International Conference on Harmonization (ICH) has given guidelines  for impurity in a drug substance and according to ICH it is chemical entity which is not defined as drug as per D & C act and having impact on the purity of active pharmaceutical ingredient or drug substances.
Every Pharmaceutical industry defines impurity by their own words so we couldn't find exact definition of the impurity. In pharma world impurity can be identified by various terms that we will see later. Preparation of the drug substance or drug products various solvents are used. Remaining solvent or residual solvent that might be present in final product is often cited as organic volatile impurities (OVI)  and the impurities associated with the inactive pharmaceutical ingredients used in formulation or as additives or adjuvants 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. Recently British Pharmacopoeia (BP), United States Pharmacopeia (USP) and Indian Pharmacopoeia (IP) starting to incorporate allowable limit of impurities present in the drug substance or drug products [3-4].
Get your grade
or your money back
using our Essay Writing Service!
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 available with information about the structural characterization and Unidentified Impurity: An impurity can only identify only on qualitative analytical values (e.g. Peak area, Retention time etc.), for which structural information is not yet available.
Impurities present in new drug substances are covered under two aspects :
Chemistry Aspects classifies and identifies impurities, generate the report for different impurities, list various impurities present in any substances, and give 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, 10]:
Organic volatile impurities (OVI)
2.1 Intermediates: The compounds formed among the middle of synthesis for the coveted product 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 unintentional compounds gave rise during the reaction are commonly called by-products. Not all by products can be easily quantified, 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 relates to expected and non-expected products that may be formed 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 occur between various chemicals involved in reaction.
2.6 Related Products: As mentioned, Impurity that is similar to active pharmaceuticals termed as related products and thus tends to play down the negativity frequently attached to the term impurity. These products can have similar chemical structure and might have standardized biological activity; however, 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: These are the materials which may be present due to contamination or adulteration, not as outcomes of synthesis, are marked foreign substances.
Always on Time
Marked to Standard
2.9 Toxic Impurities: These impurities might affect the biological activity, even at very low concentration. So that it requires identification by qualitative or quantitative means.
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: Impurity having potency to have biological activity even at trace level are called as ordinary impurities.
2.12 Organic Volatile Impurities: Solvents that may remain in the drug substance should be considered as Organic Volatile Impurities (OVIs).
3. Classification of Impurities
Organic Impurities (Process-and Drug Related)
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 intermediates, by-products, and reagents, ligands, and catalysts used in 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 , for biological medicinal products, "starting materials means any substance prevailed from the human or plant or microorganisms or any alteration to the biological origins by means biotechnological cell constructs which will have tendency to formed drug product." So measures for controlling sourcing of starting materials or intermediates must be strong.
An intermediate is a substance that is produce in reaction vessel from the starting materials might undergo further chemical modification to provide final product.
3.1.2 By Product as mention in section 2.3 of the paper, desired product commonly called the "main product" and product that is unwanted but might be useful known as by products.
3.1.3 Degradation products are the compounds formed due to chemical changes in drug products during storage. Degradants may formed due to chemically interactions with other compounds or contaminants present in to the drug substances.
In certain cases, physical degradation i.e. degradation due to change in polymorphic state of molecule, aggregation of proteinaceous material due to heat or residual solvents, absorption of water, loss of water and so forth might be present. A degradation product can be determined by short- and long-term stability studies as per ICH. For example, in treatment for common cold formulations contain acetaminophen, phenylephrine hydrochloride, and chlorpheniramine maleate. Degradation product for the formulation was isolated and found to be adduct of phenylephrine and maleic acid . The definition of degradation product in accordance with the ICH guideline is "Any chemical change occur due to overreaction or over heating or changing in condition of solution i.e., change in pH, exposure to light, etc. or reaction of final product with container or closure or excipients used in making product." [1, 16].
3.1.4 Reagents, Ligands and Catalysts are seldom present in the final products [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 concluding products as impurities at minute level. For example, carbonic acid chloromethyl tetrahydro-pyran-4-yl ester (CCMTHP) , an alkylating agent was observed as an impurity in the synthesis of a Î² lactam drug substance.
