufacturing/storage tanks gave rise to the commonly used limit test for heavy metals in the drug substance [21]. Current pipes and tanks are primarily stainless steel or glass-lined to reduce this concern, although the type of material is ultimately dependent on the nature of the reactions, the nature of the drug substance, and the nature of the manufacturing unit operations.

3.3 Residual Solvents in pharmaceuticals are defined here as organic volatile chemicals that are used or produced in the manufacture of drug substances or excipients, or in the preparation of drug products [2]. The solvents are not completely removed by practical manufacturing techniques. Therefore, the solvent may sometimes be a critical parameter in the synthetic process.

3.4 Polymorphic Forms, Polymorphism is the ability of a solid material to exist in more than one form or crystal structure. Some organic and inorganic compounds form different crystalline structures called polymorphs or polymorphic forms. The resulting change of intermolecular interactions gives rise to different pharmacokinetic properties of medical drugs as well as to different properties of organic and inorganic materials. Therefore, the unambiguous identification and characterization of polymorphs is very important especially from the economic point of view. In 2006 a new crystal form was discovered of maleic acid 124 years after the first crystal form was studied. Maleic acid is a chemical, manufactured on a very large scale in the chemical industry and is a salt forming component in medicine. The new crystal type is produced when a co-crystal of caffeine and maleic acid (2:1) is dissolved in chloroform and when the solvent is allowed to evaporate slowly [22].

3.5 Enantiomeric Impurities The enantiomeric excess (EE) of a substance is an indicator for the purity of a Chiral chemical compound. The impurity is usually the undesired enantiomer that occurs frequently as a byproduct in chemical syntheses. EE is determined by the following equation:

EE = ((R-S)/(R+S)) Ã- 100

Where R and S stand for the individual optical isomer in the mixture (and R + S = 1).

EE determinations are important in the pharmaceutical industry because undesired optical isomers of a drug can potentially alter pharmaceutical efficacy or result in toxicity.

The majority of therapeutic chiral drugs used as pure enantiomers are natural products. The high level of enantio selectivity of their biosynthesis excludes the possibility of the presence of enantiomeric impurities. In the case of synthetic chiral drugs, the racemates which are usually marketed, if the pure enantiomer is administered, the antipode is considered to be an impurity [9, 15, 16].

4. Control of impurities

According to theory, all impurities should be removed from the final product. In practice, it is generally not economically feasible to totally eliminate all impurities. However, the levels of all impurities should be controlled to provide a consistent product. In most cases, only low levels of impurities should be allowed, but in rare cases, even quite high levels of impurities are tolerated. In some cases, for example, biotechnology derived products such as macrocyclic antibiotics, or extracts of a botanical source such as some dietary supplements, the drug substance or active component contains multiple compounds, all of which have biological activity.

Most of the bulk pharmaceutical chemicals (BPCs) are obtained from various sources. Therefore, it is very crucial that impurities in BPCs be monitored and controlled very cautiously.

Various regulatory authorities [24-26] have described the maximum acceptable levels of the various impurities in the drug substance monograph or the specification.

4.1 Control of organic impurity

Generally, the reaction conditions are adjusted to reduce the amounts of by-products produced during each step of the reaction. The reaction conditions are tightly controlled to prevent varying levels of impurities, or even new impurities, from arising. High-quality starting materials may also lead to lower amounts of impurities in the final product when starting material impurities are carried through to drug substance impurities. Similarly, the use of high-quality reagents may help avoid the generation of unwanted by-products. Other options to reduce these impurities are the introduction of additional intermediate or final purification steps.

Table 1 Threshold for organic impurities

Maximum daily dosea


Reporting thresholdb,c


Identification thresholdc

Qualification thresholdc



0.10% or 1.0 mg/day intake (whichever is lower)

0.15% or 1.0 mg/day intake (whichever is lower)



0.05 %

0.05 %

Source: From ICH Q3A (R2).


aThe amount of drug substance administered per day.

bHigher reporting thresholds should be scientifically justified.

cLower thresholds can be appropriate if the impurity is unusually toxic.

