Importance Of Impurity Profiling In Drug Substances

Sponsored Links



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 various parameters to be fulfilled in an impurity profile of drug substances are 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 inclinical 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 include 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.

Rationale For The Reporting And Control Of Impurities

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 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.

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.

Residual Solvents

The control of residues of solvents used in the manufacturing process for the drug substance should be discussed. 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.

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.

General Scheme for Drug Impurity Profiling

General Scheme for Drug 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, 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.

Analytical Procedures:

Method Development

Method development usually requires the choice of columns, mobile phase, detectors, and method of quantitation etc. The factors to be considered for the method development are the following,

 Existing method may be inaccurate, artifact, or contamination prone, or they may be unreliable (have poor accuracy or precision). Existing method may be too expensive, time consuming or energy intensive, or they may not be easily automated.

Existing methods may not provide adequate sensitive or analyte selectivity in samples of interest. Newer instrumentation techniques may have evolved which can provide opportunities for improved methods, including improved analyte identification or detection limits, greater accuracy or precision and better returns on investments.

Validation Of Analytical Methods

The validation process involves confirmation or establishing a developed method by laboratory studies, procedures, systems, which can give accurate and reproducible result for an intended analytical application in a proven and established range.

The performance characteristics of the method (accuracy, precision, sensitivity, ruggedness, etc) should meet the requirements of the intended analytical applications and the process can or provide documented evidence that the system or   procedure do what it is intended for in a systematic, precise and reliable manner.

According to ICH, typical analytical performance characteristics that should be considered in the validation of all the types of methods are: 

Accuracy, Precision, Specificity, Limit of detection, Limit ofquantification, Linearity, Range Robustness, Ruggedness, Sensitivity, Repeatability, Reproducibility etc .

The limit of detection (LOD) of an individual analytical method is the lowest concentration/amount of analyte in a sample that the method can detect but not necessarily quantify under the stated experimental conditions. The LOD will not only depend on the procedure of analysis, also on the type of the instrument.

The limit of quantification of an individual analytical method is the lowest concentration/ amount of an analyte in a sample, which can be quantitatively determined with suitable precision and accuracy under stated experimental conditions.  The quantification limit is used particularly for the determination of impurities and degradation products.

The linearity of an analytical method is its ability (within a given range) to obtain test results, which are directly proportional to the concentration (amount) of analyte in the sample.

The range of an analytical method is the between the upper and lower concentration (amounts) of analyte the sample (including those concentrations) for which it has been demonstrated that analytical procedure has a suitable level of precision, accuracy and linearity.

Robustness is the measure of the analytical method to remain unaffected by small, but deliberate variations in method parameters. It provides an indication of its reliability during normal usage.   

The ruggedness is the degree of reproducibility of test results obtained by analyzing the same sample under variety of normal test conditions such as different analyst, instruments, days, reagents and, columns. The comparison of reproducibility of test results to the precision of assay is the direct measure of ruggedness of the method.


Impurity profiling is very important during the synthesis of drug substances and manufacture of dosage forms, as it can provide crucial data regarding the toxicity, safety, various limits of detection, and limits of quantitation, of several organic and inorganic impurities, usually accompany with bulk drugs and finished products.

An accurate method development and validation of the procedures make the impurity profiling task easy.


1.United States Pharmacopoeia XXIV, United States Pharmacopoeial Convention, INC, Rockville;2000.

2.Gorog S, Babjak M, Balogh G, Brlik J, Csehi A, Dravecz F, Drug impurity profiling strategies. Talanta 1977;44: 1517-26.

3.Micheael E. Swartz, Ira S. Krull. Analytical method development and validation. 3rd ed. Prentice-Hall of India Private Limited, New Delhi; 1992.

4.Zyban,welbutrin   A short one pot synthesis of bupropion HCL J.Chemical education 2000;77(11),1479

5.Juliun A Vida. Burgers Medicinal Chemistry and Drug Discovery Volume 3: Therapeutic Agents. 5th ed. John Willy and Sons, INC, New York;1997.

6. Klaus Flory. Analytical Profiles of Drug Substances Volume 16. 5th Edn. Academic press, INC, New York; 1985.

7.Ryan, Timothy W. HPLC impurity profile analysis of Pharmaceutical substances using UV photodiode array detection. Anal.Lett 1998; 31(4): 651-58.

8.Reddy KVSRK, Babu JM, Kumar YR, Reddy SV, Kumar MK, Eswaraiah S, et al. Impurity profile study of Loratadine. J. Pharm. Biomed. Anal. 2003; 32 (1): 29-39.

9.Gorog S, Herenyi B. The use of high performance liquid chromatography with diode-array UV detection for estimating impurity profiles of steroid drugs. Journal of Chromatography.1987; 400: 177-86.

10.Argekar AP, Sunil Shah. Simultaneous determination of Clonazepam and its impurities in active substances and its dosage forms by RPLC. Indian Drugs ,1997; 39(6): 342-47.

11.Radhakrishna T, Satyanrayana J, Satyanrayana A. HPLC method for the determination of Celecoxib and its related impurities. Indian Drugs 2002; 40(3): 166-171.