3.1.5 Products of over-reaction formed when reactions for the synthesis are not selective as much as necessary, so non selective interaction at undesired site will produce incorrect compound. For e.g. last step for the synthesis of nanodralone decanoate is the decanoylation of the 17 -OH group. Enol compound 3, 17 Î²-dihydroxyestra-3, 5- diene disdecanoate was formed due to overreaction at the 4ene- 3 oxo group site [11, 16].
3.1.6 Contamination organic impurities are not related to drug but might unknowingly present in to the drugs. For example drug substances derived from plants, herbicides used to protect plants may be present, for example, diquat and glyphosate, or pesticides, and carbofuran and endrin, sprayed in the environment .
3.2 Inorganic Impurities
This Essay is
a Student's Work
This essay has been submitted by a student. This is not an example of the work written by our professional essay writers.Examples of our work
Inorganic impurities includes filter aids, color removing agents like charcoal, reaction rate modifiers (catalysts), ligands and heavy metals. For example, catalyst used in substitution reaction during the synthesis of the API or raw materials. These type of impurities might have toxic effects, so it should be removed or controlled to a minimum level. Batch-to-batch variation in impurity level suggest that manufacturing or synthesis process of the drug product is not controlled [15, 18-20].
Inorganic impurities normally known and identified are as follows:
3.2.1 Contamination inorganic impurities, these are unforeseen impurities found in final product. Contaminant impurities detected in drugs has been controlled in many ways. For example, previously used glass vessel for reaction is now replaced with acid/alkali resisted glass . So, impurity that might be present due to leaching from glass vessel is minimized to safer level.
3.2.2 Reagents, Ligands and Catalysts it is well define in section 3.1.4 of this paper, however catalysts used in decomposition of intermediates (iodide catalysts), and monodentate ligand such as chloride ions might be remain in final product as inorganic impurities.
3.3 Residual Solvents in pharmaceuticals are the volatile chemicals that are produced as a result of side reaction or used in the manufacturing of API or excipients, or in the formulation . Theoretically it can be removed from the final product but practically not. Therefore, it may be a vital parameter in the process for making drug product.
3.4 Polymorphic Forms, solid material which subsist in often two or more form or crystalline structure is said to be polymorphic. 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 of maleic acid has been came up when solution of caffeine and maleic acid (2:1) in chloroform is set aside to evaporate slowly .
3.5 Enantiomeric Impurities To determine purity of the chiral compound term enantiomeric excess (EE) is used. These impurities present in the drug is due to change in critical parameter of molecule during synthesis. Following equation is used to determine enantiomeric excess (EE):
EE = ((R-S)/(R+S)) Ã- 100
Where R and S stand for the individual optical isomer in the mixture (and R + S = 1).
These determinations are important particularly when we are talking about efficacy of the drug, because in case of optical isomers of a drugs only one isomer has therapeutic efficacy while rest of them have either toxic or have no effect at all [9, 15-16].
4. Control of impurities
According to theory, all impurities should be removed from the final product, but in practice, impurities cannot be entirely abolished from the product. So, for a quality product impurities should be kept within the limits. According to study carried out for impurity, very low amount of impurities in product should be admitted, however in special cases, rather high quantity of impurities are permitted. For example, biotechnologically derived products 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 controlling authority for impurity [24-26] have mention in monograph and specification about maximum tolerable limit.
4.1 Control of organic impurity
Most often, reduction in quantity of by-products in the reaction can be carried out by tightly controlled reaction conditions at crucial steps of the reaction to preclude new impurity or diverging level of impurity. Another approach to reduce quantity of impurity in final product is to use superior quality of starting materials. Likewise, the use of high-grade solvents also impart its effort to obviate the production of by-products or any unknown entity.
Table 1 Threshold for organic impurities
Maximum daily dosea
0.10% or 1.0 mg/day intake (whichever is lower)
0.15% or 1.0 mg/day intake (whichever is lower)
Source: 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
1.0% or 50Î¼g/TDI
1.0% or 5Î¼g/TDI
#1.0% or 50Î¼g/TDI
0.5% or 20Î¼g/TDI
0.5% or 200Î¼g/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% /200Î¼g 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 carcinogens or potential contributory agents which produce 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 â‰¥10mg/day. Examples are Fe and Zn.