4.2 Control of degradation impurity

This particular impurities covers degradation products of active substance, including reaction products with excipient tor container system [1, 5]. Degradation products observed in stability studies performed at recommended storage conditions should be identified, qualified and reported when following thresholds exceeded.

Table 2 Minimum degradation threshold for daily intake of drug product

Max daily dose

Qualification threshold

Identification threshold

Reporting threshold


1.0% or 50μg/TDI

1.0% or 5μg/TDI



#1.0% or 50μg/TDI

0.5% or 20μg/TDI



0.5% or 200μg/TDI

0.2% or 2mg/TDI



0.2% or 3mg/TDI

0.2% or 2mg/TDI



0.2% or 3mg/TDI

0.2% or 2mg/TDI






#Qualification threshold for 10mg/day is 0.5% /200μg TDI

4.3 Control of inorganic impurities

Oral/parenterals concentration limits (ppm) proposed for 14 metals in active substances or excipients; Pt, Pd, Ir, Rh, Ru, Os, Mo, V, Ni. Cr, Cu, Mn, Zn and Fe.

Metals divided into 3 classes:

Class 1: Metals of significant safety concern

Some metals are known or suspected human carcinogens, genotoxic and sometimes nongenotoxic animal carcinogens or possible causative agents of irreversible toxicity e.g. neurotoxicity or teratogenicity and few of them produces significant but reversible toxicity, examples: Ir, Pd, Pt, Ru, Rh, Os, Mo, V, Cr and Ni

Class 2: Metals with low safety concern

Trace metals required for nutritional purposes which is present in foodstuffs or readily available supplements examples: Cu and Mn.

Class 3: Metals with minimal safety concern

Metals omnipresent in the environment or plant and animal kingdoms are as such having high tolerable toxic value for human. Recommended nutritional intakes of ≥10mg/day. Examples are Fe and Zn.

Table 3 Limits of inorganic impurities in oral and parenterals exposure

Classes of metals

Oral exposure

Parenteral exposure









Class 1A: Pt, Pd

Class 1B: Ir, Rh, Ru, Os

Class 1C: Mo, Ni, Cr, V













Class 2: Cu, Mn





Class 3: Fe, Zn





* Separate limits for inhalation exposure to Pt, Cr(VI) and Ni;

** Subclass limit

PDE= Permitted Daily Exposure

4.4 Control of residual solvents

The toxicity of residual solvents was recognized by the regulatory agency in the world. At that time, each pharmacopeia used various guidelines [28-30] for residual solvents control in pharmaceutical products with different categories and acceptance limits. Moreover, only 6-8 residual solvents were controlled, which was far behind from the categories that were really used in pharmaceutical industry. Internationally, a standard guideline for control of residual solvents is needed to be established. Efforts were made to harmonize the guideline for residual solvents by ICH.

Table 4 Categories and limits of residual solvents initially controlled in pharmacopoeias

Organic volatile


Limit (ppm)

USP 22 edition

3rd Supplement



EP 3rd edition


1995 edition
















Ethylene oxide






























Organic solvents are constantly present in the pharmaceutical production processes. The pharmaceutical industry is one of the largest users of organic solvents per amount of the final product [31-33]. They are usually used at any step of the synthesis pathway of an active substance or excipients, and sometimes during the drug product formulation process. Residual solvents were evaluated for their possible risk to human health and placed into one of three classes as follows:

Class 1 solvents: Solvents to be avoided

Solvents in this class should not be employed in the manufacture of drug substances, excipients, and drug products because of their Known human carcinogens, strongly suspected human carcinogens, and environmental hazards. However, if their use is unavoidable in order to produce a drug product with a significant therapeutic advance, then their levels should be restricted as shown in Table 5, unless otherwise justified.

