Impurity Profiling of Drug Substances in Pharmaceuticals : Pharmaceutical Guidelines

Impurity Profiling of Drug Substances in Pharmaceuticals

Everything about impurity profile as types impurities, impurity profiling of drugs, Classification of Impurities, Acceptance Criteria for Impurities in pharmaceutical analysis.
The description, characterization and quantitation of the identified and unidentified impurities present in new drug substances is known as impurity profile. A general scheme is set for the estimation of the impurity of bulk drug substances by the rational use of chromatographic, spectroscopic and analytical techniques. The impurity may be developed either during formulation or upon aging of both API’s and formulated API’s in medicines. 

The presence of these unwanted chemicals, even in small amount, may influence the efficacy and safety of the pharmaceutical products. Any material that affects the purity of the material of interest viz. active ingredient or drug substance. The impurities are not necessarily always inferior. From the standpoint of its usage, the drug substance is compromised in terms of purity even if it contains another material with superior pharmacological or toxicological properties. The various impurity profile of drug substances is discussed.


Impurity is defined as any substance coexisting with the original drug, such as starting material or intermediates or that is formed, due to any side reactions.
Impurity can be of three types: Impurities closely related to the product and coming from the chemical or from the biosynthetic route itself, Impurities formed due to spontaneous decomposition of the drug during the storage or on exposure to extreme conditions, or the precursors which may be present in the final product as impurities.

Impurities present in excess of 0.1% should be identified and quantified by selective methods. The suggested structures of the impurities can be synthesized and will provide the final evidence for their structures, previously determined by spectroscopic methods.  Therefore it is essential to know the structure of these impurities in the bulk drug in order to alter the reaction condition and to reduce the quantity of impurity to an acceptable level. Isolation, identification and quantification of impurities help us in various ways, to obtain a pure substance with less toxicity and, safety in drug therapy.
Quantitative determination of these impurities could be used as a method for the quality control and validation of drug substances. Regulatory authorities such as US FDA, cGMP, TGA, and MCA insist on the impurity profiling of drugs.
Impurities in new drug substances can be addressed from two perspectives, (1) the chemical aspect which includes classification and identification of impurities, report generation, listing of impurities in specifications, and a brief discussion of analytical procedures,(2) the safety aspect which includes, specific guidance for quantifying impurities, present ,substantially at lower levels, in  a drug substance used in clinical studies.

Classification of Impurities:-

Impurities can be classified as Organic impurities (process- and drug-related), Inorganic impurities and Residual Solvents. Organic impurities may arise during the manufacturing process or storage of the new drug substance which includes starting materials, by-products, intermediates, degradation products, reagents, ligands, and catalysts. Inorganic impurities include reagents, ligands & catalysts, heavy metals or other residual metals, inorganic salts, filter aids, charcoal etc. Residual Solvents are organic or inorganic liquids used during the manufacturing process. Since these are, generally of known toxicity, the selection of appropriate controls can be accomplished easily.

Organic Impurities:-

The actual and potential impurities most likely to arise during the synthesis, purification, and storage of the drug substance should be summarized, based on a sound scientific appraisal of the chemical reactions involved in the synthesis, impurities associated with raw materials that could contribute to the impurity profile of the drug substance.
The laboratory studies conducted to detect impurities in the drug substance, which include test results of materials manufactured during the development process and batches from the commercial processes. The impurity profile of the drug lots, intended for marketing should be compared with those used in development.
The spectroscopic studies (NMR, IR, MS etc. ) conducted to characterize the structure of actual impurities present in the drug substance above an apparent level of 0.1% (e.g., calculated using the response factor of the drug substance) should be described. All recurring impurities above an apparent level of 0.1% in batches manufactured by the proposed commercial process should be identified. of these studies.

By-Products Impurities:-

In synthetic organic chemistry, getting a single end product with 100% yield is very rare as there is always a chance of having some by-products along with desired end product. Eg. in case of paracetamol bulk, diacetylated paracetamol may form as a by-product.

