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  • Drug Metabolite Impurities, Pathways & Analytical Techniques

    Metabolites are the intermediate products of metabolic reactions catalyzed by various enzymes that naturally occur within cells. The term metabolite is usually used for small molecules.

    Metabolite forms may be integral or pharmacological and formed during the natural biochemical process of degrading and eliminating the compounds from chemical compounds. It is important to know how the drug products are metabolized and what are its probable side effects. e.g. Sugars are metabolites of fructose or glucose, which are both present in the metabolic pathways.

    Regulatory Guidance documents ICH Q3A (R2) and ICH Q3B (R2) state that “impurities that are also significant metabolites present in animal and/or human studies are generally considered qualified”. But no guidance is provided regarding data requirements for qualification.

    Metabolite Pathway:

    Metabolite impurities are by-products formed in the body after ingesting drug products.

    • Primary metabolites are formed directly from the parental drug. The reactions include oxidation, reduction, and hydrolysis. Enzymes that are catalyzed reactions include microsomal monooxygenases and peroxidases, cytosolic and mitochondrial oxidases, reductases, and hydrolytic enzymes. Primary oxidation enzymes are monoamine oxidase (MAO), diamine oxidase (DAO), cyclooxygenase (COX), alcohol dehydrogenase (ADH), aldehyde dehydrogenase (ALDH), and molybdenum hydroxylase. These reactions are responsible for the enrichment of drug hydrophilicity and subsequently help the excretion.
    • Secondary or successive metabolites are formed from one or more primary metabolites. Secondary reactions represent conjugating reactions and mainly further increase the hydrophilicity and enable the excretion of metabolites from the body. Secondary reactions are catalyzed by UDP glucuronosyltransferases (UGT), sulfotransferases (SULT), N-acetyltransferases (NAT), glutathione-S-transferases (GST), and methyltransferases.

    Some metabolites are formed as impurities during the drug substance or drug product development process or storage. Control of these process-related metabolite impurities in the final drug substance may not be necessary if control of other metabolites has already occurred and been taken into consideration.

    Analytical Techniques for Metabolite Identification & Quantification:

    Detection limits of the drugs and their metabolites are very much important. HPLC, HPLC-MS/MS is the main analytical method for regular analysis as well as pharmacokinetic and metabolism studies due to its inherent specificity, sensitivity, and speed.

    Metabolite quantification is essential when the metabolite is toxic or pharmacologically active or when the concentration of metabolite reaches or exceeds the parent drug concentration in plasma. Following is the list of sophisticated analytical instruments used for the identification and quantification

    HPLC with UV, Fluorescence or Electrochemical Detection, HPLC-MS/MS, MS with high-resolution detector Triple TOF and QTrap, Nuclear magnetic resonance (NMR) spectroscopy, Capillary electrophoresis (CE), GC-MS, etc.

    Metabolic changes in drugs can be responsible for problems associated with their bioavailability, interindividual variability, drug-drug interactions, pharmacologic activity, or toxicity. An effective analytical method for the identification, quantification and monitoring of drug metabolism is to be developed and validated. HPLC with MS is a very much important analytical technique for the identification and quantification of drug metabolites.


    1. Guidance for Industry ANDAs: Impurities in Drug Products, U.S. Department of Health and Human Services Food and Drug Administration Centre for Drug Evaluation and Research (CDER) November 2010 OGD,
    2. Handbook of Drug Metabolism, edited by Thomas F. Woolf, Parke-Davis Pharmaceutical Research, Ann Arbor, Michigan,
    3. ICH, Q3A(R2) Impurities in New Drug Substances,
    4. ICH, Q3B(R2) Impurities in New Drug Products,
    5. Schadt, S., et al., 2018. A decade in the MIST: Learning from investigations of drug metabolites in drug development under the “Metabolites in safety testing” regulatory guidance, Drug Metabolites, Dispos,
    6. Testa, B., Kramer, S.D., 2010. The Biochemistry of Drug Metabolism, VHCA, Verlag Helvetica Chimica Acta and Wiley-VCH9783906390543.

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