Table 3 Limits of inorganic impurities in oral and parenterals exposure
Classes of metals
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
Various regulatory authorities have been discerned about toxicity of the residual solvent in the pharmaceutical world. At the most, various pharmaceutical guidelines [28-31] for control of residual solvents with different categories in pharmaceuticals gives acceptance limits (Table 4). In addition, Solvents which are used in pharmaceuticals, there are only few residual solvents were controlled. So globally there was need for a standard guideline to be established for control of residual solvents. Therefore harmonized guideline for control of residual solvents by ICH has been released.
Table 4 limits of initially controlled residual solvents in pharmacopoeias
USP 22 3rd edition
EP 3rd edition
For the pharmaceutical production, organic solvents are invariably remain present in the processes. The pharmaceutical industry is one of the largest users of organic solvents per amount of the final product [32-34]. The synthesis of an active or inactive pharmaceutical ingredient usually requires large amount of solvent, and sometimes during the drug product formulation process. Residual solvents placed in to following classes based on their toxic effect to human health:
Class 1 solvents: Solvents to be avoided
Solvents in class 1, due to its known carcinogenicity and hazardous to environment, it should not be utilized for manufacturing of active and inactive materials, and drug products. Even so, in any circumstances if we can avoid use of this class solvents, they should be limited in final product as shown in Table 5.
Table 5 Solvents in pharmaceutical products that should be avoided
Concentration limit (ppm)
Toxic and environmental hazard
Source: ICH Q3C Impurities: Residual Solvents
Class 2 solvents: Solvents to be limited
Solvents listed in Table 6 might be less toxic than class 1 solvents, but due to its inherent toxicity it should be limited as PDEs for this class is quite higher than class 1.
Table 6 Solvents in pharmaceutical products that should be limited
Concentration limit (ppm)
Source: ICH Q3C Impurities: Residual Solvents
*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.
Class 3 solvents which should be limited by GMP are as under (As per ICH Q3C):
1-Butanol Methyl acetate
Acetone Isobutyl acetate
Anisole Isopropyl acetate
Methyl ethyl ketone
Methyl isobutyl ketone
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 (Source: ICH Q3C):
Methyl isopropyl ketone
4.5 Control of genotoxic impurities
Existing ICH Q3 guidelines doesn't provide acceptable toxicological limits of genotoxic impurities in active pharmaceuticals.
Determination of genotoxic effects of impurities without any data is very difficult for assessing impurity. For most of the pharmaceuticals and other concern industries accepted the approach of Threshold of Toxicological Concern (TTC). This approach gives an acceptable risk value (A TTC value of 1.5 Î¼g/day intake) for intake of genotoxic impurity for most pharmaceuticals.
Various classes of genotoxic impurities are as follows :
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 [35, 36].
Table 7 Permissible limit for daily intake of genotoxic impurities on clinical development
Duration of Exposure
Allowable Daily Intake (Î¼g/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., structure, physical and chemical 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.
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. Same principle is applied over here to choose solvent for dissolving 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. Moreover, organic solvents are volatile in nature so we can evaporate them under low temperature to get concentrated product.
Solid-phase extraction (SPE) [37-39] normally done with use of cartridges and disks, available with a variety of stationary phases.
184.108.40.206 Normal Phase SPE
Theory involved in normal phase SPE generally require mid- to nonpolar solvent mixture (e.g. n-hexane, methylene dichloride, acetic acid, diethyl ether etc.), a polar substrate (e.g. drug molecule, excipients etc.) and a polar stationary phase. For normal phase various stationary phase materials are used. One of them is silica, which can be further modified with polar heads (e.g. Si-C4-CN, Si-C4-NH2, etc.). Another adsorbent used are Florisil, Alumina etc. mechanism involve in retention of substrate in normal phase SPE is principally interaction with polar analyte functional group and polar heads on the stationary phase.
220.127.116.11 Reversed phase SPE
Mechanism involved in reversed phase SPE requires polar mobile phase (e.g. Methanol, ethanol, water etc.) or semi polar solvent mixture and a nonpolar stationary phase. In reverse phase SPE modified silica use as stationary phase i.e. alkyl- or aryl-bonded silicas (Si-C-18, Si-C-8, Si-C-4, and Si-C-Ph).