Table 5 Class 1 solvents in pharmaceutical products (solvents that should be avoided)


Concentration limit (ppm)





Carbon tetrachloride


Toxic and environmental hazard









Environmental hazard

Class 2 solvents: Solvents to be limited

Solvents in Table 6 should be limited in pharmaceutical products because of their inherent toxicity. PDEs are given to the nearest 0.1 mg/day, and concentrations are given to the nearest 10 ppm.

Table 6 Class 2 solvents in pharmaceutical products


PDE (mg/day)

Concentration limit (ppm)

















































Methylbutyl ketone






























*usually 60% m-xylene, 14% p-xylene, 9% o-xylene with 17% ethyl benzene

Class 3 solvents: Solvents with low toxic potential

Solvents in this class are having low toxic potential to man as these solvents have PDEs of 50 mg or more per day.

Examples of Class 3 solvents which should be limited by GMP are as under:

Acetic acid


Acetone Isobutyl acetate

Anisole Isopropyl acetate

1-Butanol Methyl acetate

2-Butanol 3-Methyl-1-butanol

Butyl acetate

Methyl ethyl ketone

Tert-Butylmethyl ether

Methyl isobutyl ketone



Dimethyl sulfoxide




Ethyl acetate


Ethyl ether


Ethyl formate

Propyl acetate

Formic acid

Other Class: Solvents for which no adequate toxicological data was found

This class lists additional solvents for which no adequate toxicological data available to generate a PDE. Some examples are:

1, 1-Diethoxypropane

Methyl isopropyl ketone

1, 1-Dimethoxymethane


2, 2-Dimethoxypropane

Petroleum ether


Trichloroacetic acid

Isopropyl ether

Trifluoroacetic acid

4.5 Control of genotoxic impurities

Assessment of genotoxic impurities and the determination of toxicological acceptable limits in active substances is a difficult issue because it is not addressed in sufficient detail in the existing ICH Q3 guidelines.

Determination of genotoxic effects of impurities without any data is very difficult for assessment. Therefore implementation of a generally applicable approach as defined by the Threshold of Toxicological Concern (TTC) is proposed. A TTC value of 1.5 μg/day intake of a genotoxic impurity is considered to be associated with an acceptable risk (excess cancer risk of <1 in 100,000 over a lifetime) for most pharmaceuticals.

Various classes of genotoxic impurities are as follows [31]:

Class 1: Impurities known to be genotoxic (mutagenic) and carcinogenic

Class 2: Impurities known to be genotoxic (mutagenic), but with unknown carcinogenic potential

Class 3: Alerting structure, unrelated to parent structure and of unknown genotoxic (mutagenic) potential

Class 4: Alerting structure, related to the parent API

Class 5: No alerting structure or indication of genotoxic potential

During the clinical trials some data that signifies the allowable daily intake is summarize in Table 7 [34, 35].

Table 7 Allowable daily intake for genotoxic impurities during clinical development

Duration of Exposure

≤1 month

>1-3 months

>3-6 months

>6-12 months

>12 months

Allowable Daily Intake (μg/day) for all Phases of development










Alternative maximum based on percentage of impurity in API





5. Isolation and characterization of Impurities

A number of methods can be used for isolating and characterizing impurities. The application of any given method depends on the nature of the impurity, i.e., its structure, physicochemical properties, and availability (the amount present in the original material from which it must be isolated). The following methods may be useful in this context.



Preparative Separations

5.1 Extraction

Extraction is one of the most useful methods for isolation of impurity. For this there are following methods can be helpful.