Degraded Products Impurities:-

Impurities can also be formed by degradation of the end product during manufacturing of bulk drugs. Such types of impurities are common in the medicines as they result from improper storage of formulation. The degradation of penicillins and cephalosporins is a well-known example of degradation product4. The presence of a ß-lactam ring, as well as that of an α-amino group in the C6/C7 side chain, plays a critical role in their degradation. In general, an individual API may contain all of the above-mentioned types of organic impurities at various levels ranging from negligible to significant amount. As the organic impurities are the most common product related impurity as well as the process related impurity, it is the responsibility of both the manufacturers of APIs and formulators to take care of these impurities according to ICH guidelines or compendia.

Organic Volatile Impurities:-

Organic volatile chemicals are produced in the manufacture of drug substances or excipients or in the preparation of drug products, they are volatile in nature and by themselves get removed out at time of storage or processing.

Inorganic Impurities:-

Inorganic impurities are normally detected and quantified using Pharmacopeial or other appropriate standards. Carryover of catalysts to the drug substance should be evaluated during development.

Heavy Metals Impurities:-

The main source of heavy metals is water which is generally used in different manufacturing processes, where acidification or acid hydrolysis takes place. These impurities of heavy metals can easily be avoided by using demineralized water and glass-lined reactors.

Other Materials (Filter Aids, Charcoal) Impurities:-

The filters or filtering aids such as centrifuge bags are routinely used in the bulk drugs manufacturing plants and in many cases, activated carbon is also used which also act as a source of impurity. Therefore regular monitoring of fibers and black particles in the bulk drugs is essential so as to avoid their contaminations.

Residual Solvents:-

Residual solvents are divided into 3 classes:-
Solvents of class I included benzene (Class I, 2 ppm limit) and carbon tetrachloride (Class I, 4 ppm limit). These are to be avoiding because of their carcinogenic, toxicity effects.
On the other hand, solvents of class II includes methylene chloride (600 ppm), methanol (3000 ppm), pyridine (200 ppm), toluene (890 ppm), N, N-dimethylformamide (880 ppm) and acetonitrile (410 ppm).These are most commonly used solvents.
Solvents of class III includes acetic acid, acetone, isopropyl alcohol, butanol, ethanol and ethyl acetate have permitted daily exposures of 50 mg or less per day. In this regard, ICH guidelines for limits should be strictly followed.
Acceptance criteria should be based on Pharmacopeial standards, or ICH guidelines or known safety data depends on the dose, duration of treatment, and route of administration.

Ordinary Impurities:-

Ordinary impurities are found in bulk pharmaceutical chemicals that are innocuous by virtue of having no significance on the biological activity of the drug substance. These impurities may arise out of the synthesis, preparation or degradation of chemicals.

Starting Materials or Intermediates Impurities:-

These are the common type of impurities which are found in almost every API unless a proper care is taken in every step involved throughout the multi-step synthesis of the drug product. Although the end products are always washed with solvents but there are chances of having the residual of unreacted starting materials unless the manufacturers are very careful about the impurities. Eg. in paracetamol bulk, there is a limit test for p-aminophenol, which could be a starting material for some manufacturer or be an intermediate for another.

Process-Based Impurities:-

Apart from bulk drug-related impurities, the formulated form of API may contain impurities that are formed in various ways during the processing of the drug like impurity obtained due to method defect, impurity obtained due to environmental defect, impurity obtained due to factor defect, impurities formed due to mutual interaction among ingredients and impurities formed due to functional group reaction degradation.

Impurity Obtained Due To Method Defect:-

Impurity related to the method may be caused by improper manufacturing processes which don’t follow the optimized conditions like pressure, the temperature during processing.9 Eg. 1-(2, 6-dichlorophenyl) indolin-2-one is formed as an impurity in the production of a parenteral dosage form of diclofenac sodium10, if it is terminally sterilized by autoclave. It was the condition of the autoclave method (i.e., 123 + 2°C) that enforced the intramolecular cyclic reaction of diclofenac sodium forming the indolinone derivative and sodium hydroxide. Formation of such impurity also depends on the initial pH of the formulation.

Impurity Obtained Due To Environmental Defect:-

The primary environmental factors that can reduce stability include the following: Exposures to adverse temperatures, there are many APIs that are labile to heat or tropical temperatures. Eg. vitamins as drug substances are very heat-sensitive and get degraded frequently leading to loss of potency in vitamin products, especially in liquid formulations. Light-especially UV light causes initiating a large number of systems that are photolyzed; and causes the formation of free radicals as end products. Several studies have reported that ergometrine, as well as methyl ergometrine injections, are unstable under tropical conditions such as light and heat.