12.Gorog S, Bihari M, Csizer E, Dravetz F, Gazdag M, Herenyi B, et al. Estimation of impurity profiles of drugs and related materials. Part 14: the role of HPLC/diode- array UV spectroscopy in the identification of minor components in various matrixes. J. Biomed. Anal 1995; 14: 85-92.

13.  Vaidya VV, Khanolkar, Mahesh, Gadre JN. Generation of trace impurity profiles for Tolnafate by reverse phase high performance liquid chromatography. Indian Drugs 2000; 37(11): 521-23.

14.White, Gregory, Katona, Thomas, Zodda, Julius P. et al. Determination of the impurity profile of g-Cyclodextrin by High-performance liquid chromatography. J. Chromatogr 1992; 625(2): 157-61.

15.Marika Kamberi, Christopher M Riley, Xiaoyan Ma, Chen-Wen C Huang. A validated, sensitive HPLC method for the determination of trace impurities in Acetaminophen drug substance. J. Pharm. Biomed. Anal. 2004; 34 (1): 123-28.

16.NageswaraRao R, Nagaraju V. An overview of the recent trends in development of HPLC methods for determination of impurities in drugs. J. Pharm. Biomed. Anal 2003; 33 (3): 84-90.

17.Halmas Zs, Szantay Cs, Brlik J, Csehi A, Varga K, Horvath P, et al. Estimation of impurity profiles of drugs and related materials. Part 15. Identification of minor impurities in Cimetidine. Journal of Bio Anal. 1996; 15: 1-5.

18.Velupula Nagaraju, Dasari Sreenath, Jammula Tirumala Rao, Ramisetti Nageswara Rao. Separation and determination of Synthetic impurities of Sildenafil by Reverse phase liquid chromatography. Analytical Sciences 2003; 19: 1007-11.

19.Randall Baker, Herenyi B. HPLC–ultraviolet method for the simultaneous determination of potential synthetic and hydrolytic impurities in Urapidil fumarate. Journal of Chromatogr. 1987; 393:447-53.

20.Olsen B A, Baertschi S W, Riggin R M.Multidimensional evaluation of impurity profiles for generic Cephalexin and Cofactors antibiotics.J. Chromatogr 1993; 648(1): 165-73.

21.Andrei Medvedovici, Florin Albu, Alexandru Farca, Victor David. Validated HPLC determination of 2-(dimethylamino) methyl] cyclohexanone, an impurity in Tramadol, using a precolumn derivatization reaction with 2,4-dinitrophenylhydrazine. J.Pharm. Biomed. Anal 2004; 34 (2): 111-15.

22.Ryan, Timothy W. Identification of four process-related impurities in the bulk drug Butalbital using HPLC-UV photodiode array detection, particle beam MS, and NMR. Anal. Lett 1998; 31(14): 2447-56.

23.Wirth David D, Miller, Marybeth S, Boini, Sathish K, Koenig, Thomas M. Identification and Comparison of Impurities in Fluoxetine Hydrochloride Synthesized by Seven Different Routes. American Chemical Society 2000; 4(6): 513-19.

24.Marshall Sittig, Pharmaceutical Manufacturing Encyclopedia Volume II. 2nd Edn. Noyes U.S.A; 1985.

25.Owies, Laila, Katona, Thomas, Monteferrante, Jo Anna, et al. Determination of the impurity profile of 1, 2-cyclohexanedione dioxime by high-performance liquid chromatography. J. Chromatogr.  1996; 719(2): 307-13.

26.Gazdag M, Babjik M, Brlik J, Maho S, Tuba Z, Gorog S. Estimation of impurity profiles of drugs and related materials. Part 18. Impurities and degradation products of mazipredone. J. Pharm. Biomed. Anal 1998; 17(6,7): 1029-36.

27.Loboz K K ,Gross AS , Roy J HPLC  determinattion of Bupropion in human plasma J .of analytical technology& Biomed. Life sciences. 2005;823 (2):115-21

28.Gorog S, Herenyi B, Renyei M. Estimation of impurity profiles of drugs and related materials. Part 9: HPLC investigation of flumecinol. J. Pharm. Biomed. Anal 1992; 10(10-12): 831-5.     

About Authors:

Dr. Madhu.C.Divakar


Pincipal,Chemist College of Pharmaceutical sciences and Research, Varikoli P.O, Ernakulam, Kochi, Kerala, India, 682308



For correspondence
Chemist College of Pharmaceutical sciences and Research, Varikoli P.O, Ernakulam, Kochi, Kerala, India, 682308

Nimmy Varghese

Nimmy Varghese

Chemist College of Pharmaceutical sciences and Research, Varikoli P.O, Ernakulam, Kochi, Kerala, India, 682308



Chemist College of Pharmaceutical sciences and Research, Varikoli P.O, Ernakulam, Kochi, Kerala, India, 682308



Chemist College of Pharmaceutical sciences and Research, Varikoli P.O, Ernakulam, Kochi, Kerala, India, 682308

Volumes and Issues: 

Similar Entries