Drug Metabolism

Metabolite Profiling & Identification

Understand the drug metabolism of your compound by identifying which metabolites are formed during in vitro or in vivo studies.

Metabolite profiling and identification is included in Cyprotex's portfolio of ADME services. Cyprotex delivers consistent, high quality data in line with regulatory guidelines, and can adapt protocols based on specific customer requirements.


Metabolite profiles of drug candidates in preclinical and clinical phases are crucial to assess the suitability of animal models for toxicological evaluations. Human drug metabolites are gaining more attention due to their impact on pharmacological activity, safety, and drug-drug interactions. Regulatory agencies emphasize early evaluation of the safety of human metabolites and recommend comparing drug metabolism in animals and humans to identify potential differences that could contribute to drug toxicity. Delayed discovery of such differences can cause development delays.

Before testing on humans, metabolite identification studies are conducted in vitro to evaluate potential differences in metabolic profiles across species. Samples from preclinical and clinical species can also be evaluated early to assess consistency between in vitro and in vivo metabolism. Metabolite Safety Testing (MIST) has become important in drug development, leading to the use of appropriate approaches to identify and semi-quantify human metabolites and assess preliminary toxicological cover. The use of high resolution mass spectrometer (HR-MS) enables semi-quantification without the need for radiolabel compounds and validated assays.

Cyprotex is able to conduct metabolite identification studies on early drug development in vitro samples and in vivo samples from animal studies and human clinical trials.

Profiling and identification of metabolites:

  • Understanding which metabolites are likely to be formed is essential for interpreting pharmacology, pharmacokinetic and toxicology data.
  • Cyprotex’s metabolite profiling and identification service uses either the Waters Vion IMS QTof or Waters Xevo® G2-S QTof to determine high resolution accurate mass of any metabolites – leading to increased sensitivity and enhanced structural characterization.
  • Critical information on the formation of metabolites including, where appropriate, both Phase I and Phase II metabolism and cross species comparisons.
  • Metabolites can be investigated in a number of different matrices including in‑vitro microsomal incubations, hepatocyte incubations, expressed enzyme incubations as well as in vivo samples (plasma, urine, and other matrices).
  • Cyprotex offers a range of metabolite profiling services depending upon the level of detail and interpretation required.


Metabolite Profiling & Identification Protocol


Data from the Metabolite Profiling & Identification Service


Why is understanding the routes of metabolism of a compound important?

There are several reasons why understanding the metabolic profile of a compound is important. Firstly, the FDA Guidance for Safety Testing of Drug Metabolites (2008)1 recommends in vitro evaluation of interspecies differences in drug metabolism between humans and those animals which are expected to be used in preclinical safety assessments. If a metabolite is formed only in humans and is absent in the animal test species, or if the metabolite is present at disproportionately higher levels in humans than in the animal species, then it may be necessary to assess the preclinical safety of the metabolite in question. Secondly, understanding the metabolic liability of a compound is important in directing chemistry. If a compound is very rapidly cleared, then identifying which functional groups are undergoing metabolism will be valuable in understanding how the chemistry may be altered to reduce compound degradation.

What do you recommend as an appropriate strategy for evaluation of the routes of metabolism of a compound?

Before embarking on an in vitro metabolite profiling study, it is recommended that the compound has previously been investigated in the in vitro drug metabolizing system of choice. This will give an indication of the extent of metabolism and whether sensitivity by mass spectrometry is likely to be a problem.

The decision to progress to metabolite profiling and/or identification, and the level of interpretation which is required is driven by the goal of the study. For example, screening multiple compounds through microsomal clearance may only require the identification of the major metabolites, in order to localize any metabolic hot-spots that can then be designed out. A single compound that is screened through multiple hepatocyte species is likely to be require a more complete interpretation if it is to aid in the decision point for preclinical toxicology models (minimize the chances of human specific metabolites). Cyprotex is able to offer services ranging from data acquisition through to full structural elucidation.

What are the advantages of high-resolution accurate mass spectrometry with ion mobility instruments for metabolite profiling studies?

By utilizing high resolution, accurate mass spectrometry and drift time information, specificity is improved without compromising on sensitivity. Both MS precursor and 'MS/MS' product ions can only be rationalized with a very small number of elemental combinations and the addition of a drift time allows for “cleaner” spectrum, overall increasing the confidence in structural assignment.


1) FDA Guidance for Industry: Safety Testing of Drug Metabolites (February 2008)


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Anna Kerins

Anna Kerins

Principal Scientist

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Cyprotex enables and enhances the prediction of human exposure, clinical efficacy and toxicological outcome of a drug or chemical. By combining quality data from robust in vitro methods with contemporary in silico technology, we add value, context and relevance to the ADME-Tox data supplied to our partners in the pharmaceutical or chemical industries.