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  • Synthesis, Isolation, & Characterisation of Impurities


    Synthesis of impurity with the predicted structure is tough and it may involve multistep synthesis. Hence for the synthesis of complex structures impurity, systematic and thoughtful consideration was involved. In-depth route design and scouting exercise performed. A systematic literature search using search tools such as SciFinder, Beilstein, and online access to most of the international journals in chemistry.

    In regular instances, the synthesis of a structure proposed on the basis of UV and mass spectrum is easy and requires less time consuming than its preparation by preparative HPLC.

    Following is the detailed requirement of the gram-scale synthesis of the impurity:

    • Once actual synthesis and full analytical study are completed then it will be matched by the chromatographic techniques with the known impurity found in the drug substance.
    • Detail study must be carried out of actual impurities by identification on the basis of chromatographic and online spectral matching with known potential impurities.
    • After matching of structure to the suggested structure, specify it with detailed evidence.
    • The spectra of synthesized material are characteristically improved than that of those taken from small isolated samples. After verification, the identity of isolated impurity spectra can be used for record-keeping.
    • This synthesized impurity can be used as the impurity standard. Selective analytical methods will be developed for the quantitative determination of the impurity. This method is routinely used for each batch analysis of the impurity standard.
    • The synthesized impurity can be subjected to toxicological study.
    • In some cases, the synthesis of impurity can be very challenging. In these exceptional cases, the synthesis can be attained by using preparative HPLC.


    Most of the time it is necessary to isolate impurities. But if the instrumental methods are used, isolation of impurities is avoided as it directly characterizes the impurities. Generally, chromatographic and non-chromatographic techniques are used for the isolation of impurities prior to their characterization. The term ‘chromatographic reactor’ refers to the use of an analytical-scale column as both a flow-through reactor and simultaneously, as a separation medium for the reactant(s) and product(s).

    A list of methods that can be used for the isolation of impurities is given below:

    • Solid-phase extraction methods
    • Liquid-liquid extraction methods
    • Accelerated solvent extraction methods
    • Supercritical fluid extraction
    • Column chromatography
    • Flash chromatography
    • Thin Layer Chromatography
    • Gas Chromatography
    • High-performance Liquid Chromatography
    • High-Performance Thin-Layer Chromatography
    • Capillary electrophoresis (CE)
    • Supercritical fluid chromatography (SFC)


    The reliability and mainly the accuracy of the quantitative determination of impurities extremely depend on whether an impurity standard is available or not. The pharmacopeias usually do not consider the availability of impurity standards and prescribe non-selective, general quantitative HPLC or GC methods for this purpose.

    Following are the highly sophisticated analytical techniques such as MS attached to a GC or HPLC used for the quantitative determination of impurities in drugs:

    • HPLC-MS
    • HPLC-MS-MS
    • GC-MS
    • NMR
    • Thermogravimetric Analyser
    • Differential Scanning Calorimeter
    • X-Ray Powder Diffractometer


    • ICH, Q3A(R2) Impurities in New Drug Substances,
    • ICH, Q3B(R2) Impurities in New Drug Products,
    • ICH, Q3C (R6) Impurities: Guideline for Residual Solvents,
    • S. Gorog, M. Babjak, and G. Balogh. Drug impurity profiling strategies, Talanta
    • Rao N.R., Mani Kiran S.S., Prasanthi N.L. Pharmaceutical Impurities: An Overview. Indian J.Pharm.Educ. Res,
    • S. Ahuja, K. M. Alsante. Handbook of Isolation and Characterization of Impurities in Pharmaceuticals, Vol. 5, Separation Science and Technology, Academic Press,
    • S. Ahuja. Impurities Evaluation of Pharmaceuticals, Marcel Dekker, Inc. New York,
    • J. Roy. Pharmaceutical Impurities–a mini review, AAPS PharmSciTech

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