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Metabolite Profiling and Identification using High Resolution Accurate Mass Spectrometry

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 deliver 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 or the 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 comparison of drug metabolism routes in different species is provided.
  • 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.
  • Cyprotex offers a range of metabolite profiling services depending upon the level of detail and interpretation required.

We encourage the identification of differences in drug metabolism between animals used in nonclinical safety assessments and humans as early as possible during the drug development process. The discovery of disproportionate drug metabolites late in drug development can potentially cause development and marketing delays.

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


Metabolite profiling and identification protocol

Matrices Analyzed In vitro: Typically, microsomal incubations, hepatocyte incubations, HµREL® co-culture, expressed enzyme incubations, and plasma (others available on request)

In vivo: Typically plasma and urine sample pooling or AUC pools (other matrices can be explored)
Instruments Wates Vion IMS QTof
Waters Xevo® G2-S QTof
Data Delivery
  • Various options are available depending on the depth and breadth of metabolite profiling or identification that is required. The resulting data are presented in a summary profile document which includes a summary table and LC-MS chromatograms of the parent and metabolites, along with their low and high energy MS spectra. 
  • In soft spot metabolite profiling, top three metabolites are reported.
  • The number of metabolites reported in comprehensive metabolite profiling depend on agreed tier:
    • Tier 1: Metabolite profiling down to 1% of drug related material
    • Tier 2: Metabolite profiling down to 1% of drug related material with cross species comparison where any observed metabolite is also reporting in any other species/ pool where observed down the limit of detection (LOD) or 0.1 % of drug related material if this is higher than the LOD
    • Tier 3: Metabolite profiling down to the LOD or 0.1 % of drug related material if this is higher than the LOD.


Data from the metabolite profiling and identification service


A summary of all potential metabolites is provided.

NameFormulaMass Differencem/z FoundMass Error (ppm)IdentifierRt (min)
Parent C18H25NO - 272.02010 -1.5 - 8.33
Dehydration -H2O -18.0118 254.1896 -4.9 M5 9.09
Demethylation -CH2 -14.0159 258.1855 -1.0 M3 8.28
Demethylation -CH2 -14.0154 258.1860 0.9 M1 5.72
Oxidation +O +15.9935 288.1949 -1.4 M4 8.85
Oxidation +O +15.9946 288.1930 -0.3 M2 6.67

Figure 1
Metabolite profiling of dextromethorphan metabolism in human liver microsomes.


Questions and answers on metabolite profiling and identification

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) 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 pre-clinical 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 instruments for metabolite profiling studies?

By utilizing high resolution, accurate mass spectrometry, specificity is improved without compromising on sensitivity. Greater confidence in structural elucidation can occur as both MS precursor and 'MS/MS' products ions can only be rationalized with a very small number of elemental combinations – therefore increasing the confidence in structural assignment.


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

Learn More

Learn more about drug metabolism in Chapter 3 of our popular Everything you need to know about ADME guide. 

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