Liquid/ Solid Extraction

Supercritical Fluid Extraction

Liquid/ Liquid Extraction or solvent extraction

5.1.1 Liquid/ Solid Extraction or Solid-phase extraction (SPE)

Solid-liquid extraction allows soluble components to be removed from solids using a solvent. By using same principle a solvent is selected that would dissolve the impurity of interest present in the solid matrix. For example, if we want to determine salt in sand, we would simply use water to dissolve it and filter the solution, which on evaporation will produce salt in a reasonably pure form. If, on the other hand, other water-soluble impurities were present in the sand, then it would be necessary to select a different solvent or it would be necessary to further manipulate the solution.

It is noticed that, when we are talking about the impurities that are present in the pharmaceuticals, then it will be harder to isolate impurity in its pure form. We have to use organic solvent or mixtures of organic solvents to get deal with impurity. Furthermore, it is generally easier to volatilize the organic solvent at low temperatures in order to concentrate the impurity.

Solid-phase extraction (SPE) [36-38] normally done with use of cartridges and disks, available with a variety of stationary phases. Normal Phase SPE

Normal phase SPE procedures typically involve a polar analyte, a mid- to nonpolar matrix (e.g. acetone, chlorinated solvents, and hexane), and a polar stationary phase. Polar-functionalized bonded silicas (e.g. LC-CN, LC-NH2, and LC-Diol), and polar adsorption media (LC-Si, LC-Florisil, and LC-Alumina) typically are used under normal phase conditions. Retention of an analyte under normal phase conditions is primarily due to interactions between polar functional groups of the analyte and polar groups on the sorbent surface. Reversed phase SPE

Reversed phase separations involve a polar (usually aqueous) or moderately polar sample matrix (mobile phase) and a nonpolar stationary phase. The analyte of interest is typically mid- to nonpolar. Several SPE materials, such as the alkyl- or aryl-bonded silicas (LC-18, LC-8, LC-4, and LC-Ph) are in the reversed phase category. Ion Exchange SPE

Ion exchange SPE can be used for compounds that are charged when in a solution (usually aqueous, but sometimes organic). Anionic (negatively charged) compounds can be isolated on LC-SAX or LC-NH2 bonded silica cartridges. Cationic (positively charged) compounds are isolated by using LC-SCX or LC-WCX bonded silica cartridges. The primary retention mechanism of the compound is based mainly on the electrostatic attraction of the charged functional group on the compound to the charged group that is bonded to the silica surface. Anion Exchange SPE

The LC-SAX material is comprised of an aliphatic quaternary amine group that is bonded to the silica surface. A quaternary amine is a strong base and exists as a positively-charged cation that exchanges or attracts anionic species in the contacting solution - thus the term strong anion exchanger (SAX). Because it binds so strongly, LC-SAX is used to extract strong anions only when recovery or elution of the strong anion is not desired. Weak anions can be isolated and eluted from LC-SAX because they can be either displaced by an alternative anion or eluted with an acidic solution at a pH that neutralizes the weak anion.

The LC-NH2 SPE material that is used for normal phase separations is also considered to be a weak anion exchanger (WAX) when used with aqueous solutions. LC-NH2 is used to isolate and recover both strong and weak anions because the amine functional group on the silica surface can be neutralized in order to elute the strong or weak anion. Cation Exchange

The LC-SCX material contains silica with aliphatic sulfonic acid groups that are bonded to the surface. The sulfonic acid group is strongly acidic (pKa <1), and attracts or exchanges cationic species in a contacting solution - thus the term strong cation exchanger (SCX). The LC-WCX SPE material contains an aliphatic carboxylic acid group that is bonded to the silica surface. The carboxylic acid group is a weak anion, and is thus considered a weak cation exchanger (WCX). LC-WCX can be used to isolate and recover both strong and weak cations because the carboxylic acid functional group on the silica surface can be neutralized in order to elute the strong or weak cation.

5.1.2 Supercritical Fluid Extraction

In the field of supercritical fluid extraction (SFE) [39-41], various researchers proposed the use of supercritical carbon dioxide (CO2) for separating one component (the extractant) from another (the matrix).