18.104.22.168 Ion Exchange SPE
The main rationale of the Ion exchange SPE is to separate oppositely charged ions in solution. Different types of exchangers have been used to separate the charged moieties. Commercially available ion exchangers contains resinous part having amine or quaternary ammonium groups or other ionic group for separation of anionic or cationic compounds. Retention mechanism for the analyte is, at the exchanger surface diffusion of ion take place. This is depends on concentration of solution and degree of cross linking of ion exchangers.
22.214.171.124.1 Anion Exchange SPE
Material used in anion exchange SPE for stationary phase is having positively charged group (e.g. an aliphatic quaternary amine group or amino group). Positively charged group like quaternary amines are strong bases which will draws in anionic molecules in the solution and strongly attach to exchanged group. As it strongly binds to the anionic group, it is termed as strong anion exchanger (SAX). Due to its strong binding capacity, it is generally used when recovery of anion is no longer required. However anions that can be displaced by another anion, shall be eluted by changing the pH of the solution.
Stationary phase containing amino group, used in normal phase SPE can be used as weak anion exchanger (WAX). Advantage of WAX utilization for separation of species, we can isolate and recover strong as well as weak anions.
126.96.36.199.1 Cation Exchange
The material used for cation exchange are high molecular weight cross linked polymer having carboxylic, phenolic or aliphatic sulfonic acid groups. Amongst these groups sulfonic acid pulls in cationic species strongly present in solution so that termed as strong cation exchanger (SCX). Moreover, material contains carboxylic or phenolic group which is a weak anion used as weak cation exchanger (WCX). By the use of WCX, strong and weak cations can be easily isolate and recover.
5.1.2 Supercritical Fluid Extraction
In the field of supercritical fluid extraction (SFE) [40-42], various researchers proposed the use of supercritical carbon dioxide (CO2) as an extractant for separating various component.
Procedure involved in SFE is very convenient to novices. Sample thimble is used to handle sample through which supercritical fluid is being pumped. The extraction of the soluble compounds is allowed to take place as the supercritical fluid passes into a collection trap through a restricting nozzle. After passing through nozzle, it is recompressed by vented in the collection trap for future use. The material left behind in the collection trap is the product of the extraction. Characteristics of gases normally used SFE are given in Table 8.
Table 8 Solvents for SFE
5.1.3 Liquid/ Liquid Extraction or solvent extraction
In Liquid-Liquid extraction we separate the components based on their solubility in two slightly miscible or completely immiscible solvent, where mass transfer occurs at interface and components separates by affinity to the solvents [9, 45].
Partition coefficient plays important role in this extraction process, by which we can easily find amount of solute which is distributed between two immiscible solvents a and b:
Kd = Ca / Cb
Where, Kd is the distribution coefficient or partition coefficient.
Ca is the concentration of component in solvent a.
Cb is the concentration of component in solvent b.
By the use of this technique we can concentrate the solution containing impurity and thus we can easily detect the impurity.
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.
5.2.1 HPLC-MS or HPLC-NMR
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 [46, 47]. 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 LC-NMR have become absolutely necessary analytical techniques for the analysis of impurities present in various drugs and drug products and have become the method of first choice. As it provides some structural information about fragments, empirical formula and molecular weight, it becomes popular and advantageous method for the impurity analysis.
Coupling of LC and NMR [47-49] is recently attracts the researcher due to reduction in tedious preparative steps and substantially acquires higher efficiency and precision, where they are handling complex mixtures.
5.2.2 TLC (THIN LAYER CHROMATOGRAPHY)
For isolation and purification of compounds, TLC gains its importance because of its simplicity and utility. No major equipment is required and method development is relatively easy [49-51]. 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 used to recover the sample from plate. Most simple and convenient method for obtaining desired material is scraping the sorbent from the adsorbent site and shifting it to extraction vessel, where different solvents used for extraction of compound.
5.2.3 CE (CAPILLARY ELECTROPHORESIS)
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.