Impurity Obtained Due To Factor Defect:-

Although the pharmaceutical companies perform pre-formulation studies, including stability studies before marketing the products, sometimes the dosage form factors influence the drug stability and force the company to recall the product. Fluocinonide Topical Solution USP, 0.05%, (Teva Pharmaceuticals USA, Inc., Sellersville, Pennsylvania) in 60-ml bottles were recalled in the United States because of degradation/impurities leading to sub-potency. In general, liquid dosage forms are very much susceptible to both degradation and microbiological contaminations. In this regard, water content, pH of the solution/suspension, compatibility of anions and cations, mutual interactions of ingredients and the primary container are also some critical factors. Microbiological growth resulting from the growth of bacteria, fungi and yeast in a humid and warm environment may result in oral liquid products that are unusable and unsafe for human consumption.
Impurities Formed Due To Mutual Interaction Among Ingredients:-
Most vitamins are very labile and on aging, they pose a problem of instability in different dosage forms, especially in liquid dosage forms. Degradation of vitamins such as folic acid, pantothenic acid, cyanocobalamin and thiamine does not give toxic impurities. However, the potency of active ingredients drops below pharmacopoeial specifications. Because of mutual interaction, the presence of nicotinamide in a formulation containing 4 vitamins (nicotinamide, pyridoxine, riboflavin, and thiamine) causes the degradation of thiamine to a sub-standard level within a 1-year shelf-life of vitamin B-complex injections. The marketed samples of vitamin B-complex injections were found to have a pH in the range of 2.8-4.0. The custom-made formulation of a simple distilled-water vehicle and in a typical formulated vehicle that included disodium edetate and benzyl alcohol was also investigated and similar mutual interaction causing degradation was also observed.

Impurities Formed Due To Functional Group Reaction Degradation:-

Degradation products of drugs are considered to be transformation products of the drug substance formed due to the effect of heat, solvents (including high and low pH), oxidizing agents, other chemical reagents, humidity and light.


Hydrolysis is a common phenomenon for ester and amide type of drugs, especially in liquid dosage forms. Certain drugs which undergo hydrolysis are benzylpenicillin, barbitol, chloramphenicol, chlordiazepoxide, lincomycin, and oxazepam.


The oxidative decomposition of pharmaceutical compounds is responsible for the instability of a considerable number of pharmaceutical preparations. These reactions are mediated either by free radicals or by molecular oxygen. Drugs which undergo oxidative degradation are hydrocortisone, methotrexate, adinazolam, hydroxyl group directly bonded to an aromatic ring (eg, phenol derivatives such as catecholamines and morphine), conjugated dienes (eg, vitamin A and unsaturated free fatty acids), heterocyclic aromatic rings, nitroso and nitrite derivatives and aldehydes (eg, flavorings).


Some dissolved carboxylic acids such as p-amino salicylic acid, lose carbon dioxide from the carboxyl group when heated. Decarboxylation also occurred in the case of photoreaction of rufloxacin.


Pharmaceutical products are bared to light while being held improperly in pharmacy shops or hospitals, or when held by the consumer for imminent use. Drugs which undergo photolytic cleavage are ergometrine, nifedipine, nitroprusside, riboflavin and phenothiazines are very labile to photo-oxidation. In vulnerable compounds, photochemical energy creates free radical intermediates, which can accomplish by chain reactions. Most compounds degrade as solutions when bared to high energy UV exposure. Fluoroquinolones antibiotics are found to be susceptible to photolytic cleavage. In ciprofloxacin eye drops preparation (0.3%), sunlight induces photocleavage reaction producing ethylenediamine analog of ciprofloxacin.