Most Supercritical Fluid Extraction processes are quite simple. A sample is placed in the Sample thimble, and supercritical fluid is pumped through the thimble. The extraction of the soluble compounds is allowed to take place as the supercritical fluid passes into a collection trap through a restricting nozzle. The fluid is vented in the collection trap, allowing the solvent to either escape or be recompressed for future use. The material left behind in the collection trap is the product of the extraction. The critical pressure, critical temperature, and density of a few compounds used for SFE are given in Table 8.

Table 8 Solvents for SFE

















5.1.3 Liquid/ Liquid Extraction or solvent extraction

Liquid-Liquid extraction is a mass transfer operation in which a liquid solution (feed) is contacted with an immiscible or nearly immiscible liquid (solvent) or is a method to separate compounds based on their relative solubilities in two different immiscible liquids, that exhibits preferential affinity or selectivity towards one or more of the components in the feed [9, 42].

In this type of extraction process, a solute is distributed between two immiscible solvents. The extraction is controlled by distribution or partition coefficient11 which defines the ratio of concentration of the solute in two solvents, a and b:

Kd = Ca / Cb

Where Kd is the distribution coefficient or partition coefficient. By the use of this technique we can concentrate the solution containing impurity and thus we can easily detect the impurity.

5.2 Chromatography

Most recently, organic drug substance impurities are measured using chromatographic procedures as it gives more accurate result. In these procedures it should involve a separation mode that allows for the resolution of impurities from the drug substance and a detection mode that allows for the accurate measurement of impurities.

Owing to the polar and nonvolatile nature of most compounds used as medicinal drugs, reversed-phase HPLC is the most common technique for monitoring the drug substance and its impurities. GC is also used, particularly for residual solvents, and capillary electrophoresis (CE) has been introduced in more recent times. Some older methods use thin-layer chromatography (TLC), but use of this methodology for the quantitative measurement of impurities is not common.


The most common technique for monitoring impurities is HPLC with UV detection. Quantification of impurities is achieved by reference standard, when available, or by area percent or height percent relative to the parent compound [43, 44]. Important application for impurity identification with HPLC is by the use of MS as detector.

Recently, HPLC-MS is the popular technique for Structural elucidation and confirmation of impurities. During the synthesis it is necessary to identify the various type of impurities for maintaining the quality of the product. Because of its selectivity, sensitivity and compatibility with LC, LC-MS and the tandem techniques of MS-MS have become vital analytical techniques for the analysis of impurities present in various drugs and drug products and have become the method of first choice. The great advantage of the technique is that it provides molecular weight information and on some instruments structural fragments and empirical formulae on line, without the need for time-consuming isolation.

The coupling of high-performance liquid chromatography and NMR (LC-NMR) is a well-established and routine technique for the study of mixtures [44-46]. Coupling the chromatographic separation and the NMR detection effectively removes a preparative step, which can result in significant gains in efficiency, particularly in situations where there are mixtures with many components. If the identities of drug impurities are inconclusive when analyzed by LC-MS then LC-NMR can be used.


This is a valuable technique for isolation and purification of compounds because of its simplicity. No major equipment is required and method development is relatively easy [46-48]. The primary limitation is the small number of theoretical plates that are obtained with this method as compared to GC or HPLC.

Detection is frequently performed visually or by UV (e.g., 366 nm). The fluorescence-quenching substances absorbing UV light in the short-wavelength region can also be detected if the layer is impregnated with a fluorescent substance. Iodine vapors can help detect most organic substances. A number of techniques can be utilized to elute the material from the plates. The simplest method is scraping the sorbent containing the material of interest and transferring it to an appropriate extraction vessel, where it is extracted with a suitable solvent. Following filtration or centrifugation, the solvent is removed to collect the desired substance.


CE is not used much for impurity identification, but it offers the advantage that CE procedures can be employed when HPLC procedures have failed to adequately measure the impurities. CE is particularly most important for the separation of chiral compounds that is having closely related structures.