Flow chart of impurity profiling
The procedure of impurity profiling begins with the detection of the impurities using the thin-layer chromatogram, high-performance liquid chromatogram or gas chromatogram. Procurement of standard impurity samples from the synthetic organic chemists which include, the last intermediate of the synthesis, products of predictable side reaction, degradation products if any etc
In the case of unsuccessful identification with standard samples, the most reasonable way to determine the structure of impurity starts with the investigation of the UV spectra, easily obtainable with the aid of the diode-array detector in the case of HPLC and the quantification with the help of densitometer.
 In exceptional cases, with full knowledge of the synthesis of drug martial, the structure of the impurity can be generated on the basis of NMR spectral data.
If the information obtained from the UV spectrum is not sufficient, the next step in the procedure of impurity profiling is to take the mass spectrum of the impurity. The major disadvantage of this method is the volatility and thermal stability problems of the impurities. The use of derivatization reactions widely used in GC/MS analysis is problematic because the side-products of the derivatization reaction can be confused with the impurities.
The next step in the impurity profiling is the synthesis of the material (impurity standard)  with the proposed structure. The retention and spectral matching of the synthesized material with the impurity in question is carried out as outlined above.
The possibilities of spectroscopic techniques in drug impurity profiling without chromatographic separation are also worth mentioning. Spectra obtained by using high-resolution, highly sensitive NMR spectrometers and mass spectrometers with APCI /ESI facilities are suitable to provide a fingerprint picture regarding the purity of the sample.


It is now getting important critical attention from regulatory authorities. The different pharmacopeias, such as the British Pharmacopoeia (BP), the United States Pharmacopoeia (USP) and the Indian Pharmacopoeia (IP) are slowly incorporating limits to allowable levels of impurities present in the APIs or formulations. Also, the International Conference on Harmonization (ICH) has published certain guidelines on impurities in drug substances, products and residual solvents. There is a significant demand for the impurity reference standards and the API reference standards for both regulatory authorities and pharmaceutical companies. According to ICH guidelines on impurities in new drug products, identification of impurities below 0.1% level is not considered to be necessary, unless potential impurities are expected to be unusually potent or toxic.

Limits for impurities in degraded products of drugs:

Degradation product impurity                                 Limits 
Each identified degraded product                          Not more than 1.0 percent
Each unidentified degraded product                      Not more than 0.5 percent
Total degraded products                                        Not more than 2.0 percent

Specifications should be set for identified and unidentified impurities expected to be present in the drug substances and drug products over the period of intended use and under recommended storage conditions. These impurities are known as specified impurities and they should be individually listed in the specifications. Stability studies, chemical development studies and routine batch analyses can be used to establish impurities likely to occur in the commercial new drug substances and new drug products. A general specification limit of not more than 0.1% for any unspecified impurity should also be included. A rationale for why impurities were included or excluded from the specifications for the drug substance and drug products should be provided. Limits for impurities should be set no higher than the level which can be justified by safety data and unless safety data indicate otherwise, no lower than the level achievable by the manufacturing process and the analytical capability.

Acceptance Criteria for Impurities:-

For newly synthesized drug substances, the specification should include acceptance criteria for impurities. Stability studies, chemical development studies, and routine batch analyses can be used to predict those impurities likely to occur in the commercial product.
A rationale for the inclusion or exclusion of impurities in the specification should include a discussion of the impurity profiles observed in batches under consideration, together with a consideration of the impurity profile of material manufactured by the proposed commercial process. For impurities known to be unusually potent or to produce toxic or unexpected pharmacological effects, the quantitation or detection limit of the analytical methods should commensurate with the level at which the impurities need to be controlled.  Appropriate qualitative analytical descriptive label included in the specification of unidentified impurities. A general acceptance criterion of not more than 0.1 % for any unspecified impurity should be included.
Acceptance criteria should be set, based on data generated on actual batches of the drug substance, allowing sufficient latitude to deal with normal manufacturing and analytical variation, and the stability characteristics of the drug substance. Although normal manufacturing variations are expected, significant variation in batch-to-batch impurity levels could indicate that the manufacturing process of the drug substance is not adequately controlled and validated.
The acceptance criteria should include limits for organic impurities; each specified identified impurity, each specified unidentified impurity at or above 0.1%, and any unspecified impurity, with a limit of not more than 0.1%, total impurities, residual solvents and inorganic impurities.

Submitted By :-
St. Mary’s College of Pharmacy

Ankur Choudhary is India's first professional pharmaceutical blogger, author and founder of Pharmaceutical Guidelines, a widely-read pharmaceutical blog since 2008. Sign-up for the free email updates for your daily dose of pharmaceutical tips.
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